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Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 843-866 / B817-B840
136. Corneal Topography and Optics Organizing Section: CO Contributing Section: LE, VI
843 - B817
Higher Order Wavefront Aberration Changes After LASIK
E.S. Loft, C.S. Banning, K.Vrochopoulos, J.B. Randleman, R.D. Stulting. Ophthalmology,
Emory University, Atlanta, GA.
Pur pose: To analyze changes in high order aber rations that occu r
a f t e r wavef r o nt- o p t i m i z e d l a s e r i n sit u ke r a t o m i le u si s ( L A SI K) .
Methods: Retrospective analysis of eyes that underwent LASIK with the Wavelight
Allegretto Wave excimer laser. Wavefront aberrations were measured preoperatively and
at 3 months postoperatively with the Nidek OPD-Scan ARK-10000 corneal analyzer.
High order aberrations measured (in microns) included total high order, total Coma,
total trefoil, total quadrafoil, total spherical aberration, and total high astigmatism.
Results: Forty-six eyes from 24 patients were included in analysis. Average patient age was
42 years (range 23-62 years). Average preoperative spherical equivalent refraction (SE) was
-3.80 D (4.38 to -9.75 D). Average postoperative SE was -0.09 D (2.13 to -1.38 D). There were
no significant differences in high order aberrations for total high order (0.195 vs. 0.194 microns,
p = 0.99), coma (0.076 vs. 0.081 microns, p = 0.68), trefoil (0.132 vs. 0.148 microns, p = 0.54),
quadrafoil (0.064 vs. 0.044 microns, p = 0.50), or spherical aberration (0.030 vs. 0.035, p =
0.55). High order astigmatism was reduced postoperatively (0.041 vs. 0.019 microns, p = 0.02).
Conclusions: LASIK with the Wavelight Alegretto Wave excimer laser did not significantly
increase any high order aberrations, and reduced high order astigmatism.
CR: E.S. Loft, None; C.S. Banning, None; K. Vrochopoulos, None; J.B. Randleman,
None; R.D. Stulting, None.
Support: None.
845 - B819
844 - B818
Corneal Elevation and Optical Zone Size Change Over Time After Conductive
J.Choremis, H.Wu. Ophthalmology, New England Eye Center, Tufts University, Boston,
Purpose: Conductive keratoplasty (CK) steepens the cornea centrally. This study is the first to
look at elevation changes of the cornea over time, as well as the optical zone size change over time.
Methods: Sixty-five patients who had CK were screened to enter the study, and 25 were excluded
due to missing data or prior surgery. All patients had BCVA of 20/20 or better. Treatments ranged
from 8 to 24 spots. Orbscan topography was performed pre-operatively as well as postoperatively at
1 week, 1 month and 2 month visits. Data collected included central anterior elevation (AE) as well as
the highest point on the anterior elevation map (AEH), posterior elevation (PE) as well as the highest
point on the posterior elevation map (PEH), spherical equivalent and optical zone size. Results:
Eighteen right eyes and 22 left eyes were entered into the study. Mean age of patients was 53 years
(+/- 3.9 years). Thirteen patients had retreats (32.5%). Mean spherical equivalent pre-operatively
was +0.58 diopters (S.D.+0.54). When comparing the highest point in the anterior elevation over
time (AEH), there was a large mean increase (0.029 mm) in elevation at 1 week (p<0.01), which
decreased slightly at 1 month by a mean of 0.003 mm (p=0.005) and then stabilizes at the 2 month
visit, with no statistically significant difference compared to the 1 month values. Similar results are
seen in the AE group. Interestingly, when groups are split into 8, 16 and 24 spots of treatment in the
AEH group, values showed a statistically significant increase in height in the 16 and 24 spot groups,
yet in the 8 spot group, the mean post-operative elevation was not statistically different compared to
pre-op values. Similar results are seen with the AE group. Results for the PE and PEH groups will be
presented. Spherical equivalent changed by a mean of - 1.7 D at 1 week, with no statistically significant
change from 1 week to 1 month. However, there was a decrease in the myopic change from 1 month
to 2 months follow-up. Again the 8 spot group had no statistically significant change in spherical
equivalent when comparing pre-operative and post-operative refraction. Effective optical zone size
increased in size from 1 week to 1 month (p=0.003), and slightly again from 1 month to 2 months
(p=0.05). Conclusions: In CK, corneal elevation increases at the 1 week post-operative visit, and
stabilizes or decreases slightly thereafter. This is only true for the 16 and 24 spot treatment groups.
Eight spot treatments did not show a statistically significant change in elevation after treatment
which may suggest a less effective treatment pattern. Loss of elevation over time correlated with
regression and the need for retreatment.
CR: J. Choremis, None; H. Wu, None.
Support: Canadian National Institute for the Blind
846 - B820
A Method for Analysing Corneal Topography Data
J.Einighammer, T.Oltrup, T.Bende, B.Jean. Division Experimental Ophthalmic Surgery,
University Eye Hospital Dept. I, Tuebingen, Germany.
Purpose:A method is presented to statistically analyse measurements of corneal topography devices
regarding trueness and reproducibility in approximation to ANSI Z80.23 draft. Methods: All
calculations are based on difference maps of topographies. These differences are weighted to get a
uniform sampling distribution. For trueness testing, differences of a series of measurements against
a known reference surface are taken. Trueness is quantified in terms of offset through the mean of
mean difference (MOM), the smaller the better. For reproducibility testing, difference maps are
calculated from corresponding measurements. Reproducibility is expressed by standard deviation
of the mean difference (SDM) and mean standard deviation of differences (MSD), the smaller the
better. All values are calculated for different zones (central c=0-3mm, middle m=3-5mm, outer
o=5-7mm, total t=0-7mm) and indexed (e.g. cMSD for central). Axial refractive power maps of test
objects in three categories were measured on two systems (A: Keratron Scout (R) and B: Technomed
C-Scan (R)): calibration spheres (42,2 & 48,2 D), test spheres (multifocal, simulated ast, simulated
central keratoconus) and 4 human corneas. Results: Both systems show small offset thus good
trueness when measuring the calibration spheres (A: tMOM=0.10/0.06; B: tMOM=-0.02/0.07). For
calibration/test spheres the SDM and MSD are smaller for system A than for B (A: tSDM=0.06/0.17,
tMSD=0.05/0.06; B: tSDM=0.26/0.28, tMSD=0.12/0.21). For human corneas the SDM of system A
is higher than for B, while the MSD is slightly smaller (A: tSDM=0.40, tMSD=0.19; B: tSDM=0.23,
tMSD=0.23). Means: system A’s reproducibility is better for calibration and test spheres than
for human corneas. System B’s reproducibility for human corneas is better than A’s, but much
worse for calibration and test spheres. On both systems the MSD decreases going to the periphery,
while the SDM increases (e.g. human corneas. A: cSDM=0.31, mSDM=0.40, oSDM=0.44; B:
cSDM=0.20, mSDM=0.22, oSDM=0.25). Conclusions: The proposed method allows an analysis
of topography data for quantification of trueness and reproducibility. The values MOM, SDM and
MSD can be useful for analysing and comparing the output of different corneal topography devices
or for analysing the behaviour of a specific corneal topography device when measuring different
test objects including human corneas. When doing trueness testing for known test objects other
than calibration spheres, with more complex surface features, this method can likewise be used to
assess a system’s resolution.
CR: J. Einighammer, None; T. Oltrup, None; T. Bende, None; B. Jean, None.
Support: None.
Ray Trace Analysis of Visual Aberrations After Laser Vision Correction Surgery
D.M. Silver1, A.Csutak2. 1Applied Physics Laboratory, Johns Hopkins University, Laurel,
MD; 2Ophthalmology, University of Debrecen Medical and Health Science Center Faculty
of Medicine, Debrecen, Hungary.
Purpose: To examine the dependence of visual aberrations on the relation between ablation diameter,
pupil diameter, other ocular geometry, and light scattering emanating from disrupted collagen
fibrils in the peripheral region of the ablated corneal stroma following laser refractive surgery.
Methods: Computer calculations are used for ray trace analysis of light passing through the
cornea, aqueous, pupil, lens, and vitreous to the retina. Light rays are affected by the transition
from the curvature of the cornea in the ablated zone to the curvature of the untreated regions
of the cornea. Laser ablation also damages the structure of collagen fibrils in the ablation
region of the stroma by terminating fibrils along the rim of the ablation circle. From the
physical dimensions of the fibrils, this change in fibril continuity is assumed to give rise to
Mie scattering, which is predominantly in the forward direction. The computational problem
is parameterized with respect to physiological and anatomical properties of the ocular path
(radii of curvature, thicknesses, indexes of refraction, pupil diameter, and relative positions of
structures within the globe) as well as forward scattering angles from damaged stromal fibrils.
Results: Graphs of the paths of parallel and oblique incident light rays illustrate
the effect of refraction and scattering and their dependence on the relation
between the ocular parameters, changes in corneal curvature and scattering
angles. Mie scattering from the perimeter of an ablation zone significantly affects
the effectiveness of the iris to block aberrant light rays from entering the pupil.
Conclusions: The uniformity and ordered arrangement of collagen fibrils in stromal lamellae
is important for the transparency of the cornea. Normally, the fibrils run parallel to the surface
of the cornea, extending limbus to limbus, within alternating layers of lamellae. Light rays
diffracted by the fibrils tend to cancel each other by destructive interference, leaving the
normal undiffracted rays unaffected. In addition to changing the curvature of the cornea, laser
ablation of stromal layers gives rise to sources of light scattering along the circumference of the
ablation zone. The present calculations have implications for the quantitative understanding of
concepts such as the effective corneal refractive diameter, the entrance pupil and the physical
pupil relative to visual aberrations that are not blocked by the iris.
CR: D.M. Silver, None; A. Csutak, None.
Support: None.
847 - B821
848 - B822
Comparison of Hartmann Shack Aberrometer and OPD Scan in Postkeratoplasty
Wavefront Analysis of Interrupted Suturing versus Combined Interrupted and
Continuous Suturing Techniques
W.M. Munir, E.Y. Tu, C.E. Joslin, T.T. McMahon. Ophthalmology, University of Illinois at
Chicago, Chicago, IL.
Purpose: To compare the wavefront aberrations after penetrating keratoplasty using a
proprietary Hartmann Shack (HS) aberrometer versus an OPD Scan (Nidek Co, Japan), and
to determine if a combined interrupted and continuous suturing technique reduces higher
order aberrations post-operatively when compared to an interrupted-only suturing technique.
Methods: Eyes were divided into two groups: eyes sutured using a combined interrupted and
continuous suturing technique (Group 1), and eyes sutured using an interrupted-only suturing
technique (Group 2). Whole eye wavefront aberrations were measured at both 3mm and 6mm
pupils using an HS aberrometer, and at a 6mm pupil using an OPD-Scan. Data was collected
at both one month and two month post-operative visits. Individual 3rd, 4th, 5th, and 6th order, as
well as total higher order aberrations were calculated for all eyes using both wavefront devices.
Results: Data collection on a total of five eyes demonstrated that the HS aberrometer generated
higher values at 6mm versus 3mm pupil size. In Group 1, one month HS total higher order RMS
was 8.355 at 6mm and 1.794 at 3mm. The Group 2 HS total higher order RMS at one month was
not measurable at 6mm, and 0.5755 at 3mm. OPD RMS error was lower than HS at 6mm pupil size,
with one month data revealing a Group 1 total RMS of 2.573 and Group 2 total RMS of 1.174. At two
months, the HS RMS error for Group 1 was 167.7 at 6mm and 1.274 at 3mm. The OPD total RMS at
two months for Group 1 was 2.476. An association in Group 1 eyes between best corrected visual
acuity and wavefront aberrations was not clearly delineated. Although manifest refraction did not
correlate with the total RMS using the HS aberrometer in Group 1 eyes, despite a range of spherical
equivalents from -0.50 to -9.50 diopters, and an astigmatism range of +0.50 to +6.00 diopters, greater
astigmatism was associated with a greater total RMS using the OPD. Over all groups, time points,
and devices, 3rd and 4th order aberrations were consistently greater than 5th and 6th order aberrations.
Conclusions:HS higher order aberrations were greater at 6mm pupil size, likely secondary to
graft edge effect, than the OPD in these eyes. No clear difference in wavefront aberrations was
noted between the two different suturing techniques. Visual acuity was not related to wavefront
aberrations, while the OPD showed greater RMS error with increasing astigmatism. The overall
most common whole eye wavefront aberrations seen in early post-keratoplasty eyes are 3rd and 4th
order aberrations.
CR: W.M. Munir, None; E.Y. Tu, None; C.E. Joslin, None; T.T. McMahon, None.
Support: None.
Variability in Retinal Image Quality With Tear Film Behavior After Blink
K.Y. Li1A, G.Yoon1B, G.Pan1C. AInst of Optics/Mathematics, BOphthalmology/Center for
Visual Science, CCenter for Visual Science, 1University of Rochester, Rochester, NY.
Purpose: Recent studies have shown that the higher order aberrations due to the changes in
the tear film can be readily measured using a Shack-Hartmann wavefront sensor (MontésMicó et al., Ophthalmology 2004). The goal of this study is to demonstrate the effectiveness
of measuring tear film dynamics quantitatively using the Shack-Hartmann wavefront sensor
and its effect on retinal image quality. Methods: The wavefront aberrations of five subjects,
4 non-contact lens users (NCL) and 4 contact lens users (CL), were measured in real time
(10 frames per second) with a Shack-Hartmann wavefront sensor. Subjects were asked to
fixate, for as long as possible voluntarily, on a point source for the entire measurement process
(NCL: 27.78±16.45 sec. CL: 17.07±10.17 sec.). CL subjects were measured immediately after
contact lens removal. Several measurement sessions were performed for each subject (at
least 30 min apart). Higher order rms and retinal image quality metrics such as Strehl ratio,
MTF and convolved image were computed to quantitatively compare the two groups on how
ocular aberrations and retinal image quality are affected by tear film dynamics. Results:
The variability in higher order rms (HORMS) and retinal image quality was measured by
means of standard deviation over time separately for the two groups. Our results show that
variability was significantly higher for CL subjects (.0446±.0219μm HORMS) than NCL
subjects (.0156±.0112μm HORMS). For HORMS, Strehl ratio and volume MTF, the variability
for the CL group was approximately three times larger than that of the NCL group. The
behavior of radially averaged MTF was nearly identical to that of the Strehl ratio. In CL
subjects, all metrics used in this study suggest a greater rate of decrease on average in retinal
image quality over time. Time-lapse image sequences of the wavefront, PSF and convolved
retinal image confirm correlations between retinal image qualities and tear film break up
as well. By observation, the behavior of the convolved retinal image correlates better with
Strehl ratio and volume MTF than HORMS. Conclusions: The real time Shack-Hartmann
wavefront sensing is capable of observing behavior differences in tear film dynamics. Strehl
ratio and volume MTF are better metrics to assess tear film dynamics. This same technique
can be used as an objective tool to diagnose dry eye symptoms.
CR: K.Y. Li, None; G. Yoon, Bausch and Lomb F, C; G. Pan, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 843-866 / B817-B840
136. Corneal Topography and Optics Organizing Section: CO Contributing Section: LE, VI
849 - B823
850 - B824
Evaluation of Customized Ablation Based on Corneal Wavefront Analysis for Myopic
Corrections in Virgin Eyes
Q.Ren1, C.Zhou1, M.Jin2, X.Wang2. 1Biomedical Engineering, Shanghai Jiao-Tong
University, Shanghai, China; 2Department of Ophthalmology, Guangzhou No.1 People’s
Hospital, Guangzhou, China.
Correlation Between Corneal and Total Wavefront Aberrations Measured at Various
Optic Zones Pre- and Six Month Post-LASIK
R.M. Hazarbassanov1, A.Grinbaum2, D.Varssano3, I.Kaiserman4. 1American Lasers Med
Ctr, Rishon-Lezion, Israel; 2Department of ophthalmology, Sheba Medical Center, TelHashomer, Tel-Aviv, Israel; 3Department of ophthalmology, Tel-Aviv Sourasky Medical
Center, Tel-Aviv, Israel; 4Department of ophthalmology, Hadassah University Hospital,
Jerusalem, Israel.
Purpose:To analyze the cor relation between cor neal and total wavefront
aber rat ions at va r ious opt ic zones (OZs) before and af ter LASI K.
Methods: Thirty-one eyes of 20 myopic- astigmatic patients (-1.25D to -7.125D) that
underwent LASIK were measured using the Oculus corneal topographer and a Tscherningtype wavefront analyzer pre-operatively and 6 month post-op. Corneal and total
wavefront aberrations (WFA) were calculated up to the 6th Zernike order and the highorder aberrations’ root mean square (RMS) was calculated in the same reference plane.
Results: There was significant correlation (P<0.05) between the LASIK induced corneal
aberrations and the total WFA for coma-like aberration at 3rd (OZs 5>4>6), 4th (OZs
5>6>4), 6th (OZs 5>6>4) order Zernike coefficients, for spherical aberrations at all OZ’s
(5>6>4) and for total WFA RMS (OZ’s 5>6>4). The fraction of LASIK induced total
WFA atributed to the cornea was largest for OZ=5. We also found a strong correlation
between the LASIK induced change in the RMS of high-order WFA (OZ=5mm) and the
changes in corneal topography’s keratoconus index, Q-mean value and mean keratometry.
Conclusions: Some LASIK induced aberrations in the mid periphery (4-6mm), such as coma
and spherical, are mostly of corneal origin while others are not. Thus, ideal customized ablations
algorithms must take both corneal and total wavefront aberrations into consideration.
CR: R.M. Hazarbassanov, None; A. Grinbaum, None; D. Varssano, None; I. Kaiserman,
Support: None.
851 - B825
852 - B826
Purpose: To evaluate the outcomes of corneal wavefront guided laser in situ keratomileusis(LASIK)
for virgin eyes in visual acuity, contrast sensitivity, corneal shape and corneal wavefront aberrations.
Methods:This study included 57 myopic virgin eyes of 30 patients with spherical equivalence 4.50±1.46D(-1.63 to -7.88D), astigmatism less than 2.5D. The corneal topography system: Keratron Scout
(Opticon 2000, Milano, Italy) was used to measure anterior corneal shape and to analyze the anterior
corneal wavefront aberrations preoperatively and/or postoperatively. The corneal wavefront aberration
data obtained from above analyses combined with manifest refraction were linked to a flying spot
scanning excimer laser refractive system: ESIRIS (Schwind Eye-Tech. Solution GmbH, Kleinostheim,
Germany) with its Optimized Refractive Keratectomy(ORK) software to treat the myopic patients
without any prior refractive treatment. In addition to visual acuity, contrast sensitivity (CSV-1000E,
Vector Vision, San Diego, CA) with and without glare was evaluated for the visual quality postoperatively.
Results: After one month of following-up, all the eyes but two got uncorrected visual acuity(UCVA)
1.0 or better. The monocular contrast sensitivity(CS) without glare didn’t change significantly except
for a slight reduction at the 12cpd frequency(P=0.002). The monocular contrast sensitivity with glare
reduced at 3,12,18cpd(P<0.05), but not at 6cpd(P>0.05). Spherical aberration(SA), coma and the higherorder root-mean-square (HO-RMS) value of the cornea increased significantly(P<0.05 paired samples
t test) after the treatment either with 6mm or 3mm pupil diameter. The amount of achieved correction
was positively correlated with the changes of SA, coma and HO-RMS(P<0.01) either with 6mm or
3mm pupil diameter, except for coma with 6mm pupil diameter(P>0.05). The changes of eccentricities
were negatively correlated with the amount of achieved correction (P<0.01). The changes of both SA
and HO-RMS(6mm pupil) value were negatively correlated with the change of either Ef or Es(P<0.01).
Conclusions: Evaluations of the early results showed that corneal wavefront guided LASIK for
myopic virgin eyes can get good result in both visual acuity and mesopic contrast sensitivity
level with and/or without glare. The increasing in corneal higher order aberrations has significant
correlation with the achieved correction after the treatment and the changes of corneal shape from
prolate to oblate shape.
CR: Q. Ren, None; C. Zhou, None; M. Jin, None; X. Wang, None.
Support: STCSM046105014
Comparison of Accuracy of Zernike Polynomials and Fourier Series in Corneal
Surface Representation
A.Alchagirov, S.D. Klyce, M.K. Smolek, M.D. Karon. Ophthalmology, LSU Eye Center,
New Orleans, LA.
Purpose: There is a controversy about the advantages of Fast Fourier Transforms (FFT) versus
Zernike series for the fitting of corneal data. Unlike the Zernike method, FFT is not an approximation,
but an exact transformation between spatial and frequency domains. FFT error is introduced during
data preparation (mapping onto an evenly spaced grid), and therefore is not comparable with the
fitting error of the Zernike series. We propose a novel method that allows direct comparison of
the accuracy of both approaches. Methods: Reconstructed elevation data was analyzed for 5
Tomey corneal topography exams for each of 4 categories: mild keratoconus (KC1), penetrating
keratoplasty (PKP), myopic refractive surgery (MRS), and normal (NRM). Best fit reference spheres
were used to obtain residual elevation data. For the Fourier approach, data was translated into the
first quadrant of the XY plane to allow the use of the even
extension of the Fourier series. Incomplete 3D Fourier series
and Zernike polynomials of various orders were fitted to
the corneal elevation data using the least-squares and the
RMS errors of the fits were compared. Mean RMS errors
and standard deviations were calculated. Results: Both
Fourier and Zernike methods exhibit good fitting accuracy;
however, at > 19 Zernike orders the design matrix of the fit
becomes singular and additional terms result in an increase
and instability of RMS error (see figure). The Fourier fit
was stable and non-singular at > 35 orders. It’s best RMS
error was ~3 times smaller than the Zernike’s for all cornea
categories, as shown in the figure. The limiting number of
fitting parameters for the Fourier series was of the same order as the Nyquist limit on the number of
frequencies for the FFT algorithm. This indicates that the Fourier series in this limit is an adequate
substitute for the FFT. Conclusions: The proposed method allows direct comparison of fitting
accuracy of the Zernike polynomial series and the Fourier cosine series. The Zernike polynomials
were shown to become unstable after a critical number of terms, which limited its accuracy. The
Fourier series was shown to be significantly more accurate at higher orders.
CR: A. Alchagirov, None; S.D. Klyce, None; M.K. Smolek, None; M.D. Karon, None.
Support: NIH EY014162 (MKS); EY003311 (SDK); EY02377
853 - B827
Change of Corneal Wavefront Parameters After Phakoemulsification - A Comparison
Between Corneal and Corneoscleral Incision
A.Kissner, M.Kohlhaas, E.Spoerl, L.E. Pillunat. Ophthalmology, University Eye Clinic,
Dresden, Germany.
Purpose: The corneal as well as the corneoscleral incision is considered to be standard in
cataract surgery. However, both incision-techniques point out advantages and disadvantages. We
investigated, if the way performing the incisions has different effects on the corneal wavefront
especially on higher order aberrations. Methods: Each of 26 patients (6 men, 20 women),
aged between 62 and 85 years (average 74.9 years), received a standard phakoemulsification
by performing a corneal incision (2.9 - 3.2 mm, av = 3.12 ± 0.12) on the right eye and a
corneoscleral incision (2.9 - 4.0 mm, av = 3.21± 0.2) on the left eye. The corneal surface
was preoperatively investigated by using the Corneal wavefront instrument KeratronScout,
(Schwind Company, Germany), as well as by the normal topography Keratometer (TechnomedCompany, Germany). After at least 6 weeks the same parameters were measured again.
Results: The following data were measured pre- and post-operatively:
(min - max; av ± SD) Group 1 corneal incision: RMS: 0.1-3.13; 0.98 ± 0.53; (-0.71 - 1.89;
1.15 ± 0.51) µm Z(2.0)=Defocus: -6.42-15.10;3.64 ± 6.20; (-1.31-5.69; 1.83±1.66) µm
Z(2±2)=Astigm.: 0.07-2.11;0.87 ± 0.53; (0.15 - 2.84; 1.14 ± 0.69) µm Z(3±1)=Coma: 0.041.12; 0.54 ± 0.28; (0.09-1.32 ; 0.54 ± 0.30) µm Z(3±3)=Trefoil: 0.07-0.91; 0.35 ± 0.22; (0.131.23; 0.64 ± 0.33) µm Z(4.0)=Sph Abe: -0.07-1.03; 0.43 ± 0.23; (0.14-0.91; 0.40 ± 0.19)
µm Group 2 corneoscleral incision: RMS: 0.44-1.89;0.96 ± 0.35; (0.53 - 1.86; 0.96 ± 0.31)
µm Z(2.0)=Defocus: -7.28-17.60;3.72 ± 6.52; (-1.48- 4.41;1.59±1.38) µm Z(2±2)=Astigm:
0.2 -2.43; 0.85 ± 0.57; (0.08 - 3.13; 1.14 ± 0.70) µm Z(3±1)=Coma: 0.03 - 1.0; 0.5 ±
0.28; (0.10 - 1.00; 0.50 ± 0.24) µm Z(3±3)=Trefoil: 0.05-0.86; 0.36 ± 0.20; (0.02 - 1.21;
0.44 ±0.27) µm Z(4.0)=Sph Aber:-0.01-0.68; 0.43 ± 0.18; (0.01- 1.03; 0.42 ± 0.22) µm
The statistic analysis of the pre- and postoperative data within the same group
indicated significant differences (p < 0.05) in group 1 for Z(3±3)=Trefoil and in group
2 for Z(2±2)=Astigmatism. Comparing the postoperative data of the corneal and the
corneoscleral incision, there was only a significant difference (p < 0.05) for Z(3±3)=Trefoil.
Conclusions: In accordance to the induced higher order aberrations both surgical techniques
can be seen equivalent.
CR: A. Kissner, None; M. Kohlhaas, None; E. Spoerl, None; L.E. Pillunat, None.
Support: None.
Correlation of Wavefront Measurements With Refraction and Corneal Topography in
Patients Submitted to Penetrating Keratoplasty
A.D. D. Oliveira, A.Hofling-Lima, R.Chalita, M.Campos. Ophthalmology, Federal
University of São Paulo, São Paulo, Brazil.
Purpose: To evaluate wavefront measurement in patients after penetrating
keratoplasty(PK) and analyze its correlation with refraction and corneal topography.
Methods: 36 eyes of patients that underwent PK at least 1 year prior to examination with
were enrolled. Ophthalmologic exam, corneal topography and wavefront maps were
compared. Wavefront analysis was performed with a Hartmann-Schack Sensor using a
6mm-pupil size, measuring up to 6th order of higher order aberrations(HOA).Results:
The wavefront device was able to capture all eyes with good reproducibility. Wavefront
refraction showed a statistically significant correlation with Manifest and Cycloplegic
refraction in those eyes. Manifest refraction cylindrical component correlated well
with wavefront refraction cylindrical component. Even eyes with high amounts of HOA
showed a good correlation between the refractions analyzed in this study. HOA values
were greater than in normal eyes, particularly trefoil, tetrafoil and secondary astigmatism.
Conclusions: Wavefront measurements were possible in highly aberrated post-PK eyes.
Wavefront analysis in post-PK eyes is a valuable tool in measuring refractive errors and
ocular aberrations.
CR: A.D.D. Oliveira, None; A. Hofling-Lima, None; R. Chalita, None; M. Campos,
Support: None.
854 - B828
Factors Affecting Repeatability of Peripheral Corneal Topography Measures
A.Cervino1,2, J.M. Gonzalez-Meijome3, A.Queiros3, J.Jorge3. 1Applied Physics, Universidad
de Santiago de Compostela, Santiago de Compostela, Spain; 2Neurosciences Research
Institute, Aston University, Birmingham, United Kingdom; 3Physics (Optometry),
Universidade do Minho, Braga, Portugal.
Purpose: Repeatability of peripheral corneal topography measures is important in
clinical practice to evaluate the progression of corneal distortions. Several studies
addressed the repeatability at the corneal center, but there are not references on the
consistency of peripheral measures, either within one session or between different sessions.
Methods: Sixty eyes from thirty young adults (9 males, 21 females), aged 21 +/- 2,46 years,
where evaluated with the Medmont E300 corneal topographer. Three readings were obtained
in each of three different sessions at weekly intervals by an experienced observer. Nine
locations on the central 3,5 mm where examined. While measuring, a second observer recorded
incidences such as lack of fixation, need of repositioning on the chin-rest or tear instability.
Results: No differences were observed for either the intrasession or intersession repeatabilities
with gender or eye examined. At the center, each individual measurement within one session
was not different from the mean value (mean diff.<0,03D; p>0,05; r2>0,99). The same happened
between sessions (mean diff.<0,025D; p>0,05; r2>0,98). For peripheral measurements, all locations
displayed significant differences between readings either intrasession or intersessions (p<0,05).
Those differences were smaller than 0,01D when comparing each measurement against both
the mean value within the same session (range 0,01 to 0,04D) and between different sessions
(range 0,02 to 0,09D). Only differences for the most peripheral superior location were higher than
0,01D. Comparing single values against the mean of three within the same session, the maximum
difference was found for the most peripheral superior measurement. Fixation stability did not play a
significant effect on repeatability. Tear instability showed an effect, particularly at the most peripheral
superior location. Head relocation was needed in 43% of the eyes due to facial physiognomy-related
problems. Severe and moderate tear instability was present in 17% and 55% of the eyes, respectively.
Conclusions: This study is of great importance for any clinical research involving corneal topography
evaluation. Although not clinically significant even for the most stringent criteria, there was some
evidence that consistency of peripheral topography measurements could be adversely affected by
external parameters as tear film instability or the need of head repositioning on the chin-rest.
CR: A. Cervino, None; J.M. Gonzalez-Meijome, None; A. Queiros, None; J. Jorge, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 843-866 / B817-B840
136. Corneal Topography and Optics Organizing Section: CO Contributing Section: LE, VI
855 - B829
856 - B830
Partial Coherence Laser Interferometry versus Autokeratometry in Measuring
Corneal Curvature in a Malay Population
J.Loo1, A.Foong2, C.Koh2,3, A.Ang2, S.-M.Saw3, D.Tan1,2, T.-Y.Wong1,2. 1Ophthalmology,
Singapore National Eye Ctr, Singapore, Singapore; 2Singapore Eye Research Institute,
Singapore, Singapore; 3National University of Singapore, Singapore, Singapore.
Purpose:Few studies have evaluated the accuracy of corneal curvature measurements
by the IOL master. The purpose of this prospective study was to compare
measurement of corneal curvature between the autokeratometer and the IOL
Master in a group of randomly selected Malays between the ages of 40-79 years.
Methods: 83 subjects (n=83, mean age=56.8, male:female=28:55) were included
in this comparative study. The corneal curvature of the right eye were measured
by automated keratometer (Canon RK 5 Auto Ref-Keratometer, Canon Inc Ltd.,
Japan), followed by IOL Master (Carl Zeiss, Germany). Two readings (one each at the
horizontal and vertical meridians) were taken with the autokeratometer, while six
readings (three each at the horizontal and vertical meridians) were taken with the IOL
Master. The mean of the readings obtained through the two methods are obtained.
Results: Measurements taken with the autokeratometer were generally higher than those
taken with the IOL Master ( mean ± SD of the difference between autokeratometer and
IOL Master readings being 0.048 ± 0.044 mm). The corneal curvature measurements
performed by both the autokeratometer and IOL Master were highly correlated
(Pearson’s correlation coefficient 0.985). A Bland-Altman plot showed that the 95%
limits of agreement between the two methods lay between 0.135 and -0.039 mm.
Conclusions:High correlation in keratometry readings between the autokeratometer and IOL
Master and the autokeratometer suggests that both instruments may be used interchangeably.
In performing biometry prior to cataract surgery. the IOL Master may be used to measure both
the axial length and corneal curvature, obviating the need to use 2 separate instruments.
CR: J. Loo, None; A. Foong, None; C. Koh, None; A. Ang, None; S. Saw, None; D. Tan,
None; T. Wong, None.
Support: NMRC
Validation of Corneal Power Following LASIK Using Tomey Topographic Modeling
System (TMS) in Pseudophakic Patients
P.Kelley. Ophthalmology, UT Southwestern, Dallas, Dallas, TX.
Purpose: To assess the validity of the keratometric ring values on the Tomey
Topographic Modeling System (TMS) as a predictor of corneal power in postLASIK patients when preoperative keratometric values are not available.
Methods: Data was retrospectively collected on 9 eyes of 6 patients who underwent
cataract surgery and had a previous history of LASIK surgery in the same eye. Data
collected included pre and post LASIK automated keratometry, post-LASIK refraction
at 3-6 months, TMS data prior to cataract surgery, power of the intraocular lens (IOL)
implanted, and spherical equivalence (SE) at 1 month after cataract surgery. The post-LASIK
K value was “back-calculated” from the IOL and postoperative SE using the double-K
SRK/T formula. The historical K method was also used to predict the post-LASIK K value.
Results: The mean “back-calculated” post LASIK K value was 39.42 +/- 5.31 D, the historical
K value was 39.54 +/- 5.80 D. The mean K value of the first, second, third, fourth, and
fifth ring on the TMS were 40.06 +/- 6.2 D, 39.64 +/- 5.77 D, 39.71 +/- 5.47, 39.92 +/- 5.22
D , and 39.98 +/- 5.14 D. The third ring correlated the most with the back-calculated K as
well as with historical K (Rsqu=0.998, p=0.24), followed by the second ring (Rsqu=0.98).
Conclusions: The TMS is a useful tool to predict post LASIK corneal power based on the
third ring value, whenever the historical method cannot be used.
CR: P. Kelley, None.
Support: None.
857 - B831
858 - B832
Modal and Zonal Analysis of Wavefront Aberrations After Lasik
A.Benito1, P.M. Prieto1, M.Redondo2, P.Artal1. 1Laboratorio de Optica, Universidad de
Murcia, Murcia, Spain; 2Clinica Ircovision, Cartagena, Murcia, Spain.
Purpose: To study the magnitude and nature of the wavefront aberrations induced after standard
LASIK refractive surgery. To compare the results provided by a modal Zernike polynomial
analysis to those obtained with a zonal fitting of the wavefront aberration in eyes after LASIK.
Methods: Ocular wavefront aberrations were measured using our own research prototype
near-infrared Hartmann-Shack sensor for a 6 mm pupil diameter, sampled with around
300 microlenses. Measurements were done before and two months after standard LASIK
surgery. Three groups of patients with different pre-op refractive errors were included:
moderate myopia (n=10), low myopia (n=10) and low hyperopia (n=4). For each eye and
condition, wavefront aberrations were analyzed both by using modal fitting to Zernike
polynomials up to the 8th order and Fourier-analysis zonal fitting of the wavefront slopes.
Results: The root mean square of aberrations after LASIK was found to be correlated to the
pre-op refractive error (r2=0.15). Coma induced by surgery was well correlated (r2=0.39).
However, we found a somehow no expected behavior of 4th-order spherical aberration (SA).
While subjects with low refractive errors showed a clear correlation of induced SA with pre-op
refraction (r2=0.68), this tendency did not hold for the larger myopic subjects (above 5 D). For
these subjects, lower than expected values of induced SA were found, while coma consistently
increased. The wavefront zonal fitting confirmed these results, excluding the possibility of
these results being an artifact of the Zernike modal fitting algorithm for this type of eyes.
Conclusions: We found an increase of aberrations after standard LASIK as previously
reported. Induced SA and coma are correlated with pre-op refractive error for a limited
range, the SA. However, induced SA in patients with larger myopia was quite small, failing
to follow the general tendency. These results were confirmed by Fourier-analysis zonal fitting.
The presence of induced coma not secondary to SA can be explained by the overlapping of a
portion of the transition zone with the subject’s pupil, caused by ablation decentration.
CR: A. Benito, None; P.M. Prieto, None; M. Redondo, None; P. Artal, None.
Support: MCyT, Spain, grant BFM20010391
A New Method to Combine Zernike Terms Into a Single Metric
A.M. Mahmoud1A, M.D. Twa1B, C.Roberts1A. AOphthalmology, BOptometry, 1The Ohio State
University, Columbus, OH.
Methods: A retrospective study of 60 eyes of 30 patients who received Zywave wavefront
measurements (Bausch&Lomb, Rochester, New York), cycloplegic and manifest refractions,
high and low contrast cycloplegic refractions, and cylcoplegic autorefractions before and six
months after LASIK using one of two lasers was conducted. Wavefront measurements were
calculated over a 6 mm diameter pupil. The set of coefficients of the resulting polynomial were
treated as a set of error vectors. Spherical terms were treated as magnitude with no direction.
All other corresponding pairs of terms were treated as components of a vector with magnitude,
cylinder, and direction. The resulting sets of vectors were then summed. The 3rd order terms ,
4th order terms, and total 3rd through 5th order tersm were analyzed. Regression analyses were
performed comparing the average error vector, the magnitude of the error vector, and the cylinder
of the error vector to the logMAR visual acuity and defocus equivalent data. Higher-order RMS
(total 3rd through 5th) were calculated and compared to the logMAR and equivalent defocus data.
Results: Pre-operatively, the cylindrical component of the 4th order error vector correlated
significantly with all defocus equivalents (p < 0.0004). There were no significant
correlations for total higher-order RMS wavefront error pre-operatively. After LASIK,
the magnitude of the 4th order error vector, the average of 4th order error vector, and RMS
error correlated significantly with logMAR acuity and defocus equivalent (p < 0.008).
Conclusions: This vector-based metric derived from Zernike coefficients is superior to total
RMS error for prediction of vision and refractive error before LASIK. After LASIK, both
metrics were similarly influenced by changes in spherical aberrations.
CR: A.M. Mahmoud, None; M.D. Twa, None; C. Roberts, None.
Support: None.
859 - B833
860 - B834
IOL Power Calculation in Eyes Having Undergone Myopic Refractive Surgery and
Cataract Surgery
C.E. Joslin, E.Y. Tu, T.T. McMahon, J.Sugar. Ophthalmology and Visual Sciences,
University of Illinois Chicago, Chicago, IL.
Purpose: To compare method accuracy in intraocular (IOL) power calculation for eyes that have undergone
myopic refractive surgery and phacoemulsification. Methods: Five consecutive eyes with previous
refractive surgery (pre-refractive surgery SE: -4.40 ± 1.90 (SD; D); RK = 4, LASIK = 1) and uncomplicated
phacoemulsification were analyzed. IOL power was estimated using our modified contact lens overrefraction
(CLO) method and the SRK/T formula. Multiple methods for IOL power calculation were analyzed by
comparing IOL prediction error, which is the difference between the implanted and predicted IOL. Methods
were analyzed using both the single-K and double-K versions of the SRK/T. Methods analyzed include manual
keratometry (K’s), clinical history, our CLO method, Hamed’s adjusted post-op K’s, Maloney’s modified
corneal topography, Wang’s modified Maloney method, and the Feiz-Mannis method. Historical and CLO
methods were calculated at the corneal (K) and spectacle (SRx) planes. Results:The mean SE was -6.77 ±
3.88 pre- and -0.10 ± 0.93 D post-phacoemulsification. Mean logMAR best corrected visual acuity was 0.42
± 0.20 (20/50-) and 0.08 ± 0.08 (20/25+), respectively. No eyes required additional surgery for refractive
complications. The IOL prediction error for each method is presented in the table below. Double-K formulas
were less likely to induce hyperopia, but had reduced consistency between cases for every method of IOL
power prediction when compared to single-K formulas.
SRx plane
IOL Prediction Error
Historical, K CLO,
plane plane
Conclusions: While this series demonstrates unpredictability in IOL power calculation, three important points
should be noted: 1) our CLO method can be used to accurately predict IOL power in eyes with dense cataracts
and poor visual acuity; 2) our CLO method can successfully be used to estimate corneal power and calculate IOL
in post-refractive surgery eyes; and, 3) our CLO method successfully predicted IOL power despite significant
refractive error. Further work in a larger series of eyes is warranted to improve our CLO method accuracy and
to determine the related confounding and interacting effects of various clinical factors.
CR: C.E. Joslin, None; E.Y. Tu, None; T.T. McMahon, None; J. Sugar, None.
Support: NIH/NEI EY 15689 (CEJ), NIH/NEI EY01792, Research to Prevent Blindness
Predicting the Rigid Contact Lens Base Curve Using Corneal Topography in
S.M. Mathews1A, J.C. Bradley1A, J.G. George1A, K.T. Xu1B. AOphthalmology & Visual
Sciences, BHealth Services Research Management, 1Texas Tech UHSC, Lubbock, TX.
Purpose: Rigid contact lenses often provide the best vision for keratoconic patients. Achieving
a comfortable stable fit that also provides both good vision and maintains corneal health can
be arduous. Previous studies have looked at pre-fit corneal topography to try to find indicators
of the proper base curve. These prior studies involved few patients and were, therefore, of
limited use as a fitting guide. We compiled topographical data from a much larger sample
of keratoconic patients and compared this data to the patients’ best fit base curves to learn
to better predict the base curve in keratoconus. Methods: A retrospective chart review was
done on all keratoconic patients successfully fit with rigid corneal contact lenses at Texas
Tech UHSC in the last 5 years. Pre-fit Keratron Scout or Tomey TMS-1 corneal topography
data and best fit contact lens base curve were gathered on 200 keratoconic eyes. Topographic
variables included: flat and steep simulated keratometry readings, flat and steep curvatures
from the normalized scale, Maloney best fit sphere, irregularity index, and asymmetry index.
Linear regression yielded a formula and R 2 value that described the relationship between each
topographical variable and the best fit base curve. Results: The best predictors of base curve
were the Maloney best fit sphere and simulated keratometric readings from the Keratron Scout
topographer. All linear regressions had non-unity slopes and non-zero Y-intercepts. R 2 values
were higher for the Keratron versus the Tomey topographer, for central versus peripheral
apices, and for simulated keratometry readings versus curvatures from the normalized scale.
Interestingly, the Tomey irregularity and asymmetry indices were better predictors than
either the Tomey simulated keratometry readings or curvatures from the normalized scale.
Conclusions: Practitioners equipped with the formulas from this study should be able to
choose an initial trial lens base curve that will be closer to the best fit base curve. Choosing
a better initial trial lens should reduce the number of trial lenses evaluated with fluorescein
per eye and shorten the time needed to arrive at the final fit.
CR: S.M. Mathews, None; J.C. Bradley, None; J.G. George, None; K.T. Xu, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 843-866 / B817-B840
136. Corneal Topography and Optics Organizing Section: CO Contributing Section: LE, VI
861 - B835
862 - B836
Changes of Corneal Aberrations in Sitting and Supine Position
T.Kawamorita1, T.Handa2, H.Uozato1. 1Ophthalmology & Visual Sciences, Kitasato
Univ Graduate Sch of Medical Sciences, Sagamihara, Japan; 2Ophthalmology & Visual
Sciences, Kitasato Univ Sch of Medical Sciences, Sagamihara, Japan.
Purpose: To examine the effect of posture change on the central corneal refractive
power and corneal aberrations. Methods: The Keratron topographer (OPTICON)
improved to measure under the supine position was used to measure corneal refractive
power and aberrations. Nine healthy volunteers (aged 20-28 years; mean 21.9±2.8)
that were free of ocular pathology took part in this study. The first was measured in
the sitting and the others immediately, 10, 20 and 30 minutes after the supine position.
Results: In central corneal refractive power a significant difference was not found through the
experimental procedure. The mean total higher order aberration in the sitting and the posture
change were 0.419±0.120 μm and 0.500±0.157μm, respectively (p < 0.05). Corneal aberration
showed a statistically significant decrease with time after posture change. After the posture change
from the sitting to the supine position, the increase in total higher order aberration may cause
slightly change of ocular structure distorted by intraocular pressure or gravitational influence.
Conclusions: These results suggest that the increase in higher order aberration from the
sitting to the supine position causes limit the improvements in visual performance after
customized refractive surgery based on wavefront measurement.
CR: T. Kawamorita, None; T. Handa, None; H. Uozato, None.
Support: None.
Higher Order Aberrations Induced by Soft Contact Lens Use for Myopic Correction
B.Roberts, G.Athappilly, B.Tinio, H.Naikoo, P.Asbell. Ophthalmology, Mt. Sinai Medical
Center, New York, NY.
Purpose: To evaluate and compare the wavefront characteristics in normal eyes
before and after being fitted with hydrogel soft contact lenses for correction of myopia.
Methods:Higher order aberrations (HOA) of 30 eyes of 15 soft contact users who had
no ocular diseases other than myopia were quantified with a Nidek Marco 3-D Wave
wavefront analyzer. Zernike’s polynomial was used to describe the wavefront measurements.
RMS values of the total HOAs, total coma, total trefoil and total spherical aberrations
were obtained in the same eyes before and after soft contact lenses were inserted.
Similar sets of data were also obtained in different brands of soft contact lenses of the
same power inserted in the same eyes. We used paired sample t-test to analyze the data.
Results: Mean values (RMS) for all higher order aberration components from before to
after soft contact lens insertion were: total HOA from 0.364μm to 0.456μm (P=0.01), total
coma from 0.203μm to 0.220μm (P=0.51), total trefoil from 0.193μm to 0.254μm (P=0.06)
and total spherical aberration from 0.126μm to 0.148μm (P=0.36). Induced HOA also
varies among different brands of soft contact lens when power and eyes were controlled.
Conclusions: Wavefront analysis showed soft contact lenses for myopia induced a significant
increase in HOA. Though total HOA with contact lens use was statistically significant,
total coma, trefoil and spherical aberrations were also higher in contact lens use but were
not statistically significant when individually evaluated. Comparison with LASIK induced
HOA will be presented.
CR: B. Roberts, None; G. Athappilly, None; B. Tinio, None; H. Naikoo, None; P. Asbell,
Support: NEI Core Grant to Dept. and Research to Prevent Blindness to PA
863 - B837
864 - B838
865 - B839
866 - B840
Serial Measurements of Higher-Order Aberrations Following Blinking in Normal
S.Koh1A, N.Maeda1A, S.Ninomiya1B, K.Bessho1B, H.Watanabe1A, T.Fujikado1B, Y.Tano1A,
Y.Hirohara2, T.Mihashi2. AOphthalmology, BVisual Science, 1Osaka University Medical
School, Suita, Japan; 2Research Institute, Topcon Corporation, Tokyo, Japan.
Purpose: To investigate the sequential changes in the optical quality of the normal eyes after blinking.
Methods: Higher-order aberrations (HOAs) were sequentially measured for 30 seconds in 20
normal subjects with a newly developed wavefront sensor. During the measurement, subjects were
forced to blink every 10 seconds. The obtained aberration data were analyzed for the central 4 mm
diameter for coma-like, spherical-like, and total HOAs up to the 6th-order Zernike polynomials.
Results: The serial changes of HOAs with blinking were classified into four groups: stable
pattern (35%), small fluctuation pattern (35%), saw-tooth pattern (20%), and others that were
not classifiable in the three patterns (10%). The fluctuation indexes of the HOAs evaluated by
the average of the standard deviation in these four groups were 0.008, 0.013, 0.024, and 0.011,
respectively. The saw-tooth pattern had a significantly higher value than the stable pattern and
the small fluctuation pattern in fluctuation index (P<0.001, P<0.001, by one-way ANOVA).
The stability indexes of the HOAs expressed by the slope of the regression line were 0.001,
0.003, 0.008, and 0.002, respectively. Saw-tooth pattern also had a significantly higher value
than the stable and the small fluctuations patterns in the stability index (P<0.001, P<0.001,
by one-way ANOVA ). In the saw-tooth group, the increased total HOAs corresponded well
with the pattern in coma-like aberrations rather than that with spherical-like aberrations,
suggesting that the asymmetric change in tear-film thickness might be mainly responsible.
Conclusions: Dynamic changes in HOAs after blinking and forced eye opening for 10 seconds
showed variations even in clinically normal subjects. Serial measurements of higher-order
aberrations might be useful to evaluate the dynamic changes in tear-film and its effects on
the quality of vision after blinking.
CR: S. Koh, None; N. Maeda, Topcon Corporation F; S. Ninomiya, None; K. Bessho,
None; H. Watanabe, None; T. Fujikado, None; Y. Tano, None; Y. Hirohara, Topcon
Corporation E; T. Mihashi, Topcon Corporation E.
Support: Japanese Ministry of Education (15591854) and Osaka Eye Bank
Higher Order Aberrations Attributable to the Cornea and Internal Ocular Components
in Myopic, Keratoconus and Cataractous Eyes
K.Yu, T.Swartz, M.Wang. Wang Vision Institute, Nashville, TN.
Purpose: To compare higher order aberration (HOA) attributable to cornea versus
internal ocular components in myopic eyes and in eyes with keratoconus and cataract.
Methods: 122 eyes of 65 patients were divided into 3 groups: myopic with normal cornea
and clear lens (N = 71), keratoconic (N = 21) and cataractous (N =30). In each patient, HOA
Root Mean Square (RMS) values derived from the whole eye, from cornea, and from internal
ocular components (lens, vitreous, retina), respectively, were obtained using a combined
wavefront sensor (iTracey) and corneal topography (EyeSys) system. In each group, the
average HOA RMS values from the whole eye, cornea and internal ocular components
were determined and compared both from within the group and in-between groups.
Results: Within the first group (myopic eyes), the average HOA RMS value from the cornea
(0.65±0.41μm) is comparable to that from internal ocular components (0.66±0.34μm);
comparing the second group (keratoconic eyes) with the first (myopic eyes with normal
corneas), the average whole-eye HOA RMS value of the keratoconic eyes (3.75±2.51μm)
is statistically higher than that from the normal myopic eyes (0.65+0.41µm ) (p<0.001);
within the second group (keratoconic eyes), corneal contribution to HOA (4.07±3.02μm) is
statistically significantly higher than that from the internal ocular component (1.83±1.41μm)
(p<0.01); comparing the third group (cataractous eyes) with the first (myopic eyes with
clear lens), the average whole-eye HOA RMS value from the cataract eyes (1.13±0.82μm) is
statistically significantly higher than of the myopic eyes with clear lens (0.65+0.41µm) (P<0.01).
Conclusions: In myopic eyes with normal cornea and clear lens, cornea and lens have equal
contribution to the whole-eye HOA; in keratoconic eyes, not only the whole-eye HOA RMS
value is increased (when compared with that of myopic eyes with normal cornea), but also the
cornea is the largest contributor to whole-eye HOA in these keratoconic eyes; in the cataractous
eyes, whole-eye HOA is also higher than that of myopic eyes with clear lens.
CR: K. Yu, None; T. Swartz, None; M. Wang, None.
Support: Wang Vision Institute
The Relationship of Corneal Curvature and Axial Length in Adults
J.C. Merriam, L.Zheng. Ophthalmology, Edward S Harkness Eye Inst, New York, NY.
Purpose: To determine if corneal curvature is related to axial length in the adult human eye.
Methods: This is a retrospective study of 1183 eyes of 761 patients, evaluated by a
single surgeon for cataract surgery. The average age was 75, with a range of 38 to
99 years. Central corneal curvature was measured with a keratometer (Bausch and
Lomb) 3 to 4 times; the mean keratometry value (K) was computed. Axial length (AL)
was measured with ultrasound (Sonomed A-2000) 5 to 7 times, and the mean was
calculated. The relationship between the K reading and axial length was established
by linear fit. The distribution of axial lengths and K readings were also documented.
Results: The average K was 43.57, with a range of 38.25 to 50. The average axial length
was 24.04, with a range of 18.4 to 31.91. More than 90% of K values were between 40.5 and
46.5; and more than 90% of the axial lengths were between 22.5 and 26.5 mm. Axial length
and corneal curvature are linearly related by: AL = 33.87 - 0.226K. The confidence interval
for the slope is 0.06. The eyes were then subdivided into those that were spherical, or had
predominantly vertical or horizontal corneal astigmatism prior to surgery. The relationship of
axial length to mean corneal curvature was not significantly different in these three groups.
Conclusions: This study of patients presenting with clinically significant cataract suggests
that mean corneal curvature is related to axial length. As axial length increases, the cornea
tends to become flatter.
CR: J.C. Merriam, None; L. Zheng, None.
Support: None.
Surgically Induced Astigmatism After 2.75 mm Clear Corneal Incisions for Cataract
F.Giansanti, G.Virgili, E.Rapizzi, R.Mencucci, L.Vannozzi, A.Bini, T.Verdina, U.Menchini.
University of Florence-Italy, Eye Clinic, Florence, Italy.
Purpose: To determine early astigmatic effect induced by 2.75 mm clear cornea
phacoemulsification cataract incisions Methods: Seventy eyes of different individuals
were included. The corneal incision was applied at different clock-hours grouped in 3
categories: at 12 in 7 right eyes and 9 left eyes, at 7-9 in 34 right eyes and at 2-3 in 20
left eyes. Corneal curvature was obtained before surgery at 1 week and 1 month using
computerized videokeratography (EyeSys Corneal Analysis System, EyeSys laboratories,
Inc.) The difference in curvature between the two orthogonal meridians was registered
as a cylinder with a given axis corresponding to the more curve meridian and assuming
orthogonality of the meridians. This cylinder was transformed into a vector using a power
vector transformation into 2 Jackson crossed-cylinders, on with 0°/90° axis (JCC0) and one
with 45°/145° axis (JCC45) in order to compute the change of each component and of the
total cylinder vector between baseline and 1 month. A paired t-test was used to compare the
change of the JCC components and a Hotelling T test was used to assess the overall change
of the cylinder vector. Regression analysis was used to assess factors influences the change
of the cylinder components, including baseline cylinder and incision site. Results: There
was no statistically significant change of the JCC0 after surgery, the mean of which changed
by -0.01D (SD=0.35D, p=0.846), whereas there was a modest but significant mean change
of the JCC45 by -0.09 (SD=0.33D, p= 0.020) for the JCC45. The overall change of the total
cylinder vector showed only a trend towards significance (p=0.066) cylinder, with a median
change of 0.27D and a change of less than 1D in 67/70 eyes (96%). There were no factors
influencing the JCC45 shift when the baseline value was considered. On the contrary the
change of the JCC0 vector was larger for the 12 o’ clock incision vs both lateral incisions with
an interaction term with the baseline JCC0 suggesting a larger increase of negative baseline
values by -1.4 D vs -0.6 D when the baseline JCC0 was -1D (p<0.001). Conclusions: Cataract
surgery with a 2.75 mm incision leads to modest change of the corneal astigmatism after 1
month. A later incision at 8 o’clock in the right eye or at 2 o’clock in the left eye causes less
increase of the counter-the-rule astigmatic component than a 12 o’clock incision in this setting.
CR: F. Giansanti, None; G. Virgili, None; E. Rapizzi, None; R. Mencucci, None; L.
Vannozzi, None; A. Bini, None; T. Verdina, None; U. Menchini, None. Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 867-903 / B841-B877
137. Corneal Epithelium Organizing Section: CO
867 - B841
A Novel Random Flexible Nanoscale Topography for Cell Culture
C.J. Murphy1, T.J. Porri2A, J.A. Dumesic2B, P.F. Nealey2B. 1Surgical Sciences, Sch of Vet
Medicine UW Madison, Madison, WI; AMaterials Science and Engineering, BChemical and
Biological Engineering, 2Sch of Engineering UW Madison, Madison, WI.
Purpose: We have documented that anisotropic nanoscale topography modulates a variety of human
corneal epithelial cell (HCEC) behaviors, including orientation, mobility, adhesion, and g-protein
signaling. However, random substrate topographies are also of interest because they more closely
mimic the topographic features found in the native corneal basement membrane. An ideal random
substrate would be comparatively easy and inexpensive to fabricate at a variety of length scales.
Methods: Gold nanotubules are fabricated by electroless deposition onto track-etched
polycarbonate membranes. This deposition process results in a 10-100nm layer of gold coating
the entire membrane. When the topmost gold and 0.5-1 micron of the underlying polymer
substrate is removed, the resulting surface consists of gold nanotubules (“gold grass”) projecting
from a solid polycarbonate surface. A self-assembled monolayer of mercapto-hexadecanoic
acid is adsorbed to the gold for improved cell attachment. Cells were plated at a density of
10,000 cells/cm 2. Assays were performed to determine the effect of topographic cueing
provided by nanoscale gold grass surfaces on cell viability, morphology, and proliferation.
Results: Gold grass suitable for cell culture was fabricated with outer diameters ranging
from 30 to 800nm with an average (center to center) spacing from 400 to 2000nm.
Viability of HCECs on gold grass surfaces after 24 hours is comparable to planar gold,
electroless gold-coated membranes and tissue culture polystyrene. HCECs extend
filopodia that attach to and bend the gold nanotubules, and exhibit time-dependent
spreading and proliferation in a manner separate from the three control surfaces.
Conclusions: Gold grass provides a viable, unique, and easy-to-fabricate substrate for
cell culture. The size and thickness of the nanotubules can be readily adjusted, and
the flexible surface is easy to chemically modify. Because a large amount of material
can be manufactured at once, these substrates may be particularly suited to the study of
topographic cueing on protein expression where large numbers of cells are required.
Support: NEI Grant RO1-12253-01, NSF MRSEC Grants DMR-9632527 and CTS-9703207
CR: C.J. Murphy, None; T.J. Porri, None; J.A. Dumesic, None; P.F. Nealey, None.
Support: NEI Grant RO1-12253-01, NSF MRSEC Grants DMR-9632527 and CTS-9703207
869 - B843
868 - B842
Immunohistochemical Evaluation of the Inhibition of Protein Nitration in Human
Corneal Epithelial Cells During Cold Storage
B.H. Jeng1A, K.G. Shadrach1A, D.M. Meisler1A, J.G. Hollyfield1A, T.Koeck1B, K.S. Aulak1B,
D.J. Stuehr1B. ACole Eye Institute, BDepartment of Immunology, 1Cleveland Clinic
Foundation, Cleveland, OH.
Purpose: We have previously demonstrated that nitric oxide (NO) production occurs in
corneal tissue during storage prior to transplantation and that this production, which leads
to protein nitration, can be inhibited with the addition of a nitric oxide synthase inhibitor to
the corneal storage media. The aim of this study was to determine if the inhibition of NO
production would lead to a decrease in immunohistochemical staining for nitrated protein
in corneal tissue during storage. Methods: Paired human corneas stored in Optisol-GS
corneal storage media were obtained from the Cleveland Eye Bank. NG-monomethyleneL-arginine (LMMA), a non-selective nitric oxide synthase inhibitor, was added to 20 ml
of storage media of one cornea from each pair of corneas to a final concentration of 2mM.
Paired corneas were stored for 3 days and then bisected. One half of each cornea was placed
in formalin fixative, and the remaining halves of each cornea were returned to their original
storage media and stored for 4 additional days. The remaining tissue was then subjected to the
same treatment as the other halves. Mouse polyclonal antibody to nitrotyrosine was used as
the primary antibody, and goat anti-mouse biotin-labeled antibody was used as a secondary
antibody. The slides were then treated with a peroxidase ABC kit and photographed with
a digital camera system. Results: Less immunohistochemical staining for nitrated protein
was observed in epithelial cells which were exposed to LMMA than in those of the control
group at day 7 of storage. No substantial differences in immunohistochemical staining
occurred at day 3 between the inhibited and control groups. Conclusions: The addition of
a nitric oxide synthase inhibitor to corneal storage media seems to decrease the amount of
immunohistochemical staining for nitrated protein in human corneal epithelial cells during
storage. This may correlate clinically to less damage to corneal cellular elements during
storage due to the toxic effects of protein nitration.
CR: B.H. Jeng, None; K.G. Shadrach, None; D.M. Meisler, None; J.G. Hollyfield, None; T.
Koeck, None; K.S. Aulak, None; D.J. Stuehr, None.
Support: Eye Bank Association of America, Cleveland Clinic Foundation Research Programs
870 - B844
Collagen XXIII Facilitates Adhesion of Corneal Epithelial Cells to Type IV Collagen and
M.K. Gordon1, P.Bhatt1, R.Song1, R.A. Hahn1, M.Shakarjian2, D.R. Gerecke1, M.Koch3. 1Dept
Pharmacology & Toxicology, E. Mario School of Pharmacy, Rutgers University, Piscataway, NJ;
Dept Pulmonary Medicine, UMDNJ, Robert Wood Johnson Medical School, Piscataway, NJ;
Institute for Biochemisty, University of Cologne, Cologne, Germany.
Purpose: Type XXIII collagen is a newly described transmembrane molecule that is expressed on
the surface of corneal epithelial and endothelial cells, but not stromal cells. As a transmembrane
molecule, it may facilitate cell attachment, migration, proliferation or differentiation. To assess
the role of collagen XXIII in cell adhesion, recombinant collagen XXIII was added to dishes
coated with various substrata, and incubated 1 hour to permit binding. Immortalized human
corneal epithelial cells were then plated on the dishes, and their ability to adhere was measured.
Methods: cDNA encoding the extracellular region of collagen XXIII was ligated into pCEP vector
and used to stably transfect 293 cells. Medium collected contained the expressed ectodomain
of the collagen. Medium from empty pCEP vector transfected into 293 cells was used as a
control. 12 well plastic culture plates were used either as is, or coated with type I collagen,
type IV collagen, or Matrigel. 2 ml of medium from either the transfected 293 cells expressing
the ecodomain of collagen XIII, or transfected with empty pCEP vector, was added to the 12
well plates, allowing interaction with the plastic or substratum for 60 minutes at room temp.
The medium was then removed, and 40,000 corneal epithelial cells were added to the wells.
One set of wells was incubated for 1 hour at 37oC before removing the medium. Adherent cells
were washed, fixed and stained with crystal violet. A second set of cells was incubated for 4
hours, and a third set for 24 hours, before removing medium and staining the adherent cells.
Attached cells were monitored by photography and by absorbance at 570 nm using a plate reader.
Results: At all time points, corneal epithelial cells attached well to plastic, collagen I, collagen IV,
and Matrigel coated plates pre-incubated with medium from empty vector-transfected 293 cells.
However, fewer corneal epithelial cells adhered to the collagen IV and Matrigel coated dishes when
the wells were pre-incubated with medium containing recombinantly expressed collagen XXIII.
Conclusions: Exogenously added type XXIII collagen competes with corneal epithelial cell
surface collagen XXIII for binding to type IV collagen and Matrigel. Our results suggest that
corneal epithelial cells use collagen XXIII to facilitate interactions with the basement membrane,
possibly through type IV collagen.
CR: M.K. Gordon, None; P. Bhatt, None; R. Song, None; R.A. Hahn, None; M. Shakarjian,
None; D.R. Gerecke, None; M. Koch, None.
Support: NIH Grant EY09056
Comparison of Cultivated Limbal Epithelial Sheets With and Without Substrates
J.Shimazaki1, K.Higa1, M.Aiba1, Y.Itabashi2A, K.Fukuda2A, K.Tsubota1,2B, S.Shimmura1.
Department of Ophthalmology, Tokyo Dental College, Ichikawa-shi, Japan; ACardiology
Division, Department of Internal Medicine, BDepartment of Ophthalmology, 2Keio University
School of Medicine, Tokyo, Japan.
Purpose: To study histological differences between cultivated limbal epithelial sheets with and without
substrates. Efficacy of transplantation of the epithelial sheets to rabbit corneas was also examined.
Methods: Rabbit limbal epithelial cells were cultivated on human amniotic membrane (AM group)
or fibrin-coated dishes (fibrin group) with mytomicin C-treated 3T3 feeder cells. In the fibrin group,
the epithelial sheet was dislodged from the underlying plastic dishes by discontinuing aprotinin, a
proteinase inhibitor in the culture medium. Limbal epithelial sheets were subjected to histological
examinations. Expression of cornea-specific keratin (AE5), p63, and basement membrane markers
including laminin and type IV collagen were examined by immunohistochemistry. Transplantation of
AM and fibrin epithelial sheets was studied using a rabbit limbal deficiency model (n=3 in each group).
Results: Both types of epithelial sheets demonstrated stratified epithelial cells with positive
staining for AE5 and p63. While the cell sheet in the AM group showed positive staining for
basement membrane markers between the basal cells and AM, there were no such staining in the
fibrin group, suggesting that the epithelial sheet in the latter group detached immediately beneath
the basal epithelium. Both types of epithelial sheets attached well on the rabbit corneas after
mechanically removing total corneal and limbal epithelium. The corneas that had sham surgery
showed delayed epithelialization with conjunctival invasion. While suturing with 10-0 nylon was
needed in the AM group, no sutures were necessary in the fibrin group. The corneas in the fibrin
group had complete epithelialization and clear visibility of the iris compared with the AM group
by 1 week following surgery. Some corneas in the AM group had localized epithelial defects
associated with loose attachment of the sheet to the underlying stroma that persisted for over a
week. Histological examination demonstrated that the epithelium remained well organized in the
both groups. Subconjunctival inflammation was more intense in the AM group than the fibrin group.
Conclusions: Both types of the cultivated limbal epithelial sheet showed histological integrity and
efficacy in the rabbit model. Sheets without a substrate may be superior to those with substrates
in terms of clarity and attachment to the corneal stroma.
CR: J. Shimazaki, None; K. Higa, None; M. Aiba, None; Y. Itabashi, None; K. Fukuda,
None; K. Tsubota, None; S. Shimmura, None.
Support: a Grant of the Ministry of Health and Welfare, Japan (H15-Saisei-013)
871 - B845
872 - B846
Cytometric Viability of Human Corneal Epithelial Cells Grown on Tissue Culture
Plastic and a Biomaterial
A.M. Wright, M.McKee, A.Renaud, P.Ranganathan. Cell Biology, CIBA Vision/Novartis
Company, Duluth, GA.
Purpose: To investigate cytometric methods to evaluate cellular viability using in vitro cell
culture of SV-40 transformed human corneal cells (HCE-T) on tissue culture plastic (TCP) and a
high oxygen permeable silicone hydrogel biomaterial (HiDk SiHy). Methods: Cell viability assay
used were, Alamar Blue™ (AB), a resazurin based dye, and the Live/Dead Viability/Cytotoxicity
Kit from Molecular Probes (LDVC) and the cytometric bead assay(CBA) for proinflammatory
cytokines. HCE-T cells were seeded on TCP and HiDk SiHy(lotrafilcon A soft contact lenses).
AB was added to the cells, incubated and fluorescence reading taken at λ exc =530nm, λ em =580
nm of the oxidized and reduced AB using a CytoFluor®, (PerSeptive Biosystems). Supernatants
were harvested and frozen for cytokine analysis; IL-6, IL-8, TNF-α, IL1-β, IL10 and IL12p70.
HCE-T cells were removed, spun and characterized by double staining with calcein and ethidium
homodimer -1 (CEH). Flow cytometric analysis was carried out at 488 nm on a BDFACSCailbur™
4 color unit cytometer. Subpopulations were identified and sorted by regions for live (green
fluorescent) and dead (red fluorescent) cells. Benzalkonium chloride (BAC) was used as a positive
control for the analysis of dead cells. Phorbol ester (PMA) was used as a stimuli to induce cytokines.
Results:HCE-T cells grown on HiDk SiHy did not show any difference visually or
with AB from controls grown on TCP. BAC exposed cells at or below 5.0 ppm showed
AB cell viability above 50% at 24 hours. Cytokines were not upregulated for HCET cells grown on HiDk SiHy compared TCP. HCE-T cells grown on TCP were found
to be 98% live cells. 10 ppm BAC treated cells were found 95% dead by region gating.
Cells grown on the HiDk SiHy were found to be 80% live and 20% damaged cells.
Conclusions: Alamar Blue and Live/Dead flow cytometry studies demonstrate the biocompatibility
of SiHy BioM with HCE-T cells. No upregulation of proinflammatory cytokines was observed
for HCE-T cells grown on HiDk SiHy further suggesting biocompatibility with the ocular
environment. These tests with other toxicological tests provide valuable information for the
evaluation of new biomaterials or solutions. HCE-T cells are biocompatible with HiDk SiHy and
support the use of lotrafilcon A; a HiDk SiHy extended wear- high oxygen permeability contact
lens; as a bandage contact lens for corneal protection and healing after insult or surgery.
CR: A.M. Wright, Ciba Vision E; M. McKee, CIBA Vision E; A. Renaud, CIBA Vision E;
P. Ranganathan, CIBA Vision E.
Support: None.
A Human Corneal Epithelial Culture Model
M.J. Powers, L.Amenuvor. Research/Development, Cambrex Bio Science Walkersville,
Walkersville, MD.
Purpose: We have developed an in vitro culture model comprising human
primary corneal epithelial cells in a physiologically relevant environment. The
system can be used as a research tool to assess ocular toxicity and irritancy
Methods: Human corneal epithelial cells were isolated and transfected with human
papilloma virus (HPV) type 16 E6 and E7 genes. Culture models were developed by
growing the cells to confluence on microporous membrane supports in Corneal Epithelial
Model Medium followed by culture at the air-liquid interface to induce stratification
and differentiation of the cells. Cultures were assessed for differentiated function via
Trans Epithelial Electrical Resistance (TEER), histological analysis, and time-totoxicity (ET50) as assessed by the MTT cytotoxicity assay for several known irritants.
Results: The culture models exhibited a histological profile similar to that observed
with in vivo corneal epithelium. Furthermore, the cytotoxic response of the system
to known irritants shows good correlation with in vivo Draize data for several
different irritant categories (severe, moderate, mild, slight and non-irritants).
Conclusions: Based on these results, the human corneal epithelial culture
model described here presents a promising in vitro system for the assessment
of ocular toxicity and irritancy.CR: M.J. Powers, None; L. Amenuvor, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 867-903 / B841-B877
137. Corneal Epithelium Organizing Section: CO
873 - B847
874 - B848
Histological Evaluation of Poly(Ethylene Glycol)-Poly(Acrylic Acid) (PEG-PAA) Double
Network Hydrogel Corneal Implant
N.Farooqui1, D.Myung1,2, M.Masek1, R.Dalal1, W.Koh2, S.Gupta1, J.Noolandi1,2, C.Frank2, C.N.
Ta1. 1Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, CA;
Department of Chemical Engineering, Stanford University, Palo Alto, CA.
Purpose: Due to their unique composition, physiologic properties, and high water content, many
hydrogel scaffolds are unable to be successfully processed for histological evaluation using
routine tissue processing methods. Our goal is to adapt novel methods for hydrogel processing
to successfully prepare a PEG-PAA double network (DN) hydrogel for histological analysis.
Methods: Three methods were assessed for their ability to produce accurate light microscopy analysis.
A modified protocol for glycol methacrylate (GMA) resin was used. The specimen, consisting of
a PEG-PAA hydrogel (water content 80%) implanted within the stroma of a bovine cornea, was
fixed overnight using 2.5% glutaraldehyde-2% paraformaldehyde. Pre-infiltration was achieved
with increasing concentrations of GMA every hour under vacuum pressure. The Technovit 7100 kit
recommendations call for dilution of the GMA monomer with ethanol. Due to the high water content
of the PEG-PAA hydrogel and to preserve the morphology, ethanol was substituted with deionized
water. Infiltration was performed overnight under vacuum with 100ml 100% GMA monomer and 1g
Hardener I (dibenzoylperoxide). The sample was embedded in gelatin capsules and polymerized at
room temperature, using 15:1 of pre-infiltration solution and Hardener II (dimethylsulfoxide). Thin
sections (2 microns) were taken using a glass knife. Sections were taken off of a water bath and placed
directly onto a dry slide and stained with toluidine blue. Bovine corneas with PEG-PAA implants were
also processed by using frozen section and the standard TEM epoxy methods as bases for comparison.
Results: Slides processed using the GMA method were evaluated under a light microscope, and the
implant was clearly visible and intact within the stroma, with excellent preservation of both tissue
and hydrogel morphology. Frozen sectioning of the implanted tissue resulted in loss of the hydrogel
due to ice crystal formation of the water in the hydrogel. Hydrogels embedded in epoxy resin showed
gross distortion of morphology due to the high ethanol concentrations required in the process.
Conclusions: The GMA method is successful in yielding light microscopy sections for the histological
evaluation of a PEG-PAA corneal implant. It proves to be a promising method for further evaluation
of our hydrogel implants in their various applications.
CR: N. Farooqui, VISX, Incorporated F; D. Myung, VISX, Incorporated F, P; M. Masek, None;
R. Dalal, VISX, Incorporated F; W. Koh, VISX, Incorporated F, P; S. Gupta, VISX, Incorporated
F; J. Noolandi, VISX, Incorporated F, P; C. Frank, VISX, Incorporated F, P; C.N. Ta, VISX,
Incorporated F, P.
Support: VISX, Incorporated
Analysis of Essential Tear Components in Umbilical Cord Serum and Its Application
for the Treatment of Ocular Surface Disease
K.-C.Yoon, B.-Y.Song, M.-S.Seo, Y.-G.Park. Department of Ophthalmology, Chonnam
National University Medical School and Hospital, Gwang-Ju, Republic of Korea.
Purpose: To analyze of essential tear components in umbilical cord serum and investigate
the efficacy of umbilical cord serum eyedrops for the treatment of ocular surface disease.
Methods: The concentrations of epidermal growth factor (EGF), transforming growth
factor-β (TGF-β), and substance P in umbilical cord serum were measured by enzymelinked immunosorbent assay (ELISA), and vitamin A concentration was measured by
high-performance liquid chromatography (HPLC). Fifty-five eyes of 31 patients with
severe dry eye syndrome and 15 eyes of 15 patients with persistent epithelial defect
were treated with umbilical cord serum eyedrops. In patients with dry eye syndrome,
symptom scoring, tear film break up time (BUT), Schirmer test, corneal sensitivity
test, and corneal fluorescein staining were performed before and 1 and 2 months after
treatment, and conjunctival impression cytology was performed before and 2 months
after treatment. In patients with persistent epithelial defect, healing time was recorded.
Results: The mean concentrations of EGF, TGF-β, and vitamin A were 0.48±0.09 ng/ml,
57.14±18.98 ng/ml, and 230.85±13.39 ng/ml in umbilical cord serum, respectively, and were stable
during 3 months. Two months after treatment, significant improvement was observed in symptom
score (from 3.07±0.54 to 0.96±0.58), tear film BUT (from 3.96±1.56 sec to 5.45±2.54 sec), and
keratoepitheliopathy score (from 4.87±3.22 to 1.71±1.84) (P<0.01). There was no statistically
significant change in Schirmer and corneal sensitivity test results. In impression cytology,
the grade of conjunctival squamous metaplasia (from 2.35±0.72 to 1.44±0.69) and goblet cell
density (from 80.91±31.53 cell/mm 2 to 154.68±43.06 cell/mm 2) improved significantly (P<0.01).
Treatment was effective or partially effective in all patients with persistent epithelial defect.
Conclusions: Umbilical cord serum contains essential tear components, and umbilical cord
serum eyedrops are effective and safe for the treatment of ocular surface disease.
CR: K. Yoon, None; B. Song, None; M. Seo, None; Y. Park, None.
Support: None.
875 - B849
876 - B850
Neurotrophic Effect of Amniotic Membrane on Neuronal Cell Cultures: An in
vitro Model to Study Underlying Action Mechanisms of Amniotic Membrane in the
Treatment of Neurotrophic Keratopathy
D.Meller1, A.Schröder2, K.P. Steuhl1, C.Theiss2. 1Ophthalmology, University of Essen,
Essen, Germany; 2Cytology, University of Bochum, Bochum, Germany.
Purpose: Transplantation of amniotic membrane (AMT) has been successfully applied to
promote corneal wound healing in neurotrophic ulcers with different aetiologies. In this line,
healing of corneal surface after AMT correlated clinically with a partial recovery of corneal
sensitivity. Moreover, AMT compared to conventional treatment strategies accelerated axonal
sprouting in an experimental animal model of keratitis induced by Herpes virus. In this study,
we investigated potentially underlying neurotrophic action mechanisms of amniotic membrane
(AM). Methods: Organ-typical cell cultures of dorsal root ganglion (DRG) neurons were
gained from 10-day-old chick embryos and cultured with MEM on the stromal or epithelial
side of intact AM for 3 to 5 days. In an additional group AM was pretretead with Dispase for
15 min at 37°C in order to remove the amniotic epithelium. Afterwards, DRG neurons were
cultured in the same manner on the exposed basement membrane side of AM. Sprouting of
neuronal axons was screened with monoclonal antibodies against cytoskeletal proteins such
as neurofilament (NF-M) and tubulin (Tub). Finally, the specimens were analyzed with a
confocal laser scanning microscope (LSEM 501, Zeiss). Results: DRG neurons cultured
on the stromal or basement membrane side of AM exhibit within few days the formation of
numerous NF-M- und Tub-positive axonal neurites. These typically run a radial fashion and are
arranged partially in nerve bundles. However, axonal sprouting of DRG neurons is drastically
inhibited when cultured directly on the amniotic epithelium of AM. Conclusions: The
basement membrane and the stromal side of AM promote in vitro extensively the outgrowth
of axonal neurites. However, a substantial inhibition of axonal sprouting is induced by the
amniotic epithelium. If soluble, neurotrophic growth factors and/or adhesion-molecules
mediated mechanisms are enrolled in these events and thereby potentially promote wound
healing in neurotrophic keratopathy, will be analyzed in further studies.
CR: D. Meller, None; A. Schröder, None; K.P. Steuhl, None; C. Theiss, None.
Support: DFG Th839/1-2
Comparison of Human Corneal Epithelial and Human Conjunctival Epithelial Cell
Line Cultures in Cytotoxicity Testing
A.M. Huhtala1, L.Salminen1, H.Uusitalo2. 1Medical School, University of Tampere,
Tampere, Finland; 2Department of Ophthalmology, University of Kuopio, Kuopio,
Purpose: Alternatives to the Draize eye test have widely been investigated for several
decades now. Despite all, no test, combination of tests, or testing strategy have been found
to be capable of replacing the Draize eye test completely, but some of the assays have shown
a considerable promise as screens for ocular irritancy. In the present study, human corneal
epithelial and human conjunctival epithelial cell lines were compared as potential pre-screen
alternatives to the Draize test. Methods: Ocular toxicity was tested by using effective multititer
techniques and two basal cytotoxicity tests, WST-1 test as an index of mitochondrial function
and cell viability/proliferation and ATP test as an index of ATP measurement. Cells were
grown in 96-well plates with different cell densities for 24 hours and consequently exposed
to BAC for one hour in similar serum-free conditions. Benzalkonium chloride (BAC),
a cationic surfactant and a known severe eye irritant, was used as a model compound.
Results: Both cell lines yielded comparable results with both test methods. ATP test showed
increasing EC50 values with increasing cell numbers while in the WST-1 test the trend was not as
clear. In the ATP test, EC50 values varied from 0.0011% to 0.0036%, and in the WST-1 test from
0.0010% to 0.0021%. In the ATP test, the EC50 values for the NHC cells were twice as high as for the
HCE cells while in the WST-1 assay the EC50 values were in the same level and more comparable.
Conclusions: The cytotoxicity assays used were simple and reproducible, and yielded a
defined endpoint. Both cell lines show considerable promise as potential pre-screen alternative
methods. As cell lines are more manageable as primary cultures these cell lines are promising
tools for toxicity testing.
CR: A.M. Huhtala, None; L. Salminen, None; H. Uusitalo, None.
Support: National Technology Agency of Finland
877 - B851
878 - B852
Distribution of Stromal and Subbasal Nerves in Fresh Human Corneas
L.J. Muller1A, E.Pels1B, Staff Cornea Bank. AOcular Signal Transduction, BCornea Bank,
Netherlands Ophthalmic R I, Amsterdam, The Netherlands.
Purpose: Based on light and electron microscopic observations and in vivo confocal
microscopy we have published a scheme for the sub-basal nerve plexus in human corneas
(Müller et al. EER, 2003). This scheme is still incomplete because the orientation of the nerves
in the periphery, especially in the 6 and 12 o’clock regions, is unknown. Furthermore, it was
recently demonstrated that in LASIK patients an eye with a nasal hinge shows less dry eye
symptoms than its fellow eye with a superior hinge (Donnenfeld, Ophthalmology 2003). This
urged us to perform a refined study on the orientation of both stromal and sub-basal nerves.
Methods: Nine fresh corneas (2 marked in the orbit), post-mortem time 11-24 hrs, were stained with
gold chloride. Five of these corneas were divided into 11 pieces which were analyzed separately.
To avoid loss of tissue the pieces were cut manually into 3-5 parallel sections from epithelium
to endothelium. One cornea was divided into an anterior and a posterior part before staining.
Results: At 6 and 12 o’clock large deep stromal nerves have essentially a vertical
orientation whereas nasal and temporal (2,3,4 and 8,9,10 hrs) large nerves have essentially
a horizontal orientation. The latter keep their orientation in the mid-anterior stroma and
their number seems to exceed that of the vertical oriented nerves. The large nerves (∅
40-60 μm) run obliquely towards the stromal surface and bifurcate into medium (∅ 10-20
μm) and subsequently into small nerves (∅ 2.5-6 μm). In the apex only few small stromal
nerves are present, indicating that most of the stromal nerves pass Bowman’s layer in the
mid-periphery to form the plexus of sub-basal nerves (SBN). These SBN run parallel to
Bowman’s layer over 1-4 mm in a vertical direction in the apex, in a vertical or a horizontal
direction in the mid-periphery and are absent near the limbus. However, below the limbal
epithelium there is a plexus of curved nerves, which was sofar found only at the nasal side.
Conclusions: The density and preferred horizontal orientation of the stromal nerves might
be as important or even more important as the orientation of the subbasal nerves to explain
the hinge related dry eye symptoms. However, a possible role of the sub-epithelial limbal
nerves cannot be excluded.
CR: L.J. Muller, None; E. Pels, None.
Support: None.
Organization of the Subbasal Nerves in a Large Group of Control Human Corneas
Visualized With the Heidelberg Retinal Tomograph (HRT II)
C.Jacobi1, K.Gottschalk1, C.Cursiefen1, F.E. Kruse1, L.J. Muller2. 1Department of
Ophthalmology, University Hospital, Erlangen, Germany; 2Netherlands Ophthalmic RI,
Ocular Signal Transduction, Amsterdam, The Netherlands.
Purpose: To establish in vivo the orientation of the subbasal nerves (SBN) in the apex
as well as nasal, temporal, superior and inferior periphery of intact human corneas.
Methods: Subbasal nerves of 110 eyes of 61 healthy volunteers (aged between 20
and 84) were recorded with the Rostock cornea module attached to the HRT II. The
resolution of this apparatus is higher than that of other in vivo confocal microscopes
and the relatively large contact plane prevents disturbances of the recordings due to
blinking. Thick as well as thin SBN in the apex, mid-periphery and periphery were easily
visualized. Also the presence of dendritic cells and of loops in the SBN were scored.
Results: Semi-quantitative observations did not show differences in density of SBN and
loops in the apex with age and no relation between the density of SBN and dendritic cells.
In the apex thick SBN of most pairs of eyes had a preferred 6-12 orientation. In addition, eyes had
a second preferred orientation which was for right eyes in the 5-11 and 6-11 direction and for left
eyes in the 7-1, 8-2 and 9-3 direction, indicating a second preference towards the temporal side.
In the periphery screening was performed in the 12, 3, 6 and 9 o’clock position and
thin SBN ran in the 6-12 direction at the superior and inferior location and in the 39 direction at the nasal and temporal location. Because such findings had not been
observed in previous publications one person was scanned along every clock hour
and these thin SBN appeared to be radially organized along the circumference.
In the mid-peripher y close to the radial f ibers many passages of stromal
ner ves th rough Bowman’s layer as well as small cur ved stromal ner ves
below the epitheliu m (subepithelial ner ves) were f requently obser ved.
Conclusions: This is the first time that important details of the corneal nerves outside the apex
were studied in vivo with confocal microscopy. Besides the 6-12 orientation pattern of SBN,
there is a second one towards the temporal side. These observations are of relevance concerning
possible changes caused by diseases or after surgical intervention such as refractive surgery.
CR: C. Jacobi, None; K. Gottschalk, None; C. Cursiefen, None; F.E. Kruse, None; L.J.
Muller, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 867-903 / B841-B877
137. Corneal Epithelium Organizing Section: CO
879 - B853
Subbasal Nerves and Highly Reflective Cells in Corneas of Diabetic Patients: In vivo
Evaluation by Confocal Microscopy
M.Popper1A,2, M.J. Quadrado1A, A.M. Morgado1B,1C, J.N. Murta1A, J.A. Van Best1A, L.J.
Muller3. AIBILI Center of Ophthalmology, BIBILI Dept of Instrumentation, CDept of
Physics, 1University of Coimbra, Coimbra, Portugal; 21st Dept of Ophthalmology,
Semmelweis University, Budapest, Hungary; 3The Netherlands Ophthalmic Research
Institute (NORI), Amsterdam, The Netherlands.
Purpose: To evaluate in vivo changes in density and morphology of subbasal nerves (SBN) and highly
reflective cells (HRC) in corneas of diabetic patients with mild and moderate diabetic retinopathy.
Methods: The left cornea of 20 type 2 diabetic patients, 10 with mild (level 20 ETDRS)
and 10 with moderate (level 35 ETDRS) retinopathy and 10 age-matched healthy controls
were examined with the Tomey Confoscan P4 scanning-slit confocal microscope.
Through focus scans were recorded from epithelium to endothelium. Images in the
epithelium and anterior stroma were analyzed in a masked fashion by two of the authors.
Results: The average number of SBN per image for level 20 (1.25 ± 0.52; mean ± SD) and
level 35 (1.22 ± 0.60) diabetic patients was about equal (p=0.996) but was significantly
lower than that in healthy controls (3.90 ± 1.13; p<0.001). The density of basal epithelial
cells in level 20 (5145 ± 237 cells/mm 2) and in level 35 (5042 ± 236 cells/mm 2) diabetics
did not differ significantly (p=0.6). Similar to the SBN, the number of basal epithelial
cells was significantly lower than that in healthy controls (5648 ± 236 cells/mm 2; p<0.001).
Furthermore, very highly reflective cells were observed immediately beneath the basal
epithelial cells’ layer. In contrast to the decrease in the number of SBN and of basal
epithelial cells, the number of HRC was significantly higher both in level 20 (2.36 ±
1.12) and level 35 (3.10 ± 0.97) diabetics than in healthy controls (1.13 ± 0.72; p<0.02).
Conclusions: The decrease in the number of SBN and of basal epithelial cells in the corneas of
diabetic patients is characteristic of the disease and these changes might be related to each other.
Since the morphology of HRC differs from keratocyte nuclei, and the HRC are located in
close vicinity of subbasal nerves, we consider them as dendritic cells.
CR: M. Popper, None; M.J. Quadrado, None; A.M. Morgado, None; J.N. Murta, None; J.
A. Van Best, None; L.J. Muller, None.
Support: FCT SFRH/BD/13710/2003
881 - B855
880 - B854
Phospholipase D in Corneal Epithelial Cell Motility
J.K. Klarlund, H.Achebe, A.Matela, E.Block. UPMC Eye Center, Ophthalmology and
Visual Science Research Center, Eye and Ear Institute, Department of Ophthalmology,
University of Pittsburgh School of Medicine, Pittsburgh, PA.
Purpose: Stimulation of cells by numerous hormones and growth factors results in
activation of phospholipase D, an enzyme that hydrolyzes phosphatidyl choline to generate
the secondary messenger phosphatidic acid. The purpose of this study was to examine
the role of phospholipase D in induction of a motile phenotype in corneal epithelial cells.
Methods: The motile state of epithelial cells was induced by acute presentation of
permissive culture area. Phospholipase D activities were assayed by labeling cells with
[3H]myristic acid, adding 1-BuOH and measuring formation of phosphatidylinositol
butanol. Directional migration was measured in Transwell ® migration chambers.
Results: Wounding of sheets of corneal epithelial and MDCK cells causes rapid activation
of phospholipase D. Short-chain analogues of phosphatidic acid were found to induce a
migratory phenotype robustly in several epithelial cell types, including corneal epithelial
cells. Addition of exogenous dioctanoyl phosphatidic acid induces marked activation of
endogenous phospholipase D suggesting the existence of a feed-forward regulatory loop.
When corneal epithelial cells were presented with phosphatidic acid as a gradient in modified
Boyden chambers, a strong chemotaxic response was observed. Epidermal growth factor
activates PLD and acts as a chemoattractant in corneal epithelial cells. When saturating
amounts of epidermal growth factor was present in both compartments of the migration
chambers chemotactic movement of corneal epithelial cells towards phosphatidic acid was
abolished. This suggests that phospholipase D and epidermal growth factor signal through
shared signaling pathways. Differential sensitivity to inhibitors and other observations strongly
suggested that the added phosphatidic acid did not act through activation of protein kinase C.
Conclusions: These data support an important role for phospholipase D in induction of
motility in corneal and other epithelial cells, and suggest that localized concentrations of
phosphatidic acid may determine direction of movement.
CR: J.K. Klarlund, None; H. Achebe, None; A. Matela, None; E. Block, None.
Support: NIH 5R01EY013463-02
882 - B856
Benzalkonium Chloride (BAK) Induces MLC Dephosphorylation in Corneal
Epithelial Cells
Y.Guo, M.Satpathy, G.Wilson, S.Srinivas. School of Optometry, Indiana University,
Bloomington, IN.
Purpose: BAK, a preservative in ophthalmic formulations, accelerates desquamation of
corneal epithelial cells with a concomitant depletion of intracellular ATP. Amongst varied
effects of ATP depletion, dephosphorylation of regulatory light chain of myosin II (MLC) has
been reported in cells undergoing hypoxic stress. Since excessive MLC dephosphorylation
is implicated in disruption of focal adhesion secondary to loss of actin contractility, we
have begun to investigate if desquamation in response to BAK is a consequence of MLC
dephosphorylation. In this study, as a first step, we examined the influence of BAK on ATP
loss and corresponding status of MLC phosphorylation. Methods: Experiments were carried
out in primarily cultured bovine corneal epithelial cells. Acute ATP release was measured by
bioluminescence of Luciferrin-Luciferase reaction. MLC phosphorylation was assayed by
urea glycerol gel electrophoresis and Western blotting. Deliberate ATP depletion was induced
by exposure to antimycin and hypoxia as a positive control. Results: Exposure to BAK at
0.003%, which is a concentration usually found in topical drug formulations, led to MLC
dephosphorylation by 54% (n=5) within 10 min and this persisted for 30 min (60%; n=5). The
extent of dephosphorylation decreased with lower concentrations of BAK as 0.001% BAK
led to MLC dephosphorylation by 16% (n=5) at 10 min and 40% (n=5) at 30 min. Exposure
to BAK (0.003%; 0.001%) also led to a transient release of ATP which was characterized
by a rapid onset and an exponential decay. Exposure to antimycin (10 μM) and hypoxia
(pO2 = 1.5% for 3 hrs in the absence of glucose) led to complete MLC dephosphorylation.
Conclusions: Exposure to BAK causes acute ATP release and this may underlie the reported
cellular ATP depletion. Consistent with the response to antimycin and hypoxia, we suggest
that BAK-induced ATP depletion leads to MLC dephosphorylation and this may form the
basis for epithelial desquamation.
CR: Y. Guo, None; M. Satpathy, None; G. Wilson, None; S. Srinivas, None.
Support: NIH Grant EY11107 (SPS) and EY14415 (SPS)
Microbiological Evaluation of a Preservative-Free Multidose Container for Eyedrops
M.Tanaka1, E.Yamada1, Y.Kajihara1, H.Mihashi2, K.Hamamoto2, T.Nishida3. 1Laboratory,
Nitten Pharmaceutical Co. Ltd., Nagoya, Japan; 2Taisei Kako Co. Ltd., Osaka, Japan;
Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University
School of Medicine, Ube, Japan.
Purpose:The addition of preservatives to eyedrops has become a standard procedure
to prevent microbiological contamination, even though allergic reactions and corneal
epithelial disorders have been attributed to these agents. We recently developed a
multidose eyedrops container fitted with a membrane filter (PF container) in order
to obviate the requirement for preservative. We have now investigated the efficacy
of this new type of container with regard to prevention of microbial contamination.
Methods: PF containers fitted with a polyethersulfone filter (pore size, 0.22 μm) were
studied. Brevundimonas diminuta (standard strain for filtration sterility test; 10 6 to 107
colony-forming units/mL) in phosphate-buffered saline was used for laboratory experiments.
PF containers containing carteolol chloride, betamethasone sodium phosphate, or sodium
cromoglicate were also provided to 76 patients for disease treatment and were recovered after
use for 1 to 2 weeks for analysis of the cap, tip, membrane filter, and solution for bacteria.
Results:No B. diminuta bacteria were detected in the solution dispensed from any of 10
bottles containing the test bacterial suspension. Similarly, no bacterial contamination
of a soybean casein digest placed in 10 containers was detected after the attempted
aspiration of B. diminuta suspension by squeezing the bottles. Analysis of the PF
containers used by patients revealed the presence of Staphylococcus, Bacillus, or other
bacterial species in the inside of the cap (9%), on the tip (30%), and on the external
surface of the membrane (14%), but no microbes were detected in the solution.
Conclusions:Use of a PF container for eyedrops prevented microbial contamination of the
solution, demonstrating that such containers are safe for clinical use without preservatives.
CR: M. Tanaka, None; E. Yamada, None; Y. Kajihara, None; H. Mihashi, None; K.
Hamamoto, None; T. Nishida, None.
Support: None.
883 - B857
884 - B858
Innate Immunity at the Ocular Surface: Spectrum of Antimicrobial Peptide
L.C. Huang1, D.Jean2, R.J. Proske1, A.M. McDermott1. 1College of Optometry, University of
Houston, Houston, TX; 2School of Optometry, Inter American University of Puerto Rico,
San Juan, PR.
Purpose: We have shown that human ocular surface epithelia express a number of antimicrobial
peptides: three β-defensins (hBD1-3) and cathelicidin (LL-37). The purpose of this study was to
explore if additional antimicrobial peptides are expressed by corneal and conjunctival epithelial
cells and to study the antimicrobial activity of the peptides against common ocular pathogens in vitro.
Methods: Expression of mRNA and protein of various antimicrobial peptides was determined
by RT-PCR and immunostaining, respectively, in scraped human corneal epithelium,
primary cultured human corneal epithelial cells (HCEC), conjunctival epithelial cells, and
corneal tissue sections. In some experiments, HCEC were treated with 10ng/ml IL-1β or
TNF-α for 6 and 24 hours. Cells treated with serum-free culture media acted as controls.
Antimicrobial assays were performed to assess peptide activity against Pseudomonas
aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE).
Results: All epithelial samples (n=3) constitutively expressed mRNA for two antimicrobial
peptides: macrophage inflammatory protein (MIP-3α/CCL20) and thymosin β-4 (Tβ4).
This expression was not modulated by IL-1β or TNF-α. None of the epithelial samples
expressed hBD-4, -5, -6, HE2β1, histatins (Hist-1, -3), or liver-expressed antimicrobial
peptides (LEAP-1, -2). Scraped corneal epithelium and cultured HCEC also expressed
MIP-3α/CCL20 and Tβ4 protein (n=2). Immunostaining showed the presence of MIP3α/CCL20 and Tβ4 throughout the entire epithelial layer (n=3). hBD-3 (EC50 = 0.9-5.3μg/
ml) inhibited growth of all bacteria in a concentration dependent manner (n=3). hBD-2
(EC50 = 1.2+0.2μg/ml), MIP-3α/CCL20 and Tβ4 (EC50 = 18.1+1.7μg/ml) were effective
against PA, but were only weakly effective against staphylococcal strains (n=3). hBD-1
(EC50 =21.4+1.5μg/ml) showed activity against PA, but was not effective against SA or SE (n=3).
Conclusions: Our data show that in addition to β-defensins and cathelicidin, the ocular surface
epithelia express MIP-3α/CCL20 and Tβ4. The presence of various antimicrobial peptides at the
ocular surface likely contributes to the innate response protecting the eye against infection.
CR: L.C. Huang, None; D. Jean, None; R.J. Proske, None; A.M. McDermott, None.
Support: NIH Grant EY13175 (AMM), UH GEAR Grant (AMM), NIH T35 EY007088-19
Prteasomal Regulation of Muc4 in Cultured Corneal Epithelium Cells
J.Lomako1A, W.M. Lomako1A, C.A. C. Carraway1B, K.L. Carraway1A. ACell Biology &
Anatomy, BBiochemistry & Molecular Biology, 1Univ Miami Sch Med, Miami, FL.
Purpose: Previous evidence indicates that the amount of Muc4/SMC in
many different epithelial cells is regulated at both transcriptional and posttranslational levels. This study was initiated to determine whether the level of
Muc4/SMC in corneal epithelial cells depends on proteasomal degradation.
Methods: Cultured rat corneal epithelial cells were treated with proteasomal inhibitors
to determine their effects on Muc4/SMC levels, measured by immunobloting.
Ubiquitination of Muc4/SMC was investigated by sequential immunoprecipitation
and immunoblotting, and localization of Muc4/SMC within the cells was determined
by confocal immunof luorescence microscopy. The effect of carbohydrate chain
processing inhibitors on Muc4 levels was also investigated by immunoblotting.
Results: The level of Muc4/SMC significantly increased in cultured corneal epithelial cells
after treatment with proteasome inhibitors, indicating that proteasomes play role in quantity
and/or quality control of Muc4/SMC in this epithelium. Transmembrane subunit (ASGP-2)
of Muc4/SMC is ubiquitinated, probably as part of the proteasomal degradation mechanism.
Inhibitors of N-linked carbohydrate chain processing increased proteasomal degradation, the
effect of which can be partially reversed by proteasome inhibitors. Cells treated with proteasome
inhibitors accumulated Muc/4SMC in intracellular inclusions resembling aggresomes.
Conclusions: This is the first report indicating that Muc4/SMC is subjected in corneal
epithelium to ubiquitination and proteosomal degradation. This mechanism may provide an
important pathway which regulates the level of Muc4 in epithelial cells.
CR: J. Lomako, None; W.M. Lomako, None; C.A.C. Carraway, None; K.L. Carraway,
Support: NIH Grant EY12343
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 867-903 / B841-B877
137. Corneal Epithelium Organizing Section: CO
885 - B859
886 - B860
Thymosin Beta 4 Inhibits Benzalkonium Chloride-Mediated Apoptosis of Human
Corneal Epithelial Cells in vitro
G.Sosne1A, A.-R.Albeiruti1B, B.Hollis1B, M.Kurpakus-Wheater2. AOphthalmology and
Anatomy/Cell Biology, BAnatomy/Cell Biology, 1Wayne State University, Detroit, MI;
3Department of Biomedical Sciences, School of Dentistry, University of Detroit Mercy,
Detroit, MI.
Purpose: Thymosin beta 4 (Tβ 4), a 43 amino acid molecule, promotes ocular
wound healing, decreases ocular inf lammation, and has anti-apoptotic effects on
corneal epithelium. In this study, the effect of Tβ 4 on the survival of cultured human
corneal epithelial cells exposed to benzalkonium chloride (BAK) was measured.
Methods: Human corneal epithelial cells at approximately 80% confluence were treated
with 0%, 0.001%, 0.01%, or 0.1% BAK for 15 minutes. After 3 and 24 hours of recovery in
culture medium, cell proliferation was measured using a colorimetric BrdU incorporation
assay. Apoptosis was measured using a colorimetric annexin-based cell death assay. Studies
were repeated in the presence of 1 μg/ml Tβ 4, an in vitro dosage demonstrated effective
in several published studies. To further assess the ability of Tβ 4 to prevent apoptosis,
corneal epithelial cells were treated with 0.01% BAK + Tβ 4 over a 5 day time course.
Results: At all BAK concentrations used, corneal epithelial cell proliferation was
inhibited, and apoptosis was increased, compared to control at 3 and 24 hours recovery
time. At the 3 and 24 hour time points, Tβ 4 did not abrogate the deleterious effects
of BAK; cell proliferation was not promoted by Tβ 4 and apoptosis was not inhibited.
However, at longer times in culture (2 to 5 days), Tβ 4 treatment significantly inhibited
the BAK-initiated epithelial cell apoptosis. In addition, Tβ 4 -treated cells demonstrated
decreased apoptosis compared to those cultured in medium alone for 5 days.
Conclusions: BAK, a preservative used in many commercially available ocular solutions,
induces corneal epithelial cell apoptosis in culture, suggesting that long-term exposure
is deleterious to corneal health. The study reported here suggests that Tβ 4 may be able to
overcome the deleterious pro-apoptotic effects of BAK. Since many BAK-containing eye
drops are typically used for extended periods of time, Tβ 4 may be a useful additive to solutions
containing this preservative.
CR: G. Sosne, None; A. Albeiruti, None; B. Hollis, None; M. Kurpakus-Wheater,
Support: NIH Grant EY13412, Career Development Award Research to Prevent Blindness
In vivo Very-High-Frequency Ultrasound Imaging and Measurement of Corneal
Epithelial Changes Induced by Chronic Exposure to BAC in Rabbit Eyes
A.H. Denoyer1A, F.Ossant1B, P.-V.Jacomet1A, B.Arbeille1C, F.Fétissof 1D, F.Patat1B, P.J.Pisella1A. AOphthalmology, BLussi FRE CNRS 2448, CCell Biology, DPathology,
University of Tours, Tours, France.
Purpose: To evaluate very-high-frequency (VHF) ultrasound in the in vivo follow-up of
corneal epithelial changes induced by chronic exposure to benzalkonium chloride (BAC).
Methods: 0.01% BAC-containing solution was applied twice a day in the tested eye of 10
rabbits during 56 days. We used a 60 MHz ultrasound device to assess ocular surface of BACexposed eyes, compared with non-exposed eyes. This comparative prospective study included
both clinical and ultrasound examinations every 10 days, and ultimate histological analysis.
Results: Clinical changes were conjunctival redness, conjunctival and corneal staining and
decreased break-up-time. In vivo VHF ultrasound revealed a decrease in absolute epithelial
thickness of tested eyes (from 40.9 ± 1.6 SD µm at D0, to 31.8 ± 3.4 SD µm at D56 ; P =
0.0006 for D0 vs D56), and a decrease in relative epithelial thickness of tested eyes compared
with controls (from -0.3 ± 1.9 SD µm at D0 to -9.1 ± 2.9 SD µm at D56 ; P = 0.0003 for D0
vs D56). VHF ultrasound epithelial thickness was correlated with corneal staining (at D34
and D56 ; P = 0.0025 and 0.0377, respectively) and histological epithelial pachymetry (P
= 0.0176 for control and 0.0505 for tested epithelium). Moreover, we reported qualitative
VHF ultrasound imaging of epithelial damages including superficial punctuate keratitis.
Conclusions: VHF ultrasound allowed an in vivo assessment of epithelial changes induced
by topical preservatives ; it could be a reproducible and reliable tool for evaluation of ocular
surface toxicity.
CR: A.H. Denoyer, None; F. Ossant, None; P. Jacomet, None; B. Arbeille, None; F.
Fétissof, None; F. Patat, None; P. Pisella, None.
Support: None.
887 - B861
888 - B862
889 - B863
890 - B864
Effects of High Glucose and Streptozotocin-Induced Diabetes on Cell Proliferation
and Protein Kinase C Activity in Rat Corneal Epithelium
R.A. Akhtar, H.Zhang. Biochemistry & Molecular Biol, Medical College of Georgia,
Augusta, GA.
Purpose: Diabetic patients are prone to delayed corneal epithelial wound repair
following vitrectomy and keratectomy. The purpose of this work was to investigate
the effects of high glucose on cell proliferation and protein kinase C (PKC) activity
in cultured corneal epithelial cells. We also examined whether streptozotocininduced diabetes has any effect on PKC activity in rat corneal epithelium.
Methods: Serum-starved, primary rat corneal epithelial cells (RCEC) were cultured in media
containing different concentrations of glucose. After 24 hrs, the cultures were terminated
and the cells trypsinized and either counted for determination of cell proliferation or used
for analysis of PKC activity. Sprague Dawley rats (200-250 g) were made diabetic by an iv
injection of streptozotocin (65 mg/kg BW). After 4 weeks of confirmed diabetes, the corneal
epithelial tissue was removed from diabetic and non-diabetic rats and used for PKC assay.
Results: RCEC exhibited a large increase in their proliferation when cultured for
24 hrs in a medium containing 7.8 mM glucose (low glucose). Increasing glucose
concentration to 37 mM (high glucose) resulted in a significant decrease in proliferation
of the cells. Under the same experimental conditions, there was a significant increase
in PKC activity in RCEC cultured in high glucose medium as compared to the control
cells cultured in low glucose medium. When analyzed for PKC activity in corneal
epithelium, a significant increase in translocation of PKC activity from soluble to the
particulate fraction was observed in the diabetic rats as compared to the non-diabetic rats.
Conclusions: The data demonstrate that high glucose inhibits RCEC proliferation but
stimulates PKC in these cells. Activation of PKC under diabetic conditions suggests a complex
role for this enzyme in delayed epithelial wound repair in diabetic cornea.
CR: R.A. Akhtar, None; H. Zhang, None.
Support: NIH EY05738
Allograft Transplantation of Functional Epithelial Corneal Cell Layers in Rabbits
F.Luengo Gimeno1,2, J.E. Gallo1, J.O. Croxatto2, L.Correa3, F.Roldán3. 1Ophthalmology,
Universidad Austral, Buenos Aires, Argentina; 2Ophthalmology, Fundación
Oftalmologica Argentina “Jorge Malbran”, Buenos Aires, Argentina; 3Bioengineering,
Laboratorios Craveri, Buenos Aires, Argentina.
Purpose: The development of corneal tissue constructs for the treatment of ocular surface
disease is still evolving. The purpose of this study was to demonstrate the usefulness of
heterologous cultivated limbal epithelial cell grafts for ocular surface reconstruction.
Methods: Limbal stem cell deficiency was induced in 5 New Zealand White rabbits
by anterior lamellar keratectomy including the limbal area. Previously, a small 2.5
mm 2 limbal sample was obtained from healthy eyes of randomly selected male rabbits.
Limbal epithelial cells were cultivated, and 21 days later epithelial sheets were ready
to be grafted. The epithelial grafts were sutured covering the exposed corneal stroma.
The control group included 5 rabbits that underwent surface keratectomy without
grafting. The animals received topical tobramicine and dexametasone during 2 weeks
after surgery. Clinical follow up was performed up to 90 days. Rabbits were euthanized
at day 2, 7, 30 and 90 and the corneas processed for histopathologic examination.
Results: Clinically, the corneas were clear in all grafted animals. A full thickness continuous
layer of stratified epithelial cells was seen in grafted corneas. Control animals showed a thinner
regenerative epithelium and more pronounced fibroblastic proliferation. Neither clinical
nor histological evidence of immune reaction was observed. Conclusions: Allografts of
bioengineered full thickness limbal epithelium were able to differentiate in corneal epithelium
and remain intact up to 3 months without significant immune reactions. Heterologous corneal
epithelial cell transplantation may be a promising procedure for bilateral ocular surface
CR: F. Luengo Gimeno, None; J.E. Gallo, None; J.O. Croxatto, None; L. Correa, None; F.
Roldán, None.
Support: None.
Assessment of Epithelial Cell Trauma Following Mechanical Separation by Means of
Trypan Blue Staining
I.Pallikaris, H.S. Ginis, V.Katsanevaki, M.Kalyvianaki, I.Naoumidi. Department of Medicine,
University of Crete, Heraklion, Greece.
Purpose: In epi-LASIK, the epithelial layer is mechanically separated by means of a specially designed
surgical instrument, is temporarily reflected and finally repositioned on the surface of the cornea
following laser ablation for the correction of ametropias. It is the purpose of the present study to employ
trypan blue staining and an image processing technique in order to quantify the degree and pattern of
trypan blue staining of epithelial sheets following mechanical separation. Moreover, to correlate the
macroscopic staining parameters to the histological appearance of the separated epithelium and to
study the effect of surgically induced trauma during separation to the postoperative clinical course.
Methods: In a group of three patients the epithelial sheets were excised, prepared by means of
trypan blue staining and photographed under controlled conditions. Additionally, the sheets were
examined microscopically both by light and electron microscopy. In ten patients, epithelium was
excised from both eyes and sheets were examined in order to evaluate staining symmetry between
fellow eyes. Following the initial observations on staining symmetry, thirty patients underwent
bilateral treatment whereas the epithelium was excised and stained from one eye (randomly
selected) while in the fellow eye the sheet was repositioned after laser ablation. A purposelydeveloped MATLAB script was used to quantify the degree and pattern of staining for the excised
sheets. The clinical course of the fellow eye was documented for a postoperative period of three
months. Study parameters were re-epithelialization time, pain score, and stability of refraction.
Results: In stained areas electron microscopy revealed discontinuities of the basal membrane
and partial separations between adjacent basal epithelial cells, while in the non-stained areas
the basal membrane and basal epithelial layer appeared intact. Degree and pattern of staining
are subject to high inter-subject variability. This is supported by the fact that there seems
to be a strong bilateral symmetry in the staining patterns. None of the study parameters of
the postoperative course in epi-LASIK eyes was correlated to the degree of staining.
Conclusions: Trypan blue staining can be used to quantify cellular level trauma in mechanical
separation of the epithelial layer. Amount of staining in intact sheets, is not correlated to clinical
CR: I. Pallikaris, Norwood Eyecare P; H.S. Ginis, Norwood Eyecare P; V. Katsanevaki,
Norwood Eyecare C; M. Kalyvianaki, None; I. Naoumidi, None.
Support: None.
PTK With an EpiLASIK Flap for the Treatment of Recurrent Erosion Syndrome
K.Hufendiek1, W.A. Herrmann1, V.-P.Gabel1, C.P. Lohmann2. 1Ophthalmology, University,
Regensburg, Germany; 2Ophthalmology, University (TU), Muenchen, Germany.
Purpose: Phototherapeutic keratectomy (PTK) is an established treatment for recurrent erosion
syndrome. After PTK many patients experience significant pain and have a slow visual recovery.
The aim of this study was to evaluate if modified PTK with an EpiLASIK epithelial flap is effective
in the treatment of recurrent erosion syndrome and might be less painfull as a conventional PTK.
Methods: 10 consecutive eyes of patients with a history of recurrent erosion (3 months
to 3 years, mean 9.3 months; age range 25 to 56 years, mean 39 years) were included in
the study. Similar to EpiLASIK, an epithelial flap with a nasal hinge was generated using
a Keratome (EpiTome, Gebauer, Germany). Photoablation with an ablation depth of 25
µm was performed with a Wavelight Concept 500 Excimer Laser. After photoablation
the epithelial flap was repositioned and secured with a soft bandage contact lens (Pure
Vision, Bausch & Lomb, USA). 3 days after surgery the contact lens was removed.
Results: During a follow-up time of 32 to 162 days (mean 83,3 days) no recurrent erosion
was observed. Best corrected visual acuity (BSCVA) recovered to preoperative values
within 14 days in all cases. Only 2/10 patients reported about a moderate postoperative pain.
Conclusions: Modified PTK with an epithelial flap like in EpiLASIK is a save and effective
procedure in the treatment of recurrent erosion syndrome. Our initial results indicate that
this procedure might be superior compared to conventional PTK regarding wound healing
time, postoperative pain and visual recovery.
CR: K. Hufendiek, None; W.A. Herrmann, None; V. Gabel, None; C.P. Lohmann,
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 867-903 / B841-B877
137. Corneal Epithelium Organizing Section: CO
891 - B865
892 - B866
Enhanced Drug Delivery to the Cornea With a Novel Peptide
D.A. Ghate1, T.Iwamoto2, B.McCarey1, J.Tomich2, H.F. Edelhauser1. 1Ophthalmology, Emory
university, Atlanta, GA; 2NaCelle Therapeutics, Manhattan, KS.
The peptide NC 1059 (developed by NaCelle therapeutics) has been shown to reduce transepithelial
resistance in cultured epithelia of different cell lines by altering protein components of tight junctions.
Purpose: To study the effect of peptide NC-1059 on corneal permeability to carboxyfluorescein(CF) in rabbit
corneas in situ and in vivo and compare the histopathology of corneas treated with the peptide and 0.01% BAC.
Methods:Transcorneal permeability(TCP) was evaluated in situ using corneal perfusion blocks. The
endothelial side solution had BSS plus. The epithelial side had BSS plus and CF solution .The peptide
(40 μM/ml) was added to the epithelial side of the test chamber. 100 μl samples were withdrawn
every 30 minutes for 6 hours. The concentrations were measured using a fluorometer .Transepithelial
permeability(TEP) was measured in vivo in unanaesthesized rabbits by non-invasive fluorometry.
Peptide Solution A( 40 μM/ml in BSS plus) or solution B(80 μM/ml in BSS plus) was exposed to the
corneal surface for 3 minutes. The contralateral eye (control) was treated with BSS. After 0 min or
30 min delay, the eyes were exposed to CF for 3 or 5 min and the corneal fluorescence was measured
on an ocular fluorometer. The histology of the corneas treated with the peptide, 0.01% BAC and
the BSS plus was evaluated using transmission electron microscopy after a ruthenium red(RR) fix.
Results: In the in situ experiments (n=7), the Ktrans (cm/sec) for CF was (mean ± SD) 9.72 ± 6.23 * 10 -8
with the peptide and 5.13±4.73 * 10 -8 with BSS plus representing an average 2.8(range 1.24-4.71) fold
increase. There were 3 sets of in vivo experiments. With solution A and CF for 3 min(n=3), the TEP
(nm/sec) of the test cornea was 0.07± 0.02 and the control was 0.03±0.01 representing an average
2.14(range1.82- 2.73)times increase. With solution A and CF for 5 min (n=4), the TEP (nm/sec) of the
test cornea was 0.29± 0.29 and the control was 0.05±0.03 representing an average 8.00 (range 1.07-18.39
)times increase. With solution B and CF for 5 min(n=5), the TEP(nm/sec) of the test cornea was 0.58±
0.32 and the control was 0.09±0.08 representing an average 15.72(range 2.13-33.02) times increase.
The corneas treated with the peptide BAC had RR penetrating paracellularly down to 3-5 epithelial
cell layers, the control corneas had no RR penetration.The peptide treated epithelial cells showed a
normal morphology and thickness. The BAC treated cells were edematous with an altered morphology.
Conclusions: The peptide NC-1059 increases the transcorneal permeability to CF without damaging
the epithelium
CR: D.A. Ghate, NaCelle therapeutics, Manhattan KS F; T. Iwamoto, Nacelle Therapeutics,
Manhattan KS P; B. McCarey, Nacelle Therapeutics Manhattan, KS F; J. Tomich, Nacelle
Therapeutics, Manhattan, KS P; H.F. Edelhauser, NaCelle Therapeutics, Manhattan, KS F.
Support: NEI grant R43-EY015606
Epidermal Growth Factor-Modified Polydimethylsiloxane for Keratoprostheses
B.Klenkler1A, J.West-Mays1B, H.Sheardown1A. AChemical Engineering, BPathology and
Molecular Medicine, 1McMaster University, Hamilton, ON, Canada.
Purpose: Lack of corneal epithelial cell coverage over the anterior surface of a
keratoprosthesis can lead to epithelial downgrowth and device extrusion. In this work,
the effect of immobilized epidermal growth factor (EGF), a mitogen for epithelial
cells, on cell growth over polydimethylsiloxane (PDMS) substrates is assessed.
Methods: EGF was bound to PDMS substrates via polyethylene glycol (PEG) tethers
by two methods. 1) Solution-first: EGF was first reacted with a bifunctional PEG in
solution, then tethered to allyamine-modified PDMS. 2) Surface-first: PDMS was first
modified with bifunctional PEG, and EGF was then reacted with the surface-bound
PEG. The amount of bound EGF was varied by altering the EGF solution concentration.
Human corneal epithelial cells were seeded onto EGF-modified and control surfaces and
cultured in keratinocyte serum free medium with and without soluble EGF. Immunofluorescence
was used to assess production of proteins including fibronectin, laminin and cytokeratin.
Results: Modification by the solution-first method resulted in 46 to 91 ng/cm 2 of bound EGF.
In this range, cells grew to confluence on the EGF-modified surfaces within two to three days
of culture, even without soluble EGF in the medium, whereas cell coverage on unmodified
PDMS surfaces remained incomplete at four days. Control surfaces with denatured EGF
resulted in lower cell coverage at one and two days compared to native EGF, indicating
that EGF is active at early time points in stimulating cell growth. Allylamine modification
alone also improved cell growth over four days vs. unmodified PDMS. Positive staining for
cytokeratin, fibronectin and laminin indicated the cells are differentiated and produce matrix
proteins on the EGF-modified surfaces at significantly higher levels vs. control surfaces.
Modification by the surface-first method resulted in 5 to 190 ng/cm 2 of bound
EGF. Cell growth was biphasic, with highest coverage at 45 to 62 ng/cm 2 of
EGF. Surface modification with PEG alone completely inhibited cell growth.
Conclusions: Tethering of EGF to PDMS significantly improves growth of corneal epithelial
cells. Effects depend on EGF concentration and underlying substrate modification.
CR: B. Klenkler, None; J. West-Mays, None; H. Sheardown, None.
Support: None.
893 - B867
894 - B868
A Randomized, Placebo-Controlled Clinical Trial of the Aldose Reductase Inhibitor
CT-112 as Management of Corneal Epithelial Disorders in Diabetic Patients
R.Nejima1, M.Nakahara2, K.Miyata2, S.Otani2, T.Miyai2, S.Amano3. 1Ophthalmology,
miyata eye hospital, Miyakonojyou, Japan; 2Ophthalmology, Miyata Eye Hospital,
Miyakonojyou, Japan; 3Department of Ophthalmology, University of Tokyo School of
Medicine, Tokyo, Japan.
Purpose: To evaluate the efficacy of topical aldose reductase inhibitor CT-112 (5-[3-ethoxy-4pentyloxyphenyl]-2,4-thiazolidinedione) on corneal epithelial barrier function in diabetic patients.
Methods:Thirty four eyes of 34 diabetic patients were randomly assigned treatment
with 0.25 % eyedrops of CT-112 (n=22) or a placebo (n=12) four times a day
for 8 weeks. Corneal f luorescein staining and corneal sensation were examined
before treatment as well as 4 and 8 weeks after administration. Corneal epithelial
permeability to f luorescence was measured with an anterior f luorophotometer.
Results: Average scores of superficial punctate keratopathy and corneal sensitivity did not differ
significantly different between the two groups at any time-point. Whereas average fluorescein
concentrations did not differ significantly for the CT-112 and placebo groups before treatment,
they did differ significantly 4 and 8 weeks after treatment (4 weeks, p = .0327; 8 weeks, p = .0143).
Conclusions: Topical ARI, CT-112 improves the corneal epithelial barrier function in diabetic
CR: R. Nejima, None; M. Nakahara, None; K. Miyata, None; S. Otani, None; T. Miyai,
None; S. Amano, None.
Support: None.
Pre-Clinical Studies of OPC-12759 Ophthalmic Suspension for Dry Eye Treatment
H.Urashima, A.Aoki, K.Fujita, T.Takizawa, S.Oshima. Ako Research Institute, Otsuka
Pharmaceutical Co Ltd, Ako, Japan.
Purpose: OPC-12759 is a novel quinolinone derivative synthesized by Otsuka Pharmaceutical
Company, Ltd., Japan. In these pre-clinical studies, we investigate the pharmacological
effect of OPC-12759 and its usefulness as a therapeutic agent for dry eye treatment.
Methods: In vivo studies; OPC-12759 ophthalmic suspension was instilled 6 times per day for
about 14 days to normal or N-acetylcysteine (NAC)-treated rabbits to evaluate the following
parameters. 1) Mucin content in the cornea and conjunctiva (Alcian blue binding method) 2)
PAS-positive cell density of the conjunctiva (impression cytology method) 3) Therapeutic effect
on corneal and conjunctival damage in NAC-treated eyes (Rose bengal scores). In vitro studies;
HCECs were cultured with or without OPC-12759, then secreted and cell-associated mucin
contents were measured by the combined methods of gel filtration and enzyme-linked lectin
assay (ELLA) and the expression of mucin gene in cultured HCECs was determined by RT-PCR.
Results: Topical application of OPC-12759 significantly increased the corneal and conjunctival
mucin contents in normal and NAC-treated eyes and conjunctival PAS-positive cell density in
normal eyes. One % OPC-12759 significantly decreased the Rose bengal scores of the cornea and
conjunctiva in NAC-treated eyes. OPC-12759 increased the mucin contents of the secreted and
cell-associated mucin from/in cultured HCECs in a concentration-dependent manner. The gene
expression levels of MUC1 and MUC4 in cultured HCECs were up-regulated by 10 -5M OPC-12759.
Conclusions: OPC-12759 increased the amount of mucin contents in the ocular surface due to
its effect on corneal epithelial cells and conjunctival PAS-positive cells, and improved ocular
surface damage. For corneal epithelial cells, it directly increased secreted and membranespanning mucin contents. Therefore, OPC-12759 may be of therapeutic value in the treatment
of dry eye patients by increasing the mucin contents in the ocular surface.
CR: H. Urashima, Otsuka Pharmaceutical Co Ltd E; A. Aoki, Otsuka Pharmaceutical Co
Ltd E; K. Fujita, Otsuka Pharmaceutical Co Ltd E; T. Takizawa, Otsuka Pharmaceutical
Co Ltd E; S. Oshima, Otsuka Pharmaceutical Co Ltd E.
Support: None.
895 - B869
896 - B870
Active Form of Gelatinase in the Tear Fluid of Recurrent Corneal Erosion Patients
T.Sakimoto1,2, J.Shoji2, A.Ikeda2, M.Sawa2. 1Schepens Eye Research Institute/MEEI,
Department of Ophthalmology, Harvard Medical School, Boston, MA; 2Nihon University
School of Medicine, Department of Ophthalmology, Tokyo, Japan.
Purpose: To investigate gelatinase {Matrix metalloproteinase (MMP) -2 and
MMP-9} expression in the patients with recurrent corneal erosion (RCE).
Methods: The subjects comprised patients with RCE, RCE group (10 cases of total 25 eyes); the
patients with conventional corneal erosion caused by trauma, erosion group (3 cases of 3 eyes);
and normal subjects, normal group (10 cases of 10 eyes). In RCE group, tear was taken during
the onset period (7 cases of 7 eyes) or latency period (4 cases of total 6 eyes) and in the fellow eye
(9 cases of total 12 eyes), the tear was also taken essentially. Latency period of RCE was termed
as the absence of erosion occurence at least 4 weeks. After obtaining the informed consent, tear
samples were collected by the method of Schirmer test I and analyzed by gelatin zymography.
Results: In normal group and erosion group, neither active form of MMP-2 (62kDa) nor
active form of MMP-9 (82kDa) was detected. In RCE group, active MMP-2 and active
MMP-9 were both detected in all 7 eyes in onset period. In latency period, active MMP-2
(5/6) and active MMP-9 (3/6) were detected. In fellow eye, active MMP-2 (3/12) and active
MMP-9 (3/12) were detected even though there was no history of recurrent corneal erosion.
Conclusions: In the tear fluid of RCE, gelatinase expression was up-regulated. The existences
of gelatinase in latency period and fellow eye indicate that gelatinese expression in the tear
fluid may have relationship with its recurrence.
CR: T. Sakimoto, None; J. Shoji, None; A. Ikeda, None; M. Sawa, None.
Support: None.
Pseudomonas Aeruginosa Exposure Regulates Surfactant Protein D Production by
Human Corneal Epithelial Cells
M.Ni1, D.J. Evans1,2, S.M. J. Fleiszig1. 1Optometry and Vision Science, University of
California-Berkeley, Berkeley, CA; 2Touro University-California, Vallejo, CA.
Purpose: We previously showed that SP-D was present in human tear fluid and that it protected corneal
epithelial cells against Pseudomonas aeruginosa invasion in vitro. In this study, we explored the
expression of SP-D in corneal epithelium and then examined the effect of P. aeruginosa exposure.
Methods: Primary cultures of mouse corneal epithelial cells were prepared from female 8-12
week old wild type C57BL/6 mice and gene-targeted SP-D deficient mice. SDS-PAGE and
Western blot were performed to detect the SP-D level in cultured corneal epithelial cell lysates or
cell growth media. To determine whether P. aeruginosa exposure can regulate SP-D expression,
SV 40-immortalized human corneal epithelial cells were stimulated with 2 x 107 or 2 x 1010
heat killed bacteria (invasive strain PAK) or were sham inoculated. After stimulation, cells
were lysed and SP-D quantified using Western Blot. The effect of bacterial lipopolysaccharide
(LPS) on SP-D expression was explored using two different mutants with defects in LPS
core and O antigen. A fliC mutant was used to examine the role of bacterial flagellin.
Results: SP-D was detected in primary cultured mouse corneal epithelial cells derived from
C57BL/6 mice, but not in lysates of corneal epithelial cells derived from SP-D deficient mice.
SP-D was also detected extracellularly in cultured corneal epithelial cell growth media. Cells
treated with heat killed wild type P. aeruginosa showed a strong dose-dependent upregulation
of SP-D production in both cell lysates and cellular secretions. LPS and flagellin mutants,
however, were each defective in their ability to upregulate SP-D in cell lysates and cell secretions.
Conclusions: Corneal epithelial cells were found to make and secrete SP-D and as such could
contribute to tear fluid SP-D level. SP-D expression in human corneal epithelial cells was
strongly upregulated when cells were exposed to P. aeruginosa, which involved bacterial
LPS and flagellin. These results suggest that SP-D is an inducible factor involved in innate
immunity against P. aeruginosa invasion, and that induction could involve TLR signaling.
CR: M. Ni, None; D.J. Evans, None; S.M.J. Fleiszig, None.
Support: NIH grant EY11221
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 867-903 / B841-B877
137. Corneal Epithelium Organizing Section: CO
897 - B871
898 - B872
Changes in Tear Matrix Metalloproteinases-9 and-2 From Patients With Climatic Droplet
Keratopathy in the Argentine Patagonia
H.M. Serra1, M.C. Sánchez1, E.G. Knoll2, P.F. Barcelona1, P.Maccio2, E.A. Urrets-Zavalia2,3, J.A.
Urrets-Zavalia2,3. 1Cibici, Fac Cs Quimicas, UNC, Cordoba, Argentina; 2Ophthalmology, Clínica
Universitaria Reina Fabiola, Cordoba, Argentina; 3Ophthalmology, UCC, Cordoba, Argentina.
Purpose: Matrix metalloproteinases (MMPs) are upregulated during degradation of epithelial and stromal
tissues, and participated in remodelling of extra cellular matrix. We investigated the activity and molecular
forms of MMP-9 and -2 in tear fluid of patients with climatic droplet keratopathy (CDK) in Argentine.
Methods: 18 patients with CDK and 10 control subjects living in a semi deserted barren plain of
Argentine Patagonia, were investigated. Complete ocular examination [slit-lamp biomicroscopy,
external eye tests (total and basal Schirmer test, BUT, staining with lisamina green) and corneal
aesthesiometry] was performed. Tear samples (residual + reflex) were collected with micro capillaries.
MMP-9 and -2 activities were determined by gelatin zymography loading 30 ug of tear proteins
into the gel and quantified by densitometry (pixels) using digital image analysis. The data were
analyzed by use of the Mann-Whitney and Spearman tests being considered significant a p<0.05.
Results: 17/18 patients had a bilateral disease, being asymmetric in 5 of them. There were
not significant differences between patients and controls in regard to external eye evaluation
tests. Corneal aesthesiometry showed that the more advanced the disease, the more profound
was the corneal hypoesthesia (r=-0.53, p=0.029). In regard to corneal alterations, patients
were grouped as: 1 (confluent translucent micro droplets localized in the limbic region of the
horizontal meridians in 8 patients); 2 (5 patients with sub epithelial haziness in a band-shaped
fashion from limbus-to-limbus through the central cornea); and 3 (5 patients with the previously
described lesions and numerous evident amber-like sub epithelial droplets forming clusters).
Most of the MMPs detected in patients and controls represented the latent forms. There was
not significant differences in levels of pro-MMP-2 between both groups (p=0.11). Pro-MMP-9
median was found to be significantly higher in CDK patients than in controls (2891 pixels vs 1360,
p=0.03). No correlations could be found between levels of pro-MMP-9 and corneal alterations.
Conclusions: Both MMP-9 and -2 are present mainly in their latent forms in the tears of patients
and controls. In spite of the increased levels of pro-MMP-9 in CDK patients, the lack of association
with disease progression would suggest no fundamental role of this gelatinase in the evolution
of CDK.
CR: H.M. Serra, None; M.C. Sánchez, None; E.G. Knoll, None; P.F. Barcelona, None; P.
Maccio, None; E.A. Urrets-Zavalia, None; J.A. Urrets-Zavalia, None.
Support: SECYT UNC 123/04
Effects of Environmental Oxygen Concentration on Paracellular Permeability and
the Expression and Localization of Zonula Occludens-1 in Human Corneal Epithelial
S.Teranishi, K.Kimura, K.Kawamoto, K.Fukuda, R.Yanai, K.Seki, T.Nishida. Biomolec
Recog & ophthal, Yamaguchi Univ Sch Med, Ube City, Japan.
Purpose: Atmospheric oxygen plays a role in maintenance of the structure and
function of the cornea as a result of the anatomic location of this tissue. The effect of
oxygen concentration on the barrier function of corneal epithelial cells was examined.
Methods: Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured for
4 days to allow them to form an epithelial barrier. The cells were then deprived of serum and
subsequently incubated for 24 h in the presence of various concentrations (1, 21, or 60%) of oxygen
gas. The barrier function of the cells was monitored by determination of transepithelial resistance.
HCE cells incubated in the presence of 1, 21, or 60% oxygen were also either fixed and subjected
to immunofluorescence staining with antibodies to zonula occludens-1 (ZO-1), a component of
tight junctions, or lysed and subjected to immunoblot analysis with antibodies to this protein.
Results: Transepithelial resistance decreased during incubation of HCE cells under the
hypoxic condition (1% oxygen) but was stable in cells cultured under normoxic (21%) or
hyperoxic (60%) conditions. Immunofluorescence analysis revealed that the abundance
and localization of ZO-1 at tight junctions decreased in response to exposure of cells
to hypoxia. Immunoblot analysis showed that the amount of ZO-1 was reduced in cells
after culture with 1% oxygen for 24 or 48 h, and was increased in cells cultured with
60% oxygen for 48 h, compared with that apparent in cells exposed to 21% oxygen.
Conclusions: Hypoxia impaired the barrier function of corneal epithelial cells, likely as a
result of down-regulation and redistribution of the tight-junction protein ZO-1.
CR: S. Teranishi, None; K. Kimura, None; K. Kawamoto, None; K. Fukuda, None; R.
Yanai, None; K. Seki, None; T. Nishida, None.
Support: None.
899 - B873
900 - B874
Effects of Desiccation on Inflammatory Cytokines Production in Cornea
A.Higuchi1A, Y.Takahashi1A, K.Tsubota1B. A6N9 Research Park, BOphthalmology, 1Keio
University School of Medicine, Tokyo, Japan.
Purpose: Cornea epithelial cells are always affected by various physical factors, such as,
temperature, humidity, ultraviolet irradiation, and airflow. Desiccation is significantly affected on
these cellular conditions. We investigated the influence of desiccation on inflammatory cytokine
production in corneal cells using human corneal epithelial (CEPI) cell line and dry eye model rats.
Methods: CEPI was grown in keratinocyte growth medium 2 (KGM2) to approximately
80% confluence in dishes. The medium is discarded by aspiration and plates were left
for 0 to 30 minutes with opening the cover to dry the cells (short term desiccation).
KGM2 was poured into the dishes. Fifteen minutes later, the medium was collected to
measure the concentration of the cytokines in medium by EIA. Viability of the cells
was estimated with alamer Blue. To study the effect of long term desiccation, we use
transwell (Corning Inc., corning, NY). CEPI was grown on membrane of transwell.
KGM2 in upper-layer was discarded to dry CEPI. After desiccation, viability of the cells,
expression of cytokines, and concentration of the cytokines in medium were measured. The
expression of cytokines in cornea of dry eye model rat was measured by real time PCR.
Results: In short term desiccation, CEPI started to die after 20 minute of desiccation. Secretion
of IL-6 from CEPI increased by 15-20 minutes desiccation but that of TNFα did not change.
In long term desiccation, secretion of IL-6 and IL-8 from CEPI increased but that of TNFα
did not change. Expression of IL-6 was also increased. In dry eye model rat, the mRNA of
IL-6 obtained from cornea also increased significantly, but that of TNFα did not change.
Conclusions: The cell death induced by desiccation is suggested to related IL-6 and IL-8
secretion but not TNFα.
CR: A. Higuchi, None; Y. Takahashi, None; K. Tsubota, None.
Support: None.
Pathophysiological Corneal Epithelium Changes After Prolonged Low-Dk RGP Lens
Wear or Eyelid Closure-Induced Hypoxia
N.Yamamoto1, N.Yamamoto1, J.V. Jester2, H.D. Cavanagh1. 1Ophthalmology, UT SW
Medical Center, Dallas, TX; 2Ophthalmology, UC Irvine, Irvine, CA.
Pur pose: Deter mine pathophysiological effects of low-Dk RGP CTLwe a r o r eyel id clo s u r e i n d u c e d - hy p ox i a o n c o r n e a l e pit h el iu m .
Methods: 37 NZW rabbits (15-20 weeks old) were used for this study and treated according to
the ARVO statement for the use of animals in ophthalmic and vision research. One randomly
chosen eye was fitted with a low-Dk RGP lens (EOP=5.8) or was sutured closed (EOP=7.7) ; the
other served as a control. After 72-hour wear or 24, 72, 168 hour of closure, rabbits were humanly
sacrificed. Western blots for Bcl-2 were performed (n=4). Epithelial and stromal thickness
and surface cell size after 72-hour low-Dk RGP CTL-wear or eyelid closure were measured
by in vivo confocal microscopy. To assess the susceptiblity to Pseudomonas aeruginosa
ATCC27853 (PA) binding, lipid rafts were stained with β-cholera toxin with or without bacteria.
Results: 24-hour eyelid closure showed no changes of Bcl-2 expression. After 72-hour,
Bcl-2 expression decreased in both closed eye and low-Dk CTL-wearing corneal epithelium.
At 72-hours, closed eye decreased Bcl-2 expression as much as low-Dk RGP. 72-hour lowDk RGP CTL-wear and eyelid closure increased stromal thickness (low-Dk RGP CTL :
325.1μm ± 32.7 to 375.2μm ± 41.5 p<0.01, eyelid closure : 319.5μm ± 14.2 to 355.8μm ±
31.8 p<0.05) but with no significant changes in epithelial thickness or surface cell size.
72-hour closed eyes showed many lipid raft forming cells in central corneal epithelium;
however, low-Dk RGP CTL-wearing eyes and control eyes showed no lipid raft forming
cells centrally but occasionally showed raft formation in the peripheral cornea. β-cholera
toxin staining with PA showed colocalization of PA with those lipid raft forming cells.
Conclusions: Bcl-2 expression changes, epithelial or stromal thickness, and sureface cell size
indicate identical effects of low-Dk CTL-wear or eyelid closure. However β-cholera toxin
staining suggests low-Dk CTL-wear or eyelid closure increase susceptibility to bacteria in
a different manner.
CR: N. Yamamoto, None; N. Yamamoto, None; J.V. Jester, None; H.D. Cavanagh,
Support: EY10738 and an unrestricted grant from R.P.B
901 - B875
902 - B876
Corneal Epithelial Homeostasis Following Laser in situ Keratomileusis (LASIK)
D.Parmar1, P.M. Ladage2, S.Awwad1, J.P. McCulley1, R.W. Bowman1, H.D. Cavanagh1.
Cornea & External Disease, UT Southwestern Medical Center, Dallas, TX; 2Optometry,
University of Houston College of Optometry, Houston, TX.
Purpose: To determine the corneal epithelial response following LASIK.
Methods: 14 corneas of 7 patients undergoing routine LASIK were prospectively enrolled,
3 male and 4 female. Corneal epithelial exfoliation rate (EXFR) was assessed using a
non-contact corneal irrigation chamber (NCIC) preoperatively and at 1, 3 and 6 months
postoperatively. Collected corneal epithelial cells were stained with Acridine Orange dye and
counted by fluorescence microscopy. Corneal epithelial thickness was measured by tandem
scanning corneal confocal microscopy preoperatively and at 6 months postoperatively.
Results: At an irrigation rate of 9mL/min, mean preoperative EXFR was 38.5 ±15.6
cells/min, increasing significantly to 53.9 ±16.9cells/min at 1 month postoperatively
(Student’s t-test, P≤0.005), but returning to preoperative levels by 3 months (36.7 ±12.4
cells/min) and 6 months (33.4 ±13.2 cells/min). An increase in mean central corneal
epithelial thickness was seen 6 months postoperatively (50.2 +/- 4.5 µm) compared
to preoperative values (46.2 +/- 4.8 µm), although this was not statistically significant.
Conclusions: Corneal epithelial desquamation rate increases in the first month following
LASIK, returning to normal levels by 3 months, without any significant change in central
corneal epithelial thickness. This may be related to altered lid-shearing forces at the edge of
the stromal ablation zone causing a temporary increase in apoptotic-driven corneal epithelial
CR: D. Parmar, None; P.M. Ladage, None; S. Awwad, None; J.P. McCulley, None; R.W.
Bowman, None; H.D. Cavanagh, None.
Support: Research to Prevent Blindess, New York, NY.
Comparison of the Effects of Latanoprost and Timolol Maleate 0.5% on the Corneal
Epithelium Using Confocal Microscopy
J.M. Benitez Del Castillo, M.A. Wassfi, J.Garcia-Feijoo, J.M. Martinez-de-la-Casa,
J.Garcia-Sanchez. Unidad Superficie Ocular, Hospital Clinco San Carlos, Madrid, Spain.
Purpose: The aim of the study was to compare the effects of the two most
frequently used antiglaucomatous agents (latanoprost (Pf izer) and timolol
maleate 0.5% (MSD)) on the corneal epithelium using confocal microscopy.
Methods: A Tomey confoscan slit-scanning confocal microscope was used to examine the
cornea of 40 eyes; 20 eyes of 15 patients treated by latanoprost and 20 eyes of 16 patients treated
by timolol maleate 0.5%. The interpretation of the images was performed in a masked manner.
Results: The density of the superficial and basal epithelial cells as well as the number of
subbasal nerves was similar in both groups. However, the latanoprost group showed highly
reflective superficial epithelial cells with absence of nuclei and dark intercellular spaces.
Conclusions: Latanoprost produces more changes in the superficial corneal epithelium than
does timolol maleate.
CR: J.M. Benitez Del Castillo, None; M.A. Wassfi, None; J. Garcia-Feijoo, None; J.M.
Martinez-de-la-Casa, None; J. Garcia-Sanchez, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 867-903 / B841-B877
137. Corneal Epithelium Organizing Section: CO
903 - B877
Comparison of Two in vitro Models to Assess Ocular Irritation
S.Khoh-Reiter, K.Rittenhouse. Safety Sciences, Pfizer Inc, San Diego, CA.
Purpose: To compare and contrast two commercial in vitro models to understand
their potential to serve as an early predictive tool to assess ocular irritation of selected
topical ophthalmic compounds. Mat-Tek’s EpiOcular™ system consists of normal
human-derived keratinocytes while SkinEthic’s Human Corneal Epithelium model
consists of immortalized human corneal epithelial cells. Both systems are 3-dimensional
cultures grown on inserts at the air-liquid interface in a chemically defined medium.
Methods: A series of compounds were chosen based on literature reports of their irritancy
potential. Upon receipt of the in vitro model inserts, they were transferred to plates
containing medium and incubated at 37°C overnight prior to treatment with test compound.
The test article was applied to the apical surface of the culture at the appropriate volume
to deliver the desired concentration. After the 60-minute incubation at 37°C, the cultures
were washed with PBS and the MTT assay was performed to assess tissue viability. Each
compound was tested in duplicate and negative controls were run in parallel. Data were
expressed as a percentage of viable cells relative to the corresponding negative control.
Results: Based on this side-by-side comparison of the assays, both assays appear equivalent
in their ability to detect irritancy of the compounds tested. There is a linear dose response
curve for both the systems with concentrations ranging from 0.001 percent to 2 percent.
Conclusions: SkinEthic’s model was preferred due to decreased material requirements, along
with fewer steps prior to and during the experimental procedure. The fact that SkinEthic’s
model employs human corneal cells is also an important consideration for ophthalmic
products. As an early screen, an assay that is predictive, has fewer steps, and requires less
bulk material, is highly desirable.
CR: S. Khoh-Reiter, None; K. Rittenhouse, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 904-932 / B878-B906
138. Contact Lens I Organizing Section: CO
904 - B878
Minimum Contact Lens Oxygen Transmissibility (Dk/L) With Monod Kinetics for the
Corneal Oxygen Consumption Rate
C.J. Radke1A, M.Chhabra1B. AChemical Engineering and Vision Science, BChemical
Engineering, 1University of California, Berkeley, CA.
P u r p o s e :To p r e d i c t t h e m i n i m u m c o n t a c t l e n s t r a n s m i s s i b i l i t y
needed to avoid cor neal edema in open and closed eye conditions.
Methods: A 4-layer (3 layers of cornea, epithelium, stroma and endothelium, and a contact
lens) diffusion-reaction model was used to calculate the steady oxygen partial pressure
(oxygen tension) profile in the cornea-lens system. Previous estimates of the minimum Dk/L
adopt a constant oxygen consumption rate yielding aphysical negative oxygen tensions.
As reported by Fatt et al, oxygen consumption must decrease to zero when oxygen tension
approaches zero. This metabolic behavior is rigorously described by classical Monod
kinetics. Here the Monod expression is used for oxygen consumption rates in each layer of
the cornea. Necessary parameters (oxygen permeabilities, thicknesses, maximum oxygen
consumption rate, and oxygen tensions at the boundaries) were obtained from accepted
literature values. With Monod kinetics, the oxygen tension no longer falls to zero. Hence,
we adopt a criterion for the minimum oxygen transmissibility of the contact lens as the
critical oxygen tension needed at the anterior corneal surface to avoid corneal edema.
Results: The minimum Dk/L of the contact lens was found to be 9 and 29 Barrer/
cm in open and closed eye conditions, respectively. These values are lower than the
corresponding experimentally observed values of 24 and 87 Barrer/cm by Holden and
Mertz. Acidosis in the cornea is known to increase the maximum oxygen consumption rate,
and apparently, must be accounted for. Although Monod kinetics for oxygen consumption
correctly avoids the aphysical negative oxygen partial pressures in the cornea, a more
complete metabolic model of corneal respiration seems necessary. Such a model must
include coupled transport of glucose, lactate and hydrogen ions, and carbon dioxide.
Conclusions:Adoption of Monod kinetics for oxygen consumption predicts the minimum
Dk/L of a contact lens to be lower than the experimentally observed values to avoid corneal
edema. However, the effect of acidosis on oxygen consumption rate is not included in our
analysis. A more rigorous metabolic model of oxygen diffusion and reaction in the cornea
is called for.
CR: C.J. Radke, None; M. Chhabra, None.
Support: None.
906 - B880
905 - B879
Does the Blink Alter the Apparent Physiologically Effective Transmissibility (peDk/t)
of Silicone Hydrogel (SH) Combination (“Piggyback”) Contact Lens Systems?
B.A. Fink, L.N. Florkey, G.Mitchell, R.M. Hill. College of Optometry, The Ohio State
University, Columbus, OH.
Purpose: For combination systems: (1) How does the transmissibility of an SH carrier lens (vs.
other materials) affect post-lens oxygenation? (2) How do cap lenses of various transmissibilities
and thicknesses affect post-lens oxygenation? (3) How does blinking affect post-lens oxygenation?
Methods: Corneal oxygen uptake rates were measured polarographically for 10 subjects
(right eye only). Dk values for two GP cap lenses Fluoroperm 30 and 151, and three carrier
lenses (PureVision, Permalens, and Optima 38) were 30, 151, 99, 34, and 8.4 respectively.
Each cornea was measured one time and averaged with the others to obtain mean responses
to 26 different exposure conditions immediately after 300 sec of wear: (1-6) FL30(0.12 mm),
FL151(0.60 mm), FL151(0.12 mm), PureVision (PV), Permalens (CP), Optima 38 (Opt38)
alone, no blink; (7-24) each carrier lens PV, CP, Opt38 in combination with each FL type
cap lens, both with and without blinking; (25, 26) no lens and PMMA wear as controls. All
responses are given as physiologically effective transmissibility (peDk/t) values based on
responses ratioed to the non-wear rate. Repeated measures analysis of variance was used to test
for significant difference, and Tukey’s test for post-hoc comparisons at the p = 0.01 threshold.
Results: Differences in response between non blink and blink conditions were greatest
within the highest Dk/t (PureVision) carrier series, in which: (a) the lowest Dk/t (FL30)
cap lens case benefited most (relatively by 3.5X) with the blink (vs. no blink); (b) a
cap lens (FL151, 0 .60) of the same Dk/t as in (a), but 5X thicker, was found with that
same carrier to benefit nearly identically with and without the blink; and (c) a high Dk,
but thin, cap lens (FL151, 0.12) with that same carrier showed a reduced performance
with vs. without blink, although both of those performances exceeded all responses
in (a) and (b) Yet, none of the combination cases manifest statistically significant
differences between their blink and non-blink outcomes (p value range = 0.263 to 0.924).
Conclusions: Differences, both in total thickness and in material permeability, among the
combination systems observed here, appear fundamental to the relative effectiveness of the
blink on oxygenation of the covered corneal surface.
CR: B.A. Fink, None; L.N. Florkey, None; G. Mitchell, None; R.M. Hill, None.
Support: None.
907 - B881
The Effect of Tear Components on the in vitro Wettability of Silicone Hydrogel
D.L. Meadows, H.A. Ketelson, N.McQueen, R.P. Stone. Consumer Products Research,
Alcon Laboratories, Inc., Fort Worth, TX.
Release of Wetting Agents From Nelfilcon Contact Lenses
M.A. Princz1, L.W. Jones2, H.Sheardown1. 1Chemical Engineering, McMaster University,
Hamilton, ON, Canada; 2Centre for Contact Lens Research, School of Optometry,
University of Waterloo, Waterloo, ON, Canada.
Purpose: The release of various wetting agents from a hydrogel contact lens
material was examined to assess whether the use of different wetting agents
could be used to improve patient comfort with daily wear soft contact lenses.
Methods: Nelfilcon A (CIBA Vision Focus DailiesTM ) contact lenses were swollen in
varying concentrations of 2500 and 10000 MW polyvinyl pyrrolidone (PVP), as well
as high concentrations of carboxymethyl cellulose (CMC), hyaluronic acid (HA) and
dextran (10K and 40K MW) in different solvents at room temperature for 24 hours.
The solvents included Milli-Q water, PBS and varying dilutions of ethanol in MilliQ water. Release was measured by soaking the lenses in phosphate buffer solution
(PBS) followed by UV-Vis spectrophotometry or fluorometric analysis. Advancing
and receding sessile drop contact angles and swelling studies were also performed.
Results: First order release profiles were observed for all wetting agents. Little, or no
release of PVP, CMC and dextran were observed after 1.5 hours, while HA was released
from the lenses for more than 4 days. Swelling in ethanol solutions resulted in a slightly
greater solvent uptake and presumably an increased amount of the wetting agent in the
lens. Incorporation and release of PVP initially decreased contact angles but water contact
angles returned to values equal to control contact angles within the first hour of sampling.
Conclusions: These results suggest that high molecular weight compounds may be released
from contact lenses for clinically useful wear periods, potentially improving the comfort of
hydrogel lenses. The water contact angle results suggest that release of the wetting agents
during the period of wear will improve the wettability of the lenses.
CR: M.A. Princz, CIBA Vision F; L.W. Jones, None; H. Sheardown, CIBA Vision F.
Support: CIBA Vision
908 - B882
909 - B883
Purpose: A sessile drop technique was utilized to measure in vitro advancing contact angles as an
indicator of silicone hydrogel lens wettability in the presence of artificial tear solutions (ATS). The
influence of lysozyme, albumin and mucin were studied for their effect on the wettability of Acuvue ®
Advance™ (AA), Focus N&D and O2 Optix lenses presoaked in three commercial multipurpose
solutions (MPS), Opti-Free® Express®, Renu® MoistureLoc™, Aquify ® and an investigational MPS.
Methods: Advancing contact angles were determined via a sessile drop method using a high speed
video system with curve fitting software. The lenses were cycled through ATS-air exposures to simulate
clinical blinking conditions. MPS products were used according to the manufacturers’ listed regimen.
Results: Silicone hydrogel lens materials displayed different wettabilities and it was believed
that their differences in surface chemistry modification technologies were one of the main factors
responsible for this. The contact angles of AA lenses increased to over 100° during lens cycling in
saline or ATS solutions. Comparatively, new Focus and Optix lenses displayed lower contact angles
and this indicated improved wettability relative to the AA lens material. The ATS components
mucin and lysozyme had no effect on the contact angles of the lenses whereas albumin played
an important role when lenses were preconditioned with particular surfactants. The effect of
preconditioning the lenses in MPS showed different effects on the material wettabilities. This
was believed to be a reflection of the different polymers and surfactants in the marketed products
and their interactions with the lens materials. Specific physico-chemical interactions taking place
between the contact lens material, tear components and formulation components were believed
to be important aspects that play a role in the wettability of the silicone hydrogel materials.
Conclusions: This study indicated that SiH lens materials display different wettabilities depending
on the type of surface modification technology and preconditioning solution. The effect of ATS
components such as lysozyme and mucin showed no impact on improving the wettability of the
lenses. Comparatively, albumin had a significant impact on improving the wettability of the lens
materials when used with preconditioning agents.
CR: D.L. Meadows, Alcon Laboratories, Inc. E; H.A. Ketelson, Alcon Laboratories, Inc. E; N.
McQueen, Alcon Laboratories, Inc. E; R.P. Stone, Alcon Laboratories, Inc. E.
Support: None.
Wetting Agent Release From Contact Lenses
L.Liu1, L.Jones2, H.Sheardown1. 1Chemical Engineering, McMaster University, Hamilton,
ON, Canada; 2Centre for Contact Lens Research, University of Waterloo, Waterloo, ON,
Purpose: End of day dryness is a major complaint of contact lens wearers. One
potential solution is to incorporate ophthalmic compatible wetting agents into
the contact lens matrix. The slow release of the incorporated wetting agents
will provide a continuously wetted surface, reducing end of day discomfort.
Methods: Polyvinyl pyrrolidone (PVP), hyaluronic acid (HA) and dextran were incorporated
during curing of PVA based daily wear contact lenses. Wetting agents of different molecular
weights were used to investigate the effect of molecular weight on the release profiles. The
incorporation process, release kinetics and surface wettability of the lenses were studied.
Release was monitored by UV spectrophotometry. Water contact angles at different
release times were measured to test surface wettibility. Transparency of lens materials was
examined by scanning the sample between 300-700nm using UV-visible spectrophotometry.
Results:0.5% to 20% wt/wt (wetting agent/lens formulation) of the wetting agents were
introduced into the contact lens matrix. Materials containing PVP showed relatively rapid
release, with a plateau within 20 hours followed by slow release for another 50-70 hours.
This was likely due to the lower molecular weight and flexible nature of the polymer chains.
The release percent increased with the increasing of percent of incorporated wetting agent.
Dextran and HA demonstrated more sustained release, with release plateaus after 40
hrs. Contact angle results over 72 hours indicated an improvement in surface wettibility
independent of wetting agent. While the incorporation of HA and PVP into the lenses did
not alter the transparency, dextran incorporation significantly decreased transparency.
Conclusions:Wetting agents were successfully incorporated in contact lenses. Release
resulted in significant decreases in the contact angles over 72 hours with all wetting agents.
Given the release profiles and the transparency measurements, HA showed the most promise
for improving the wetting properties of the lenses.
CR: L. Liu, None; L. Jones, None; H. Sheardown, None.
Support: CIBA Vision
Microscopic Observations of Silicone Hydrogels With Three Different Techniques
J.Gonzalez-Meijome1, A.López-Alemany2, J.Almeida3, M.Parafita4. 1Physics-Optometry,
University of Minho, Braga, Portugal; 2Optics, University of Valencia, Valencia, Spain;
Physics, University Minho, Braga, Portugal; 4Surgery (Ophthalmology), University of
Santiago de Compostela, Santiago de Compostela, Spain.
Purpose: The mechanical impact of silicone hydrogel contact lenses (SHCL) on corneal surface has
been considered and the surface structure of these lenses could be important in several aspects of
clinical response of the ocular surface. Scanning electron microscopy (SEM) has been previously
applied to contact lens polymers, as well as atomic force microscopy (AFM). The purpose of this study
was to observe different silicone hydrogel contact lens materials by three microscopic techniques in
order to identify which one give us more representative information of the surface polymer structure.
Methods: Contact lens materials included lotrafilcon A, balafilcon A and galyfilcon
A. Microscopic techniques used in this study were AFM, SEM and cryo-scanning
electron microscopy (cryoSEM). AFM statistics (tapping mode) including mean
roughness (Ra) and maximum high (Rmax) were explored for the three lens materials.
Results: The three materials showed a different surface configuration when observed by either method
of microscopy. However, AFM technology allows us to study the sample in the hydrated state which
is very important with hydrogels. AFM also allow us to study portions of the surface as small as 0.25
square microns with high resolution. Resolution at high magnification was one of the major limitations
of SEM and CryoSEM, which also induce serious damage to the sample during the preparationfixation process. However other features have been observed with these two methods that had not been
reported before, particularly with cryoSEM. AFM statistics revealed a significantly more irregular
surface of the Balafilcon A material (Ra=6,44nm;Rmax=96,82) when compared with Lotrafilcon
A (Ra=2,40nm;Rmax=40,89) and Galyfilcon A (Ra=1,40nm;Rmax=15,33), for a 1 μm 2 area.
Conclusions: The present study addresses the microscopic comparison of current SHCL pointing
out the benefits of each technique. It also shows the value of cryoSEM for the study of polymeric
samples from contact lenses. Present observations could have implications in clinical aspects of
contact lens wear as material spoliation, resistance to bacterial adhesion or mechanical interaction
with the ocular surface.
CR: J. Gonzalez-Meijome, None; A. López-Alemany, None; J. Almeida, None; M. Parafita,
Support: Science and Technology Foundation (FCT-MCES) contract 8281/2002 from the European
Social Funding
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 904-932 / B878-B906
138. Contact Lens I Organizing Section: CO
910 - B884
911 - B885
Kinetics of in vitro Lysozyme Deposition on Silicone Hydrogel, Group II and Group
IV Contact Lens Materials
L.N. Subbaraman1, M.Senchyna1,2, L.Jones1. 1School of Optometry, Centre for Contact
Lens Research, University of Waterloo, Waterloo, ON, Canada; 2Alcon Research Ltd, Fort
Worth, TX.
Purpose: To determine lysozyme deposition as a function of time in group IV, group II and silicone
hydrogel (SH) contact lens materials by artificially doping lenses with 125I labeled lysozyme.
Methods: 4 individual Acuvue (AV; Gp IV), Sof Lens 66 (SL; Gp II) and SH lenses
(Focus Night&Day (FND), PureVision (PV) and Acuvue Advance (AA)) were doped in
1mL of simple lysozyme solution (1.9 mg/ml) containing 125I labeled lysozyme. The lenses
were doped for time periods ranging from 1 hr to 28 days at 37o C with constant shaking.
Following the specified doping period, the lenses were rinsed briefly with phosphate
buffered saline to remove unbound protein and were then placed in polypropylene tubes
and counted in a Beckman Gamma Counter. The amount of protein adsorbed to the lenses
was calculated by dividing the counts on the lenses by the specific activity of the protein.
Results: Lysozyme accumulated rapidly on AV lenses (1 hr, 98±8 µg/lens), reached a maximum
on the 7th day (1386±21 µg/lens) and then reached a plateau, with no further increase occurring
(p=NS). Lysozyme accumulation on Gp II and SH lenses continued to increase across all
time periods, with no plateau being observed (p<0.05). At 7 days, FND lenses deposited
1.8±0.4 µg of lysozyme per lens while PV and AA lenses deposited 5.9±2 and 3.6±1 µg of
lysozyme respectively. After 28 days of doping, FND lenses deposited 4.2±1 µg of lysozyme
per lens while PV and AA lenses deposited 19.4±3 and 16.8±4 µg of lysozyme respectively.
Conclusions: Radiochemical analysis is a sensitive and effective technique to determine the
small quantities of lysozyme deposited on SH lenses. The kinetics of contact lens deposition
depends on the chemical structure of lens material under consideration. Lysozyme deposition
occurs rapidly with Gp IV materials before reaching a maximum, while SH and Gp II materials
progressively accumulate lysozyme, with no plateau occurring.
CR: L.N. Subbaraman, None; M. Senchyna, Alcon Research Ltd, Fort Worth, TX, USA
F; L. Jones, Alcon Research Ltd, Fort Worth, TX, USA F.
Support: NSERC and Alcon
In situ Activity of Lysozyme Deposited on Hydrogel Lenses
S.Zhang1, R.N. Borazjani2, J.C. Salamone2, D.G. Ahearn1, S.A. Crow1, G.E. Pierce1.
Biology Department, Georgia State University, Atlanta, GA; 2Research Group, Bausch &
Lomb, Rochester, NY.
Purpose: Deposition of lysozyme on hydrogel contact lenses is known to involve both
adsorption and absorption. The overall deposition is dependent upon characteristics of the
lens such as hydrophobicity, surface charge and water content. Lysozyme deposition on the
HEMA-type etafilcon A lens (58% water content) is reported to be significantly greater than
that to a silicone hydrogel lens (36% water content). Deposited lysozyme upon extraction
from a lens demonstrated activity with a micrococcyl assay, but the degree of activity
in-situ is unclear. Herein we report the in-situ activity of lysozyme on various lens types.
Methods: Unworn etafilcon A lenses (SureVue), balafilcon A silicone lenses (Pure Vision)
and a rigid gas permeable lens were soaked in various concentrations of lysozyme (5 ml of
2 to 100 ug/ml) for time periods up to 22 h. In-situ activity and amount of lysozyme were
determined with a direct (without extraction) micrococcyl assay and a micro-BCA assay.
Results: Screening of concentrations of lysozyme between ~ 2 to 100 ug/ml indicated
that concentration of 2 or 20 ug/ml in 5 ml phosphate buffer were most suitable for the
assay system for etafilcon A lens. The total concentration (extracted) was higher than the
concentration detected on the surface of lens. With repeated rinses (2 h intervals) of the lens,
the concentrations on the surface decreased slowly. Markedly lower levels of lysozyme were
deposited on silicone vs. etafilcon A lens, and about 90% was removed with the first rinse.
Deposition on the RGP lens was low and no lysozyme was detected upon subsequent rinsings.
Conclusions: Concentrations of lysozyme deposited on etafilcon A lenses appeared mostly
absorbed and inactive but capable of replenishing a stable surface activity after repeated
rinses. Total lysozyme deposited on the balafilcon A silicone lens from initially comparable
concentrations to those used for the etafilcon A lens were markedly reduced and mostly
CR: S. Zhang, Bausch & Lomb R; R.N. Borazjani, Bausch & Lomb E; J.C. Salamone,
Bausch & Lomb C; D.G. Ahearn, Bausch & Lomb C; S.A. Crow, Bausch & Lomb C; G.E.
Pierce, Bausch & Lomb C.
Support: Bausch & Lomb Research Grant
912 - B886
913 - B887
In vitro Uptake and Release Interactions of Benzylkonium Chloride (BAK) by SiliconContaining and p-HEMA Hydrogel Contact Lens Materials
A.Dracopoulos, V.Bantseev, J.G. Sivak. Optometry, School of Optometry; University of
Waterloo, Waterloo, ON, Canada.
Purpose: The in vitro uptake and release behavior of benzylkonium chloride (BAK)
with silicon-containing (lotrafilcon and galyifilcon) and p-HEMA-containing
(etafilcon and vifilcon) hydrogel contact lenses was studied via extract analysis with
the Bovine Lens Assay and the Sodium Fluorescein Permeability Assay (SFPA).
Methods: Contact lenses were soaked in a solution at varying concentrations of BAK (1%,
0.1%, 0.01%, and 0.001%) for more than 24 hours at room temperature. After exposure
contact lenses were placed in a saline solution a total of 7 days at 37° C. Bovine lenses were
exposed to the extract for 15 minutes, rinsed with saline and M199 and then incubated in
culture medium at 37° C and 4-5% CO2. The Bovine Lens Assay measures the potential
for ophthalmic irritancy of an extract by evaluating its effect on the optical properties of
the lens with a scanning laser system for 72 hours after exposure. With the SFPA, Mandin
Darby Canine Kidney (MDCK) cells were grown to 80% confluence and were prepared
for treatment in 24 well plate inserts. The SFPA evaluates cell membranes and tight
junctions of a cultured MDCK monoloayer. Results are obtained spectrophotometrically.
Results: The Bovine Lens Assay demonstrates that exposure to extracts obtained from
silicon-containing lenses were significantly more damaging to the lens at higher concentrations
then that obtained from the p-HEMA-containing contact lenses. Compared to controls
(n=5, p‹0.05) loss of sharp focus was evident with extract obtained from silicon-containing
1% BAK increasing from 0.29±0.03 mm (SEM) to 1.09±0.21 mm (SEM), whereas pHEMA-containing at 1% BAK had no effect. Similar results were noted for SFPA.
Conclusions: This experiment demonstrates that the Bovine Lens Assay and the SFPA can be
used with extracts to determine the potential effects of an ophthalmic agent and its behavior
with various contact lens polymers on the physiological and functional properties of the lens
and on the integrity of an epithelium in culture.
CR: A. Dracopoulos, None; V. Bantseev, None; J.G. Sivak, None.
Support: NSERC Grant
Comparative Cytotoxicity of Silicone Hydrogel Contact Lenses With Various Contact
Lens Care Products Using Human Corneal Epithelial Cells and Flow Cytometry
M.F. Mowrey-McKee, A.Wright, A.Renaud. Toxicology/Microbiology, CIBA Vision
Corporation, Duluth, GA.
Purpose:To evaluate cell viability of SV-40 transformed human corneal epithelial cells
(HCE-T) exposed to high oxygen permeable silicone hydrogel contact lenses (HiDk SiHy)
and contact lens care products (LCP) using flow cytometry. Methods: HCE-T cells were
exposed to LCP and to HiDk SiHy treated with LCP and analyzed for cell viability. Cell
viability was evaluated using Alamar Blue™ (AB), a resazurin based dye, and flow cytometry
of calcein and ethidium homodimer-1 (CEH) stained cells (Live/Dead Viability/Cytotoxicity
Kit from Molecular Probes). Three different cell exposure conditions were evaluated. 1) HiDk
SiHy soaked in LCP were seeded with HCE-T, incubated, trypsinized, stained with CEH
and analyzed by flow cytometry (BDFACSCailbur™ 4 color unit cytometer). 2) HCE-T in
suspension were exposed 5 and 30 minutes to 50% dilutions of LCP; the cells were stained
with CEH and analyzed using flow cytometry. 3) HCE-T were seeded into 96 well plates,
exposed to 50% dilutions of three LCP for 5 and 30 minutes, rinsed and evaluated for viability
with AB using a CytoFluor® (PerSeptive Biosystems) plate reader. The LCP used were:
AQuify™ MPS (AQ), Clear Care® (CC), and OPTI-FREE® Express® with Aldox™ (OF).
Using flow cytometry, subpopulations of cells were identified and sorted by regions for live
(green fluorescent) and dead (red fluorescent) cells. Benzalkonium chloride was used as a
positive control for the analysis of dead cells. Results: Alamar Blue cell viability was above
50% for HCE-T exposed to all three LCP for 5 minutes; however, at the 30 minute exposure
time cell viability for OF was less than 50%. Flow cytometric analysis demonstrated that
CC and AQ are much less cytotoxic to HCE-T in suspension than OF at both exposure times.
HCE-T cells grown on HiDk SiHy lenses soaked for one day in CC and AQ were found to
be 70% alive and 30% damaged. No live HCE-T cells were detected on HiDk SiHy lenses
exposed to OF for one day. Conclusions: HCE-T cells are significantly more viable when
grown on HiDk SiHy contact lenses cycled in Clear Care® or Aquify MPS compared to HiDk
SiHy contact lenses cycled in OPTI-FREE Express with Aldox.
CR: M.F. Mowrey-McKee, CIBA Vision Corporation E; CIBA Vision Corporation E;
CIBA Vision Corporation E; A. Wright, CIBA Vision Corporation E; A. Renaud, CIBA
Vision Corporation E. Support: CIBA Vision Corporation
914 - B888
915 - B889
Assessments of Cleaning Efficacy for Contact Lens Multi-Purpose Disinfecting
B.-S.Hong, P.Stauffer, D.L. Meadows. Consumer Products Research, Alcon Laboratories,
Inc., Fort Worth, TX.
Purpose: To assess the passive cleaning efficacy of various multipurpose solutions
(MPS) for hydrogel contact lenses using an in vitro lysozyme deposition model.
Methods: The solutions assessed included AQuify ®, ReNu® MoistureLoc™, ReNu MultiPlus®,
Complete®, Complete® Moisture Plus, Solocare™, Solocare™ Plus, Opti-Free®, Opti-Free®
Express® and a test formulation. Acuvue 2 lenses (FDA Group IV) were used to deposit with an
in vitro lysozyme deposit model. Each lens was soaked with a 5 ml lysozyme solution (1.5mg/
ml in phosphate buffered saline, pH 7.4) at 37oC for 24 hours. No rubbing/rinsing was applied
to the cleaning regimen. The lysozyme remained in the lens after the passive soaking with
MPS was extracted with an extraction solvent composing of acetonitrile/trifluoroacetic acid/
water in a volume ratio of 500/1/500. Quantitation of the extracted lysozyme was conducted
by a fluorescence spectrophotometer with an excitation/emission setting at 280nm/346nm.
Results: Time course studies demonstrated that the cleaning efficacy increased with the duration
of soaking; 6 hr and overnight (16 hr) soaking achieved more significant cleaning than 2 and 4 hr
soaking. Differences in cleaning efficacies were observed among various MPS; they ranged from
approximately 6% to 44 %. The overall cleaning efficacy for all MPS were increased after 7-day
cleaning cycle. In both cases, Opti-Free® Express® exceeded all other MPS in cleaning efficacy,
whereas AQuify® and ReNu MoistureLoc™ provided the lowest cleaning. These in vitro results
agree with ex vivo clinical studies that have tested the cleaning efficacies of the various products.
Conclusions: Using Acuvue hydrogel lenses deposited with an in vitro lysozyme deposition
model, we are able to differentiate the cleaning efficacy of various MPS. Opti-Free® Express®
was demonstrated to exceed all other MPS in cleaning of the contact lenses.
CR: B. Hong, Alcon Laboratories, Inc. E; P. Stauffer, Alcon Laboratories, Inc. E; D.L.
Meadows, Alcon Laboratories, Inc. E.
Support: None.
Correlating Biocide Uptake and Release Profiles With Corneal Staining and Subjective
N.L. Dassanayake, R.Garofalo, C.Carey, R.David, D.L. Meadows, R.P. Stone. Consumer
Products Research, Alcon Laboratories, Inc., Fort Worth, TX.
Purpose: To determine if the interactions of lens care preservatives with contact lenses is an
important factor in clinical signs and symptoms. Methods: This study evaluated uptake and
release profiles of two preservatives used in MPS products, alexidine contained in ReNu® with
MoistureLoc™ and POLYQUAD® (polyguaternium-1) contained in OPTI-FREE® Express®.
Three lens materials were used in a cross over study design: SofLens 66® (alphafilcon A),
Acuvue® 2 (etafilcon A) and Acuvue® Advance™ (galyfilcon A, with HYDRACLEAR™).
Asymptomatic, adapted daily wear soft lens users wore groups I and IV soft hydrophilic
or silicone hydrogel lenses for a maximum of 2, 4 and 6 hrs each day. New lenses were
dispensed for each wear period. The worn lenses were analyzed for preservative content
using HPLC and release profiles calculated. Short-term changes in corneal staining and
ocular symptoms were assessed at defined time intervals following lens insertion in order to
compare these ocular findings with laboratory uptake and release properties. Results: All
three lens types uptake alexidine during an overnight soaking period. The rate of preservative
release during wear depended on the lens type. After 2 hours, Acuvue® 2 and SofLens 66®
released 67% and 78% of alexidine, respectively. Acuvue® Advance™ released 29% of
the preservative in two hours. Clinically significant corneal staining was observed at 2
and 4 hours when subjects wore Acuvue® 2 and Acuvue® Advance™ lenses soaked in the
alexidine-based system. At the 2 hour interval, extent of corneal staining increased in all
subjects with Acuvue 2 lenses, and 50% of the subjects with Acuvue Advance lenses. Only
minimal staining was observed for SoftLens 66® at 2, 4 and 6 hours. Symptoms were not
correlated with the extent of staining. Lenses cycled in the POLYQUAD ® - based product
showed minimal uptake into lenses and corneal staining was low with all the lens materials
tested. Conclusions: The study demonstrated the difference in uptake/release profiles for
MPS products preserved with Alexidine or POLYQUAD ® using 3 soft lens materials. For
some lens materials these differences in biocide uptake/release appear to be correlated with
corneal staining patterns but not symptoms. CR: N.L. Dassanayake, Alcon Laboratories,
Inc. E; R. Garofalo, Alcon Laboratories, Inc. E; C. Carey, Alcon Laboratories, Inc. E; R.
David, Alcon Laboratories, Inc. E; D.L. Meadows, Alcon Laboratories, Inc. E; R.P. Stone,
Alcon Laboratories, Inc. E. Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 904-932 / B878-B906
138. Contact Lens I Organizing Section: CO
916 - B890
Varying Susceptibility of Corneal and Environmental Strains of Acanthamoebae to
Multipurpose Solutions using a Novel Method
D.V. Seal1, M.Shoff 2, A.Rogerson3, S.Schatz4, H.Park4, T.Beattie5, A.Tomlinson6,
G.Booton7. 1Ophthal/Applied Vision Res Ctr, City University, London, United Kingdom;
Oceanographic Center, Nova Southeastern University, Dania, FL; 3Oceanographic Center,
Nova Southeastern Univeristy, Dania, FL; 4College of Optometry, Nova Southeastern
University, Ft. Lauderdale, FL; 5Glasgow Caledonian University, Glasgow, United
Kingdom; 6Glasgow Caledonian UNiversity, Glasgow, United Kingdom; 7Molecular
Genetics, Ohio State University, Columbus, OH.
Purpose: A novel method has been developed to compare Acanthamoeba strain differences by testing their
susceptibility to multipurpose contact lens cleaning solutions (MPS). Methods: This method uses surface
attached protozoa that mimic cells attached to a contact lens. In brief, acanthamoebae were grown on non
nutrient agar plates with E. coli prey. Blocks of agar with cysts or trophs (ca. 50 cells) were cut out and
transferred to MPS (ReNu, Complete, and Opti-free) for up to 24 h treatment. After neutralizing in Dey Engley
Broth (Difco), blocks were washed in amoeba saline and reinoculated onto fresh prey bacteria. Postitve growth
was indicative of survival. Results: Testing showed that the efficacy of the 3 MPS was different. ReNu was the
most effective, followed by Opti-free while Complete was relatively ineffective. Not surprisingly, trophozoites
were more susceptible than cysts. Overall strain differences are summarized in the table. Conclusions:
Findings for individual MPS complement previous work with other methods.This study has also identified that
environmental strains vary in their sensitivity to disinfecting solutions. Overall, T3, T5 and T11 environmental
isolates were more resistant than the T4 isolates from the cornea and beach. This resilience supports previous
work on temperature tolerance, where T3 and T5 acanthamoebae survived up to 41 C. Investigators should
consider the strain genotype and its source before embarking on sensitivity testing.
Genotype and Source
T3, tapwater, Hong Kong
T4, Corneal Scrape (P6)
T4, dry sand, FL
T4, Troon beach, UK
T4, Wet Sand, FL
T4 ,Wet Sand, FL
T5, Soil, FL
T5, tapwater, FL
T5, dry sand, FL
T11, soil, FL
% Survival of Acanthamoebae
0-6 hr
24 hr
0-6 hr
24 hr
917 - B891
The Impact of Post-Insertion Time on Corneal Staining and Comfort With Group II
Hydrogel Materials Disinfected With Various Lens Care Regimens
L.W. Jones, N.Keir, P.Situ, D.Fonn. Cclr, School of Optometry, Waterloo, ON, Canada.
Purpose: To determine if corneal staining and comfort associated with FDA group II soft
contact lenses disinfected by various care regimens vary with time post-insertion. Methods:
20 soft lens-wearing subjects were enrolled into this investigator-masked, randomized,
cross-over study. Subjects were fitted with Bausch & Lomb Sof Lens 66 (Alphafilcon;
FDA group II) lenses and randomly used three multipurpose solutions (MPS) - OPTIFREE ® EXPRESS® Multi-Purpose Disinfecting Solution (OFX; Alcon); Complete ®
MoisturePLUS™ MPS (COM; Advanced Medical Optics) and ReNu MultiPlus® MPS (RNU;
Bausch & Lomb) for consecutive 2-week periods, with new lenses dispensed with each
regimen. Subjects were examined at baseline and at 1 day, 1 week and 2 weeks with each
regimen. On each visit day, staining and comfort were rated after 2 and 6 hours of lens
wear. In addition, lens comfort was graded at the beginning, middle and end of the day.
Results: Corneal staining occurred most frequently and at the greatest severity with RNU and
least with OFX (p<0.001). OFX showed no significant change in staining (p=NS), with RNU
and COM showing greater staining at 2 hours than at 6 hours (p<0.001). At all follow-up visits,
COM and RNU demonstrated more staining than OFX (p<0.001). The staining varied from a
low grade, micropunctate pattern to clinically significant diffuse staining requiring cessation
of lens wear. Subjective comfort ratings decreased significantly from beginning to end of the
day (p<0.001) with no significant differences between the three regimens (p=NS). There was
no significant correlation between corneal staining and comfort at any time of the day (p=NS).
Conclusions: Significant corneal staining can occur with certain FDA group II lenses when
used with certain MPSs, but this staining does not appear to influence comfort levels. Lens
comfort is not a predictor of corneal health. Patients who experience good lens comfort
should have a comprehensive slit lamp examination to evaluate corneal staining. This is
particularly relevant after two hours of lens wear, when maximal solution sensitivity appears
to be represented.
CR: L.W. Jones, Alcon F; N. Keir, None; P. Situ, None; D. Fonn, Alcon F.
Support: Alcon Research Ltd
CR: D.V. Seal, None; M. Shoff, None; A. Rogerson, None; S. Schatz, None; H. Park, None; T. Beattie,
None; A. Tomlinson, None; G. Booton, None. Support: None.
918 - B892
In vitro and ex vivo Wettability of Phema and Siloxane-Based Contact Lens Polymers
R.Rogers, L.W. Jones. School of Optometry, CCLR, University of Waterloo, Waterloo, ON,
Purpose: Wettability of contact lens materials can impact in-eye comfort. A contact
angle measuring device was used to determine the in vitro wettability of daily
disposable (DD) and silicone hydrogel (SH) lenses. Ex vivo measurements were made
on Etafilcon A following 8 hours of in-eye wear after soaking in various care regimens.
Methods: Lens materials (DD: Etafilcon A, Hilafilcon A, Ocufilcon B, Nelfilcon A; SH:
Lotrafilcon A, Lotrafilcon B, Galyfilcon A, Balafilcon A) were analysed directly from the packing
solution and after soaking in care regimens for 12 hours. Contact angles were also measured after
soaking lenses for 5 minutes in saline for 8 cycles to determine the residence time of the regimen
and/or packing solution on the lens. Regimens included: Sensitive Eyes® (Bausch & Lomb),
Complete® MoisturePLUS™ (Advanced Medical Optics), OPTI-FREE® EXPRESS® (OFX;
Alcon), ReNu MultiPlus® (RMP; Bausch & Lomb) and SoloCare® Plus (SCP; CIBA Vision).
Results: Lens material and rinsing time significantly impacted contact angles (p<0.001).
Initially, Ocufilcon was the least wettable DD (60°). After rinsing, all DD materials
rapidly developed high contact angles (>60°). Group II materials (Hilafilcon/Nelfilcon)
retained the lowest contact angles. Balafilcon had the highest SH contact angles (115°)
through all cycles. Galyfilcon was the most wettable SH initially, but rapidly developed
contact angles similar to Balafilcon. Lotrafilcon materials retained the most wettable
surfaces (75° - Lotrafilcon A; 50° - Lotrafilcon B). Contact angles of SH lenses were
significantly lower after soaking in SCP and OFX. Other solutions had minimal impact
on wettability. Acuvue lenses soaked in OFX and examined ex vivo after 8 hours
of wear had low contact angles (<10°) compared to lenses soaked in RMP (>80°).
Conclusions: SH materials are less wettable than pHEMA-based hydrogels and can be
modified by soaking in various care regimens. Contact angle analysis provides valuable
data on in vitro and ex vivo wettability of hydrogel polymers and could provide a method
for development of materials with more wettable surfaces and in-eye rewetting agents.
Study funded by Alcon
CR: R. Rogers, None; L.W. Jones, None.
Support: None.
920 - B894
The Effect of Organic Soil on Serratia Marcescens Susceptibility to Contact Lens
C.Lakkis, M.Boyd. Clinical Vision Resarch Australia, University of Melbourne,
Melbourne, Australia.
Purpose: The presence of organic material, such as tear film components, may affect the
antimicrobial efficacy of some chemical contact lens disinfectants. We have shown that
P. aeruginosa isolates vary in their resistance to chemical disinfectants in the presence
of organic soil. S. marcescens is a common causative organism in CL-associated
microbial keratitis. The aim of this study was to investigate the effect of organic soil,
using an artificial tear model, on disinfection susceptibility of S. marcescens strains.
Methods: Disinfection susceptibility of five S. marcescens isolates (1 lab and 4
clinical strains) was investigated with and without the addition of organic soil. The
ISO 14729 organic soil (artificial tear) model was utilized; heat killed S. cereviseae in
inactivated fetal bovine serum. Three disinfectants were assessed: A) preserved with
polyquaternium-1 0.001% and myristamidopropyl dimethylamine 0.0005%, B) and C)
preserved with polyhexamethylene biguanide (PHMB) 0.0001%. Testing was carried out
in CL cases at room temperature using an initial inoculum of 106 CFU/ml. The number
of bacterial survivors was determined at 4 and 6 h post-inoculation via viable counts.
Results: Resistance to disinfection varied significantly between the strains for solutions
A & B, with poorer efficacy observed with the addition of organic soil compared
to unsoiled samples for at least one of the isolates (P<0.05). Overall, resistance to
solution B was significantly greater with the addition of organic soil at both 4 and 6 h
(P<0.05). Susceptibility to solution C did not vary significantly between the strains in
the presence or absence of organic soil (P>0.05) and solution C was the only disinfectant
to achieve log reductions of at least 3.0 for all strains under all test conditions.
Conclusions: S. marcescens isolates varied in susceptibility to two out of three chemical
contact lens disinfectants in the presence of organic soil, with the majority of isolates showing
greater disinfection resistance when tested with organic soil. The presence of organic material,
such as tear film components, during CL disinfection may increase bacterial resistance to
disinfection and result in subsequent contamination of CLs and CL care systems. Further
investigation of factors contributing to S. marcescens resistance is warranted.
CR: C. Lakkis, None; M. Boyd, None.
Support: None.
919 - B893
Effect of Growth Media on the Kill of Staph. aureus by Contact Lens Multipurpose
K.S. Ambrus, N.Azizi, W.Chang, T.Denbo. Eye Care R&D Microbiology, Advanced
Medical Optics, Santa Ana, CA.
Purpose: Contact lens multipurpose solutions sold in the US and in Europe are tested according to
the FDA (510(k)) guidelines, or ISO guidelines. This testing includes disinfection efficacy testing
of a panel of five test organisms. Staph aureus, ATCC 6538 is one of the five organisms. Both
guidelines require growth of this organism on Tryptic Soy Agar (TSA) prior to testing. Various
journal publications have reported growing the organism in the liquid version of the above media
instead - Tryptic Soy Broth (TSB). The scope of our study was to establish the effect of the growth
media on the outcome of disinfection efficacy studies vs. S. aureus. Methods: We prepared the test
culture in two ways: 1) We grew the test organism on TSA slant for 18-24 hours, harvested it with
sterile saline, and adjusted on a spectrophotometer to result in a final inoculum in the product of
1-10 x 105 CFU/mL. 2) We grew the test organisms in a test tube containing 10 mL TSB for 18-24
hours, then centrifuged the cells at 4000 rpm for 10 minutes. We washed the cells once more with
Dulbecco’s PBS, using centrifugation as above. We suspended the final pellet in Dulbecco’s PBS,
and adjusted the culture spectrophotometrically as above. We then inoculated three marketed contact
lens multipurpose solutions with the two culture preparations to contain the final inoculum level of
1-10 x 105 CFU/mL: Solution A - 1 ppm polyhexamethylene biguanide, taurine. Solution B - 0.001%
POLYQUAD®, 0.0005% ALDOX®. Solution C - MoistureLocTM, 0.00045% alexidine. We evaluated
the levels of survivors at 4 and/or 6 hour contact time points, using Letheen Broth. Results: The
results are presented in terms of log-drops at the 4 and/or 6 hour contact time points.
Contact time (hrs)
3.6, 3.5
3.1, 3.4
1.6, 2.3
3.7, 4.1
1.6, 1.7
>4.1*, 3.7
NT = not tested * no recovery of test organism (<10 CFU/mL) Conclusions: These data show that
the growth medium affects the test results to the degree where it could affect the outcome of the
performance criteria set by the FDA or ISO. To claim “stand-alone” disinfection, the log-drop for S.
aureus must be at least 3.0 logs at the soak time. The guidelines also prescribe the growth medium
prior to testing, and specify that this must be Tryptic Soy Agar (TSA), and not the broth (TSB).
CR: K.S. Ambrus, None; N. Azizi, None; W. Chang, None; T. Denbo, None.
Support: None.
921 - B895
Efficacy of Contact Lens Disinfecting Solutions in Inhibition of Environmental Yeast
S.Schatz1A, H.Laubach1B, A.Rogerson1C. ACollege of Optometry, BCollege of Medical
Sciences, COceanographic Center, 1Nova Southeastern Univ, Fort Lauderdale, FL.
Purpose: While the efficacy of contact lens disinfecting solutions (CLS) is tested against laboratory
or clinical isolates, little is known about their ability to inhibit microbial growth when tested against
environmental isolates. This study tested the killing effect of five CLS against eleven environmental
yeast isolates obtained from beach sand in south Florida. Methods: The tested CLS were Clear
Care, COMPLETE Moisture PLUS, OPTI-FREE Express, ReNu MultiPlus and SOLO-care PLUS.
Sabouraud’s dextrose broth cultures of Candida albicans, C. krusei, C. parapsilosis, C. tropicalis,
Metschnikowia bicuspidata, Pichia anomala, P. ohmeri, P. onychis, Rhodotorula mucilaginosa,
Torulaspora delbrueckii and Yarrowia lipolytica were incubated at 23 C, while shaking, for 10 days.
Cultures were centrifuged at 1400 rpm for 5 min, resuspended in phosphate buffered water (PBW),
centrifuged and resuspended in PBW. One ml of each species containing approximately 107 cells/ml
was placed into 9.0 ml of each CLS. Samples were removed from each mixture after 1 min, 30 min,
2 hrs and 4 hrs incubation times, diluted with PBW, spread on 3 Sabouraud’s dextrose agar plates,
incubated at 23 C for 72 hrs and colony forming units (CFU) were counted. Results: All of the CLS
eliminated 99% of the yeasts within 2 hrs. Clear Care, Opti-Free and Renu eliminated 100% of the
organisms within 4 hours. The differences between the killing of the isolates by the CLS treatments
were significantly different (p<.05, students t-test) at one min exposure time when compared to 0.5,
2 or 4 hrs post-treatment. Differences were not observed after one min. C. parapsilosis was most
resistant to the five CLS following initial one min exposure but resistance was overcome after 30 min
treatment. Complete was the least effective CLS with six of the yeast isolates displaying an increase
in numbers above the original inoculum after one minute of exposure. Conclusions: Environmental
yeast isolates proved to be as susceptible as standard challenge organisms to commercially available
CLS. The initial resistance of C. parapsilosis may be attributed to the formation of pseudohyphae
by C. parapsilosis before treatment thus prolonging the ability of the yeast to resist the effect of the
CLS. The increase in the initial inoculum after treatment by Complete may be due to the removal
of budding yeast cells from their mother cells before cell death. These data confirm that the CLS
are effective against a broad spectrum of environmental yeast isolates.
CR: S. Schatz, None; H. Laubach, None; A. Rogerson, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 904-932 / B878-B906
138. Contact Lens I Organizing Section: CO
922 - B896
923 - B897
Water Retention in Dry Eye Subjects of Soft Contact Lenses Treated With Several
Multi-Purpose Solutions
M.W. Robboy1A, S.Groemminger1B, A.Dobie1B, K.Pierce1A, L.Stiffler1A. AResearch Clinic,
Formulations Department, 1Bausch & Lomb, Rochester, NY.
Purpose: Three clinical evaluations were undertaken to assess whether changes
in water content can be demonstrated in dry eye subjects following the wearing
of Acuvue® 2 lenses soaked in Bausch & Lomb ReNu® with MoistureLoc™,
when compared to Acuvue® 2 lenses soaked in Alcon Opti-Free® Express®,
Ciba AQuify®, and AMO Complete® Moisture Plus™ multi-purpose solutions.
Methods: 18, 20, and 20 dry eye subjects, all habitual soft spherical contact lens wearers
with a computer-based visual analog dryness score of 60 or less on a 100-point scale, were
each enrolled in a clinical evaluation. Test and control solution lenses were pre-treated in
ReNu® with MoistureLoc™ and in Alcon Opti-Free® Express®, Ciba AQuify®, and AMO
Complete® Moisture Plus™, respectively, in pre-treated lens cases. Each subject wore a
test ReNu with MoistureLoc/ Acuvue® 2 lens and a control solution/ Acuvue® 2 lens on
contralateral eyes for approximately 1 hour. After one hour, lenses were removed from the eye,
and were immediately weighed on an analytical balance. Lenses were placed into a vacuum
chamber overnight under pressure at ambient temperature and subsequently re-weighed.
Results: In each of the three studies, the test lenses exhibited statistically less mean relative
percent total water loss compared to the control lenses. The ReNu® with MoistureLoc™
soaked test lenses lost (1) 5.4 percent of their total water content, while the Alcon Opti-Free®
Express® soaked control lenses lost 7.6 percent of their total water content, (2) 5.1 percent
of their total water content, while the Ciba AQuify® soaked control lenses lost 6.6 percent
of their total water content, and (3) 5.7 percent of their total water content, while the AMO
Complete® Moisture Plus™ soaked control lenses lost 6.8 percent of their total water content.
Conclusions: The results of this study indicate that ReNu® with MoistureLoc™ exhibits the
capacity to decrease the level of lost water, in lenses worn by contact lens dry eye subjects.
CR: M.W. Robboy, Bausch & Lomb E; S. Groemminger, Bausch & Lomb E; A. Dobie,
Bausch & Lomb E; K. Pierce, Bausch & Lomb E; L. Stiffler, Bausch & Lomb E.
Support: None.
Contact Lens Intolerance: Role of Oxidative Stress and Mitochondrial Injury Induced
by Mutlipurpose Solutions
M.Dutot1,2A, C.Baudouin2B,3, J.-M.M. Warnet1,2A, P.Rat1,2A. 1Laboratoire de Toxicologie,
Faculté de Pharmacie, Université René Descartes-Paris5, Paris, France; AUnité
de Pharmaco-Toxicologie Cellulaire, BService 3, 2Centre Hospitalier National
d’Ophtalmologie (CHNO) des XV-XX, Paris, France; 3Institut des Cordeliers, INSERMU598, Paris, France.
Purpose: Millions of people wear contact lens and they widely use multipurpose solutions
(MPS) containing preservatives for contact lens disinfection and rinse. At the same time,
contact lens intolerance is increasing maybe due to adsorption of MPS on contact lens leading to
MPS releasing on ocular surface with chronic cytotoxic effects. Our purpose was to investigate
apoptosis through oxidative stress and mitochondrial injury induced by MPS on conjunctiva.
Methods: Four MPS were investigated (Optifree™, Renu™, Solocare™ and Complete™)
compared to 0.9% NaCl. Cytotoxicity was evaluated using a cytofluorometer (Safire™, Tecan,
France) adapted to microplates after a 15-minute and 3-hour incubation on a human WKD
conjunctival cell line. Membrane integrity, reactive oxygen species and anion superoxide
overproductions, intracellular metabolism, mitochondrial mass and mitochondrial activity
were assessed using neutral red, DCFH-DA, dihydroethidium, alamar blue, nonyl acridine
orange and JC-1 tests, respectively. Apoptosis was confirmed with the cell death receptor
and caspase activations evaluated using immunochemistry and rhodamine 110-DEVD dye.
Results: Different cytotoxicity pathways dependent on the preservative (quaternary
ammonium or PHMB-biguanide) were observed. Some MPS induced oxidative
stress with increased mitochondrial mass, others induced a decrease in reactive
oxygen species production with mitochondrial alterations, but every MPS
stimulated apoptosis with cell death receptor activation and caspase induction.
We can modulate cell death receptor activation with unpreserved sea derivates.
Conclusions: Preservatives as quaternary ammonium and PHMB, used in ophthalmic
formulations are known to be cytotoxic and could explain the cytotoxic mechanism induced
by MPS. MPS should not be considered as rinse products because they can induce damage
to ocular surface and then contact lens intolerance. The implication of cell death receptor is
interesting because unpreserved sea derivates could be used as contact lens rinse products.
CR: M. Dutot, None; C. Baudouin, None; J.M. Warnet, None; P. Rat, None.
Support: None.
924 - B898
925 - B899
PVA Containing Hydrogel Lens Affects Bacterial Adherence
S.G. Sickler, J.W. Carter, L.C. Winterton, J.M. Lally, M.M. Gabriel. Lens Business R & D,
CIBA Vision Corporation, Duluth, GA.
Purpose: To compare the adherence of Pseudomonas aeruginosa and other bacteria to the
leading daily disposable and frequent replacement (Groups II and IV) lenses. Nelfilcon A
and HEMA-like material lenses including etafilcon A, omafilcon, vifilcon A, vasurfilcon A,
alphafilcon A, hilafilcon, and ocufilcon D were tested. A comparison of the relative adherence
to these hydrogel lenses using invasive and cytotoxic strains of Pseudomonas was investigated.
Methods: Lenses were suspended in 1x10E8/ml challenge organisms for 14 hours at 350C
while shaking. A sonication and release procedure was performed on unworn lenses and
lenses pre-conditioned with an artificial tear fluid. Results: Nelfilcon A lenses allowed the
lowest number of adherence among all substrata tested both in the presence or absence of a
film induced from artificial tear fluid. The bacterial adherence to different types of hydrogel
lenses by Pseudomonas aeruginosa invasive strain was much higher than with the cytotoxic
strain. Maximal adherence by Staphylococcus species to hydrogels was typically lower than
of Pseudomonas. Conclusions: The PVA containing nelfilcon A hydrogel may help lower
the number of bacteria adhered to the lens. This, in turn, may reduce the potential risk of
microbial-related adverse events.
CR: S.G. Sickler, CIBA Vision Corp. E; J.W. Carter, CIBA Vision Corp. E; L.C. Winterton,
Ciba Vison Corporation E; J.M. Lally, Ciba Vison Corporation E; M.M. Gabriel, Ciba Vison
Corporation E.
Support: None.
Microbial Keratitis in Contact Lens Related Ulcers
C.J. Clark1, W.H. Ayliffe1, A.Dharmasena1, S.Khemika1, M.Guillon2, N.Mahalingham2.
Department of Ophthalmology, Mayday University Hospital, London, United Kingdom;
Contact Lens Research Consultants, London, United Kingdom.
Purpose:To evaluate risk factors contributing to the occurrence and severity of microbial
keratitis in contact lens wearers, its management and effect on corneal endothelium. Methods:
32 patients with contact lens related corneal ulcers (CLRU) were studied prospectively over
two years. Ulcers were classified as mild, moderate or severe using: presence or absence of
anterior chamber reaction; position of the ulcer in relation to the visual axis; and size of the
ulcer. A case study questionnaire was employed and the subjects were referred for Confocal
Microscopy of the corneal endothelium after ulcer healing. Results: CLRU: 12.5% extended
wear, 4.2% annual, 4.2% 3-monthly disposable, 70.8% monthly disposable and 8.3% daily
regimen. A one-step care system led to an increased risk of complications. 44.4% CLRU were
mild, 33.3% moderate and 22.2% severe. 59% were central and 41% were peripheral. GPs
had commenced antibiotic therapy in 18.5% of the ulcers. Eight culture positive results from
the corneal scrapes: 5/8 Pseudomonas, 1/8 Klebsiella, 1/8 Serratia Liquefaciens, 1/8 Herpes
Simplex. 90% were treated with Ofloxacin (40% in combination with Cefuroxime; 18% with
Predsol). One patient was prescribed oral Ciprofloxacin and one Acyclovir. On treatment
68% showed an improvement of visual acuity, 19% remained unchanged and 14% showed
deterioration. On resolution, 2 patients had Deep Lamellar Keratoplasty and 1 LASIK. Six
patients underwent Confocal Microscopy. Useful data was only obtained from 5, as 1 patient
had dense central corneal scarring. A significantly lower endothelial count was recorded in
the affected eye (2244 ± 688 cells/mm2) compared to the fellow eye (2964 ± 505 cells/mm2).
Two eyes, both with Pseudomonas infection using Lotrafilcon A on an extended wear regimen,
suffered very large losses (1555 vs 3669 and 1380 vs 3127) Conclusions: Compared with
daily disposable, extended wear and monthly replacement contact lenses (even high oxygen
transmissibility silicone hydrogel lenses) pose a higher risk of developing microbial keratitis.
Confocal Microscopy revealed that microbial keratitis produces endothelial cell loss, which
can be greater in the more severe cases.
CR: C.J. Clark, None; W.H. Ayliffe, None; A. Dharmasena, None; S. Khemika, None; M.
Guillon, None; N. Mahalingham, None.
Support: None.
926 - B900
927 - B901
Risk Factors for Contact Lens Related Microbial Keratitis in Australia
K.Edwards1,2, L.Keay1,2, T.Naduvilath1,3, G.Brian1, F.Stapleton2,3, Microbial Keratitis
Study Group. 1Vision CRC, Sydney, Australia; 2School of Optometry and Vision Science,
University of New South Wales, Sydney, Australia; 3Institute for Eye Research, Sydney,
Purpose: To establish risk factors for and the relative risk of microbial
kerat it is ( M K) for dif ferent cont act lens (CL) t y pes i n Aust ralia.
Methods: New cases of CL related presumed MK were identified in a 12 month prospective
study. Cases were interviewed by phone to establish risk factor data. Seven hundred CL
wearing controls were identified using a population based telephone survey. CL wearers were
grouped as follows: daily wear soft CL (including silicone hydrogel and hydrogel) (DWS),
soft extended wear CL (EWS), silicone hydrogel extended wear (EWSH), rigid CL daily
wear (RGP), and daily disposable CL (DD). The association of independent risk factors with
corneal infection was estimated using odds ratios (OR) and their 95% confidence intervals.
Results: 151 cases were reported in the first 6 months with data on CL type and risk factors
available in 93 and 70 cases respectively. Males had a higher risk of MK compared to females
(OR=1.6, 95% CI=1.01-2.6). The mean age of cases was 33.6±12.8 years, compared with
36.7±12.1 years in controls (p=0.015). Compared to DD wear, the relative risk of MK was 0.2x
(95% CI: 0.0-2.4) with RGP, 1.4x (0.4-4.8) with DWS, 24.6x (5.8-103.8) with EWS and 25.5x
(7.0-93.1) with EWSH. Behaviours associated with a higher risk of MK included: occasional
overnight use of daily wear CL (OR=4.7x, 95% CI=2.1-10.3), not storing CL cases dry (OR=2.4,
95% CI=1.2-5.1), showering in lenses (OR=2.7x, 95% CI=1.2-5.9), smoking (OR=2.5x, 95%
CI=1.1-5.5), and inappropriate treatment of CL while swimming (1.3, 95% CI=1.1-1.5). Of the
117 cases where information on final visual outcome was available, there were 7 cases of vision
loss of at least 2 lines of best corrected visual acuity: 5 in DWS, 1 in EWS and 1 in EWSH.
Conclusions: While the data have yet to be independently reviewed, overnight wear of both
hydrogel and silicone hydrogel CLs has a higher risk of MK infection compared to DD, RGP
and DWS use. Showering in lenses, smoking, occasional overnight use in DW and inappropriate
handling of lenses while swimming are associated with an increased risk of infection.
CR: K. Edwards, None; L. Keay, None; T. Naduvilath, None; G. Brian, None; F. Stapleton,
Support: Australian Government Cooperative Research Centres Grant
Incidence and Morbidity of Contact Lens-Associated Keratitis and Relevant Risk
Factors: A 12 Month Hospital-Based Survey
N.Efron1, P.B. Morgan1, E.A. Hill1, M.Raynor2, M.Whiting2, A.B. Tullo2, N.A. Brennan3.
Dept Optometry, Univ Manchester, Manchester, United Kingdom; 2Royal Eye Hospital,
Manchester, United Kingdom; 3Brennan Consultants, Melbourne, Australia.
Purpose: To determine the incidence and morbidity of keratitis among wearers of current generation
contact lenses and to explore relevant risk factors. Methods: We conducted a 12-month, prospective,
hospital-based epidemiological study in which the clinical severity of each presenting case of contact
lens-associated keratitis was scored. The size and location of corneal infiltrates was documented. The
hospital catchment population, and the wearing modalities (daily wear [DW] or extended wear [EW])
and lens types being used, were estimated from relevant demographic and market data. Risk factors
were assessed using logistic regression analysis. Results: Over the 12 month survey, there were 38
cases of severe keratitis (SK; traditionally termed ‘microbial keratitis’) and 80 cases of non-severe
keratitis (NSK). The annual incidence (cases per 10,000 wearers) of all forms of keratitis, SK and NSK
among contact lens wearers was 21.3 (95% confidence interval 17.8 to 25.5), 6.9 (5.0 to 9.4) and 14.4
(11.6 to 18.0), respectively. The incidence of SK stratified for wearing modality and lens type was:
DW rigid 2.9 (0.8 to 10.4); DW hydrogel daily disposable 4.9 (2.5 to 9.6); DW hydrogel (excluding
daily disposable) 6.4 (4.1 to 9.9); DW silicone hydrogel 0.0 (0.0 to 210.1); EW rigid 0.0 (0.0 to 1759);
EW hydrogel 96.4 (37.5 to 245.2); and EW silicone hydrogel 19.8 (6.7 to 58.0). The difference between
EW hydrogel and EW silicone hydrogel was significant (p = 0.04). Corneal scrapes were performed
in 23 cases of SK; 43% were culture-positive, with Pseudomonas aeruginosa identified as the main
pathogen in 30% of cases. More infiltrates occur in the peripheral cornea with DW hydrogel lenses,
in the central cornea with DW hydrogel daily disposable lenses and in the superior cornea with EW
silicone hydrogel lenses (p < 0.05 in all cases). Infiltrative events that occur in the corneal periphery
are less clinically severe. No patients suffered significant visual loss (95% confidence interval 0.0 to
9.4%). Significant risk factors in addition to lens type/modality were: gender, smoking, relevant eye
and health problems, and season. Conclusions: There is a significantly higher incidence of severe
keratitis in wearers who sleep in contact lenses. Silicone hydrogel lenses carry a 5X lower risk of
severe keratitis for extended wear compared with hydrogel lenses. Overall, morbidity (visual loss)
is low. Significant risk factors are identified but are of little predictive value.
CR: N. Efron, None; P.B. Morgan, None; E.A. Hill, None; M. Raynor, None; M. Whiting,
None; A.B. Tullo, None; N.A. Brennan, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 11:00 AM - 12:45 PM Hall B/C Poster Session Program Number/Board # Range: 904-932 / B878-B906
138. Contact Lens I Organizing Section: CO
928 - B902
Factors Influencing the Morbidity of Contact Lens Related Microbial Keratitis
L.Keay1A,2, K.Edwards1A,2, T.Naduvilath2,3, G.Brian2, K.Forde1B, F.Stapleton1A,3, Microbial
Keratitis Study Group. AOptometry/Vision Science, BSchool of Public Health and
Community Medicine, 1University of New South Wales, Sydney, Australia; 2Vision CRC,
Sydney, Australia; 3Institute for Eye Research, Sydney, Australia.
Purpose: To examine factors inf luencing the severit y of contact lens (CL)
related microbial keratitis (M K) managed in hospit als and pr ivate clinics
Methods: Cases of CL related MK in 15-64 year olds, wearing CLs for the correction of simple
refractive error were detected through surveillance of ophthalmic practitioners in Australia and
New Zealand. Clinicians provided information on treatment and patients were interviewed by
telephone where possible at least one month after the event. Morbidity was assessed in terms
of direct and indirect costs, days before symptoms resolved (up to 28 days) and percentage of
cases with 2 or more lines of vision loss. The effect of pathogen, CL modality (DW/EW), CL
material (low/high Dk) and delay in CL removal or seeking consultation were examined.
Results: The mean age was 37±13 years (mean±SD) and 59% were female (n=139). One third (60%)
delayed CL removal by >4 hours and 67% delayed >24 hours before seeking medical advice. Medical
visits and hospital bed days were used to calculate the direct costs [$799(1810)], indirect costs included
days off work for patients [2(5)] and their carers [1(2)] combining to $553(1671), patients were
symptomatic for 7(9) days of the first month [median(IQR)] and >2 lines of vision loss was documented
in 7.3% of cases. Culture proven cases appeared to be more costly to treat (p<0.2) and had longer
duration of symptoms (14±10 vs 6±5 days, p=0.02). 50% of cases were in DW (25% high Dk, 38%
occasional overnight wear) and the remainder EW (95% high Dk). DW cases had longer duration of
symptoms (11±9 vs 7±7 days, p=0.02) and rate of visual loss was higher but this did not reach statistical
significance (6/52 vs 1/43, p=0.12). There was no difference in costs (p>0.4). Delay in seeking
consultation was associated with longer duration of symptoms (11±9 vs 7±5 days, p=0.04) but no
difference in cost or rate of vision loss. Multivariate analysis will more fully explore these relationships.
Conclusions: Both positive identification of a microbial pathogen and CL modality/material are
determinants of morbidity. However modifiable factors such as delays in treatment appear to have some
influence on disease outcome. Cost analysis and symptom duration are useful measures of morbidity
for a condition, which ranges widely in severity but is rarely associated with vision loss.
929 - B903
Bacterial Transmission From Contact Lenses to ex vivo Porcine Eyes
J.M. Hooymans1, P.B. J. Vermeltfoort2, T.G. Van Kooten2, G.M. Bruinsma2, H.C. Van der
Mei2, H.J. Busscher2. 1Ophthalmology, University Hospital, Groningen, The Netherlands;
Biomedical Engineering, Faculty of Medical Sciences, University of Groningen,
Groningen, The Netherlands.
Purpose: This study aimed to quantify the transmission of the hydrophilic Staphylococcus
aureus 835 and hydrophobic Pseudomonas aeruginosa #3 from three types of contact
lenses, differing in surface hydrophobicity and roughness, to ex vivo porcine eyes.
Methods: Contact lenses were inoculated in a bacterial suspension for 30 min
and then placed on an ex vivo porcine eye. After 16 h of contact between lens and
eye, confocal laser scanning microscopy was used to determine the number of
bacteria on the lens and eye surface for the calculation of transmission percentages.
Results: Transmission percentages were significantly different for both bacterial
strains from an etafilcon A lens and balafilcon A lens (p=0.006 and 0.04, respectively).
Percentages varied from 51 to 68% for the hydrophobic P. aeruginosa and from 54 to
82% for the hydrophilic S. aureus strain, depending on the contact lens type involved.
Both strains were transferred the least from the most hydrophilic and roughest lens,
made of lotrafilcon A, although this was only statistically significant for S. aureus.
Conclusions: Bacterial transmission to a porcine eye was proven to differ for various types
of contact lenses and was the least from a hydrophilic and rough lens type.
CR: J.M. Hooymans, None; P.B.J. Vermeltfoort, None; T.G. Van Kooten, None; G.M.
Bruinsma, None; H.C. Van der Mei, None; H.J. Busscher, None.
Support: None.
CR: L. Keay, None; K. Edwards, None; T. Naduvilath, None; G. Brian, None; K. Forde,
None; F. Stapleton, None.
Support: Australian Government via CRC Program, NHMRC post graduate student scholarship
930 - B904
Influence of Day and Night Wear on Surface Properties of Silicone Hydrogel Contact
Lenses and Bacterial Adhesion
P.Vermeltfoort1, M.Rustema-Abbing1, J.De Vries1, G.M. Bruinsma1, H.J. Busscher1, M.L.
Van der Linden2A, J.M. M. Hooymans2B, H.C. Van der Mei1. 1Department of Biomedical
Engineering, Faculty of Medical Sciences, University of Groningen, Groningen, The
Netherlands; AOculenti, BDepartment of Ophthalmology, 2University Hospital Groningen,
Groningen, The Netherlands.
Purpose: The aim of this study is to determine the effect of continuous wear on physico-chemical
surface properties of silicone hydrogel contact lenses and their susceptibility to bacterial adhesion.
Methods: In this study volunteers wore two pairs of either “lotrafilcon A” or ‘balafilcon
A” silicone hydrogel lenses. The first pair was worn continuously for a week, the second
pair for 4 weeks. One lens out of each pair was used for surface characterization,
the other one for bacterial adhesion experiments. Lens surfaces were characterized by
examinination of their wettablilty, roughness and elemental composition. Adhesion of
the hydrophilic Staphylococcus aureus 835 and hydrophobic Pseudomonas aeruginosa
#3 bacterial strains to a lens was studied using a parallel plate f low chamber.
Results: Before use, the lotrafilcon A lens was rougher, was more wettable and had a higher
susceptibility for S. aureus adhesion than the balafilcon A lens. After wear, both lens types
became more and equally wettable, whereas the differences in elemental surface composition
decreased as well. S. aureus adhered more on worn balafilcon A lenses, while the opposite
was seen for P. aeruginosa. The initial deposition rates of both bacteria to lotrafilcon A lenses
decreased by wearing and were found to correlate with the surface roughness of worn lenses.
Conclusions: In this study the differences in surface properties between 2 types of silicone
hydrogel lenses were found to change after 1 week of continuous wear. Generally, bacteria
adhered in lower numbers and less tenaciously to worn lenses, except S. aureus, adhering in
higher numbers to worn balafilcon A lenses.
CR: P. Vermeltfoort, None; M. Rustema-Abbing, None; J. De Vries, None; G.M.
Bruinsma, None; H.J. Busscher, None; M.L. Van der Linden, None; J.M.M. Hooymans,
None; H.C. Van der Mei, None.
Support: None.
931 - B905
The Effect of Patient Wear on the Attachment of Acanthamoeba to Acuvue Advance,
Second Generation, Silicone Hydrogel Contact Lenses
A.Tomlinson1A, T.K. Beattie1A, A.K. McFadyen1B. ADepartment of Vision Sciences,
Division of Mathematics, 1Glasgow Caledonian University, Glasgow, United Kingdom.
Purpose: To determine if patient wear had any effect on the attachment of Acanthamoeba
to the new second-generation silicone hydrogel (S-H) lens, Acuvue Advance. Attachment
was compared to that of the first generation S-H lenses and a conventional hydrogel lens.
Methods: Unworn and worn (daily for one week) Acuvue Advance (Johnson & Johnson), Focus
Night & Day (Ciba Vision), Purevision (Bausch & Lomb), and Acuvue (Johnson & Johnson)
lens quarters were incubated for 90 minutes in suspensions of plate-cultured Acanthamoeba
castellanii trophozoites. Trophozoites attached to one surface of each quarter were counted
by direct light microscopy. Sixteen replicates of each lens type were examined. Logarithmic
transformation of data allowed the use of parametric ANOVA for statistical analysis.
Results: Patient wear produced a signif icant increase in the number of
Acanthamoeba attaching to the second-generation lens (p< 0.001) and the
conventional hydrogel lens (p=0.009). No such increase in attachment was found
with the first generation S-H lenses, but attachment was significantly greater than
to the unworn or worn second generation and conventional hydrogel lenses (p<0.001).
Conclusions: Acanthamoeba demonstrated a significantly higher affinity for worn second
generation S-H lenses compared with unworn lenses. Patient wear may therefore be considered
as a risk factor for Acanthamoebal infection. However, as attachment to the worn second
generation S-H was similar to that found with the worn conventionally hydrogel, this new
lenses is at no more risk of promoting infection with Acanthamoeba than a conventional
hydrogel. The same cannot be said for the first generation S-H lenses, which had significantly
greater attachment than both the unworn and worn second generation S-H, and may be at
greater risk of initiating infection through lens colonisation.
CR: A. Tomlinson, None; T.K. Beattie, None; A.K. McFadyen, None.
Support: None.
932 - B906
Microbial Attachment to Second-generation, Silicone Hydrogel Contact Lenses
T.K. Beattie1A, A.Tomlinson1A, A.K. McFadyen1B. AVision Sciences, BMathematics, 1Glasgow
Caledonian Univ, Glasgow, United Kingdom.
Purpose: To investigate the attachment of Acanthamoeba to the new secondgeneration silicone hydrogel (S-H) lens, Acuvue Advance, and determine if the
presence of a bacterial biofilm coating affects attachment. Amoebal attachment will
be compared to that of first generation S-H lenses and a conventional hydrogel lens.
Methods: Unworn and Pseudomonas aeruginosa biofilm coated Acuvue Advance
(Johnson & Johnson), Focus Night & Day (Ciba Vision), Purevision (Bausch & Lomb),
and Acuvue (Johnson & Johnson) lens quarters were incubated for 90 minutes in
suspensions of plate-cultured Acanthamoeba castellanii trophozoites. Trophozoites
attached to one surface of each quarter were counted by direct light microscopy. Sixteen
replicates of unworn and biofilm coated lenses of each type were examined. Logarithmic
transformation of data allowed the use of parametric ANOVA for statistical analysis.
Results: The presence of a bacterial biofilm on the Acuvue Advance lens (p<0.001) and
the conventional hydrogel (p=0.009) lens produced a significant increase in attachment,
compared with the unworn state. No such effect was noted with the first generation SH lenses. Amoebal attachment to unworn or biofilm coated second generation S-H and
the conventional hydrogels did not differ significantly, but both had significantly fewer
attached organisms than the unworn or biofilm coated first generation S-H lenses (p<0.001).
Conclusions: The similarity in amoebal attachment to the second generation S-H lens and the
conventional hydrogel lens suggests that the Acuvue Advance lens poses no more of a risk of
ocular infection with Acanthamoeba, as a result of amoebal colonisation, than a conventional
hydrogel lens. The risk may actually be less due to the improved corneal oxygenation and
metabolism achieved with this new lens material. However, the first generation S-H lenses,
which had significantly more organisms attached, may provide a greater risk of infection if
Acanthamoebal exposure occurs, for instance when showering, swimming or through noncontinuous wear and inefficient lens care regimes.
CR: T.K. Beattie, None; A. Tomlinson, None; A.K. McFadyen, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 933-947 / B907-B921
139. Ocular Allergy/Conjunctivitis Organizing Section: CO
933 - B907
Enhancement of Conjunctival Inflammation by Corneal Epithelial Ablation in
Experimental Allergic Conjunctivitis
N.Kumagai1A, K.Fukuda1B, T.Nishida1A. ABiomolecular Recognition and Ophthalmology,
Ocular Pathophysiology, 1Yamaguchi Univ School of Medicine, Ube City, Japan.
Purpose: Corneal epithelial lesions are a characteristic of vernal keratoconjunctivitis. Studies on
cultured corneal cells have suggested that corneal fibroblasts may enhance allergic inflammation
through the release of chemokines and the expression of adhesion molecules, and that corneal
epithelial cells might reduce the extent of such inflammation by separating corneal fibroblasts
from cytokines present in tear fluid. The effects of corneal epithelial ablation and consequent
exposure of the corneal stroma to tear fluid on conjunctival inflammation were investigated in rats.
Methods: Brown Norway rats were sensitized to short ragweed pollen and alum. The corneal
epithelium of the right eye was mechanically removed 21 days after sensitization, and the animals
were challenged by topical instillation of pollen. Early-phase and late-phase clinical symptoms
were scored at 20 min and 24 h after challenge, respectively. Histopathologic changes and the
abundance of transcripts for adhesion molecules (ICAM-1, VCAM-1) and chemokines (TARC,
IP-10, eotaxin, RANTES, MCP-1) in the conjunctiva were examined 24 h after challenge.
Results: Allergen sensitization and corneal epithelial ablation induced a synergistic
increase in the late-phase clinical score ( p < 0.05, two-way ANOVA), without
affecting that of the early phase, in challenged rats. Corneal epithelial ablation
markedly increased eosinophil infiltration as well as the abundance of ICAM1, TARC, RANTES, and MCP-1 mRNAs in the conjunctiva of sensitized rats.
Conclusions: Corneal epithelial ablation enhances the late phase of conjunctival allergic
inflammation by promoting eosinophil infiltration and expression of chemokines and adhesion
molecules in the conjunctiva.
Effect of corneal epithelial ablation on the late phase clinical score of conjunctival inflammation
Ablation (-)
Ablation (+)
Sensitization (-)
Sensitization (+)
CR: N. Kumagai, None; K. Fukuda, None; T. Nishida, None.
Support: None.
935 - B909
934 - B908
Human Cord Blood Mast Cell IgE Cross-Linking: An in vitro Model of Conjunctival
Mast Cell Responses?
V.L. Calder, G.Galatowicz. Ophthalmology, UCL, London, United Kingdom.
Purpose: Conjunctival mast cells are important effector cells in ocular allergy and secrete a range
of mediators, but can only be isolated in low cell numbers. The aim of this study was to characterise
human cord blood-derived mast cells (HCBMC) phenotypically and functionally in response to IgE
stimulation.The effects of two anti-allergy drugs - epinastine & olopatadine - were also investigated.
Methods: Human cord blood CD34+stem cells (105) were cultured in 200ul Stemspan™ serumfree medium containing SCF [100ng/ml] and IL-6 [50ng/ml], with IL-3 [1ng/ml] added during the
first 14 days. On week 9-11, 10% fetal calf serum was added. At week 11, cells were characterised
by flow cytometry for CD117 (c-kit) and FcεR1, and cytospins prepared for tryptase (AA1) and
chymase (CCI) staining. For cross-linking, cells were incubated with IgE [4μg/ml; 16 hr] before
anti-IgE Ab[25μg/ml] was added and histamine release determined by ELISA. Intracellular
cytokine expression for IL-4 and IL-13 was investigated at 4, 24 & 48hr. Multiplex cytokine assays
were performed to detect IL-2, IL-4, IL-5, IL-6, IL-10, TGFα & β by flow cytometry. Results: At
week 10, HCBMC were immunophenotyped as 100% c-kit+ and 90% FcεR1+, >95% AA1+ and
>58% CCI+. Following FcεR1 stimulation, significant histamine secretion was detected at 1hr
[35.9±5.9 ng/ml; p<0.01], which was inhibited by 51.4% with epinastine [0.1μg/ml] (p<0.05) and
by 35.1% with olopatadine [10μg/ml] (p<0.05). Intracellular expression of IL-4 was 90.6±1.6% in
unstimulated cells, which decreased to 47.3±0.3% at 48hr post stimulation (p<0.0001). Epinastine
[10μg/ml] reduced IL-4 expression (39.1±2.9%; p<0.05). In contrast, basal expression of IL-13
was 8.2±0.3%, which increased to 14.0±1.5% at 48hr post stimulation. Production of IL-1β was
induced by 1hr and peaked at 4hr [95.4±14.6 ρg/ml]. IL-8 was significantly increased at 1hr and
peaked at 24hr (7.5±0.08 ng/ml; p<0.003). IL-5 was detected even in unstimulated cells [55.8±2.7
ρg/ml]. The effect of the anti-allergic drugs on production of these cytokines is being investigated.
Conclusions: The HCBMC were phenotypically comparable to conjunctival mast cells and
demonstrated a differential cytokine response following IgE cross-linking. The anti-allergic drugs
effectively reduced the release of histamine and had some effect on IL-4 expression. The data suggests
these cells provide an in vitro system with which to investigate mast cell cytokine responses and
we are currently investigating human conjunctival mast cells (in collaboration with Prof. J. Dart,
Moorfields Eye Hospital).
CR: V.L. Calder, Allergan Inc. F; G. Galatowicz, None.
Support: Fight For Sight (UK); Allergan Inc.
936 - B910
Inhibition of Matrix Metalloproteinase-3 Synthesis in Human Conjunctival
Fibroblasts by IL-4 or IL-13
K.Fukuda1A, Y.Fujitsu1B, K.Yamamoto1B, K.Seki1A, N.Kumagai1B, T.Nishida1B. AOcular
Pathophysiology, BBiomolecular Recognition and Ophthalmology, 1Yamaguchi Univ Sch
of Medicine, Ube City, Japan.
Purpose: Fibroproliferative lesions known as giant papillae are a characteristic of vernal
keratoconjunctivitis (VKC). The abundance of T helper 2 (Th2) cells and Th2 cytokines is
increased in the giant papillae and tear fluid of individuals with VKC. We previously showed that
the Th2 cytokines interleukin (IL)-4 and IL-13 each stimulate the synthesis of extracellular matrix
(ECM) proteins in conjunctival fibroblasts. The role of Th2 cytokines in the development of giant
papillae was examined further by determining the effects of IL-4 and IL-13 on the production
by these cells of matrix metalloproteinase (MMP)-3, a key enzyme in ECM degradation.
Methods: Human conjunctival fibroblasts were cultured with Th2 cytokines in the
absence or presence of IL-1β. The amount of MMP-3 released into the culture medium was
determined by enzyme-linked immunosorbent assay, and the intracellular abundance of
MMP-3 mRNA was quantitated by reverse transcription and real-time polymerase chain
reaction analysis. The phosphorylation of IκBα and c-Jun and the subcellular localization
of NF-κB were evaluated by immunoblot and immunofluorescence analyses, respectively.
Results: Of the Th2 cytokines tested, only IL-4 and IL-13 (not IL-5, IL-9, or IL-10) inhibited
basal and IL-1β-induced MMP-3 release by conjunctival fibroblasts. These effects of IL-4
and IL-13 were dose dependent and inhibited by neutralizing antibodies to the common
IL-4Rα chain of both IL-4 and IL-13 receptors. IL-4 and IL-13 also each reduced the
basal abundance of, as well as inhibited the IL-1β-induced increase in, MMP-3 mRNA
in conjunctival fibroblasts. IL-4 or IL-13 did not affect either the phosphorylation of IκBα
and c-Jun or the translocation of NF-κB to the nucleus induced in these cells by IL-1β.
Conclusions: IL-4 and IL-13 inhibit MMP-3 synthesis in human conjunctival fibroblasts,
suggesting that these cytokines contribute to the excessive deposition of ECM in giant papillae
by preventing ECM degradation by this enzyme.
CR: K. Fukuda, None; Y. Fujitsu, None; K. Yamamoto, None; K. Seki, None; N. Kumagai,
None; T. Nishida, None.
Support: a Grand-in-Aid for Young Scientists (B) (KAKENHI) (no. 16791050)
Differential Expression of Matrix Metalloproteinases in Vernal Keratoconjunctivitis,
Allergic Asthma and Nasal Polyps
A.A. Leonardi1A, P.Brun1B, A.DiStefano2, L.Motterle1A, C.F. Donner2, G.Abatangelo1B, A.G.
Secchi1A. ADepartment of Neuroscience, Ophthalmology Unit, BDepartment of Histology,
Microbiology and Medical Biotechnology, 1University of Padova, Padova, Italy; 2Irccs,
Fondazione S. Maugeri, Veruno, Italy.
Purpose: Allergic conditions in different organs share many similarities in their
inf lammatory response. Vernal keratoconjunctivitis (VKC), asthma, and nasal
polyps exhibit several similar, but site-specific mucosal structural changes.
We hypothesized that a differential expression of matrix metalloproteinases
(MMP) may contribute to different tissue remodeling aspects in different organs.
Methods: Mucosal biopsies from different sources were obtained from: 1) conjunctiva
of healthy donors (cCT); 2) tarsal conjunctiva of VKC patients; 3) bronchii of nonasthmatic subjects (bCT); 4) bronchii of mild stable asthmatic patients; 5) nasal
mucosa of healthy donors (nCT); 6) nasal polyps of allergic patients. Expression
of MMP-1, 3, 9, 13 and TIMP-1 was evaluated by immunohistochemistry.
Results: In the normal conjunctiva, tissue expression of MMPs was low or absent, while
TIMP-1 was highly expressed. In conjunctival stroma of VKC patients, MMP-1, -9, and
-13 were significantly higher expressed compared to respective cCT. MMP-9 and -13 were
significantly more expressed in conjunctival epithelium of VKC patients compared to
cCT. MMP expression was higher in normal bronchial tissues than in normal conjunctiva.
In bronchial tissues, only MMP-13 was significantly more expressed in asthmatic
compared to non-asthmatic subjects. In nasal mucosa, MMP-9 staining was higher in
polyps then normal nasal mucosa. Compared to respective controls, abnormalities in
the epithelial and sub-epithelial structure were more evident in pathologic conjunctiva.
Conclusions: MMP-s may play an important role in inducing the structural changes seen in
VKC, nasal polyps and asthma. Tissue remodeling was more dramatic in giant papillae of
VKC patients compared to other tissue sites of chronic allergic inflammation.
CR: A.A. Leonardi, None; P. Brun, None; A. DiStefano, None; L. Motterle, None; C.F.
Donner, None; G. Abatangelo, None; A.G. Secchi, None.
Support: MIUR 4277/03 and 1488/04
937 - B911
938 - B912
Patient Evaluation of Azelastine HCl versus Olopatadine HCl in the Treatment of
Allergic Conjunctivitis
A.-M.Oliva1A, C.B. Slonim1B. ACollege of Medicine, BOphthalmology, 1University of South
Florida, Tampa, FL.
Purpose: The use of a combination H1 antagonist and mast cell stabilizer anti-allergy product
(e.g., olopatadine HCl and azelastine HCl) can not only inhibit the release of histamine
and other mediators from the mast cells but also block the action of histamine that has
already been released, thus decreasing the severity of the allergic response. In this study, we
compare the subjective responses of previous olopatadine (Patanol, Alcon) users with allergic
conjunctivitis who were treated for five (5) days with azelastine HCl (Optivar, MedPointe).
Methods: 1428 ocular allergy patients in 75 ophthalmologist/optometrist offices were surveyed.
Patients were asked 6 questions regarding the effectiveness of azelastine for the treatment of
allergic conjunctivitis (i.e., onset, duration, and overall satisfaction) and compare it to previous
treatments. The answers were graded on a scale of 0 to 10 for onset and duration and yes or no for
overall satisfaction. The groups were further divided into previous olopatadine users or nonusers.
Results: In patients who had used a previous ophthalmic anti-allergy medication
in the past and those who had not, 89% claimed azelastine effectively treated
their eye allergy. In the subset of patients who had previously used olopatadine,
60% claimed that azelastine was more favorable (scored 7-10) than olopatadine.
Conclusions: In this patient survey, azelastine was favored to be more effective and yield higher
patient satisfaction scores than olopatadine in the treatment of allergic conjunctivitis.
CR: A. Oliva, None; C.B. Slonim, Bauch and Lomb, Inc. C; MedPointe Pharmaceutical
C, R.
Support: Bauch and Lomb, Muro, MedPointe
The Efficacy of FK506 Eye-Drop in the Treatment of Ocular Surface Complications in
Patients With Severe Allergic Patients With Corneal Complications
M.Tanaka, D.Murat, K.Fukagawa, N.Okada, N.Asano-Kato, Y.Takano, A.Igarashi,
A.Kujira, K.Tsubota, H.Fujishima. Ophthalmology, Keio University School of Medicine,
Tokyo, Japan.
Purpose: It is well-known that FK506 strongly inhibits cytokine production by T cells. ECP is a
tissue damage protein which is secreted from activated eosinophils. Tear ECP levels in allergic
patients are known to be related to the severity of clinical findings. To examine the clinical
efficacy and anti-inflammatory effects of FK506 eye drops, we studied the changes in clinical
ocular findings and measured tear ECP levels of allergic patients before and after the treatment.
Methods: Six eyes of 4 patients diagnosed AKC or VKC were enrolled in this study (all
males: mean 12.5±2.6 years old) and treated with FK506. The study was placebo controlled
where 8 eyes of 4 patients received saline drops in a masked fashion. Tear samples were
collected from each patient using a capillary micropipette before and after the treatment.
All patients received 0.1% FK506 or placebo eye drops 4 times a day for 1 month. A clinical
score of corneal findings (0-4: 0=absent; 4=severe) was determined by slitlamp examination.
Tear ECP measurement and corneal findings were evaluated before and after the treatment.
Results: Clinical corneal scores improved significantly in patients treated
with FK506 eye-drops compare to the placebo drop. Post-treatment tear ECP
levels were also significantly reduced compared to the pretreatment level.
Conclusions: FK506 eye drops were found to be safe and effective in the treatment of ocular
complications in severe ocular allergy. Tear ECP level was observed to be a useful marker in
assessing the severity of ocular allergy and efficacy of treatment with FK506.
CR: M. Tanaka, None; D. Murat, None; K. Fukagawa, None; N. Okada, None; N. AsanoKato, None; Y. Takano, None; A. Igarashi, None; A. Kujira, None; K. Tsubota, None; H.
Fujishima, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 933-947 / B907-B921
139. Ocular Allergy/Conjunctivitis Organizing Section: CO
939 - B913
Tear Function and Ocular Surface Mucin Alterations in Atopic Dermatitis Patients
M.Dogru1, N.Okada1, N.Asano Kato1, A.Igarashi1, M.Tanaka1, Y.Takano1, K.Fukagawa1,
K.Tsubota1, J.Shimazaki2, H.Fujishima1. 1Dept of Ophthalmology, Keio University School
of Medicine, Tokyo, Japan; 2Dept of Ophthalmology, Tokyo Dental College, Ichikawa,
Purpose:To study the tear functions and ocular surface mucin alterations in patients with
atopic dermatitis ( AD) Methods: Twenty eyes of 10 patients (9 males, 1female; mean age:
24 years) with AD as well as 14 eyes of 7 age and sex matched healthy control subjects were
recruited. All patients had atopic keratoconjunctivitis (AKC).None of the patients or controls
had any other ocular or systemic disease. All patients had the same treatment including
topical antiallergic and steroids. Subjects underwent corneal sensitivity measurements,
Schirmer test -I, BUT measurements, Rose-Bengal and fluorescein vital stainings, upper
tarsal conjunctival impression and brush cytology(BC). Informed consents and ethic board
review for the procedures were also obtained. Impression cytology (IC) specimens were
evaluated by PAS and immunohistochemical staining employing MUC 1 and 4 antibodies.
BC specimens obtained from upper tarsal conjunctiva were studied for expression of
inflammatory cells and with Real Time RT PCR to assess the MUC 1 and 4 mRNA expression.
Results: The mean corneal sensitivity and BUT values were significantly lower
in atopic eyes which also had higher staining scores, compared to control eyes (
p<0.001). BC revealed significantly higher numbers of inflammatory cells in atopic
eyes . IC showed significant goblet cell loss and squamous metaplasia in eyes with
AKC compared to control eyes. Real Time RT PCR showed decreased MUC 1 and
4 mRNA expression in eyes with AKC. Immunohistochemistry staining for MUC 1
and 4 in eyes with AKC showed a scanty staining for mucin compared to control eyes.
Conclusions: The inflammatory process, tear instability and decreased MUC 1 and 4 mRNA
expression were thought to be important factors in the pathogenesis of ocular surface disease
in atopic dermatitis.
CR: M. Dogru, None; N. Okada, None; N. Asano Kato, None; A. Igarashi, None; M.
Tanaka, None; Y. Takano, None; K. Fukagawa, None; K. Tsubota, None; J. Shimazaki,
None; H. Fujishima, None.
Support: JSPS Grant 02261
941 - B915
940 - B914
Quality of Life in Rhinoconjunctivitis Sufferers and the Effects of Topical Ocular
N.MacDonald1, P.J. Gomes1, M.B. Abelson1,2, W.Berger3, M.Beck4, S.Kimura5,
T.Westbrook6, W.Storms7, S.Galant8. 1Ophthalmic Research Associates, North Andover,
MA; 2Schepens Eye Research Institute and Harvard Medical School, Boston, MA;
Southern California Research, Mission Viejo, CA; 4Asthma & Allergy Associates,
Miami, FL; 5Cordova Allergy, Pensacola, FL; 6Allergy & Astma Center of NW Florida,
Pensacola, FL; 7Asthma & Allergy Associates, PC & Research Center, Colorado Springs,
CO; 8Clinical Trials of Orange County, Inc., Orange, CA.
Purpose: To analyze impact on quality of life (QOL) in rhinitis patients when topical ocular
therapy is added to systemic and/or nasal medication regimens. Signs & symptoms of
allergic rhinoconjunctivitis impact patients’ QOL, affecting work and school performance.
Medication choice can inf luence degree of QOL improvement in these patients.
Methods: Environmental, multi-center, open-label, crossover study design was used in
this 3-visit 4-week study. 2 QOL instruments, the RQLQ and ACQLQ served as evaluatory
criteria at visit 1 (baseline) and visits 2 & 3 (evaluation). Patients had prior diagnosis of
rhinitis and were on stable doses of prescribed rhinitis treatment (nasal and/or systemic),
which was continued throughout study. No patients were prior users of prescription ocular
allergy therapy. Paired t-tests compared changes from baseline for RQLQ and ACQLQ.
Results: Rhinitis patients experienced benefit with addition of topical ocular therapy (N=200). Prior
medication regimens consisted of systemic antihistamine (AH) (~50%), AH & nasal spray (33%), or
nasal spray alone (16%). Clinically & statistically significant improvements in all 5 domains of ACQLQ
were observed following the 2 week period of added ocular therapy (P<0.001). The largest improvements
were evident in eye symptoms (1.39 unit change). Correlations betwen RQLQ and ACQLQ in
overlapping domains did exist, with the highest correlation in the Eye Symptoms category (R=0.89).
Conclusions: Allergic rhinitis patients experienced significant improvements in quality of life with
addition of topical ophthalmic medication; this was most evident in ocular parameters of the RQLQ
and ACQLQ, but was also evident globally, and in nasal symptom categories. The ocular component
of the disease, and the broad surface area the ocular surface provides for the collection of allergens
should not be overlooked when prescribing therapy regimens.
CR: N. MacDonald, None; P.J. Gomes, None; M.B. Abelson, Alcon Laboratories F; W. Berger,
None; M. Beck, None; S. Kimura, None; T. Westbrook, None; W. Storms, None; S. Galant,
Support: None.
942 - B916
Managing Symptoms of Allergy and Dry Eye in Patients With Keratoconus
C.V. Sundarraj1, G.N. Foulks2. 1Opthalmology Department, UPMC Eye Center, Eye and
Ear Institute, Univ. of Pittsburgh School of Medicine, Pittsburgh, PA; 2Opthalmology
Department, University of Louisville, Louisville, KY.
Purpose: Although many of the keratoconus patients successfully wear rigid gas
permeable (RGP) lenses for several years without any discomfort, recently we have
noticed an increased number of keratoconus patients whose allergic symptoms of
itching, redness, tearing, chemosis in combination with strings of mucous need
intervention. Differentiation between dry eye patients and those who suffer from
allergy is important for management of allergy and dry eye keratocounus patients.
Methods: Patients with allergic symptoms were distinguished from dry eye patients by
meibomian gland function, tear break up time, fluroescein staining and Schirmer test.
We have reported (CLES, 2004) the advantages of piggy back lenses in combination
with RGP lenses for keratoconus and post corneal transplant surgery (PK) patients
in maintaining the best visual acuity.Recently, (ICER, 2004) we reported the use of
Restasis, ( Cyclosporine A, 0.05%) in some of the piggy back contact lens wearers who
exhibit dry eye syndrome. Treatment with Patanol (olopadine)or Alamast(pemirolast
sodium) twice a day , prior to insertion of the contact lenses and after removalof the
lenses at the end of the day reduces the allergy symptoms and extends visual comfort.
Results: Among the ten keratoconus patients, five with allergy were treated with mast cell
stabilizers. Three of the keratoconus post-PK patients have mild seasonal allergies and benefit
from this treatment.Specialized contact lenses such as Proclear and Focus Night & Day which
have been used as piggy back lenses in some of these patients have decreased allergy symptoms.
Conclusions: The combination use of Patanol and Alamast, the mast cell stabilizers in the
allergypatients is very useful in management of allergy among the keratoconus patients.
The long term care of the post PK patients depends upon the proper testing to diagnose the
allergy from dry eye patients.
CR: C.V. Sundarraj, None; G.N. Foulks, None.
Support: unrestricted research grants from RPB and Eye and Ear Foundation, Pittsburgh,
Omalizumab Improves Symptoms of Severe Ocular Allergy
P.B. Williams, D.J. Simon, J.D. Sheppard, Jr., F.A. Lattanzio, Jr.. TR Lee Ctr Optical
Pharmacology, Eastern Virginia Medical Sch, Norfolk, VA.
Purpose: The recently FDA approved asthma drug, omalizumab (XolairTM), shows promise in
relieving allergy symptoms. Omalizumab is a humanized FC fragment with murine IgE receptor
binding sites. It forms IgE immune complexes, which interrupt the immune response cascade
that causes allergic symptoms. This project examines the relationship between IgE levels, RAST
titers, and systemic clinical response in patients with severe ocular allergic conjunctivitis.
Methods: Inclusion criteria included elevated IgE and serum RAST titers for at least one antigen
and symptoms consistent with allergic conjunctivitis. Patients were evaluated for ocular allergic
symptoms, topical and oral steroid use, FEV1, and systemic allergic symptoms (asthma, eczema,
rhinitis, itchy or watery eyes, hives, sneezing, and coughing). The patients periodically graded
their allergic symptoms for each symptom on a scale of 0-10 (10 being most severe) and a cumulative
score was calculated such that the most severe allergy symptoms would receive a score of 80.
Omalizumab was administered subcutaneously 1-2 times per month with dose
based on weight and total serum IgE. Each visit included an interval history and
slit lamp exam (SLE), with additional attention to systemic allergic symptoms.
Results: Omalizumab requires from 1-3 months to show effect. Patients with the
highest serum levels of IgE had the highest cumulative allergy score (CAS) at baseline
prior to treatment (range 25 - 51). Those same patients with the highest levels of
IgE showed the most dramatic decrease in CAS (range 4 - 25) with a particularly
striking improvement in ocular and upper respiratory symptoms and SLE findings.
There was also a trend toward decreased dependence on ophthalmic topical steroids.
Conclusions: Preliminary data indicates that in patients with marked serum IgE elevation,
omalizumab shows a proportionate clinical response when compared to patients with lower
serum IgE levels. Drugs with this novel mechanism show promise for severe ocular surface
allergic disease, atopic keratoconjunctivitis, and concomitant systemic allergic morbidity.
CR: P.B. Williams, None; D.J. Simon, None; J.D. Sheppard, Jr., None; F.A. Lattanzio,
Jr., None.
Support: institutional funds
943 - B917
944 - B918
Ocular Reactions During Aspirin Challenge in Aspirin-Exacerbated Respiratory
M.H. Friedlaender1A, P.K. Mehra1B, G.Siuzdak2, D.D. Stevenson1B. AOphthalmology,
Allergy, 1Scripps Clinic, La Jolla, CA; 2Mass Spectrometry Laboratory, The Scripps
Research Institute, La Jolla, CA.
Purpose: Clinical grading of ocular reactions during aspirin challenges is problematic.
We attempted to objectively evaluate these reactions using new methodology.
Methods: Six patients with aspirin-exacerbated respiratory disease (AERD) were studied
at baseline and at the time of aspirin challenge using a symptom score, photography, and
measurement of conjunctival erythema with spectroradiometry. Tear samples were collected on
Schirmer strips and labeled using ProlyticaTM reagent (Stratagene) with either heavy (O18) or
light (O16) isotopic tags. LC separation was performed on a laser pulled 100 um ID C18 column.
The MS/MS analysis was performed on an Agilent LC/MSD Trap ion mass spectrometer.
Results: The ocular rating score showed that from baseline to aspirin reaction,
symptoms of itching, burning, and tearing were highly variable and independent of
each other. Spectroradiometer measurements in 3/6 patients demonstrated a significant
increase in conjunctival erythema. Data obtained from mass spectrometry of tears was
searched with Mascot using the NCBInr data base. this resulted in the identification
of 10 proteins such as lacrimal proline-rich protein 4, lactoferrin, and prolactininducible protein. This proteomics profiling strategy helped identify several proteins
that were expressed differentially at baseline and at the time of aspirin challenge.
Conclusions: The ocular reactions during aspirin challenge are clinically variable and
molecularly complex. Protein profiling of tears provides quantitative characterization of
protein expression in AERD.
CR: M.H. Friedlaender, None; P.K. Mehra, None; G. Siuzdak, None; D.D. Stevenson,
Support: None.
Clinical Efficacy and Preference of Visine-A® and Patanol® in Preventing the Signs and
Symptoms of Allergic Conjunctivitis in the Conjunctival Allergen Challenge Model
S.I. Frisch1, H.Druce1,2. 1Medical/Clinical Development-Eye Care/ Tobacco Dependence/
Upper Respiratory, Pfizer Consumer Healthcare, Morris Plains, NJ; 2New Jersey Medical
School, University of Medicine and Dentistry of New Jersey, Newark, NJ.
Purpose: To evaluate efficacy of Visine-A® as compared to Patanol® in prevention of the
signs and symptoms of allergic conjunctivitis (AC) induced by the conjunctival allergen
challenge (CAC) model and to determine subject preference using a forced choice query.
Methods: This was a prospective, randomized, double-masked, contralateral, active and placebocontrolled study of asymptomatic subjects with a history of AC. In the CAC model, the allergen
solution and dose required to elicit a positive allergic response were determined for each subject
at visit 1 and confirmed at visit 2, with treatment evaluation taking place at visit 3. Subjects were
randomized to receive one of the following at visit 3: Visine-A (Vis)/Patanol (P), Vis/Placebo
(Pl) or P/Pl. CAC was performed 10 minsutes following instillation of masked study medication.
Ocular itching, redness (conjunctival, ciliary, and episcleral), chemosis, and lid swelling
assessments were taken at 7, 12, and 20 mins. post-CAC at visits 2 and 3. At the end of the visit,
subjects were asked which eye treatment they preferred in terms of overall efficacy. Results:
A total of 83 subjects completed this study. Both actives had lower mean itching scores than Pl
(clinically and statistically significant at all time points). Vis had lower mean redness scores than
Pl (clinically and statistically significant in all vessel beds at all 3 time points). P had lower mean
redness scores than Pl (clinically significant in ciliary at 7 mins; statistically significant for all
vessel beds at all time points). Both Vis and P had lower mean chemosis and lid swelling scores than
Pl (p<0.05 at all time points). When compared to each other, there were no clinically significant
differences in any parameter between Vis and P. Vis had lower mean redness scores (p<0.05 at
9/9 timepoints) and lower chemosis scores (p<0.05 at 2/3 time points) than P. P had a lower mean
itching score (p<0.05 at 1/3 time points) than Vis. No statistically significant differences were seen
between Vis and P in lid swelling scores. Most subjects (57.1%) in the Vis/P group preferred Vis.
Conclusions: Visine-A was more effective than Patanol (at majority of assessments) and placebo
in the prevention of the signs and symptoms of acute AC when administered 10 mins. prior to onset
of the reaction. Efficacy data generated from the CAC model are generally recognized as the basis
to support ocular allergy treatment indications.
CR: S.I. Frisch, Pfizer Consumer Healthcare E; H. Druce, Pfizer Consumer Healthcare E.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 933-947 / B907-B921
139. Ocular Allergy/Conjunctivitis Organizing Section: CO
945 - B919
Evaluation of the Efficacy of an Association of Hyaluronic Acid and Ginkgo Biloba
Extract (TRIUM® Eye Drops) in the Treatment of Allergic Conjunctivitis
V.Russo1, A.Stella1, M.Marchetti2, G.Luciani2, N.Delle Noci1. 1Institute of Ophthalmology,
University of Foggia, Foggia, Italy; 2University of Camerino, Biosooft, Camerino, Italy.
Purpose: Many research studies have shown that the dry Ginkgo Biloba extract
(GbE) ha s a nt i-i n f la m mator y, a nt iox id a nt a nd neu roprot e ct ive prop er t ies.
Hyaluronic acid (HA) is well known for its mucomimetic and cytoprotective properties.
A r e c e n t l y p r o p o s e d e y e d r o p s f o r m u l a t i o n (T R I U M ® e y e d r o p s,
I t a l y)
( 0 .15 % ) .
The objective of this study has been to test the efficacy and tolerability of this association in
the treatment of allergic conjunctivitis. Methods: We assessed 40 patients of both sexes
(26 males and 14 females), more than 18 years old, suffering from allergic conjunctivitis.
At the basal control, after one week of wash-out, we observed the symptomatology,
we carried out the systemic laboratory tests (skin reaction, PRIST and RAST) and we
carried out specific eye tests (conjunctival scraping and cytology of the lacrymal fluid).
For inclusion in the study all the patients had to give their informed consent.
For the evaluation of the symptoms we used a semiquantitive scale with a score from 0 to 4 for the
signs (hyperaemia, chemosis and secretion) and from 0 to 3 for the symptoms (hitching, photophobia,
burning sensation and lacrymation). The posology of the GbE-HA association was 2 drops per eye 3
times a day. We carried out two controls: at basal conditions and at the end of the treatment (after 15
days). The statistical analysis was carried out using the non-parametric Wilkoxon’s test. Results: At
basal conditions all the patients had conjunctival hyperaemia, 50% had secretion, 25% had conjunctival
chemosis, whereas 20% of the patients had a papillary reaction. All the patients reported hitching,
whereas photophobia was present in 87.5% of the cases, lacrymation in 90% and burning in 77.5%.
We found the treatment to be effective in reducing all the symptoms with a statistically
significant difference (p<0.001). Hitching went from a score of 2.21 to 0.3; photophobia
from 1.61 to 0.34; burning from 0.98 to 0.07; lacrymation from 1.10 to 0.15.
The objective examination showed a decrease in hyperaemia (score from 1.33 to 0.8; p<0.05), secretion
(score from 0.7 to 0.34; p<0.05) and chemosis. The speed of healing was found to be good in 81.3% of the cases.
No topical side effects ascribable to the GbE-HA association were reported. Conclusions: Analysis
of the data showed a statistically significant decrease in the symptoms and signs ascribed to allergic
conjunctivitis, hence suggesting a positive pharmacological effect from the combination of GbE
and HA.
CR: V. Russo, None; A. Stella, None; M. Marchetti, None; G. Luciani, None; N. Delle Noci,
None. Support: None.
946 - B920
Objective Measurement of Human Conjunctival Erythema by Spectroradiometry
After Instillation of Irritants
L.Sharf, D.Breshears, M.H. Friedlaender. Ophthalmology Research, Scripps Clinic, La
Jolla, CA.
Purpose: To objectively measure conjunctival erythema before and after instillation
of irritant solutions. Methods: Conjunctival erythema was measured objectively
with a spectroradiometer in 8 normal human subjects before and after instillation of
1:10 dilutions of pH balanced and non-pH balanced shampoos. Erythema of the bulbar
conjunctiva was determined using the u’ coordinate of the 1976 CIE chromaticity
scale. Measurements were recorded 5, 10, and 15 minutes after instillation of irritants.
Results: Conjunctival erythema, measured by u’, increased after instillation of both pH
balanced (2.4%) and non-pH balanced (8.2%) shampoo. The greatest amount of erythema
was detected at the 5 minute interval. The increase in erythema was greater for nonpH balanced shampoo than for pH balanced shampoo at all time intervals (p<0.05).
Conclusions: Increased conjunctival erythema can be measured objectively with
spectroradiometry after instillation of irritant solutions.
CR: L. Sharf, None; D. Breshears, None; M.H. Friedlaender, None.
Support: None.
947 - B921
Using a Neural Network to Study a Screening Questionnaire for Ocular Allergy in
D.A. Goulart1A, M.A. Tacla1A, A.Paranhos Jr.1B, P.M. F. Marback1A, D.Sole1C, D.Freitas1A,
E.H. Sato1A. ACorneal and external diseases, BGlaucoma, CPediatrics, 1Unifesp Paulista
School of Medicine, São Paulo, Brazil.
Purpose: To evaluate sensibility and specificity of a screening questionnaire
for ocular allergy using an artif icial neural network and to elaborate a
primary artificial neural network to be useful for other future screenings.
Methods:Observational transversal study including 2 groups: [1] 48 from our Corneal
and External diseases sector (subject); [2] 54 healthy children from “Escola Paulistinha de
Educação without signs of ocular allergy (control). Questionnaire was applied to their mothers
or tutors and was composed by eight questions about allergic ocular disease based on data
from our sector and seven items about systemic allergy from the validated questionnaire of the
International Study of Asthma and Allergy Steering Committee (ISAAC). An artificial neural
network was created to identify the most important and to exclude possible redundant items. The
study objective was to predict ocular allergy diagnosis as less as possible amount of questions.
The model of network used was the multilayer perceptron with the algorithm backpropagation.
Results: It was possible to develop an artificial neural network using only seven of usual
fifteen questions (Q5, Q7, Q9, Q10, Q11, Q14 and Q15). The remaining questions did not
add any information to the model. The most important question in this particular neural
network was number five, in a manner that if this item was missed, the error achieved 0,634.
Conclusions: The artificial neural network was very useful on eliminating unnecessary and
redundant items of our questionnaire. Just one question may be enough to sort the patients
more prone to present ocular allergy in a population survey.
CR: D.A. Goulart, None; M.A. Tacla, None; A. Paranhos Jr., None; P.M.F. Marback,
None; D. Sole, None; D. Freitas, None; E.H. Sato, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 948-965 / B922-B939
140. Pterygium Organizing Section: CO
948 - B922
949 - B923
Recurrent Pterygia With Cilia
V.V. Mootha1, A.Suarez2. 1Dept of Ophthalmology, UT Southwestern Med Ctr, Dallas, TX;
Dept of Ophthalmology, University of New Mexico, Albuquerque, NM.
Purpose: To report a series of patients with recurrent pterygia with cilia previously unreported.
Methods: Clinical presentations of six patients (seven eyes) with recurrent
pterygia and overlying cilia were reviewed. Slit-lamp photos were obtained.
Results: A total of six patients were included in the study. Seven of these eyes were noted
to have recurrent pterygium with overlying cilia. Mean age of the patients was 59 years,
ranging from 46-70 years. All of the pterygia with cilia were nasal except in one eye with
a temporal pterygium after childhood surgery. Three patients had bilateral pterygia but
only one of these patients had overlying cilia bilaterally. Four of the patients had had their
primary pterygium excision with amniotic membrane grafts by VVM. Histopathology
evaluation of the tissue submitted was consistent with pterygium in these four cases.
Conclusions: Recurrent pterygia may rarely have overlying cilia. In the nasal cases, the
authors speculate that contiguous caruncular tissue which normally has cilia may be dragged
by the residual base of the pterygium.
CR: V.V. Mootha, None; A. Suarez, None.
Support: Research Prevent Blindness
Ultraviolet Photography to Detect Early Sun Damage in the Eyes of School-Aged Children
J.-L.Ooi1,2, N.Sharma1,2, M.Oakey3, P.Dawes3, D.Papalkar1,2, S.Sharma1,2, M.T. Coroneo1,2.
Department of Ophthalmology, Prince of Wales Hospital, Sydney, Australia; 2The University of
New South Wales, Sydney, Australia; 3Medical Illustration Unit, UNSW, Sydney, Australia.
Purpose: Ophthalmohelioses, or sun-related eye conditions, pose a significant problem to the eye
health of the Australian community. It is not currently possible to detect early changes of disease
due to chronic ocular sun exposure before they are clinically evident on slit-lamp examination.
We aimed to develop a method to detect preclinical changes of ocular sun damage
using ultraviolet f luorescence photography (UVFP) and to investigate the sensitivity
of our system compared to standard photography. We also aimed to determine
the age at which changes become detectable and the prevalence in children.
Methods: We developed a photographic system using UVF to detect evidence
of early ocular sun damage. This concept is based on the established technique
of UVF in the detection of dermatological diseases resulting from UV exposure.
Significant advances were made including refining the UV light source, ensuring portability
and utilising digital technology to allow a greater depth of field compared to film.
The UVF system was initially tested on established pingueculae. We then screened
71 children aged 3 to 15 from schools in Sydney, Australia. UV and standard (control)
photographs were taken of the nasal and temporal interpalpebral regions of each eye.
Results: Established pinguecula consistently fluoresced using UVFP, demonstrating the ability
of our method to detect ocular UV damage. High quality, reproducible images were obtained.
Of the 71 children studied, 23 (32%) had changes on UVFP. Importantly, of these,
16/23 (70%) had changes seen only on UVFP; 7/23 (30%) had changes that were
also detected on control. Also, no changes visible on control photographs were
undetected by UVFP. Thus UVFP was considerably more sensitive than control.
Changes became detectable on UVFP from 9 years of age. Prevalence increased
with chronological age, such that the presence of changes was 0/27 (0%) for children
aged 3-8, 6/23 (26%) for those aged 9-11 and 17/21 (81%) of those aged 12-15.
Conclusions: We hypothesise that the areas seen to fluoresce on UVFP but not detectable on control
photographs represent precursors for clinical conditions such as pinguecula and pterygium. We
have developed the first UVF photographic system to be used successfully to detect such changes,
which may occur years before the development of clinical manifestations.
CR: J. Ooi, None; N. Sharma, None; M. Oakey, None; P. Dawes, None; D. Papalkar, None; S.
Sharma, None; M.T. Coroneo, None.
Support: Rebecca L. Cooper Foundation grant
950 - B924
951 - B925
Comprehensive Allelotyping of Human Pterygium
P.C. Lu1, L.S. Wu2, P.Wu1. 1Ophthalmology, Chang Gung Memorial Hospital, Pu Tz,
Taiwan Republic of China; 2RD, Vita Genomics Lab., Taipei, Taiwan Republic of China.
Purpose: To search for specific alterations in human pterygiae and understand the molecular
events leading to the pathogenesis of human pterygiae by examination of loss of heterogenesity
(LOH) in human pterygiae.Methods: Forty eight pairs of samples were collected from 48
patients from the department of ophthalmology , Chiayi Chang Gung Memorial Hospital,
Taiwan. For those who have bilateral pterygiae, only one eye will be used in the study.
The pterygium specimens were obtained by excision. We matched pterygium tissue and
corresponding peripheral blood cell (PBC) DNA samples by using the ABI PRISM Linkage
Mapping Sets MD-10 (400 makers) and PCR. Data of genotypes are scored for LOH using
Gene Mapper (ABI) software.Results: The highest percentage of LOH was found for a locus
in 4q35 (STR marker D4S426, 17%, 5 in 30 informative cases) This region was not reported in
previous studies and nearby the FAT and caspase 3 tumor suppression genes. The other aberrant
locus is 13q14 (STR marker D13S153, 7%, 3 in 45 informative cases) which region close to
RB gene. This result should help in identifying new genes whose loss of function contributes
to the development of pterygium. Conclusions: LOH in pterygium are less than that in other
tumor. Chromosome abnormality may not most important cause for pterygium. DNA repair
genes polymorphism or mutation should be considered as a major genetic factor.
CR: P.C. Lu, None; L.S. Wu, None; P. Wu, None.
Support: CMRPG62002
Conjunctival Autografting Combined With Low-Dose Mitomycin C for Prevention of
Primary Pterygium Recurrence
F.Orucov, A.Solomon, F.Raiskup, M.Ilsar, J.Frucht-Pery. Ophthalmology, Hadassah
University Hospital, Jerusalem, Israel.
Purpose: To compare the clinical outcome of pterygium surgery combining
intraoperative mitomycin C (MMC) with a free conjunctival autograft, with three
other surgical methods of pterygium surgery including intraoperative MMC
alone, conjunctival autograft alone, and bare sclera without adjunctive treatment.
Methods: One hundred and twenty patients underwent pterygium excision surgery. These
patients were divided into 4 treatment groups. In group 1 (30 patients) 0.2 mg/ml MMC was
applied for three minutes. In group 2 (30 patients) conjunctival autografting was performed.
Group 3 (30 patients) received 0.9%NaCl only leaving the sclera bare, and group 4 (30 patients)
underwent conjunctival autografting combined with one minute application of 0.2 mg/ml MMC.
Results: Pterygium recurred in 2 patients (6.6%) in group 1, in 6 patients (13.3%) in
group 2, in 14 patients (46.6%) in group 3 and in none of the patients in group 4. Chisquare analysis revealed a significantly lower recurrence rate when group 4 was
compared with group 2 (p=0.038) and with group 3 (p<0.0001). Epithelialization of the
wounds was complete within 14 days of surgery. No complications were demonstrated
in any of the study groups except one case of minor melting of the flap in group 4.
Conclusions: This study indicates that pterygium excision with a free conjunctival autograft
combined with intraoperative low-dose MMC is a safe and effective technique for prevention
of recurrence of a primary pterygium.
CR: F. Orucov, None; A. Solomon, None; F. Raiskup, None; M. Ilsar, None; J. FruchtPery, None.
Support: None.
952 - B926
953 - B927
Primary Pterygia Removal With Conjunctival Autograft, Cauterized Punctal
Occlusion, and Temporary Tarsorraphy
D.R. Hardten, A.M. Fahmy. Ophthalmology, Minnesota Eye Consultants, Minneapolis,
Purpose: To evaluate the clinical outcomes and postoperative symptoms of patients after primary
pterygia excision with conjunctival autograft, punctal occlusion, and temporary tarsorrhaphy.
Setting Minnesota Eye Consultants, Minneapolis, Minnesota.
Methods: In this retrospective study, forty-four eyes with primary pterygia were treated by
five physicians using different methods. Variations in treatment plans after excision included
use of AmbioDry (OKTO Costa Mesa, California) dehydrated amniotic membrane allograft,
conjunctival autograft, punctal occlusion by cautery, temporary tarsorrhaphy, and post operative
medical treatment regimen. The mean follow-up period was 6.53 months (range 1-13 months).
Results: Mean best corrected visual acuity (BCVA) results showed significant improvement from
0.65 preoperatively to 0.90 postoperatively. Cases treated with the combination of conjunctival
autograft, punctal occlusion by cautery, temporary tarsorrhaphy, and aggressive postoperative
lubrication resulted in significant long-term patient comfort and very low recurrence rate. There
were no intraoperative complications yet one patient treated with an AmbioDryTM allograft
developed corneal and scleral melt with the need to apply a patch graft and temporary tarsorrhaphy.
Conclusions: Utilizing the treatment combination of conjunctival autograft, punctal occlusion
by cautery, temporary tarsorrhaphy, and aggressive postoperative lubrication results in good
patient comfort and low incidence of recurrence of primary pterygia.
CR: D.R. Hardten, None; A.M. Fahmy, None.
Support: None.
Amniotic Membrane Patch Reduces Pain and Recurrence Rate After Pterygium
J.Kim, J.Lee, Y.Song, Y.Kwon, M.Shin. Ophthalmology, Yongsan Hosp Chung-Ang Univ,
Seoul, Republic of Korea.
Purpose: Bone marrow-derived stem cells (BMSCs) involved in wound healing via both
systemic and local chemotactic factors after pterygium removal and pain acts as a trigger
signal. Temporary amniotic membrane patch (TAMP) could reduce pain and recurrence rate.
Methods: Post-operative pain and recurrence rate in TAMP (n=62) and bare sclera
excision groups (BS, n=57) were compared over 12 month period. Substance-P (SP), a
pain-related neuropeptide, and related VEGF and SCF were measured both in serum and
tear using ELISA and migrating CD34 + and c-kit+ mononuclear cells (MNCs) by flow
cytometry. BMSCs and SP absorbed in TAMP were confirmed by immunohistochemistry.
Correlation between chemotactic factors and migrating BMSCs were analyzed statistically.
Results: In TAMP group, 30.6% of patients experienced a foreign body sensation or
mild pain with marked decrease in serum SP whereas 94.7% complained of severe pain
with markedly increased SP in BS group (pain: p<0.05, SP: p<0.05). The systemic and
local VEGF, SCF showed a similar pattern to SP in serum ( p<0.05; VEGF, p<0.01;
SCF, respectively). In contrast to BS group, CD34 +MNCs count was not increased in
TAMP group after surgery (5.19% p=0.62 VS. 22.3%, p<0.05, respectively). Decreased
systemic SP and CD34+MNCs showed a close correlation in TAMP group showed close
relationship (r=0.91, p<0.05). BMSCs infiltration was confirmed with removed TAMP.
Conclusions: Clinically, these results have led to a markedly lowering of recurrence rate in
TAMP group (TAMP: 3.2%, BS: 15.8%, p<0.05, respectively). TAMP might be an effective
procedure for preventing recurrence of pterygium.
CR: J. Kim, None; J. Lee, None; Y. Song, None; Y. Kwon, None; M. Shin, None.
Support: Stem Cell Grant SC13132
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 948-965 / B922-B939
140. Pterygium Organizing Section: CO
954 - B928
955 - B929
956 - B930
957 - B931
958 - B932
959 - B933
“Sutureless” Pterygium Surgery, an Alternative Method of Fibrin Sealant Application
M.J. Gallardo, D.Johnson, F.Trujillo, T.Starck. Ophthalmology, University of TX HSC San
Antonio, San Antonio, TX.
Purpose: Fibrin glue, the product of mixing fibrin sealant and thrombin, has been shown
to be effective for adhering conjunctival grafts in pterygium surgery. The use of the
fibrin sealant alone, without using the thrombin simplifies the technique of “sutureless”
pterygium surgery. The purpose of this review is to determine the conjunctival autograft
(CAG) stability following pterygium excision that were secured solely with fibrin sealant.
Methods: Seventeen patients with clinically significant pterygium underwent pterygium excision
and harvesting of CAGs. The CAGs were placed onto the scleral bed maintaining limbal orientation.
The fibrin sealant component of the tissue adhesive [Tisseel VH Fibrin Sealant (Baxter Healthcare
Corporation, Glendale, CA)], was infused beneath the CAG and adjacent conjunctival margins without
thrombin. All grafts adhered within 60 seconds. At the end of the procedure, patients were given
subconjunctival injections of cefazolin and dexamethasone, and the eyes patched with neomycinpolymixin-dexamethasone combination ointment. Post-operatively, patients were treated with
topical antibiotics until fully epithelialized and a tapering dose of prednisolone acetate for 3 months.
Results: Between March 2004 and November 2004, 28 eyes of 28 patients underwent
pterygium surgery with fibrin sealant application. No sutures were used in 17 eyes of 17
patients. Of these, 16 involved primary pterygia and 1 a double pterygium. Mean limbal
dimension of the pterygia was 5.9 mm (range 4.5-8.0 mm). Mean corneal extension was
3.84 mm (range 1.0-5.4mm). Mean autograft size was 22.27 mm 2 (range 20-32.5mm 2). Mean
follow up was 11.25 weeks (range 5-19 weeks). In these 17 cases, there were no episodes
of CAG dehiscence, retraction, infection, allergic reaction, or pterygium recurrence.
Conclusions: Tisseel VH Fibrin Sealant is a tissue adhesive composed of two components: fibrin
sealant and thrombin. Both are either combined with a dual injector syringe upon application,
or one component is applied to the scleral bed and the second to the conjunctival graft. Upon
mixing, the resultant solution provides immediate adhesion of the graft onto the scleral bed that
resists subsequent manipulation. Use of the fibrin sealant alone simplifies application since the
solution is less viscous and allows several seconds for graft manipulation. In our series, we had
no cases of graft loss or dehiscence suggesting that the fibrin sealant component alone provides
sufficient adhesive strength for autograft surgery. Given our limited follow-up, no Conclusions:
can yet be made regarding recurrence rates.
CR: M.J. Gallardo, None; D. Johnson, None; F. Trujillo, None; T. Starck, None.
Support: Research to Prevent Blindness
Sutureless Conjunctival Transplantation With Tisseel VH Fibrin Sealant
V.Panday1A, S.B. Hannush1B. 1Wills Eye Hospital, Philadelphia, PA; BCornea Service,
Wills Eye Hospital, Philadelphia, PA.
Pur pose: To repor t the t wo -yea r results of sut u reless conju nct ival
transplantation using Tisseel VH fibrin sealant at the time of pterygium excision.
Methods: Thir ty-nine eyes with primar y pter ygium under went lamellar
keratectomy for excision of the lesion. A free conjunctival autograft was
then placed and secured using Tisseel VH fibrin sealant, without sutures.
conju nctival
t ransplants
fol low-up
per iod
(1-2 3
mont h s) .
Conclusions: Tisseel VH fibrin sealant is as effective as sutures for securing conjunctival
transplants after pterygium excision. It has several advantages over traditional suturing
techniques including decreased operative time, less post-operative patient discomfort, less
inflammation, and more rapid healing.
CR: V. Panday, None; S.B. Hannush, None.
Support: None.
Histopathological Differences Between Primary and Recurrent Pterygium
H.Obata1, T.Usui2, T.Tsuru1. 1Department of Ophthalmology, Jichi Medical School,
Tochigi, Japan; 2Department of Ophthalmology, University of Tokyo School of Medicine,
Tokyo, Japan.
Purpose: Pterygia have been considered to originate from pingueculae. However, most
pterygia develop in the nasal bulbar conjunctiva close to the limbus, while pingueculae
can occur on both the nasal and temporal sides. To explore the pathogenesis of pterygium,
histopathological differences between primary and recurrent pterygium were examined.
Methods: Samples of primary (n=37) and recurrent (n=8) pterygial tissues were taken from
pterygium surgery with free conjunctival transplantation. Tissues were placed on filter paper to
avoid shrinkage before fixation. Tissues were subsequently cut into halves along the long axis of
the pterygium, then embedded in paraffin. Sections were stained using hematoxylin and eosin,
and Elastica van Gieson (EVG). Elastotic degeneration and basophilic degeneration, which
are known as characteristic findings in pingueculae, were examined under light microscopy.
Results: In 35 of 37 primary pterygia (94.6%), both elastotic degeneration and basophilic
degeneration were present, indicating that most primary pterygia display the same
histopathological characteristics as pinguecula. However, all 8 recurrent pterygia displayed
abnormal elastic fibers and hyalinized connective tissues, with no basophilic degeneration.
Conclusions: The present results suggest that abnormal elastogenesis and dysfunction of
barriers in the limbus might be involved in the pathogenesis of pterygium, in addition to the
presence of pinguecula.
CR: H. Obata, None; T. Usui, None; T. Tsuru, None.
Support: None.
Pterygium Recurrence After Excision and Conjunctival Graft Fixation With Tissue
R.M. Portela, S.Stoleru, S.Butrus, H.Peracha, M.Helfgott. Ophthalmology, GeorgetownWashington Hospital Center, Washington, DC.
Purpose: Evaluation of pterygium recurrence rate and patient satisfaction after
surgical excision with autologous autograft transplant using fibrin tissue adhesive
Methods: A retrospective chart review of twenty-five patients that had undergone pterygium
excision and autologous conjunctiva transplant with tissue adhesive between the months
of April 2004 and August 2004 was performed. Patients who had proper post operative
follow up for a total of three months were selcted. Total of ten eyes of ten patients were
included in the study. Pterygium recurrence rates and patient satisfaction were analyzed.
Results: Nine of the ten patients evaluated were satisfied with the cosmetic outcome and
denied any discomfort. One patient had pterygium recurrence (> 1mm of corneal invasion)
in the area of the graft and was not satisfied with the cosmetic appearance. This patient had
more glue applied at the time of surgery compared to the others. Nine out of ten had no
recurrence in the area of the graft and were satisfied with the cosmetic outcome. Three of the
nine presented a small regrowth of fibrovascular tissue inferiorly and at the margin of the graft.
These were not causing symptoms of discomfort and were only evident on slit lamp magnification
Conclusions: Tissue adhesive is a proper alternative for graft placement during pterygium
surgery with advantages of less surgical time and patient discomfort. Although tissue adhesive
has become more popular, pterygium recurrence rates after surgery have not been documented.
One of our patients showed recurrence in the area of the graft. Recurrence may be accounted by
the improper alignment of the graft with the native conjunctiva and /or the excess of adhesive
placed in the bed. Despite surgical success, some small fibrovascular strands may find alternate
routes and grow towards the cornea, as was seen in three patients. They are not considered
recurrences since they are were not visually or cosmetically significant (less than 1mm of corneal
invasion) and were not causing discomfort. This may have occurred secondary to any possible
gaps between the graft and the native conjunctiva. Successes rates may vary depending on the
proper placement of the graft and amount of adhesive applied therefore, surgical experience
add proper techniques are necessary. Long term follow up needs to be assessed.
CR: R.M. Portela, None; S. Stoleru, None; S. Butrus, None; H. Peracha, None; M. Helfgott,
Support: None.
Efficacy and Safety of Pterygium Excision With Mini-Conjunctival Graft Combined
With Amniotic Membrane Graft
D.J. Agriantonis, D.S. Chu. Ophthalmology, UMDNJ- NJMS, Newark, NJ.
Purpose: To determine the efficacy and safety of pterygium excision with a
mini-conjunctival autograft coupled with amniotic membrane graft (AMG).
Methods: We conducted a retrospective chart review of consecutive patients treated for pterygium
between April 2001 and October 2004 at our institution. The pterygium was excised from the
ocular surface in conventional manner. Then the conjunctival defect was covered with amniotic
membrane. The membrane was secured with interrupted sutures. A mini conjunctival graft
harvested from the limbus, usually superior, was secured on the globe over the amniotic membrane.
Charts were reviewed for any intraoperative or postoperative complications and recurrences.
Results: 16 eyes of 16 patients (19 pterygia total) underwent pterygium excision with miniconjunctival graft combined with AMG. Nineteen pterygia were excised with the following
distribution: 12 eyes nasal, 1 eye temporal, 3 eyes both. Eight of these were recurrent while
11 were primary. The average patient age was 56.8 years old. No intraoperative complications
were noted. Postoperative complications consisted of one case of symblepharon and one
case of slipped AMG. The procedure was successful in 17 pterygia (89.5%) with a lack
of recurrence at follow-up that ranged up to 31 weeks with a mean follow-up of 13 weeks.
Recurrence of 2mm or more occurred for 2 of the pterygia (10.5%) during this follow up period.
Conclusions: Pterygium excision with combined mini-conjunctival autograft and AMG is
a safe and effective technique in the management of pterygium.
CR: D.J. Agriantonis, None; D.S. Chu, None.
Support: None.
Doxycycline’s Effect on the Angiogenic Potential of Pterygial Epithelial Cells
C.A. Cox1, A.Martinez2, T.W. Reid3, N.Dushku4, C.Jaworski1, M.John-Aryankalayil2, D.A.
Carper1. 1Section of Molecular Therapeutics, National Eye Institute, Bethesda, MD;
National Cancer Institute, Bethesda, MD; 3Ophthalmology and Visual Sciences and Cell
Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX;
Kaiser Permanente Medical Center, Sacramento, CA.
Purpose: To determine the angiogenic potential of human pterygial epithelial cells and
doxycycline’s effect on neovascularization induced by these cells using the directed in vivo
angiogenesis assay (DIVAA). Methods: The angiogenic potential of fourth passage human
pterygial epithelial cells was compared with basic fibroblast growth factor (bFGF) in Matrigel
for a positive control and PBS in Matrigel for a negative control using DIVAA (approved
NCI animal protocol). In this assay, silicone capsules (angioreactors) containing Matrigel
with PBS, bFGF or 10,000 human pterygial epithelial cells were implanted under the skin
of anesthetized nude mice. After 11 days, fluorescein isothiocyanate-dextran (FITC-d) was
injected into the tail veins of the mice, and ten minutes later they were euthanized and the
extent of neovascularization was quantified using a spectrofluorimeter (excitation 485 nm,
emission 510 nm.). The mean relative fluorescence for six replicates was determined; Student’s
t-test was used to analyze the data. In a separate DIVAA experiment, doxycycline at three
different concentrations was added to the angioreactors in combination with pterygial cells to
determine the effect on angiogenesis. Six replicates were compared for each group as above.
Results: Human pterygial epithelial cells were significantly angiogenic when compared to
Matrigel with PBS (p<0.01) and Matrigel with bFGF (p<0.05). Doxycycline significantly
inhibited angiogenesis in the mid-concentration (500 micrograms/ml) p=0.003, but not in
the lowest or highest concentrations. Conclusions: Human pterygial epithelial cells were
very angiogenic using an in vivo model, and a mid-range dose of doxycycline inhibited
neovascularization in this model. This may eventually be beneficial to patients’ vision by
reducing the growth and/or recurrence of not only pterygia, but other angiogenic ocular
CR: C.A. Cox, None; A. Martinez, None; T.W. Reid, None; N. Dushku, None; C. Jaworski,
None; M. John-Aryankalayil, None; D.A. Carper, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Sunday, May 1, 2:30 PM - 4:15 PM Hall B/C Poster Session Program Number/Board # Range: 948-965 / B922-B939
140. Pterygium Organizing Section: CO
960 - B934
Proliferative Activity and Cholesterol Ester Metabolism in Primary Culture of
Human Pterygium Fibroblasts
M.Galantuomo1A, M.F. Mulas1B, P.Baire1A, C.Abete1C, E.Peiretti1A, S.Dessì1C,
M.Fossarello1A. AOphthalmology, BDepartment of Biomedical Sciences and
Biotechnologies Section of Experimental Pathology, CDepartment of Biomedical Sciences
and Biotechnologies Section of Experimental Pathology, 1University of Cagliari, Cagliari,
Purpose:There is now increasing evidence that pterygium is a tumor-like tissue,
and that cell growth, as well as DNA replication, is closely linked to cholesterol
ester metabolism. Therefore, in the present study, primary cultures of human
pterygium fibroblasts in vitro were utilized to investigate a possible correlation
between cell growth and cholesterol ester metabolism in pterygial formation.
Methods: Primary cultures of pterygium and normal conjunctiva fibroblasts were obtained
from patients classified above grade 3 using micro-dissection surgery and from healthy donors,
respectively. Expression of p53 and Ki-67 oncogenes was evaluated by immunostaining techniques.
Growth kinetic studies were evaluated by growth curve, and 3H-thymidine incorporation.
Cholesterol esterification was evaluated by 14C- oleate incorporated into cholesterol esters.
Results: Pterygium fibroblasts revealed an increased expression of P53 and Ki-67 compared
to normal cells. In addition they grow at faster rate than normal cells. In pterygium fibroblasts,
cholesterol esterification increased as cells progressed from resting to proliferating
phase. These results provide evidence that cholesterol esterification “per se” may be a
limiting factor in determining pterygium cell cycle progression. These Conclusions: are
consistent with the fact that, when fibroblasts are treated with potent inhibitors of cell
growth such as pioglitazone and everolimus, the reduction of DNA synthesis caused
by the drugs is accompanied by an extensive decrease of cholesterol esterification.
Conclusions: This study indicates that pterygium has an altered metabolism of cholesterol
esters, and thus that cholesterol esterification could be a potential pharmacological target
for prevention and treatment of pterygium.
CR: M. Galantuomo, None; M.F. Mulas, None; P. Baire, None; C. Abete, None; E. Peiretti,
None; S. Dessì, None; M. Fossarello, None.
Support: None.
962 - B936
961 - B935
Substance P Induction of Pro-Inflammatory Cytokines in Pterygium
J.J. Y. Chui1, T.Hampartzoumian1, N.Di Girolamo1, M.T. Coroneo2, D.Wakefield1.
Dept of Pathology, University of New South Wales, Kensington, Australia; 2Dept of
Ophthalmology, Prince of Wales Hospital, Randwick, Australia.
Purpose: We hypothesis that neurogenic inflammation mediated by the sensory neuropeptide
substance P (SP), is involved in the pathogenesis of pterygium. Given pterygium is
characterised in part by inflammation and expressed elevated IL-6 and IL-8, we investigate
if pro-inflammatory cytokines expressed in pterygium could result from stimulation by SP.
Methods: SP (10 -10 to 10 -6M) was added to primary epithelial cell cultures established from
pterygium (n=2) or normal limbal (n=1) explants. After 24 hours, cell culture supernatants
were collected and stored at -80oC. The supernatants were assayed for pro-inflammatory
cytokines using the Bio-Plex suspension array system and a human cytokine assay
purchased from Bio-rad. RT-PCR was used to detect IL-8 transcripts on SP stimulated cells.
Results: SP induced secretion of IL-6, IL-8 and MCP-1 from both pterygium and normal limbal
epithelial cell cultures. When normalised to basal conditions, SP induced a minimal 3-fold increase
in IL-6, 14-fold increase in IL-8 and 4-fold increase in MCP-1 in the supernatants of pterygium
epithelial cells. For normal limbal epithelial cells, a 2-fold increase in IL-6, 6-fold increase in
IL-8 and 3-fold increase in MCP-1 was detected in the supernatants. IL-8 mRNA was not upregulated by SP. Pterygium derived epithelial cell lines were more responsive to SP stimulation
as compared to epithelial cells derived from normal limbus when measured by protein assays.
Conclusions: SP induces secretion of the pro-inflammatory cytokines IL-6, IL-8 and MCP-1
in both normal limbal and pterygium epithelial cells in culture. Elevated cytokine secretion by
pterygium epithelial cells in response to stimulation by SP could play a role in the pathogenesis
of pterygium.
CR: J.J.Y. Chui, None; T. Hampartzoumian, None; N. Di Girolamo, None; M.T. Coroneo,
None; D. Wakefield, None.
Support: None.
963 - B937
Tissue Transglutaminase (tTgase) in Native Pterygium and Primary Pterygium
M.Grueterich1, S.Priglinger1, A.May2, K.Eibl1, C.Alge1, A.Kampik1, U.Welge-Lussen1.
Ophthalmology, LMU, Munich, Germany; 2Anatomy, FAU, Erlangen, Germany.
Introduction: Pterygium is characterized by inflammatory fibrovascular overgrowth of
abnormal conjunctival tissue onto the clear cornea. A number of intrinisic and extrinsic
factors have been identified in the pathogenesis of pterygium formation (i.e. UV radiation,
IL-1, 6, 8, TNF-alpha, bFGF, TGF-beta, PDGF and others). Altered extracellular matrix
(ECM) expression and composition appear to be relevant for pterygial stromal changes. In this
context investigation of tTgase expression known to catalyse irreversible cross-linking of ECM
proteins may allow further insite into pterygium pathogenesis and potential future therapies.
Methods: Fresh pterygium specimen (n=3) and primary pterygium body fibroblast cultures
were analyzed for co - expression of tTgase and fibronectin (Fn) as well as tTgase and epsilongama-glutamyl-lysine by immunhistochemistry using confocal laser scanning microscopy.
Pterygium body fibroblast cultures and normal conjunctival fibroblast cultures (n=3 lines each)
were treated with 10ng/mL IL-beta, PDGF-BB, EGF, bFGF, TNF-alpha and TGF-beta2 for
24h. mRNA and protein levels of tTgase were measued by RT-PCR and Western blot analysis.
Results: Fresh pterygium samples showed tTgase expression in the stroma with a
perivascular predominance. In confluent pterygium body fibroblast cultures a network like
pattern of tTgase was identified. The enzyme showed co-expression with Fn and epsilongama-glutamyl-lysine. Expression of tTgase was increased by PDGF-BB and TGF-beta in
pterygium fibroblast cultures but not in normal conjunctival fibroblast cultures as shown
by RT-PCR (2 fold increase for PDGF-BB and 1.5 fold increase for TGF-beta) Western
blot analysis showed similar results. All other substances did not alter tTgase expression.
Conclusions: We were able to demonstrate that tTgase and its reaction product epsilon-gammaglutamyl-lysine are expressed in the stroma of pterygium sections as well as in confluent
pterygium fibroblast cultures. Furthermore an increase of tTgase was induced by PDGF-BB
and TGF-beta, fibroangiogenic growth factors identified in different pterygium cell types.
Intervention in this pathway may allow future approaches in the treatment of pterygium.
CR: M. Grueterich, None; S. Priglinger, None; A. May, None; K. Eibl, None; C. Alge,
None; A. Kampik, None; U. Welge-Lussen, None.
Support: DFG WE 2577/2-1 and Vera & Volker Doppelfeld Stiftung
UVB Activated Pathways in Pterygium Pathogenesis
M.T. Coroneo1, N.Di Girolamo2, D.Wakefield2. 1Department of Ophthalmology, UNSW
Prince of Wales Hospital, Sydney, Australia; 2Pathology, University of New South Wales,
Sydney, Australia.
Purpose: A central process in pterygium pathogenesis is thought to be matrix
metalloproteinase (MMP) activation by ultraviolet light (UV) and subsequent MMP activity
against interstitial tissue. A number of MMP’s are involved but MMP-1 is abundantly
expressed in pterygia. The purpose of this study was to identify the pathways responsible
for the enhanced expression of MMP-1 in pterygium epithelial cells (PEC) following UVB
exposure and to determine whether cell-surface receptors transmit the UV stress signal.
Methods: : Immunohistochemical analysis was performed on diseased and normal ocular
tissue. PEC were cultured and exposed to UVB and/or treated with inhibitors of mitogen
activated protein kinase (MAPK), pertussis toxin or an epidermal growth factor receptor
(EGFR) inhibitor. Conditioned medium and cell lysates were analyzed by gelatin zymography,
Western blotting, and ELISA. Total RNA was reverse transcribed and analyzed by PCR.
Results: MMP-1 protein was strongly expressed in pterygium specimens compared to normal
conjunctiva, limbus, and cornea. The UVB-mediated induction of MMP-1 was completely
inhibited following the treatment of PEC with PD98059, a specific ERK1/2 MAPK inhibitor.
SB203580, an inhibitor of JNK and p38 was not able to reduce the production of MMP-1.
UVB radiation increased levels of phosphorylated ERK1/2 in a time-dependent manner and
the addition of PD98059 decreased this induction by at least 12-fold. c-fos transcripts were
detected as early as 2-hrs, returned to basal levels 24-hrs post-UVB and were inhibited by
PD98059 in cultured PEC. Immunohistochemical analysis revealed both total and active/
phosphorylated EGFR in pterygium tissue and in UVB irradiated PEC. Pertussis toxin reduced
the UVB-mediated induction of MMP-1 in PEC by at least 30% suggesting the involvement of
a G-protein coupled receptor and PD153035 (a specific EGFR inhibitor) had a similar effect.
Conclusions: A UV sensitive cell-surface receptor and a specific intracellular signaling
pathway responsible for the enhanced production of a key enzyme that denatures corneal
collagens have been demonstrated. These results advance our knowledge as to how pterygia
may develop and may allow consideration of non-surgical strategies to treat this disease.
CR: M.T. Coroneo, None; N. Di Girolamo, None; D. Wakefield, None.
Support: None.
964 - B938
965 - B939
Expression of Beta-Microseminoprotein in Pterygium
Y.Wong1, J.Chew1, L.Ang1,2, D.T. Tan1,2, R.W. Beuerman1,2. 1Singapore Eye Research
Institute, Singapore National Eye Center, Singapore, Singapore; 2Department of
Ophthalmology, National University of Singapore, Singapore, Singapore.
Purpose: Beta microseminoprotein (PSP94), a prostate associated protein, has been shown to be
up-regulated in both pterygium tissues and the tears of pterygium patients. In this study, we have
examined PSP94 expression by real time PCR, Western analysis and immunohistochemistry.
Methods: Pterygium and conjunctiva (normal control) tissues were snap frozen in liquid
nitrogen immediately upon surgical excision from patients. Total RNA and protein were
extracted for both PCR and Western blotting procedures. Pterygium samples and their
matched conjunctiva samples for immunohistochemistry were embedded in OCT medium and
sectioned longitudinally at 5μm. Custom made rabbit polyclonal antibodies against PSP94 and
cytokeratin 4 (Acris Antibodies GmbH) were used in the immunohistochemistry experiments.
Results: Both real time PCR and Western blot analysis showed that PSP94 was up-regulated
in pterygium. Immunohistochemical staining showed a preferential expression of PSP94
in the pterygium epithelium. Interestingly, the expression of PSP94 was higher in the
posterior part of the pterygium closer to the bulbar conjunctiva compared to the anterior
epithelium growing over the cornea. Further investigation showed that the pterygium
epithelium of the anterior tip, growing over the cornea, stained negative for cytokeratin-4.
Conclusions: Expression of PSP94 in messenger RNA level as well as the protein level was
shown to be up-regulated in the pterygium epithelium compared to the conjunctival epithelium.
Cytokeratin 4, which is expressed in normal conjunctival epithelium and mature corneal
epithelium, was negatively stained in pterygium anterior epithelium. This result indicates
that the anterior epithelium had lost its conjunctival epithelial characteristics. The unique
localization of PSP94 in pterygium epithelium remains to be elucidated.
CR: Y. Wong, None; J. Chew, None; L. Ang, None; D.T. Tan, None; R.W. Beuerman,
Support: Singapore BMRC 03/1/35/19/231
Antibody Profile Studies of Human Pterygium
V.K. Sharma1, S.Kashani2, D.Hopster2, R.Sandhu2, P.A. Hunter2. 1Ophthalmology, kings
college london, London, United Kingdom; 2Ophthalmology, Kings College London,
London, United Kingdom.
P u r p o se : To eva lu at e t he a nt ib o dy prof i le of pt e r yg iu m t iss ue.
Methods: 21 human pterygium tissue and 6 conjunctival controls were stained
using direct immunof luorescence for the different types of antibodies present
Results: An increased amount of IgE and IgA ( in both plasma cells and
perivascular ) was found in pterygium tissue than in the controls. IgG was
present in all specimens but very little IgM was found in the pterygium tissue.
Conclusions: An immunological mechanism, possibly a type 1 hypersensitivity reaction,
may be involved in the pathogenesis of pterygium
CR: V.K. Sharma, None; S. Kashani, None; D. Hopster, None; R. Sandhu, None; P.A.
Hunter, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Ballroom BCD Paper Session Program Number Range: 1077-1083
209. Corneal Optics, Topography and Imaging Organizing Section: CO
1077 - 8:30AM
Mapping LASIK Flap Thickness With High-speed Optical Coherence Tomography
D.Huang1, Y.Li2. 1Doheny Eye Institute, Univ.of Southern California, Los Angeles, CA;
Dept. of Biomedical Engineering, Case Western Reserve Univ., Cleveland, OH.
Purpose: To map LASIK f lap thickness with a high-speed cor neal and
a nter ior seg ment opt ical coherence tomog r aphy (CAS - OCT) system.
Methods: The CAS-OCT prototype operated at 1.3 micron wavelength, 17 micron FWHM axial
resolution in cornea and 2000 a-scan/sec. The cornea is mapped with 8 mm radial lines (256
a-scans) on 4 meridians centered on the apex reflection. The map acquisition time is 0.5 second.
Twenty seven eyes undergoing primary LASIK were studied. LASIK flap was created using either
the Hansatome or the Intralase femtosecond laser. Intraoperative pachymetry was performed
using a 50 MHz ultrasound (US) probe. The LADARWave system was used for ablation. Three
OCT scans were done on each visit, 1 day and 1 week postoperatively. An automated algorithm
was developed to process the OCT images and map flap thickness. The maps were divided
into central (r<1mm), pericentral (r=1-2.5mm) and transitional (r=2.5-3.5) zones for analysis.
Results: The flap interface is best detected in the pericentral zone (r=1-2.5mm). Interpolation
is necessary in the central and transitional zones. The automated flap boundary detection was
accurate in all eyes by visual inspection. The 1 week flap thickness measurements are reported
below in microns. The central flap thickness in 8 Hansatome Z18 eyes was 148+/-17 (mean+/-SD)
microns by OCT and 118+/-18 microns by US. In the 8 Intralase cases with 120 micron setting, it
was 158+/-10 microns by OCT and 160+/-19 microns by US. Eleven cases with other depth settings
were also analyzed. Regional analysis of flap thickness maps showed the flaps are significantly
thinner in the central region compared to the transitional regions in both the Hansatome
and Intralase groups. The repeatability is 7 microns (SD) in central and pericentral zones.
Conclusions: We have developed a method for using high-speed OCT to map LASIK flap
thickness postoperatively. The measurement is non-contact, rapid, and repeatable. Mapping
provides more information than point and profile measurements previously demonstrated.
This could be valueble for the planning of LASIK enhancement and characterization of
microkeratome performance.
CR: D. Huang, Carl Zeiss Meditec F, P; Y. Li, Carl Zeiss Meditec R.
Support: NIH EY13015 and Carl Zeiss Meditec, Inc.
1079 - 9:00AM
Conjunctival Tumors Evaluated by in vivo Confocal Microscopy
M.J. Mackert, D.M. Zapp, A.Kampik, E.M. Messmer. Dept. of Ophthalmology, LudwigMaximilians-University, Munich, Germany.
Pur pose: T he Rostock Cor nea Modul (RCM)/ Heidelberg Retinog raph
(HRTII) system operates with a 670 nm diode laser and therefore allows
visualization of conjunctival st r uct u res in a resolution up to 1 µm.
Methods: Confocal microscopy was performed in 59 eyes with conjunctival lesions
using the RCM/HRTII. Conjunctival disease analyzed included benign lesions (9
pterygia, 9 pingueculae, 1 papilloma, 1 dermoid, 1 lymphangioma, 6 nevi, 11 primary
acquired melanoses-PAM, 3 secondary acquired melanoses) as well as malignant
tumors (6 melanoma, 2 carcinoma in situ-CIN, 3 lymphoma). Between 61 and 551
images (ø 203 images) were obtained of each eye. In 24 of these patients histological
sections of the same lesion were compared with confocal in vivo microscopy.
Results: Pterygia and pingueculae demonstrated typical histological features such as increased
vascularization, derangement of collagen fibers, calcium deposits and rare inflammatory cells.
The papilloma showed multiple fronds of proliferated epithelium surrounding fibrovascular
cores. Pigmented lesions could be differentiated by their typical in vivo microscopic
features: Nevi showed highly reflective nevus cell nests and pseudocyst formation. PAM was
characterized by highly reflective dendritic cells, single large atypical cells with prominent
nucleoli and clumps of pigment in the conjunctival epithelium as well as pigment dispersion
into the corneal epithelium. Melanoma exhibited multiple large, partly highly reflective cells
with unusually prominent nucleoli and large tumor vessels. CIN lesions were acanthotic with
highly reflective keratinized superficial cells, atpyical epithelial cells and intraepithelial
dendritic cells. Lymphoma could not be visualized due to its subconjunctival localization.
Histology highly correlated with the images obtained by in vivo confocal microscopy.
Conclusions: In vivo confocal microscopy using the RCM/HRTII allows direct imaging of
conjunctival lesions and is able to differentiate between benign and malignant conjunctival
CR: M.J. Mackert, None; D.M. Zapp, None; A. Kampik, None; E.M. Messmer, Heidelberg
Engineering GmbH R.
Support: None.
1081 - 9:30AM
Multiphoton Imaging of Porcine Eyes
H.-Y.Tan1A,2, S.-W.Teng1B, J.-L.Peng1C, H.-H.Lin1B, H.-Y.Wu1B, W.Lo1B, Y.Sun1B, W.-C.Lin3A,
S.-J.Lin3B, C.-Y.Dong1B. AInstitute of Medical Engineering, BPhysics, CLife Science,
National Taiwan University, Taipei, Taiwan Republic of China; 2Ophthalmology, Chang
Gung Memorial Hospital, TaoYuan, Taiwan Republic of China; APathology, BDermatology,
National Taiwan University Hospital, Taipei, Taiwan Republic of China.
Purpose: To demonstrate the feasibility of using multiphoton microscopy in imaging ocular surface.
Methods: The porcine eyes used for imaging were immersed in PBS buffer for
viewing. The home-built multiphoton microscope used for this study was constructed
from a commercial upright microscope (Nikon E800). Using a 40x, NA 0.8
water immersion objective (Fluor), the 780 nm photons from a titanium sapphire
laser were used to induced multiphoton autof luorescence and second-harmonic
generation(SHG) signals from different positions and depths of porcine ocular surface.
Results: Without extrinsic f luorescent molecules, we were able to image the
ocular surface. The cornea and limbal epithelial cells (autof luorescence), as
well as the well-organized corneal stromal collagen fibers (SHG) are visible
. The imaging results were compatible with the histological mor phology.
Conclusions: We demonstrate an excellent imaging of porcine ocular surface using
multiphoton induced fluorescence and SHG signals, without the introduction of extrinsic
fluorescent molecules. With additional developements, multiphoton microscopy may in future
be applicated for in vivo investigation of ophthalmologic pathologies.
CR: H. Tan, None; S. Teng, None; J. Peng, None; H. Lin, None; H. Wu, None; W. Lo,
None; Y. Sun, None; W. Lin, None; S. Lin, None; C. Dong, None.
Support: NSC 93-3112-B-002-033 (national science council, Taiwan)
1078 - 8:45AM
Ultrasound Determination of Anterior and Posterior Corneal Curvature
R.H. Silverman, J.Ellison, D.J. Coleman. Ophthalmology, Weill Medical College of
Cornell University, New York, NY.
Purpose: Arc-scan very high frequency ultrasound (VHFU) allows visualization of the entire
cornea in a single image. Because of the relatively small difference in speed of sound between the
normal saline coupling medium and the cornea, and because the arc-scan maintains approximate
normality of the beam axis relative to the corneal surface, refraction is negligible, facilitating
measurement of posterior curvature. Our aim was to use the Artemis 2 high-frequency ultrasound
system to acquire scan data on test objects of known radius and the corneas of human subjects,
with comparison of ultrasound determined anterior radii with manual and automated keratometry.
Methods: We developed software for measurement of anterior and posterior corneal radii of curvature
for successive zones from 3 to 10 mm in diameter. After calibration with an 8 mm radius glass sphere,
radii of 10 human subjects (20 eyes) were determined. In each case, five scans were acquired and analyzed
in both vertical and horizontal planes, allowing determination of repeatability. Ultrasound determined
anterior curvature values were then compared with manual and automated optical keratometry.
Results: Standard deviations of successive ultrasound curvature measurements were 0.20 mm and 0.16 mm for
anterior and posterior corneal surfaces respectively. Root mean square errors for mean ultrasound determined
anterior radius of curvature versus automated and manual optical keratometry were 0.12 mm and 0.11 mm,
respectively. The root mean square difference between automated and manual keratometry was 0.075 mm.
Conclusions: Ultrasound determination of corneal radius of curvature is based on tracking the corneal
surface in a scan plane and fitting a circle to the contour. Although it takes only a fraction of a second
to scan a single plane, microsaccades may introduce uncertainties in curvature measurements. Our
findings show greater variation in curvature measurements between ultrasound and optical keratometry
than between automated and manual optical methods. However, because arc-scan ultrasound is largely
unaffected by refraction, posterior corneal curvature values are readily determined with the same
accuracy as anterior curvature. The ability to measure corneal thickness and the curvature of both
corneal surfaces can allow improved assessment of corneal power. Because ultrasound also allows
visualization and measurement of Bowman’s layer, it is also conceivable that an ultrasound-based ray
tracing model can be developed based on the thickness, curvature and individual refractive indices of
the epithelium and stroma.
CR: R.H. Silverman, Cornell Research Foundation P; Ultralink, LLC I; J. Ellison, None; D.J.
Coleman, Cornell Research Foundation P; Ultralink, LLC I.
Support: NIH Grants EY01212, EY012320 and Research to Prevent Blindness
1080 - 9:15AM
Non-Invasive Corneal Imaging by Second Harmonic Microscopy
M.Han1, G.Giese2, J.Bille1. 1Kirchhoff Institut fuer Physik, Heidelberg, Germany; 2Max
Planck Institute for Medical Research, Heidelberg, Germany.
Purpose: Second harmonic microscopy is a novel method to non-invasively image the intrastromal
structure of corneal tissue. Since fixation, slicing and labelling are not required, the corneal structure
can be probed under the conditions closest to its physiological states. We are interested to investigate
the micro- and macroscopic structure of collagen fibre in cornea and the influences of temperature,
fixation, dehydration rate and ultrafast laser surgery to the corneal intrastromal structures.
Methods: Excised porcine corneas were probed by a Zeiss multiphoton laser scanning
microscope (LSM 510 NLO). Excited by a commercial mode-locked femtosecond Ti:
sapphire laser (Coherent Chameleon), second harmonic signals generated by collagen fibres
were collected in the transmission direction. Temperature and hydrated rate of corneal tissue
were controlled via a custom tissue chamber allowing real time second harmonic imaging.
The laser ablation experiments was conducted by a home-made all-solid-state Nd:glass
femtosecond laser designed for next generation mini-invasive intrastromal corneal surgery.
Results: Second harmonic microscopy enables high resolution, strong contrast and large sensing
depth corneal imaging. Inside the full thickness of the cornea, the orientations and distribution
of collagen fibres are nicely visualized. The shrinking and swelling of cornea in responding to
temperature, dehydration rate and ultrafast laser ablations can be quantitatively characterized.
Conclusions: Based on the intrinsic properties of collagen, second harmonic corneal imaging is well
suited to non-invasively investigate the intrastromal structures of corneal tissue. As a complementary
imaging modality for laser scanning confocal and multiphoton fluorescence microscopy, second
harmonic corneal imaging is valuable for physiological and pathological corneal studies.
CR: M. Han, None; G. Giese, None; J. Bille, None.
Support: BMBF - FST project
1082 - 9:45AM
Automated Decision Tree Classification of Keratoconus From Videokeratography
M.D. Twa1A, S.Parthasarathy1B, C.Roberts1C, A.Mahmoud1C, M.A. Bullimore1A. ACollege
Optometry, BComputer Science and Engineering, CDepartment of Ophthalmology and
Biomedical Engineering, 1The Ohio State University, Columbus, OH.
Purpose: We describe an application of automated decision tree induction--a quantitative
classification method of machine learning--to facilitate pattern classification of videokeratography
data by quantitative analysis of corneal surface features and compare this approach with established
classification methods. We also compare hold-out and cross validation methods of model error estimation.
Methods: We fit a 7 mm diameter area of corneal surface data with a 7th order Zernike polynomial
for 132 normal eyes and 112 eyes diagnosed with keratoconus. We then induced a decision tree
classifier using the C4.5 algorithm. Model prediction error for the decision tree based classifier was
estimated by both ten-fold cross validation and hold-out methods. Using the decision tree classifier as
the gold standard, we compared the area under the Receiver Operator Characteristic (ROC) curve to
five other classification indices: Rabinowitz McDonnell index (RM), Schwiegerling’s Z3 index (Z3),
Keratoconus Prediction Index (KPI), KISA%, and Cone Location and Magnitude Index (CLMI).
Results: Model prediction error estimates based on ten-fold cross validaion were significantly
less than error estimates by the hold-out method (P = .003). The decision tree based classifier had
significantly greater area underneath the ROC curve (0.93) than other classification methods except
CLMI (0.89; P = 0.30) and Z3 (0.89; P = 0.19), KPI (0.76; P < .0001) , RM (0.72; P < .0001) , KISA%
(0.61; P < .0001). Only 5 of 36 Zernike polynomial coefficients-- C(3,-1), C(0,0), C(3,3), C(2,-2),
C(6,-6)-- were needed to distinguish between normal and keratoconus eyes using our decision
tree classification method with an accuracy of 93%, sensitivity of 90% and specificity of 96%.
Conclusions: Our automated decision tree induction method of corneal shape classification from
Zernike polynomials is an accurate quantitative approach that is interpretable and can be generated
from any instrument platform capable of elevation data output. Cross validation methods are a
desirable method of model error estimation. This generic method of pattern classification is extendable
to other classification problems.
CR: M.D. Twa, None; S. Parthasarathy, None; C. Roberts, None; A. Mahmoud, None; M.A.
Bullimore, None.
Support: NIH EY13359 and AOF Ocular Sciences William C. Ezell Fellowship (MDT); NSF
Career IIS-95515 (SP).
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Ballroom BCD Paper Session Program Number Range: 1077-1083
209. Corneal Optics, Topography and Imaging Organizing Section: CO
1083 - 10:00AM
Estimation of Corneal Scatter by Analysis of Tscherning Images
D.De Brouwere, H.Ginis, K.Giannakoudaki, I.Pallikaris. Medicine, University of Crete,
Voutes, Greece.
Purpose: Corneal scattering is assumed to be a consideral drawback of refractive
surgery. At present, only subjective methods are available to estimate haze
development. The purpose of this study is to get a quantitive relation between corneal
scattering and the broadening of peaks in tscherning images after refractive surgery
Methods: Images of the Wavelight aberrometer were analysed. They can be seen as the
result of a double pass experiment in which light gets scattered twice at the corneal plane,
once localised (way-in) and once non-localised (way-out). This scattering causes broadening
of the incoming laser profile. These profiles were measured before refractive surgery, 1
day, 1 week and 1 month, 3 months and 6 months after surgery. In the scattering model,
only anomalous scattering is taken into account due to the similar optical properties of
activated myofibroblasts observed in confocal microscopy. Analysis was performed in Matlab.
Results: 60 eyes following Lasik, Epi-Lasik or PRK have been examined before surgery,
one day post-op, 1 week post-op one month post-op, 3 months post-op and 6 months post-op.
All spots of the tscherning images have been compared and interpollated to corneal scatter
maps. We observed very low scatter befory surgery, serious increased scatter 1 day until 1
week after surgery. From 1 month until 6 months scatter tends to go back to the initial value.
Conclusions: In this study we correlated the peak broadening of tscherning images of eyes
following refractive surgery with the increased corneal scattering. The results are appropriate
according morphologic changes of the cornea after refractive sugery.
CR: D. De Brouwere, None; H. Ginis, None; K. Giannakoudaki, None; I. Pallikaris,
Support: HPRN-CT-2002-00301
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Ballroom BCD Paper Session Program Number Range: 1203-1209
228. Corneal Genome and Proteome Organizing Section: CO
1203 - 3:00PM
1204 - 3:15PM
Distinctive Gene Expression Profiles of the Mouse Cornea and Discovery of Novel Cornea
M.J. Schumacher, F.Wu, A.Jun, L.Roberts, L.Savino, S.Chakravarti. Medicine, Johns
Hopkins Univ, Baltimore, MD.
Purpose: To elucidate global and differential gene expression patterns of the adult and postnatal day 10 (P10) mouse cornea and discover novel cornea genes, using the tendon and lens as
comparative tissues, to further understand post-natal maturation and maintenance of the cornea.
Methods: Microarray analysis (MG-U74Av2, Affymetrix) was performed on
the adult cornea, P10 cornea, tendon, and lens. Expression data were analyzed by
Microarray Suite v5.0 and dChip2004 software. RT-PCR, Northern blot analysis, and
immunohistochemistry were used to confirm the presence of selected genes in the cornea.
Results: Forty percent of the genes queried (12,488 per microarray) were expressed in at least
one of the four tissue types. Functional clustering of the expressed genes revealed skewed overrepresentation of distinct functional groups in each tissue type; tendon (ECM), lens (crystallins),
adult cornea (biosynthesis and metabolism), and P10 cornea (development). Some of these genes
have known functions in the cornea (Aldh3a1, Col7a1, Muc1, Npy, Pax6), whereas a majority
are currently uncharacterized (Arhgdib, Cx3cl1, Efnb2, Mal, Sfn, Tacstd2, Xlr4). A direct
comparison of the adult and P10 cornea expression data identified nine genes over expressed in
the adult cornea (Cyp24a1, Ca3, Npy, Psca) that may represent genes with a corneal maintenance
function whose proteins undergo rapid turnover requiring a considerable up regulation of their
message levels. In contrast, many more genes are over expressed in the P10 cornea indicative
of collagen build-up and crosslinking (Col1a2, Col3a1, Col4a2, Col8a1, Col14a1, Lox), Tgf-β
signaling (Ctgf, Ltbp4), cell-matrix interaction (Ddr1, Sparc), ECM assembly and remodeling
(Fbn, Timp2), and regulation of transcription (Cri1, Rest). We confirmed the presence of 16
genes by RT-PCR, one gene by Northern blot analysis, and two genes by immunohistochemistry.
Conclusions: Analysis of the adult and P10 cornea revealed consistent and differential gene
expression patterns in the maturing cornea. By comparing genes expressed in the cornea to those
expressed in the lens and tendon, we identified a group of novel cornea genes whose functions
have yet to be fully understood. Knowledge of gene expression patterns in the developing and
mature cornea will help pave the way toward a greater understanding of the forces contributing
to the maturation and maintenance of this remarkable tissue.
CR: M.J. Schumacher, None; F. Wu, None; A. Jun, None; L. Roberts, None; L. Savino,
None; S. Chakravarti, None.
Support: None.
Characterization of Molecular Features and Pathway Profiles for Partially Enriched
Putative Human Limbal Stem Cells by Affymetrix Microarray
D.-Q.Li, Z.Chen, S.C. Pflugfelder. Ocular Surface Center, Cullen Eye Institute, Department of
Ophthalmology, Baylor College Medicine, Houston, TX.
Purpose: The concept that corneal epithelial stem cells reside in limbus has been recognized for more than a
decade, but identification of these stem cells is challenging. This study characterized molecular features
and pathway profiles of partially enriched putative human limbal stem cells by Affymetrix microarray.
Methods: Primary cultured human limbal epithelial cells were selected into three
populations by adhesion to collagen IV: 1) the rapidly adherent cells (RAC) within the first
20 minutes, 2) the slowly adherent cells (SAC) from 20 minutes to 2 hours, and 3) the nonadherent cells (NAC) after 2 hours. Five μg total RNA for each population was used for
Affymetrix microarray with whole human genome GeneChip ® U133 Plus 2.0. The array
images were analyzed using Affymetrix GCOS software v1.2. The gene expression profiling
was analyzed using the R package multcomp, GenMAPP 2.0 and MAPPFinder software.
Results: Based on their phenotypes and growth potential, the RAC, SAC and NAC represent partially
enriched putative stem cells, the transient amplifying cells (TAC) and terminally differentiated cells
(TDC), respectively. The array data from duplicate experiments were highly reproducible (R 2>0.95).
Among 47,000 transcripts on a chip, about 19,500 transcripts (41.6±1.6%, n=6) were expressed
by human limbal epithelial cells. 1958 transcripts, accounting for 10% of expressed genes, were
significantly regulated among these 3 populations. When clustering the 292 genes filtered by 5-fold upor down-regulation, the heatmap showed a unique expression pattern by RAC. There were 192 genes
regulated in the same trend (46 up and 146 down) in RAC compared with both SAC and NAC. These
genes include up-regulated growth arrest-specific 1, collagens, PDGF receptor and kinesin family,
and down-regulated protein kinases, ubiquitin, SPRRs, interleukins and hypothetical proteins. Novel
uses of gene ontology allowed functional validation and pathway profiling. In RAC, only 2 genes,
CDKN1C and CDKN2B, were down- and 17 genes were up-regulated in cell cycle group; while 6
genes down- and 5 genes up-regulated in anti-apoptosis group, when compared with SAC and NAC.
Conclusions: These findings reveal the molecular features and pathway profiles of the partially
enriched putative human limbal stem cells, distinguishable from TAC and TDC. Further
characterization of these exclusive regulated genes may facilitate identification of limbal stem
CR: D. Li, None; Z. Chen, None; S.C. Pflugfelder, None.
Support: NIH Grants, EY014553 (DQL), EY11915 (SCP), RPB Grant, Oshman Foundation,
William Stamps Farish Fund
1205 - 3:30PM
1206 - 3:45PM
1207 - 4:00PM
1208 - 4:15PM
Gene Expression Profiles of Freshly Isolated Mouse Limbal and Corneal Epithelial Basal
M.Zhou1, J.Xu1, T.-T.Sun2, R.M. Lavker1. 1Dermatology, Northwestern University, Chicago,
IL; 2Dermatology, New York University Medical School, New York, NY.
Purpose: It is well accepted that corneal epithelial stem cells are preferentially located in the
basal layer of the limbal epithelium, whereas the corneal epithelial basal layer contains the
transit amplifying cells (stem cell progeny). A lack of specific markers for limbal epithelial
stem cells is a major impediment in their isolation and biochemical characterization. To identify
molecular markers for limbal epithelial stem cells, we have analyzed the gene expression
profiles of limbal and corneal epithelial basal cells that were freshly isolated from cryosections.
Methods: Frozen sections (6 µm) of mouse limbal and corneal epithelia were subjected to laser
capture microdissection (LCM) using a PALM MicroBeam system. As few as 50 limbal and
corneal epithelial basal cells isolated from each mouse (n=6) yielded sufficient material for RNA
extraction, 2 rounds of amplification, biotin labeling, and hybridization to Affymetrix cDNA
GeneChips, which contained ~30,000 mouse genes. Arrays were analyzed using Gene Ontology
and dChip software to determine sample and gene clustering. The criteria used for gene selection
was 2 fold or greater change and a p-value of 0.05, identifying ~ 1000 expressed transcripts.
Results: The LCM method that we employed obviated the need for tissue dehydration prior to
microdissection. This enabled us to isolate quiescent limbal and corneal epithelial basal cells
directly from frozen sections, thereby increasing the yield and quality of RNA. Sixty percent
of the genes were up-regulated, and 40% were down-regulated in limbal basal comparing
with corneal basal cells. Analysis of microarrays indicated that observed changes in transcript
levels could be clustered into groups with specific functions (e.g., cell-cell adhesion, cell
cycle regulation, signal transduction and transcription factors). Some of the up-regulated
genes in limbal basal cells were expected (e.g., enolase), while others had not been previously
characterized (e.g., transmembrane proteins, cell cycle inhibitors and anti-apoptosis genes).
Conclusions: This LCM methodology allows us to obtain gene expression profiles from individual
cells that are not affected by extrinsic manipulations (e.g., tissue processing, dissociation,
culturing and/or cell sorting). Several of the genes that were preferentially expressed in limbal
basal cells have also been reported in epidermal cell populations enriched in stem cells. This
suggests the existence of a “signature” set of genes that may help define epithelial stem cells.
CR: M. Zhou, None; J. Xu, None; T. Sun, None; R.M. Lavker, None.
Support: NIH Grants EY13711, EY06769
Human Corneal Keratocytes: Microarray Analysis of the Phenotypic Shift to Fibroblasts
S.A. K. Harvey, Y.Du, J.L. Funderburgh, E.DeGarmo, N.SundarRaj. UPMC Eye Center,
Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Department of
Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Purpose: Wounding activates the quiescent dendritic keratocytes of the corneal stroma, which
undergo a phenotypic shift to proliferative fibroblasts. Similarly, isolated keratocytes cultured in
defined serum-free medium are activated to fibroblasts on exposure to serum. We used this serumexposure model to compare human keratocyte gene expression with that of matched fibroblasts.
Methods: Keratocytes isolated from four human corneas were cultured with or without serum.
Total RNA was extracted from these 8 independent samples, and processed using appropriate
products (Affymetrix: Santa Clara, CA). First, 35 - 40 ng was used as a source of mRNA for
two-cycle cDNA amplification. The resultant cDNA was subjected to in vitro transcription,
yielding 75 - 100 μg of biotinylated cRNA. Analysis of cRNA on HG-U133 Plus 2.0 GeneChips
characterized 54,675 probe sets, representing 47,000 transcripts and variants. Signal prior
to normalization was 304 ± 63 (mean ± SD, n=8) with 33 ± 2 % of probe sets called present.
Results: We found 810 panels which were consistently changed in the same direction in all four paired
comparisons. Of these the strongest candidates had > 2-fold change for every paired comparison:
120 unique, characterized transcripts were relatively increased in fibroblasts and 45 were relatively
increased in keratocytes. Fibroblast increases included transcripts (HGNC designation) associated
with cell cycle control or mitosis (ASPM, CCNA2, CCNB1, CCNB2, CDC2, CDC20, CDCA8,
CDKN2C, CENPF, also BUB1B, CUL4B, KIF23, MCM5, NEDD9, SKP2, SMC4L1, STK6); altered
extracellular matrix synthesis (COL1A1, COL3A1, COL5A1, COL5A2, COL8A1, EFEMP1, LOX,
LOXL2, TNC); and altered cytoskeletal function (COTL1, FSCN1, MYH10, PFN1, TAGLN, TPM1,
TPM2). Keratocyte increases included presumptive corneal crystallins (ADH1B, ALDH3A1) and
metallothioneins (MT1A, MT1F, MT1G, MT1H, MT1X) which can protect against oxidative stress.
Conclusions: The subset of strong candidates described above includes known or plausible changes
associated with the keratocyte shift to fibroblast; i.e., decreases in crystallins and in an oxidative
stress protection system and increases in systems supporting proliferation and remodeling of the
cytoskeleton and extracellular matrix. A closer review of other candidates, and comparison of the
present study with equivalent published murine data, will further our understanding of corneal
responses to wounding.
CR: S.A.K. Harvey, None; Y. Du, None; J.L. Funderburgh, None; E. DeGarmo, None; N.
SundarRaj, None.
Support: NIH Grants EY08098, EY03263, EY009368, Research to Prevent Blindness, Eye and
Ear Foundation of Pgh
The Matricellular Protein SPARC Is Constitutively Expressed by Limbal Fibroblasts
and Inhibits Intercellular Adhesion of Corneal Epithelial Cells in vitro
S.Shimmura1A, H.Miyashita1B, K.Higa1B, S.Yoshida1B, J.Shimazaki1A, K.Tsubota2.
Department of Ophthalmology, BCornea Center, 1Tokyo Dental College, Ichikawa, Japan;
Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
Purpose:To identify and characterize proteins preferentially secreted by limbal fibroblasts in vitro.
Methods:Human donor limbal and corneal fibroblasts were explant-cultured in DMEM
(15% serum) until confluence, and condition in serum free medium. The supernatant from
each cell was condensed and subjected to proteomic analysis. The predominant protein
identified (SPARC) was further examined by real-time PCR, western blot analysis and
immunohistochemistry of limbal tissue. The effects of mouse recombinant SPARC (10
µg/ml) on cell adhesion and morphology was observed using an immortalized human
epithelial cell line (HCEC) cultured in EpiLife® medium supplemented with 0 to 1 mM Ca 2+.
Results:Proteomic analysis of supernatants revealed greater levels of SPARC, TIMP 2, TIMP 2
precursor, vimentin and alpha 2 pro-collagen secreted by limbal fibroblasts compared with corneal
fibroblasts. Western blot analysis and real-time PCR confirmed the higher levels of SPARC in
cultured limbal fibroblasts. Anti-SPARC staining was strongest in the subepithelial region of the
limbus in immunohistology. Recombinant SPARC (10 µg/ml) significantly inhibited intercellular
adhesion of HCEC in serum-free EpiLife® medium with 1 mM Ca2+ for up to 48 hours (p<0.05).
Conclusions: SPARC secreted constitutively by limbal fibroblasts may regulate cell to cell
interaction in the basal limbal epithelium.
CR: S. Shimmura, None; H. Miyashita, None; K. Higa, None; S. Yoshida, None; J.
Shimazaki, None; K. Tsubota, None.
Support: None.
Gene Expression Profile Studies of Human Keratoconus Cornea for NEIBank: A Novel
Cornea Expressed Gene and the Absence of Transcripts for Aquaporin 5
L.Dong1, Y.S. Rabinowitz2, G.Wistow1. 1Section on Molecular Structure and Function, NEI,
NIH, Bethesda, MD; 2Cornea Genetic Eye Institute, Cedars-Sinai Medical Center, Los
Angeles, CA.
Purpose: To increase the database of genes expressed in human cornea
and to gain insights into the molecular basis of keratoconus (KC).
Methods: A cDNA library was constructed from KC corneas harvested at
keratoplasty and used for expressed sequence tag (EST) analysis. Data were
analyzed using GRIST. Expression of selected clones was examined by RT-PCR.
Results: A total of 7680 clones were sequenced from the 5’ end. After bioinformatics analysis,
4090 clusters of clones, each potentially representing individual genes, were identified.
Of these, 887 genes were represented by more than one clone. The five most abundant
transcripts, represented by more than 60 clones each, were Keratin 12, TGFBI (BIGH3),
decorin, ALDH3, and enolase 1, all known markers for cornea. Many other markers for
epithelial, stromal and endothelial expressed genes were also present. One cluster of 6
clones came from an apparently novel gene (designated ‘KC6’) located on chromosome
18p12.3. RT-PCR of RNA from several human tissues detected ‘KC6’ transcripts only in
cornea. In addition, no clones were observed for the usually prominent corneal epithelial
cell marker aquaporin 5 (AQP5), a water channel protein. Semi-quantitative RT-PCR
confirmed that expression of AQP5 is much lower in KC cornea than in non-KC cornea.
Conclusions: This analysis increases the database of genes expressed in the human cornea
and provides insights into KC. ‘KC6’ is a novel gene of unknown function that shows corneapreferred expression while the suppression of transcripts for AQP5 provides the first clear
evidence of molecular defect identified in KC.
CR: L. Dong, None; Y.S. Rabinowitz, None; G. Wistow, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Ballroom BCD Paper Session Program Number Range: 1203-1209
228. Corneal Genome and Proteome Organizing Section: CO
1209 - 4:30PM
2-D Proteomic Analysis of Cultured Human Corneal Endothelial Cells (HCEC) From
Old and Young Donors
C.Zhu, J.Kurtz, N.C. Joyce. Department of Ophthalmology, Harvard Medical School,
Schepens Eye Research Institute, Boston, MA.
Purpose: To illustrate the similarity and difference in protein expression of
cultured human corneal endothelial cells (HCEC) from different aged donors.
Methods: Five pairs of donor human corneas with ages younger than 30-years-old (<30 yo),
as well as another 5 pairs from donors older than 50-years-old (>50 yo) were obtained from
National Disease Research Interchange and formed 2 age comparison groups. Primary culture
and subcultures were performed following published protocols. Confluent passage 2 cells were
rinsed with PBS to remove residual culture medium. Cell scrapers were used to remove cells
from the culture plates. Harvested cells were centrifuged at 5K rpm for 10 min to form firm
pellets. Bio-Rad Extraction Buffer III (ER3) was added and cells were homogenized for 1-2
min. Soluble proteins were harvested after centrifugation at 36K rpm at room temperature
for 1 hr, then stored at -80°C until further analysis. Five samples from each age group were
pooled and protein concentration determined by a modified Bio-Rad protein assay. 125ug of
protein were loaded onto 17cm IPG strips with different pH ranges for iso-electric focusing
(IEF). Proteins were then separated on19 cm Pre-Cast gels (8-16 % & 10-20% acrylamide)
run at 1440 Vhr. After fixing and staining with Sypro-Ruby, protein spots were imaged by
ProXPRESS, then analyzed using Nonlinear Dynamics PRO Finder 2005 version software.
Results: Comparison of combined samples from the < 30 yo and > 50 yo groups showed similar
protein patterns, as well differences. Analysis of protein separated on pH 3-10 and pH 5-8 IEF
strips showed differences in approximately 10% of the protein spots between the two age groups.
Conclusions: The majority of proteins were similarly expressed in confluent passage 2 HCEC
from both young and older donors. However, age-related differences were also detected in
relative protein levels, as well as in specific protein expression. These findings could lead to
a greater understanding of important functional differences between HCEC from old and
young individuals.
CR: C. Zhu, None; J. Kurtz, None; N.C. Joyce, None.
Support: NEI R01 EY 05767, 12700 (NCJ)
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2016-2049 / B785-B818
260. Dry Eye Diseases: Treatment Organizing Section: CO
2016 - B785
A Comparison of Performance Attributes Between a New Concept Artificial Tear and
Systane® Lubricant Eye Drops
M.T. Christensen1, D.L. Meadows1, J.M. Stein1, M.R. Tudor1, R.P. Stone1, M.B. Abelson2,
C.Michaelson2. 1Consumer Products Clinical, Alcon Labs Inc, Fort Worth, TX;
Ophthalmic Research Associates, North Andover, MA.
Purpose: To evaluate the perfor mance characteristics of a new ar tif icial tear
(Concept Tear) against Systane® Lubricant Eye Drops under acute dosing regimens.
Methods: Two clinical studies were conducted to compare performance attributes of the Concept
Tear and Systane. The Concept Tear is multi-dose with buffer ingredients that work in concert to
provide preservative efficacy without using a traditional preservative. Like Systane, the Concept
Tear contains PEG-400 and propylene glycol as demulcents and HP-Guar as a gelling agent.
Study 1: 20 dry eye patients were enrolled in this single center, randomized, double masked,
two-period crossover study. To be eligible, patients had to have a diagnosis of dry eye and answer
that they needed artificial tears at least “some of the time” due to their dry eye condition. Drops
were administered OU per randomization. Drop instillation comfort (10 point scale), overall
acceptability (10 point scale) and 3 minute blur profile (50 point scale) comparisons were made.
Study 2: 60 patients were enrolled in this single center, randomized, double masked, two periodcrossover study. To be eligible, patients had to have a Tear Film Break-Up Time (TFBUT) < 5 sec
and demonstrate a deficient Ocular Protection Index (TFBUT/Inter-Blink Interval). 40 μL of the
assigned tear were instilled OU per randomization. A masked observer instilled 1μL of sodium
fluorescein and measured TFBUT at 5, 10, 15, 20, 30, 45 and 60 minutes post drop instillation.
Results: Study 1: No statistically significant differences between the Concept Tear and Systane
were seen for drop instillation comfort (Concept Tear mean = 1.0; Systane mean = 1.0), overall drop
acceptability (Concept Tear mean = 1.0; Systane mean = 0.8) or 3 minute blur profile (@ t 0 Concept
Tear Mean = 20.2; Systane Mean = 21.5; both diminishing to < 0.1 @ 3 minutes). Study 2: TFBUT
average at baseline was 2.25. Comparisons between treatments showed that the Concept Tear
and Systane were not statistically different (@ t 5 Concept Tear Mean = 3.8; Systane Mean = 4.4).
Conclusions: These studies demonstrated that the Concept Tear was similar to Systane under acute
dosing conditions.
CR: M.T. Christensen, Alcon Research LTD E; D.L. Meadows, Alcon Research LTD E; J.M.
Stein, Alcon Research LTD E; M.R. Tudor, Alcon Research LTD E; R.P. Stone, Alcon Reseach
LTD E; M.B. Abelson, Alcon Research LTD C, R; C. Michaelson, None.
Support: Alcon Research LTD
2018 - B787
2017 - B786
Treatment of Lid Wiper Epitheliopathy With a Metastable Lipid Emulsion or a
J.P. Herman1, D.R. Korb2, J.V. Greiner3,4, R.C. Scaffidi4, C.A. Blackie2. 1Pittsfield
Eye Associates, Pittsfield, MA; 2Korb Associates, Boston, MA; 3Department of
Ophthalmology, Harvard Medical School, Boston, MA; 4Schepens Eye Res Institute,
Boston, MA.
Purpose: Lid wiper epitheliopathy (LWE), a clinically observable alteration of that portion of the
marginal conjunctival epithelium that wipes the ocular surface during blinking, is diagnosed by
vital staining. LWE occurs with patients presenting with dry eye symptoms, both in the presence
and absence of conventional ocular signs. The purpose of this study was to evaluate the efficacy of
2 treatment regimes for LWE: 1.0 % prednisolone acetate eye drop and a metastable lipid emulsion
eye drop. Methods: Subjects with LWE were divided into two treatment groups: (1) a steroid group
(n=30) treated with 1.0% prednisolone acetate (Pred Forte ®, Allergan, Inc., Irvine, CA) qid x 30 days
and (2) a lipid group treated with a metastable lipid emulsion eye drop (Soothe™, Alimera Sciences,
Inc., Alpharetta, GA) qid x 30 days. Inclusion criteria included: the presence of > grade 1.5 LWE
determined by staining with 2% fluorescein and 1% lissamine green and a symptom score >10 out
of a possible 24 points using a custom symptoms(SPEED) questionnaire (Korb et al: Eye & Contact
Lens 2005) to grade four symptoms. The upper eyelid was everted and lid wiper (LW) staining was
graded as 0 (no staining), 1 (mild), 2 (moderate) and 3 (severe) [Korb et al: Eye & Contact Lens
2005]. After the treatment, examination of the LW and the questionnaire were repeated. Results:
The mean symptom scores prior to treatment were 13.2 points for the steroid group and 12.6 points
for the lipid group, and following treatment 4.9 points for the steroid group and 6.2 points for the
lipid group. The difference in symptom scores following treatment was significant for both groups
(p < 0.01 for the steroid group and p < 0.01 for the lipid group). The mean LWE staining grades prior
to treatment were 2.1 for the steroid group and 1.9 for the lipid group, and following treatment 0.9
for the steroid group and 0.8 for the lipid group. The difference in LWE staining grades following
treatment was significant for both groups (p < 0.01 for the steroid group and p < 0.01 for the lipid
group). Conclusions: Treatment with either 1.0% prednisolone acetate or the metastable lipid emulsion
was effective in diminishing the severity of the LWE and the associated symptoms. These results
suggest a mechanical etiology is associated with LWE. The duration of the decrease in LWE upon
cessation of treatment was not addressed in this study.
CR: J.P. Herman, None; D.R. Korb, Ocular Research of Boston, Inc. P; J.V. Greiner, Ocular
Research of Boston, Inc. P; R.C. Scaffidi, Ocular Research of Boston, Inc. E; C.A. Blackie, None.
Support: Ocular Research of Boston, Inc., and The Walter and Valerie Winchester Research
2019 - B788
Assessment of Signs and Symptoms of Dry Eye After Treatment With Emulsion-Based
Eye Drops
J.G. Vehige, P.A. Simmons, C.Carlisle-Wilcox. Consumer Eye Care R&D, Allergan Inc,
Irvine, CA.
Effects of Lubricants on Ocular Surface Functions
S.-I.Hirai, M.Kawahara, K.Sakamoto, Y.Sugihara, A.Kimura, Y.Horibe, M.Nakamura.
Research and Development Division, Santen Pharmaceutical Co., Ltd., Nara-Osaka,
2020 - B789
2021 - B790
Purpose: In dry eye disease, patient-reported severity of symptoms may not agree with objective
findings; however relief of patient symptoms is an important treatment objective along with
preservation of ocular surface health. In a clinical trial of a new dry eye treatment, specific symptoms
and signs are selected on the basis of the known or proposed mechanism of action of the agent
tested. This study investigates the usefulness of several different subjective and objective variables
used to assess the safety and efficacy of two emulsion-based artificial tears for dry eye treatment.
Methods: A new multi-dose emulsion-based artificial tear formula was evaluated in comparison to
an existing unit-dose emulsion in a parallel, multi-center, randomized, investigator-masked clinical
study. Entry criteria included presence of dry eye symptoms, current
need for artificial tears, and reduced Schirmer or Tear Break-up
(TBUT) scores. After entry, subjects were instructed to use one of
the two formulas as needed, but at least 2 times a day. After 7, 30, 60,
and 90 days, signs and symptoms of dry eye disease were measured.
Results: 158 subjects were enrolled in the study, of which 132
completed the 90-day visit. The figure illustrates the results as %
change from baseline for the multi-dose emulsion (the unit-dose was
similar). By day 90, there were significant decreases in symptoms
of dryness, OSDI score, and conjunctival staining (p<0.001), and
a significant increase in TBUT (p<0.001), with an improvement in
Schirmer score and corneal staining (not shown) at some time points.
Conclusions: Treatment with an emulsion-based artificial tear
produced consistent reduction in symptoms as measured with
two methods. Signs also improved, with TBUT and conjunctival
staining having the most consistent improvement across treatments and time points. This result
is supportive of the proposed mechanism of action of these emulsions, in stabilizing the tear film
through enhancement of the lipid layer. Further, these data stress the importance of selection of
appropriate outcome variables for different types of dry eye treatments.
CR: J.G. Vehige, Allergan E; P.A. Simmons, Allergan E; C. Carlisle-Wilcox, Allergan E.
Support: None.
Efficacy and Compatibility of an HP-Guar Based Lubricant Eye Drop When Used as
Supportive Therapy With a Cyclosporine-Based Ophthalmic Emulsion
K.N. Sall1, S.M. Cohen2, M.T. Christensen3. 1Sall Research Med Ctr Inc, Bellflower, CA;
Stephen M. Cohen, O.D., Scottsdale, AZ; 3Alcon Research LTD, Fort Worth, TX.
Purpose: To evaluate the efficacy and compatibility of two marketed artificial tears in relieving dry
eye signs/symptoms when used as supportive therapy to a cyclosporine based ophthalmic emulsion.
Methods: 60 patients were evaluable by intent-to-treat analysis for this randomized,
investigator masked, parallel study of 6-months duration. Enrollment criteria included
corneal staining of > 3 (NEI grid), Schirmer w/o anesthesia of < 7mm and subjects
had to answer that they needed artificial tears at least “some of the time”. Subjects
were randomized to one of 3 treatment groups. Treatment (Tx)1: Restasis® (0.05%
cyclosporine) BID w/Systane® (PEG 400/propylene glycol w/HP-Guar as a gelling agent)
used a minimum of 1/day as supportive therapy. Tx2: Restasis® BID w/Refresh Tears®
(carboxymethylcellulose) used a minimum of 1/day as supportive therapy. Tx3: Systane
used alone QID. Signs and symptoms were measured at Days -7, 0, 7, 14, 28, 42, 120 and 180.
Results: A statistical difference was seen in favor of Tx1 (Restasis+Systane) vs Tx2
(Restasis+Refresh) for greater reduction in corneal staining (p=0.0048) and a trend (p=0.0725)
for increased TFBUT at 6 months. Schirmer showed non-significant increases from baseline at
6 months: Tx1=1.41, Tx2=2.15, Tx3=1.42 mm. Significant differences were seen in favor of Tx1
vs Tx2 for less frequent ocular burning (p=0.0210), stinging (p=0.0314), grittiness (p=0.0128)
and dryness (p=0.0132). Tx3 (Systane alone) was better than Tx2 (Restasis+Refresh) for
less frequent ocular burning (p=0.0288), dryness (p=0.0480) and scratchiness (p=0.0294).
Conclusions: The choice of artificial tears used as supportive therapy with Restasis has
significant indications for outcome measures. There were significant clinical advantages
with Restasis+Systane vs Restasis+Refresh Tears. There were no clinical or statistical
differences seen between Restasis+Systane vs Systane used alone. Both supportive therapies
were compatible with Restasis.
CR: K.N. Sall, None; S.M. Cohen, None; M.T. Christensen, Alcon E.
Support: None.
Purpose: Certain lubricants increase tear film stability in humans. However, the effects
of such lubricants on ocular surface functions are not well characterized. The effects
of various lubricants on tear film stability and corneal epithelial integrity as well as the
precorneal residence times of these agents were determined in experimental animals.
Methods: All experiments were conducted in accordance with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research. Eyedrops containing 0.1% hyaluronan (HA, 3.74 mPas
), 0.2% hydroxyethylcellulose (HEC, 3.68 mPas), 0.25% hydroxypropylmethylcellulose (HPMC, 3.44
mPas), 0.5% carboxymethylcellulose (CMC, 5.36 mPas), 1.0% dextran 70 (1.11 mPas), or saline (control)
were tested. The corneal surface regularity index (SRI) was determined with a corneal topographic
modeling system as an index of tear film stability after the instillation of eyedrops in anesthetized
rabbits. Corneal epithelial damage was evaluated from fluorescein staining after repeated instillation
of eyedrops in a rat model of dry eye. Precorneal residence time of each eyedrop containing 0.002%
fluorescein sodium as a tracer, was evaluated with an anterior fluorophotometer in anesthetized rabbits.
Results: Instillation of saline, HPMC, or CMC, but not that of HA, dextran, or HEC, in
rabbits resulted in a time-dependent increase in the corneal SRI, representing a decrease
in tear film stability. Instillation of HA, but not that of HEC or dextran, reduced the extent of
fluorescein staining in the rat cornea compared with that observed after instillation of saline.
With regard to precorneal residence time, the area under the fluorescein intensity time curve
from 0 to 30 min (AUC 0-30) for HA or HEC was significantly greater than that for saline.
Conclusions: The lubricants tested manifested different effects on ocular surface functions, with no
correlation apparent among the stabilizing effect on the tear film, amelioration of corneal epithelial
damage, and precorneal residence time. The amelioration of corneal epithelial damage may be due
to a direct effect of each lubricant, whereas the precorneal residence time may be related to eyedrop
viscosity. The effect on tear film stability may be attributable to multiple factors.
CR: S. Hirai, Santen Pharmaceutical Co., Ltd. E; M. Kawahara, Santen Pharmaceutical Co., Ltd.
E; K. Sakamoto, Santen Pharmaceutical Co., Ltd. E; Y. Sugihara, Santen Pharmaceutical Co., Ltd.
E; A. Kimura, Santen Pharmaceutical Co., Ltd. E; Y. Horibe, Santen Pharmaceutical Co., Ltd. E;
M. Nakamura, Santen Pharmaceutical Co., Ltd. E.
Support: None.
Effect of Artificial Tears on Vision in Dry Eye Subjects
W.H. Ridder1, A.Tomlinson2, J.Paugh1. 1Basic & Visual Science, Southern CA Coll
of Optometry, Fullerton, CA; 2Department of Vision Sciences, Glasgow Caledonian
University, Glasgow, United Kingdom.
Purpose: Disruption of the anterior refracting surface of the eye (i.e., the tear layer) reduces
visual performance. Tear layer break up occurs soon after a blink in contact lens wearers and
dry eye patients. This study determined if artificial tears stabilize the tear film and improve
visual performance in contact lens wearers who also exhibit a mild to moderate dry eye.
Methods: Five subjects with complaints of mild to moderate dry eye (due to evaporative dry
eye) during spectacle and contact lens wear were fitted with a Focus Night & Day hydrogel lens
for this study. A temporal, 2-alternative, forced choice paradigm was employed to measure
contrast sensitivity. The stimuli were vertically oriented sine wave gratings (between 0.5 and
14 cpd) presented for 16.67 msecs. The stimuli were presented at two different times after blink
detection: 2 seconds after blink detection (i.e., before tear layer break up) or 4 seconds after tear
film break up. Four conditions were investigated at 4 seconds after tear layer break up; 1) without
artificial tears added, 2) with Clerz2 (Alcon), 3) with Sensitive Eyes (Bausch & Lomb) or 4)
with GenTeal (Novartis) applied. The artificial tears were instilled at 10 minute intervals during
the data collection. The short term visual effects of drop instillation were also investigated by
continually monitoring contrast sensitivity for a 14 cpd grating after a single drop administration.
Results: High spatial frequency contrast sensitivity and visual acuity were found to be reduced
following tear film break up in the absence of supplementation with artificial tears. For the group data
(and 4 out of 5 subjects), the instillation of B & L Sensitive Eyes improved the contrast sensitivity
and visual acuity to the level attained before tear break-up, thus prolonging visual performance.
Clerz2 and GenTeal did not produce any enhancement in visual performance. A short term decrease
in contrast sensitivity was also observed with a single administration of Clerz2 and Genteal.
Conclusions: This study indicates that there was a benefit of B & L Sensitive Eyes tear supplementation
on visual performance in subjects with an evaporative dry eye. This may be due to 1) aqueous
supplementation in these subjects and 2) the minimal tear layer disruption found with B & L Sensitive
Eyes drop administration. The results suggest that practitioners need to identify those patients who
can benefit from the use of appropriate artificial tear supplements.
CR: W.H. Ridder, None; A. Tomlinson, None; J. Paugh, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2016-2049 / B785-B818
260. Dry Eye Diseases: Treatment Organizing Section: CO
2022 - B791
2023 - B792
The Effect of Artificial Tear Therapy on Aqueous Tear Production in Normal
A.Nemi1, J.Kuo2, P.E. Carvounis1, S.Grewal1. 1Department of Ophthalmology, The George
Washington University, Washington, DC; 2The George Washington University School of
Medicine, Washington, DC.
Purpose: A r tif icial tears are routinely recommended for patients with
dry eye syndrome. The goal of this pilot study is to evaluate the effect of
artif icial tear therapy on aqueous tear production in healthy volunteers.
Methods: Fifteen healthy individuals (11 female, 4 male; mean age 30.2, range 2445), without signs or symptoms of dry eye syndrome, participated in the study.
Volunteers were instructed to use artificial tears in both eyes, three times per day.
The Schirmer test, without anesthesia, was performed at baseline and at the end of
weeks 1, 2, and 4. The artificial tears were discontinued after four weeks of therapy.
Results: Mean tear production, as measured by the Schirmer test, without anesthesia, was
14.6 mm (SEM ± 1.96 mm) at baseline, 15.9 mm (SEM ± 2.62 mm) at week 1, 12.2 mm
(SEM ± 1.58 mm) at week 2, and 10.9 mm (SEM ± 2.08) at week 4. Therefore, the mean
decline in tear production from baseline to week 4 was 3.7 mm (p = 0.037, Student’s t-test).
Conclusions: This study suggests that artificial tear therapy may decrease tear production
in normal subjects. This may result from a physiological feedback mechanism or from
preservative toxicity. If the results of our pilot study generalize to patients with dry eye
syndrome, physicians and their patients may need to apply caution regarding overzealous
use of artificial tears as a therapeutic measure.
CR: A. Nemi, None; J. Kuo, None; P.E. Carvounis, None; S. Grewal, None.
Support: None.
Therapeutic Ocular Surface Medium: A Novel Therapy for Moderate to Severe Dry
S.L. Watson1, G.Geerling2, J.K. G. Dart1. 1Cornea and External diseases, Moorfields Eye
Hospital, London, United Kingdom; 2Department of Ophthalmology, University Hospital
Schleswig-Holstein, Campus Lübeck, Lübeck, Germany.
Purpose: To evaluate the clinical efficacy of a new novel therapy, Therapeutic
Ocular Surface Medium (TOSM), for the treatment of moderate to severe dry eye.
Methods: A non-comparative prospective pilot study enrolled 20 eyes of 10 patients, with
moderate to severe dry eye, who had failed standard therapy. Patients with a Schirmer’s
test ≤ 5mm at 5 minutes or tear break-up time ≤ 5 seconds were recruited from Corneal
and External disease clinics at Moorfields Eye Hospital. One drop of TOSM was applied
8 times a day. Patients were assessed at enrolment and at one week, two weeks and four
weeks. Objective signs and subjective symptoms of dry eye were graded at each visit
and slit lamp photographs taken. In addition, conjunctival impression cytology was
performed at enrolment and at the final visit. The Wilcoxon Signed Ranks test was used
to analyse the outcome measures for the worse eye at baseline (the study eye), except
for safety data, which was analysed using both eyes. Moorfields Eye Hospital Ethics
committee approved the study and informed consent was obtained from all patients.
Results: Significant improvement in rose Bengal staining from baseline to
completion of the trial at 4 weeks was found in 7 of 10 patients in the study eye
(p=0.0104). Subjective symptoms (dryness, FB sensation, discomfort, photophobia)
improved in all 10 study eyes over this period (p= 0.0049). Safety data analysis
showed no significant change in the study or fellow eye for BCVA, IOP, or cataract.
Conclusions: TOSM was effective in improving the subjective symptoms and objective
signs of moderate to severe dry eye in patients who had failed standard therapy. TOSM is
being evaluated in a randomized controlled trial to confirm its potential as a novel dry eye
CR: S.L. Watson, None; G. Geerling, None; J.K.G. Dart, None.
Support: Gustav Nossal Scholarship National Health and Medical Research Council
2024 - B793
2025 - B794
Randomised Control Trial Comparing the Effectiveness of Methylcellusose Eye
Dressings (Geliperm) and Ocular Lubricants in the Prevention of Exposure
Keratopathy in the Critically Ill
D.G. Ezra1A, M.P. Y. Chan1A, L.Solebo1A, A.N. J. Malik1A, M.Healy1B, A.Coombes1A.
Ophthalmology, BIntensive Care Medicine, 1St Bartholomew’s and the Royal London
Hospitals, London, United Kingdom.
Evaluation of Pimecronlimus Tablets 30 mg Bid for the Treatment of Dry Eye in
Primary Sjögren’s Syndrome Patients
M.Tomsic1, M.Gekkieva2, A.Weichselberger2, N.C. Yannoulis2. 1Rheumatology, Univ Med
Ctr Ljubljana, Ljubljana, Slovenia; 2Novartis Ophthalmics, Basel, Switzerland.
Purpose: To evaluate the efficacy and safety of pimecrolimus tablets (30 mg bid) as compared to
placebo on keratoconjunctivitis sicca (KCS) in patients with primary Sjögren’s syndrome (pSS).
Methods: This was a prospective, randomized, double-masked, placebo-controlled,
parallel-group, multi-center proof-of-concept trial. pSS patients with at least moderate
signs and symptoms of KCS were randomized to pimecrolimus or placebo bid for 12 weeks.
Results: Thirty-eight patients (19 pimecrolimus; 19 placebo) completed the 12-week treatment
period according to protocol. For both corneal staining (Oxford Grading Scheme, 0-5 scale)
and the composite ocular symptoms score (average of three ratings 0-4 for sandiness/grittiness,
itching, and burning/pain), greater mean decreases from baseline were observed in the
pimecrolimus group than in the placebo group at all 3 visits where efficacy was assessed.
For the reduction in corneal staining, the mean difference vs placebo at Week 12 was -0.45
score units in favor of pimecrolimus (P=0.2833). For the reduction in composite ocular
symptoms score, the mean difference vs placebo at Week 12 was -0.26 score units, again in
favor of pimecrolimus (P=0.4025). Numerically favorable results with pimecrolimus were
also observed for tear-film break-up time, Schirmer’s test, and the investigators’ and patients’
global assessments of dry eye and pSS conditions. Results of the safety assessments conducted
during the 12-week double-masked treatment period and a follow-up visit performed one year
after discontinuation of therapy, indicate pimecrolimus 30 mg bid was safe and well tolerated.
Conclusions: The results of this exploratory study are suggestive of a favorable treatment
effect with pimecrolimus 30 mg bid on the signs and symptoms of KCS in patients with pSS,
albeit statistical significance was not reached due to the limited sample size of the study.
Further evaluation of the potential effect of pimecrolimus for the treatment of KCS appears
to be warranted.
CR: M. Tomsic, Department of Rheumatology, Univ Med Ctr Ljubljana F; M. Gekkieva,
Novartis Ophthalmics E; A. Weichselberger, Novartis Ophthalmics E; N.C. Yannoulis,
Novartis Ophthalmics E.
Support: None.
2026 - B795
2027 - B796
Purpose: . Microbial keratitis, particularly pseudomonas, has been reported among critically ill
patients and the need for effective eye care in ICU (Intensive Care Unit) has been recognised for
some time. However, there is no clear consensus defining the best form of eye care. A recent survey
in the UK found that 75% of ICUs used Geliperm routinely as eye care, with 25% using ocular
lubricants such as Lacrilube or Lubritears. Although Geliperm is the most widely used eye care
measure on ICU, its efficacy has never previously been adequately assessed. Geliperm was originally
designed as a wound dressing and there is no evidence to support its use in eye protection. This
randomised control trial attempts to provide direction regarding the best practice for ICU eye care.
Methods: This study was conducted in accordance with the Declaration of Helsinki and was
approved by the East London and the City Regional Research Ethics Committee. All patients
admitted to the ICU at the Royal London Hospital were considered for inclusion. Exclusion criteria
included primary orbital injury, or length of stay likely to be less than 48 hours. Each patient
had each of their eyes randomly assigned to either Geliperm or Lacrilube such that each patient
received both treatments concurrently in different eyes. Independent randomisation sequencing and
statistical support was provided by the Wolfson Centre for preventative disease, University of London.
An ophthalmologist examined all eyes of patients using a portable slitlamp. record
was made of grade of exposure keratopathy, chemosis, palpebral aperture, sedation
score, length of stay on ITU, APACHE disability score and reason for admission.
Results: At the time of submission 30 patients have been recruited to the study. This is the
midpoint of the sample size projection and our data thus far show no significant difference
between the efficacies of the two treatments in all grades of exposure keratopathy (p>0.05).
No significant variance in palpebral aperture or conjunctival chemosis was noted between groups. Nor
was significant variance detected in general health indicators such as sedation scores or APACHE scores.
Conclusions: Our preliminary data suggest that both treatments are equally effective. We project that
we shall have the full results of our study in 3 months and will present them in their entirety.
CR: D.G. Ezra, None; M.P.Y. Chan, None; L. Solebo, None; A.N.J. Malik, None; M. Healy,
None; A. Coombes, None.
Support: None.
Efficacy of Cyclosporine a (Restasis) for the Treatment of Dry Eye Symptoms in the
First 30 Days of Therapy
L.A. Herrygers1, R.Noecker2. 1Ophthalmology, Private Practice, Belgrade, MT;
Ophthalmology, University of Pittsburgh, Pittsburgh, PA.
Purpose: To evaluate the effect of topical cyclosporine A (Restasis ® ,
Allergan) on patients’ daily dry eye symptoms in the first 30 days of use.
Methods: Patients (n=10) with a history of at least moderate dry eye symptoms and evidence
of corneal staining were provided printed patient diary forms and asked to record their
symptom severity daily for 30 days. Patients were queried as to how the cyclosporine A
drops felt upon instillation, if their eyes felt better than when they started the drops, and
how many times they thought about their eyes each day. Patients were also asked to rate, on
a scale of 1 to 5 (where 5 = dry eyes greatly impact my activity and 1= dry eyes don’t impact
my activity) how their dry eye impacted their work, outdoor activities, ability/desire to
watch TV, ability/desire to use a computer, ability/desire to read, and ability to drive a car.
Results: Overall, 30 days of cyclosporine A therapy improved patient quality of life
scores in 5 of the 6 measurements. Patient scores improved for working, outdoor activities,
watching TV, reading, and driving. The improvement in ability/desire to participate in
outdoor activities achieved statistical significance at days 9, 12, and 13 (P<.035). Most
patients (67%) reported that their eyes felt better after 30 days of cyclosporine A than before
starting treatment and 60% found cyclosporine A to be soothing or only minimally stinging.
Conclusions: Cyclosporine A is an effective therapy for the relief of patient dry eye
symptoms. Moreover, patients reported improvement in symptoms after only 30 days of
therapy, suggesting that cyclosporine A provides a faster onset of symptom relief than
previously reported.
CR: L.A. Herrygers, None; R. Noecker, Allergan C.
Support: None.
Comparison of Cyclosprine to Punctal Plugs in Relieving the Signs and Symptoms of
Dry Eyes
C.W. Roberts, P.E. Carniglia, B.G. Brazzo. Ophthalmology, Weill Medical College of
Cornell University, New York, NY.
Purpose: To compare the efficacy of cyclosporine ophthalmic emulsion 0.05% to punctual
plugs in relieving the signs and symptoms of dry eyes. Methods:30 patients with chronic
symptoms of dry eye in both eyes (scratchy or burning eyes relieved at least in part by
artificial tears) and conjunctival staining with rose bengal were randomized to one of three
treatment groups. 1. Cyclosporine ophthalmic emulsion 0.05% twice a day; 2. Punctal plugs
to lower lids only; 3. Both cyclosporine and punctual plugs (Combination). Patients were
evaluated at initial visit and then one, three, and six months after initiation of treatment by
Schirmer measurement without anesthesia (three minutes),rose bengal staining of the cornea
and conjunctiva, and patient reporting of the number of times per day they apply artificial
tears. Results: Increased Schirmer measurements: Combination = Plugs > Cyclosprine.
Improvement in conjunctival staining: Combination = Cyclosporine > Plugs. Decreased
frequency of artifical tears: Combination > Cyclosporine > Plugs. Conclusions: While
punctual plugs increase Schirmer measurement of tear production, cyclosporine is more
effective in decreasing the conjunctival staining and need for artificial tears seen in dry eyes.
There is a synergy of these two modes of improving dry eyes such that the use in combination
was the most effective in relieving both the signs and symptoms of dry eyes.
CR: C.W. Roberts, Allergan Pharmaceuticals C; P.E. Carniglia, None; B.G. Brazzo,
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2016-2049 / B785-B818
260. Dry Eye Diseases: Treatment Organizing Section: CO
2028 - B797
2029 - B798
A Novel Episcleral Cyclosporine Implant for Keratoconjunctivitis Sicca
J.P. Dunn1, K.G. Csaky2, H.Kim2, B.C. Gilger3, M.Tremblay2, Monasterio2, G.Tansey2,
P.Yuan4, M.R. Robinson2. 1Ophthalmology, Johns Hopkins University, Baltimore, MD;
National Institutes of Health/National Eye Institute, Bethesda, MD; 3North Carolina State
University, College of Veterinary Medicine, Raleigh, NC; 4Pharmacy/Clinical Center,
National Institutes of Health, Bethesda, MD.
Purpose: Treatment with topical ophthalmic cyclosporine formulations for keratoconjunctivitis
sicca related to Sjogren’s syndrome or graft-versus-host disease is limited by poor tissue penetration
and ocular surface toxicity. We developed a cyclosporine episcleral implant to provide therapeutic
drug concentrations to the lacrimal gland and ocular surface to treat keratoconjunctivitis sicca.
Methods: Episcleral cyclosporine implants were manufactured with a silicone-based
matrix design, and in vitro release rates were performed. Preclinical evaluation included
toxicology (clinical examination, serial electroretinography and histopathology) in
normal rabbits and dogs, pharmacokinetics in normal rabbits, and pharmacodynamics
in a canine model of aqueous tear deficiency and keratoconjunctivitis sicca.
Results:The cyclosporine implants showed sustained release of drug over time with in
vitro assays for 1-year. Histopathology showed normal ocular tissues in both dogs and
rabbits 6-months after implantation. The cyclosporine implant produced lacrimal gland,
corneal, and conjunctival drug levels 1- to 2-log units higher than those reported with a
variety of topical cyclosporine formulations and oral dosing. The cyclosporine implant was
effective in a canine model of keratoconjunctivitis sicca with all animals able to discontinue
topical cyclosporine and maintain normal Schirmer tear scores over a 6-month follow-up.
Conclusions: This preclinical evaluation showed that the episcleral cyclosporine implant was
safe, delivered potentially therapeutic cyclosporine levels to the lacrimal gland, cornea, and
conjunctiva, and showed efficacy in a clinically relevant model of keratoconjunctivitis sicca.
The episcleral cyclosporine implant shows promise in the treatment of keratoconjunctivitis
sicca associated with Sjogren’s syndrome and graft-versus-host disease following allogeneic
hematopoietic stem cell transplantation.
CR: J.P. Dunn, None; K.G. Csaky, None; H. Kim, None; B.C. Gilger, None; M. Tremblay,
None; F. de Monasterio, None; G. Tansey, None; P. Yuan, None; M.R. Robinson, None.
Support: None.
Canaliculitis Secondary to SmartPLUGs
S.Cho1A, I.Raber1B. 1Wills Eye Hospital, Philadelphia, PA; BCornea, 1Wills Eye Hospital,
Philadelphia, PA.
Purpose: To report three cases of canaliculitis caused by SmartPLUGs. The SmartPLUG,
an acrylic punctal plug, was introduced in 2002 and was designed to decrease the potential
for irritation, migration, and extrusion that is often seen with other types of punctal
plugs. There have been no other reported complications associated with these plugs.
Methods: Retrospective case series. Results: All three patients were being treated
for corneal and surface disease by a cornea specialist. In all cases, the SmartPLUG
were chosen secondary to either intolerance of or repeated loss of silicone plugs. The
canaliculitis presented 2 months to 1 year after insertion of the SmartPLUG. Cultures
were performed on one patient and were negative. All cases resolved after flushing out
the plug via nasolacrimal irrigation and treating the infection with antibiotic eye drops.
Conclusions: This is the first report of canaliculitis associated with SmartPLUGs.
CR: S. Cho, None; I. Raber, None.
Support: None.
2030 - B799
2031 - B800
Conjunctival Scar Excision in Severe Ocular Surface Disease
S.C. Wilker, D.S. Bardenstein. Ophthalmology, University Hospitals of Cleveland,
Cleveland Heights, OH.
Purpose:To describe a new treatment option for severe ocular surface
disease in patients with conjunctival scarring associated with graft-versushost-disease (GVHD) following hematopoietic stem cell transplantation.
Methods:A 50-year-old man with chronic myelogenous leukemia in complete remission,
following allogeneic stem cell transplantation, complained of unilateral severe ocular
surface pain. Examination revealed extensive corneal punctate staining in a superior
and central pattern with conjunctival scarring of the upper eyelid in an area overlying
the corneal staining. Conservative treatment with lubrication, topical steroids, punctal
plugs, punctal cauterization and a brief trial of topical cyclosporine provided no relief.
Placement of a bandage contact lens provided sustained subjective relief with improvement
of the corneal surface and vision. He was not, however, able to tolerate the absence of
the contact lens for more than an hour. Based on a presumed mechanical etiology of
the overlying conjunctival scar causing the corneal surface disease, excision of the scar
was offered after informed consent was obtained regarding the risks of operating in the
setting of ocular surface disease. The conjunctival scar was excised microsurgically.
Post-operatively, a bandage contact lens, topical steroids and antibiotics were used.
Results:After healing there was an 80% subjective improvement in symptoms.
Examination showed marked reduction of corneal staining and stabilization of
his vision. Most importantly, dependency on bandage contact lens wear ended.
Conclusions:There may be a mechanical basis for ocular surface disease in the setting of stem
cell transplant and GVHD. In patients with severe ocular surface disease and conjunctival
scarring, simple scar excision may provide benefit from excision of the scar.
CR: S.C. Wilker, None; D.S. Bardenstein, None.
Support: None.
Comparison of Pimecrolimus 1%, 0.3% and 0.1% With Vehicle for the Treatment of
Dry Eye in the Controlled Adverse Environment (CAE) Model
G.W. Ousler1, R.Haque2, A.Weichselberger3, N.C. Yannoulis3, M.B. Abelson4,1. 1ORA, N.
Andover, MA; 2Novartis, E. Hanover, NJ; 3Novartis, Basel, Switzerland; 4Schepens Eye
Inst, Harvard Med School, Boston, MA.
2032 - B801
2033 - B802
Symptomatic Relief of Dry Eye Assessed With the OSDI in Patients Using 5%
Testosterone Cream
C.G. Connor. Optometry, Southern College of Optometry, Memphis, TN.
Purpose: The present study examines symptomatic relief based on OSDI scores from
dry eye patients treated with 5% testosterone cream. Previously in a masked placebo
controlled study we showed that testosterone cream increased tear production in dry eye
patients based on Schirmer values. The OSDI (Ocular Surface Disease Index) appears
to be a valid and reliable instrument for measuring the severity of dry eye disease.
Methods: Patients were enrolled based on a decreased TBUT ( less than 10 seconds)
and accompanying symptoms. These patients were then administered the OSDI.
Then these patients were instructed on how to apply the 5% testosterone cream to
their eyelids twice a day for three weeks. After three weeks of cream use the OSDI
and TBUT were repeated. Twenty patients were included in this study: 17 females
and 3 males. Mean age of the patients was 45.8 yrs. with a range of 22 to 65 yrs.
Results: The baseline TBUT was 4.22 seconds with an OSDI score of 39.7. This
OSDI score corresponds to a dry eye of moderate severity. After 3 weeks of treatment
with the testosterone cream, the TBUT increased to 6.02 seconds and the OSDI score
declined to 20.33. The use of the testosterone cream twice daily reduced the severity of
the treated dry eye patients from moderate to mild based on OSDI scores. None of the
patients showed an increased OSDI score after treatment, and most of the patients had a
dramatically reduced OSDI score. The observed change in OSDI score was statistically
significant at the p=.001 level. The change in TBUT was not statistically significant.
Conclusions: The dramatic reduction in OSDI score suggests treating dry eye patients with
5% testosterone cream twice a day reduces the patient’s perception of dry eye symptoms.
CR: C.G. Connor, None.
Support: None.
Purpose: To compare safety & efficacy of pimecrolimus 1%, 0.3% and 0.1% ophthalmic suspensions
with vehicle for treating dry eye in the CAE model. Methods: Randomized, double-masked,
parallel group, 16-week, single-center study. Dry eye patients entered a 4-week run-in period with
vehicle treatment and underwent screening in the CAE, an environmentally regulated room that
standardizes humidity, temperature, airflow, and visual tasking. Subjects with worsened dry eye
symptoms & corneal staining post CAE were randomized to dose BID with pimecrolimus or vehicle
for 12 weeks. Follow-up CAE exposure and pre & post exposure exams occurred at weeks 2, 4, 8,
12. Results: 105 (1%: 26, 0.3%: 28, 0.1%: 24, vehicle: 27) patients completed per protocol. Mean
differences between pimecrolimus & vehicle for inferior corneal staining (0-4 scale) after 12 weeks
of treatment (primary efficacy variable - inferior corneal staining post CAE) are shown. Negative
values favor pimecrolimus.
Before CAE
P-Value *
Mean Diff. vs. Vehicle
0.046 / 0.095
0.85 / 0.534
0.417 / 0.364
After CAE
P-Value *
Mean Diff. vs. Vehicle
0.055 / 0.102
0.036 / 0.043
0.29 / 0.163
*unstratified / baseline stratified CMH-test
Pimecrolimus 1% results suggested a treatment effect on corneal staining pre & post CAE. The
maximum effect of pimecrolimus 1% occurred after 4 weeks and was sustained for the study. Similar
trends were observed for sum corneal and conjunctival staining pre & post CAE. All treatments were
well tolerated. Conclusions: The data suggest a treatment effect for pimecrolimus in the reduction
of corneal staining under both environmental and adverse conditions. Further evaluation of safety
& efficacy of pimecrolimus for dry eye therapy is warranted. CR: G.W. Ousler, None; R. Haque,
Novartis Ophthalmics E; A. Weichselberger, Novartis Ophthalmics E; N.C. Yannoulis, Novartis
Ophthalmics E; M.B. Abelson, Novartis Ophthalmics F. Support: None.
Evaluation of Ketorolac (Acular LS) During the Induction Phase of Cyclosporine a
(Restasis) Therapy to Improve Patient Comfort
B.A. Schechter1, J.R. Wittpenn2. 1Cornea/External Diseases, Rand Eye Institute, Pompano
Beach, FL; 2Private Practice, Stony Brook, NY.
Purpose: Cyclosporine A (Restasis, Allergan) has been proven to effectively relieve the
signs and symptoms of dry eye. A small percentage of patients, however, experience stinging
with initial use. Since it is imperative to ensure compliance with cyclosporine A therapy to
effectively treat dry eye disease and helpful to provide patients with immediate relief of
symptoms during the induction phase, the purpose of this study was to determine if use
of ketorolac 0.4% (Acular LS, Allergan) improves patient comfort during the initiation
of treatment with topical cyclosporine A for the treatment of chronic dry eye disease.
Methods: Single center, randomized, 6-week, open-label clinical trial. Patients with a history of
dry eye (n=23) were randomized to cyclosporine A monotherapy or to an adjunctive regimen of
ketorolac followed by cyclosporine A instillation 10 minutes later. Study visits were at baseline,
week 2, and week 6. Outcome measures included evaluation of corneal staining, ocular comfort
on a 4-point scale (1= mild, 4=severe), OSDI and Schirmer scores, and tear break-up time.
Results: After 6 weeks of therapy, the concomitant use of ketorolac with cyclosporine A provided
increased ocular comfort compared with cyclosporine A monotherapy. The mean ocular
comfort score of the adjunctive patients improved 2.1 points, compared with an improvement
of 1.3 points for cyclosporine A alone. The adjunctive regimen also provided significantly
greater reductions in corneal staining than cyclosporine A monotherapy, with a mean
improvement in staining of 1.9 with adjunctive therapy, compared with 1.4 with monotherapy.
Conclusions: In this small pilot study, the combination of ketorolac with cyclosporine A
improved the signs and symptoms of dry eye in the induction phase of cyclosporine A therapy.
This induction combination may provide a viable method of increasing compliance and
reducing discomfort in patients during the first few weeks of cyclosporine therapy.
CR: B.A. Schechter, Allergan C; J.R. Wittpenn, Allergan C.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2016-2049 / B785-B818
260. Dry Eye Diseases: Treatment Organizing Section: CO
2034 - B803
In vitro Evaluation of Solutions Prepared From Mucoadhesive Polymers
R.Herrero-Vanrell1A, M.Vicario1A, las Heras1B, A.Rincon1A, I.T. Molina-Martinez1A.
Department of Pharmacy & Pharmaceutical Technology, BDepartment of Pharmacology,
School of Pharmacy,Complutense University, Madrid, Spain.
Purpose: Ocular mucoadhesive systems involve dosage forms containing polymers which
are able to adhere to the mucins present on the ocular surface. Topical treatments of ocular
surface disorders include the use of mucoadhesive polymers, being the ocular surface exposed
to these agents for a long time. The aim of this work was to evaluate in-vitro mucoadhesion
of hypotonic and isotonic solutions containing hyaluronic acid (sodium salt) (HA-Na) and
polyacrlylic acids (PAA) of different molecular weights. Methods: Hypotonic and isotonic
solutions of Hyaluronic acid sodium salt (HA-Na Mw1,3x 106 Da) and polyacrylic acids
(Mw 450,000 and 1,25 x 106 Da) were prepared at concentrations: 0.1; 0.2 and 0.3 % (w/v).
Isotonicity was adjusted with ClNa (HA-Na) or Mannitol (PAA). The vehicle employed was
an aqueous solution of phosphate buffer pH (6,0) being used as reference (AS). Mucoadhesion
measurements were carried out using a tensiometer. The adhesion values obtained for the
formulations (n=5) were expressed as a percentaje of mucin-mucin standard measurements
(100% mucoadhesion). Cell viability assays were performed by the MTT method based on
cellular mitochondrial respiration. Results: High molecular weight PAA rendered the higher
mucoadhesion values (133.21% ± 4.89). HA-Na (0.1%) presented a similar value of mucinmucin adhesion (106.6% ± 10) while the formulation without polymer addition (AS) resulted
in less mucoadhesiveness (87.27%± 3.5). Cell viability was decreased for PAA formulations
depending on the molecular weight of polymer and its concentration. Only treatment with PAA
(1,25 x 106Da) solution resulted in a significant decrease of cellular respiration. No significant
differences were observed between isotonic and hypotonic solutions. Conclusions: In vitro
mucoadhesion properties allow characterizing bioadhesive polymers. Taking into account that
formulations containing mucoadhesive polymers are widely employed to hydrate the ocular
surface, further studies are necessary to evaluate their effects on long- term treatments.
CR: R. Herrero-Vanrell, None; M. Vicario, None; B. de las Heras, None; A. Rincon,
None; I.T. Molina-Martinez, None.
Support: FIS Grant (PI030910) and MCyT Grant (SAF2004-06119-C02-02)
2036 - B805
Lipid Layer Thickness Changes Following the Instillation of Two Novel Lubricant Eye
D.R. Korb1, R.C. Scaffidi2, J.V. Greiner2,3, K.R. Kenyon2,3, J.P. Herman4, C.A. Blackie1,
T.Glonek5, C.L. Case1, V.M. Finnemore1, T.Douglass1. 1Korb Associates, Boston, MA;
Schepens Eye Research Institute, Boston, MA; 3Department of Ophthalmology, Harvard
Medical School, Boston, MA; 4Pittsfield Eye Associates, Pittsfield, MA; 5Midwestern
University, Downers Grove, IL.
2035 - B804
Evaluation of the First Metastable Lipid Emulsion on Symptomatic Dry Eye Patients
J.V. Greiner1,2, T.Glonek3, R.Scaffidi2. 1Department of Ophthalmology, Harvard Medical
School, Boston, MA; 2Schepens Eye Research Institute, Boston, MA; 3Midwestern
University, Downers Grove, IL.
Purpose: To evaluate the first metastable lipid emulsion on patients with signs and symptoms of dry
eye syndrome. Methods: After informed consent, subjects (n=20) presenting consecutively with one
or more of the following dry eye symptoms (dryness, grittiness or scratchiness; soreness or irritation;
burning or watering) and signs of decreased tear-film break-up time (TFBUT) [<10 sec] and lipid layer
thickness (LLT) [<90 nm] were selected. Frequency and severity of symptoms were tabulated and
also quantified using a scored [SPEED (Korb, Herman, Greiner et al.: Eye & Contact Lens, 2005)]
questionnaire; subjects with symptom scores >5 of 24 were enrolled. Statistical comparisons were
computed from the numeric data using the paired t-test (2-tailed). TFBUT was determined using the
DET™ method (Korb, Greiner, Herman: Cornea 2001). LLT was evaluated at 0 (baseline), 1, 5, and 15
min using methods standard in our laboratory (Korb, Baron, Herman et al: Cornea 1994). Symptoms and
signs were evaluated for each subject on day 1 and after using the metastable lipid emulsion (Soothe™,
Alimera Sciences, Inc., Alpharetta, GA) a minimum of twice daily OU for a >1-month duration.
Results: Regardless of the degree of dry eye symptoms, after one month, symptoms improved in all
subjects (Frequency measures: dryness, grittiness or scratchiness, sig=0.000; soreness or irritation,
sig=0.000; burning or watering, sig=0.012. Severity measures: dryness, grittiness or scratchiness,
sig=0.000; soreness or irritation, sig=0.000; burning or watering, sig=0.058. Scored SPEED
questionnaire, sig=0.000). There was a dramatic improvement in LLT over 0 min at the 1, 5, and 15 min
time intervals at both the baseline visit and 1-month visit evaluations (Baseline visit: 0-1 min time interval,
sig=0.000; 0-5, sig=0.000; 0-15, sig=0.000; 1-5, sig=0.024; 1-15, sig=0.012; 5-15, sig=0.350. 1-month
visit: 0-1, sig=0.000; 0-5, sig=0.000; 0-15, sig=0.000; 1-5, sig=0.003; 1-15, sig=0.032; 5-15, sig=0.508).
Conclusions: This new technology, the first metastable emulsion for the treatment of dry eye, proved
highly effective in reducing dry eye symptoms after one month and in increasing the thickness of the
tear film lipid layer within 1 minute of instillation. This is especially important when considering
the correlation between tear film lipid layer thickness and dry eye syndromes (Isreb, Greiner, Korb
et al: Eye 2003).
CR: J.V. Greiner, Ocular Research of Boston, Inc. P; T. Glonek, Ocular Research of Boston,
Inc. P; R. Scaffidi, Ocular Research of Boston, Inc. E.
Support: Ocular Research of Boston, Inc. and The Walter and Valerie Winchester Research
2037 - B806
Multicenter, Randomized, Double-Masked, Dose-Response, Placebo-Controlled,
Parallel-Group Study of the Safety and Efficacy of Rebamipide (OPC-12759) Sterile
Ophthalmic Suspension in the Treatment of Dry Eye
P.C. Donshik1, G.Foulks2, M.Monica3, P.Zhang4, A.Tano5, S.Nakatsu4, S.Bramer4.
University of Connecticut Health, Bloomfield, CT; 2Ophthalmology, University of
Louisville, Louisville, KY; 3Tulane, New Orleans, LA; 4Otsuka Maryland Reseach
Institute, Rockville, MD; 5Otsuka Pharmaceutical Company, Osaka, Japan.
Purpose: Tear film lipid layer thickness (LLT) has been correlated to the presence of dry eye
symptoms. Two novel products, Soothe™ (Alimera Sciences Inc., Alpharetta, GA) and Systane™
(Alcon Laboratories, Inc., Fort Worth, TX), have been introduced for the lubrication of dry eye and
relief of symptoms. This study was conducted to determine if a single eye drop of either product
produced a significant increase in LLT for subjects reporting symptoms indicative of dry eyes.
Methods: A double-blind, internally-paired study was performed. A custom lipid layer interferometer,
enabling characterization of lipid layer interference patterns, was used to quantify LLT (OU) of
eligible subjects. Subjects (n=50) received a single drop of Soothe™ in one eye and a single drop of
Systane™ in the other eye. Test drops were supplied in masked identical opaque eye dropper bottles.
The LLT of all subjects, following the instillation of each test drop, was analyzed. Inclusion criteria
included: 1) presence of dry eye symptoms and 2) LLT < 75 nm, with variability no greater than ± 15
nm in each eye or between an individual pair of eyes, over a 15 minute observation period. Results:
The mean ± standard error baseline LLT findings pre-eye drop instillation were 61.4 ± 1.6 nm for
eyes treated with Soothe™, and 60.3 ± 1.7 nm for eyes treated with Systane™. These means were
not significantly different (p > 0.5). Post-eye drop instillation, the mean LLT for eyes treated with
Soothe™ more than doubled from 61.4 nm to 146.2 ± 4.7 nm, and the mean LLT for eyes treated
with Systane™ increased from 60.3 nm to 83.7 ± 4.8 nm. The mean increase in LLT for Soothe™
was significantly greater than that produced by the weakest measurable blink response (p < 0.0001).
The mean increase in LLT for Systane™ was not significantly greater than that produced by the
weakest measurable blink response (p = 0.07). Conclusions: In subjects with symptoms indicative
of dry eye states and with LLT < 75 nm, one eye drop of Soothe™ resulted in a significant increase
in LLT, more than doubling LLT, while the increase for Systane™ was not significantly greater than
that produced by the weakest measurable blink response.
CR: D.R. Korb, Ocular Research of Boston, Inc. P; R.C. Scaffidi, Ocular Research of Boston, Inc.
E; J.V. Greiner, Ocular Research of Boston, Inc. P; K.R. Kenyon, None; J.P. Herman, None; C.A.
Blackie, None; T. Glonek, Ocular Research of Boston, Inc. P; C.L. Case, None; V.M. Finnemore,
None; T. Douglass, None. Support: Alimera Sciences, Ocular Research of Boston, and Walter and
Valerie Winchester Research Grant
Purpose: To evaluate the safety and efficacy of rebamipide ophthalmic suspension 0.5%, 1% and
2% for the treatment of dry eye. Methods: This was a multicenter, randomized, double-masked,
dose-response, placebo-controlled, parallel-group study that evaluated the safety and efficacy of
multiple doses of Rebamipide ophthalmic suspension instilled into both eyes for 12 weeks. After a
two week run-in period a total of 200 subjects with signs and symptoms of dry eyes were randomized
to 4 treatment groups. The primary objective endpoint was fluorescein corneal staining (FCS).
Secondary objective endpoints included lissamine green conjunctival staining (LGCS) and Schirmer’s
test. The primary subjective endpoint was the subject’s primary ocular discomfort (POD), defined
as the subject’s most bothersome symptom, identified at screening. Severity of individual dry-eyerelated ocular symptoms and the subject’s overall treatment impression at week 12 were secondary
subjective endpoint. Results: Compared to placebo at week 12, subjects taking 2% rebamipide
showed superiority (p-value<0.05) or favorable trend (p-value<0.1) in mean change from baseline
(CFB) for 1)FCS score (p=0.076; p=0.015 at week 6). 2)POD (p=0.044). 3) LGCS (p=0.070). and
4) the individual symptom severity scores of gritty/sandy sensation (p=0.015), burning/pain (p0.003) as well as the overall treatment impression scores (p=0.044). The 2% rebamipide group was
statistically superior to placebo group in mean CFB for Schirmer’s test at Week 2(p=0.002) and
Week 8 (p=0.034). The safety profile of rebamipide was favorable. No deaths or treatment-related
SAE were reported. The incidence of treatment-related eye disorders in the rebamipide treatment
groups was less than the incidence of treatment-related eye disorders in the placebo group. Results
from other safety assessments were not clinically meaningful. Conclusions: All three concentrations
of rebamipide ophthalmic suspension were well-tolerated and efficacy was demonstrated in dry eye
subjects. The concentration of 2% rebamipide appears to be the most effective concentration. There
were no serious safety issues in this trial.
CR: P.C. Donshik, Otsuka Maryland Reseach Institute F, C, R; G. Foulks, Otsuka Maryland
Reseach Institute F, C, R; M. Monica, Ostuka Maryland Reseach Institute F, C, R; P. Zhang, Otsuka
Maryland Reseach Institute E; A. Tano, Otsuka Pharmaceutical Company E; S. Nakatsu, Otsuka
Maryland Reseach Institute E; S. Bramer, Otsuka Maryland Reseach Institute E.
2038 - B807
2039 - B808
Efficacy of an Emulsion Eye Drop in the Management of Mild to Moderate Dry Eyes
S.Khanal, A.Tomlinson, E.I. Pearce. Vision Sciences, Glasgow Caledonian University,
Glasgow, United Kingdom.
Purpose: To determine the efficacy of an oil-in-water emulsion eye drop (EED) in the
management of dry eye compared to a traditional dry eye supplement (0.32% hypromellose)
Methods: A longitudinal, randomised, single-masked and parallel study of the efficacy
of EED and hypromellose solution was carried out. A total of 58 mild to moderate dry eye
patients (29 in each group) were recruited for the study. Patients were enrolled if at least
two symptoms were reported positively in McMonnies Dry Eye Questionnaire together
with one of the following screening tests: non-invasive TBUT (<10 secs to 5 secs) and
Schirmers test (<5 mm to 2 mm in 5 mins). Patients were instructed to use either EED or
hypromellose solutions three times a day for 30 days. Tear production, evaporation, structure
and osmolality were measured at baseline and after 30 days. Compliance tests (non-invasive
TBUT and Schirmers test) was carried out at 7 days from the start of the use of the drops.
Results: A statistically significant decrease in tear evaporation rates with both EED (7.24 ±5.43 g/
m2/h) and hypromellose (2.0 2 ±4.74 g/m2/h) was found after a one month period of use of the drops.
However, the decrease with emulsion was significantly greater than with hypromellose (p<0.001).
No significant changes were seen in tear production and osmolality with either of the drops.
Conclusions: The oil water emulsion was more effective in reducing evaporation of the tears
than hypromellose following chronic application over a 1 month period. This signifies its
potential in the management of evaporative dry eyes.
CR: S. Khanal, None; A. Tomlinson, Allergan F; E.I. Pearce, Allergan F.
Support: None.
Support: None.
Changes in Tear Evaporation Rates After Topical Low-Dose Lipid Eye Ointment
Application to Lid Margin
M.Uchino1, E.Goto1,2, Y.Matsumoto2, M.Dogru1,2, M.Saiki1, K.Fukagawa1, K.Tsubota1.
Ophthalmology, Keio University, Shinjyuku Tokyo, Japan; 2Ophthalmology, Tokyo
Dental College, Ichikawa Chiba, Japan.
Purpose: To evaluate the change of tear evaporation rates after low-dose lipid eye ointment
application to lid margin. Methods: Subjects with dry eye sensations(DE group; 7eyes, 36.0
± 8.3 years old), soft contact lenses user(SCL group; 7eyes, 28.4 ± 6.7 years old), and normal
subjects without dry eye sensation(NE group; 12 eyes, 34.5 ± 8.4 years old) were enrolled in
this study. From commercially available topical lipid drugs, ofloxacin eye ointment(Tarivid,
Santen Pharmaceutical, Co, Japan) was chosen, and applied to lid margin with minimal low
dose. After 5 minutes, the evaporation rates was measured using our new evaporimeter.
Results: After the application, tear evaporation rates decreased significantly in all groups; in DE
group, from 9.1 ± 4.0 to 6.3 ± 3.9 (10 -7g/cm.sec , p<0.05), in SCL group,10.9 ± 5.6 to 4.1 ± 2.8 (10 -7g/
cm 2.sec, p<0.047), and in NE group, 4.8 ± 1.8 to 3.1 ± 2.0 (10 -7g/cm 2.sec , p<0.0001), respectively.
Conclusions:Low-dose ofloxacin eye ointment application to lid margin effectively suppressed
tear evaporation rates from the ocular surface in all subjects groups. This treatment may help
in suppressing excessive evaporative tear loss leading to decrease dryness possibly due to
forming stable lipid layer.
CR: M. Uchino, None; E. Goto, None; Y. Matsumoto, None; M. Dogru, None; M. Saiki,
None; K. Fukagawa, None; K. Tsubota, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2016-2049 / B785-B818
260. Dry Eye Diseases: Treatment Organizing Section: CO
2040 - B809
2041 - B810
2042 - B811
2043 - B812
Topical Eye Ointment Application to Lid Margin for the Treatment of Dry Eye
E.Goto1,2, M.Dogru1,3, Y.Matsumoto1,3, K.Fukagawa1,4, M.Saiki1, M.Uchino1, T.Kojima1,5,
Y.Yamamoto1, M.Kawashima1,3, K.Tsubota1,3. 1Ophalmology, School of Med. Keio
University, Tokyo, Japan; 2Tokyo Dry Eye Center, Iidabashi Eye Clinic, Tokyo, Japan;
Tokyo Dental College, Ichikawa, Japan; 4Ryogoku Eye Clinic, Tokyo, Japan; 5Social
Insuarance Chukyo Hospital, Nagoya, Japan.
Purpose: To assess the efficacy of minimal-dose lipid eye ointment application to lid margin
for the treatment of dry eye. Methods: Sixty two eyes of 31 consecutive dry eye patients were
examined in the sub-specialty clinic in Tokyo Dry Eye Center in Iidabashi Eye Clinic. Four
cases were performed LASIK surgery. Dry eye patients were firstly treated with conventional
topical treatment such as artificial eye drops or steroid eye drops. In the next visit after 2
weeks, DR-1 (Kowa, Nagoya, Japan) tear interfeometry was performed and subjects with thin
precorneal lipid layer were included in the present study. To supply missing lipid on the ocular
surface, from commercially available topical lipid drugs, ofloxacin eye ointment (Tarivid,
Santen Pharmaceutical, Co, Japan) was chosen since it spread well over the cornea to form lipid
layer, and was applied to lid margin with minimal low dose 3 times a day for 2 weeks. Informed
consent was obtained from all the subjects, and IRB approval was also obtained. Dry eye
symptom using visual analogue scale, DR-1 tear interferometry to assess lipid layer thickness,
fluorescein corneal vital staining, tear break-up time (BUT), and meibum expressibility were
investigated. Results: After 2 weeks of treatment, symptom scale scores improved from 79.0
± 38.2 to 38.2 ± 18.3 (P < 0.0001). Precorneal lipid layer thickness increased from 35.0
± 6.1 nm to 74.5 ± 27.8 nm (P < 0.0001). BUT also improved significantly. Conclusions:
Dry eye patients with thin precorneal lipid layer were successfully treated with minimal-dose
ofloxacin eye ointment application to lid margin. DR-1 tear interferometry indicated smooth
spread of eye ointment on the cornea and increase of lipid layer thickness after the application,
accompanied with the improvement of the symptoms. This treatment was effective to the dry
eye with thin surface lipid layer, and may help to suppress excessive evaporative tear loss
from ocular surface leading to decreased dryness.
CR: E. Goto, None; M. Dogru, None; Y. Matsumoto, None; K. Fukagawa, None; M.
Saiki, None; M. Uchino, None; T. Kojima, None; Y. Yamamoto, None; M. Kawashima,
None; K. Tsubota, None.
Support: None.
Microbiology Assessment of a Multi-Dose Preservative Free Tear Product
R.P. Stone, R.A. Rosenthal, S.L. Buck, B.A. Schlech. Consumer Products, Alcon Research
Ltd, Fort Worth, TX.
Purpose:Preservatives are added to products to prevent contamination. Multi-dose
products are required to meet preservative efficacy standards. But sometimes these useful
preservatives cause irratation to the eye, and a preservative-free product may be desirable.
A new preservative free product in a multi-dose container (MDPF product) was developed to
meet these needs. The purpose of this study was to show that a new preservative free product
is capable of controlling and preventing contamination under extreme microbial conditions.
Methods:The new preservative free product was repeatedly challenged with
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans
and Aspergillus niger throughout a 6-month period. Additionally, multiple lots of
the new formulation were stored at elevated and room temperatures for over 6 months.
Results:The results consistently showed that the new product is capable of preventing contamination
of the product. The new product showed greater than a 3 log reduction of the challenge bacteria.
Conclusions:Though the MDPF product contains no preservatives, it is formulated with
a unique blend of ingredients that allow it to maintain efficacy when exposed to extreme
microbial conditions.
CR: R.P. Stone, Alcon Laboratories E; R.A. Rosenthal, Alcon Laboratories E; S.L. Buck,
Alcon Laboratories E; B.A. Schlech, Alcon Laboratories E.
Support: Alcon Laboratories
Dry Eyes Syndrome: Psychovegetative Complaints Influenced by Acupuncture
J.Nepp1, L.Linzmayer2, K.Jandrasits1, G.Schild1, J.Schauersberger1, S.Richter-Müksch1,
A.Wedrich1. 1Ophthalmology, Medical University, Vienna, Austria; 2Psychophysiology,
Psychiatry, Medical University, Medical University of Vienna, Austria.
Purpose: This study was perfor med to evaluate if acupuncture reduce
t he psychovegeat ive t en sion of pat ient s w it h d r y eye sy nd romes.
Methods: In a randomized double blind study patients with dry eyes were observed. The
tension of the sympathetic system was measured by skin contact level, and habituation,
and the pupillary diameter by a TV pupillometer 1050 (whittaker cooperation).
Evaluation of mood and the psychic condition was by means of the Zerssen patient selfrating questionnaire. The tear film was evaluated by slit lamp, Schirmer test(I), break
up time and Rosa Bengal staining. Laser acupuncture and sham-laser were carried out
double blind randomized for 10 weeks. The paired t-test served for statistic analysis.
Results: 50 patients were observed. After acupuncture with the laser the diameter of
the pupil reduced 1,2 average, 0,1 mm in the sham-laser-group. Perspiration reduced
and skin tension values improved from 42,2 to 48 after laser and 37,1 to 37,8 after shamlaser. The emotional well-being improved with a statistically significant difference
between the first and second measurement in the examinations of both groups.
The parameters of dry eyes improved after laser, the difference was significant (p<0,05). RosaBengal-staining and Schirmer tests however, produced no statistically significant difference.
Conclusions: The elevated psychovegetative tension in dry eyes syndrome lowered after
acupuncture, which is well known as a relaxing method.
CR: J. Nepp, None; L. Linzmayer, None; K. Jandrasits, None; G. Schild, None; J.
Schauersberger, None; S. Richter-Müksch, None; A. Wedrich, None.
Support: None.
The Effect of Medium Chain Triglycerides-Containing Tear Substitute on the
Dynamics of Lipid Layer Interference Patterns (DLIP) in Dry Eye Patients
A.Mastromarino1, M.Papadia1, V.Amico2, S.Giuffrida2, M.Rolando1. 1Dept Neurosciences
Ophthalmology, University of Genova, Genova, Italy; 2Global Research and Development,
Bausch&Lomb, Catania, Italy.
2044 - B813
2045 - B814
Clinical Evaluation of New Punctal Plug (Smart PlugTM ) for the Treatment of Dry
Eye Patient That Conventional Plugs Doesn’t Suit
T.Kojima1,2, S.Hara1,2, R.Ishida2, E.Goto2, D.Murat2, K.Tsubota2. 1Dept of Ophthalmology,
Social Insurance Chukyo Hospital, Nagoya, Japan; 2Dept of Ophthalmology, Keio
University, Tokyo, Japan.
Purpose: Smart Plug is a new generation of thermo-sensitive punctum plug which is solid
at room temperature but becomes a soft gel with expansion in size at body temperatures.
We evaluated the efficacy of this new punctual plug in the treatment of conventional plug
non-suitable dry eyes. Methods: 18 eyes of 10 dry eye patients (Sjögren syndrome:6
patients ,Non-Sjögren syndrome: 3 patients, GVHD: 1 patient ) who previously
received conventional plug insertion were enrolled in this study. Conventional punctal
plugs were not suitable in these patients because of severe foreign body sensation,
recurrent extrusion, granuloma formation and conjunctival laceration. Schirmer 1
test, vital staining scores (fluorescein and Rose-Bengal), tear clearance tests were
performed before plug insertion, 2 weeks, 1 month, and 3 month after insertion.
Results: Although Schirmer test values were not significantly different before and
after plug insertion, (Before: 4.4±2.6 mm,After: 5.2±4.3 mm) the tear clearance rate
significantly decreased after plug insertion (Before: 14.8±11.4,After: 8.2±6.6) (P <
0.05). Rose-Bengal score improved significantly after plug insertion (Before 6.4±2.0,
After 3.3±1.6 points) (P < 0.05). Fluorescein score also improved significantly with
plug insertion (Before 4.8±2.3, After 2.1±1.3) (P < 0.05). No complications including
granulation and conjuntival laceration after smart plug insertion were observed.
Conclusions: Smart PlugTM proved to be a safe and an efficient option in the treatment of dry
eyes. This plug seems to be suitable for patients with complications of conventional plugs
such as frequent extrusion, granuloma formation and conjunctival laceration.
CR: T. Kojima, None; S. Hara, None; R. Ishida, None; E. Goto, None; D. Murat, None; K.
Tsubota, None.
Support: None.
Purpose: The lipid layer of the tear film in keratoconjunctivitis sicca (KCS) shows the
inability to maintain a consistent interference pattern with repeated blinking while in the
normal healthy eye it maintains a consistent pattern of interference among several blinks.
The aim of the study was to evaluate the possibility of improving the consistency of dynamic
lipid interference patterns (DLIP) in dry eyes by means of a lipid-containing tear substitute.
Methods: Twenty patients with bilateral definite clinical diagnosis of dry eye (positive to at least
two out of the following three tests: Schirmer I<5 mm/5 min, BUT<7 sec, positive lissamine
green staining of the ocular surface) were enrolled for the study. All subjects underwent DLIP
test which measures the number of blinks during which the precorneal tear film lipid layer
maintains a definite interference pattern. The DLIP test was performed in a randomly chosen
eye of each subject at three different times: 1) in basal condition (without instillation of any tear
substitute), 2) 5 minutes after the instillation of a 0.2% carbopol tear substitute and 3) 5 minutes
after the instillation of a 0.2% carbopol+medium chain triglycerides-containing tear substitute.
The differences in the average number of blinks for each eye showing a consistent lipid layer interference
pattern at the three different times of the experiment was statistically evaluated using Student t-test.
Results: The number of blinks providing the same consistent lipid layer interference pattern of
the tear film measured by means of DLIP test was increased both by carbopol only tear substitute
and carbopol+triglycerides-containing tear substitute. In particular the KCS eyes showed
an average of 2.3±1.5 blinks with consistent DLIP, KCS eyes after 0.2 carbopol instillation
showed an average of 4.2±3.2 blinks with consistent DLIP(p<0.01) and KCS eyes after 0.2
carbopol+triglycerides containing tear substitute instillation showed an average of 8.2±3.4 blinks
with consistent DLIP (p<0.001 vs. untreated KCS and p<0.01 vs. 0.2% carbopol treated KCS eyes).
Conclusions: The presence of the medium chain triglycerides component in a carbopol tear substitute
significantly improved the results of the DLIP test in dry eye patients. These results could be possibly
due to the ability of triglycerides to be included in the polar structural portion of the tear film lipid
layer with consequent improvement of its stability.
CR: A. Mastromarino, None; M. Papadia, None; V. Amico, Bausch&Lomb E; S. Giuffrida,
Baush&Lomb E; M. Rolando, None.
Support: None.
Undiluted versus Diluted Autologous Serum Eye Drops (ASED): A Prospective,
Randomized, Double-Blind Study in Patients With Refractory Dry Eye-Syndrome
A.Jaksche1A, Z.Sbeity1A, E.Domeier1A, R.Fimmers1B, F.G. Holz1A, K.U. Loeffler1A.
Ophthalmology, BMedical Biometry, Informatics, Epidemiology, 1University of Bonn,
Bonn, Germany.
Purpose: ASED have been used in the treatment of severe dry eye-disease. We have
evaluated the efficacy of undiluted (ud) ASED compared to a 50% concentration
(di) of ASED for refractory dry eye-syndrome in a prospective, randomized and
double-blind fashion. Here, we present our 3-months-results of an ongoing study.
Methods: Patients fulfilling ophthalmological and medical entry criteria were randomized to 3
months of udASED and diASED (1:1 in sodium chloride) to the right or left eye, respectively. Clinical
assessment, including Schirmer’s test, break-up time, and fluorescein staining was performed at
baseline, 2 weeks, 2 and 3 months. Examination and grading was carried out by two ophthalmologists.
Subjective comfort and improvement was recorded during each visit using the ‘‘faces’’-scale
(FS; 7 grades with 1 being best and 7 worst). Results are presented as the arithmetic mean.
Results: Eleven patients were enrolled into the study, and 16 eyes from 8 patients could be evaluated
so far. Eight eyes were treated with diASED and 8 eyes with udASED. Baseline result on the FS was
in average 5.63 for udASED and 5.25 for diASED. The FS showed improvement in 14 eyes after the
first 2 weeks: eyes treated with udASED improved by 1.5 (to 4.13), and eyes with diASED by 1.38 (to
4). The results of the 2 months’ visit included 10 eyes of 5 patients. Compared to the baseline results
there was a decrease by 3 (to 2.75) in the eyes treated with udASED and by 2.4 (to 3) in the eyes with
diASED. After 3 months, no obvious further improvement was seen (“face”-score 2.75 in 4 eyes with
udASED and 3 in 4 eyes with diASED). Objective clinical criteria did not correlate well with the FS
but also improved based on an obvious reduction of corneal staining, conjunctival folds and hyperemia.
Conclusions: The results provide further evidence for the beneficial effects of ASED in dry eyesyndrome. Within the first 2 weeks of treatment there was a distinct increase in subjective comfort
for both diASED and udASED. This improvement continued during the first 2 months and remained
stable during the last month. Although udASED showed only little numerical improvement compared
to diASED, most patients preferred the use of udASED. In general, ASED was considered superior
to conventional treatment for improving ocular surface health and subjective comfort.
CR: A. Jaksche, None; Z. Sbeity, None; E. Domeier, None; R. Fimmers, None; F.G. Holz,
None; K.U. Loeffler, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2016-2049 / B785-B818
260. Dry Eye Diseases: Treatment Organizing Section: CO
2046 - B815
2047 - B816
Tear Function and Ocular Surface Alterations With Lactoferrin Treatment in Severe
Dry Eyes
Y.Yamamoto1, M.Dogru1, Y.Matsumoto2, M.Saeki1, E.Goto1, M.Ishioka2, K.Tsubota1.
Ophthalmology, Keio University, Tokyo, Japan; 2Ophthalmology, Tokyo Dental College,
Chiba, Japan.
Purpose: To assess the changes in tear functions, ocular surface findings
and dry eye symptomatology in severe dry eye patients with oral lactoferrin
supplementation and compare the results with those of healthy control subjects.
Methods: Fourteen eyes of 7 Sjogren’s syndrome patients( 4 males, 3 females; mean
age:63.5 years) who had no improvement in tear functions and ocular surface vital staining
scores after at least 8 weeks of conventional treatment with preservative free artificial
tears, autologous serum eye drops and punctum plug applications as well as 10 eyes of
5 age and sex matched healthy control subjects were enrolled in this study. All subjects
received lactoferrin supplement (270mg/day) for 2 months and underwent tear film lipid
layer interferometry examinations with assessment of lipid layer thickness(DR-1, Kowa,
Tokyo), Schirmer test-1, tear film break up time and corneal sensitivity measurements as
well as ocular surface vital staining with fluorescein and Rose Bengal dyes before and after
supplement therapy. Dry eye symptomatology was assessed with visual analog score scales.
Results: There were no considerable changes in Schirmer test values before and 2
months after lactoferrin supplementation in all subjects. Pre treatment tear stability,
interferometry dry eye grades and ocular surface staining scores fared worse in dry
eye patients compared to controls. Tear stability, interferometry dry eye grades and
ocular surface staining scores improved in all dry eyes with thickening of the tear film
lipid layer(p<0.05). Lactoferrin supplementation was not associated with considerable
thickening of the lipid layer in the control subjects. Dry eye symptomatology improved in
all patients with significance. The need for artificial tear drops in the patients decreased
from an average of 12 drops/day to a mean value of 5.5 drops / day within 2 months(p<0.05).
Conclusions: Lactoferrin supplementation in severe dry eye patients is associated with
improvement of dry eye symptoms, tear stability and vital staining scores owing to alterations
in the tear film lipid layer.
CR: Y. Yamamoto, None; M. Dogru, None; Y. Matsumoto, None; M. Saeki, None; E.
Goto, None; M. Ishioka, None; K. Tsubota, None.
Support: None.
Evaluation of a Novel Dry Eye Oral Supplement for the Treatment of the Signs and
Symptoms of Dry Eye in the Controlled Adverse Environment (CAE) Model
S.G. Pratt1, G.W. Ousler2A, M.Schindelar2A, M.J. Chapin2B, M.B. Abelson3. 1Scripps
Memorial Hospital, La Jolla, CA; ADry Eye Department, 2Ophthalmic Research
Associates, Inc., North Andover, MA; 3Department of Ophthalmology, Schepens Eye
Research Institute, Harvard Medical School, Boston, MA.
Purpose: To evaluate the efficacy and safety of a novel Dry Eye Oral Supplement compared
to a standard multivitamin in relieving the signs and symptoms of dry eye as induced by
the controlled adverse environment (CAE). The use of the CAE allows for assessment
before, during and following acute exacerbation of ocular signs and symptoms, allowing
for a more comprehensive assessment of efficacy. Methods: This was a double-masked,
randomized, controlled, parallel-group, 5-visit, single-center, CAE study evaluating signs
and symptoms associated with dry eye. 24 dry eye patients entered a 3-week washout period.
Following CAE screening, eligible patients were randomized to receive either the Dry Eye Oral
Supplement or multi-vitamin placebo BID, in conjunction with the same standard artificial
tear product TID for 10 weeks. Ophthalmic examinations and follow-up CAE challenges
took place at weeks 3, 6 and 10. Results: 23 patients completed the study. Patient reported
symptom scores (0-4 on a standardized scale) during CAE exposure were compiled as follows:
Mean Total Discomfort during 90-Minute CAE Exposure
2048 - B817
2049 - B818
Ocular Surface in vivo Tolerance to New Nanoparticulate Polymer Systems Designed
for Drug Delivery
Y.Diebold1, A.Enríquez de Salamanca1, M.Calonge1, A.Vila2, E.L. S. Carvalho2,
la Fuente2, B.Seijo2, M.J. Alonso2. 1IOBA-University of Valladolid, Valladolid, Spain;
Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela,
Santiago de Compostela, Spain.
Purpose: To evaluate the in vivo acute tolerance of ocular surface structures to new classes
of nanoparticulate colloidal systems previously developed and tested in vitro by our group
and designed as potential drug delivery systems for ocular administration. Methods: Three
different nanoparticulate systems were prepared by ionotropic gelation, followed by freeze
drying in the presence of 5.0 % trehalose, and carrying bovine serum albumin as a marker
molecule: chitosan-based nanoparticles (CH-NPs), hyaluronic acid-based nanoparticles (HANPs) and lipids-chitosan nanoparticle complexes (LNPs). In vivo acute tolerance of 0.5 mg/ml
CH-NPs, HA-NPs and LNPs was studied in 15 female albino rabbits. Each formulation was
instilled in OD every 30 minutes, for a total of 13 times, leaving OS untouched as a control.
Clinical signs were evaluated previously and 3, 6, and 24 h after the first instillation and
statistically analyzed using the Fisher’s exact test. Conjunctival impression cytology (CIC),
collected 6 days before starting and 24 h after the first instillation, were done in all animals.
Ocular structures were removed, fixed and processed for a pathology study. Experiments
were performed in a masked fashion. Results: Neither irritation nor damage was observed in
vivo after ocular surface exposure to any class of nanoparticles and complexes. CIC showed
normal characteristics related to size and distribution of goblet cells and N/C ratio. Ocular
surface epithelia were not altered and lid tissues had no signs of edema or inflammation.
Inflammatory cells were basically absent, although some scattered polymorphonuclear cells,
also present in control eyes, were seen. Conclusions: Tested CH-NPs, HA-NPs and LNPs
were well tolerated and the ocular surface tissues remained normal after in vivo exposure.
These results, along with previous results showing virtually no in vitro toxicity, add further
support to the potential use these nanoparticulate systems as drug carriers to treat ocular
surface disorders.
CR: Y. Diebold, None; A. Enríquez de Salamanca, None; M. Calonge, None; A. Vila,
None; E.L.S. Carvalho, None; M. de la Fuente, None; B. Seijo, None; M.J. Alonso, None.
Support: FEDER-CICYT MAT2003-09713-C02-01 and MAT2004-04792-C02-01, Ministry
of Science, Spain.
Baseline p-value Week 3 p-value Week 6 p-value Week 10 p-value
Dry Eye Oral
< 0.0001 2.32
< 0.0001
0.195 2.86
0.482 2.44
Multivitamin (placebo) 3.13
13 eyes (7 active, 6 placebo) presented at baseline with lid edema >0 (0-4 scale). 71% of eyes
on active test article compared with no eyes on placebo, resolved completely after 10 weeks.
Conclusions: The active Dry Eye Nutritional Supplement was significantly more effective
than placebo in this study for the treatment of the ocular symptoms associated with Dry Eye
in the CAE. This controlled study showed that the novel Dry Eye Nutritional Supplement
tested has potential for treating dry eye patient.
CR: S.G. Pratt, Steven Pratt P; G.W. Ousler, ORA E; M. Schindelar, ORA E; M.J. Chapin,
ORA E; M.B. Abelson, ORA E.
Support: None.
Change of Nerve Growth Factor After 01% Prednisolone Instillation in Dry Eye
Syndrome Patients and Its Correlation With Clinical Parameters
I.Ryu, H.K. Lee, K.R. Seo, E.K. Kim. Ophthalmology, Institute of Vision Research, Yonsei
Univ College of med, Seoul, Republic of Korea.
Purpose: To evaluate the change of the tear nerve growth factor concentration after 0.1%
prednisolone treatment in dry eye syndrome patients and its correlation with clinical parameters
Methods: Patients with dry eye received 0.1% prednisolone and hyaluronic acid eye drops
three times a day each on right and left eye respectively. After 2, 4 weeks after treatment,
clinic visits were followed, and BUT, schirmer, IOP and symptom scale were measured
on both eyes. And on every visits, by collecting tear from both eyes, concentrations
of nerve growth factor were measured and impression cytology was performed.
Results: Total of 17 patients enrolled the study, and their mean age was 49.9 years old. The
initial tear NGF concentrations were 862.28 ± 144.58 pg/mL and 812.75 ± 124.24 pg/mL on right
and left eye respectively. On day 14, concentration changed to 731.92 ± 128.51 pg/mL on right
eye with a statistical significance with correlation of impression cytology. Clinical parameters
such as BUT, schirmer, IOP didn’t show any significant changes during the treatment. But,
the symptom scale decreased on day 14 and 28 on right eye with a statistical significance.
Conclusions: 0.1% prednisolone eye drop treatment on dry eye patients decreased the
concentration of nerve growth factor, and would be an effective treatment as it discontinues
anti-inflammatory process in short period of time without serious complications.
CR: I. Ryu, None; H.K. Lee, None; K.R. Seo, None; E.K. Kim, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2050-5732 / B819-B844A
261. Contact Lens II Organizing Section: CO Contributing Sections: CL, VI
2050 - B819
Simulation of Retinal Images in Eyes Wearing Bifocal HCL Using a Point Spread
Function Analyzer
M.A. Goto1, T.Tachikawa1, M.Matsubara1, T.Kobuchi2, K.Uno3, M.Shibutani4,
K.Kobayashi4, K.Ohnuma5, K.Ohno6, T.Noda 6. 1Department of Ophthalmology, Tokyo
Women’s Medical University Daini Hospital, Tokyo, Japan; 2Itoi Eye Clinic, Tokyo,
Japan; 3Seed Corporation, Tokyo, Japan; 4Topcon Corporation, Tokyo, Japan; 5Faculty
of Engineering, Chiba University, Chiba, Japan; 6National Institute of Sensory Organs,
Tokyo, Japan.
2051 - B820
Effect of Dynamic Movement of Customized Contact Lens on Visual Benefit of
Correcting Higher Order Aberrations in Keratoconic Eyes
G.-Y.Yoon1A,1B, T.Jeong1B. ADept Ophthalmology, BCenter for Visual Science, 1University of
Rochester, Rochester, NY.
Purpose: To predict the optical function in eyes wearing bifocal rigid gas permeable contact
lens (RGPCL) by simulating its retinal image using a point spread function (PSF) analyzer.
Methods: Simulation of retinal images was obtained using a PSF analyzer (Topcon, Tokyo). The
optical characteristics of the eye were evaluated on the basis of the PSF of a light beam reflected from
the retina when a point light is projected on the ocular optic system. A model eye was used for the
experiment which had anterior radius of curvature and refraction equivalent to 7.70mm and -5.12D,
respectively. The trial lens utilized was monofocal or bifocal concentric RGPCL, the added refraction
for near vision in the latter being +0.75, +1.25 or +1.75D. The center of the lens and the visual axis for
light projection in the model eye was either concurrent, or the lens was worn 0.5mm to 2mm lower on
the model eye. The PSF was measured with an artificial pupil 4mm in diameter. Simulation of the retinal
images of Landolt’s rings formed on the retina of the model eye was synthesized from the PSF data.
Results: With monofocal RGPCL, little influence on the simulated retinal image due to the shift in
lens position was recognized. With bifocal RGPCL adding +0.75D correction for near vision, the
image quality did not deteriorate significantly when the lens position was shifted down 1.5mm or
less. The image blurred when the shift in lens position was 2mm or more. With those adding +1.25
D or +1.75 D for near vision, the simulated retinal images blurred in the form of the Landolt’s rings
dragged toward the superior direction when the lens position was shifted down 0.5mm or 1mm,
retinal images becoming remarkably indistinct when the lens was shifted down 1.5mm or more.
Conclusions: Predicting the retinal image seen with shifting in multifocal RGPCL position by
simulation of the retinal image using a PSF analyzer may provide guidance in prescribing bifocal
contact lens.
CR: M.A. Goto, None; T. Tachikawa, None; M. Matsubara, None; T. Kobuchi, None; K. Uno,
Seed Corporation E; M. Shibutani, Topcon Corporation E; K. Kobayashi, Topcon Corporation E;
K. Ohnuma, Topcon Corporation R; K. Ohno, None; T. Noda, None.
Support: None.
Purpose: Dynamic movement of customized contact lens after blinks is one of the factors that reduces
visual benefit of correcting higher order aberrations in the eye. The goal of this study is to theoretically
investigate the feasibility of improving vision with customized contact lenses in keratoconic
eyes when dynamic decentration and rotation of customized contact lens are taken into account.
Methods: Wavefront aberrations of fifteen keratoconic eyes were measured with the large dynamic
range Shack-Hartmann wavefront sensor. With the measured wavefront aberration, modulation transfer
functions under the white light condition were calculated to estimate visual benefit. Visual benefit was
defined as the ratio of the volume under the modulation transfer functions (vMTF) with second and
higher order correction to that with second order correction only. Visual benefit was also calculated with
dynamic decentration and rotation of customized contact lens. To simulate this dynamic movement,
Monte-Carlo method was used to combine dynamic decentration and rotation of customized contact lens
weighted by Gaussian distribution. The same analysis was performed in 30 normal eyes for comparison.
Results: When perfectly correcting higher order aberrations with customized contact lens, keratoconic
eyes experienced the visual benefit of 4.4 ± 2.0 for a 6 mm pupil. This visual benefit is approximately
2 times larger than that obtained in normal eyes. When considering the dynamic movement of
customized optics, the visual benefit in keratoconic eyes was decreased to 1.5 ± 0.2 with the standard
deviations of 300 μm decentration and 7 degree rotation in Gaussian distribution which covers a
range of ±600 μm and ±14 degree, respectively. On the other hand, normal eyes experienced 1.3 ±
0.1 with the same amount of contact lens movement. Visual benefit in keratoconic eyes was reduced
more largely with an increase in the amount of lens movement compared to that in normal eyes.
Conclusions: Correcting higher order aberrations in keratoconic eyes provides larger visual benefit
than in normal eyes. Visual benefit for keratoconic eyes is more sensitive to decentration and
rotation of customized optics compared to the normal eyes. Our calculation demonstrates that
correcting higher order aberration with customized optics improves retinal image quality even with
the dynamic movement of customized contact lens although actual perceived vision improvement
needs to be evaluated.
CR: G. Yoon, Bausch & Lomb F, C; T. Jeong, None.
Support: NIH/NEI R01 EY014999, NYSTAR/CEIS, Research to Prevent Blindness (RPB), Bausch
& Lomb
2052 - B821
2053 - B822
Power Profiles and Short Term Visual Performance of Soft Contact Lenses
E.B. Papas1,2, A.Dahms1,3, N.Tahhan1, N.Carnt1. 1Vision CRC, Sydney, Australia; 2School
of Optometry & Vision Science, University of New South Wales, Sydney, Australia;
Technische Fachhochschule, Berlin, Germany.
The Change of High Order Aberration in Keratoconic Eyes and Myopic Eyes With
Rigid Gas Permeable Contact Lenses Wearing
J.Choi1,2, W.R. Wee3,2, J.H. Lee1,2, M.K. Kim1,2. 1Department of Ophthalmology, Seoul
National University College of Medicine, Seoul, Republic of Korea; 2Artificial Eye Center,
Seoul National University Hospital Clinical Research Institute, Seoul, Republic of Korea;
Seoul National University Bundang Hospital, Seoul, Republic of Korea.
Purpose: To investigate the change of high order aberration(HOA) in keratoconic
eyes and myopic eyes with rigid gas permeable(RGP) contact lenses wearing.
Methods: In 18 keratoconic eyes which average keratometric index was 44.27±2.65diopter
and 41 myopic eyes, the root mean square(RMS) values of high order aberrations(HOAs) were
obtained using wave scan(Visx WS1, Santa Clara, USA) before and after wearing of RGP.
Results: Total HOA, coma, and trefoil aberration RMS values decreased by 0.029, 0.042,
0.056, respectively, and spherical aberration increased by 0.139 in keratoconus with RGP
wearing. However, these changes were not statistically significant. Total HOA, trefoil, and
spherical aberration RMS values were decreased by 0.018, 0.065, 0.147, respectively, and coma
aberration was increased by 0.327 in myopic eyes. Only the trefoil aberration was significantly
changed(p=0.001). The change of spherical aberration was significantly different between the two
groups(p=.016). In myopic eyes with total HOA equal to or greater than 0.30 before the wearing,
total HOA and trefoil aberration significantly decreased by 0.137 and 0.127(p=0.000) after RGP
wearing. On the contrary, total HOA and coma aberration significantly increased with RGP
wearing by 0.085 and 0.101(p=0.000) in eyes with total HOA lower than 0.30 before the wearing.
Conclusions: Wearing of the RGP did not influence the HOAs in keratoconic eyes. However,
RGP wearing seemed to reduce total HOA by decreasing trefoil in eyes with primarily high
HOA, and RGP wearing seemed to augment total HOA by increasing coma in eyes with
originally low HOA.
CR: J. Choi, None; W.R. Wee, None; J.H. Lee, None; M.K. Kim, None.
Support: None.
2054 - B823
2055 - B824
Purpose: To determine the effect on vision of refractive power distribution in the optic zone of
soft contact lenses. Methods: Twenty subjects wore each of five contact lens types in one eye only.
Lenses were ACUVUE® 2, ACUVUE® ADVANCE™ (Vistakon), O2OPTIX™, NIGHT & DAY®
(CibaVision) and PureVision™ (Bausch & Lomb). Presentation order was randomized and masked.
In-eye lens power was adjusted to achieve zero spherical over-refraction prior to carrying out the
following visual assessments: High (HCVA) and low contrast visual acuity (LCVA) in normal
illumination (1700 Lux), high contrast acuity in reduced illumination (HCLIVA) (17 Lux), subjective
visual quality (VQ) using a numerical rating scale and visual satisfaction rating (VS) by Likert scale.
Refractive power across the central 5mm was measured at the back surface of all lenses with both
Nikon focimeter and Visionix VC 2001. Results: Group mean responses, for selected variables,
are shown in the table below. Comparison between lens types made using analysis of variance with
repeated measures and Friedman’s test for Likert scale data showed no significant differences for
any of the visual performance variables (p > 0.05). Group mean back vertex power error (BVPE),
defined as the difference between labeled power and focimeter measured power, was significantly
more negative for ACUVUE® 2 and ACUVUE® ADVANCE™ than for other lens types (p < 0.0005).
Comparison of power distributions across the optic zone indicated marked differences between
the lens types. Conclusions: Variations in power profile between these lens types did not result in
measurable short term visual performance differences. Lenses of equivalent labeled power did not
necessarily produce identical refractive effects on-eye. Over-refraction to establish the appropriate
back vertex power, for each lens type, is a pre-requisite for optimum vision.
CR: E.B. Papas, CibaVision F; A. Dahms, CibaVision F; N. Tahhan, CibaVision F; N. Carnt,
CibaVision F. Support: Australian Government CRC Scheme
The Effects of Overnight Orthokeratology Lens Wear
N.Stuebiger1, M.Ronecker2, R.Michels2, H.Specht3. 1Ophthalmology, University of
Tuebingen, Tuebingen, Germany; 2Ophthalmic Optics, University of Applied Sciences,
Aalen, Germany; 3Ophthalmology, University of Heidelberg, Heidelberg, Germany.
Purpose: Over night or thokeratolog y (Or tho-K) is a method for temporar ily
eliminating myopia. A special geometr y of rigid gas per meable (RGP) lenses
(double reverse back curve) enables transformation of the cornea to a f latter form.
The aim of our study was to evaluate the effectiveness of Ortho-K for treatment of myopia, to
evaluate possible pathologic corneal alterations and changes of the intraocular pressure (IOP).
Methods: A group of 20 volunteers (m:f=9:11) with a mean age of 25.92 ± 5.86 years were included in our
study. Both eyes were fitted with Ortho-K lenses, and uncorrected visual acuity, refractive correction,
corneal curvature and corneal changes, including measurements of corneal endothelial alterations,
were achieved. The control group consisted of 6 volunteers, who have worn glasses as vision correction.
Neither group exhibited any eye disease or any pathological symptom at the beginning of the study.
Results: Before fitting the Ortho-K lenses, the wearing group had a mean spherical correction
(oculus dexter/oculus sinister) of -2.06/-2.25 ± 0.90/0.84 diopter (D), a mean uncorrected vision
of 0.64 ± 0.31 (visus logmar) and a mean central corneal radius of 7.78/7.77 ± 0.31/0.30 mm. After
one week of wearing contact lenses the majority of the volunteers had such a good response, that no
statistical difference in visual acuity between the two groups was detectable (the mean spherical
correction was +0.14/-0.02 ± 0.25/0.14 D, the mean uncorrected vision was -0.12 ± 0.06 and the
mean central corneal radius was 8.09 / 8.08 ± 0.35/0.34 mm). Corneal irritations which occurred at
the beginning of the study in the wearing group included corneal staining (40%, Grading scale 1.1),
acute corneal edema (10%), microcystes (5%). Problems with halos were frequently noticed (90%),
but the wearers get accustomed to that. The study was completed after a mean observation period of
8 months and, . At that time no corneal edema could be observed, and in the whole observation period
no corneal endothelial alterations, no infectious corneal complications, in none of the volunteers IOP
changes occured, and altogether, five volunteers discontinued the study from the wearing group .
Conclusions: Overnight orthokeratology, as an alternative to refractive corneal surgery, is a very safe
method for temporarily eliminating minor myopia. We achieved in our study stable remaining visual
changes for all walking hours of the day, which allow patients enjoy excellent device-free vision.
CR: N. Stuebiger, None; M. Ronecker, None; R. Michels, None; H. Specht, None.
Support: None.
Reverse Geometry Lenses: Are Changes in Corneal Epithelium Responsible for
Myopia Reduction?
A.Minavi, N.Leach, W.Miller. College of Optometry, University of Houston, Houston, TX.
Purpose: To investigate central and peripheral corneal epithelial changes in neophyte
orthokeratology patients after three months of treatment. Methods: Confocal microscopy
(ConfoScan3) and ultrasonic pachymetry (Sonogage Corneo-Gage Plus 2) were used to obtain
baseline central and peripheral corneal epithelium thickness (CET, PET) measurements
on 8 healthy myopic subjects (28 + 4 years; 6 females and 2 males) with a mean spherical
equivalent refractive error of -2.31+ 0.89 D; range -1.50 to -4.38 D. Both eyes were fit according
to protocol with CKRTM (Boston Equalens II; Dk/t= 85) reverse geometry gas permeable
lenses and patients were instructed to wear the lenses overnight for three months. Central
measurements were taken with the patient in primary gaze and peripheral measurements were
taken inferiorly with the subject in superior gaze (28o above primary gaze). All measurements
were repeated after one and three months of continuous lens wear. One eye was chosen for
statistical analysis. Measurements were compared using 2-tailed t-tests using the Bonferroni
correction factor for multiple comparisons. Results: While individual differences were
observed, average mean differences (AMD) between baseline, one month and three month
CET and PET measurements showed no statistical difference with the ConfoScan3 or with
the Sonogage instruments (p>0.05). For the ConfoScan3, CET and PET at one month was
7.5 + 17.2 µm (AMD + SD) and 2.9 + 17.3 µm and at three months 0 + 10.73 and 4.6 + 15.83
µm respectfully. As for the Sonogage, AMD for CET and PET at one month was -1.8 + 2.7
and 0.2 + 0.1 µm; at three months - 0.4 + 0.96 and 0.1+ 0.79 µm respectfully. All subjects
showed significant myopia reduction (p<0.05) at the one month visit and maintained this
reduction during the three month visit. Conclusions: Our findings show that myopia reduction
after orthokeratology does not appear to be a result of epithelial change as reported by other
investigators. It is possible that structural changes within the cornea are not detectable by
the methods used. Differences in instrument sensitivity and/or operator error may also
partially explain our findings.
CR: A. Minavi, None; N. Leach, None; W. Miller, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2050-5732 / B819-B844A
261. Contact Lens II Organizing Section: CO Contributing Sections: CL, VI
2056 - B825
2057 - B826
Overnight Corneal Edema Can Modulate the Short-Term Clinical Response to
Orthokeratology Lens Wear
H.A. Swarbrick, J.Jayakumar, W.Co, D.He, C.Siu, B.Yau. School Optometry/Vision Sci,
University of New South Wales, Sydney, Australia.
Pur pose: To i nvest igate the i n f luence of over n ig ht cor neal edema
on t he cl i n ical re sp on se to ove r n ig ht or t hoke r atolog y len s wea r.
Methods: Twelve young adult subjects (age 20 to 25 years) were fitted with reverse-geometry
lenses for orthokeratology (BE; UltraVision, Brisbane, Australia) in both eyes. Matched design
lenses in Boston ES (nominal Dk/t = 8) and Boston XO materials (nominal Dk/t = 45) were
worn in the two eyes for eight hours overnight. A separate overnight trial was conducted to
examine corneal changes in the absence of lens wear in the contralateral eye. LogMAR visual
acuity, apical corneal radius (ro, mm; Medmont E-300 topographer) and corneal eccentricity
(e; Medmont) were measured before and immediately after overnight lens wear. The overnight
corneal edema response was monitored using optical pachometry. Changes from baseline
were examined using ANOVA and post hoc protected t-tests, with a critical p-value of 0.05.
Results: Overnight central corneal edema averaged 11.5 ± 5.8% and 3.3 ± 3.7% in the ES and XO
lens-wearing eyes respectively (ES > XO; p < 0.001), compared to 2.4 ± 2.1% with no lens wear.
Despite the matched reverse-geometry lens designs, changes in unaided visual acuity and corneal
topography were much less evident in the ES lens-wearing eyes compared to those wearing the
XO material (change in logMAR VA: -0.09 ± 0.25 vs. -0.33 ± 0.16, p < 0.001; change in ro: 0.02
± 0.06 vs. 0.18 ± 0.11 mm, p < 0.001; change in e: 0.00 ± 0.09 vs. -0.19 ± 0.13; p < 0.001). No
significant differences in these clinical outcomes were found between ES lenses and no lens wear.
Conclusions: High levels of overnight corneal edema appear to limit the clinical effects of
overnight reverse-geometry lens wear, at least in the short term. This suggests that the use of
high Dk materials for overnight orthokeratology not only provides physiological advantages,
but may also optimize clinical outcomes.
CR: H.A. Swarbrick, UltraVision Capricornia (Australia) Pty Ltd F; J. Jayakumar,
None; W. Co, None; D. He, None; C. Siu, None; B. Yau, None.
Support: None.
The Relationship Between the Treatment Zone Diameter With Visual and Optical
Performance in Hyperopic Corneal Refractive Therapy Lens Wearers
F.Lu, T.L. Simpson, L.Sorbara, D.Fonn. School Optometry, CCLR University Waterloo,
Waterloo, ON, Canada.
Purpose: To investigate the stability of the treatment zone (TZ) after one
night of hyperopic Corneal Refractive Therapy (CRT ®H) lens wear and to
determine the association between the TZ with visual and optical performance.
Methods: Paragon CRT®H lenses (Dk=100) were fit on one eye of 20 ametropes (mean±SD
spherical equivalent: -2.09±2.53D) with no lens on the contralateral eye (randomly
selected). High/Low contrast visual acuity (HCVA/LCVA), refractive error, aberrations
(LADARWaveTM), and corneal topography were measured at baseline, and immediately after
lens removal and 1, 3, 6, 12 hours later on the following day. The TZ was defined by the change
in curvature from negative to positive and vice versa, using the tangential difference map
from the Atlas TM and Orbscan II TM corneal topographers. Root mean square (RMS) wavefront
errors and zernike coefficients of spherical aberration (SA) were collected using 4.5mm pupils.
Results: During the day, the central steepened zone (CSZ, ±SE) was statistically constant (both
p≥0.118) and was 2.50±0.11mm using the Atlas and 3.30±0.18mm using the Orbscan. During
the same period, the total treatment zone (TTZ) ranged from 7.18±0.13mm to 6.65±0.24mm
using Atlas, from 6.11±0.10mm to 5.82±0.13mm using the Orbscan (both p≤0.011). There
was a positive correlation between CSZ and signed SA using Orbscan (r=0.676, p=0.002);
there were negative correlations between TTZ and RMS SA using Atlas and CSZ and HCVA/
LCVA using Orbscan (r =-0.480~-0.584, all p≤0.044). There were also marginal correlations
between TTZ and signed SA and LCVA using Orbscan (r=0.432, p=0.073; r=-0.441, p=0.067).
Conclusions: CSZ was approximately constant after one night of CRT®H lens wear, while
TTZ reduced. The treatment zone concept (both central and total) is a useful indicator of
visual and optical performance of CRT®H lens wear.
CR: F. Lu, Paragon Vision Sciences F; T.L. Simpson, Paragon Vision Sciences F; L.
Sorbara, Paragon Vision Sciences F; D. Fonn, Paragon Vision Sciences F.
Support: Paragon Vision Sciences
2058 - B827
2059 - B828
Histological Findings of Rabbit Cornea Produced by an Orthokeratology Lens
M.Matsubara1, S.Takeda1, T.Hirowatari1, K.Mukai2, Y.Ishii2. 1Ophthalmology, Daini
Hospital, Tokyo Womens Medical Uni, Arakawa-Ku, Japan; 2New Vision Institute, TaitoKu, Japan.
Pur pose:To repor t the topog raphical epithelial changes histologically
after the use of or thokeratology lens (or tho-K lens) in rabbit cor nea.
Methods:An ortho-K lens (target reduction; -3.0 diopter) was continuously inserted into right
eyes of white rabbits. Eyes were enucleated after 4 weeks, and served to histological examination
using hematoxylin-eosin staining and electron microscopic study. Left eyes were used as controls.
Results:Panoramic view of cornea showed apparent topographical changes of epithelial
thickness corresponding to portions of Ortho-K lens. In the area that corresponds to the
base curve zone, epithelial layers showed almost normal configuration. In the reverse curve
zone, epithelium was thick with 7 to 9 layers. Epithelial layers were thinnest in the alignment
curve zone. Electron microscopic study revealed slightly increased proteoglycans in deep
stroma at the center. Epithelial cells in any area showed no abnormal staining pattern.
Conclusions: Continuous wear of Ortho-K lens gives topographical epithelial changes in
CR: M. Matsubara, None; S. Takeda, None; T. Hirowatari, None; K. Mukai, None; Y.
Ishii, None.
Support: None.
Orthokeratology-Like Effects of Everted Soft Contact Lenses: A Mechanical Model
S.R. Evans1,2, A.Ho1,2, J.D. Choo1,2. 1Institute for Eye Research, Sydney, Australia; 2Vision
Cooperative Research Centre, Sydney, Australia.
2060 - B829
2061 - B830
Short-Term Corneal Changes in Closed Eye Condition With Orthokeratology Lenses
Y.Kamei, K.Cassar, J.Shen, P.S. Soni. School of Optometry, Indiana University,
Bloomington, IN.
P u r p o s e : : To d et e r m i ne t he shor t- t e r m c or ne a l cu r vat u r e a nd
ref ractive changes with CRT and Contex OK or thokeratolog y lenses.
Methods: Both eyes of 10 subjects were fitted, using the fitting guidelines provided
by the manufacturer, with CRT® and Contex OK orthokeratology lenses. Each lens
design was worn by each subject for 60 minutes. The eyes were closed and covered
with a mask and the subject was asked to rest in a quite, dark room. Uncorrected visual
acuity (UVA), spherical (SPH) and spherical equivalent (SPE) refractive correction,
central flat (FK) and steep (SK) curvature were measured before and after lens wear.
A three week wash-out period was used in between the tests with the two designs.
Results: : Results: The average UVA, SPH, SPE, FK and SK readings before using CRT®
and Contex OK lenses was 0.9 (±0.34) LogMAR, -3.63 (±1.74) D, -3.89 (±1.72) D, 44.05
(±1.32) D, and 44.86 (±0.88) D. Post treatment with CRT® and Contex OK orthokeratology
lenses the UVA, SPH, SPE, FK and SK was 0.71 (±0.36) LogMAR, -2.99 (±1.84) D, -3.28
(±1.74) D, 43.48 (±1.43) D, and 44.24 (±0.98) D and 0.64 (±0.31) LogMAR, -2.85 (±1.72) D,
-3.17 (±1.69) D, 43.59 (±1.3) D, and 44.22 (±0.94) D respectively. There was a statistically
significant (p=0.001) difference in the data collected prior to and after each of he lenses were
worn. While there is no statistical difference between the refractive and corneal responses
to the two lens designs, the Contex OK lens consistently demonstrated a greater change
Conclusions: The data shows that on the average, the two lenses produced significant but
similar refractive and corneal change when used under a short-term closed eye conditions.
CR: Y. Kamei, None; K. Cassar, None; J. Shen, None; P.S. Soni, Polymer Technology
F, R.
Support: EY015504-01-Soni
Purpose: Recent work has shown that soft contact lenses (SCLs) can produce corneal reshaping when worn
everted (inside-out). Orthokeratology traditionally relies on rigid contact lenses to reshape the cornea.
The purpose of this study is to examine the mechanisms by which everted SCLs produce similar effects.
Methods: Finite-element analysis (FEA) was used to simulate the eversion of various SCLs, and their
on-eye pressure profiles. The model uses 500-1500 axisymmetric planar elastic elements, depending on
lens shape. The lenses were a simple generic design in a range of powers (-30, -20, -10, -6, plano and +6
dioptres) and elastic moduli (0.2, 1.0 and 2.0 MPa). First, the everted lenses are compared off-eye, since
the internal stresses produced by eversion may be important on-eye. Secondly, each lens is pressed against
a rigid cornea by imposing a uniform constant ‘eyelid pressure’ of 200 Pa, in order to derive an estimate
for the on-eye pressure profile. The changes in internal stress produced by pressing the everted lens
onto the eye are analysed, and the on-eye pressure profiles are compared to those of right-side-in lenses.
Results: On eye, all high minus lenses showed a low-pressure area near the optic zone margin, and
high pressures (>800 Pa) in the mid-periphery. In the central zone, pressure is nearly independent
of power for minus-powered lenses (around 300 Pa), while the +6D lens showed central pressure
of about 550 Pa. The pressure pattern compares favourably with images obtained clinically using
Fluoresoft 0.35%. The everted pressure profiles are in dramatic contrast to those of non-everted
lenses. The effect of lens modulus is to adjust the position and magnitude of peaks in the pressure
profile for a given power. For example, a -10D lens with a modulus of 1.0 MPa gives a central
treatment zone of 3 mm, while a modulus of 2.0 MPa gives a central treatment zone of 5.5 mm.
The difference between the off-eye (equilibrium) and on-eye (non-equilibrium) stress
states of everted lenses show an alternating pattern of ‘push-down’ and ‘springoff’ forces which modify the pressure profile. On-eye performance of an everted
lens is thus different to that of an identically shaped lens without prestressing.
Conclusions: Both geometry and stress preload play a role in the on-eye performance of everted
SCLs. The position of pressure peaks and stand-off zones compare well with clinical results. FEA
provides a useful tool for the analysis of everted lenses, whose shape and preload states would be
difficult to determine otherwise.
CR: S.R. Evans, Institute for Eye Research P; A. Ho, Institute for Eye Research P; J.D. Choo,
Institute for Eye Research P.
Support: Australian Government Cooperative Research Centre Program
Topographic Keratometric Effects of Corneal Refractive Therapy for Hyperopia
After One Night of Lens Wear
L.Sorbara, F.Lu, D.Fonn, T.Simpson. School of Optometry, University of Waterloo,
Waterloo, ON, Canada.
Purpose: To examine the topographical corneal shape change across the horizontal meridian
after one night of wearing CRT™ corneal reshaping contact lenses for the correction of hyperopia.
Methods: Twenty participants wore a CRT™ HDS 100 contact lens in one eye designed to
reduce hyperopia .The other eye served as the control. The lens was worn during 8 hours
of sleep. Topography and refractive error was measured using the Humphrey Atlas™
corneal topographer the night prior to lens insertion, immediately after lens removal on
the following morning and 1, 3, 6 and 12 hours afterwards. Recovery data was gathered
28 hours later. Topographic changes were measured over a 6-8mm chord in 1mm steps.
Results: There were significant differences in corneal curvature over time; the central cornea
was steepened by 0.85±0.67D (range 0.00 to 2.20D) and at the mid-periphery (3mm temporal
from the centre) the cornea flattened 1.70±1.11D (range 0.00 to 4.30D) in experimental eyes
(both p=0.000) compared to baseline, while no change was found in control eyes (p=0.139). The
central corneal steepening regressed over time 50% centrally and 56% mid-peripherally. (both
p=0.000) and did not recover to baseline after 12 hours. At the 28 hour (post-discontinuation)
visit, the central cornea recovered to baseline, although it was still steeper (approx. 0.12D)
and the mid-periphery was still flatter by 0.25D representing a 98% recovery (both p>0.05).
Conclusions: After one night of lens wear, the CRT™ for Hyperopia lens wearing eye
showed a moderate although significant central steepening which translates into hyperopic
correction . The shape of the cornea did not recover to baseline at 12 hours after one night,
but, recovered after 28 hours of no lens wear.
CR: L. Sorbara, None; F. Lu, None; D. Fonn, None; T. Simpson, None.
Support: Paragon Vision Sciences
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2050-5732 / B819-B844A
261. Contact Lens II Organizing Section: CO Contributing Sections: CL, VI
2062 - B831
2063 - B832
Soft Contact Lenses Can Induce Orthokeratology-Like Topographical Changes
J.D. Choo1,2, P.J. Caroline3, B.A. Holden1,2, S.R. Evans1,4, A.Ho1,4, P.D. Bergenske3.
Institute for Eye Research, Sydney, Australia; 2School of Optometry, University of New
South Wales, Sydney, Australia; 3College of Optometry, Pacific University, Forest Grove,
OR; 4Vision Cooperative Research Centre, Sydney, Australia.
OSEIRT/Ortho-K Indication for the Hyperopia Patients
I.Mitsui, Y.Yamada, Y.Yagi. Ophthalmology, Mitsui Medical Clinic, Minato-ku, Japan.
Purpose: Ortho-K is usually known as non-invasive technique to reduce myopia and
astigmatism. Traditionally, it was not indicated before for the hyperopia patients because of the
difficulties of lens design. However, two years ago we started Ortho-K like procedure to reduce
hyperopia, with the newly developed lens design called OSEIRT(Ocular Surface and External
Integrated Remodeling Therapy). We report the results from twenty-one cases of hyperopia.
Methods: OSEIRT was indicated for thirty-eight eyes of twenty-one hyperopia patients;
age form six to fifty-eight. The average of their Uncorrective Visual Acuity (UCVA)
before OSEIRT was 20/30 or worse, and mean Spherical equivalent (SE) was +2.43D
(range +0.75 to +4.25D). They were followed at least one years wearing of OSEIRT
lenses during night. The following studies were examined on their auto-refraction,
auto-keratometry, uncorrected and corrected visual acuity, intra-ocular pressure,
corneal thickness and curvature, corneal shape (topography), and corneal endothelium.
Results: The mean SE improved to +2.04±1.33D during six months, +1.86±0.92D during
nine months, and +1.02±0.72D during one year. Astigmatism also slightly decreased.
68% of the patients improved in UCVA up to 20/30 or better, 46% of them improved
up to 20/20 or better, and 28% of them improved up to 20/15. Intra-ocular pressure and
endothelium remained normal. Topographically, cornea changed its shape to convexity.
Ophthalmologic examinations showed no abnormalities throughout the therapeutic period.
Conclusions: OSEIRT/Ortho-K was so effective to reduce hyperopia and evaluated to be
safe enough for the patients of all age. However, in order to get the effective results, the lens
design of OSEIRT for hyperopia was needed to be completely customized.
CR: I. Mitsui, None; Y. Yamada, None; Y. Yagi, None.
Support: None.
2064 - B833
2065 - B834
Purpose: Traditionally, orthokeratology has relied on rigid contact lenses to achieve the desired
corneal reshaping effects. However, recent work has shown that soft lenses worn everted (inside
out) can create similar changes to the corneal surface. The purpose of this study was to examine
the topographical changes associated with the wearing of everted soft lenses of various designs and
powers. Methods: One subject was used in this study. CIBA Focus Night and Day (lotrafilcon-A)
24% water lenses with base curves of 8.4mm, diameters of 13.8mm and powers of +6.00D, -0.25D,
-6.00D and -10.00D were each everted and placed on one eye of the subject. Also evaluated were
two custom soft lenses made in the Benz-G5X, (hioxifilcon-A) 59% water material with a base
curve of 8.15mm, a diameter of 14.5mm and powers of -20.00D and -30.00D. The subject wore
each of the everted lenses overnight for 6.5 hours and separately during the day for 6.5 hours.
Each wearing period was separated by an appropriate period of non-lens wear to allow the cornea
to return to baseline. All lens fits were evaluated with Fluoresoft 0.35% and photographed. The
topographical changes between baseline and post-lens removal were analysed using the Medmont
topographer. Results: Soft contact lenses worn everted resulted in corneal topographic changes
generally consistent with traditional rigid lens orthokeratology; soft lenses worn without inversion
resulted in very small topographic changes. Everted lenses of higher minus power showed more
distinct Fluoresoft patterns and greater amounts of corneal topographic changes. For example, a
-10D everted lens gave up to 2.50D of central flattening during daily wear, while a -20D everted lens
gave up to 5.40D of central flattening. By comparison, the -10D lens produced 1.30D of flattening
during overnight wear. The higher minus lenses were also found to induce a mid-peripheral ring of
epithelial erosion. The Fluoresoft patterns of all everted lenses compare favourably with pressure
profile patterns obtained mathematically using finite element analysis. Conclusions: It appears
that everted soft contact lenses are capable of inducing significant changes in corneal topography,
with daily wear resulting in greater topographic changes than overnight wear. Further work must
be done to help understand these changes to develop a predictable and effective way of using soft
contact lenses for corneal reshaping.
CR: J.D. Choo, Institute for Eye Research C, P; P.J. Caroline, Paragon Vision Sciences P; B.A.
Holden, Institute for Eye Research E, P; S.R. Evans, Institute for Eye Research E, P; A. Ho, Institute
for Eye Research E, P; P.D. Bergenske, None.
Support: Australian Government Cooperative Research Centre Scheme
The Incidence of Local and General Contact Lens Induced Papillary Conjunctivitis in
Silicone Hydrogel Contact Lenses
C.C. Skotnitsky1,2, D.F. Sweeney1,2, T.J. Naduvilath1, P.R. Sankaridurg1,2. 1Vision
Cooperative Research Centre, The University New South Wales, Sydney, Australia;
School of Optometry and Vision Science, The University of New South Wales, Sydney,
Purpose: To describe the incidence of contact lens associated papillary conjunctivitis
(CLPC) in subjects wearing low Dk and silicone hydrogel contact lenses (CL) extended
wear (EW); and to determine the recurrence rate of CLPC in silicone hydrogel CL EW.
Methods: In retrospective clinical trials at both Australia and India from 1993 to 2003, 1,894
subjects wore either low Dk hydrogel CL on a 6 night (N) or silicone hydrogel CL on either a 6
N or 30N EW schedule. The incidence of CLPC per 100 eye years (%) for each lens type was
determined for first events only. CLPC was classified by location of papillae. Papillae confined
to ≤ 2 areas of the tarsal conjunctiva were classified as local and papillae spread over more than 2
areas were classified as general. Fisher’s Exact Test assessed differences between the two groups.
Results: The incidence of CLPC in low Dk CL wearers was 3.9 per 100 eye years (95% CI
= 2.9 - 5.0) and in silicone hydrogel CL wearers 5.2 per 100 eye years (95% CI = 4.3 - 6.2).
There was a significantly greater incidence of local CLPC events compared to general events in
silicone hydrogel CL (3.6% vs. 0.7%, p<0.0001) and a significantly greater incidence of general
CLPC in low Dk hydrogel CL compared to local CLPC events (3.4% vs. 1.7%, p=0.003).
63% of all eyes had a recurrence of CLPC. 28% of eyes with prior CLPC events recurred as
a general event and 49% of eyes recurred as a local event with silicone hydrogel CL wear.
Conclusions: The incidence of local CLPC is higher in silicone hydrogel CL wear than in
low Dk CL wear. This difference in incidence between local and general CLPC events may
be caused by different mechanisms. At least 60% of eyes will have a recurrence of a CLPC
event with silicone hydrogel EW.
CR: C.C. Skotnitsky, None; D.F. Sweeney, None; T.J. Naduvilath, None; P.R.
Sankaridurg, None.
Support: None.
Survey on the Lens Complaints of One-day Disposable Soft Contact Lenses
M.Itoi1, H.Shioya2, M.Kajita3, M.Inaba4. 1Itoi Eye Clinic, Tokyo, Japan; 2Shioya Eye Clinic,
Fukushima, Japan; 3Kajita Eye Clinic, Tokoyo, Japan; 4Inaba Eye Clinic, Osaka, Japan.
Purpose: : We conducted a survey on the occurrence of lens complaints
of c om me r cia l ly- ava i lable one - d ay d is p os able sof t c ont a c t le n se s.
Methods: The survey was conducted at 4 facilities during the period from October
2003 to April 2004. The subjects were 290 patients who wanted to use contact lenses
and were fitted with Focus Dailies (CIBA Vision) or 1day Acuvue (Johnson & Johnson)
lenses. We asked them to record any lens complaints of one-day disposable soft contact
lenses in survey forms, which we collect and compile at the time of regular inspection.
Results: Complaints (most frequently lens torn) were noted with the both types of one-day
disposable contact lenses. The occurrence of lens complaints was 28 per 10,942 lenses
(0.256%) for Focus Dailies and 20 per 5,207 lenses (0.378%) for 1day Acuvue. The causes of
lens torn were: lens torn during the removal of lenses from blister packs (0.055% for Focus
Dailies and 0.250% for 1day Acuvue), lens torn during the removal of lenses from the eyes
(0.119% and 0.019%), initial defects at the time of delivery (0.037% and 0.040%), and lens
torn during wear (0.037% and 0.080%). There were no cases of eye injury due to lens torn.
Conclusions: A difference was observed in the causes of lens complaints between different
brands of one-day soft contact lenses. While this survey identified no cases of eye injury
due to lens torn, wearing broken lenses may be a cause of eye injury. We recommend that
contact lens wearers should be instructed the proper handling of lenses according to the
type of lenses they use. We would like to request to lens manufacturers that they completely
prevent the shipping of lenses with initial defects through adequate quality control efforts
and they develop lens torn-resistant lenses.
CR: M. Itoi, CIBA Vision Corporation R; H. Shioya, CIBA Vision Corporation R; M.
Kajita, CIBA Vision Corporation R; M. Inaba, CIBA Vision Corporation R.
Support: None.
2066 - B835
2067 - B836
Hydrogel Contact Lens (HCL) Wear in Controlled Adverse Environment (CAE)
M.J. Gonzalez-Garcia1A, A.Gonzalez-Saiz1A, J.M. Herreras1A, A.Mayo1A, la Fuente1B, J.San
Jose1B, J.Feijo1B, M.E. Stern2, M.Calonge1A. AOcular Surface Group, IOBA, BCAE Laboratory,
School of Architecture, 1University of Valladolid, Valladolid, Spain; 2Biological Sciences,
Allergan, Inc., Irvine, CA.
Purpose: Hydrogel contact lenses (HCL) comfort decreases with wearing time, which exacerbate in adverse
(ie low humidity) environments. We exposed low and high water content HCL symptomatic wearers to a
controlled adverse environment (CAE) to test influence of low humidity on ocular surface health and HCL
tolerance. Methods: Ten symptomatic HCL wearers with 2 out of 4 dry eye tests altered were recruited
and exposed to CAE (22 ± 2ºC, 20% ± 5% humidity, no air flow, reading activity) for 2 h. In visit 1 subjects
wore no HCL. In VISIT 2, subjects were fitted with LOW water content (silicone hydrogel) HCL. In VISIT 3
subjects wore HIGH water content HC. Visits 2 and 3 was randomised and double masked. Comfort, dry eye
symptoms, NIBUT, BUT, bulbar and limbar hyperemia, fluorescein staining, phenol red thread test, Schirmer
test and tear lysozime concentration were evaluated before and after CAE exposure. Results: Significant
differences (Student’s t test) before and after CAE exposure in the different visits were found (table1). Table
1: Significant (p<0.05) changes after CAE exposure (mean difference ± standard deviation)
Visit 1 (no ↓ 0.8 ± 1.6
2 (low
↑ 1.1 ± 1.2
Visit 3
↓ 1.0 ± 0.7 ↑ 0.4 ± 0.7
↑ 0.09 ± 0.1
↑ 0.1 ± 0.14
↑ 0.1 ± 0.2
↑ 1.0 ± 0.7
red thread
↓ 3.6 ± 7.6 ↓ 3.5 ±
↑ 5.6 ± 3.7
↑ 0.5 ± 0.5
↑ 1.1 ± 1.9
↓ 3.9 ±
In addition, low and high water content HCL behaved similarly in this low humidity environment. Conclusions:
Symptoms and signs (conjunctival hyperemia and fluorescein staining) increased and comfort decreased when
HCL were worn in CAE. Lens water content made no difference in this type of dry environment. Additionally,
even without wearing HCL, low humidity had an impact on ocular surface health.
CR: M.J. Gonzalez-Garcia, None; A. Gonzalez-Saiz, None; J.M. Herreras, None; A. Mayo, None; B. de
la Fuente, None; J. San Jose, None; J. Feijo, None; M.E. Stern, Allergan, Inc. E; M. Calonge, None.
Support: None.
Risk of Infiltrates and CLPC With Traditional Hydrogel and Silicone Hydrogel
Extended Wear: A Meta Analysis
L.B. Szczotka-Flynn1A, M.Diaz-Insua1B. AOphthalmology, BEpidemiology & Biostatistics,
Case Western Reserve Univ, Cleveland, OH.
Purpose: High Dk silicone hydrogel (SH) lenses have been shown to significantly
decrease the risk of hypoxic complications compared to traditional low Dk hydrogels
(H). However, the risks of inf lammatory and mechanical complications with SH
compared to H lenses are not as clear. A meta analysis was performed to combine
the relevant literature to evaluate the risks of corneal inf lammatory events and
contact lens papillary conjunctivitis (CLPC) between SH and H extended lens wear.
Methods: Twenty-two studies published or presented on either or both arms by November 2004
were selected for analysis. Six studies were published in the 1990s. A total of 6,343 subjects
and 12,139 eyes comprised the entire sample. Nineteen studies were prospective studies, of
which 89% used a randomized scheme to assign lenses. Nine studies were performed in the
US, nine in Europe, three in Australia, and one in India. The follow-up ranged from 4-36
months, with a median of 12 months. Infiltrates were defined as any occurrence of CLPU,
CLARE, or IK, or as defined by a given study. A generalized mixed model framework
was employed to combine the information from the 22 studies. Values were converted
from persons to eyes in studies that only reported results based on number of participants.
Results: The probability of an event per 100 eyes in the traditional hydrogel group was
4.34 (2.68, 6.94) and 2.24 (1.27, 3.81) for infiltrates and CLPC, respectively. The pooled
estimates of the occurrence of infiltrates were not significantly different between the lens
groups (odds ratio 0.99 (0.75, 1.32) for SH compared to H lenses). There was an increased
risk for CLPC in the SH group compared to the H group (odds ratio 1.48 (1.09, 2.01)).
Conclusions: SH lenses have the same overall risk for corneal infiltrates as H lenses, however,
there is a greater risk for CLPC with SH lenses.
CR: L.B. Szczotka-Flynn, Vistakon C; Ciba R; Bausch & Lomb R; M. Diaz-Insua,
Support: NIH EY015145-02 and K23 EY015270-01, RPB, Ohio Lion’s
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2050-5732 / B819-B844A
261. Contact Lens II Organizing Section: CO Contributing Sections: CL, VI
2068 - B837
2069 - B838
Wearer Retention and Disposition in the Post Approval Evaluation of Lotrafilcon a
in the US
R.L. Chalmers1, J.J. McNally2, O.Schein3, J.Tielsch4, J.Katz4, E.Alfonso5, M.A. Bullimore6,
D.O’Day7, J.Shovlin8. 1Clinical Trial Consultant, Atlanta, GA; 2CIBA Vision Corp.,
Duluth, GA; 3Johns Hopkins Medical Institute, Baltimore, MD; 4Johns Hopkins
Bloomberg School of Public Health, Baltimore, MD; 5Bascom Palmer Eye Institute,
Miami, FL; 6The Ohio State University College of Optometry, Columbus, OH; 7Vanderbilt
Eye Institute, Nashville, TN; 8Northeastern Eye Institute, Scranton, PA.
Purpose: To report the one year retention and disposition of wearers prescribed 30 night
continuous wear who were registered in the post-market evaluation of lotrafilcon A lenses in
the US. Methods: 6,245 wearers who intended to use a 30 night continuous wearing schedule
of lotrafilcon A lenses registered for the study in 131 practices in the US and Canada. Practices
were selected based on volume of lens sales and included private practices, corporate practices,
and retail settings. After registration, wearers were contacted directly at 3 and 12 months
and asked, among other questions, whether they had experienced an episode of painful,
red eyes which caused them to seek eye care. Any events that presented to the registering
practice were reported directly by the practice. All events were reviewed for the presence
of a corneal infiltrate and forwarded to an Endpoint Adjudication Committee. Results: Of
the enrolled cohort, 5,903 (94.5%) wearers completed final exit questionnaires and only 342
(5.5%) were lost to follow-up. 4,995 wearers (80.0%) completed the one year period wearing
lenses and 908 (14.5%) discontinued wear of the study lenses. Of the reported events, 167
(2.7%) corneal infiltrates of varying severity have been confirmed in 158 wearers (2.5%).
At the 12 month survey 79.3% of wearers reported overnight wear of lenses for >21 nights
in a row; 9.3% wore them 1-2 weeks in a row; 7.6% for 1-6 nights and 3.2% for daily wear
only. Conclusions: The design of this study has resulted in a very high follow-up rate. The
infiltrate event rate in this post-approval evaluation was similar to that reported in the FDA
pre-market approval study, indicating robust performance of the lenses when released for
wider use outside of clinical trials.
CR: R.L. Chalmers, CIBA Vision C; Alcon C; Rose Biomedical C; Hydrogel Vision C; J.J.
McNally, CIBA Vision E; O. Schein, CIBA Vision C; J. Tielsch, CIBA Vision C; J. Katz,
CIBA Vision F, C; E. Alfonso, CIBA Vision C; M.A. Bullimore, Eyeonics C; Refractec C;
Alcon C; CIBA Vision C; Paragon Vision Sciences C; Staar Surgical C; D. O’Day, CIBA
Vision C; J. Shovlin, CIBA Vision C. Support: CIBA Vision, a Novartis Company
The Relationship Between Corneal Chemical Thresholds Estimated at Different Flow
Rates Using a Belmonte Pneumatic Esthesiometer
T.L. Simpson1A,1B, L.Henderson1A. ASchool of Optometry, BCentre for Contact Lens
Research, School of Optometry, 1University of Waterloo, Waterloo, ON, Canada.
Purpose: To examine the effects of different f low rates on cor neal
ch e m ic a l t h r e s h old s e s t i m a t e d u si ng a p n e u m a t ic e s t h e sio m e t e r.
Methods: 20 control subjects had chemical (burning/stinging) detection thresholds (%CO2)
estimated in the centre of their corneas. Stimulus temperature, flow rate and %CO2 were regulated
by a computer controlled Belmonte esthesiometer and subject responses were collected by the
computer using a button box. Initial mechanical thresholds were determined using a preliminary
ascending method of limits and a subsequent method of constant stimuli and then chemical
thresholds were estimated using an ascending method of limits. Flow rates for the delivery of the
CO2 mixtures were set at 100%, 75% and 50% of the mechanical thresholds (in random order).
Results: As the stimulus flow rate went down, CO2 detection threshold went up (repeated
measures ANOVA p<0.0001). When the flow rates were 75% and 50% of the threshold, the CO2
thresholds were 83% and 68% of the respective chemical threshold at the maximum flow rates.
Conclusions: The results illustrate the difficulty in measuring chemical thresholds with a
Belmonte esthesiometer because flow rate must be selected beforehand and this selection
has a direct effect on the measurement. The results also demonstrates the importance of
standardizing novel techniques to make comparison between studies possible. Finally,
because the thresholds are not simply related to the flow rates at which the chemical stimuli
are delivered, the results point to possible interactions between the mechanical and chemical
stimuli and/or interactions between the corneal chemical nociceptive channels.
CR: T.L. Simpson, None; L. Henderson, None.
Support: NSERC Canada operating grant
2070 - B839
2071 - B840
Corneal Sensitivity Change After Wearing Contact Lens
S.Xu1, F.Lu2, J.Jiang2, X.Mao2, W.Jin2, J.Qu2. 1SUNY State College of Optometry, New
York, NY; 2Eye Hospital, Wenzhou Medical College, Wenzhou, China.
The Effect of Soft Toric Contact Lens Wear on Corneal and Conjunctival Sensitivity
Measured With a Belmonte Esthesiometer
Y.Feng1, T.L. Simpson1, D.Fonn1, S.Hickson-Curran2. 1Centre for Contact Lens Research,
School of Optometry, University of Waterloo, Waterloo, ON, Canada; 2Johnson &
Johnson, Jacksonville, FL.
P ur pose : To i nvest igat e how cor neal a nd conju nct ival mecha n ical
s e n s o r y c h a n n e l s r e s p o n d t o t o r i c s of t c o n t a c t l e n s we a r.
Methods: 48 non-contact lens wearing healthy subjects (M=17, F=31) participated in this study.
Central corneal and inferior conjunctival mechanical sensitivities of both eyes were measured
at baseline (without contact lens wear) and after wearing lens for one, three, six hours and one
and two weeks. A Belmonte pneumatic esthesiometer was used to deliver stimuli at ocular
surface temperature. The ascending method of limits was used to estimate the threshold.
Results: At each time point during this experiment, corneal mechanical sensitivity
was higher than that of the conjunctiva. There was no difference in both corneal and
conjunctival sensitivities between eyes. After one, three, and six hours wearing of
contact lenses, corneal and conjunctival mechanical sensitivities increased. Repeated
measures ANOVA showed a main effect of time on corneal sensitivity (F (5,235) =
5.362, p = 0.000) and conjunctival sensitivity (F(5,235) = 4.752, p = 0.000). Both corneal
and conjunctival sensitivities gradually returned to baseline level by two weeks.
Conclusions: This study showed that corneal and conjunctival sensory channels respond to
contact lens wear with an initial transient increase in corneal and conjunctival mechanical
sensitivity before adapting to contact lens wear. It suggests that the ocular surface sensory
system response to contact lens wear is more complex than simple adaptation.
CR: Y. Feng, None; T.L. Simpson, None; D. Fonn, None; S. Hickson-Curran, Johnson
& Johnson E.
Support: NSERC Canada and Johnson & Johnson.
2072 - B841
2073 - B842
Purpose: To evaluate corneal sensitivity variation after wearing different types of contact lens in
children and adults. To determine the association of corneal sensation change with contact lens wear
period. Methods: A total of 80 subjects (160 eyes) were recruited. Subjects completed an extensive
questionnaire regarding age, type of contact lens, wearing periods, daily wearing time, diseases
associated with corneal sensitivity change; Corneal sensitivity at central, temporal, nasal, superior
and inferior locations was assessed using Cochet and Bonnet aesthesiometer. Contact lens wearer
subjects were grouped according to age (≤15y, >15y), wearing duration(≤1y,1-5y), and lens type (soft
contact lens (SCL) vs. rigid gas permeable (RGP) lens). Results: 1.In children (≤15yrs) wearing RGP
lenses we found that (1) Central and inferior corneal sensitivity was significantly decreased in subjects
wearing lenses for less than one-year or one-to-five-years (central: ≤1y P=0.000, 1-5y P=0.000;
inferior: ≤1y P=0.002, 1-5y P=0.000). No significant difference was found between one year and
1-5 years wear group (central P=0.997, inferior P=0.056); (2)Nasal , temporal and superior corneal
sensitivity significantly decreased for 1-5 years wear group(temporal=3.2350±1.53838, P=0.000;
nasal= 3.1650±1.5742, P=0.000; superior=3.8625±2.7623, P=0.001) but was not affected in subjects
wearing RGP lenses for one year or less (temporal=2.0167± 1.62527, P=0.06; nasal=1.9192±1.62197,
P=0.200; superior=2.2583±2.11207, P=0.207). 2.In adults (>15yrs) wearing RGP lenses we found
that (1)Central corneal sensitivity significantly decreased in subjects wearing lenses for less than
one-year or one-to-five-years(P=0.000).No significant change between these two groups was obs
erved(P=0.921);(2)There was no significant difference in CTT(cornea touch threshold) between
high-Dk RGP lens and low-Dk RGP lens for the one-year-wear group(P=0.263);(3)The effect of
soft lens on central corneal sensitivity was similar to RGP lens(1y P=0.239,1-5y P=0.366). 3.There
was no significant difference between children and adults for central corneal sensitivity change
after wearing RGP lens(1y P=0.343,1-5y P=0.105) Conclusions:The loss of sensitivity induced by
wearing RGP appears to plateau after the first few months. The effect of wearing RGP lenses on
corneal sensitivity loss in children and adults is similar. Both soft contact lens and RGP lens produce
a similar type of corneal sensitivity loss.The mechanism for changes in sensitivity resulting from
RGP wear is sensory adaptation to mechanical stimulation.
CR: S. Xu, None; F. Lu, None; J. Jiang, None; X. Mao, None; W. Jin, None; J. Qu, None.
Support: Natural Science Fund
Contact Lenses as a Diagnostic Tool I: The Uptake and Release of Steroid Hormones
by Commercial Hydrogel Lenses
B.J. Hughes1, P.Segu1, S.Narayanan1, C.Morris2, M.Venugopal3, L.Chapoy4, A.M.
McDermott1, J.P. Bergmanson1, F.P. Carney3. 1Texas Eye Research and Technology
Center, University of Houston, Houston, TX; 2Southern Cross University, Nsw, Australia;
CIBA Vision, Duluth, GA; 4HPM, Barrington, IL.
Purpose:The presence of female hormones in the tear film has been documented
and correlated to serum levels (Carney et al. 2005, The Ocular Surface, 3, s52). The
purpose of this study was to determine if contact lenses can be used as a collection
device for such analytes in tears allowing the monitoring of fertility through tear fluid.
Methods:Nelfilcon A lenses (-0.50 diopters) were incubated with either 3H-estradiol (23000pg/ml) or 3H-progesterone (5-100nM) diluted in phosphate buffered saline (PBS) or
artificial tear solution (ATF) for 2 hrs at room temperature with shaking. The lenses were
stored over night at 4oC, then the amount of hormone bound was determined by liquid
scintillation counting. This data was then confirmed by soaking lenses in non-radiolabelled
estradiol (0-500pg/mL) and progesterone (1-100nM) and detected via competitive
immunoassay. Release of progesterone from lenses after absorption was investigated
by soaking in 200μl of 5% ethanol solution for 2 minutes, 10 minutes and 3 hours.
Results:Both hormones were significantly bound to the contact lenses in a concentration
dependent manner (up to 350pg/lens estradiol and 870pg/lens progesterone, n=2).
Maximum uptake was achieved by 2 hours of incubation with no further increase being
noted with longer incubation times. For estradiol, binding was greatest when PBS (0.4 to
350pg/lens) was used rather than ATF (0.4 to 159pg/lens). These results were confirmed
by the competitive immunoassay technique. 95% of progesterone absorbed by the nelfilcon
A lens was recovered within 2 minutes of soaking in 200ul of 5% ethanol solution.
Conclusions:The results of this preliminary study indicate that contact lenses can successfully
be used as a collection device for diagnostics through tear fluid. Progesterone and estradiol
are taken up by contact lenses in a concentration dependent manner and can be released
within 2 minutes.
CR: B.J. Hughes, CIBA Vision F; P. Segu, CIBA Vision F; S. Narayanan, CIBA Vision
F; C. Morris, Ciba Vision E; M. Venugopal, CIBA Vision E; L. Chapoy, CIBA Vision F;
A.M. McDermott, CIBA Vision F; J.P. Bergmanson, CIBA Vision F; F.P. Carney, CIBA
Vision E. Support: None.
Contact Lenses as a Diagnostic Tool II: Hormone Uptake by Poly-Electrolyte Coated
Surfaces and Contact Lenses
P.Segu1, B.Hughes1, S.Narayanan1, F.P. Carney2, C.Morris3, M.Venugopal2, L.Chapoy4,
Y.Lvov5, A.McDermott1, J.P. Bergmanson1. 1Tertc, University of Houston, Houston,
TX; 2CIBA Vision, Duluth, GA; 3Southern Cross University, Nsw, Australia; 4HPM,
Barrington, IL; 5LA Tech, Ruston, LA.
Purpose: Contact lenses may be useful collection devices for detecting various analytes
in the tear film. With a view to determining their potential to assist in monitoring fertility,
we have investigated if hormones are taken up by poly electrolyte coated slides and lenses.
Methods: Glass slides were coated with different charged coatings prepared in the presence or
absence of salt using layer-by-layer technology. The slides were incubated with either 3H-estradiol
(2-3000pg/ml) or 3H-progesterone (5-100nM) diluted in phosphate buffered saline or artificial
tear solution for 2 hours at room temperature with shaking. The slides were stored over night at
4oC, then the amount of hormone bound was determined by liquid scintillation counting. Nelfilcon
A lenses with or without coatings were soaked in lutenizing hormone (0.1ug/ml - 0.5mg/ml)
overnight, washed, and the amount of hormone bound was detected by enzyme immunoassay.
Results: All coated slides showed higher hormone binding than control uncoated slides except
for progesterone uptake by poly(dimethyldiallyl ammonium chloride) (PDDA )/poly (acrylic
acid) (PAA) coated slides. Estradiol binding was greatest with poly(allylamine) hydrochloride
(PAH)/PSS (0.03+/-0.01 to 1.54 pg/slide) and PDDA/ poly(styrenesulfonate) (PSS) (0.03+/-1
to 1.3+/-0.07 pg/slide) slides prepared in the presence of salt. For progesterone, binding was
greatest with PAH/PSS (4+/-0.2 to 97+/-12 pg/slide), PAH/PAA (5+/-1 to 64+/-10 pg/slide)
and PDDA/PSS (5+/-0.8 to 74+/-7 pg/slide) slides prepared in the presence of salt. Lutenizing
hormone was taken up by the lenses and uptake was increased 2-3 fold in PAA coated lenses.
Conclusions: Uptake of steroid and peptide hormones is possible with polyelectrolyte coated
surfaces. Coated lenses showed enhanced hormone uptake. Contact lenses with specialized
coatings may be a viable, noninvasive, in vivo option for monitoring hormone levels.
CR: P. Segu, CIBA Vision F; B. Hughes, CIBA Vision F; S. Narayanan, CIBA Vision F;
F.P. Carney, CIBA Vision E; C. Morris, CIBA Vision E; M. Venugopal, CIBA Vision E;
L. Chapoy, CIBA Vision F; Y. Lvov, CIBA Vision F; A. McDermott, CIBA Vision F; J.P.
Bergmanson, CIBA Vision F.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2050-5732 / B819-B844A
261. Contact Lens II Organizing Section: CO Contributing Sections: CL, VI
2074 - B843
Ocular Responses To Contact Lens Wear on an Extended Wear Schedule in a Guinea
Pig Model for Lens Wear
A.Vijay1, P.Sankaridurg2, M.D. P. Willcox3, D.F. Sweeney2. 1Optometry, School of
Optometry and Vision Science, Sydney, Australia; 2Optometry, Vision CRC, Sydney,
Australia; 3Optometry, IER/School Of Optometry and Vision Science, Sydney, Australia.
Purpose: To develop an animal model for contact lens wear and to determine whether the
ocular responses to contact lenses were similar to the responses seen in human eye. Methods:
Guinea pigs were divided into 2 groups of 5 animals each and fitted with specially designed
soft contact lenses in one eye. Group 1 was fitted with low Dk hydrogel lenses and group
2 was fitted with high Dk hydrogel lenses. Low Dk lenses were worn on a 6 day and night
extended wear and disposal schedule and high Dk lenses were worn on a 30 day and night
continuous wear schedule. The study was conducted for 3 months and ocular assessments
(bulbar and limbal redness, endothelial polymegethism) conducted following 1 night, 1 week,
1 month, 2 months and 3 months of lens wear. Ocular assessments were conducted with slit
lamp biomicroscopy and confocal microscopy. Results: Bulbar redness was increased at all
the examination time points (p<0.05) in both low and high oxygen permeable lens wearing
eyes compared to control eyes; there was a clinical (Grade 2) and statistical increase in the
limbal redness response in low Dk lens wearing eyes when compared to control eyes after 3
months of lens wear (p<0.05). There was a significant increase in the co-efficient of variation
of endothelial cell size in low Dk lens wearing eyes after just 1 week of lens wear compared
to control eyes (p<0.05) and remained elevated through out the period of the study. There
were no significant differences in the limbal redness (p>0.5) or the co-efficient of variation
of endothelial cell size (p>0.1) between control and high Dk lens wearing eyes. These results
are in good agreement with the responses reported in human eyes with extended wear of high
and low Dk lenses. Conclusions: Guinea pig ocular responses following extended wear of
contact lens are consistent with human ocular responses. The guinea pig is a reliable model
that can be used for contact lens extended wear studies.
CR: A. Vijay, None; P. Sankaridurg, None; M.D.P. Willcox, None; D.F. Sweeney,
Support: None.
2075 - B844
Options And Limits of Visual Rehabilitation Using Contact Lenses in Patients After
Complicated Laser in situ Keratomileusis
U.Kloevekorn-Fischer1, K.Kloevekorn-Norgall1, G.I. W. Duncker2, C.GruenauerKloevekorn2. 1Institute of Optometry Trothe, Trothe-Optik, Halle, Germany; 2Department
of Ophthalmology, University of Halle, Halle, Germany.
Purpose: Certain changes in the anterior corneal surface and especially in the eccentricity
can be found in eyes following LASIK making the fitting of contact lenses difficult.
In our study we try to determine which special back surface design should be used in which case and
whether fitting of contact lenses could improve the visual acuity in complicated post LASIK cases.
Methods: The choice of the contact lens design depending on the corneal eccentricity was analysed
in 12 eyes of six patients who had undergone contact lens fitting following complicated LASIK.
Results: In all cases we chose the back surface of the initial lens depending on the measured
eccentricity. Using this strategy we managed to get optimal contact lens fit in 8 out of 12 cases
(67%). In 2 cases with negative eccentricity we had to change the back surface design from
reverse to aspheric with a good result. Although in 4 cases eccentricity was bigger than 0.7
we could fit an aspheric design. In 10 of 12 cases (83.3%) contact lens fitting was successful
with regard to an improvement of the visual acuitiy, loss of ghost images or correcting residual
refractive error. In two cases there was no improvement of visual acuity because of a central haze.
Conclusions: Contact lens fitting after LASIK is a safe and reliable procedure to improve
visual acuity and reduce complications such as ghost images or irregular astigmatism. Fitting
of rigid gas permeable contact lenses and the choice of a special back surface design depending
on the measured eccentricity minimize problems in contact lens fitting after LASIK and can
improve the tolerance and the visual results.
CR: U. Kloevekorn-Fischer, None; K. Kloevekorn-Norgall, None; G.I.W. Duncker,
None; C. Gruenauer-Kloevekorn, None.
Support: None.
5732 - B844A
Punctate Fluorescein Corneal Staining Observed Using Polyhexamethylene Biguanide
Containing Disinfecting Solution Not Indicative of Corneal Surface Damage
R.P. Barrett1, M.Mowery-McKee2, L.D. Hazlett1. 1Anatomy and Cell Biology, Wayne State
Univ Sch of Med, Detroit, MI; 2CIBA Vision, A Novartis Company, Duluth, GA.
Purpose: Punctate corneal staining with fluorescein is frequently observed in patients
wearing soft contact lenses (SCL) that have been disinfected in a polyhexamethylene
biguanide (PHMB)-containing anti-microbial solution. The purpose of this study was to
determine whether or not such staining is indicative of damage to the corneal epithelium
by this agent. The hypothesis tested was that staining with fluorescein is an artifact.
Methods: SCL (nelfilcon A) were soaked for 24 hours in either a PHMB-containing solution or in
phosphate buffered saline (PBS). SCL were fitted onto rat eyes (24 total) and then removed at 0, 1,
3 and 5 hours. Fluorescein was instilled into the eye of each rat and the eyes were observed using
slit lamp and the degree of fluorescence staining observed and photographed. Animals were then
sacrificed and the eyes enucleated and processed for scanning electron microscopic observation.
Results: Slit lamp observation showed differences in fluorescein staining between PBS
and PHMB soaked SCL wearing eyes. At 0 time, the surface of the cornea of PBS soaked
SCL wearing rats showed smaller, fewer punctuate fluorescent dots compared with PHMB
soaked SCL wearing rats. At 1 hour, the PHMB vs. PBS group showed large aggregates of
fluorescein. By 3 and at 5 hours, little to no punctuate fluorescent staining was detectable in
either group of eyes. Scanning EM of these corneas showed no morphologically detectable
effect of PHMB that would correlate with the slit lamp information and confirmed that the
cytoarchitecture of the corneal epithelial cells did not differ between the two treatment groups.
Conclusions: We conclude that corneal staining with fluorescein following contact lens
disinfection with PHMB is not indicative of damage to the surface of the cornea and that the
staining is an artifact that disappears within hours.
CR: R.P. Barrett , None; M. Mowery-McKee, CIBA Vision, A Novartis Company E; L.
D. Hazlett, None.
Support: CIBA Vision Novartis and P30EY04068
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2076-2101 / B845-B870
262. Stem Cells I Organizing Section: CO
2076 - B845
2077 - B846
Comparison of Growth Rates for ex vivo Expanded Limbal Epithelium on Amniotic
Membrane, Laminin, Hyaluronic Acid, Collagen I, Collagen IV and Fibronectin
D.S. A. Swaby1, S.Myriknas1, N.Forraz2, C.McGuckin2, C.Rostron1. 1Keratec Eye Bank, St
Georges Medical School, United Kingdom; 2School of Life Science, Kingston University,
United Kingdom.
Purpose: To compare the growth rate of human limbal explant cell cultures
established on human amniotic membrane with cell cultures established
on laminin, hyaluronic acid, collagen I, collagen IV and f ibronectin.
Methods: Six human limbal explants were prepared from a single corneo - scleral rim.
The cell cultures were established under identical cell culture conditions and grown within
cell culture inserts on human amniotic membrane, hyaluronic acid, collagen I, collagen
IV, fibronectin, laminin and co-cultured with mitomycin C inactivated 3t3 fibroblasts.
The cell cultures were examined by light microscopy and serial digital photographs were
obtained. The mean cell culture growth distance after twenty one days was calculated by
novel computer software developed by the stem cell research group at Kingston University.
Results: The computer software calculated mean growth distances after twenty one
days were: amniotic membrane - 0.43 mm; Hyaluronic acid - 0.22 mm; Collagen IV
- 0.24 mm; Laminin - 0.22 mm; Fibronectin - 0.09 mm and Collagen I - 0.14 mm.
Conclusions: Amniotic membrane produced cell cultures with the highest mean cell culture
growth distances. The release of intrinsic growth factors and the structure of the basement
membrane may explain the observed results. Collagen IV, laminin and hyaluronic acid all
produced similar growth distance values, however the mean growth distances were almost half
that observed with amniotic membrane. The use of Collagen I and fibronectin produced very
low growth distances and these were considered sub-optimal substrates for cell culture.
CR: D.S.A. Swaby, None; S. Myriknas, None; N. Forraz, None; C. McGuckin, None; C.
Rostron, None.
Support: None.
Conjunctival and Limbal Epithelial Side Population and Low Side Light Scattering
Flow Cytometry Cohorts Contain a High Proportion of Stem Cells
J.M. Wolosin1, R.M. Lavker2, M.Zhou2, L.Cong1, M.Taveras1. 1Ophthalmology, Mt Sinai
School of Medicine, New York, NY; 2Dermatology, Northwestern University, Chicago, IL.
2078 - B847
2079 - B848
Purpose: After staining cells with Hoechst 33342, a DNA-binding fluorescent dye, flow cytometry plots
of blue vs. red emissions reveal the presence of a rare subset of cells (0.1-1.0 %), positioned to the blue
side of the blue/red norm. This cohort, present in many tissues, is known as a side population (SP) and
in several cases has been shown to be highly enriched in stem cells. We have previously demonstrated
SPs in the limbal and conjunctival epithelia (Budak, ARVO 2003 # 859; Int. J. Dev. Biol., 48:981). Now
we have assessed whether these SPs contain slow cycling cells, a main characteristic of stem cells.
Methods: Rabbits were maintained for 2 weeks under 5-bromo-2’-deoxyuridine (BrdU; a
thymidine analog) infusion by osmotic pumps and sacrificed at the end of the infusion or 6 weeks
later. Tissue BrdU uptake was monitored by indirect immuno-fluorescence. Freshly dissociated
conjunctival epithelial cells were sorted by flow cytometry into SP and non-SP cohorts, cytospun
onto slides, and stained with FITC-conjugated anti-BrdU. For the limbus, we analyzed a cohort
defined by extremely low light side scattering (LSSC), which markedly overlaps with the SP.
Results: SPs accounted for > 1 % of the total limbal or conjunctival epithelia cell populations.
In all experiments at least 50 % of the SP cells displayed the LSCC phenotype. Conversely, more
than 50 % of the LSSC cells were SP cells. The relatively stem cell-free corneal epithelium was
devoid of either SP or LSSC cells. The BrdU infusion labeled a great proportion of the basal
and suprabasal limbal-corneal and conjunctival epithelial cells. Consistent with this labeling,
more than 80 % of the cells in the main cytometer-sorted fractions (contain > 90 % of the cells)
were BrdU-positive. In contrast, at least 70 % of the cells in the small conjunctival SP or limbal
LSSC were BrdU-negative, indicative that these cells were mostly quiescent during the twoweek labeling period. After a 6-week chase, label-retaining cells (LCRs) were observed only
and sparsely within the basal limbal and conjunctival zones. Consistent with this result, the main
(i.e., non-SP, non-LSSC) cytometer fractions contained very few LCRs. In contrast, 22 % of the
conjunctival SP cells and 16 % of the limbal LSSC cells were BrdU-rich, indicative that cells
labeled during the infusion period underwent minimal cell division over the long chase period.
Conclusions: These results indicate that the conjunctival and limbal SP and LSSC flow cytometry
cohorts are highly enriched in slow cycling stem cells.
CR: J.M. Wolosin, None; R.M. Lavker, None; M. Zhou, None; L. Cong, None; M. Taveras,
Support: EY014878; EY015132
Basement Membrane Formation of Limbal Corneal Epithelial Cells Expanded on
Intact or Denuded Amniotic Membrane
W.Li1, H.He2, T.Kawakita1, S.C. G. Tseng1. 1Ocular Surface Center, South Miami, FL;
TissueTech Inc., South Miami, FL.
Purpose: Previously we reported that limbal epithelial phenotype is preserved
by intact amniotic membrane (iAM), while corneal epithelial phenotype is
preserved by denuded AM (dAM). We wonder whether the basement membrane
(BM) formation may be different during ex vivo expansion on iAM or dAM.
Methods: Human limbal explants or limbal corneal epithelial sheets isolated by dispase from
eyebank limbal rings, were cultured on iAM or dAM under a submerged manner in SHEM
medium for 4 weeks. Cryosections of the limbal explant and the epithelial outgrowth were
immunostained for collagen IV, collagen VII, laminin 5, and perlecan, i.e., BM components.
Results: In controls, collagen IV, collagen VII, laminin 5, and perlecan were all present
in the BM of human corneal and limbal epithelia, but all except for perlecan were present
in the BM of human AM. After 4 W culturing, all four BM components were degraded in
limbal explants, more so when cultured on dAM than on iAM. Collagen IV, collagen VII, and
laminin 5 were strongly stained underneath the epithelial outgrowth when limbal explants
or epithelial sheets were cultured on iAM. In contrast, collagen IV, laminin 5, and perlecan
were weak and diffuse while collagen VII was negative underneath the epithelial outgrowth
when limbal explants and epithelial sheets were cultured on dAM. Perlecan staining was
negative when limbal corneal epithelial sheets were cultured on either iAM or dAM, but
was much more strongly positive when limbal explants were cultured on iAM than dAM.
Conclusions: Intact AM, which retains devitalized amniotic epithelial cells, prevents
degradation, thus helping BM formation during ex vivo expansion of corneal limbal epithelial
cells. The presence of perlecan in cultured explants but not pure epithelial sheets strongly
supports that new BM sythesis is further facilitated by stromal fibroblasts in the explant.
This new information helps future refinement of ex vivo expansion.
CR: W. Li, TissueTech Inc. E; H. He, TissueTech Inc. E; T. Kawakita, TissueTech Inc. E;
S.C.G. Tseng, TissueTech Inc. I, C, P.
Support: NIH EY06819 (to SCGT)
The Stable and Ectopic Presence of Conjunctival Epithelial Stem Cells in a
Conjunctivalized Cornea
T.Nagasaki, J.Zhao. Ophthalmololgy, Columbia University, New York, NY.
2080 - B849
2081 - B850
Isolation of Adult Stem Cell From Corneal Limbal Epithelium and Autologous
Transplantation for Limbal Stem Cell Deficiency in Rabbit
T.Mimura1A, S.Yamagami1B, T.Usui1A, S.Yokoo1B, K.Ono1A, M.Araie1A, S.Amano1A.
Departmemt of Ophthalmology, BDepartment of Corneal Tissue Regeneration,
University of Tokyo Graduate School of Medicine, Tokyo, Japan.
Pur pose: To isolate adu lt stem cells of cor neal li mbal epit helial cells
a nd i nvest igate a novel st r ateg y for tot al li mbal stem cell def iciency.
Methods: An ocular surface injury was created in right eye of each of thirty adult New Zealand
white rabbits by a lamellar keratectomy. Autologous adult stem cells, isolated from rabbit corneal
limbal epithelial cells by sphere-forming assay were cultivated for 2 weeks on a denuded amniotic
membrane. The sphere colonies and their progeny were examined by immunocytochemistry and
RT-PCR. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces
were surgically removed and the autologous cultivated adult stem cells were transplanted (sphere
group). The no transplantation, AM (rabbits with an amniotic membrane transplantation only),
and limbs-2 and 4 weeks (rabbit with transplantation of corneal limbal epithelial cells cultured
on amniotic membrane for 2 or 4 weeks) groups were the controls. Each group, observed for 8
weeks after surgery, was underwent external examination and immunhistochemical analysis.
Results: Immunocytochemistry and/or RT-PCR shows the sphere colonies expressed BrdU, P63,
P75NGFR and nestin and the progeny expressed cytokeratins 3, cytokeratin 12, vimentin, α-SMA,
NSE, and MAP2. The cultivated epithelial stem cells for 2 weeks were completely stratified over
ten layers on denuded amniotic membrane and expressed both P63 and P75NGFR throughout the
epithelium, whereas cultured limbal epithelium on amniotic membrane for 4 weeks consisted of 4-5
layers and expressed these markers at only the basal layer 8 weeks after transplantation. In the limbs-4
weeks and the sphere groups, injured corneas showed no epithelial defects, no neovascularization
into the cornea, and improved corneal clarity. Histologically corneal epithelium formed monolayer
in the AM, double layers in the limbs-2 weeks, 4-5 layers in the limbs-4 weeks, and approximately
10-12 layers in the sphere group. The new stratified epithelium showed the highest expression levels
of P63 and P75NGFR in the sphere group as compared with the other groups. Conclusions: We have
isolated adult stem cells of rabbit corneal limbal epithelium by sphere-forming assay. Autologous
adult stem cell transplantation can be an effective strategy to supply a large number of possible stem
cell marker, P63-postive cells, for corneal epithelial cell deficiency.
CR: T. Mimura, None; S. Yamagami, None; T. Usui, None; S. Yokoo, None; K. Ono, None; M.
Araie, None; S. Amano, None.
Support: HOYA Healthcare Corporation
Purpose: Limbal destruction has been known to trigger conjunctivalization of the
corneal surface. This study was initiated to determine whether epithelial homeostasis in
a conjunctivalized cornea is maintained by the presence of conjunctival epithelial stem cells.
Methods: Ubiquitous GFP mice were used. The limbal epithelium was removed in the entire
circumference and the injury was allowed to heal naturally, which lead to conjunctivalization of the
corneal surface. Epithelial cell movement in a conjunctivalized cornea was determined by monitoring
epithelial GFP patterns by in vivo time-lapse fluorescence microscopy. The presence of stem cells
was determined by mapping label-retaining cells (LRCs) in whole-mounts after pulse-chase with
bromodeoxyuridine (BrdU). In the first experiment, newborn mice were pulse-labeled with BrdU
for 4 days, and at 6 weeks the conjunctivalization was triggered by limbal destruction. Animals were
sacrificed 2 weeks later to determine LRCs in the conjunctivalized cornea. In the second experiment,
the animals, whose cornea had been conjunctivalized for at least 10 weeks, were pulse-labeled with
BrdU for 2 weeks, followed by a chase of >8 weeks to determine LRCs in the conjunctivalized cornea.
Results: Epithelial GFP patterns in a conjunctivalized cornea were stationary for at least 20 weeks,
and much longer in some occasions, suggesting that epithelial cells were generally immobile
in a lateral direction. Labeling with BrdU demonstrated that stationary GFP positive cells were
mitotically active, suggesting that they were capable of self-renewal in situ. In normal ocular
surface, LRCs were present in the conjunctiva and the limbus, but absent in the cornea. After
total conjunctivalization of the cornea, however, LRCs were now found throughout the cornea
proper, suggesting that conjunctival LRCs migrated to the cornea during corneal resurfacing.
Corneal LRCs could be also demonstrated by BrdU pulse-chase with corneas that had been
conjunctivalized for at least 10 weeks. Taken together, these results suggest that epithelial
homeostasis of conjunctivalized cornea is maintained by stationary conjunctival epithelial stem cells.
Conclusions: Conjunctivalization of the cornea is associated with stable, ectopic presence of
conjunctival epithelial stem cells, suggesting that conjunctival stem cells and their niches can
establish their presence in the corneal environment and continue to express their native, non-corneal
phenotype. Thus, eradication of conjunctivalization-associated pathology may require removal of
conjunctival epithelial stem cells from the cornea.
CR: T. Nagasaki, None; J. Zhao, None.
Support: NIH EY00431, RPB
Defining the Limbal Stem Cell Niche: The Role of Extracellular Matrix Components
F.E. Kruse, T.Dietrich, B.Seitz, U.Schlötzer-Schrehardt. Department of Ophthalmology,
University of Erlangen-Nuernberg, Erlangen, Germany.
Purpose: A specialized microenvironment (niche) is one of the key prerequisites for a stem cell
phenotype. In contrast to other tissues, the niche parameters of corneal epithelial stem cells have
not been defined. We therefore wanted to characterize the role of basement membrane components
in the generation of a microenvironmental niche for human corneal epithelial stem cells.
Methods: Corneal sections from 20 donors (age 61-91 years, post mortem times
1.5-15 hours) were stained by indirect immunofluorescence using a broad panel of
antibodies against integral and associated basement membrane components. In double
labeling experiments, commercially available antibodies against putative stem cell and
differentiation markers, e.g. p63, ABCG-2, connexin 43 (Cx43), were used in addition.
Results: Basement membrane components that were homogenously distributed in ocular
surface epithelia include laminin-1, laminin-5, laminin (α3, α5, ß3, γ1, γ2, and γ3 chains),
nidogen-1, perlecan, collagen type IV (α5 and α6 chains), collagen types VII, XV, XVII, and
XVIII, endostatin, fibronectin, fibrillin-2, matrilin-2 and -4, bamacan, and thrombospondin4. As compared to the corneal and conjunctival basement membrane, the limbal basement
membrane showed increased immunoreactivity for agrin, BM40/SPARC, laminin (α1, α2
and ß1 chains), collagen type IV (α1 and α2 chains), nidogen-2, collagen type XVI, versican,
and tenascin-C, whereas reduced immunoreactivity was observed for collagen type IV
(α3 and α4 chains), collagen type V, fibulin-2, fibrillin-1, and clusterin. Patchy staining
patterns of agrin, BM40/SPARC, tenascin-C, and versican appeared to co-localize with
p63/ABCG-2-positive and Cx43-negative cell clusters in the limbal basal epithelium. No
evidence was found for the presence of laminin α4 and ß2, fibulin-1,-3,-4, and -5, matrilin-3,
Smoc-1 and -2, testican-1,-2, and -3, in basement membranes of ocular surface epithelia.
Conclusions: Basement membrane heterogeneity in the limbal area may be involved
in providing a unique microenvironment for corneal stem cells. The characteristics of
this microenvironment could serve as tools for their selective enrichment and in vitro
CR: F.E. Kruse, None; T. Dietrich, None; B. Seitz, None; U. Schlötzer-Schrehardt,
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2076-2101 / B845-B870
262. Stem Cells I Organizing Section: CO
2082 - B851
2083 - B852
Expression of Keratinocyte Differentiation Markers in Rabbit Cornea During Wound
M.-H.Kao1, C.-C.Cheng1, D.Wang1,2, J.Chen1. 1Physiology, College of Med Chang Gung
Univ, Taoyuan, Taiwan Republic of China; 2Drug Biology, Bureau of Food and Drug
Analysis, Taipei, Taiwan Republic of China.
Purpose: Limbal epithelial stem cells (LSC) are the progenitors of corneal epithelium. In normal
cornea, the p63 positive cells are localized to the basal cells of the limbus but not in the central
cornea. In wound-healing cornea, p63 positive cells are present in both corneal and limbal
epithelium. The purpose of this study is to explore the possible roles of p63 in corneal wound repair.
Methods: Immunofluorescent staining was used to examine the expression patterns of
p63, PCNA and keratins 14 and 3 in freshly prepared wound-healing or normal corneal
tissues. Corneal and limbal explants derived from normal and wound-healing cornea/
limbus were cultured on amniotic membrane (AM) to examine their proliferative potential.
Results: It has been well established that corneal epithelium is strongly positive with
Keratin 3 and limbal epithelial basal layer is the site where Keratin 14-, PCNA- or p63positive cells are resided. Here we show that in the epithelium of wound-healing cornea,
the basal cells are positive with p63, keratin 14 and PCNA, and are negative with Keratin
3, characteristics of that of limbal basal cells. In contrast, Keratin 3 is expressed in cells
located from suprabasal to superficcal layers. Interestingly, immunofluorescent staining of
serial sections show that all p63 positive cells are also keratin 14 and PCNA positive. Similar
results were obtained with ex vivo cultured cornea with or without wounding. In vivo wounded
corneas were allowed to undergo repair to complete the reepithlization. On days 7th and
30th post-reepithelization, central corneas were excised and explanted on AM to evaluate
their growth potential. Central corneal epithelium taken from day 7th post-reepithelization
grew vigorously to form an epithelial outgrowth with a rate that was faster than unwounded
limbal tissue. In contrast, central corneas taken from day 30th post-reepithelization were
unable to grow and no epithelial outgrowth was formed. The growth potential of the wound
repaired central cornea is correlated with the expression of p63, Keraint 14 and PCNA.
Conclusions: Our results suggest that p63 is an important regulator for the proliferation of
corneal epithelial cells.
CR: M. Kao, None; C. Cheng, None; D. Wang, None; J. Chen, None.
Support: None.
In vivo Limbal Epithelial Culture and Graft for Limbal Deficiency
S.Ha, W.Park, D.Lee, S.Rho, K.Yoo, H.Ahn. Ophthalmology, Dong-a University Hospital,
Busan, Republic of Korea.
Purpose: To treat limbal stem cell deficiency, the epithelial characteristic in rabbits
with total limbal stem cell deficiency(LSCD) after reconstruction with autologous
corneal epithelial cells in vivo expanded on amniotic membrane was investigated..
Methods: The right eye of 10 rabbits were rendered total LSCD, verified by impression
cytology. The left eye of 10 rabbits, in vivo corneal etithelial cells culture was achieved. In
group A(n=4), we evaluate cultured corneal epithelial cells characteristic and in group B(n=6)
we evaluate transplanted corneal epithelial cells characteristic at postoperative 1, 2, 4 weeks.
Results: Successful epithelial growth was observed on amniotic membrane in all eyes of group A.
Transplanted epithelial cells was well remained in five eyes of group B. Each epithelial cells of group
A and B were confirmed with corneal specificity by immunohistochemical staining (AK-2, AE-5).
Conclusions: Transplantation with autologous corneal epithelial cells in vivo expanded
on amniotic membrane can successfully reconstruct corneal surface with unilateral total
CR: S. Ha, None; W. Park, None; D. Lee, None; S. Rho, None; K. Yoo, None; H. Ahn,
Support: None.
2084 - B853
2085 - B854
Experimental Transplantation of Corneal Epithelium Induced by PAX6 Gene
Trnsfection to Mouse Embryonic Stem (ES) Cell
R.Homma1A, H.Yoshikawa1B, M.S. Kurokawa1B, C.Masuda1B, E.Takada1B, H.Ueno1A,
K.Tsubota2, S.Ueno1A, N.Suzuki1B. ADept. of Ophthalmology, BDepts. of Immunology and
Medicine, 1St.Marianna Univ. School of Med., Kawasaki, Japan; 2Dept. of Ophthalmology,
Keio Univ. School of Med., Tokyo, Japan.
Characterization of Human Limbal Side Population (SP) Cells
H.Miyashita1A, S.Shimmura1B, Y.Matsuzaki2A, K.Higa1A, H.Okano2A, J.Shimazaki1B,
K.Tsubota2B. ACornea Center, BOphthalmology, 1Tokyo Dental College, Ichikawa, Japan;
Physiology, BOphthalmology, 2Keio University School of Medicine, Tokyo, Japan.
P u r po se : To i nve st igat e t he mole cu la r m a rke r expre ssion prof i le
a n d c olo ny fo r m i n g ef f ic i e n c y of l i m b a l e pit h el i a l SP c el l s .
Methods: Four to ten limbal segments from human eyebank corneas were treated enzymatically
to obtain single cell suspensions. Cells were incubated with Hoechst 33342 solution (4 ~ 5 μg
/ ml) with or without reserpine at 37 °C for 60 min, stained with propidium iodide (PI), and
analyzed by flow cytometry. Dead (PI +) cells were gated out. SP cells were defined as reserpine
sensitive Hoechst blue (low) and red (low) population. Cytospin samples of SP cells and main
population (MP) cells were stained with anti-keratin 19, anti-keratin 3, anti-vimentin, and
anti-MART1 (melanocyte marker) antibodies. Unsorted limbal cells were used as control. SP
and MP cells were cultured with mitomycin C treated 3T3 cells to observe colony formation.
Results: Percentage of SP cells in viable (PI-) cells ranged from 0.01 % to 0.92 %
(average=0.25 % ± 0.32 %, n= 10). K19, K3, vimentin, and MART1 positive cells
were 52.8 %, 10.8 %, 19.3 %, and 6.4 % in SP cells, 53.8 %, 26.6 %, 8.5 %, and 5.0 %
in MP cells, and 44.1%, 44.6 %, 9.7 %, and 2.0 % in unsorted limbal cells, respectively.
Colony forming efficiency (CFE) of SP and MP cells was 2.7 % and 2.0 %, respectively.
Conclusions: SP fraction of limbal epithelial cells contained fewer K3 (+) cells and higher
levels of vimentin (+) cells compared with the MP fraction, however, there was no difference
observed in CFE.
CR: H. Miyashita, None; S. Shimmura, None; Y. Matsuzaki, None; K. Higa, None; H.
Okano, None; J. Shimazaki, None; K. Tsubota, None.
Support: None.
2086 - B855
2087 - B856
Purpose: Embryonic stem (ES) cells can differentiate into any cell type and may be suitable for use
in allogeneic corneal transplantation because cornea is an immune privileged organ. To determine
whether transfection with the eye development-associated transcription factor, pax6, gene of ES
cells leads to their preferential differentiation to corneal epithelium, we transfected pax6 gene
into mouse ES cells, and the ES-derived epithelium was transplanted onto damaged mouse cornea.
Methods: Pax6 cDNA was ligated to a stable expression vector having green fluorescence protein (GFP)
gene and was electrotransfected to ES cells. G418 resistant cells were recovered for subsequent analysis
and transplantation. Corneal injury was developed by n-heptanol treatment of corneal epithelium of mice.
The corneal epithelial cells derived from pax6 transfected ES cells were transplanted onto a damaged
mouse cornea. Reconstitution of corneal epithelium was evaluated by confocal laser microscopy.
Results: The ES cells transfected with pax6 have differentiated exclusively, if not all, into epithelial
cells and formed a sheet of the epithelial cells. These cells expressed mRNA for cytokeratins and
Pax6. Thus the cells were applicable for corneal transplantation. When the pax6-transfected cells
were transplanted onto corneal lesion, they overlaid the corneal surface for at least 12 hours and
formed a mono-layer and bi-layers of epithelial cells. GFP positive ES-originated cells were detected
on the recipient cornea by confocal laser microscope, suggesting that the transfected ES-derived
epithelial cells have kept alive on the cornea. Contamination of other germ cells was not detected.
Conclusions:We have successfully transfected pax6 into mouse ES cells, that led to the differentiation
of ES cells into corneal epithelial cells exclusively. Pax6 gene transfection and subsequent
selection made purified corneal epithelial cells available for the transplantation, that were free
from contamination of other germ cells. Thus, the ES-derived epithelial cells were applicable for
transplantation of corneal injury in mice. ES cells may become an unlimited donor source of corneal
epithelial cells for clinical transplantation.
CR: R. Homma, None; H. Yoshikawa, None; M.S. Kurokawa, None; C. Masuda, None; E.
Takada, None; H. Ueno, None; K. Tsubota, None; S. Ueno, None; N. Suzuki, None.
Support: Ministry of ECSST,Japan
Experimental Study of Inducing Embryonic Stem Cell to Differentiate Into Corneal
Z.Wang, J.Ge, B.Huang, L.Wang, Z.Fan, L.Yu, B.Liu, J.Liu, X.Lu. Zhongshan Ophthalmic
Center, Zhongshan Ophthalmic Center, Guangzhou, China.
Purpose: Our project was to determine whether embryonic stem (ES) cells could be
induced to differentiate into corneal epithelia with superficial corneoscleral limbal stroma.
Methods: To achieve this, ES-GFP cells line D3 were pre-induced with retinoic
acid (RA). Then, the pre-induced cells were seeded on deepithelialized superficial
corneoscleral slices (SCSS). At last, one group of SCSS with confluent differentiated
cells in vitro were passaged for detection, another group of SCSS with confluent
differentiated cells were exposed to air-liquid interface for 10 days, then implanted
into subcutaneous layer of nude mice for 2 weeks for further induction in vivo.
Results: No teratomas were found 2 weeks after the implantation of differentiated ES cells
into nude mice. The differentiated cells showed an appearance of epithelia both in vivo and
in vitro. Expression of CK3, P63 and PCNA were detected by immunohistochemical stain
in the differentiated cells in both groups. Microvillis and zonula occludens were observed
on the surface of the differentiated cells under electronic microscope. In control group, ES
cells differentiated freely without any inducing factors. Most cells were shedding, majority of
survival cells formed a dendrite-like structure as neurons, and minority appeared polymorphic.
Conclusions: These results demonstrate that ES cells can differentiate into corneal epithelia
on the surface of SCSS under controlled condition. Differentiated ES cells can be used as
epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering
in the future.
CR: Z. Wang, None; J. Ge, None; B. Huang, None; L. Wang, None; Z. Fan, None; L. Yu,
None; B. Liu, None; J. Liu, None; X. Lu, None.
Support: 973 Program Grant G1999054309)
Stem Cells in the Central Cornea in Mammals
F.Majo1,2, A.Rochat2, T.Hoang-Xuan3, Y.Barrandon2. 1Ophthalmology, Hôpital Jules
Gonin, Lausanne, Switzerland; 2Ecole Polytechnique Fédérale de Lausanne / Centre
Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 3Ophthalmology, Hôpital
Bichat Claude Bernard / Fondation A. de Rothschild, Paris, France.
The cornea is supposed to contain only transient amplifying cells (TAC) with limited growth
capabilities generated by stem cells located at the limbus. Using a mouse model and a surgical
approach, we have demonstrated that the corneal epithelium of the mouse self renews for
months and can be serially transplanted and that the limbus does not normally contribute
to corneal renewal under physiological conditions. We have also demonstrated the central
cornea of several mammals contains clonogenic epithelial cells with extensive self renewal
capacities. Collectively, our results demonstrate that the corneal epithelium of many mammals
is self-maintained and contains stem cells. This implies that the mechanism of the renewal
of the human cornea is an exception in mammals or that it should be revisited. Our results
have significant implications in physiology, in pathology and for the therapeutic of ocular
surface diseases.
CR: F. Majo, None; A. Rochat, None; T. Hoang-Xuan, None; Y. Barrandon, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2076-2101 / B845-B870
262. Stem Cells I Organizing Section: CO
2088 - B857
2089 - B858
Transplantation of Autologous Cultivated Conjunctival Epithelium on Amniotic
Membrane for Total Limbal Stem Cell Deficiency in a Rabbit Model
K.Ono, S.Yamagami, T.Mimura, T.Usui, S.Yokoo, M.Araie, S.Amano. Ophthalmolgy,
University of Tokyo Graduate School of Medicine, Tokyo, Japan.
Purpose: To evaluate the feasibility of autologous cultivated conjunctival
epithelium on amniotic membrane for total limbal stem cell def iciency..
Methods: Lamellar keratectomy to create total limbal stem cell deficiency was performed
in the right eye of adult New Zealand white rabbit. Autologous conjunctival epithelium,
obtained from the left eye, was cultivated by co-culture with 3T3 fibroblasts on a denuded
human amniotic membrane for 3 to 4 weeks. Conjunctival epithelial sheets were examined
histologically and immunohistochemically. At 3 to 4 weeks after the lamellar keratectomy,
the conjunctivalized corneal surfaces were surgically removed and the autologous
cultivated conjunctival epithelial sheets were transplanted (Conj-AM group, n=10). The
no transplantation (n=6) and AM (rabbits with an amniotic membrane transplantation
only, n=10) groups were the controls. Each group, observed for 2 months after surgery,
was evaluated by the scoring systems of neovascularization and corneal opacity.
Results: The cultivated conjunctival epithelial sheets formed 3-5 layers on denuded
amniotic membrane in HE staining. Transmission electron microscopic examination
revealed hemidesmosome, desmosome, and microvilli on the cultivated conjunctival
epithelium. Averaged scores of corneal neovascularization and corneal opacity in the
Conj-AM group were significantly suppressed as compared with those in the AM and no
transplantation groups 2 months after transplantation. Immunohistochemical study showed
cytokeratin 3 and 12 expressions on the conjunctival epithelial sheets in the Conj-AM group.
Conclusions: Our findings indicate that transplantation of autologous cultivated conjunctival
epithelial sheet can be an effective strategy for total limbal stem cell deficiency in a rabbit
CR: K. Ono, None; S. Yamagami, None; T. Mimura, None; T. Usui, None; S. Yokoo,
None; M. Araie, None; S. Amano, None.
Support: None.
Methods to Transduce Primary Cultured Human Limbal Epithelial Cells
M.A. Barry1A,2, H.Mok2, L.Chen1B, D.-Q.Li1B, S.C. Pflugfelder1B. ACenter for Cell and Gene
Therapy, BOcular Surface Center, Cullen Eye Institute, Department of Opthalmology,
Baylor College of Medicine, Houston, TX; 2Bioengineering, Rice University, Houston,
Purpose: To develop targeted gene therapy vectors to specifically modify limbal stem
cells to treat ocular surface diseases. Limbal stem cells play a pivotal role in maintaining
ocular surface function and repairing damage to the corneal epithelium. Recent work
suggests that putative stem cells in the basal epithelium of the limbus express p63,
ABCG2, α9 integrin, β1 integrin, and the epidermal growth factor receptor (EGFR).
Methods: To determine the feasibility of targeting gene therapy vectors to these putative
stem cells, we have tested the ability of untargeted and targeted adenoviral (Ad) vectors to
genetically modify human limbal cells in vitro. Human corneal epithelial cells expanded
by limbal explant culture or limbal single cell suspension culture were exposed to Ad5
vectors expressing the green fluorescent protein (GFP). EGF conjugated to Ad using
bifunctional polyethylene glycol (PEG) molecules to specifically target EGFR positive cells
was also evaluated. The efficiency was compared with liposome transfection on these cells.
Results: Unmodified Ad5 vectors transduced approximately 50% of K3 keratin-positive
primary cultured limbal epithelial cells, while Ad5 conjugated to EGF transduced 7%
of these epithelial cells. In comparison, liposomes transfected 21% of these cells.
Conclusions: Adenovirus is the most efficient mean to transduce cultured human limbal
epithelial cells. Additional work will be required to determine if PEG or biotin-mediated
targeting of Ad vectors will enhance transduction of limbal stem cells in culture and in vivo.
CR: N. Support: NIH Grants, EY014553 (DQL) and EY11915 (SCP), an unrestricted Grant
from Research to Prevent Blindness, the Oshman Foundation and the William Stamps
Farish Fund.
CR: M.A. Barry, None; H. Mok, None; L. Chen, None; D. Li, None; S.C. Pflugfelder,
Support: None.
2090 - B859
2091 - B860
2092 - B861
2093 - B862
Defining the Limbal Stem Cell Niche: Role of Cell-Cell- and Cell-Matrix Adhesion
U.M. Schloetzer Schrehardt, T.Dietrich, B.Seitz, F.E. Kruse. Department of
Ophthalmology, Univ of Erlangen-Nuernberg, Erlangen, Germany.
Purpose: To characterize the role of cell surface receptors involved in cellcell and cell-matrix interaction as well as anchoring junctions in the generation
of a microenvironmental niche for human cor neal epithelial stem cells.
Methods: Cryosections of corneoscleral specimens from 20 fresh human donor eyes were stained
by indirect immunofluorescence using commercially available antibodies against various cellcell and cell-matrix adhesion molecules including integrins, cadherins, catenins, syndecans,
dystroglycan, CD44, and plectin. In double labeling experiments, antibodies against putative stem
cell and differentiation markers, e.g. p63, ABCG2, connexin 43 (Cx43), were additionally used.
Results: Basal cells of ocular surface epithelia including the limbus showed preferential
immunoreactivity for the cell surface receptors cadherin-P, CD44, and integrins α3, αv, ß1, and
ß5; dystroglycan, plectin, and integrins α6 and ß4 were restricted to their basal cell membranes.
In contrast, cadherin-E, ß-catenin, and syndecan-1 and -4 were predominantly localized to
suprabasal cells. Immunostaining for cadherin-P and -E, syndecan-1 and -4, and integrin α2,
which are involved in cell-cell adhesion, revealed small clusters of negative cells in the basal
limbal epithelium and appeared to co-localize with p63/ABCG2-positive and Cx43-negative
cells. These cell clusters were also associated with distinct gaps in the basal cell membrane
staining for dystroglycan, plectin, integrins α6 and ß4, which are involved in hemidesmosome
formation. Reduced numbers of hemidesmosomes were also observed along small immature
cells with a high nuclear:cytoplasmic ratio in the basal limbal epithelium by electron microscopy.
Instead, integrin α3ß1 was more abundantly present along the basal cell membranes at the limbus.
Conclusions: The absence of intercellular adhesion molecules may be an inherent feature of
limbal stem cells reflecting the need for the uniqueness of their microenvironment. Adherence
to their extracellular niche appears not to be primarily mediated by hemidesmosomes, but
rather by increased expression of the basement membrane receptor integrin α3ß1, enabling
individual stem cells to exit rapidly from their niche for self-renewal.
CR: U.M. Schloetzer Schrehardt, None; T. Dietrich, None; B. Seitz, None; F.E. Kruse,
Support: None.
Relationship Between Stem Cell Phenotypes and p63 Levels in Rabbit Conjunctival
and Limbal Epithelial Cells
S.P. Epstein, M.Taveras, P.A. Asbell, J.M. Wolosin. Ophthalmology Box 1183, Mount Sinai
Medical Center, New York, NY.
Purpose: The developmental morphogen p63 has been proposed to be a marker for
stem cells in the limbal/corneal epithelium (PNAS, 98:3156), a view undermined by
studies showing p63 staining within the stem cell-free corneal domain (DNA Cell
Biol. 21:443). Here, we examine our hypothesis (Epstein, ARVO 2004 #3770) that
the level of p63, rather than its mere presence, reflects the degree of cell stemness.
Methods: Flow cytometry was used to isolate Hoechst 33342 side population (SP) cells and
extremely low side light scattering (LSSC; see Results) cells from enzymatically dissociated
rabbit conjunctival and limbal epithelial cells, respectively. Two animals were infused with
BrdU prior to sorting to assess cell cycling frequency according to BrdU labeling index. SP cells
and control non-SP cells, representing the majority of the cell population, were cytospun onto
glass slides and fixed in formalin. These cells and human and rabbit limbo-corneal cryosections
were stained for p63 by indirect immunofluorescence using two anti-p63 clones. The intensity
of nuclear staining, used as a measure of p63 concentration, was measured by image analysis.
Results: SP and LSSC cells accounted for less than 1% of the total limbal or conjunctival epithelia
cell populations. In all experiments, at least 50% of the SP cells displayed the LSSC phenotype
and, conversely, more than 50% of the LSSC cells were found to be SP cells. Low levels of BrdU
incorporation after 2-week nucleotide analog infusion demonstrated the slow cycling nature
of the SP and LSSC cells. The average intensity of p63 staining for the conjunctival SP and
LSSC cells was 2.5-fold higher than their respective non-SP or non-LSSC control populations.
Limbal LSSC cells, in turn, were 3 times higher than their controls. In the tissue sections all
basal limbal cells were p63+. Yet 5-7% of them expressed p63 at clearly unique high levels.
Conclusions: Our results are consistent with a relationship in which the level of p63
nuclear concentration is maximal in stem cells and decrease gradually as cells become
more differentiated.
CR: S.P. Epstein, None; M. Taveras, None; P.A. Asbell, None; J.M. Wolosin, None.
Support: EY014878(JMW), EY015132(JMW), NEI#5P30EYO1867(JMW,PAA) and Research
to Prevent Blindness (JMW,PAA)
Reconstruction of Corneal Epithelium Using Corneal Limbal Stem Cell on Amniotic
J.-A.Lim, H.-J.Kim, C.-K.Joo. Ophthalmology and visual Science, Catholic University of
Korea, Seoul, Republic of Korea.
P ur pose : To exa m i ne t he cha r act er ist ics of recon st r uct ed cor neal
epithelium cultured on denuded amniotic membrane (AM) using cor neal
epithelial cells having differential propor tions of limbal stem cells.
Methods: Corneal epithelial cells were taken from human corneoscleral rims. After primary
culture, limbal stem cells were separated from corneal epithelial cells through Hoechst dye
efflux assay by flow cytometry (FACSVantage). The corneal epithelial cells having the
differential proportions with 1% and 4.5% of limbal stem cell were prepared to reconstruction,
respectively. Then, 3-dimensional (3D) culture for reconstruction of corneal epithelium
was cultured on 12-mm insert tied denuded human AM on bottom. Three weeks containing
airlifting later, reconstructed corneal epithelium was examined morphological research by
immunohistochemistry, confocal microscopy, electron microscopy, western bolt, and RT-PCR.
Results: Limbal stem cells were separated from corneal epithelial cells using
hoechst 33342 dye and verapamil by side population (SP). The reconstruction of
corneal epithelium was predominantly formed 5-7 layers on Millipore™ membrane
and amniotic membrane in vitro 3D culture system. The reconstruction efficiency
was enhanced on 3D culture of corneal epithelial cells having more limbal stem cells.
Moreover, the proportion of limbal stem cells in reconstr uction process
is ver y impor t ant to differentiation, proliferation, and st ratif ication.
Conclusions: The reconstructed corneal epithelium on amniotic membrane (AM) is healthier
than epithelium on other membrane. Also, the reconstruction of corneal epithelium having
more limbal stem cells on amniotic membrane was observed better differentiation, proliferation
and stratification, when compare with other having less in the same of period and culture
condition. This finding supports the important of stem cell number on AM for reconstruction
and clinical approach.
CR: J. Lim, None; H. Kim, None; C. Joo, None.
Support: The Cernsan Foundation for Eye Research
Comparative Analysis of Basement Membrane Composition of the Human Limbal and
Amniotic Membrane Epithelia
T.Dietrich, U.Schlötzer-Schrehardt, C.Hofmann-Rummelt, B.Seitz, F.E. Kruse.
Department of Ophthalmology, University of Erlangen-Nuernberg, Erlangen, Germany.
Purpose: Amniotic membrane has been widely used as a graft in ocular surface
reconstruction and as a substrate for the in vitro expansion of corneal limbal epithelial cells.
To determine, whether the specific extracellular niche parameters required for maintaining
an undifferentiated phenotype are reflected by this substrate, we comparatively analyzed the
basement membrane composition of the human limbal and amniotic membrane epithelia.
Methods: Cryosections of human corneoscleral specimens from 10 donor eyes and of 5
amniotic membrane specimens obtained at Cesarean sections were stained by indirect
immunofluorescence using a broad panel of antibodies against basement membrane components.
Results: Both the limbal epithelial and the amniotic epithelial basement membranes showed
positive immunoreactivity for collagen type IV (α1, α2, α5, and α6 chains), laminin (α3,
ß1, γ1, and γ2 chains), laminin-1 and -5, perlecan, nidogen-1 and -2, agrin, fibronectin,
collagen types VII, XV, XVI, and XVII, matrilin-4, and tenascin-C. Similarly, both basement
membranes were negative for collagen type IV (α3 and α4 chains), laminin (α4, ß2, ß3, and
γ3 chains), and collagen type V. Differences in distribution patterns of basement membrane
components were observed for laminin (α1, α2, and α5 chains), BM40/SPARC, collagen type
XVIII, endostatin, and matrilin-2, which revealed positive immunoreactivity in the limbal
basement membrane, but negative immunolabeling along the amniotic basement membrane.
Conclusions: In spite of minor differences, the basement membrane composition of amniotic
membrane largely resembles that of the corneal limbal epithelium suggesting that amniotic
membrane provides a proper microenvironment for the maintenance of an undifferentiated
phenotype. The findings, therefore, support the concept that epithelially denuded amniotic
membrane represents a suitable substrate for the in vitro expansion and transplantation of
limbal epithelial cells.
CR: T. Dietrich, None; U. Schlötzer-Schrehardt, None; C. Hofmann-Rummelt, None; B.
Seitz, None; F.E. Kruse, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2076-2101 / B845-B870
262. Stem Cells I Organizing Section: CO
2094 - B863
Spatial Distribution of Label-Retaining Cells in the Mouse Limbus
J.Zhao, V.Mo, T.Nagasaki. Ophthalmology, Columbia University, New York, NY.
Purpose: Corneal epithelial stem cells are believed to be localized within the basal layer of
the limbus, but their precise distribution is not known. This study was initiated to determine
the spatial distribution of individual label-retaining cells (LRCs) in the entire circumference
of the limbus and also in the cornea. Methods: Mouse pups were injected subcutaneously
with 5-bromo-2-deoxy-uridine (BrdU) twice daily for 4 days and were sacrificed after a chase
period of 6 to 11 weeks. An eyeball and skinless eyelids were removed together to prepare
a globe with continuous ocular surface from the cornea to the mucocutaneous junction. An
anterior segment was dissected to prepare a superior and an inferior piece in reference to
inner and outer canthus. BrdU immunoreactivity was determined in whole-mount specimens.
DAPI nuclear stain was used to differentiate epithelial cells of the cornea and the limbus,
and also vertical layers of the epithelium. BrdU positive cells (LRCs) in the cornea and the
entire circumference of the limbus were identified under a fluorescent microscope and plotted
manually. Since LRCs tended to cluster together, both individual cells and cell clusters
were counted and their distribution in different regions of the limbus compared. Limbal
blood vessels were identified by CD31 immunostaining. Results: A boundary between the
cornea and the limbus could be discerned by nuclear profile of basal epithelial cells; nuclei
of peripheral corneal cells were smaller, irregularly shaped, and contained barely detectable
nucleoli while limbal cell nuclei were larger, uniformly oval, and exhibited prominent nucleoli.
BrdU positive cells were found exclusively in the basal epithelium just outside the cornealimbus boundary, along the entire limbal circumference, but within a distance of roughly ten
cells from the boundary. Most of them were located near the cornea side of the limbal vessel
arcade, frequently forming a cluster of several cells. The total number ranged from 200 to
500 per eye. There were about 50%, on average, more cells in the temporal half than the
nasal half, and about 50% more in the superior than the inferior, in both right and left eyes.
Distribution of LRC clusters, about 150 per eye on average, was similar to that of individual
cells. In the cornea, a handful of LRCs were found in the suprabasal layer, but none in the
basal layer. Conclusions: Limbal LRCs are present in a narrow zone within the limbus that
can be defined precisely in reference to an anatomical boundary between the cornea and the
limbus. The results suggest that limbal stem cells are distributed asymmetrically within the
limbus. Epithelial stem cells appear to be absent in the cornea proper. CR: J. Zhao, None; V.
Mo, None; T. Nagasaki, None. Support: NIH grant EY0431 and RPB
2096 - B865
2095 - B864
Culture of Limbal Stem Cells-Measures to Reduce Cost and Widen Scope for
Developing World
R.Tandon1, N.Singh2A, T.Saxena1, H.Sharma2A, S.Kashyap2B, R.B. Vajpayee1.
Ophthalmology, All India Institute of Medical Sciences, Delhi, India; ADepartment of
Biochemistry, BOcular Pathology, 2AIIMS, Delhi, India.
Purpose: Limbal stem cells (LSC) have been expanded ex-vivo using growth media containing
Dulbecco-modified Eagle’s medium (DMEM) and various additives. Some of the adjuvants are not
readily available and others too expensive or practically inaccessible in developing countries. Limbal
stem cells are known to proliferate easily. The substantia propria of human amniotic membrane
(AM) contains many of the factors used in the culture media. The aim of this study is to try ex-vivo
expansion of LSC on AM using minimal adjuvants in the culture media and other cost effective
modifications. Methods: Cadaveric or autologous limbal tissue was divided in pieces of 1-2mm and
placed on AM. To stabilize the AM, various techniques were tried i.e. wrapping around a sterile
glass slide and suturing the edges, placing AM and denuded AM over a cover slip, on nitrocellulose
paper and directly on the surface of the culture plate. Limbal tissue was placed on the plate without
AM for comparison. The tissue pieces were allowed to adhere on the surface and then submerged
in the specially prepared culture medium containing DMEM, fetal bovine serum, insulin, adenine,
hydrocortisone, gentamicin and amphotericin B and incubated. The medium was changed every 2
days and extent of outgrowth monitored. After standardization of technique, clinical application
was done. Cells cultured on AM were transplanted in 5 patients with stem cell deficiency. Three had
localized stem cell deficiency in the form of pterygium, two had total stem cell deficiency. Patients
with allografts, were given topical 2% Cyclosporine A and systemic corticosteroids for 6 months.
Impression cytology was done after 6 months in all cases. Results: Visible outgrowth of epithelial
cells was seen in 2-3 days. The growth reached confluence and spread uniformly on the AM in 14-21
days except in the well in which the limbal tissue was kept without AM. The donor tissue kept on the
denuded amniotic membrane showed initial slow rate of growth (5-6 days) as compared to that grown
on non- denuded AM, but reached confluence in 14-21 days. None of the patients with pterygium had
recurrence in the follow up period. Both patients of chemical burn had a decrease in corneal scarring,
vascularization and improvement in vision. In all cases impression cytology done after 6 months
revealed absence of goblet cells on the cornea. Conclusions: Limbal epithelial cells can be cultured
on human AM for successful clinical use with minimal adjuvants in the culture media.
CR: R. Tandon, None; N. Singh, None; T. Saxena, None; H. Sharma, None; S. Kashyap, None; R.
B. Vajpayee, None.
Support: None.
2097 - B866
The Analysis of Human Limbal Epithelial Cells Cultured on Several Extracellular
Matrix Components
S.Ahmad1,2A, M.Lako2A, F.C. Figueiredo1,2B. 1Department of Ophthalmology, Royal
Victoria Infirmary, Newcastle upon Tyne, United Kingdom; AInstitute of Human Genetics,
Department of Ophthalmology, 2University of Newcastle upon Tyne, Newcastle upon
Tyne, United Kingdom.
Expression and Tissue Distribution of P63-Isoforms on Ocular Surface Epithelia
S.Kawasaki1, H.Tanioka1, K.Yamasaki1, C.J. Connon2, S.Kinoshita1. 1Ophthalmology,
Kyoto Prefectural Univ of Med, Kawarama Chi, Japan; 2School of Optometry & Vision
Sciences,, Cardiff University, Cardiff, United Kingdom.
Purpose: The functional significance of p63 in maintaining cell proliferative
capacity especially in the stem cells of various epithelial tissues has previously
been established. Although functional differences are still unknown, more than
six isoforms have been reported for this protein. We studied the expression and the
spatial distribution of the 6 p63-isoforms within human ocular surface epithelia.
Methods: Individual layers (basal, intermediate, superficial) of human ocular surface
epithelia (cornea, limbus, conjunctiva) were separately obtained using a laser microdissection device. These samples were equally amplified using a SMART™ PCR
cDNA Synthesis Kit and subjected to RT-PCR analysis with primer pairs, which
specifically recognize five isoform-determining regions and the six p63-isoforms.
Results: ΔNp63α was detected in the basal to intermediate layers of limbus and
conjunctiva. The other 5 isoforms were not detected in any layers of any epithelia.
Conclusions: These results suggest that ΔNp63α is the most dominant isoform in the human
ocular surface epithelia. This protein may contribute, at least in part, to the maintenance of
putative stem cells and early progenitor cells in limbal or conjunctival epithelium possibly
via its dominant negative effect on p53-dependent apoptosis.
CR: S. Kawasaki, None; H. Tanioka, None; K. Yamasaki, None; C.J. Connon, None; S.
Kinoshita, None.
Support: Grants-in-Aid (15791001) for scientific research from the Japanese Ministry
2098 - B867
2099 - B868
Purpose: The extracellular environments, and in particular the basement membrane zones,
of the limbal and corneal epithelium vary considerably. Because of this, we investigated
the culture of limbal epithelial cells on several extracellular matrix (ECM) components.
Methods: 3 adult human limbal rings (donor ages 74-80 years) donated for research were obtained
from UK Transplant. Limbal epithelial cells, retrieved from these rings by trypsinisation, were cocultured with mitotically inactivated mouse 3T3-J2 fibroblasts. Prior to confluence of these primary
limbal epithelial cultures, the 3T3 fibroblasts were removed, and the cultured limbal epithelial
cells were subcultured onto plates coated with collagen 1, collagen 4, fibronectin, laminin, or
Matrigel™ using 3T3-J2 fibroblast conditioned medium, and also onto 3T3 fibroblasts for comparison.
These cultures were then investigated using the following parameters - culture morphology
by phase contrast microscopy; p63 localisation in limbal epithelial colonies using fluorescent
immunohistochemistry; and cytokeratin (CK) 3/12, CK19 and p63 expression by flow cytometry.
Results: Limbal epithelial cells established and formed colonies on the ECM components in an
identical time period to the 3T3 fibroblast co-cultures (3-5 days). The morphology of colonies on
the ECM components however lacked the tight circular looking appearance of the colonies in the
3T3 co-cultures. Despite this difference in morphology, p63 expression by immunohistochemistry
was still observed in the colonies on the ECM components. Flow cytometric analysis revealed
the following changes in ECM, as compared to 3T3, established cultures - collagen 1 (+0.03%
CK3/12, +34.65% CK19, -1.24% p63); collagen 4 (+11.32 CK3/12, 35.87% CK19, +7.60% p63);
fibronectin (+32.50% CK3/12, +30.08% CK19, +12.83% p63); laminin (+12.32% CK3/12,
+15.21% CK19, +5.80% p63); and Matrigel™ (+22.06% CK3/12, + 33.72% CK19, -0.14% p63).
Conclusions: Although the limbal epithelial cells cultured on the various ECM components have
a more differentiated appearance morphologically as compared to 3T3 co-cultures, for collagen 4,
fibronectin, and laminin there is a significant increase in the expression of p63, a marker for corneal
epithelial progenitors. The preservation and expansion of limbal stem cells on these three ECM
components will require further investigation.
CR: S. Ahmad, None; M. Lako, None; F.C. Figueiredo, None.
Support: Newcastle Healthcare Charity
Melanocytes in the Corneal Limbus Interact With K19-Positive Basal Epithelial Cells
K.Higa1A, S.Shimmura1B, H.Miyashita1A, J.Shimazaki1B, K.Tsubota2,1A. ACornea center,
Ophthalmology, 1Tokyo Dental Coll, Ichikawa, Japan; 2Ophthalmology, Keio University
School of Medicine, Tokyo, Japan.
identif y
dist r ibution
melanocy tes
Methods: Human limbal tissues were examined by whole mounts and serial histological
sections to localize epithelial cells containing melanin granules. Immunohistochemistry
in the basal epithelial layer of the limbus was done for cytokeratin 19 (K19), and
melanocytes were identified by vimentin and the melanocyte-specific marker MART1. Tyrosinase activity, the enzyme responsible for melanin formation, was assayed
in non-pigmented donor tissue by the reaction with the enzyme substrate DOPA.
Immunocytochemistry with anti-K19 and anti-MART-1 was done in dissociated
limbal cells to analyze the ratio of MART-1 (+) melanocytes to K19 (+) epithelial cells.
Results: Whole mount tissue from a pigmented donor revealed densely pigmented tissue
corresponding to the palisades of Vogt in the limbal area. K19 positive cells were observed
lining the limbal basal epithelium, which were highly associated with melanin granules. A
superimposed image revealed that melanin granules were oriented towards the apex of each
basal cell, acting as a pigmented cap facing the ocular surface. Melanocytes identified by
vimentin and MART-1 were shown to exist as sporadic cells with dendritic processes that
extend to pigmented basal cells. K19 (+) epithelial cells comprised an average of 48.7 ± 10.4 %,
and melanocytes were found in the order of 5.3 ± 2.7 % of total cells in cytospin samples from
3 different donors. Tyrosinase assay confirmed melanocytes in non-pigmented limbal tissue.
Conclusions: Melanocytes were sporadically located in the basal limbal epithelium in both
pigmented and non-pigmented tissue. Tyrosinase positive melanocytes produce melanin,
which are transported predominantly to K-19 positive basal epithelial cells.
CR: K. Higa, None; S. Shimmura, None; H. Miyashita, None; J. Shimazaki, None; K.
Tsubota, None.
Support: None.
Interleukin-1 Receptor Antagonist (IL-1ra) Prevents Apoptosis in ex vivo Expanded
Human Limbal Epithelial Cells Cultivated on Human Amniotic Membrane
C.-C.Sun1, J.-H.S. Pang2A, C.-Y.Cheng2B, H.-F.Cheng2B, C.-S.Chien2B, Y.-S.Lee2A, W.-C.Ku1,
C.-H.Hsiao1, C.-M.Yang2A. 1Ophthaomology, Chang Gung Memorial Hospital, Keelung,
Taiwan Republic of China; AGraduate Institute of Clinical Medical Sciences, BDepartment
of Pharmacology, 2Chang Gung University, Taoyuan, Taiwan Republic of China.
Purpose: To investigate the differential expression of interleukin-1 receptor antagonist (IL1ra) in human limbal epithelial cells expanded on intact amniotic membrane (AM) or on plastic
dishes, and to demonstrate the anti-apoptotic function of IL-1ra protein in this co-culture model.
Methods: Corneoscleral buttons from human donor eyes were cut into 2 x 3 mm2 pieces
and cultured on intact AM, or plastic dishes for 3 weeks. Cultures of either condition
were subjected to phase contrast microscopic examination and annexin V staining to
detect apoptotic cells. Differential expression of genes in between these two conditions
was determined by cDNA microarray analysis, which was further confirmed by reverse
transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent
assay (ELISA). Cultures on plastic dishes at day 14 were added with media with or
without 200 ng/ml of IL-1ra protein for one week, followed by apoptotic studies.
Results: Limbal epithelial cells grown on intact AM demonstrated fewer apoptotic
cells as compared with those on plastic dishes. IL-1ra over-expression in cultures on
intact AM in both transcriptional and translational level was confirmed by cDNA
microarray analysis, RT-PCR and ELISA. The prominent apoptosis detected in cultures
on plastic dishes could be reversed by adding recombinant IL-1ra protein into the media.
Conclusions: Human AM may prevent cultured human limbal epithelial cells from undergoing
apoptosis. In addition to its well-recognized anti-inflammatory effect, IL-1ra may function as
an anti-apoptotic molecule during the interaction between limbal epithelial cells and AM.
CR: C. Sun, None; J.S. Pang, None; C. Cheng, None; H. Cheng, None; C. Chien, None; Y.
Lee, None; W. Ku, None; C. Hsiao, None; C. Yang, None.
Support: CMRPG 23002
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2076-2101 / B845-B870
262. Stem Cells I Organizing Section: CO
2100 - B869
Expression of p63 Transcription Factor by Limbal Keratinocytes Indicates Low
Differentiation and High Proliferative Potential
C.-C.Cheng1, D.-Y.Wang1,2, M.-H.Kao1, J.-K.Chen1. 1Physiology, College of Medicine
Chang Gung Univ, Taoyuan, Taiwan Republic of China; 2Drug Biology, Bureau of Food
and Drug Analysis, Taipei, Taiwan Republic of China.
Purpose: Limbal epithelial stem cells (LSC) are the progenitors of corneal epithelium and
p63 has been suggested to be the putative marker of the limbal epithelial stem cell. P63 protein
consists of six isoforms belonging to either the transcriptional activation (TA) from or the
delta N (ΔN) form. The possible roles of p63 protein isoforms in the regulation of corneal
differentiation lineage and the maintenance of limbal epithelial stem cell remained to be explored.
Methods: Real-time quantitative reverse transcription PCR (real-time Q-RTPCR), immunof luorescent staining, p63 antisense oligo blockage, and confocal
microscopy were employed to exam the expression patterns of p63, keratin 3
and keratin 14 in freshly prepared corneal and limbal tissues, and in limbal
explants and epithelial outgrowth cultured on amniotic membrane (AM).
Results: P63 positive cells are unevenly distributed in different quadrants of the
limbus and it is positively correlated with the potential of the epithelial outgrowth
when explanted on AM. Blockage of p63 expression with antisense oligos, especially
the ΔNp63, effectively, suppresses epithelial outgrowth. Immunostaining shows
that blockage of TAp63 with antisense oligo enhances Keratin 3 expression.
Conclusions: Our results show that the abundance of p63-positive cells is positively correlated
with the potential of the epithelial outgrowth of the limbal explant. In vitro study show that
ΔNp63 expression by antisense indicates that ΔNp63 is important for the proliferation of limbal
epithelial cells. In contrast, TAp63 seems important for limbal epithelial cells to stay in a less
differentiated state as the blockade of its expression enhances Keratin 3 expression.
CR: C. Cheng, None; D. Wang, None; M. Kao, None; J. Chen, None.
Support: None.
2101 - B870
Differential Expression of Type IV Collagen Isoforms: Implications for Stem Cell
P.Charukamnoetkanok1, Y.Sado2, N.SundarRaj1. 1Ophthalmology Department, UPMC Eye
Center, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Univ.
of Pittsburgh School of Medicine, Pittsburgh, PA; 2Ophthalmology Department, Division
of Immunology, Shigei Medical Research Institute, Okayama, Japan.
Purpose: Basement membrane is an important component of the stem cell
microenvironment (niche). Type IV Collagen is an essential constituent of
corneal basement membrane. This study evaluated differential expression of type
IV collagen isoforms (α-1,2,3,4,5,6) in the human central cornea and limbus.
Methods: Frozen sections of normal human cornea were treated with acetone
and 6 M urea in 0.1 M glycene-HCl buffer (pH 3.5). Immunohistochemistry was
performed using rat antihuman monoclonal antibodies against specific isoforms of
type IV collagen. Observations were made using confocal fluorescence microscopy.
Results: α1 and α2 (IV) collagens were detected only in the limbus and perivascular
region. α3 and α4 (IV) collagens were most abundantly located in the central cornea.
Interestingly, α3 isoform showed unique punctate pattern of expression in the limbal
region. The regions devoid of α3 isoform also lacked K3 keratin expression. α5 and
α6 collagens were expressed in both the central cornea and limbus. All isoforms
were present in the Descemet’s membrane but had different distribution pattern.
Conclusions: Isoforms of type IV collagen exhibited differential expression in the central
cornea and limbus. The absence of K3 expression in the cells located over the regions of
basement membrane devoid of α3 (IV) collagen raises a possibility that these specialized
regions may represent the stem cell niche, and the lack of α3 (IV) expression may be another
distinguishing feature of corneal epithelial stem cells.
CR: P. Charukamnoetkanok, None; Y. Sado, None; N. SundarRaj, None.
Support: NIH EY03263 and Core Grant EYO08098; grants from RPB and Eye and Ear
Foundation, Pittsburgh, PA
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2102-2128 / B871-B897
263. Corneal Epithelium: Signaling Organizing Section: CO
2102 - B871
2103 - B872
2104 - B873
2105 - B874
Caspase Activation in Human Corneal Epithelial Cells Using FRET
D.M. Robertson1, W.M. Petroll1, J.V. Jester2, H.D. Cavanagh1. 1Ophthalmology, UT
Southwestern Medical Center, Dallas, TX; 2Ophthalmology, UC, Irvine, CA.
Purpose: Activation of caspase 3, a cysteine protease involved in the apoptotic cascade,
precedes nuclear degradation and cell death. The purpose of this experiment was to
develop a fluorescence-resonance energy transfer (FRET) probe to dynamically evaluate
caspase 3 activation in telomerase-immortalized human corneal epithelial (hTCEpi) cells.
Methods: Enhanced yellow fluorescent protein, EYFP, was amplified from pEYFP by
PCR using a forward primer containing a 4 amino acid sequence specific for the caspase
3 proteolytic cleavage site DEVD. The fragment was directionally cloned into the N
terminus of an enhanced cyan fluorescent protein expression plasmid, pECFP-N1, with
the resulting sequence ECFP-DEVD-EYFP. ECFP-DEVG-EYFP, a mutant substituting
a Glycine for Aspartic Acid at the fourth amino acid residue and ECFP-EYFP, cleavage
site omitted, were used as controls. hTCEpi cells were plated and transiently transfected
using Lipofectamine. 1µM staurosporine was used to induce apoptosis. Cells were imaged
using a Nikon microscope equipped with individual CFP and YFP excitation and emission
filters and digitally acquired using a Photometrics CoolSnap Camera. The average regional
pixel intensity for CFP and YFP emission using the CFP excitation filter was measured for
each cell using MetaMorph Software and the ratio of YFP/CFP emission was calculated.
Results: Prior to induction of apoptosis, ratio imaging analysis of cells transfected
with either the DEVD, DEVG, or CFP-YFP FRET probe, showed a 2.43 ± 0.68 ratio
of YFP/CFP emission with CFP excitation. After addition of staurosporine to activate
caspase 3, the DEVD transfected cells showed a marked decrease in the ratio of
YFP/CFP emission to 1.01 ± 0.06 in single cells. Cells transfected with the control
FRET probes containing either the DEVG or CFP-YFP linker showed no change.
Conclusions: These findings indicate that FRET can be used to study dynamic activation of
caspase 3 in living hTCEpi cells. Stable cell lines expressing FRET probes for caspase 3 and
the upstream effector caspase 9 will enable caspase activation to be evaluated temporally
and spatially in a 4D epithelial cell culture model.
CR: D.M. Robertson, None; W.M. Petroll, None; J.V. Jester, None; H.D. Cavanagh,
Support: NIH Grant K08 EY015713, NIH Grant EY10738 and Research to Prevent Blindness,
CTCF Mediates Cytokine- and Stress-Induced Alterations in Pax6 Transcription
L.Lu, T.Li, Y.Xu. Molecular Medicine, Harbor UCLA Medical Ctr, Torrance, CA.
Purpose: Our previous study found that CTCF negatively controls Pax6 gene
expression through its interaction with a repressor element located at its upstream
promoter. The present work explored the molecular mechanism of this interaction.
Methods: Human and rabbit corneal epithelial cells (HCE and RCE cells) were cultured in DMEM/
F12 medium containing 10% FBS in 37 °C incubator gassed with 5% CO2. Western analysis
was used to determine target protein expressions. Pax6 transcriptional activity was measured
with the luciferase reporter assay. The CTCF binding activity to the Pax6 promoter repressor
was determined with the gel retardation assay for DNA-binding proteins. The gel retardation
assay used normal DNA repressor fragment or its various mutants with CTCF binding sites.
Results: UV irradiation reduced CTCF protein content in corneal epithelial cell, resulting
in decrease in CTCF DNA binding activity. In contrast, EGF (0 ng/ml) stimulated CTCF
expression and attenuated Pax6 repressor DNA-binding activity. The protein-binding ability
of the Pax6 promoter repressor was dependent on its 5 binding sites (CCCTC sequence) to the
CTCF protein. When the 5 CTCF-binding sites repeats were mutated, respectively with single,
double, triple and quadruple site mutations, the protein-binding activity of the Pax6 promoter
repressor was progressively reduced. When all 5 CTCF binding sites were mutated, its proteinbinding activity was no longer detectable. The results clearly indicate that the Pax6 promoter
repressor interacts with the CTCF protein. Additionally, the reporter assay showed that the
transcriptional activity of the Pax6 promoter was undoubtedly increased when its CTCF
binding sites in the repressor element were mutated, which indicates the functional significance
of this physical interaction between the Pax6 promoter repressor and the CTCF protein.
Conclusions: Our results reveal a regulatory mechanism that involves Pax6 transcription
regulation. This suggests that CTCF protein regulation of Pax6 transcription plays a significant
role in controlling corneal epithelial cell growth and fate in response to cytokine and stress
CR: L. Lu, None; T. Li, None; Y. Xu, None.
Support: NIH grants EY12953 & EY 15282
Stimulation of Corneal Epithelial Migration by a Peptide Corresponding to the
PHSRN Sequence of Fibronectin
A.Hattori1, Y.Usui1, K.Kitazawa1, M.Naganuma1, K.Kimura2, T.Nishida2. 1Laboratory,
Nitten Pharmaceutical Co., Ltd., Nagoya, Japan; 2Department of Biomolecular
Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube, Japan.
Purpose: Fibronectin and its integrin receptor play an important role in the migration
of corneal epithelial cells. In addition to its RGD sequence, the sequence PHSRN
constitutes another cell-binding site of fibronectin. We investigated whether
the PHSRN peptide stimulates corneal epithelial migration in vitro and in vivo.
Methods: The effect of PHSRN peptide on corneal epithelial wound closure was evaluated in
rabbits after epithelial debridement with 1-heptanol. The effect of PHSRN peptide on corneal
epithelial migration in vitro was assessed with an organ culture system of the rabbit cornea.
Results: The PHSRN peptide (2 nanoM to 2 micro M) increased the length of the path of
epithelial migration in the rabbit cornea in organ culture in a concentration dependent manner.
The control peptide NRSHP did not affect corneal epithelial migration in this system at any
concentration examined. The administration of eyedrops containing PHSRN peptide (20 micro
M to 2 milliM) in phosphate-buffered saline also facilitated corneal epithelial wound closure
in rabbits in a dose-dependent manner. Again, the control peptide NRSHP had no such effect.
Conclusions: These results demonstrate that the PHSRN peptide stimulates corneal epithelial
migration in vitro and in vivo.
CR: A. Hattori, None; Y. Usui, None; K. Kitazawa, None; M. Naganuma, None; K.
Kimura, None; T. Nishida, None.
Support: None.
Regulation of Epithelial Membrane Protein-2 (EMP2) Expression in Human Corneal
L.Luo1,2, M.Wadehra2, P.Coulam2, R.Levinson1, L.Gordon1,3. 1Ophthalmology, Jules Stein
Eye Institute, Los angeles, CA; 2Department of Pathology and Laboratory Medicine,
Jonsson Comprehensive Cancer Center, Los Angeles, CA; 3Ophthalmology, Greater Los
Angeles VA Heathcare System, Los Angeles, CA.
Purpose: Epithelial membrane protein- 2 (EMP2) is highly expressed in ocular tissues. EMP2
has an important role in modulation of cell surface protein expression including specific integrin
isoforms and changes in expression levels may coordinately alter biologic behavior in specific
epithelial cells. The purpose of this study is to explore expression of epithelial membrane
protein-2 (EMP2) after exposure to estrogen or the inflammatory mediators, interleukin1alpha (IL-1α) and interleukin-1beta (IL-1β), in cultured human corneal epithelial cells.
Methods: A human transformed corneal epithelial cell line was exposed to estrogen or
inflammatory mediators and analyzed for EMP2 expression by quantitative Western blot analysis.
Expression of estrogen receptor alpha (ERα) in the cell line was confirmed by Western blot
analysis. For estrogen exposure, the cells were cultured in charcoal treated serum supplemented
either with 10 uM/L, 20 uM/L estrogen or vehicle control for 72 hours. Other cells were cultured
for 72 hours in the presence of either 10 ng/ml, 20 ng/ml IL-1α or 10 ng/ml, 20 ng/ml IL-1β.
Results: Expression of ERa was found in human transformed corneal epithelial
cell line by Western blots. Compared with untreated cells and vehicle treated cells,
estrogen markedly stimulated expression of EMP-2. Exposure to IL-1b markedly
decreased the expression of EMP-2 to about 10% of the control, while IL-1a did
not significantly affect expression of EMP2 when compared with the control.
Conclusions: These findings demonstrate that estrogen and IL-1β may modulate expression
of EMP-2 in human corneal epithelium. The consequence of increased EMP2 may directly
or indirectly modulate cell-cell or cell-extracellular matrix interactions and regulate cell
adhesion. Stimulation of EMP-2 by estrogen is hypothesized to play a role in the pathogenesis
of dry eye.
CR: L. Luo, None; M. Wadehra, None; P. Coulam, None; R. Levinson, None; L. Gordon,
Support: Research to Prevent Blindness
2106 - B875
2107 - B876
In vitro Contact Lens Exposure Attenuates Pseudomonas aeruginosa-mediated Tolllike Receptor Gene Expression
I.Maltseva1, C.Basbaum2, S.Fleiszig1. 1Vision Science, UC Berkeley School of Optometry,
Berkeley, CA; 2Anatomy, UC San Francisco, San Francisco, CA.
Purpose: We previously reported that contact lens exposure (CLE) of corneal cells in
vitro inhibits the induction of human beta defensin-2 (hbd-2) by Pseudomonas aeruginosa
(PA). This seemed to derive from a failure of PA to stimulate JNK in the presence
of CLE. Hbd-2 expression requires both NF-kB and JNK activation, both of which are
downstream effectors of Toll-like receptor (TLR) signaling. To further understand the effect
of CLE on hbd-2 expression, we investigated its effect on TLR signaling induced by PA.
Methods: To determine whether TLRs were involved in hbd-2 expression, we co-transfected
HCE cells with a dominant negative construct for the common TLR adaptor protein MyD88
(MyD C) and an hbd-2 reporter plasmid. Cells were exposed to PA supernatant in serum-free
medium for 6 hours prior to luciferase assay. HCE were grown with or without CLE for 3.5
days. CLE was terminated and cells were exposed to PA for 6 hours before Real-Time PCR
analysis. Results: Cells expressing dominant negative MyD88 lost responsiveness to PA.
Real-Time PCR analysis revealed that P. aeruginosa induced mRNA expression of TLR2
4.8x, TLR4 9x and TLR5 2.5x. This response to the pathogen was decreased to 1.7x, 2x and
1.3x respectively in cells pre-exposed to the contact lens. Conclusions: PA stimulation of
hbd-2 promoter is TLR-mediated. In addition, PA exposure increased TLR 2, 4 and 5 gene
expression. This induction of expression is reduced in the presence of CLE. These data might
explain the observed decrease in JNK activity and subsequent decrease in hbd-2 expression
in cells co-cultured with a contact lens. They might also at least partly explain why contact
lens wearers are more susceptible to serious infection by PA.
CR: I. Maltseva, None; C. Basbaum, None; S. Fleiszig, None.
Support: K08 EY014473-01
Promotion of Corneal Epithelial Cell Migration by PHSRN, a Peptide Corresponding
to a Cell Binding Domain of Fibronectin
K.Kimura1, K.Kawamoto1, S.Teranishi1, K.Fukuda1, A.Hattori2, T.Nishida1. 1Biomolec
Recog & Ophthal, Yamaguchi Univ Sch Med, Ube city, Japan; 2Laboratory, Nitten
Pharmaceutical Co., Ltd., Nagoya city, Japan.
Purpose: Fibronectin plays an important role in the migration of corneal epithelial cells.
The RGD sequence in the cell binding site of fibronectin mediates the interaction between
fibronectin and its integrin receptor, whereas the PHSRN sequence, which constitutes
another cell binding domain of fibronectin, contributes to cell adhesion. We examined the
possible effect of a synthetic PHSRN peptide on corneal epithelial cell migration in vitro
Methods: Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured,
harvested by exposure to trypsin-EDTA, and replated in 96-well plates in the absence or presence
of PHSRN or the control peptide NRSHP at concentrations of 0.2 nM to 200 µM. The number
of attached cells was counted after incubation for 1 hour. The effect of PHSRN on HCE cell
proliferation in vitro was assessed by monitoring of mitochondrial metabolic activity with the
MTS substrate. The behavior of cells plated on glass-bottom dishes with or without PHSRN or
NRSHP was monitored by video microscopy. Cell migration was evaluated with a transwell assay.
Results: The PHSRN peptide had no effect on the adhesion or proliferation of HCE cells. In
contrast, PHSRN, but not NRSHP, promoted HCE cell migration in the transwell assay in
a concentration-dependent manner. Video microscopy also revealed that PHSRN induced
ruffling at the cell periphery and promoted cell migration, whereas NRSHP had no such effects.
Conclusions: These results demonstrate that the fibronectin-derived PHSRN peptide
stimulates the migration of HCE cells in vitro.
CR: K. Kimura, None; K. Kawamoto, None; S. Teranishi, None; K. Fukuda, None; A.
Hattori, None; T. Nishida, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2102-2128 / B871-B897
263. Corneal Epithelium: Signaling Organizing Section: CO
2108 - B877
2109 - B878
EGF Antagonizes TGFbeta Signaling in Human Corneal Epithelial Cells
A.E. Hutcheon1,2, X.Q. Guo1,2, J.D. Zieske1,2. 1Schepens Eye Research Institute, Boston,
MA; 2Department of Ophthalmology, Harvard Medical School, Boston, MA.
Purpose: It has been observed in non-ocular systems that EGF antagonizes TGFβ signaling. In
addition, we have observed that the expression of p15INK4B, a cell cycle inhibitor, is upregulated
in corneal epithelial cells by TGFβ, but blunted by EGF. Since Smad is a major pathway in
TGFβ signaling, we examined the effects of TGFβ and EGF on Smad 2 and 4-activation
(translocation). Methods: Primary human corneal epithelial cells were plated in four-well
chamber slides, grown to approximately 70% confluence, and then serum starved overnight.
Fresh media containing either EGF (0.5 or 5 ng/ml) or no growth factor was applied to the
cells for 15 minutes. After 15 minutes, the media was removed and fresh media containing
either EGF (0.5 or 5 ng/ml), TGF-β1, -β2, or -β3 (0.2 or 2 ng/ml), a combination of EGF
and TGFβ, or no growth factors (control) was applied for 30 minutes. The media was then
removed, cells were fixed with methanol, and indirect immunofluorescence was performed
with antibodies against Smad 2 and Smad 4. Results: Upon stimulating the cells with TGF-β1,
-β2 or -β3 only, Smads 2 and 4 were activated; however, upon EGF stimulation, neither Smad
translocated. When TGF-β1 was combined with EGF, both Smad 2 and 4 translocated into the
nucleus. This was also observed when TGF-β2 or -β3 high concentration was combined with
either EGF concentration. However, when TGF-β2 or -β3 low concentration was combined
with either EGF concentration, the amount of translocation of both Smad 2 and 4 was
greatly diminished. Conclusions: EGF does appear to blunt TGFβ signaling. Surprisingly,
there appears to be more of an effect on TGF-β2 and TGF-β3 than there is on TGF-β1. We
hypothesize that relative levels of EGF and TGFβ isoforms released in corneal wounds may
influence the wound response.
CR: A.E. Hutcheon, None; X.Q. Guo, None; J.D. Zieske, None.
Support: NIH Grant EY05665 to JDZ
Small Interfering RNA Identifies the Role of p57KIP2 and p15INK4b in TGF-β1
Inhibited Proliferation of Primary Cultured Human Corneal Epithelial Cells
L.Z. Chen, P.Stewart, C.Chu, L.M. Tong, S.C. Pflugfelder, D.Q. Li. Ophthalmology,
Baylor College of Medicine, Houston, TX.
Purpose: Small interfering RNA (siRNA) has become a powerful tool for silencing
gene expression and function in mammalian cells. This work was to evaluate a role of
cyclin-dependent kinase (CDK) inhibitors, p57KIP2 (p57) and p15INK4b (p15), in TGFβ1
inhibited proliferation of primary cultured human corneal epithelial cells using siRNA.
Methods: Primary cultured human corneal epithelial cells were treated with 1ng/ml of TGFβ1
for 6 and 24 hours, and subjected to total RNA extraction for RT-PCR with primers for CDK
inhibitors, p21, p27 and p57 (CIP/KIP family) and p15 and p19 (INK4 family). The effect
of TGF-β1 on cell proliferation was evaluated by BrdU incorporation and colony forming
efficiency (CFE) on a mouse 3T3 fibroblast feeder layer. For RNA interference assay, primary
cultures were passaged at 4x10 4 cells/cm 2 into 12-well plates in Keratinocytes Serum-Free
Medium for 24 hours, and transfected by annealed double-stranded siRNA (2 µg/well) for p57,
p15, or siRNA-Fluorescein (siRNA-F) as a negative control with Qiagen RNAiFect reagent
for 48 hours, followed by treatment with or without adding TGFβ1 for additional 6-24 hours.
Results: TGFβ1 significantly inhibited the cell proliferation showing a decreased BrdU
incorporation and CFE. TGFβ1 up-regulated the expression of p57 and p15 mRNA, while
did not effect the expression of p19, p21 and p27 after treatment for 6 and 24 hours. About
80% cells were transfected with these siRNAs in 6 hours without visible cell damage, as
indicated by siRNA-F and cell morphology. The TGF-β1 stimulated expression of p57 and p15
mRNA was markedly blocked by siRNA-p57 or -p15, respectively, but not by siRNA-F. The
TGFβ1 suppressed BrdU incorporation was increased to near normal by siRNA-p57 or -p15.
Conclusions: These findings demonstrate that the siRNA can be successfully transfected
into primary cultured human corneal epithelial cells and temporally knocked down the target
gene. The siRNA targeted to p57KIP2 and p15INK4b identifies their role in the inhibitory
effect of TGF-β1 on epithelial cell growth.
CR: L.Z. Chen, None; P. Stewart, None; C. Chu, None; L.M. Tong, None; S.C. Pflugfelder,
None; D.Q. Li, None.
Support: EY014553,EY11915, RPB & FFS grant,Oshman Fund, William Stamps Farish
Fund, Lion Eye Bank Fund
2110 - B879
2111 - B880
Wnt 7a Promotes Matrix Metalloprotainase 12-Dependent Proliferation in Human
Corneal Epithelial Cells
C.-K.Joo, J.Lyu. Ophthalmology & Visual Sci, Catholic Univ Korea/Coll Med, Seoul,
Republic of Korea.
Purpose: During wound healing, proliferation and migration are two different cell behaviors
in two different regions of the epithelium. The cell migration and proliferative responses
are believed to be regulated by several cues. However, the extracellular cues that induce the
compartmentalized responses to these events during wound healing are poorly understood.
Methods: Corneoscleral rims taken from human donor were used to culture the primary
human corneal epithelial (HCE) cells. Each scleral rim removed endothelial layer was treated
with Dispase II for 15 min, and epithelial cells were then isolated. For the assays used in this
study, the cells were plated on a diluted Matrigel matrix, which is similar to the basement
membrane of corneal epithelium, and incubated in a growth factor- and serum-free for 24 hrs.
Results: Here, we found that Wnt 7a is rapidly induced in a wounded cornea, and Wnt 7a
promotes the proliferation of corneal epithelial cells and enhances the wound closure. In
addition, matrix metalloproteinase (MMP)-12 expression was detected in the peripheral
epithelium, where the cells enhanced the rate of proliferation, but diminished in the migrating
central epithelium. The transcriptional activity of MMP-12 was found to be responsive to Wnt
7a. In particular, Wnt 7a induced the accumulation of β-catenin and the activation of Rac, and
β-catenin and Rac synergistically induced the transcription of this gene. The effect of MMP-12
on cell proliferation was also examined in order to evaluate functional consequences of MMP12 induction. The function-blocking of MMP-12 delayed the Wnt 7a-induced wound closure.
Conclusions: These results indicate that in addition to the β-catenin pathway, Wnt 7a
might induce the β-catenin-independent pathway, and Wnt 7a and MMP-12 can regulate the
proliferation of the corneal epithelial cells, which can contribute to the coverage of a defective
area during corneal wound healing.
CR: C. Joo, None; J. Lyu, None.
Support: Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea
Role of ROCK in G1/S Transition of Corneal Epithelial Cells: Regulation of CDK4
and Cyclins D1 and D3
N.SundarRaj, E.DeGarmo, L.DiCesare. Ophthalmology Department,, UPMC Eye Center,
Ophthalmology and Visual Science Research Center, Eye and Ear Institute,, Univ. of
Pittsburgh School of Medicine, Pittsburgh, PA.
Purpose: We have observed that the inhibition of a Rho-associated serine/threonine kinase
(ROCK) delayed cell cycle progression in corneal epithelial cells. The purpose of the present
study was to identify the cell cycle modulators that are regulated by Rho/ROCK pathway.
Methods: Rabbit corneal epithelial cells in culture were arrested in the G 0 phase of the
cell cycle by TGFβ1 treatment. TheTGFβ1 block was then removed to allow the cells to
progress through G1 and S phase, in the presence or absence of the ROCK inhibitor (Y27632).
BrdU labeling was employed to estimate the number of cells in the S phase and western
blot analyses was performed to determine relative levels of specific cyclins and CDKs.
Results: TGFβ1 treatment of P1 cultures of rabbit corneal epithelial cells resulted in arrest of the
cells in G0 with less than 1% of the cells in the S phase. While without ROCK inhibition 25+3.5%
of the cells had entered the S phase at 24 hours after the removal of the TGFβ1 block, with ROCK
inhibition only 8.1+ 2.6% were in the S phase. The difference was less apparent at 36 hours.
ROCK inhibition during the progression of G1/S, resulted in decreased levels of cyclins D1 and
D3 and CDK4 as compared to control cells which were not exposed to Y27632. The differences
in the relative levels of these proteins between the ROCK-inhibited and non-inhibited cells
were evident at 12 hours and maximum at 36 hours after the removal of the TGFβ1 block.
Conclusions: The Rho/ROCK pathway is involved in the control of G1/S progression of the cell
cycle of corneal epithelial cells in culture. Intracellular levels of at least three components of
G1 progression, including CDK4 and cyclins D1, D3, are regulated by Rho/ROCK activity.
CR: N. SundarRaj, None; E. DeGarmo, None; L. DiCesare, None.
Support: NIH grants EY03263 and Core Grant EYO08098; RPB, New York and Eye and
Ear Foundation, Pittsburgh
2112 - B881
2113 - B882
UVB Reduces Cornified Envelope Proteins and Barrier Function Through
Transglutaminase and C-Jun N-Terminal Kinase Pathways in Human Corneal
Epithelial Cells
L.M. Tong1,2, R.Corrales2, Z.Chen2, C.S. de Paiva2, D.Q. Li2, S.C. Pflugfelder2. 1Singapore
National Eye Center, Singapore Eye Research Institute, Singapore; 2Ocular Surface
Center, Cullen Eye Institute, Dept of Ophthalmology, Baylor College of Medicine,
Houston, TX.
Purpose: Stratified squamous epithelial cells assemble a specialized protective barrier structure
on their periphery termed the cornified cell envelope (CE). This study evaluated the expression and
regulatory signaling pathways of CE precusors (loricrin, involucrin and small proline-rich proteins
[SPRR1A, 1B, 2A, 2B, 3 and 4]) and later envelope proteins (LEP1, 6, 16 and filaggrin) by human
corneal epithelial cells in response to ultraviolet radiation (UVB). Methods: Primary epithelial cell
cultures were grown from human cadaveric limbal explants. A uniform single dose (20 mJ/cm 2) of
UVB was employed. Gene expression was evaluated by semi-quantitative RT-PCR. Western blotting
was performed using antibodies against transglutaminase (TG)-1 and phospho-c-Jun N-terminal
kinase (p-JNK). The incorporation of fluorescein-cadaverine (FC), a TG substrate, was used to
assess TG activity. The trans-epithelial electrical resistance (TER) was measured to evaluate the
barrier function of corneal epithelial cells. Results: Among 12 CE proteins studied, 9 genes including
involucrin, SPRR (1A, 1B, 2A, 2B and 3), LEP (1, 16) and filaggrin were expressed by human corneal
epithelial cells. A single dose of UVB down-regulated SPRR (1A, 2A, 2B), LEP (1, 16) and filaggrin
at 6 hours with reduced TER observed after 24 hours. UVB stimulated TG-1 production, which was
detected only in the insoluble fraction of cell lysate, and FC incorporation in the cell membrane as
early as 3 hours, as well as p-JNK within 1 hour. Monodansyl-cadaverine (MDC), a competititve
TG inhibitor, reversed the UVB induced changes in mRNA expression of certain CE components
(LEP1 and 16) and production of TG-1 protein. SP600125, a JNK inhibitor, opposed the stimulated
TG-1 production and FC incorporation, as well as the reduced expression of SPRR1B and filaggrin
by UVB. Either MDC or SP600125 restored the UVB reduced TER. Conclusions: These findings
demonstrate for the first time that acute UVB stress reduces expression of cornified envelope
proteins, such as SPRR1A, 2A, 2B, LEP1, 16 and filaggrin, and barrier function in human corneal
epithelial cells. This effect is mediated by stimulated TG-1 and activated JNK signaling pathways,
which could be potential therapeutic targets for ocular surface diseases.
CR: L.M. Tong, None; R. Corrales, None; Z. Chen, None; C.S. de Paiva, None; D.Q. Li, None; S.
C. Pflugfelder, None.
Support: NIH EY11915;EY014553; ASTAR Singapore; Res Prevent Blindness, Oshman Foun,William
Stamps Parish Fund
Characterization of the Promoter Region of Connexin 26 and Its Regulation by
Transcription Factors
A.R. Djalilian1A, D.M. McGaughey1A, S.Patel1B, J.A. Segre1B. ANational Eye Institute,
National Human Genome Research Institute, 1National Institutes of Health, Bethesda,
Purpose: The expression of connexins (gap junction proteins) are spatially and
temporally controlled in the corneal epithelium during normal and wound healing
conditions. This study was performed to characterize the promoter region of
connexin 26 and identify transcription factors that may regulate its expression.
Methods: Multiple primers and RT-PCR were used to localize the transcription start site
for mouse connexin 26 more precisely. The conserved sequences upstream of the human
and mouse connexin 26 were identified using the Pipmaker program. Potential transcription
factor binding sites were identified using TRANSFAC program. The regulation of
the mouse connexin 26 promoter by transcription factors was assessed by a luciferase
reporter assay following transient transfection into a mouse keratinocyte cell line.
Results: The connexin 26 first exon was found to begin at least 250 bp upstream
to that previously reported in Refseq. Besides the coding sequence, conserved
sequences were identified within the intron regions and up to 6 kb upstream of the
transcription start site. Both a 250 bp and 1 kb fragment immediately upstream of the
first exon were found to have promoter activity. A transcription factor was identified
with repressor activity on both the 250 bp and 1kb presumed connexin 26 promoter.
Conclusions: This study demonstrates the use of bio-informatics tools for promoter analysis
and provides insight into the regulation of the connexin 26 gene and its role in epithelial
CR: A.R. Djalilian, None; D.M. McGaughey, None; S. Patel, None; J.A. Segre, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2102-2128 / B871-B897
263. Corneal Epithelium: Signaling Organizing Section: CO
2114 - B883
2115 - B884
pRb2/p130 and PAI-2 Interaction in the Cytoplasm and Nucleus of Normal Human
Corneal and Conjunctival Cells
M.Massaro-Giordano1, M.Montanari2, C.M. Marshall3, A.Gambone4, C.Cinti5, G.Tosi6,7A,
A.Giordano4,7B, M.Macaluso4,7B. 1Ophthalmology, University of Pennsylvania ,Scheie Eye
Institute, Philadelphia, PA; 2Pathology, Catholic University of the Sacred Heart, Rome,
Italy; 3Dermatology, University of Pennsylvania, Philadelphia, PA; 4Biology, SHRO,
College of Science and Technology, Temple University, Philadelphia, PA; 5Physiology,
Institute of Clinical Physiology, CNR, Siena, Italy; 6Ophthalmology, NY Presbyterian,
Columbia Campus, New York, NY; AOphthalmology, BPathology and Oncology,
University of Siena, Siena, Italy.
Purpose: Under physiological conditions, the expression of the human plasminogen
activator inhibitor type-2 (PAI-2) gene is maintained at low to undetectable levels
in most cells. The molecular mechanisms regulating the PAI-2 basal levels are
unknown. However, we previously showed the PAI-2 and the pRb2/p130 proteins
are able to bind in vivo the same fragment of the PAI-2 proximal promoter, and
suggested that these proteins could play a role in controlling PAI-2 transcription.
Methods: Here we investigate the significance of PAI-2 and pRb2/p130 interactions in
normal cornea and conjunctiva cells. We performed immunoprecipitation experiments
using nuclear and cytoplasmic fractions from cycling normal cornea and conjunctiva cells.
Results: We found that both proteins interact in the nuclear and cytoplasmic fractions
of conjunctiva cells, whereas in the cornea cells pRb2/p130 and PAI-2 interact only in
the cytoplasm. Conclusions: Our hypothesis is that the pRb2/p130-PAI-2 interaction is
controlled by a biochemical balance between pRb2/p130 and PAI-2 proteins. Then, we suggest
t h a t t h e t r a n s c r ip t io n of t h e PA I-2 ge n e i s c o nt r ol le d by a
feedback loop t r iggered by the PA I-2 concent ration in the nucleus.
Our goal will be to understand, in a physiological setting, the biochemical
and biological significance of the pRb2/p130-PAI-2 interaction, and how they
carry out the molecular signals regulating this mechanism.
CR: M. Massaro-Giordano, None; M. Montanari, None; C.M. Marshall, None; A.
Gambone, None; C. Cinti, None; G. Tosi, None; A. Giordano, None; M. Macaluso,
Support: None.
AP-1 and COX-2 Expression in Corneal Epithelium Following Anti-Glaucomatous
Y.Okada1A, S.Saika1A, K.Shirai1A, T. Miyamoto1A, O.Yamanaka1A, T.Ueyama1B, Y.Ohnishi1A.
Department of Ophthalmology, BDepartment of Anatomy, 1Wakayama Medical
University, Wakayama, Japan.
Purpose: To examine the expression patter n of stress-related genes, cfos and c-jun, both the major components of AP-1, and cyclooxygenase2(COX-2) in rat corneal epithelium treated with anti-glaucomatous eyedrops.
Methods: Seventy-six male Wistar rats were used. We dropped anti-glaucomatous eyedrops
(0.5%timolol, 0.005%latanoprost or 0.12%unoprostone) on the one eye of rat. The affected eyes
were enucleated after various intervals. Frozen sections were processed for in situ hybridization
with c-fos, c-jun and COX-2 mRNAs or were stained with anti-c-Fos and anti-COX-2 antibodies.
Results: Thirty min after dropping 0.5%timolol, signals for c-fos and c-jun mRNAs were
detected in the corneal epithelium. Thirty to 60 min after dropping 0.005%latanoprost or
0.12%unoprostone, signals for c-fos and c-jun mRNAs were detected in the corneal epithelium.
Thirty to 90 min after dropping 0.005%latanoprost or 0.12%unoprostone, signals for COX2 mRNA was detected in the corneal epithelium. These signals were no longer evident
at 120 min. c-Fos protein was detected in the corneal epithelium 120 min after dropping
0.5%timolol, 0.005%latanoprost or 0.12%unoprostone. COX-2 protein was detected in
the corneal epithelium 120 min after dropping 0.005%latanoprost or 0.12%unoprostone.
Conclusions: Corneal epithelial cells are transiently transcriptionally activated at a very
early phase following anti-glaucomatous eyedrops. Expression of COX-2 following these
eyedrops may potentially induce inflammatory response in cornea.
CR: Y. Okada, None; S. Saika, None; K. Shirai, None; T. Miyamoto, None; O. Yamanaka,
None; T. Ueyama, None; Y. Ohnishi, None.
Support: None.
2116 - B885
2117 - B886
IGF-1 Activates EGF Receptor in Human Corneal Epithelial Cells
K.-S.Lee, J.Lyu, C.-K.Joo. Ophthalmology & Visual Science, Catholic Univ of Korea,
Seoul, Republic of Korea.
Purpose: Wound healing requires a complex processes to cover defect area and quickly
re-establish the barrier function of the injury. These include the proliferation and
migration of epithelial cells. The several growth factors involved in corneal wound
healing. Insulin-like growth factor 1 (IGF-1) of these factors has been known as a
stimulator the proliferation and migration of epithelial cells to coverage the defected
area. However, the cellular mechanism that regulates the separated process is unclear.
Methods: To investigate the cellular responses for IGF-1 in corneal epithelial cells, we
tested the proliferation and migration using the SV40 transformed cells (HCET). HECT
cells (4X102) were seed, incubated for 24 hrs in serum free-medium, and treated with
IGF-1. The rate of growth was determined by using the Proliferation Reagent WST-1.
The migration assays were performed by the 8 mm-pore Tissue Culture Inserts. HCET
were treated with IGF-1(50ng/ml) or EGF(25ng/ml), with a neutralizing antibody to
anti-EGFR or anti-IGF-1R to establish the intracellular mechanism. Western blotting
was then subjected with antibodies to anti-phospho-ERK and anti-phospho-Akt.
Results: ERK and AKT were rapidly activated by IGF-1. Interestingly, the activation
of ERK was reduced by the inhibition of EGFR, but not by the inhibition of IGF-1R. In
contrast, Akt activation was not changed by their inhibition. HCET cells treated with
IGF-1 revealed the increased proliferation and migration rate compared with control cells.
However, the proliferation and migration caused by IGF-1 showed the different signal
pathways. The growth rate of the HCET cells incubated with IGF-1 and a neutralizing
antibody to anti-EGFR was decreased compared with cells incubated with IGF-1 and
IgG. The migration of HCET cells was regulated by the IGF-1R, rather than EFGR.
Conclusions: In this study, we found that IGF activates both IGF-1R and EGFR in corneal
epithelial cells, and that IGF can promote the compartmentalized responses through different
pathway during wound healing. Our finding provides, to our knowledge, for the first time the
dual function of IGF in corneal epithelial cells.
CR: K. Lee, None; J. Lyu, None; C. Joo, None.
Support: Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea(03PJ1-PG10-20700-0002)
Signaling Transduction Pathways Required for ex vivo Expansion of Human Limbal
Explants on Amniotic Membrane
H.He1, H.-T.Cho2, W.Li1, T.Kawakita1, L.Jong3, S.C. G. Tseng1. 1TissueTech, Inc. and Ocular
Surface Center, Miami, FL; 2Ocular Surface Center, Miami, FL; 3SRI International, Menlo
Park, CA.
2118 - B887
2119 - B888
Loss of FGF2 Retards Proliferation of Epithelial Cells in an Uninjured or a Penetrating
Injured Cornea in Mice
T.Tanaka1, S.Saika1, Y.Ohnishi1, C.-Y.Liu2, M.Azhar3A, T.Doetschman3A, W.W. Kao3B.
Ophthalmology, Wakayama Medical University, Wakayama, Japan; 2Bascom Palmer
Eye Institute, University of Miami School of Medicine, Miami, FL; AMolecular Genetics,
Ophthalmology, 3University of Cincinnati Medical Center, Cincinnati, OH.
Purpose: To examine the roles of f ibroblast growth factor 2 (FGF2) in
regulating proliferation of cor neal epithelial cells in vivo and in vitro.
Methods: Experimental mice (34 Fg f2+/+ and 42 Fg f2-/-) were under both general
and topical anesthesia. A penetrating wound was created with a hypodermic needle
in one eye. At days 2, 5, and 10 post-injury the mice were sacrificed following a 2 hrlabeling period with bromo-deoxyuridine (BrdU). The number of BrdU-labeled cells
and those positive in TUNEL staining in epithelium was determined. The effects
of exogenous FGF2 and an inhibitor of MAP kinase, PD98059, on cell proliferation
of SV40-immortalized human corneal epithelial cells (HCECs) were determined.
Results: Penetrating injury in the central cornea did not stimulate nor inhibit cell
proliferation of corneal epithelium. The number of BrdU-labeled corneal epithelial
cells was statistically significantly less in Fgf2-/- mice than in Fgf2+/+ mice in both
uninjured, and injured, conditions. There was no difference in number of cells
undergoing apoptosis of epithelial cells between both groups as determined by TUNEL.
In vitro studies showed that addition of 5.0 ng/ml FGF2 enhanced proliferation of
HCECs and this growth-promoting effect was abolished by adding 50 μg/ml PD98059.
Conclusions: FGF2 is required to support proliferating activity of corneal epithelium in mice.
MAP kinase cascade is involved in FGF2’s cell proliferation-stimulating effect.
CR: T. Tanaka, None; S. Saika, None; Y. Ohnishi, None; C. Liu, None; M. Azhar, None; T.
Doetschman, None; W.W. Kao, None.
Support: None.
Purpose: Ex vivo expansion of limbal epithelial progenitor cells by amniotic membrane
(AM) is a new surgical approach to treat eyes with limbal stem cell deficiency. Previously,
we reported that NGF-TrkA mediated signaling is required. Herein, we sought to
further delineate the downstream signaling pathways involved during such expansion.
Methods: The human corneolimbal tissue was briefly incubated with 1.2 units/ml Dispase II, cut
into explants of approximately 1x1.5x2.5 mm, and cultured on intact AM in SHEM. At day 0 or
day 10, different concentrations of small MW inhibitors of PI3K, phospho-AKT, p38, JNK, and
p42/p44 MAPK were added, while the control group received the same amount of the vehicle, i.e.,
DMSO. The epithelial outgrowth was monitored daily for 17 days by digitizing the surface area.
The epithelial cells in the outgrowth and the explant were collected for western blotting analysis.
Results: In the control, expansion of human limbal epithelial cells was more rapidly from the limbal
than the corneal and scleral area during day 5 to 7 and reached ~80 % confluence at day 17 on a 20
mm diameter AM insert. Compared to the control, LY294002 (PI3K inhibitor) at 50 µM, SR13668
(phosphor-AKT inhibitor) at 50 µM completely, and U0126 at 10 µM significantly suppressed the
expansion of limbal epithelial cells on AM (p=0.0006, 0.0005, and 0.0008, respectively). However,
the outgrowth was not affected by 10 µM of either SB203580 (MAPK p38 inhibitor) or JNK inhibitor
1 (JNK inhibitor) (p=0.2 and 0.3, respectively). The inhibition of outgrowth by LY294002, SR13668,
and U0126 was reversible but with a much slower rate. Western blotting analysis showed that
SR13668 abolished phosphorylation at Ser473 and Thr308, while LY294002 and U0126 abolished
AKT phosphorylation at Thr 308 but down-regulated it at Ser473. All three of them down-regulated
FKHRL1 phosphorylation at Thr32. In contrast, U0126 abolished, LY294002 down-regulated,
while SR13668, SB203580 and JNK inhibitor 1 did not affect MAPK p42/44 phosphorylation.
Conclusions: Both survival signaling pathway mediated by AKT-FKHRL1 and mitogenic pathways
mediated by p42/44, but not by p38 and JNK, MAPK are involved in ex vivo expansion of human
limbal epithelial progenitor cells on AM.
CR: H. He, TissueTech, Inc. E; H. Cho, None; W. Li, TissueTech, Inc. E; T. Kawakita, TissueTech,
Inc. E; L. Jong, None; S.C.G. Tseng, TissueTech, Inc. and Ocular Surface Center I, C, P.
Support: NIH EY06819 (to SCGT)
Tumor Necrosis Factor-Induced Apoptosis in Corneal Epithelial Cells Is Attenuated
by Novel Lacrimal Glycoprotein, Lacritin
A.K. Sharma1, R.Raab2, R.McKown2, G.W. Laurie1. 1Cell Biology, University of Virginia,
Charlottesville, VA; 2James Madison University, Harrisonburg, VA.
Purpose: Cell survival is critical during corneal epithelial regeneration after infection or
injury and novel lacrimal glycoprotein, lacritin, could be fundamental in cytoprotection and
cell proliferation. We set out to investigate the role of lacritin in the stimulation of a putative
G-protein coupled receptor (GPCR) dependent pathway regulating corneal epithelial cell
turnover- possibly via a increased calcium signaling involving protein kinase C (PKC), and
its crosslink with a tumor necrosis factor (TNF-α)-induced apoptosis in a human corneal
epithelial cell line (HCE). Methods: Lacritin-induced proliferation and calcium signaling
was studied in the human corneal epithelial (HCE) cell line (Araki-Sasaki et al, 1995).
HCE cells were grown at 370C in DMEM F/12 supplemented with 10% FBS and 50 μg/ml
gentamicin. Each experiment was done in a dose- and time- dependent manner using TNF-α
(5-20 ng/ml) and lacritin (1-10 nM). Cell viability was measured spectrophotometrically at an
absorbance of 570 nm using the MTT assay. Caspase-8 actvity was detected fluorometrically
by measuring cleavage of the fluorogenic substrate IETD-pNA at an excitation wavelength of
405 nm. Lacritin-induced cell proliferation was studied by H3-thymidine incorporation and
via fluorescence-based detection of PKC, PLC and cAMP. Calcium signaling was detected
by confocal microscopy of Fluo-4 loaded cells. Results: A) TNF-α- induced cell death in
HCE cells (32.2 ± 2.6% after 18 hr and 73.7 ±10% after 30 hr) was attenuated by lacritin (10
nM) pretreatment. B) TNF-α induces a three-fold increase in the proapoptotic caspase-8
activity which was completely blocked by lacritin pretreatment. C) Lacritin and its deletion
construct (N-24) promote cell proliferation as measured by the thymidine incorporation in
HCE cells. Conclusions: The novel prosecretory mitogen, lacritin, promotes cell proliferation
and offers cytoprotection to the corneal epithelial cells against TNF-α-induced cell death.
Signal transduction events related to this cytoprotection and proliferation of the corneal
epithelial cells are under investigation.
CR: A.K. Sharma, None; R. Raab, None; R. McKown, None; G.W. Laurie, None.
Support: NIH Grant EY13143
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2102-2128 / B871-B897
263. Corneal Epithelium: Signaling Organizing Section: CO
2120 - B889
Effect of Type IV Collagen on the Alpha5 Integrin Subunit Gene Promoter Function in
Rabbit Corneal Epithelial Cells: Differential Regulatory Influences Dictated by Cell
M.-E.Gingras1, C.Fugère2, S.Leclerc1, L.Germain2, S.L. Guerin1. 1Endocrinologie
Moleculaire, CHUL, Sainte-Foy, PQ, Canada; 2LOEX, Hopital du Saint-Sacrement du
CHA, Quebec, PQ, Canada.
Purpose: Upon injury of the cornea, both the stromal keratocytes and the basal epithelial cells
surrounding the wound secrete components from the extracellular matrix (ECM), mostly fibronectin
(FN), that help promote epithelial cells adhesion and migration. Early during this process, type IV
collagen (CIV), a major component of the basement membrane, disappears until the denuded area
is completely covered, and then sequentially reappears beneath the newly regeneratedconstructed
epithelium. The FN binding integrin a5b1 plays a major role in corneal wound healing by promoting
epithelial cell adhesion and migration over the FN matrix. We recently showed that transcription
directed by the a5 promoter is induced by FN when rabbit corneal epithelial cells (RCECs) are grown
on FN-coated culture dishes. Here, we examined whether the activity directed by the a5 promoter
is similarly altered when RCECs are grown at different densities on CIV. Methods: Recombinant
constructs bearing various fragments from the a5 promoter fused to the CAT gene were transfected in
RCECs grown to different densities with or without CIV. Electrophoretic mobility shift assays (EMSA)
and Western blot analyses were conducted to monitor the influence of CIV on both the expression
and DNA binding properties of the transcription factors Sp1, Ap1 and NF1, which have been reported
to influence transcription of the a5 gene. The influence of CIV on the proliferative properties of
RCECs was evaluated by BrDU incorporation and flow cytometry. Results: CIV exerted a strong
positive influence on the a5 promoter activity at a low cell density but acted negatively at a high cell
density. Both the expression and DNA binding properties of the transcription factors Sp1, Ap1 and
NF1 were also differently affected by CIV in a cell-density dependent manner. CIV also increased
the proliferative rate of RCECs but only at low cell density. Conclusions: These results suggest that
the regulatory influence of CIV (either positive or negative) on the a5 promoter activity is dictated
by subtle alterations ofin the cell density in primary cultured RCECs. As expression of CIV changes
during corneal wound healing, it is also likely that its influence on gene expression (and consequently
on cell adhesion and migration as well) will also vary considerably during this process.
CR: M. Gingras, None; C. Fugère, None; S. Leclerc, None; L. Germain, None; S.L. Guerin,
Support: Canadian Institutes of Health Research (CIHR)
2122 - B891
2121 - B890
EGF-Induced Down-Regulation of Pax6 to Promote Corneal Epithelial Cell
T.Li, L.Lu. Division of Molecular Medicine, Harbor-UCLA Medical Ctr, Torrance, CA.
Purpose: Epidermal Growth factor (EGF) plays important roles in regulating
cor neal epithelial proliferation/differentiation during wound healing. The
purpose of the study is to investigate the requirement of suppressing corneal
epithelial lineage-specif ic Pax 6 activity in EGF-induced proliferation.
Methods: Human and rabbit corneal epithelial cells (HCE and RCE cells) were cultured
in in DMEM/F12 medium contained 10% FBS in 37 °C incubator gassed with 5% CO2..
Northern blot and Western analysis were used to determine target gene and protein
expressions. Expression levels of genes were manipulated by gene transfection and
siRNA techniques. Corneal epithelial cell proliferation was measured by MTT assay.
Results: We found in the study EGF stimulated corneal epithelial cell growth through
inhibition of Pax6 activity. 1) EGF-induced CTCF activation subsequently inhibited pax6
expression by interacting with a CTCF-specific region upstream of Pax6 P0 promoter.
2) Suppression of EGF-induced Erk activation by specific inhibitor or by the dominant
expression of a silent Erk mutant effectively abolished the effects of EGF stimulation
on regulations of CTCF and Pax6. 3) Down-regulation of Pax6 expression induced by
EGF is required for corneal epithelial proliferation because overexpression of Pax6 in
these cells attenuated EGF-induced proliferation. 4) Knockdown of Pax6 mRNA with
siRNA significantly promoted EGF-induced proliferation of corneal epithelial cells.
Conclusions: Our results revealed a new regulatory mechanism that involves cellular signaling
events and Pax6 transcription regulation in growth factor-mediated proliferation. This suggests
in corneal epithelial cells that inhibition of Pax6 expression is a prerequisite for EGF to elicit
controls of cell growth and fate.
CR: T. Li, None; L. Lu, None.
Support: EY12953 and EY15282
2123 - B892
Internalization of Pseudomonas Aeruginosa in Corneal Epithelium is Mediated by
Lipid Rafts
N.Yamamoto1, N.Yamamoto1, J.V. Jester2, H.D. Cavanagh1. 1Ophthalmology, UT
Southwestern Med Ctr, Dallas, TX; 2Ophthalmology, University of California at Irvine,
Irvine, CA.
Purpose: We previously showed that the internalization of P. aeruginosa (PA) in corneal
epithelial cells (in vivo, in vitro) appears to involve the formation of sphingolipidrich plasma membrane domains forming lipid raft platforms (ARVO, 2004). The
purpose of this study was to investigate whether the internalization of PA through lipid
raft (LR) formation is a common mechanism among corneally invasive PA strains.
Methods: Two corneally invasive isolates, 6294 and 6487 (gifts of Dr. Suzanne M. Fleiszig),
and one non-corneal isolate, known to be infectious to the cornea (ATCC27853) of PA were
used in this study. LR formation was visualized in an immortalized human corneal epithelial
cell line (hTCEpi) before and after PA infection by staining with the FITC-conjugated
β-subunit of cholera toxin (β-CT) known to bind to LR component, ganglioside GM1,
followed by confocal microscopy. Bacterial internalization was quantified by gentamicin
survival assay. The role of LR in PA internalization was evaluated by pretreatment of hTCEpi
cells with cholesterol metabolism inhibitors, methyl-β-cyclodextrin, filipin and nystatin
prior to PA infection. Following exposure of PA to hTCEpi cell lysate and complexing
with FITC-conjugated β-CT, the specific interaction of PA with LR was tested by FACS.
Results: Infection with all PA strains produced LR reorganization, aggregation and
PA internalization at the same LR membrane sites in hTCEpi cells. Quantification
of PA internalization showed that all cholesterol metabolism inhibitors significantly
decreased PA internalization of 3 strains in a dose-dependent manner (p<0.01).
FACS analysis showed that exposure of PA to cell lysate significantly increased
bacterial f luorescence (p<0.05), confirming specific binding of LR to PA.
Conclusions: These findings with 3 infectious strains of PA, one non-corneal but infectious
and two corneal isolates, suggest that LR formation is required for infection of invasive PA
strains in corneal epithelium.
CR: N. Yamamoto, None; N. Yamamoto, None; J.V. Jester, None; H.D. Cavanagh,
Support: EY10738, an unrestricted grant from R.P.B.
P63 Expression in hTERT-Immortalized Corneal Epithelial Cells
S.R. Fisher, D.M. Robertson, H.D. Cavanagh. Ophthalmology, University Texas
Southewestern, Dallas, TX.
Purpose: p63 has been reported as a marker for corneal epithelial stem cells. The
purpose of this experiment was to evaluate p63 expression in hTERT-immortalized
human corneal epithelial cells (hTCEpi) throughout the cell cycle in primary culture
conditions and during subsequent air-lifted epithelial differentiation and in normal cornea.
Methods: To assess nuclear changes with the cell cycle, hTCEpi cells were plated and grown for
3 days on collagen-coated coverslips in serum-free media containing 0.15 mM calcium. Cells
were fixed in 4% paraformaldehyde and double-labeled with a mouse monoclonal antibody to
Ki-67 and a rabbit polyclonal antibody recognizing an epitope specific for the ΔNp63α isoform.
To assess changes in p63 levels during subsequent differentiation, cells were plated on collagen
coated tissue culture inserts in 1.15mM calcium and grown in sequential submersed and airlifted culture. Cells were double-labeled with a p63 antibody and phalloidin and evaluated at 3
time points; 3 day low calcium, 7 days submerged, and after an additional 7 days air-lifted. Whole
mount fresh (organ donor) human corneal tissue was used to establish patterns of p63 expression
in vivo. All images were obtained with a Leica SP2 laser scanning confocal microscope.
Results: Double-labeling with Ki-67 and p63 demonstrated the expected characteristic
cell cycle changes for Ki-67 in nuclear staining; however, there was no change in nuclear
p63 expression at any stage of the cell cycle defined by concomitant Ki-67 staining.
Both confluent cell cultures and 7 day submerged conditions demonstrated robust p63
nuclear staining. p63 nuclear staining was also observed to persist in 7 day air-lifted
constructs and was detected in fresh normal whole-mount human corneal controls.
Conclusions: Taken together, these data show: (1) that all hTCEpi immortalized cells express
p63 in cultured, submersed, and air-lifted conditions. There appears to be a tendency to lose
p63 expression as multi-layering occurs in both air-lifted and normal human controls; and,
(2) nuclear p63 expression does not appear to vary during the cell cycle as shown by Ki-67
indicating that it is not a cyclin.
CR: S.R. Fisher, None; D.M. Robertson, None; H.D. Cavanagh, None.
Support: NIH Grant K08 EY015713, NIH Grant EY10738 and Research to Prevent Blindness,
2124 - B893
2125 - B894
Protection of UV Irradiation-Induced Cornea Epithelial Cell Apoptosis by Caffeine
Through Suppression of JNK Activation
L.Wang, L.Lu. Div of Molecular Medicine, Harbor-UCLA Research & Educ Inst,
Torrance, CA.
Purpose: The purpose of the study is to define the role of molecular interaction between
caffeine and UV-induced JNK cascades in promoting corneal epithelial cell survival from UV
irradiation. Methods: Rabbit and human cornea epithelial cell were cultured in DMEM/F12
medium containing 10% FBS and 5 μg/ml insulin at 370C, 5% CO2. DNA fragmentation and
A/O nuclear staining were performed to detect cell death. Western blot, immunoprecipitation
and kinase assays were employed to measure UV-induced MAP kinase activity.
Results: 1) UV irradiation induced corneal epithelial cell death was prevented by caffeine
in a dose-dependent manner. 2) JNK activity activated by UV irradiation was suppressed
by caffeine in the similar dose range. 3) The inhibitory effect of caffeine on JNK activity
was not affected by alterations of either cAMP levels or intracellular Ca++ concentrations.
4) Inhibition of PI3K pathway using wortmannin had no effect on caffeine mediated protection.
5) Theophylline, an adenosine receptor antagonist, mimics the role of caffeine in UV-induced
JNK activation. 6) Neither caffeine nor theophylline affects EGF- and sorbital-induced
ERK and p38 activation, respectively. Conclusions: Caffeine is the world’s most popular
consumption because of present in coffee, tea, chocolate, soft drinks, and pain and appetite
suppressant. We have found a new role of caffeine that promotes survival of cornea epithelial
cells from UV irradiation-caused damage by specific suppression of JNK activation.
CR: L. Wang, None; L. Lu, None.
Support: EY12953 and EY15282
Expression of 15-lipoxygenases in the Human Cornea
S.Presley1A, F.R. Haselton1B, M.S. Chang1C. ADivision of Cardiovascular Medicine,
Department of Biomedical Engineering, CDepartment of Ophthalmology and Visual
Sciences, 1Vanderbilt University, Nashville, TN.
Purpose: 15S-hydroxyeicosatetraenoic acid (15S-HETE) is a major arachidonic
acid metabolite produced by human corneal epithelium. There are two human
15-lipoxygenases (LOX), 15-LOX-1 and 15-LOX-2, which convert arachidonic
acid to 15-HETE. The presence of both 15-lipoxygenases in the human cornea
prompted this study to better delineate their roles in the human corneal epithelium.
Methods: Human corneal epithelium from eye banked corneas and a human corneal epithelial
cell line (HCE) were used in [1-14C]arachidonic acid incubations, Western analysis, and
quantitative real time RT-PCR. Cell cultures of HCE were treated with 15S-HETE to
measure its effect on cellular proliferation (BrdU uptake) and apoptosis (TUNEL assay).
Results: 15-LOX-2 but not 15-LOX-1 was detected by Western blot analyses, although we
were able to detect similar levels of both 15-LOX mRNAs by real-time quantitative RTPCR. [1-14C]Arachidonic acid incubations revealed 15S-HETE as the major lipoxygenase
product in the human corneal epithelium. Treatment with 15S-HETE (5-10 µM) reduced
growth rate and induced apoptosis of cultured HCE cells in a dose dependent manner.
Conclusions: These findings indicate that 15-LOX-2 is the predominant 15-lipoxygenase
protein in human corneal epithelium, and its product, 15S-HETE, may play a role in
maintenance of the corneal epithelium through cellular apoptosis.
CR: S. Presley, None; F.R. Haselton, None; M.S. Chang, None.
Support: NIH grant EY13592 and Research to Prevent Blindness
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2102-2128 / B871-B897
263. Corneal Epithelium: Signaling Organizing Section: CO
2126 - B895
Wounding-Induced Epidermal Growth Factor Receptor Activation Is Mediated by Src
Tyrosine Kinase in Human Corneal Epithelial Cells
F.X. Yu, K.-P.Xu. Dept Opthalmology, Wayne State Univ School of Med, Detroit, MI.
Purpose: We previously demonstrated that corneal epithelial wound healing
is mediated by activation of epidermal growth factor receptor (EGFR). In this
study, we sought to identify the underlying mechanism for EGFR activation
in response to wounding in cultured human corneal epithelial cells (HCECs).
Methods: SV40-immortalized HCECs were wounded and allowed to heal in the presence
or absence of PP2, a selective inhibitor of Src kinase and methyl-β-cyclodextrin (MβCD), a
cholesterol sequestering drug. The activation of EGFR was analyzed by immunoprecipitation
of EGFR, followed by Western blotting with phosphotyrosine antibody. Phosphorylations of
extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3’-kinase (PI3K) were
analyzed by Western blotting with antibodies specific to phosphorylated proteins. Lipid rafts were
isolated from Triton-insoluble material using equilibrium density gradient centrifugation. The
presence of phosphorylated Src and EGFR in raft fractions was assessed using Western blotting.
Results: Wounding of HCECs induced phosphorylation of Src at tyrosine 418. The Src
kinase inhibitor PP2 blocked wound-induced Src phosphorylation and attenuated epithelial
migration and wound closure with or without exogenously added heparin-binding EGF-like
growth factor. Concomitant with the inhibition of epithelial wound healing, presence of PP2
in the culture media also impaired the wound-induced EGFR activation and its downstream
signaling pathways, ERK and PI3K, in a concentration-dependent manner. Phosphorylated
Src at tyrosine 418 and EGFR at tyrosine 845 (a Src dependent phosphorylation site) were
preferential distributed in the lipid raft fractions. Depletion of cholesterol by MβCD retarded
wound-induced activation of Src and EGFR and disrupted their localization in lipid rafts.
Conclusions: Non-receptor tyrosine kinase Src plays an important role in corneal epithelial
wound healing via regulating EGFR phosphorylation. Membrane microdomains are important
for the formation of signaling complexes including Src and EGFR in response to wounding
in HCECs.
CR: F.X. Yu, None; K. Xu, None.
Support: NIH/NEI R01 EY10869 & 14080, Research to Prevent Blindness
2127 - B896
Protein Kinase C Alpha and Epsilon Gene-Knockdown Differentially Affects
HGF-Induced Cell Proliferation and Migration in Human Corneal Epithelial Cells
G.D. Sharma, H.E. P. Bazan. Ophthalmology & Neuroscience Center, LSU Health
Science Center, New Orleans, LA.
Purpose: Previous studies have shown that hepatocyte growth factor (HGF) translocates PKCα
and PKCε to the plasma membrane, inducing their activation in HCEC, and that inhibition
of PKCα by Go6976 decreases cell proliferation (Sharma et al. ARVO. 2003). Since there
are no available PKC inhibitors specific for one isoform, in the present study we have used
PKCα and PKCε-siRNA to knockdown these genes and investigate more precisely their roles
in HGF-induced cell proliferation and migration in HCEC. Methods: HCEC were seeded in
60-mm dishes (5 x 105 cells/well) or 96-well plates (10,000 cells/well), allowed to grow to
50-60% confluence, and then transfected with PKCα- and PKCε-siRNA overnight in KBM
(without growth supplements). Cells were then fed with fresh KGM medium before they were
serum-starved and stimulated with HGF for migration studies using a scrape-wound method
(Sharma et al. J. Biol. Chem. 21989, 2003). For proliferation, transfected cells were treated
with HGF and allowed to grow for 48 h before being analyzed for proliferation using a DNAbinding fluorescent dye. Results: PKCα and PKCε genes were knocked down up to 60-70%
with more than 80% of the cells transfected. HCEC transfected with PKCα-siRNA showed a
significant (p<0.005) inhibition in cell proliferation compared to non-transfected control at 48
h. Similarly, when transfected cells were incubated with HGF, there was a significant (p<0.001)
reduction in cell proliferation compared to HGF-stimulated non-transfected cells. On the other
hand, cells transfected with PKCε-siRNA showed no significant inhibition in cell proliferation
with or without HGF stimulation. Proliferation was unaffected by transfection reagent alone.
Cell migration was inhibited by more than 80% in PKCε-siRNA-transfected cells compared
to controls, with or without HGF stimulation. In contrast, PKCα-siRNA-transfection did not
affect the migration of cells stimulated by HGF. Conclusions: Our results suggest that the
two isoenzymes, PKCα and PKCε, had functional selectivity in proliferation and migration
stimulated by HGF. This elucidates a mechanism involving both PKCs to regulate different
stages of corneal epithelial repair (Supported by NIH-NE1 EY06635).
CR: G.D. Sharma, None; H.E.P. Bazan, None.
Support: NIH Grant EY06635
2128 - B897
Global Expression Analysis of the MMP-TIMP Gene Families in Corneal Epithelial
J.S. Austin, G.M. Gordon, M.E. Fini. Bascom Palmer Eye Institute, University of Miami,
Miami, FL.
Purpose: Matrix metalloproteinases (MMPs) are key regulators of tissue remodeling.
Expression of the MMP gelatinase B (gelB, MMP-9) is induced at the migrating epithelial
front and blocking its activity by “knock-out” of the MMP-9 gene results in faster epithelial
resurfacing (Mohan et al., JBC, 2002). In contrast, broad-spectrum, synthetic MMP inhibitors
retard the rate of epithelial resurfacing, suggesting role for other MMPs. The purpose of this study
was to identify candidate MMPs, as well as members of the TIMP family of MMP inhibitors,
involved in epithelial regeneration in the mouse cornea by global gene expression analysis.
Methods: Corneal debridement was performed by demarcating a 1mm circular region
of the cornea with a trephine and removing the corneal epithelium in that region with an
algebrush. The corneal stromal integrity was visualized and ensured by topically applying
2% Fluorescein Disodium. The migrating corneal epithelium was harvested 24 hours later
by scraping with a scalpel dipped in TRIzol reagent (Invitrogen), and frozen at -80°C. The
unwounded epithelium from the contralateral eye was also harvested for comparison. Total
RNA was purified from both sets of tissues the next day. Quantitative RT-PCR was performed
using SYBR green as a probe. The PCR conditions were as follows: one step of denaturation
for 2 min at 95°C followed by 50 cycles of amplification at 95°C for 15 sec, 60°C for 1 min.
Results: In our first set of experiments, seven MMPs were assayed: MMP-1a and 1b, MMP-2,
MMP-3, MMP-7, MMP-8, and MMP-10. The following MMPs were up-regulated in migrating
epithelium: MMP-1a (2.9 fold), MMP3 (3.4-fold), MMP-7 (2.8-fold) and MMP-10 (2.7-fold). The
mRNA for MMP-1b and MMP-8 was detected only in the samples from migrating epithelium.
We are in the process of assaying the remaining sixteen members of the MMP family, eight
members of the related ADAM family, the four TIMPs, and the related inhibitor, RECK.
Conclusions: These preliminary data identify several MMPs which may be involved in corneal
epithelial regeneration. The role of these enzymes will be assayed using gene knock-out and
knock-down methodologies.
CR: J.S. Austin, None; G.M. Gordon, None; M.E. Fini, None.
Support: NIH Grant EY012651, NIH Grant EY014801, Research to Prevent Blindness
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2129 - B898
2130 - B899
Lumican Regulates Wound Healing and Innate Immune Responses in the Cornea
S.Chakravarti, N.Vij, L.Roberts, F.Wu, L.Savino. Medicine, Johns Hopkins Univ Sch of
Medicine, Baltimore, MD.
Purpose: Lumican is a keratan sulfate proteoglycan found in barrier tissues such as the cornea.
Its biologically active leucine-repeat rich (LRR) domain binds collagen, and possibly other
proteins, with as yet unknown biological implications. We hypothesize that lumican through
interactions of the core protein with other cell surface receptors and microbes, play a critical role
in the host’s first line of defense. To test this hypothesis we examined corneal healing of wounds
exposed to bacterial lipopolysacharides (LPS) in lumican-null (Lum-/-) and wild type (WT) mice.
Methods: Circular and incision type stromal wounds, were exposed to P. aeruginosa
lipopolysaccharide (LPS) and healing was assessed by (1) following wound closure by fluorescent
and bright field microscopy, (2) histology to quantify inflammatory infiltrates by immunostaining
for macrophages (F4/80) and neutrophils (NIMP-R14) and Myeloperoxidase (MPO) levels by
ELISA to further quantify neutrophils, (3) measuring pro-inflammatory cytokines by ELISA.
Results: Circular Stromal wounds showed little change in diameter 72 hours after
injury in Lum -/- mice, in contrast wounds healed completely in wildtypes by this time
(n=5 animals per genotype). Corneas immunostained for macrophages (F4/80) and
neutrophils (NIMP-R14), 4, 8, and 24 hours after stromal incision wounding and
exposure to LPS, showed significantly lower numbers of macrophages and neutrophils
in the Lum -/- corneas compared to wildtype controls. Measurement of inflammatory
cytokines in the injured corneas showed poor induction of TNFα and IL1β in the Lum /mouse. We also noted a disruption in Fas-FasL mediated signaling in the Lum -/- mouse.
Conclusions: Lumican is required for optimal induction of inflammatory cytokines,
recruitment of inflammatory cells and efficient healing of stromal wounds. Impaired induction
of inflammatory cytokines in the Lum-/- mouse may be due to disruptions in Fas-signaling
in the absence of lumican.
CR: S. Chakravarti, None; N. Vij, None; L. Roberts, None; F. Wu, None; L. Savino,
Support: NIH Grant EY11654
Lumican Serves as a Substratum for PMN Formation and Migration during
Myelopoiesis and Corneal Wound Healing
C.Kao1, T.-I.Chikama1, C.-Y.Liu2, S.A. K. Harvey3, M.L. Funderburgh3, J.L. Funderburgh3,
W.W. Kao1. 1Ophthalmology, University of Cincinnati, Cincinnati, OH; 2Ophthalmology,
University of Miami, Miami, FL; 3Ophthalmology, University of Pittsburgh, Pittsburgh,
Purpose: Lumican null mice suffered delayed healing of corneal epithelium debridement.
Our present studies further investigate the role of lumican on inflammatory response during
corneal wound healing and formation of PMN (polymorphonuclear neutrophils) during
myelopoiesis. Methods: Inflammatory responses of corneal epithelium debridement (2 mm in
diameter) were compared in 2 months-old knock out mice lacking lumican (Lum-/-), keratocan
(Kera -/-), and Kera-Lum/Lum -/- bitransgenic mice. Wild type (Lum+/+) mice were used as
control. A two chamber assay using fMLP as a chemotactant was used to elucidate the role
of lumican on the cell migration of PMN from Lum+/+ and Lum-/- mice. Microarray analysis
(Affymetrix GeneChip) was performed to compare the mRNA profiles of PMN isolated from
Lum+/+ and Lum-/- mice. Results: The epithelium debridement of Lum-/- mice healed (in 48 h)
slower than the wild type mice (in 24 h). Histological examination revealed that in wild type
and Kera -/- mice, inflammatory cells appeared in stroma at the leading edge of the migrating
epithelium at 12 hours after wounding and the number of PMN reached a peak in 24 hours. The
PMN invasion was significantly retarded in the injured corneas of Lum-/- mice in comparison
to wild type mice. Whereas in compound Kera-Lum/Lum-/- transgenic mice, infiltration of
inflammatory cells was delayed and could be found at the central denuded cornea in 24 h at
a time lumican was detected by immunohistochemistry. Chemotactic analysis with fMLP
indicated that lumican coated surface facilitated the migration of both Lum+/+ and Lum-/- PMN,
but the latter showed impaired response to the chemotactant. Microarray analysis revealed
the down regulation of Gpr33, a receptor of formyl peptides.Conclusions: Our results suggest
that lumican modulates inflammatory response by serving as a substratum for PMN migration
and a regulatory factor for the formation of PMN during myelopoiesis.
CR: C. Kao, None; T. Chikama, None; C. Liu, None; S.A.K. Harvey, None; M.L.
Funderburgh, None; J.L. Funderburgh, None; W.W. Kao, None.
Support: NIH Grant EY11845, EY09368, RPB; OLERF
2131 - B900
2132 - B901
Role of Keratocan in Corneal Epithelium During Wound Healing
C.-Y.Liu1, T.-I.Chikama2,3, E.Carlson4, V.L. Perez4, W.W. Kao2. 1University of Miami School
of Medicine, Bascom Palmer Eye Institute, Miami, FL; 2Ophthalmology, University of
Cincinnati, Cincinnati, OH; 3Biomolecular Recognition &Ophthalmology,, Yamaguchi
University, Ube, Japan; 4Ophthalmic Research, Cole Eye Institute/ Cleveland Clinic
Foundation, Cleveland, OH.
P u r p o se : Ke r at o ca n h a s b e e n def i ne d a s st r uc t u r al ext r a c el lu al r
kerat an sulfate proteoglycan (KSPG) in the nor mal cor nea. I n this
st udy, we investigate the role of keratocan in cor neal wound healing
Methods: Keratocan-null (Kera-/-) mice and wild-type littermate were employed in epithelium
debridement wound healing experiments. The central cornea was marked by a trephine of 2 mm
in diameter and the epithelium was debrided by an Algerbrush IITM corneal rust ring remover
with a 0.5 mm burr under a stereo-microscope. The corneal wound healing was evaluated both in
vivo and in vitro in an organ culture system. The extent of corneal wound closure was examined
at 6~48 hr after debridement by fluorescein staining and photographed using Axiovision4
digital camera (ZEISS). The animals were sacrificed and the eyeballs were enucleated
for histological and biochemical analysis. Keratocan expression was detected by western
blotting and immunohistochemistry with an epitope-specific anti-mouse keratocan antibody
Results: Corneal epithelial wound healing experiment showed that most of corneal epithelium
healed at 48 hr post-debridement in the wild-type mouse corneas and less than 20% remain small
punctuate defect, while 80% of the Kera-/- cornea still remain defect at 48 hr post-debridement.
Interestingly, keratocan, expressed only by the keratocyte in normal cornea, was indeed
transiently and ectopically detected by the corneal epithelial cells during wound healing. Western
blotting analysis showed that keratocan exists as proteoglycan form in the unwounded cornea,
but as glycoprotein form in the healing epithelial cells. .Adding anti-keratocan antibody at 50
μg/ml in an organ culture system was able to retard the rate of corneal epithelial wound healing.
Conclusions: These results indicated that in addition to being a structural extracelluar matrix
protein, keratocan may actively involve in facilitating corneal epithelial wound healing.
CR: C. Liu, None; T. Chikama, None; E. Carlson, None; V.L. Perez, None; W.W. Kao,
Support: NIH RO1EY12486, EY11845, EY 13755, K08EY014912-01, P30-EY014801, RPB
Analysis and Quantification of Sulfated Glycosaminoglycan (GAG) Dissacharides
in Human LASIK Corneal Wounds Using Single Sections Microdissected by Laser
Capture Microdissection (LCM) and Evaluated by Electronspray Ionization Tandem
Mass Spectrometry (ESI-MS/MS)
I.Schmack1,2, D.G. Dawson1, Y.Zhang3, H.E. Grossniklaus1, G.W. Conrad3, H.F.
Edelhauser1. 1Ophthalmology, Emory Univ Eye Clinic, Atlanta, GA; 2Ophthalmology,
Ruprecht-Karls-University, Heidelberg, Germany; 3Biology, Kansas State University,
Manhattan, KS.
Purpose: To analyze and quantify the concentrations of sulfated keratan sulfate (KS) and
chondroitin/dermatan sulfate (CS/DS) disaccharides in human LASIK corneal wounds.
Methods: Human corneas with prior LASIK surgery stored in Optisol-GS (4°C, mean
5 days) were obtained from eye banks. The specimens were fixed in 70% ethanol for 24
hours, dehydrated through a graded series of alcohol, embedded in paraffin, and sectioned.
Single sections were de-waxed in xylene. The LASIK scar was microdissected using
the LCM with laser spots set at a diameter of 7.5µm. The samples collected contained
either 1, 10, 25, 50, 100, or 200 spots/samples. They were then digested with 50µl droplets
of keratanase II or chondroitinase ABC, and analyzed by ESI-MS/MS as described by
Zhang et al., Analytical Chemistry 2004 (in press). Normal corneas served as controls.
Results: Sulfated KS and CS/DS disaccharides were detectable in the samples containing ≥
50 and 200 spots/sample, respectively. The average (±SD) concentration of mono-sulfated
and di-sulfated KS dissacharide was 0.0018 ± 0.0002 and 0.0034 ± 0.0002 pmol/µm 3.
The total average concentration of sulfated CS/DS dissacharides was 0.00036 ± 0.00004
pmol/µm 3 (54.6% Δdi-4S and 45.4% Δdi-6S). Controls averaged 0.0023 ± 0.0003 pmol/
µm 3 of mono-sulfated KS, 0.0032 ± 0.0002 pmol/µm 3 of di-sulfated KS, and 0.00058 ±
0.00003 pmol/µm 3 of total sulfated CS/DS dissacharides (85.0% Δdi-4S and 15.0% Δdi-6S).
Conclusions: GAGs can now be analyzed and quantitatively compared using small
microdissected samples (≤ 8,835 µm 2) from single ethanol-fixed slide sections. These sections
show that LASIK corneal stromal wounds have a 21% reduction in mono-sulfated KS, similar
concentrations of di-sulfated KS, and a 38% reduction in CS/DS compared to controls. The
clinical significance of these results requires further study.
CR: I. Schmack, None; D.G. Dawson, None; Y. Zhang, None; H.E. Grossniklaus, None; G.
W. Conrad, None; H.F. Edelhauser, None.
Support: NIH Grants R1-EY00933, P30-EY06360, EY00952, RPB Inc.
2133 - B902
2134 - B903
Laminin 5, Netrin-4 and Lumican have Potential to Serve as Counterreceptors of
Z.Cao1A,1B, Y.Li1B, D.D. Hunter1B, M.Koch2, C.Liu3, L.Yeh3, F.-T.Liu4, D.K. Hsu4, W.J.
Brunken1B, N.Panjwani1A,1B. ANew England Eye Center, Dept of Ophthalmology, BCenter
for Vision Research, 1Tufts University School of Medicine, Boston, MA; 2Center for
Biochemistry, University of Cologne, Cologne, Germany; 3Bascom Palmer Eye Institute,
University of Miami, Miami, FL; 4Dermatology, University of California Davis School of
Medicine, Davis, CA.
Purpose: We have previously shown that : (i) a carbohydrate-binding protein, galectin-3, is expressed
in corneal epithelium, (ii) re-epithelialization of corneal wounds is significantly slower in galectin-3
deficient mice compared to the wild type mice, and (iii) the exogenous addition of galectin-3 stimulates
re-epithelialization of corneal wounds in a mouse animal model (J. Biol. Chem. 277:42299-42305,
2002). Cell-matrix interactions play a key role in re-epithelialization of corneal wounds, and it is
well established that galectin-3 contains binding sites for some ECM molecules such as laminin 1.
Laminin 5 (α3β3γ2), netrin and lumican are among ECM molecules known to be present in corneal
epithelial basement membrane and play a role in re-epithelialzation of corneal wounds or epithelial
cell migration. Since these ECM molecules are glycosylated, it is logical to hypothesize that they
may serve as counterreceptors of galectin-3 and that, galectin-3 may influence re-epithelialization of
corneal wounds by binding to and modulating the function of one or more of these counterreceptors.
Therefore, the goal of the present study was to determine whether galectin-3 binds to laminin 5, netrin4 or lumican. Methods: Dot blots and/or electrophoresis blots of purified laminin 5, heterologously
expressed netrin-4, a fragment of the laminin γ2 chain and affinity-purified lumican from amniotic
membranes were probed with peroxidase-conjugated galectin-3 in the presence and absence of a
competing disaccharide, β-lactose. The blots were developed by a chemiluminescence detection
system (PerkinElmer, Life Sciences). Results: Galectin-3 bound to laminin 5, γ2 chain of laminin
5, netrin-4 and lumican. The binding was abolished by a competing disaccharide, β-lactose, but
not by a non-competing disaccharide, sucrose. Also, as expected, galectin-3 did not bind to bovine
serum albumin, which was used as a negative control. Conclusions: Galectin-3 may influence reepithelialization of corneal wounds by modulating the function of key ECM molecules (laminin 5,
netrin-4 and/or lumican) known to play a role in cell migration.
CR: Z. Cao, None; Y. Li, None; D.D. Hunter, None; M. Koch, None; C. Liu, None; L. Yeh, None; F.
Liu, None; D.K. Hsu, None; W.J. Brunken, None; N. Panjwani, None.
Support: NIH Grant EY09349, Ey12676, EY12486 and P30 EY13078
Heparan Mimetics (RGTA) Promote Corneal Wound Healing in vivo and in vitro
Y.Takesue1, E.Huet2, E.Gabison3,2, L.Racine3, T.Hoang-Xuan3, D.Barritault2, J.Caruelle2,
S.Menashi2. 1Department of Ophthalmology, Fukuoka Univ Chikushi Hosp, Chikushinoshi, Japan; 2CRRET/CNRS FRE 2412, Faculte des Sciences, Universite de Paris 12,
Creteil Cedex, France; 3Fondation Ophtalmologique A. de Rothschild, Hopital Bichat,
Paris, France.
Purpose: Regenerating Agent (RGTA) are heparan sulfate mimetics, that
are known to stimulate tissue repair and reduce fibrosis wound healing in
several models. We evaluated their activity in the cornea in vivo and in vitro.
Methods: We assessed the effect of topical application of RGTA on corneal wound healing
after alkali burn injury in C57BL6 mice. Slit-lamp examination, with fluorescein staining
was performed on a daily basis after 1 N NaOH corneal injury. Epithelial defect, corneal
opacity, and neovascularization were evaluated in a masked fashion. Corneal tissue sections
were evaluated for inflammatory cell infiltration. In Vitro, cultured corneal epithelial cells
were treated with RGTA or PBS as control to assess the effect of epithelial cell migration.
Results: RGTA treated mice showed decreased corneal opacity as compared with
non-treated group. Histological evaluation for inf lammatory cell infiltration did
not show any difference between both groups on days 3 after injury. RGTA treated
corneal epithelial cells displayed enhanced migration rate as compared with PBS.
Conclusions: Topical application of RGTA represents a new therapeutic approach in corneal
injury. Further investigations are needed to assess the molecular events involved in this
improved wound healing.
CR: Y. Takesue, None; E. Huet, None; E. Gabison, None; L. Racine, None; T. HoangXuan, None; D. Barritault, None; J. Caruelle, None; S. Menashi, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2135 - B904
2136 - B905
The Effect of MB500 on Corneal Epithelium and Keratocyte as Hyaluronan
Biosynthesis Promoter: in vitro and in vivo Study
K.C. Shin1,2, Y.S. In3,2, J.-H.Ko2, M.-G.Park4, H.-G.Kim4, W.R. Wee3,2, J.H. Lee3,2, M.K.
Kim3,2. 1Department of Ophthalmology, Seoul National University collage of medicine,
Seoul, Republic of Korea; 2Clinical Research Institute, Seoul National University
Hospital, Seoul, Republic of Korea; 3Department of Ophthalmology, Seoul National
University College of Medicine, Seoul, Republic of Korea; 4MD Bioalpha, Daejeon,
Republic of Korea.
Pur pose: To investigate the effect of M B500 (M Dbioalpha, Daejeon,
Korea) on cultured human cor neal epithelial cells and keratocytes and
to evaluate the effect of MB500 on cor neal wound healing in rabbit.
Methods: Various concentrations(1-200uM) of MB 500(10ul) was applied to the cultured
human epithelial cells and keratocytes. Supernatant was collected after 48 hours of incubation
with MB 500. MTT assay was performed to evaluate toxicity of MB 500 to the cells.
Hyaluronan(HA) synthesis assay with biotinylated hyaluronan binding protein(b-HABP),
glycosaminoglycan(GAG) synthesis assay and nitric oxide(NO) assay were performed using
ELISA. Epithelial defects were made using 7.0 mm trephine and application of 20% alcohol
for 20 seconds in 24 rabbits. MB500(200uM) or phosphate buffer saline(PBS) as a control was
topically instilled four times a day to each 12 rabbit’s eye. The ratio of the epithelial defect size
to the total corneal area was calculated using Image Pro PlusTM(Media Cybernetics, MD, USA)
every day until the wound was completely healed. Hyaluronan was stained with b-HABP and 3,3’diaminobenzidine dihydrochloride at 1,2 and 5 days after injury in both treated group and control
Results: Cell death was not increased in MB500-treated cells(1uM and 200uM) compared with
that in control. HA increased by 1.15 folds in epithelial cell with 5uM of MB500 and by 1.35 folds
in keratocytes with 5uM of MB 500. 50uM of MB500 increased the concentration of GAG by 2
folds. MB500 decrease NO concentration by 43% at 50uM compared with that in positive control
with IL-1ß. Corneal wound healing is rapid in study group with MB500 compared with control
group with PBS and statistically significant difference(p=0.034) is observed at 3 days after injury.
Conclusions: The instillation of MB500 shows no cell toxicity. MB 500 can promote corneal
wound healing possibly by increasing HA and GAG and by decreasing NO.
CR: K.C. Shin, None; Y.S. In, None; J. Ko, None; M. Park, MD bioalpha E; H. Kim, MD
bioalpha E; W.R. Wee, None; J.H. Lee, None; M.K. Kim, None.
Support: SNUH Grant 04-2004-019
Expression of Thrombospondin 1 and 2 in Scarred and Vascularized Corneas
M.Batterbury, A.Choudhary, S.B. Kaye, I.Grierson, P.Hiscott. Unit of Ophthalmology,
University of Liverpool, Liverpool, United Kingdom.
Purpose: Thrombospondin (TSP) 1 and 2 are multifunctional matricellular glycoproteins. They
are involved in wound healing and are potent anti-angiogenic agents. TSP 1 is present during the
early (inflammatory) phase of skin repair whereas TSP 2 is found in the late (remodelling) phase. The
purpose of this study was to evaluate TSP 1 and 2 expression in scarred and vascularized corneas.
Methods: Archived frozen normal, fibrotic and vascularized human corneal specimens
were used for immunohistochemical staining with polyclonal antibodies to TSP 1 and
2. In addition specimens were stained with IgG fragments, which served as a control.
Results: TSP 1 and 2 were expressed in the basal epithelium and endothelium of normal
corneas but the stroma and stromal cells (keratocytes) were devoid of the proteins. However,
TSP 1 and 2 were expressed in the stroma and stromal cells of established (late) corneal
scars. They were also found in the endothelium of blood vessels in addition to the corneal
epithelium and endothelium. No staining was observed in sections stained with IgG fragments.
Conclusions: These results demonstrate that TSP 1 and 2 are expressed in the stroma of
vascularized and scarred human corneas but are absent in normal corneas. The persistence
of TSP 1 into the late (remodelling) phase of corneal repair suggests that the mechanisms of
stromal repair differ between cornea and skin.
CR: M. Batterbury, None; A. Choudhary, None; S.B. Kaye, None; I. Grierson, None; P.
Hiscott, None.
Support: Royal College of Surgeons of Edinburgh
2137 - B906
2138 - B907
Suppression of NF-Kappa B Signaling by SN50 Potentiates TNF-Alpha/JNK-Driven
Cell Proliferation in Healing Corneal Epithelium and Has Therapeutic Effects on
Corneal Alkali Burns in Mice
S.Saika1A, T.Miyamoto1A, K.C. Flanders2, O.Yamanaka1A, Y.Ohnishi1A, K.Ikeda3,
Y.Nakajima3, W.W. Y. Kao4, A.Ooshima1B. AOphthalmology, BPathology, 1Wakayama
Medical College, Wakayama, Japan; 2Lab. Cell Regul. Carcinogenesis, NCI/NIH,
Bethesda, MD; 3Anatomy, Osaka City University, Osaka, Japan; 4Ophthalmology,
University of Cincinanti Med, Ctr., Cincinnati, OH.
Purpose: To evaluate the therapeutic efficacy of topical administration of SN50,
an inhibitor of nuclear factor-kappa B (NF-kB), in a corneal alkali burn model
in mice and to examine the role of NF-kB signal in epithelial cell proliferation.
Methods: A central alkali burn was produced with 1N NaOH in right cornea of
C57BL/6 mice (n=68) under general and topical anesthesia. SN50 (10 micro g/micro l)
or vehicle was topically administered daily for up to 12 days. The eyes were processed
for western blot, histology, immunohistochemistry after BrdU labeling and real-time
RT-PCR. To elucidate the role of NF-kB in epithelial cell proliferation, mouse corneas
were organ-cultured with either TNF-alpha, SN50, or an inhibitor of JNK and epithelial
proliferation was evaluated. Examinations of cell proliferation in healing epithelium
in TNF-alpha-null mice (n=8) treated with SN50 or vehicle were also conducted.
Results: Western blot and immunostaining showed that SN50 suppressed NF-kB activation
in tissue and reduced the incidence of epithelial defects/ulceration in healing corneas.
Myofibroblast generation, inflammation, basement membrane destruction and mRNA expression
of cytokines were all less in SN50-treated corneas as compared with control. Experiments
using an organ-culture and TNF-alpha-null mice showed that acceleration of epithelial
cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling.
Conclusions: Topical SN50 is effective in treating corneal alkali burns in mice. The mechanisms
may include suppression of activation of stromal cells and invasion of inflammatory cells, as
well as acceleration of epithelial cell proliferation by blocking NF-kB signaling.
CR: S. Saika, None; T. Miyamoto, None; K.C. Flanders, None; O. Yamanaka, None; Y.
Ohnishi, None; K. Ikeda, None; Y. Nakajima, None; W.W.Y. Kao, None; A. Ooshima,
Support: MESSCJ C15591871, C16590150, NIH Grant EY 13755
Differential Expression of Emmprin in Normal and Ulcerated Corneas: Role in
Epithelio-Stromal Interactions and MMP Induction
E.E. Gabison1,2, T.Hoang-Xuan1, S.Mourah3, E.Steinfels2, L.Yan4, M.A. Watsky5, B.De
Wever6, A.Mauviel2, S.Menashi2. 1Service du Pr T. Hoang-Xuan, Fondation A. de
Rothschild, Paris, France; 2Institut de Recherche sur la Peau Hôpital St Louis, INSERM
U 532, Paris, France; 3Institut de Génétique Moléculaire, Hôpital St Louis, Paris, France;
Oncology Research, Centocor, Inc., Malvern, PA; 5Department of Physiology, The
University of Tennessee Health Science Centre,, Memphis, TN; 6SkinEthic Laboratories,,
Nice, France.
Purpose: Extracellular matrix metalloproteinase inducer (EMMPRIN) was originally
identified on the tumor cell surface as an inducer of matrix metalloproteinases (MMPs)
production in neighboring fibroblasts. Here we demonstrate a role for EMMPRIN in MMPs
induction during corneal wound healing Methods: MMPs and EMMPRIN expression was
analyzed in normal and ulcerated human corneas, as well as in corneal epithelial and stromal
cells in culture using confoncal microscopy, zymography, immunoblots and real-time PCR
Results: In normal cornea EMMPRIN was predominantly expressed in the epithelium but
was markedly induced in the anterior stroma of ulcerated corneas. This coincided with MMP-2
induction which colocalized with EMMPRIN at the epithlio-stromal boundary. The role of
epithelial-stromal interaction in MMP induction was investigated in an in-vitro coculture
system and demonstrated an induction and colocalization of EMMPRIN and MMP-2 in the
fibroblasts at the interface with epithelial cells. Direct contact of fibroblasts with EMMPRINcontaining purified epithelial cell membranes also induced MMP-1, MMP-2 and EMMPRIN
and this was inhibited by a blocking anti-EMMPRIN antibody, suggesting that EMMPRIN
was largely responsible for this induction Conclusions: In this study we demonstrate a role
for EMMPRIN in wound healing and suggest that sustained local upregulation of EMMPRIN
and MMPs in chronic situations where healing is delayed may lead to excessive matrix
degradation and corneal melts.
CR: E.E. Gabison, None; T. Hoang-Xuan, None; S. Mourah, None; E. Steinfels, None; L.
Yan, None; M.A. Watsky, None; B. De Wever, None; A. Mauviel, None; S. Menashi,
Support: Fondation de France, Association Francaise des Amblyopes Unilateraux
2139 - B908
2140 - B909
The Effects of CTGF on HCE Cell Re-epithelialisation and Migration
G.Secker1A, S.L. Watson1B, A.J. Shortt1B, G.R. Grotendorst2, G.S. Schultz3, P.T. Khaw1B, J.T.
Daniels1B. AOcular Repair and Regeneration Biology, BOccular Repair and Regeneration
Biology, 1Institute of Ophthalmology, London, United Kingdom; 2Department of Anatomy
and Cell Biology, University of Miami, Miami, FL; 3Department of Obstetrics and
Gynecology, University of Florida, Gainsesville, FL.
Purpose: Connective tissue growth factor (CTGF) is a cysteine-rich, heparin-binding protein
thought to be a downstream mediator of the profibrotic action of transforming growth factor
beta (TGFβ) in fibroblasts. CTGF’s effect on human corneal epithelial cells (HCEC) has
not been completely characterised. Previously, we have found that HCEC produce CTGF
in a temporal manner during differentiation into multi-layered epithelium and that CTGF
stimulates proliferation. This study investigated the effects of CTGF and CTGF antisense
oligonucleotides on HCEC wound healing and migration in comparision with TGFβ1 and TGFβ2.
Methods: Confluent monolayers of HCE cell line (HCE-T) were wounded using a pipette
tip. Digital photographs were taken immediately after wounding and at 5, 8, and 11 hours
post wounding. Image tool was used to determine wound width at each time point. HCE-T
migration was assessed using a colony dispersion assay. Using these two assays, under
serum free conditions, the effects of various concentrations of CTGF, CTGF antisense,
TGFβ1 and TGFβ2on re-epithelialisation and migration was compared with controls.
Results: Percentage wound closure of HCE-T was increased significantly with
CTGF and TGFβ2 in a time dependant manner, with no effect on migration. CTGF
antisense inhibited wound closure at concentrations of 10 & 100µM when examined
at 5 & 11 hours and it did not affect migration. Comparatively, TGFβ1 had no
significant effect on wound closure and but induced a marked decreased in migration.
Conclusions: CTGF and TGFβ2 promote re-epithelialisation in vitro but did not affect
migration of HCE-T cells. On this basis, we suggest that these growth factors may have an
important role in human corneal epithelial wound healing.
CR: G. Secker, None; S.L. Watson, None; A.J. Shortt, None; G.R. Grotendorst, None; G.
S. Schultz, None; P.T. Khaw, None; J.T. Daniels, None.
Support: Eranda Foundation
Protein Tyrosine Phosphatase 1B (PTP1B) Is Activated During Corneal Epithelial
Wound Healing and Modulates HGF Signaling
A.H. Kakazu, G.D. Sharma, H.E. P. Bazan. Ophthalmology and Neuroscience Center,
LSU Health Sciences Center, New Orleans, LA.
Purpose: During corneal epithelial wound healing, activation of the PI-3Kinase signaling by HGF
is maintained for some time and then switched off, probably to avoid overactivation (Curr Eye Res,
18:168,1999) Protein tyrosine phosphatases (PTPs) are a family of enzymes that catalyze the dephosphorylation of tyrosyl phosphorylated proteins. The purpose of this study was to investigate
whether there are changes in the expression or activity of three non-transmembrane PTPs, PTP1B,
1C, and 1D, during corneal wound healing, and how they affect signal-transduction pathways
stimulated by HGF. Methods: Total de-epithelialization was performed in New Zealand rabbits and
epithelium was collected at 1, 2, 3, and 7 days after injury. In corneal organ-culture experiments,
the epithelium was removed from a 7-mm-diameter central area and corneas were incubated with
HGF for 1, 2, or 3 days. In additional experiments PTP inhibitors were added and the wound was
monitored. Corneal epithelial cells in culture (CEC) were stimulated with HGF with or without the PTP
inhibitor bpV(phen) (10 μM). Total cell lysate and cytosolic and membrane fractions were collected
and proteins were analyzed by Western blot using specific antibodies. PTP 1C and 1D activity was
measured in immunoprecipitates with p-NPP as substrate. For PTP1B a specific peptide substrate was
used. Results: Expression of PTP1C and 1D was found mainly in the cytosolic fraction. PTP1B was
mostly expressed in the membrane fraction. In vivo, 48 and 72 hours after injury there was increased
PTP1B expression that returned to control levels at 72 hours. In organ culture, HGF stimulation
of the injured corneas induced the expression of PTP1B in the membrane fraction of the epithelial
cells at 24 and 48 hours after injury. No changes were noted with PTP1D or 1C.Stimulation of CEC
with HGF for 5 min increased the activity of PTP1B by 25 %. There was no change in PTP1C or 1D
activity under the same conditions. Blocking the activity of PTP1B with bpV(phen) increased the
phosphorylation of the HGF receptor c-Met, the p85 subunit of PI3-K, and the downstream kinases
Akt and p70S6K. The PTP inhibitor significantly increased the rate of epithelial wound healing in
corneas in organ culture. Conclusions: PTP1B is the main PTP in corneal epithelium. Our results
suggest that during HGF stimulation, the phosphatase could be complexed to the HGF receptor to
control its activity. The PTP inhibitor bpV(phen) mimics the effect of HGF by activating the PI-3K
signal involved in wound healing.
CR: A.H. Kakazu, None; G.D. Sharma, None; H.E.P. Bazan, None.
Support: NIH Grant EY06635
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2141 - B910
Role of PKC-epsilon in Regulating Human Corneal Epithelial Wound Healing and
Barrier Function
J.Yin, F.-S.X. Yu. Departments of Ophthalmology and Anatomy and Cell Biology, Wayne
State University, Detroit, MI.
Purpose: Our previous study suggested that protein kinase C-epsilon (PKCε)
is one of the isozymes responsible for phorbol 12-myristate 13-acetate-induced
human corneal epithelial cell (HCEC) activation. In this study, we sought
to elucidate the role of PKCε in HCEC wound healing and barrier function.
Methods: HCEC lines expressing dominant negative (dn) and constitutive active (ca) PKCε,
along with control cell line transfected with pcDNA3, were generated using cDNA-mediated
transfection and colony selection. The effects of these mutants on cell migration and wound healing
were determined by Boyden chamber and scratch wound assays, respectively. The rate of scratch
wound healing was determined by the distance between the leading edge of migrating cells and
the scraped edge. Measurement of trans-epithelial resistance (TER) was performed to assess the
barrier function. Tight junction was evaluated by immunohistochemistry using ZO-1 antibody.
Results: Compared with the control cell line, cells expressing dn-PKCε displayed an
accelerated wound closure whereas the healing of a scratch wound was greatly retarded by
the expression of ca-PKCε. Cells expressing dn-PKCε also exhibited an increased migrating
capability than control cells in Boyden chamber assay. A significant increase in TER was
observed in dn-PKCε expressing cells; while TER of ca-PKCε expressing cells was decreased,
suggesting compromised barrier function. At confluence, cells expressing dn-PKCε appeared
to be larger in size, whereas cells expressing ca-PKCε were morphologically similar to
control cells. The staining of ZO-1 in dn-PKCε expressing cells was more pronounced and
had a sharper-staining pattern as compared with control cells and cells expressing ca-PKCε.
Conclusions: PKCε may play a role in corneal physiology/pathophysiology by inducing
differentiation and barrier formation and/or by modulating epithelial migration and wound
CR: J. Yin, None; F.X. Yu, None.
Support: NIH/NEI RO1 EY10869, EY14080, Research to Prevent Blindness
2143 - B912
2142 - B911
Inhibitor of FAK Blocks Activation of Corneal Fibroblasts Induced by TGFβ
K.Nakamura1,2, D.Kurosaka2, M.Yoshino2, K.Negishi2, K.Tsubota2. 1Ophthalmology,
Tanashi Nakamura Eye Clinic, Nishitokyo-shi, Japan; 2Ophthalmology, Keio University,
School of Medicine, Tokyo, Japan.
Purpose: To determin whether inhibitor of focal adhesion kinase (FAK)
can block activation of corneal fibroblasts induced by transforming factorβ(
TGF β ), in ter ms of proliferation, collagen synthesis, and contraction.
Methods: Human corneal fibroblasts (HCFs) obtained from eye bank eye were cultured.
Then cultured HCFs were exposed to PP2 (inhibitor of FAK), PP3 (inactive analog of
PP2), PP2 and TGF β2, or PP3 and TGF β2, separately. To evaluate proliferation, the
total number of cells on the culture dish was counted with Coulter counter at 48hr after
exposure. To evaluate collagen synthesis, Type 1 collagen in the conditioning medium
was measured with enzyme-linked immunosorbent assay kit at 24hr after exposure. To
evaluate the corneal fibroblasts-derived contraction, HCFs were cultured on collagen gel,
the thickness of the gel was measured daily for 3 days and compared with original thickness.
Results: The total cell number of the dishes exposed PP2 and TGF β2(0.8 ± 0.3 ×10 4
cells) were significantly less than of the dishes exposed PP3 and TGF β2(1.4 ± 0.2 ×10 4
cells) (p =0.0013). The amount of type 1 collagen of the dishes exposed PP2 and TGF
β2(520 ± 81 ng/ml) were significantly less than of the dishes exposed PP3 and TGF
β2(869 ± 187 ng/ml) (p =0.0066). The gels exposed PP3 and TGF β2 (69.5 ± 4.9 %)
significantly contracted than the gels exposed PP2 and TGF β2(99.9 ± 0.6 %)(p < 0.0001) .
Conclusions: Inhibitor of FAK significantly blocked activation of corneal fibroblasts induced
by TGF βin terms of proliferation, collagen synthesis, and contraction. These findings
suggest that Inhibitor of FAK may be useful for suppressing excessive corneal stromal
wound healing.
CR: K. Nakamura, None; D. Kurosaka, None; M. Yoshino, None; K. Negishi, None; K.
Tsubota, None.
Support: None.
2144 - B913
c-Jun Activates Wound Healing-Associated Gene Promoters and Is Transcriptionally
Regulated By Thymosin β 4 (Tβ4) in Human Cornea Epithelial Cells
P.Qiu, G.Sosne. Ophthalmology, Wayne State University, Detroit, MI.
Purpose: This study investigated the c-Jun mediated transcriptional regulation of wound-healing
related genes and the potential modulation of c-Jun expression by Τβ4 in human cornea epithelial cells.
Methods: Transient transfection was performed in cultured human cornea epithelial cells
(HCET) with vectors expressing wild type and mutants of c-Jun and luciferase-reporters driven
by the promoter for the transcription of matrix metalloproteinases (MMPs), tissue inhibitors of
metalloproteinases (TIMPs), plasminogen activator inhibitor type-1 (PAI-1) and laminin-5 (LM5). The effects of different signaling pathways were analyzed during c-Jun mediated promoter
activation. The dynamics of c-Jun mRNA and protein levels were assayed in Tβ4-treated HCET.
In addition, Τβ4-promotion of cell migration was evaluated in an in vitro scrape wound model.
Results: Overexpression of c-Jun, but not c-Fos, increased MMP-1 and LM-5’s promoter
activity 30 and 10 fold respectively. This stimulatory function depends on both c-Jun’s
domains for DNA binding, dimerization and transcriptional activation and cis-elements in
the promoter regions for AP-1 binding. The c-jun mediated promoter activation was partially
blocked by SP600, an inhibitor for the JNK pathway, and overpression of the dominant negative
type of c-Fos. c-Jun mildly enhanced MMP-9 and PAI-1 promoter activity. c-Jun’s dominant
negative elevated TIMP-1 promoter activity. Tβ4 promoted migration of cultured HCET and
transiently stimulated c-Jun expression at least 2 fold at 30 minutes and 1 hour after treatment.
Conclusions: In HCET, overexpression of c-Jun dramatically increases MMP-1 and LM-5
promoter activation. C-Jun’s function involves direct binding to the promoter’s targeted
region, forming hetero- and homodimer, and JNK’s phosphorylation. Tβ4 promotes cell
migration during wound healing and increases c-Jun expression. Our data suggests that Tβ4
may promote epithelial cell migration through c-Jun mediated gene expression.
CR: P. Qiu, None; G. Sosne, None.
Support: NIH EY 13412, Research to Prevent Blindness
Role of TGF-β Signaling in Wound Healing of Corneal Epithelium
K.Terai, Y.Hayashi, T.-I.Chikama, N.Terai, W.W. Kao. Ophthalmology, University of
Cincinnati, Cincinnati, OH.
Purpose: To examine the roles of TGF-β signaling pathways in regulating cell
migration and proliferation of the healing of corneal epithelium debridement.
Methods: TGF-β type II receptor (TbR2) floxed mice were bred with Krt12-Cre mice to generate
bitransgenic mice in which the TGF-β receptor 2 gene was disrupted selectively in the corneal
epithelial cells. Corneal epithelial debridement (2 mm diameter) was created in 2-month-old
bitransgenic Krt12Cre/CreTbR2f/f mice and their littermates as controls Krt12Cre/CreTbR2f/+ and
Krt12Cre/CreTbR2+/+. The healing of corneal epithelium defect was assessed by fluorescein staining
at different intervals of debridement. The experimental mice were administrated by i.p. injection
of BrdU two hours prior to sacrifice to determine cell proliferation. Immunohistochemistry
was performed to elucidate potential signal transduction molecules involved.
Results: The corneal epithelium of Krt12Cre/CreTbR2f/f mice lost TbR2 as judged by western blot
analysis with anti-TbR2 antibodies and exhibited delayed healing of debridemnt, in comparison
to control littermates that were heterozygous floxed and wild typeTbR2. The naïve uninjured
corneal epithelium of Krt12Cre/CreTbR2f/f mice exhibited higher cell proliferative activities than
controls. Corneal epithelium debridement caused cessation of epithelial cell proliferation in
all three groups of experimental mice in 6-12 h. Immunohistochemistry using anti-phosphop38 revealed a significant decrease of phosphorylation of p38 in 6-12 h of debridement.
Conclusions: Our results indicate that the TGF-β signals play a critical role in the corneal
epithelial wound healing, especially in the migration of corneal epithelial cells.
CR: K. Terai, None; Y. Hayashi, None; T. Chikama, None; N. Terai, None; W.W. Kao,
Support: NIH Grant EY13755, EY14207; RPB; OLERF
2145 - B914
2146 - B915
NGF Treatment and Corneal Wound Healing Process
T.Blanco-Mezquita1A, J.Merayo-LLoves1A, M.Martínez-García.1B, R.Torres1A, H.MartinezOsorio1A, M.González-Parra.1A, S.Bonini2, S.Mar-Sardaña1C, A.Lambiase2. AIoba, BCell
biology department, COptics and Applied physics department, 1University of Valladolid,
Valladolid, Spain; 2Laboratory of ophthalmology, University of Rome, Rome, Italy.
Purpose: The purpose of this research is to evaluate the effectiveness of topical administration
of nerve growth factor in the wound healing process after refractive surgery and to prove
that NGF-TrkA interaction decrease earlier apoptosis inhibiting mitochondrial cytochrome
c release, in both culture and in vivo conditions. Methods: Iber Brown hens underwent PRK
were divided into different groups treated with topical administration of 0.2% murine NGF
group A, balanced salt solution (BSS) group B and group C received no treatment. Clinical
monitoring and transparence measurements were taken. Eyes were exenterated at sequential
time points. Sections were stained with H-E. TUNEL technique was used for apoptosis
detection. TrkA receptors and cytochrome c were detected by immunohistochemistry and
westerm blot assay. Culture keratocytes was performed in presence of 0,1 µg/ml of Murine
NGF (NGF group) and absence of NGF (CONTROL group). Proliferation in culture keratocytes
was mean by incorporation of 3H-timidine and keratocyte apoptosis was evaluated by flow
cytometry with annexin V FITC. Results: The appearance and disappearance of the Haze
process was more quickly in time in group A than both B and C groups. TrkA receptor was
found in epithelium, endothelium and keratocytes in all the corneas. In the first hours after
surgery, apoptosis and mitochondrial cytocrhrome c release were lesser in group A than
both B and C groups. Proliferation in culture keratocytes was higher in NGF group than
CONTROL group. Apoptosis and mitochondrial cytocrhrome c release were lesser in NGF
group than CONTROL group. Conclusions: TrkA receptors are present in the cornea of
hens NGF inhibits mitochondrial cytocrome c release, in both culture and in vivo assays
which probably causes a decreases of keratocyte apoptosis. NGF stimulates proliferation in
culture cells NGF accelerates the wound healing procces. The hen is a very good model to
study corneal wound healing after refractive surgery
CR: T. Blanco-Mezquita, None; J. Merayo-LLoves, None; M. Martínez-García., None; R.
Torres, None; H. Martinez-Osorio, None; M. González-Parra., None; S. Bonini, None; S.
Mar-Sardaña, None; A. Lambiase, None.
Support: None.
The Cytokine Response of a Wounded Corneal in vitro Model
M.A. Radburn-Smith, M.Berry. Ophthalmology, University of Bristol, Bristol, United
Purpose: Interactions between epithelial cells and keratocytes are important to maintain
a healthy cornea and its ability to respond to an insult. We have assessed cytokine
production, as an indication of intercellular communication, following chemical insults
in tissue culture models of stratified epithelia with and without a stromal construct.
Methods: Using immortalized human corneal cells, epithelial models were built and
stratified at the air-liquid interface in a fully defined medium. Models were also built with
the epithelium stratified on top of a collagen gel seeded with primary human keratocytes
from donor eyes. The stratified constructs were treated for 10 min with NaOH (Sigma,
Seelze, Germany), sodium dodecyl sulfate (SLS, Merck, Darmstadt, Germany) and
Tomadol™ 45-7 (Tomadol, Tomah3, Los Angeles, USA) at a concentration of 0.66%.
Cytokine production was assessed using Human Cytometric Bead Array kits (BD
Biosciences) as well as fluorescein leakage, LDH release, protein and metabolic assays.
Results: IL-8 and IL-6 were detected in the Araki-Sasaki cell line, whilst IL-10, 8, 6
and 12p70 was produced by the USA line. The cytokine responses were different with
different toxicants. On addition of a stromal layer to the USA epithelium, IL-8 production
increased ten-fold, whilst there was a four-fold increase in IL-6. The stromal constructs
without an epithelium did not produce any measurable cytokines. The response following
chemical injury was also changed if the epithelium was grown on a stromal layer.
Conclusions: Different corneal cell lines behave differently with respect to cytokine
production. Upon addition of further layers to the constructs cytokine patterns changed,
implying communication between the layers.
CR: M.A. Radburn-Smith, None; M. Berry, None.
Support: Colipa and the National Eye Research Council
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2147 - B916
2148 - B917
Synergistic Effect of PAF and TNF-α on Corneal Myofibroblast Apoptosis
J.He, H.E. P. Bazan. Department of Ophthalmology and Neuroscience Center of
Excellence, LSU Health Sciences Center, New Orleans, LA.
Purpose: After injury, the quiescent keratocytes of corneal stroma become activated
and transform to fibroblasts and myofibroblasts. Previous work in our laboratory
has shown that platelet-activating factor (PAF) is a strong inducer of apoptosis in
corneal keratocytes. More recently we found that PAF also induced apoptosis in
corneal myofibroblasts, although these were more resistant than keratocytes. Here we
investigated how PAF, combined with TNF-α, induces corneal myofibroblast apoptosis.
Methods: Pig corneal myofibroblasts were obtained from subcultured fibroblasts plated at a
low density (5cells/mm 2). Mouse anti-α-SMA antibody was used to identify the cell phenotype
of myofibroblasts. Immunofluorescence was performed to localize PAF and TNF-α receptors
in those cells. To induce myofibroblast apoptosis, 7-day-cultured corneal myofibroblasts
were treated with cPAF (300 nM), TNF-α (20 ng/ml), or TNF-α plus cPAF with or without
LAU0901(150 nM), a novel PAF antagonist, for 24, 48, or 72 hours. Apoptosis was assayed
by Hoechst 33258 and TUNEL staining and DNA laddering. DAPI was used for nuclear
counterstaining. Images were recorded by fluorescence microscope, Nikon Eclipse TE200
equipped with a Nikon digital camera DXM1200 using the MetaVue imaging software.
Results: Immunofluorescence with a rabbit anti-PAF-receptor (N-terminus) polyclonal
antibody showed that PAF receptor was expressed in both plasma and nuclear membranes
of myofibroblasts. TNF-α receptor 2 was localized in the cytoplasm, while TNFreceptor 1 was found in both cytoplasm and plasma membrane. Treatment with TNF-α
for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the
myofibroblasts. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells,
respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and
86%, respectively, of the cells, suggesting a synergistic action between PAF and TNF-α.
Blocking the PAF receptor with LAU0901 inhibited the synergistic effect induced by PAF.
Conclusions: Although it has been reported that corneal keratocytes and fibroblasts both
express TNF receptors, to our knowledge, this is the first study that shows the expression of
TNF receptors in corneal myofibroblasts. The synergistic effect on myofibroblast apoptosis
by PAF and TNF-α suggests that during prolonged inflammation, PAF acting in conjunction
with other cytokines could delay stromal wound healing.
CR: J. He, None; H.E.P. Bazan, None. Support: NIH grant EY04928
Topical Combination of NGF and DHA Increases Corneal Nerve Regeneration After
PRK in Rabbits
H.Bazan, S.Esquenazi, V.Bui, J.He, D.B. Kim, N.G. Bazan. Ophthalmology and
Neuroscience Center, LSU Health Sciences Ctr, New Orleans, LA.
2149 - B918
2150 - B919
Prolonged Impairment in Corneal Innervation Following Sulfur Mustard Exposure
Contributes to the Development of Partial Limbal Defficiency
T.Kadar1A, S.Dachir1B, M.Cohen1B, H.Gutman1B, L.Cohen1B, E.Fishbine1B, R.Brandeis1B,
A.Amir1B. APharmacology Dept., BPharmacology, 1Israel Institute for Biological Research,
Ness Ziona, Israel.
Purpose: Ocular injuries induced by sulfur mustard (SM) are characterized by acute
corneal erosions, anterior segment inf lammation and delayed keratopathy, leading to
irreversible visual deficits. Corneal nerves were shown to play an important role in the
maintenance of corneal metabolism and healthy ocular surface. The present study focused
on the role of corneal nerves in the pathogenesis and healing processes of SM ocular injuries.
Methods: Animal Care and Use Committee approval at IIBR was obtained. Rabbit eyes
were exposed to SM vapor for a period of four minutes. A clinical follow-up was carried out
for one month. Corneal sensitivity was determined using the Cochet-Bonnet esthesiometer.
Animals were euthanized at different time points, eyes enucleated and processed for
histochemistry and histology. The effect of SM on the structure and density of nerves was
studied in whole mount corneas, stained for cholinesterase (ChE) activity, using both light
microscopy and morphometric analysis. Corneal sections were stained with H&E and PAS for
general morphology and with the monoclonal antibodies AE1 and AE3 for keratin phenotype.
Results: Typical signs of SM toxicity were observed within 3-4 hrs after exposure associated
with photophobia, inflammation and corneal erosions. The erosions healed spontaneously within
one week. Following an asymptomatic latent period 50-90% of corneas displayed clinical signs,
characterized by neovascularization and recurrent erosions. The regenerating epithelium was
abnormal in respect to adhesion and corneal phenotype. Corneal nerves displayed a typical
Wallerian degeneration beginning few hours after exposure and lasting for weeks associated with
alterations in corneal sensitivity. Only partial regeneration was noted. Thus, two simultaneous
opposing processes were taking place at the cornea for weeks after SM exposure: healing of the
epithelium, concomitant with degeneration of corneal nerves and a significant decline in their density.
Conclusions: The improper regeneration of corneal epithelium and the delayed clinical signs are
typical for partial limbal deficiency (PLD). The prolonged damage of corneal nerves may induce
deficit of factors such as SP and CGRP that are essential for normal function of corneal epithelium
and for limbal stromal microenvironment, contributing to loss of limbal stem cells and development
of PLD. A supplement of trophic factors may be beneficial.
CR: T. Kadar, None; S. Dachir, None; M. Cohen, None; H. Gutman, None; L. Cohen, None; E.
Fishbine, None; R. Brandeis, None; A. Amir, None. Support: U.S. Army Medical Research and
Material Command, contract No. DAMD17-03-C-0064.
2151 - B920
Corneal Epithelial Cell Reaction to Sulfur Mustard Exposure Contributes to the Acute
Anterior Segment Inflammation and to the Development of Epithelial Lesions
A.Amir, S.Dachir, L.Cohen, M.Cohen, H.Gutman, Y.Shalem, T.Kadar. Pharmacology,
Israel Institute for Biological Research, Ness Ziona, Israel.
Purpose: Sulfur Mustard (SM) induced ocular lesions are clinically characterized, by acute and late
corneal epithelial defects, anterior segment inflammation and deterioration of corneal nerves. This
work aimed to study the reaction of corneal epithelial cells to SM exposure using three in vitro models:
1.SIRC corneal cell line. 2. Primary corneal epithelial cells grown from explant cultures of naïve
rabbit corneas (PRCEC). 3. PRCEC taken from explants of in vivo exposed rabbit corneas- ex vivo.
Methods: Animal Care and Use Committee (ACUC) approval at IIBR was obtained. SIRC
cells were from ATCC. Rabbit eyes were exposed to SM vapor and a clinical follow up was
carried out. At 24 hrs, 1,2 and 3 weeks following exposure, corneas (4-8/time point) were taken
for culture. PRCEC were grown using adaptation of published methods. Cell proliferation
following in vitro SM exposure was quantitated by 3H-thymidine incorporation and culture
growth by MTT reaction. Secretion of PGE was measured by RIA. Proliferative capacity of exvivo explants was measured from explant outgrowth, and from growth of 1st passage cultures.
Results: In vitro SM exposure caused a dose dependent inhibition of proliferation with 50% inhibition
at ~150uM and 10uM at 2-6hrs and at 24 hrs respectively for SIRC and PRCEC. MTT growth curves
also showed that SIRC cells were less sensitive to SM than PRCEC cells. Both SIRC and PRCEC
reacted to SM exposure by releasing PGE in a dose dependent manner. PGE release in PRCEC
was enhanced by the neurogenic factor CGRP (5-10nM). Ex- vivo explants obtained from severely
damaged corneas 24 hrs after exposure practically did not grow to form monolayers. Explants obtained
from corneas 1-3 weeks after SM exposure, when corneal erosions seemed clinically healed, had
a decreased rate of cell outgrowth. 1st passage ex vivo cultures also showed attenuation of growth.
Conclusions: Cell growth arrest and cell death following SM exposure could explain the postponed
appearance of corneal erosions observed clinically. Epithelial cells contribute to the anterior segment
inflammation by releasing PGE upon SM exposure. This secretion can be augmented by nerve
deterioration releasing neurogenic factors. The damage and lower proliferation potential of the
regenerated epithelium suggests that long term effects following SM exposure derived in part from
improper regeneration of corneal epithelium.
CR: A. Amir, None; S. Dachir, None; L. Cohen, None; M. Cohen, None; H. Gutman, None; Y.
Shalem, None; T. Kadar, None.
Support: U.S. Army Medical Research and Material Command, Contract No. DAMD 17-03-C0064.
Purpose: The polyunsaturated fatty acid docosahexaenoic acid (DHA) is enriched in neurons including
nerve fibers and nerve endings (J Lipid Res,44:2221, 2003). A DHA-derived messenger, neuroprotectin
(NPD1), promotes cell survival by inhibiting proinflammatory gene expression and modulating Bcl-2
pro- and anti-apoptotic protein expression (PNAS,101:8491, 2004). Here, we tested in a rabbit PRK
model the hypothesis that DHA in combination with NGF promotes corneal nerve regeneration.
Methods: Unilateral PRK was performed in 21 New Zealand albino rabbits. Three groups, each consisting
of six rabbits, were randomized to receive twice-per-week treatments with DHA, NGF, or NGF plus DHA,
delivered by collagen shields. A fourth control group received treatment with albumin. Rabbits were
monitored for 8 weeks and tear-secretion tests were conducted every 15 days. The eyes were prepared
for immunostaining with monoclonal antibodies for class III β tubulin, calcitonin gene-related peptide
(CGRP), substance P, and Ki-67. The nerve areas were calculated with an Image Pro Plus 4.5 program.
Results: There were no significant differences in the tear-secretion tests among the four groups.
However, no eyes treated with DHA + NGF developed rose Bengal-positive staining at 1 month
after PRK, compared with 50% in the control group and 33% in the DHA-treated group. A higher
percentage of Ki-67-positive cells was observed in the DHA+NGF- and NGF-treated groups, compared
with DHA alone or controls. The tubulin-positive sub-basal nerve bundle area was 3.1 mm/mm² in
the DHA+NGF group, 2.15 mm/mm 2 in the NGF group, 1.4 mm/mm 2 in the DHA group, and 0.85
mm/mm 2 in the control group. The CGRP-positive sub-basal nerve bundle areas were 1.9 mm/mm 2,
1.45 mm/mm 2, 0.85 mm/mm 2, and 0.62 mm/mm 2 for the DHA+NGF, NGF, DHA, and control groups,
respectively. No differences were noted in the SP-positive nerve bundles with the treatments.
Conclusions: Topical NGF plus DHA treatment after PRK in rabbits is associated with increased
corneal nerve surface area and less rose bengal staining compared with NGF, DHA, or vehicle control
alone. A growth factor-mediated DHA utilization, as a component of corneal nerve cell membrane
phospholipids and/or as a precursor of pro-survival NPD1, may modulate corneal nerve regeneration
after PRK. NGF plus DHA yielded faster functional nerve recovery after PRK, and may be used to
treat post-PRK dry eye or other neurotrophic keratopathies
CR: H. Bazan, None; S. Esquenazi, None; V. Bui, None; J. He, None; D.B. Kim, None; N.G.
Bazan, None.
Support: NIH grants EY04928 and EY06635 (HEPB) and EY05121 (NGB).
A Novel Role for the 15-Lipoxygenase (Alox15) in Promoting Re-Epithelialization of
the Mouse Cornea
I.R. Hassan1A, N.Maheshwari1B, N.Khan1B, M.Dunn1B, K.Gronert1A. APharmacology, 1New
York Medical College, Valhalla, NY.
Purpose: 12/15-lipoxygenases (LOX) are key enzymes in the formation of the eicosanoid
lipoxin A4 (LXA4). We have recently demonstrated that LXA4 and another 12/15-LOX product,
DHA-derived neuroprotectin D1, are formed in mouse corneas and that these anti-inflammatory
lipids have an epithelial bioaction that limits the sequelae of corneal injury. Mice express
at least 4 distinct 12/15 lipoxygenases. We set out to identify the LOX that initiates LXA4
formation in mouse corneas and to define its endogenous role in maintaining corneal function.
Methods: Corneal injury was induced in 15-LOX deficient mice and in age and gender
matched congenic C57BL/6J mice by epithelial removal up to the corneal limbal border using
an Algerbrush. Wound size and degree of injury were determined by slit lamp biomicroscopy
and documented for quantitation and analysis by a CCD camera. RNA expression of 12/15lipoxygenases (Alox12, Alox15, Alox15b, Aloxe3) and 5-LOX were determined by RT-PCR
analysis. Endogenous LXA4 levels were quantitated by a specific ELISA and RP-HPLC analysis.
Results: The mouse cornea predominantly expressed the leukocyte-type 12/15-LOX
(Alox15) and expression was restricted to the epithelium. In addition, 5-LOX mRNA was
also expressed at low levels in healthy corneas and dramatically increased with corneal injury.
Targeted deletion of Alox15 in the 15-LOX deficient mice demonstrated a significant 65± 5%
(p=0.0009, ANOVA, n=4) and 38± 4% (p=0.04, ANOVA, n=4) decrease in re-epithelialization
24 and 48 hrs, respectively, after epithelial removal. This defect in re-epithelialization
was associated with impaired endogenous formation of LXA4 (43% decrease, n=3).
Conclusions: Our findings identify a previously unknown phenotype of delayed wound
healing in 15-LOX-/- mice and provide direct in vivo evidence for a novel role of the 15LOX (Alox15) and its products in epithelial wound healing. Taken together, these findings
identify a constitutive lipid pathway in corneal epithelial cells that attenuates inflammation
and promotes re-epithelialization.
CR: I.R. Hassan, None; N. Maheshwari, None; N. Khan, None; M. Dunn, None; K.
Gronert, None.
Support: None.
2152 - B921
Melatonin Increases the Rate of Corneal Re-epithelialisation in New Zealand White
J.J. Pintor1A, G.Carracedo1A, A.Mediero1A, A.Guzman-Aranguez1A, M.Irazu1A, T.Pelaez1A,
A.Peral1B. ABioquimica, BOptica, 1E U Optica Universidad Compluten, Madrid, Spain.
Purpose: Melatonin is a biologically relevant substance that can affect multiple ocular processes
such as the retinal functioning or the aqueous humour hydrodynamics. We have investigated if the
topical application of melatonin to rabbit corneal wounds can modify the rate of re-epithelialisation.
Methods: Corneal wounds were made in both eyes by anaesthetising New Zealand rabbits
with 1.5 mg/kg propofol. After topical anaesthesia with oxibuprocaine and tetracaine (0.4
and 1% respectively), corneal wounds were made to the epithelia of both eyes by applying
a 3-mm disc of Whatman no. 1 paper soaked in n-heptanol. The wounds were stained with
2% fluorescein every 2 h and eyes were examined with a Topcon SL-8Z slit lamp. Melatonin
and analogues were assayed at single doses of 10 nmol (volume instilled 10 µL) to the wound
every 6 h. The contralateral eye received the same volume of saline. Migration rates were
determined by linear regression of the decrease in wound radius during the healing phase (1034 h) and were obtained by the slope of the regression line expressed as micrometers per hour.
Results: In the absence of any added compound the rate of healing was 75 ± 5 µm h-1. Melatonin
(10 nmol/10 µl) accelerated the rate of healing to 110 ± 7 µm h-1 (n=8). The application of the nonselective melatonin antagonist luzindole (10 nmol/10µL), reversed the effect of melatonin to values
lower than the control (52 µm h-1). Melatonin effect was also antagonised by the MT3 receptor
antagonist prazosin sugesting the involvement of this receptor in the re-epithelialisation process.
Conclusions: Melatonin receptors seem to be present on the corneal epithelial cells and they
may be responsible for the control of the epithelial cell migration.
CR: J.J. Pintor, None; G. Carracedo, None; A. Mediero, None; A. Guzman-Aranguez,
None; M. Irazu, None; T. Pelaez, None; A. Peral, None.
Support: SAF-2003-00338, SAF-2004-06119-C02-01
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2153 - B922
2154 - B923
2155 - B924
2156 - B925
Gelatin Hydrogel as Controlled-Release Carrier of Growth Factor on Ocular Surface
K.Hori1, C.Sotozono1, K.Yamasaki1, M.Ozeki2, Y.Kimura2, Y.Tabata2, S.Kinoshita1.
Ophthalmology, Kyoto Prefectural Univ of Med, Kyoto, Japan; 2Dept. of Biomaterials,
Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Purpose: We have prepared several variations of biodegradable hydrogels from gelatin that has
the ability to physicochemically interact with some growth factors, enabling their controlled
release, have thereby successfully enhanced growth factor biological activities. Growth factor is
released as a result of hydrogel degradation. The object of this study is to investigate the feasibility
of using gelatin hydrogel for the controlled release of growth factor on the ocular surface.
Methods: We used epidermal growth factor (EGF) as a representative growth factor. A
biodegradable hydrogel was prepared from a cationic derivative of gelatin that can interact
with EGF. The cationized gelatin hydrogel, with 125I-labelled EGF incorporated, was placed
in the conjunctival sacs of mice, and the residual radiation was measured at different
time intervals to evaluate the in vivo profile of EGF release. Hydrogel incorporating 5
μg of EGF was placed on a round defect of rabbit cornea (177 mm 2) and the defect area
was measured by image analyzing software to evaluate the healing process (n=6). In
addition, epithelial proliferation was assessed immunohistochemically by counting the
number of Ki67-positive cells. EGF-free cationized gelatin hydrogel was used as control.
Results: Cationized gelatin hydrogel incorporating EGF remained over 7 days in the conjunctival
sacs. When hydrogel incorporating EGF (E+) was applied to the corneal epithelial defect, defect
area decreased to 10.0 ± 13.7 mm 2 at 4 days after wounding; this was a significantly greater
decrease than that seen with EGF-free hydrogel (E-) (49.3 ± 22.4 mm 2) (p=0.01). The number
of Ki67-positive cells at the peripheral unwounded cornea was 11 (E+) and 1.4 (E-) cells/mm.
Conclusions: Cationized gelatin hydrogel is a promising carrier for controlled release of
biologically active EGF on the ocular surface. This growth factor controlled-release system
can be applied to other growth factors and may provide a novel treatment option for severe
ocular surface diseases.
CR: K. Hori, None; C. Sotozono, None; K. Yamasaki, None; M. Ozeki, None; Y. Kimura,
None; Y. Tabata, None; S. Kinoshita, None.
Support: None.
Effects of a Phospholipid-Based Microemulsion (TBX-024) on Re-Epithelization
Processes in Patients Suffering From Non Infectious Corneal Erosions: A Randomised
Single Blind Pilot Study
C.Barozzi1, F.De Gregorio2, R.Falchetti3, M.Rolando4. 1R&d, Tubilux Pharma S.p.A,
Pomezia (Rome), Italy; 2Corneal PhysioPathology, La Sapienza University, Rome, Italy;
Inmm, CNR, Rome, Italy; 4Eye Clinic, University of Genoa, Genoa, Italy.
Purpose: The primary objective of this pilot study was to evaluate the effect of a
phospholipid-based microemulsion named TBX-024 on both the extent of the erosion
and the recovery time. The secondary objective of the study was to evaluate the effect of
TBX-024 on either objective or subjective symptoms and on ocular and systemic tolerance
Methods: Study design: randomised single blind clinical trial. Thirty patients suffering
from non infectious corneal erosion were included in the study: 15 were treated with TBX024 and 15 with 0.2% Sodium Hyaluronate (HA). Each patient instilled one drop of either
TBX-024 or HA four times a day for five days. The following criteria for evaluation were
used: Primary efficacy variable: measure of the size of lesion by fluorescein staining;
complete recovery time (in hours). Secondary efficacy variable: Visual Analogic Scales
(VAS) for subjective (pain, lachrymation, photophobia, foreign body sensation) and
objective symptoms (conjunctival hyperemia, chemosis, oedema). Tolerability: global
local tolerance assessment by the investigator on a VAS and global local tolerance
assessment by the patient on a VAS; systemic safety variables; evaluation of adverse events.
Results: Efficacy: The results of the study demonstrated that: 1) the patients treated with TBX-024
had a faster recovery time when compared with HA-treated patients; 2) the patients treated with
TBX-024 had faster reduction of the severity of both objective and subjective symptomatology;
3) Both study products were judged efficacious by both investigator and patients. Tollerability
and safety: Both local and systemic tolerance appeared to be very good with both study products.
Conclusions: The results of this pilot study showed a “trend” of a better efficacy of TBX-024
vs HA in accelerating the recovery time and in reducing the severity of symptomatology,
although not yet statistically significant. Further studies to demonstrate the clinical efficacy
of TBX024 in wound healing processes are on going.
CR: C. Barozzi, Tubilux Pharma E; F. De Gregorio, Tubilux Pharma C; R. Falchetti,
Tubilux Pharma C; M. Rolando, None.
Support: None.
Corneal Epithelial Cells Are Essential for Prevention of Myodifferentiation of Corneal
Fibroblasts in a Coculture System
K.Izumi1, D.Kurosaka1, T.Iwata2, K.Nakamura1, Y.Ohashi3, Y.Oguchi1, Y.Tanaka2,
K.Tsubota1, Y.Mashima1. 1Ophthalmology, Keio University School of Medicine, Tokyo,
Japan; 2National Institute of Sensory Organs, National Hospital Organization Tokyo
Medical Center, Tokyo, Japan; 3Ophthalmology, Ehime University School of Medicine,
Ehime, Japan.
Purpose: To establish a human corneal epithelial cell-fibroblast coculture system and
to demonstrate corneal epithelial-mesenchymal interactions and the influence of growth
factors on myofibroblast differentiation of corneal fibroblast using our coculture model.
Methods: Corneal fibroblasts were cultured on collagen gel with or without confluent
corneal epithelial cells. To evaluate myodifferentiation of fibroblasts, collagen gel
contraction was measured over 6 days with or without antibody against transforming
growth factor (TGF)-β. Amounts of mRNA encoding TGF-β2 and α-smooth muscle actin
(SMA) were determined by real-time quantitative PCR. Procedures were repeated with
cell exposure to antibody against basic fibroblast growth factor (bFGF) antibody. Basic
FGF was assayed in media on days 3 and 6. To evaluate the nature of fibroblast interaction
with epithelial cells, fibroblasts cultured in conditioned medium collected from epithelial
cells were subjected to gel contraction measurement and real-time quantitative PCR.
Results: Collagen gel contraction was increased on gels with fibroblasts alone, but suppressed
when fibroblasts were cocultured with epithelial cells. Anti-TGF-β antibody eliminated gel
contraction almost completely. TGF-β2 and α-SMA mRNA expression increased with time
in fibroblasts cultured alone, but changed little in cocultured fibroblasts. Coculture media
contained significant amounts of bFGF. Anti-bFGF antibody increased gel contraction of
cocultured fibroblasts, and expression of TGF-β2 and α-SMA mRNA. Conditioned medium
suppressed gel contraction and α-SMA mRNA expression in fibroblasts, but less than coculture.
Conclusions: Our coculture model is useful for the investigations of corneal epithelial cellfibroblast interaction. Corneal epithelial cells suppress differentiation of corneal fibroblasts into
myofibroblasts and prevent corneal stromal scarring via soluble factors including bFGF.
CR: K. Izumi, None; D. Kurosaka, None; T. Iwata, None; K. Nakamura, None; Y. Ohashi,
None; Y. Oguchi, None; Y. Tanaka, None; K. Tsubota, None; Y. Mashima, None.
Support: None.
Purpose: Matrix metalloproteinases (MMPs) are key regulators of tissue remodeling in cornea and
other organs. MMPs such as MMP-1 and -3 are expressed only on demand, typically stimulated by
autocrine or paracrine cytokines. In contrast, we have found that expression of gelatinase B (GelB;
MMP-9), a MMP expressed by corneal epithelial cells (CECs), is relatively refractory to cytokine
stimulation. Nevertheless, reports from other labs suggest that there may be physiologically relevant
conditions under which cytokines could act as significant stimulators of MMP-9 expression by
CECs. The purpose of this study was to investigate some of these possibilities. Methods: CECs
were harvested from rabbits, plated in 24 well culture dishes, and allowed to adhere overnight,
when cells were thoroughly washed to abrogate all receptor-mediated signaling. Many labs use
cholera toxin in their CEC cultures because it stimulates cell growth by up-regulating intracellular
cAMP levels. We hypothesized that this provides a secondary signal needed for cytokine induction
of GelB. Cultures were treated with IL-1β, TNF-α, TGF-β, EGF, HB-EGF, or FGF-2 at 10ng/
mL, in the presence or absence of 30ng/mL of cholera toxin. PMA (1μM) was used as a positive
control. Conditioned media was then collected to quantify GelB secretion by zymography. To
ensure a CE phenotype, immunocytochemistry was performed using antibodies against keratin 12.
Results: GelB expression is induced at the migrating front of the CE in healing wounds. GelB
is also expressed constitutively by CECs in culture, with highest expression levels at low cell
density, mimicking the conditions of low cell contact at the migrating wound front. GelB can
also be induced to high levels by agents such as PMA that activate signal transduction pathways
downstream of cytokine receptors. None of the cytokines, alone or with cholera toxin, stimulated
GelB expression above constitutive levels. As EGF is endogenously produced by migrating CECs,
we treated with AG1478, an EGFR antagonist, to inhibit endogenous EGFR activity; this did not
reduce constitutive expression. Additionally, EGF combined with IL-1beta was unable to elicit
upregulation of GelB expression, with or without cholera toxin. Experiments were performed multiple
times at different plating densities (5*10 4, 1*105, 2*105, 4*105, and 8*105 cells/well) with unvarying
results. In contrast, GelB expression was stimulated to high levels by the positive control PMA.
Conclusions: The results presented here provide further support for the idea that GelB expression is
not under significant paracrine or autocrine regulation, but is instead controlled by cell-cell contact
or physical forces at the migrating wound edge.
CR: G.M. Gorden, None; M.E. Fini, None.
Support: NIH Grants EY012651, EY014801 and Research to Prevent Blindness
2157 - B926
2158 - B927
Expression of Matrix Metalloproteinases 2 and 9 in Chronic Wounded Canine Corneas
and Spontaneous Chronic Corneal Epithelial Defects (SCCED)
R.T. Carter, R.Kambampati, C.J. Murphy, E.Bentley. Dept of Surgical Science, UW - Sch
of Veterinary Medicine, Madison, WI.
Purpose: Spontaneous chronic corneal epithelial defects (SCCED) are non-healing corneal
erosions characterized by redundant, non-adherent epithelium similar to recurrent erosions
in humans. Morphology of samples shows sheets of poorly adherent epithelial cells, absence
of basement membrane components, and the presence of a superficial hyalinized stromal
acellular zone. Matrix metalloproteinases (MMP) remodel the extracellular matrix and are
elevated in various pathological processes, including recurrent erosions. Previous work
in our lab has shown that expression of MMP 9 is upregulated following acute epithelial
wound healing and down-regulated as re-epithelialization is completed in normal dogs;
no change is MMP 2 was identified. We evaluated chronic, experimentally wounded
corneas and keratectomy samples from SCCED patients for MMP 9 and MMP 2 activity.
Methods: Left eyes of 5 normal beagle dogs were used for study. A 10 mm region of
the axial cornea underwent mechanical epithelial debridement once a week for 8 weeks.
The dogs were humanely euthanized and corneas collected. Superficial keratectomies
(n=17) were performed on canine SCCED patients for therapeutic reasons. Samples were
homogenized and supernatants collected. Protein concentrations for each sample were
obtained to standardize samples. Zymography was performed as previously described.
Gels were imaged and statistical analysis was performed to assess for significance.
Results: There was no sig nif icant difference in values for M M P 9
or M M P2 i n ch ronically wou nded cor neas versus SCCED samples.
Conclusions: Preliminary results show no significant difference in MMP 2 and MMP 9
activity between SCCED and chronically wounded corneas. These data suggest that these
MMPs are not involved in the pathophysiology of SCCED and are more likely the response
to chronic epithelial injury. These findings emphasize the importance of using a model of
chronic wound healing to determine whether changes noted in SCCED patients are due to
the underlying pathophysiology or merely due to the presence of a spontaneous chronic
corneal epithelial defect.
CR: R.T. Carter, None; R. Kambampati, None; C.J. Murphy, None; E. Bentley, None.
Support: Resident Grant American College of Veterinary Ophthalmologists
Regulation of GelB During Corneal Resurfacing
G.M. Gorden, M.E. Fini. Ophthalmology, University Miami, Miami, FL.
Effects of MMP-10 and a Cathepsin Inhibitor, Cystatin C, on Organ-Cultured Normal
and Diabetic Human Corneas
A.V. Ljubimov1,2, A.M. Aoki1, A.A. Kramerov1. 1Ophthalmology Research Laboratories,
Cedars-Sinai Medical Center, Los Angeles, CA; 2David Geffen School of Medicine at
UCLA, Los Angeles, CA.
Purpose: We have recently documented an increased expression of matrix metalloproteinase10 (MMP-10) and cathepsin F in the epithelium of human corneas from patients with diabetes
and diabetic retinopathy (DR). The purpose of this work was to (1) examine the action of
MMP-10 on wound healing and DR marker expression in organ-cultured normal corneas, and
(2) determine the effects of cathepsin inhibition on organ cultured corneas from DR patients.
Methods: Human organ-cultured corneas were wounded using n-heptanol to preserve epithelial
basement membrane (BM). Purified MMP-10 was added to air-exposed top of normal wounded
corneas at 1 μg/ml in 20 μl, 1-2 times a day until wound closure. Wounded DR corneas were
treated with broad-spectrum MMP inhibitors, galardin at 5 μM, or a chemically modified
tetracycline, CMT-3 (from Collagenex), at 50 μg/ml, in medium. Some DR corneas were also
treated with a cathepsin inhibitor, cystatin C, at 50 μg/ml. After wound healing was complete,
corneas were cultured for another week and processed for immunohistochemical analysis
of diabetic markers, epithelial integrin α 3β1, and α5 chain of BM component, laminin-10.
Results: Analysis of 26 normal corneas showed that epithelial defects healed on average by 3.4±0.8
(mean±SEM) days without MMP-10 and by 5.5±1.9 days with MMP-10 present. In MMP-10-treated
corneas, immunofluorescent staining for laminin α5 chain was discontinuous in the epithelial
BM, and integrin α 3β1 staining in the epithelium was weak and disorganized, very similar to DR
corneas. Compared to vehicle treatment, galardin or CMT-3 did not show any effect on healing of
organ-cultured and wounded DR corneas. Possibly, these MMP inhibitors also blocked other MMPs
that are needed for corneal wound healing, e.g., MMP-9. Cystatin C treatment reduced cathepsin
F staining in DR corneal epithelium. Moreover, integrin α 3β1 staining increased and became more
regular, similar to normal corneas. This result was obtained in all three pairs of DR corneas analyzed.
Wound healing rates, however, were not significantly different with or without cystatin C treatment.
Conclusions: The data support the hypothesis that increased activity of certain proteinases may be
responsible for clinically observed diabetic corneal abnormalities. Specific inhibition of proteinase
expression using antisense oligonucleotides or siRNA may be an effective way to restore normal
wound healing and marker patterns in corneas of diabetic and DR patients.
CR: A.V. Ljubimov, None; A.M. Aoki, None; A.A. Kramerov, None.
Support: NIH Grant EY13431 and the Skirball Program in Molecular Ophthalmology
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2159 - B928
2160 - B929
Myofibroblast Generation in the Anterior Stroma Is Triggered by Surface
S.E. Wilson, M.V. Netto, R.R. Mohan. Cole Eye Institute, The Cleveland Clinic, Cleveland,
Purpose: To determine the relationship between the level of stromal surface irregularity
and myofibroblast generation. Methods: A fine mesh screen was used to mask excimer laser
ablation using a refractive ablation of -4.5 diopters (D) by placing the screen for varying
percentages of pulses at the end of the ablation in 35 New Zealand rabbits, divided into five
different groups: -4.5 D PRK with no screening (group I), -4.5 D PRK with screening of
10% of pulses (group II), -4.5 D PRK with screening of 30% of pulses (group III), -4.5 D
PRK with screening of 50% of pulses (group IV) and -4.5 D PRK with screening of 50% of
pulses followed by smoothing phototherapeutic keratectomy using either hyaluronic acid or
1% methylcellulose as masking agents (group V). Slit lamp analysis and haze grading was
performed in all five groups. Rabbits were sacrificed at 4 weeks after treatment and corneas
underwent immunohistological staining for myofibroblast marker alpha-smooth muscle actin
(SMA). Results: Slit-lamp examination revealed significant haze formation in corneas in
groups III and IV, less in group II, and little haze in groups I or V. Analysis of SMA staining
at 4 weeks after surgery showed the lowest myofibroblast formation in groups I (3.0 ± 5.1
cells/400x field) and V (3.2 ± 5.7), with progressively more in groups II (7.1 ± 6.5 ), III (11.8
± 7.2) and IV (19.5 ± 10.0). Group I was significantly different from groups II, III, and IV,
and groups II and III were significantly different from group IV ( p < 0.05). Groups I and V
were not significantly different. Conclusions: Objective and subjective analysis demonstrated
a correlation between the level of stromal surface irregularity and the level of corneal haze
formation after surface ablation. We hypothesize that surface irregularity leads to abnormal
basement membrane regeneration and, therefore, greater TGF beta and PDGF penetration
into the stroma from the epithelium, associated with trans-differentiation of keratocytes to
myofibroblasts. Alternatively, greater surface area of contact between epithelium and the
underlying stroma may be the key factor associated with greater TGF beta and PDGF access
to the stroma and resulting myofibroblast generation. EY10056
CR: S.E. Wilson, None; M.V. Netto, None; R.R. Mohan, None.
Support: NIH Grant EY10056
Non-Fibrotic Wound Healing in the Cornea: Preliminary Results of Intrastromal
Corneal Ring Surgery
O.Abdiu1, G.Schultz2, G.Van Setten1. 1Dept Ophthalmology, St Eriks Eye Hosp, Stockholm,
Sweden; 2Institute of Wound Research and Ophtalmology, University of Florida,
Gainesville, FL.
Purpose: Intrastromal corneal ring (ICR) surgery is considered an attractive option
for correcting minor refractive errors. Besides reported low risk of complications
and surgical side effects, the option of taking out the ICR does enhance the value of
this operation technique compared to others such as LASIK. In the present study,
we investigated the occurrence of markers of fibrosis in eyes with ICR implants.
Methods: Eight rabbits were operated on with ICR (INTACS), in accordance with
ARVO guidelines, and corneas were harvest at 6 days (4 rabbits) and 13 days (4
rabbits) after surgery. The corneas were then prepared and immunohistologically
stained with specific antibody to connective tissue growth factor (CTGF).
Results: The corneal specimens showed staining in the epithelium at the basal layer, which
intensified close to the area of the base of the created corneal stromal ridge created by
the ICR. No staining was observed inside the wound channel or on the top of the ridge.
Conclusions: It appears that ICR implantation does not induce a strong stromal wound
healing reaction leading to fibrosis via the CTGF associated pathways. With the surgical
method used, corneal stromal shaping appears to be possible without inducing permanent
scar formation. This may reflect on other recent corneal surgery techniques such as partial
corneal transplantation.
CR: O. Abdiu, None; G. Schultz, None; G. Van Setten, None.
Support: None.
2161 - B930
2162 - B931
High Affinity Targeting of Activated Corneal Keratocytes in vitro: Subtractive
SELEX; New Frontiers for in vivo Cell Labelling and Targeted Drug Delivery
R.I. Angunawela1, D.H. J. Bunka2, P.Stockley2, J.Marshall1. 1Ophthalmology, Rayne
Institute, St Thomas Hospital, London, United Kingdom; 2School of Biochemistry and
Molecular Biology, Astbury Centre for Structural Molecular Biology, University of Leeds,
United Kingdom.
Viral Gene Delivery Methods for the Cornea
J.Liu1A, S.S. Tuli1B, A.S. Lewin1A, D.C. Bloom1A, W.W. Hauswirth1A,1B, G.S. Schultz1C.
Molecular Genetics and Microbiology, BOphthalmology, CObstetrics and Gynecology,
University of Florida, Gainesville, FL.
Purpose: Both inherited and viral diseases affecting the cornea might be treated
through gene therapy. Adeno-associated virus (AAV) has the advantages of long term
delivery of genes to both dividing and non dividing cells. The goal of this study
includes (1) to compare gene transfer efficiency of different AAV vectors in the rabbit
cornea, and (2) to locate transgene expression in different cell layers of corneal tissue.
Methods: An AAV2 vector expressing GFP driven by chicken- β-actin promoter and
CMV enhancer was packaged in 5 different capsid types (1, 2, 5, 7, 8). Both eyes of NZ
white rabbits were infected with the same serotype. Equal numbers of AAV particles
(1011 vector genomes) were diluted in buffer and applied to the cornea for 10 minutes
following excimer ablation of the corneal epithelium to 25 microns. Seven days were
allowed for gene expression before corneas were removed. The corneas were fixed in 4%
formaldehyde before cryosectioning at 10um thickness. One sample was analyzed at every
third section. Immunostaining using biotinylated antibody against GFP and an alkaline
phosphatase detection as well as a peroxidase detection system were employed. Bright
field and fluorescence micrographs were taken for each section using a morphometric
microscope, and the level of GFP immunostaining was measured using MCID software.
Results: By 7 days all the serotypes of AAV led to expression of the transgene in all layers of the
corneal tissue. Epithelial cells were heavily stained compared with stroma and endothelial cells.
AAV type 1 was 20 -30% more efficient in gene delivery in comparison with other serotypes,
followed by AAV type 8 which exhibited the highest level of penetration to deep layers of the cornea.
Conclusions: Among all the serotypes that have been tested AAV type 1 and 8 can provide
long lasting and highly efficient transduction. These experiments point the way for delivery
of potentially therapeutic genes to the cornea.
CR: J. Liu, None; S.S. Tuli, None; A.S. Lewin, None; D.C. Bloom, None; W.W. Hauswirth,
None; G.S. Schultz, None.
Support: None.
2163 - B932
2164 - B933
Purpose: To determine if the SELEX (Systematic Evolution of Ligands by Exponential
enrichment) technique could be used to evolve high aff inity ssRNA aptamers,
capable to differentiating activated, from quiescent cor neal keratocyte in-vitro.
Methods: Cells were liberated by collagenase digestion of fresh bovine corneas. Quiescent and
activated bovine keratocytes were cultured in serum free and serum containing medium respectively.
The highly random ssRNA starting pool was incubated with quiescent cells, to eliminate aptamers
binding to non specific shared cellular components of both cell types. The remaining ssRNA pool
was then exposed to the activated cell phenotype, and binding RNA’s isolated (product). These were
amplified and this iterative process was repeated with the selectively reduced ssRNA pool (product)
of the previous round. After 10 rounds, the final product was coupled to a fluorophore for in-vitro
detection by fluorescent microscopy of cultured cells. Both cell phenotypes were incubated with either
the final high affinity aptamer or with the random fluorophore coupled non specific ssRNA pool. As a
further control we also used bovine skin fibroblasts to determine the specificity of the final product.
Results: In the high affinity aptamer group, fluorescence was only detected with the activated
corneal keratocyte phenotype. There was no fluorescence of either the quiescent cell type
or of skin fibroblasts. In contrast, fluorescence was detected with all cells incubated with the
random fluorophore coupled ssRNA pool. These results indicate that this subtractive SELEX
technique yields highly specific ligands, that can distinguish between cells of homologous
origin, and hence differentiate activated, from quiescent corneal keratocytes in-vitro
Conclusions: Through a subtractive SELEX selection method, we have developed a ssRNA aptamer
with high affinity for activated bovine corneal keratocytes. Applying a similar protocol, we have now
begun the synthesis of an aptamer for activated human corneal keratocytes. The novel properties of
aptamers have enormous potential for in-vivo application; in the critical appraisal of new therapeutic
products, corneal wound healing events and significantly, for specific, and targeted drug delivery
in the human cornea.
CR: R.I. Angunawela, None; D.H.J. Bunka, None; P. Stockley, None; J. Marshall, None.
Support: British Eye Research Foundation
Controlled and Site-selective Gene Delivery Into Mouse Keratocytes by Adenoassociated Viral, Lentiviral and Plasmid Vectors
R.R. Mohan1, G.S. Schultz2, A.Sharma1, S.Sinha1, M.V. Netto1, S.E. Wilson1. 1Cole Eye
Institute, The Cleveland Clinic Foundation, Cleveland, OH; 2OBGYN and Institute for
Wound Research, University of Florida, Gainesville, FL.
Purpose: Controlled and site-selective gene delivery techniques provide an alternative approach to
study gene’s functions and pathological conditions in the cornea and prevent undesired side effects
of surgery. The goals of this study were to: i) identify optimal viral and non-viral vectors for gene
delivery in keratocyte; ii) determine changes in the quantity and/or location of transgene delivery
mediated by vector-application methods; and iii) characterize the first point in time and duration of
expression, and localization of transgene with different vectors in cornea. Methods: The efficacy
of three vectors, adeno-associated virus serotype 5 (AAV5), lentiviral and plasmid, and two vectordelivery techniques, were investigated. Two microliters of a) AAV5-EGFP (9.6x1010 particles/µl),
b) Lentiviral-EGFP (1.2x1010 particles/µl), c) pTR-β-gal plasmid (50µg DNA mixed with 200nM of
DDAB and DOPE) or d) BSS were injected into central stroma using a Hamilton microinjection
system or applied topically on the exposed and dried stromal surface after removal of epithelium in the
mouse cornea. Virus-treated eyes were collected at 2d, 3d, 1w, 2w, 4w, and 6w, and plasmid-treated
eyes were collected at 2hrs, 4hrs, 24hrs, 36hrs, 3d and 7d. Transgene expression was analyzed with
immunostaining, ELISA assay, and Confocal microscopy. Velocity software was used for generating
3-D reconstructions. Results: All three vectors delivered reporter genes selectively into keratocytes.
Viral vectors delivered with higher efficiency and for longer duration compared to the plasmid vector.
In virus-treated tissues, reporter gene expression was first detected at day 3. However, in plasmid
treated tissues expression was first detected at 2 hrs. AAV- or lentivirus-exposed tissues expressed
transgene up to 6 weeks (last tested time point), whereas no expression in plasmid-treated tissues
was observed after 3 days. Vector-delivery techniques also influenced efficiency and the area of
gene delivery. Topical application delivered reporter genes to the stromal surface exposed to the
vector, in contrast to microinjection where transgene delivery was localized to the site of injection.
Conclusions: AAV5, lentiviral, and pTR-plasmid vectors can be used for selective gene delivery into
mouse keratocytes and may be useful for gene therapy in humans. Appropriate vectors and delivery
techniques can be selected to achieve desired levels and duration of gene expression.
CR: R.R. Mohan, None; G.S. Schultz, None; A. Sharma, None; S. Sinha, None; M.V. Netto,
None; S.E. Wilson, None.
Support: EY10056
Transport of Poly (lactic-co-glycolic acid) Particles Containing Antisense
Oligonucleotides by Iontophoresis: An in vitro Study
T.D. Washington1A, K.S. Burney1B. AMaterials Science & Engineering, BBiomedical
Engineering, 1University of Florida, Gainesville, FL.
Purpose: Research in corneal drug delivery has shown transport across the corneal tissue layers
to be a barrier for effective genetic therapeutic treatment. Transport of the CTGF antisense
oligonucleotides to treat corneal scarring can benefit from using polymeric carriers to extend
bioavailability and protect from enzymatic degradation. The rate of particle diffusion across
the cornea is further enhanced by the addition of iontophoresis. The current study was designed
to investigate the permeability of the cornea to antisense oligonucleotides within carriers and
to demonstrate an increase in transport efficiency of the oligonucleotides with iontophoresis.
Methods: Eight rabbit corneas were mounted between two horizontal diffusion cells.
Half were exposed to a 1mM solution of fluorescently labeled oligonucleotides and half
were exposed to a 0.60 % solution of PLGA particles at a constant temperature of 35°C.
Two corneas from each group were subjected to iontophoresis at 1.5mA for 10 minutes. A
fluorescent plate reader was used to determine the concentration of particles present. The
confocal microscope was used to confirm the presence of oligonucleotides and microspheres.
Results: There is a higher concentration of oligonucleotides present in the receiver solution
after iontophoresis. Conclusions: The results suggest that iontophoresis increases the rate
of particle transport across the cornea.
CR: T.D. Washington, None; K.S. Burney, None.
Support: NIH Grant EY005587-17
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2165 - B934
2166 - B935
Correlation Between Wound Healing Response and Refractive Regression After
Conductive Keratoplasty (CK)
S.Esquenazi, H.E. P. Bazan, D.B. Kim, J.He, V.Bui, N.G. Bazan. Ophthalmology, LSU Eye
and Neuroscience Centers, New Orleans, LA.
P u r po se : To def i ne t he st abi l it y of t he ref r a ct ive re su lt s af t e r
conductive keratoplasty (CK) with cellular responses in a rabbit model.
Methods: CK was performed in eight eyes of New Zealand albino rabbits. On each eye,
24 spots were placed in a cross-corneal manner using three optical zones at 6, 7, and 8
mm. Eyes were monitored by corneal topography weekly. Rabbits were humanely
euthanized at 2, 4, 6, or 8 weeks postoperatively. The eyes were then enucleated and
processed for histopathology, TUNEL staining and immunohistochemical analysis using
antibodies against ∀-smooth muscle actin (∀-SMA), chondroitin sulfate, and collagen III.
Results: All eyes showed an initial mean steepening of the corneal curvature of 2.07
diopters (D) at 2 weeks postoperatively. Corneal topography revealed a 23%, 37%, and
42% regression of the refractive results at 4, 6, and 8 weeks postoperatively, respectively.
Immunohistochemical analysis showed evidence of keratocyte apoptosis, myofibroblast
transformation, and up-regulation of chondroitin sulfate and collagen III in the area
surrounding the tip in each spot. The percentage of positive ∀-SMA in the area of CK was
5.4% at 2 weeks and decreased to 1.65% at 8 weeks. The up-regulation of myofibroblasts had a
high correlation with the refractive results and with the short-term stability of the procedure.
Conclusions: The histologic changes that occur after CK may be responsible for the regression
observed, and the maintenance of myofibroblasts over time may be associated with better
long-term stability.
CR: S. Esquenazi, None; H.E.P. Bazan, None; D.B. Kim, None; J. He, None; V. Bui,
None; N.G. Bazan, None.
Support: NEI EY 04928
Effects of RGTA OTR4131 on Ocular Surface: in vivo Evaluation on a Rabbit Corneal
Wound Healing Model and in vitro Toxicological Studies
F.Brignole1,2, L.Potron1, C.Martin1, P.Rat1,2, L.Riancho1,2, J.P. Caruelle3, D.Barritault4,
J.M. Warnet1,2, C.Baudouin2. 1Dept. of Toxicology, Faculty of Pharmaceutical and
Biological Sciences, University Paris 5, France; 2Quinze-Vingts National Ophthalmology
Hospital and INSERM U598, Paris, France; 3CNRS FREA-2412, University Paris 12,
France; 4OTR3, Paris, France.
2167 - B936
2168 - B937
Purpose: Regenerating Agent, RGTA, is an engineered biopolymer mimicking heparan sulfates
as a protector and stabilizer of the actions of heparin-binding growth factors and is known to
stimulate wound healing in different in vivo systems. In this study, we analyzed its effects both in
vivo and in vitro in order to assess its potential interest in ocular surface diseases. Methods: 1/ In
vivo, we investigated the wound healing activity of a single application of RGTA 1h after an alkali
burn using NaOH in 1 eye of 20 rabbits. During 7d, we scored the clinical signs and quantified the
corneal opacity and the deepithelialization. Histology was performed at day 7. 2/ In vitro, we studied,
on human conjunctival epithelial cells and rabbit corneal fibroblasts, the effects of RGTA on cell
proliferation, apoptosis using flow cytometry and oxidative stress (OS) with a previously validated
technique of cold light cytometry on live cells. We looked also after its eventual protective effect in
an in vitro model of Benzalkonium Chloride (BAC)-induced cell toxicity. Results: In vivo, RGTA
was efficient in enhancing reepithelialization, reducing clinical signs, corneal opacity and improving
histological patterns. In vitro, it did not induce any alteration of cell viability nor apoptosis on both
cell lines. On corneal fibroblasts but not on epithelial cells, it reduced cell proliferation in a dosedependent manner. Used in combination with a toxic dose of BAC, it showed a protective effect
on conjunctival epithelial cells. RGTA did not induce by itself OS, but could protect conjunctival
cells from OS induced by BAC through a reduction of H 2O2 production and glutathione uptake.
Conclusions: RGTA is a promising drug in controlling ocular surface inflammation and promoting
corneal wound healing, and is not toxic both in vivo and in vitro. Its antioxidative and antifibroblast
proliferation effects also suggest that it could be interesting for reducing uncontrolled corneal
scarring. These properties may deserve further interest for future therapeutic applications in patients
suffering of various ocular surface diseases.
CR: F. Brignole, None; L. Potron, None; C. Martin, None; P. Rat, None; L. Riancho, None; J.P.
Caruelle, None; D. Barritault, OTR3 I, P; J.M. Warnet, None; C. Baudouin, None.
Support: INSERM U598 and CNRS supports
Cohesive Tensile Strength of Human Laser in situ Keratomileusis Wounds With
Histologic and Ultrastructural Correlation
D.G. Dawson, I.Schmack, B.E. McCarey, H.E. Grossniklaus, H.F. Edelhauser.
Ophthalmology, Emory University, Atlanta, GA.
Purpose: The purpose of this study was to measure the cohesive tensile strength of human
LASIK corneal wounds. Histology and ultrastructural correlations from the separated and intact
portions of the specimens were evaluated to help better understand the biomechanical results.
Methods: Twenty-one human corneosceral specimens from 11 eyebank donors with previous
LASIK surgery were obtained from U.S. eye banks. Four-mm limbus-to-limbus corneoscleral
strips were cut and dissected by manual lamellar dissection to expose the LASIK interface
wound. Using a motorized pulling device with an attached force transducer, the force
required to separate the wound in LASIK corneas was measured (grams/mm). Intact and
torn portions of the specimen were processed for histologic and ultrastructural evaluations.
Eleven normal control corneoscleral specimens from 7 eyebank donors served as controls.
Results: The mean tensile strength averaged 2.5% (0.75 +/- 0.33 gram/mm) of normal (30.21
+/- 3.03 grams/mm) in wound regions (central and paracentral) that contained a hypocellular
primitive stromal scar and 24.9% (7.52 +/- 4.30 grams/mm) of normal in the flap wound margin,
which contained a hypercellular fibrotic stromal scar. Although the hypocellular primitive
scar demonstrated no gain in strength over time, the peak tensile strength of the hypercellular
fibrotic scar gradually increased until maximum values were reached by 3.5 year postoperatively; averaging 28.0% (8.46 +/- 4.56 grams/mm) of normal. There was no significant
difference in wound strength between mechanical and laser microkeratome LASIK cases.
Histologic and ultrastructural correlations demonstrated that wound margins with persistent
epithelial ingrowth were on average one third the strength of those without epithelial ingrowth.
Conclusions: Human corneal stroma typically heals in a limited and incomplete fashion that
results in a very weak, transparent hypocellular primitive scar. The LASIK wound margin
is different in that epithelial-stromal interactions appear to augment the normal corneal
stromal wound healing response typically resulting in a 10-fold stronger and 30% more
hazy hypercellular fibrotic scar.
CR: D.G. Dawson, None; I. Schmack, None; B.E. McCarey, None; H.E. Grossniklaus,
None; H.F. Edelhauser, None.
Support: NIH Grants EY-000933, P30-EY06360, T32-EY07092, and RPB
Inflammatory Responses After Ferrara Ring Segments™ Implantation Coated and
Non-Coated With Chondroitin Sulphate in Rabbits Corneas
F.B. D. D. Silva, P.F. A. Cunha, D.B. Miranda, D.Miranda. Cornea, Center of Advanced
Ophthalmology, Belo Horizonte, Brazil.
Purpose: To compare the inflammatory responses after Ferrara Ring Segments™
implantation coated and non-coated with chondroitin sulphate in rabbits corneas.
Methods: Fifteen rabbits underwent Ferrara Ring Segments™ procedure at the same
day (Ferrara Ophthalmics Inc., Belo Horizonte, MG, Brazil). Each rabbit received, in its
right eye, a non-coated segment and, in its left eye, a chondroitin sulphate coated segment.
The rabbits had been sacrificed 1 month after surgery. The corneas of the enucleated
eyes were analyzed through a histological routine. Histological evaluation of the induced
morphological changes was additionally described and both groups were compared.
Results: Epithelial thinning located above the coated and non-coated segment and
hyperplasia of the adjacent cells layers, fibroblastic proliferation and edema were
related after 30 days. There was no histopathological difference between the rabbit
corneas with the non-coated and those with the coated Ferrara Ring Segment™.
Histology of the corneas of the implanted eyes in both groups revealed good
biocompatibility, with only one major complication such as stromal neovascularization.
Conclusions: In summary, chondroitin sulphate coating does not alter inflammatory reaction
in rabbit’s corneas and the structural changes founded in both groups were the same.
CR: F.B.D.D. Silva, None; P.F.A. Cunha, Ferrara Ophthalmics P; D.B. Miranda, None; D.
Miranda, None.
Support: None.
2169 - B938
2170 - B939
Clinical Efficacy of Eyedrops Containing FGLM-NH 2 and IGF-1 for Treatment of
Neurotrophic Keratopathy
N.Morishige, T.-I.Chikama, R.Yanai, N.Yamada, T.Nishida. Biomol Recog Ophthalmology,
Yamaguchi Univ School of Med, Ube City, Japan.
Purpose: We have previously demonstrated the successful treatment in a case of
neurotrophic keratopathy with eyedrops containing insulin-like growth factor-1 (IGF-1)
and the substance P-derived peptide FGLM-NH 2 .We have now performed an uncontrolled
case series study of this treatment in 22 patients with neurotrophic keratopathy.
Methods: A total of 22 patients (7 men, 15 women; age range, 27 to 78 years; mean age ±
SD, 56.9 ± 13.4 years) with neurotrophic keratopathy was enrolled in the study between
January 1997 and December 2003. Fifteen and seven of the subjects manifested persistent
epithelial defects (PED) or superficial punctate keratopathy (SPK), respectively. Among
the patients with PED, neurotrophic keratopathy was attributable to neurosurgery in five,
diabetes in six, keratoplasty in two, and other causes in two; among those with SPK, it was
attributable to neurosurgery in five and other causes in two. Administration of eyedrops
containing FGLM-NH 2 (1 mg/ml) plus IGF-1 (10 µg/ml) as well as either Levofloxacin or
Ofloxacin eyedrops was performed four times a day for 4 weeks. For the PED cases, clinical
efficacy was determined from the decrease in the area of fluorescein staining on slitlamp
photographs. For SPK cases, clinical efficacy was determined from the decrease in SPK
grade as defined by fluorescein staining (Miyata K et al. Arch Ophthalmol. 2003;1537-9).
Results: The epithelial defects of nine patients with PED (five due to neurosurgery, three due
to diabetes, and one due to other causes) disappeared within 2 weeks of treatment onset. At the
end of the 4-week treatment period, the epithelial defects of 11 PED patients had disappeared
and those of two had not disappeared; the remaining two PED patients dropped out from
the study. SPK improved by two grades or more in three SPK cases due to neurosurgery. No
worsening of clinical findings or adverse effects were observed in any of the study subjects.
Conclusions: Administration of eyedrops containing FGLM-NH 2 plus IGF-1 is highly effective
for the treatment of PED due to neurosurgery in patients with neurotrophic keratopathy. It
is also effective for the treatment of PED due to diabetes and of SPK due to neuosurgery
in such patients.
CR: N. Morishige, None; T. Chikama, None; R. Yanai, None; N. Yamada, None; T.
Nishida, None.
Support: None.
Risk Functions for the Prediction of Specific Eye Injuries Using Projectile Data
E.A. Kennedy1, S.M. Duma1, T.P. Ng1, J.D. Stitzel1, F.P. Kuhn2. 1Center for Injury
Biomechanics, Virginia Tech - Wake Forest, Blacksburg, VA; 2American Society of
Ocular Trauma, Birmingham, AL.
Purpose: The purpose of this study is to determine the most significant factors for predicting ocular
injuries or tissue lesions based on a parametric analysis of experimental data from eye impact tests.
Methods: Data from nine existing studies consisting of 71 total experiments were sorted
according to projectile characteristics such as type, mass, and diameter. Five eye injury groups
were established: corneal abrasion, hyphema, lens dislocation, retinal damage, and globe rupture.
Projectiles used to impact the eye included BB’s, foam, metal rods, baseballs, and squash balls.
Impact velocities ranged from 2 m/s to 122 m/s. Statistical values were generated from logistic
regression to determine significant projectile characteristics for predicting ocular injury.
Results: Among all of the predictors tested, normalized energy (energy/projected area)
yielded the best injury risk curve with the most significance (p=0.001 in all cases). A 50%
risk of corneal abrasion and lens dislocation were found at 1,479 kg/s2 and 18,450 kg/s2,
respectively, for globe rupture and hyphema at 23,767 kg/s2 and 20,183 kg/s2, respectively, and
at 30,869 kg/s2 for retinal damage. Kinetic energy alone was not as significant a predictor for
ocular injury as normalized energy, yielding less significant p-values. Both mass and velocity
were considered poor predictors, with higher p-values in comparison with other measures.
Conclusions: This study is the first to derive injury risk functions based on a pooling and
reevaluation of data from previously published eye impact experiments. Such data pooling
yields a larger database able to cover a broader range of projectile characteristics and observed
injuries. Normalized energy was the most significant predictor of injury type and tissue
lesion. The development of these risk functions allow automobile, sports equipment, and other
consumer product designers to evaluate the potential for eye injuries from the outset of the
design process, seriously reducing the eye injury potential for a variety of products.
CR: E.A. Kennedy, None; S.M. Duma, None; T.P. Ng, None; J.D. Stitzel, None; F.P.
Kuhn, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2129-2173 / B898-B942
264. Corneal Wound Healing I Organizing Section: CO
2171 - B940
Keratocyte Response Following INTACS®, Intracorneal Lens Inserts, and Flap
Creation by Femtosecond Laser Ablation (Intralase®) for LASIK
D.F. Goldberg, H.D. Cavanagh, W.Bowman, P.Kelley, D.Parmar, M.Petroll, S.M. Verity,
J.P. McCulley. Ophthalmology, UT Southwestern Med Ctr, Dallas, TX.
Purpose: Several different surgical techniques have emerged in recent years which rely
on either mechanical stress (e.g INTACS), tissue removal (e.g. LASIK) or tissue addition
(e.g. intracorneal lens inserts) to correct refractive errors. The purpose of this study
was to compare the corneal response to these insults using in vivo confocal microscopy.
Methods: A total of 14 patients (17 eyes) were evaluated: 5 eyes that had undegone LASIK
with flap creation by Intralase® (3-4 months post-op), 7 eyes with intracorneal lens inserts
(PermaVision®, 1-6 months post-op), and 5 patients with INTACS® (5 years post-op).
Tandem scanning confocal microscopy (TSCM) was used to measure epithelial thickness,
and identify morphologic changes in the corneal stroma following these procedures.
Results: TSCM identified keratocyte activation and deposition of extracellular matrix (fibrosis)
in two of five eyes that had undergone LASIK with Intralase®; the other three eyes had interface
particles but no cell activation or detectible ECM deposition. Interestingly, the patient with
the largest amount of fibrosis (40 *m thick) also exhibited delayed light sensitivity syndrome
clinically. Following intracorneal lens insertion, 4 of 7 eyes had keratocyte activation and
fibrosis adjacent to both the anterior and posterior surface of the implant. Interestingly, epithelial
thickness was reduced in these patients as compared to the Intralase® patients (34.8 + 6.4 *m vs.
45.2 + 6.4 *m, p < 0.05). Following long-term use of INTACS, abnormal ECM deposition (haze)
was observed in varying degrees in all five patients adjacent to the implant. Interestingly, bright,
crystal-like structures consistent with lipid deposition were also observed in three patients.
Conclusions: Both Intralase® and intracorneal lens insertion can induce keratocyte activation
and ECM deposition in some patients; in general this response is greater than that previously
observed following traditional LASIK, consistent with clinical observations of haze and/or
delayed light sensitivity in some cases. In addition to ECM deposition, long-term mechanical
perturbation following INTACS appears to result in lipid synthesis by corneal keratocytes
in a subset of patients.
CR: D.F. Goldberg, None; H.D. Cavanagh, None; W. Bowman, None; P. Kelley, None; D.
Parmar, None; M. Petroll, None; S.M. Verity, None; J.P. McCulley, None.
Support: None.
2172 - B941
Tissue Transglutaminase Promotes Cell Adhesion and Collagen Gel Contraction
Induced by Keratocytes
N.Taenaka1, T.Hibino2, K.Abe1, M.Fukuda1, H.Mishima1, H.Mishima3, Y.Shimomura1.
Ophthalmology, Kinki Univ. School of Medicine, Osaka-Sayama, Japan; 2Ophthalmology,
Kinki Univ. Sakai Hospital, Sakai, Japan; 3Ophthalmology, Kinki Univ. Nara hospital,
Ikoma, Japan.
Purpose: Cell-surface tissue transglutaminase (tTG) affects cell-matrix interactions
in cell adhesion, migration and extracellular matrix reorganization. However, the
inf luence of tTG on keratocytes has not been well understood yet. In this study,
to understand the role of tTG in corneal stromal wound healing, we investigated
the effect of tTG on cell adhesion and collagen gel contraction by keratocytes.
Methods: Human keratocytes were seeded on fibronectin-coated or BSA-coated plates, and
were incubated with tTG (0.01-0.1U/ml) with or without RGD or RGE peptide(200-400μM)
for 90 minutes. The number of attached cells was subsequently measured by MTT assay. In
other experiments, keratocytes in collagen gels were cultured with serum-free MEM medium
containing tTG (0.1-0.3U/ml) with or without anti-tTG antibody(10μg/ml ), fibronectin(10μg/
ml), TGF-β(0.1ng/ml) and PDGF(10ng/ml) for 5 days. The gel diameters were then measured.
Results: tTG enhanced cell adhesion of keratocytes in a dose-dependent manner. The
tTG-induced cell adhesion was inhibited not by RGE peptide but by RGD peptide on both
BSA-coated and fibronectin-coated plates. On the other hand, tTG stimulated collagen gel
contraction by keratocytes in a dose-dependent manner as well. The stimulative effect on
collagen gel contraction was antagonized by anti-tTG antibody. Moreover, fibronectin,
TGF-β and PDGF also contributed to the stimulation to collagen gel contraction by
keratocytes. tTG also enhanced these stimulative effects on collagen gel contraction.
Conclusions: These findings suggest that tTG promotes both the RGD-dependent cell adhesion
and collagen gel contraction by keratocytes.
CR: N. Taenaka, None; T. Hibino, None; K. Abe, None; M. Fukuda, None; H. Mishima,
None; H. Mishima, None; Y. Shimomura, None.
Support: Grant 15791019 from Japanese Ministry
2173 - B942
Experimental Model of LASIK and Pharmacological Modulation of Corneal
R.M. Torres1, J.M. Merayo-Lloves1, J.T. Blanco-Mezquita1, S.Mar2, C.P. Günther1,
G.Rodríguez1, A.Mayo1, C.Martínez-García3. 1IOBA, Valladolid, Spain; 2Physics and
Optics Applied, Valladolid, Spain; 3Biology Department, Valladolid, Spain.
Purpose: Develop an experimental animal model of LASIK and assess
pharmacological modulation of cornel transparency with an objective method.
Methods: 32 adult hens were cared follow ARVO guidelines, underwent LASIK
surgery (right eyes) and were divided in 4 groups with different post-operative
treatment: A- no treatment; B- carboxymethyl chitosan 0.5%; C- carmellose sodium
0.5%; D- f luorometholone 0.20%. Left eyes from group A were used as control
(no surgery, no drugs): group E. Masked treatments were applied for 10 days.
Surgical procedure: animals were anesthetised (intramuscular and topical) and
corneal f laps were created with a special microkeratome (60 μm plate). Stromal
photoablation was performed with a 193nm excimer laser, programmed for -4D.
Surgical outcome and follow up was performed 1, 15 and 30 days after surgery,
with a surgical microscope, and haze was clinically evaluated (grade 0-4).
Day 30, animals were euthanasized, corneas were carefully excised and corneal
transmittance was objectively assessed with an experimental scatterometer.
f i xed
s t u d y.
Results: Surgical outcome shows intra-operative (12.5%) and postoperative
(18.7%) complications and these animals were excluded from the study. Haze
was not clinically detected in the 22 eyes (68.7%) that finished the study.
The higher corneal transmittance value was found in group E and lower in group B.
Statistically significance difference was not found between groups B, C and D (treated groups) but
significance difference (P < 0.05) was found between groups B, C comparing that with group E.
Conclusions: an experimental animal model of LASIK was developed and was used to assess
pharmacological modulation of corneal transparency, which was evaluated with an objective
method. Corneal transparency shows no differences between treated groups.
CR: R.M. Torres, None; J.M. Merayo-Lloves, None; J.T. Blanco-Mezquita, None; S.
Mar, None; C.P. Günther, None; G. Rodríguez, None; A. Mayo, None; C. MartínezGarcía, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2174-2180 / B943-B949
265. Corneal Development Organizing Section: CO
2174 - B943
2175 - B944
Temporal and Spatial Expression of Matrix Metalloproteinases and ECM as Well as
Adhesion-Related Proteins During Corneal Development
J.-C.Jung, Y.Lee, M.Huh, S.Seo, K.Kim, S.Kang. Biology, Kyungpook National University,
Daegu, Republic of Korea.
Connective Tissue Growth Factor Function in the Developing Eye
V.B. Mahajan1, A.Assefnia1, S.Tsang2, D.Farber1, P.J. McDonnell3, B.J. Mondino1.
Jules Stein Eye Institute, UCLA, Los Angeles, CA; 2Harkness Eye Institute, Columbia
University, New York, NY; 3Wilmer Eye Institute, Johns Hopkins University, Baltimore,
Purpose: To better understand the molecular mechanisms by which distinct ECMs are formed
in the eye, transcriptional profiling was used to identify candidate genes that might contribute
to corneal transparency. To confirm the biological significance of these results, we studied
a mouse knockout model for the Connective Tissue Growth Factor (CTGF) candidate gene.
Methods: Transcriptional profiling was performed on cultured corneal and scleral
fibroblasts using Affymetrix DNA microarrays and a Bayesian statistical framework.
A CTGF knockout mouse was examined by standard histological methods.
Results: CTGF RNA expression was elevated in corneal fibroblasts when compared with
scleral fibroblasts. Examination of E18.5 CTGF knockout eyes revealed several developmental
abnormalities. While CTGF-/- sclera appeared normal, CTGF-/- corneas demonstrated
hypertrophic cells and a poorly organized lamellar structure resulting in corneal opacity.
Furthermore, the anterior chamber showed a persistent pupillary membrane. Fetal hyaloid
vessels also showed a delayed regression pattern in the vitreous cavity with more numerous,
large diameter vessels. Immunohistochemistry studies showed a normal number of localized
macrophages. Finally, consistent with the major CTGF effect on chondrogenesis, orbital
bone abnormalities were observed. Conclusions: Comparative expression profiling is an
important method to identify candidate genes for ECM remodeling, and mouse knockouts can
provide functional confirmation of gene activity. Exploration of CTGF signaling pathways
will help reveal the molecular mechanisms underlying corneal opacity and persistent fetal
CR: V.B. Mahajan, None; A. Assefnia, None; S. Tsang, None; D. Farber, None; P.J.
McDonnell, None; B.J. Mondino, None.
Support: Research To Prevent Blindness
2176 - B945
2177 - B946
Purpose: Previous our work shows that MMPs were involved in the process of corneal
development. The purpose of this study was to investigate the spatial expression patterns
of MMPs and ECM as well as adhesion-related molecules during corneal development.
Methods: Corneas were isolated from developing day 5, 7, 10, 14, 18 embryos, and 2 days and
2 months old chick. Immunohistochemistry was also performed to detect the localization of
MMPs (stromelysin-1, collagenase-3, MT-MMP) and TIMP-2, adhesive proteins (CD44v6, Ncadherin, and ADAM10), and fibronectin as well as tenascin expression. Epithelial and stromal
cells isolated from developing cornea were primary cultured, and MT-MMP and ADAM10
expressions were confirmed by western blot analysis and immunof luorescence staining.
Results: Based on the important roles of CD44 for stromal cell migration, CD44v6 and its cleaved
forms of CD44v6 were highly expressed in the early corneal development. Furthermore, its
expression was strongly detected invading neural crest cells and neural crest-derived stromal
cells as well as developing epithelium in early corneal development. However, its expression was
dramatically decreased in stromal layer 14 days after corneal development, but strongly detected
in maturating corneal epithelium. Both MT-MMP and ADAM10 cleave the ectodomain of CD44v6
at the leading edge of migrating cells, and facilitate cell migration and reorganization of the
extracellular matrix. As expected, active form of MT3-MMP was strongly expressed in the all
layer of developing cornea at day 7, 14, and 18. Stromelysin-1 expression pattern showed similar
to MT3-MMP expression patterns. ADAM10 was expressed in the all layer of early developing
cornea, but its expression was not detected in the epithelium at day 14 and 18. Furthermore, by in
vitro culture, we found that 50-KDa of ADAM10 was secreted form and 82-KDa was cell surface
bound form. Interestingly, tenascin and fibronectin as well as N-cadherin expression was distinctly
expressed in the epithelium, stroma, and endothelium depends on the developing day of cornea.
Conclusions: Our results suggest that temporally and spatially expressed ECM as well as CD44v6
and MMPs/TIMP, and MT3-MMP and ADAM10 may act function together to effect the migration
of neural crest-derived mesenchymal cell into the stroma and the proliferation and differentiation
of epithelium and endothelium as well as remodeling of cornea.
CR: J. Jung, None; Y. Lee, None; M. Huh, None; S. Seo, None; K. Kim, None; S. Kang, None.
Support: KRF-2003-070-C00033
Role of FGF7 in Maintenance of Corneal Homeostasis
L.Wang, T.-I.Chikama, Y.Hayashi, C.W. Kao, W.W. Kao. Ophthalmology, University of
Cincinnati, Cincinnati, OH.
Purpose:To elucidate the effects of excess FGF7 expression by corneal epithelium on corneal
morphogenesis during embryonic development and in maintenance of homeostasis in adult.
Methods: Bitransgenic Krt12rtTA/rtTA /tetO-FGF7 and Krt12rtTA/+ /tetO-FGF7 mice were obtained
by breeding Krt12rtTA/rtTA and tetO-FGF7 mice. Single transgenic Krt12rtTA/rtTA, tetO-FGF7
mice were used as controls. The experimental animals were fed doxycycline in water and chow
to induce the expression of FGF7 from conception or in adult for various periods of times.
Histology and immunohistochemistry were used to evaluate the pathology caused by excess
FGF7 synthesized by corneal epithelium. Results: Overexpression of tetO-FGF7 induced
by doxycycline during embryonic development of resulted in the formation of vascularized
cornea with epithelium hyperplasia in Krt12rtTA/rtTA /tetO-FGF7 and Krt12rtTA/+ /tetO-FGF7
bitransgenic mice. Postnatal induction of FGF7 overexpression also leads to lesion in the
cornea similar, albeit the pathological changes were not as severe to the findings of induction
commenced during embryogenesis. Immunofluorescent staining revealed both keratin 12
and keratin 14 were expressed by all cell layers of corneal epithelium consistent with the
keratin expression pattern found in corneal epithelium hyperplasia. Removal of doxycycline
from the diet for 4 weeks following a 4 weeks induction the corneal epithelium hyperplasia
reduced but small blood vessels could still be seen.Conclusions:Excess FGF7 synthesized by
corneal epithelium of Krt12rtTA/rtTA /tetO-FGF7 and Krt12rtTA/+ /tetO-FGF7 mice caused partially
reversible pathological changes in corneas. The strategy of inducible overexpressing growth
factors and/or cytokine is useful for studies of corneal cell biology in vivo.
CR: L. Wang, None; T. Chikama, None; Y. Hayashi, None; C.W. Kao, None; W.W. Kao,
Support: NIH Grants EY14207, EY13755; RPB;OLERF
Role of cJun in Eyelid Morphogenesis during Mouse Embryonic Development
Y.Hayashi1, C.-Y.Liu2, D.Y. Weng1, W.W. Kao1. 1Ophthalmology, University of Cincinnati,
Cincinnati, OH; 2Ophthalmology, University of Miami, Miami, FL.
Purpose: We previously reported that the reduction of cJun activation in periocular
mesenchymal cells resulting from interrupted TGF-α gradient by excess biglycan perturbed
eyelid morphogenesis of Kera-Bgn transgenic mice. In present study, we further examined
the role of cJun in eyelid morphogenesis. Methods: The Cre-loxP system was adapted for
eyelid-specific gene ablation. Keratocan promoter (Kera5) containing 3.2 kb 5’ flanking
sequence, exon 1 and intron 1 of Kera gene was used to prepare transgenic mouse lines
expressing Cre. KC9, one of 22 Kera5-Cre founder mouse lines, were crossed with ROSA26R
(LacZ reporter mice) and cJun-floxed mice (cJunf/f). The distribution of cells derived from
periocular mesenchyme was mapped by determining β-galactosidase activities in eyelid with
whole mount X-gal staining. The effect of cJun on eyelid morphogenesis was examined by
histology. Results: β-Galactosidase activity was detected in most, if not all eyelid stromal
cells, suggesting that they are derived from periocular mesenchyme. The enzyme activity
was also detected in tarsal muscle, but absent in orbicularis oculi. KC9 mouse line was mated
with cJun- floxed mice to prepare KC9/cJunf/f bitransgenic mice. Histology examination
revealed that the KC9/cJunf/f mice had thinner eyelid with fewer stromal cells than that of
KC9/cJunf/+ (heterozygous cJun- floxed alleles) and wild type mice. However, the KC9/cJunf/f
did not show premature eye open phenotype.Conclusions:cJun signaling has important role
for normal eyelid morphogenesis. The ablation of cJun in periocular mesenchymal cells may
alter cell migration, proliferation and apoptosis required for normal eyelid morphogenesis
during embryonic development.
CR: Y. Hayashi, None; C. Liu, None; D.Y. Weng, None; W.W. Kao, None.
Support: NIH EY13755, EY14207; RPB; OLERF
2178 - B947
2179 - B948
Two Gelsolin Genes in Zebrafish: Differences in Structure and Expression Pattern
M.Spencer, J.Kanungo, S.Swamynathan, J.Piatigorsky. Lmdb, National Eye Institute,
Bethesda, MD.
Purpose: In zebrafish, gelsolin-1 (previously called C/L-gelsolin) is expressed highly in cornea
(50% of water-soluble protein) and slightly in lens and early cleavage-stage embryos. Since this
is an atypical expression pattern for vertebrate gelsolin, we searched for a second gelsolin gene.
Methods: Gelsolin-2 was cloned by PCR based on EST data available from the GenBank
database. Sequences for in situ hybridization probes were constructed in pBS-sk(-) for
gelsolin-2 and pCS2 for gelsolin-1. Probes were labeled with the DIG kit from Roche.
Multiple alignments were generated with clustalW from sequences obtained from GeneBank.
Dendograms were created from the clustalW results and formatted with Tree View.
Results: We cloned a second zebrafish gelsolin (gelsolin-2) which exhibits 56% identity
to corneal-preferred zebrafish gelsolin-1 at the protein level. The gelsolin-2 protein has
71% identity to human gelsolin compared to 58% for gelsolin-1. As expected, gelsolin-2
exhibits six conserved gelsolin motifs. Phylogenetic analysis demonstrates that gelsolin-1
and gelsolin-2 diverged early in vertebrate evolution and that gelsolin-2 gave rise to the other
vertebrate gelsolins. Gelsolin-2 mRNA is widely expressed at a low level in the embryo and
adult; it does not accumulate in the cornea as gelsolin-1. Comparison of up to 10 kb of the
upstream regions of these two genes indicated that only a few short stretches of sequence are
comparable. In addition, the gene structures of the gelsolin-1 and gelsolin-2 differ, with 18
introns and 20 exons, and 16 introns and 17 exons for gelsolin-1 and gelsolin-2, respectively.
Conclusions: The differences in gene structures and upstream putative regulatory sequences
driving their diverse patterns of expression suggest that the two zebrafish gelsolin genes
have undergone adaptive changes in regulation, consistent with functional differences for
the proteins, following gene duplication.
CR: M. Spencer, None; J. Kanungo, None; S. Swamynathan, None; J. Piatigorsky,
Support: NEI Intramural Support
Overexpression of TGF-ß1 by Periocular Mesenchymal Cells Perturbs Ocular Surface
Tissue Morphogenesis
S.H. Yi, K.Terai, Y.Hayashi, W.W. Kao. Ophthalmology, University of Cincinnati,
Cincinnati, OH.
Purpose: We investigated the effect of excess transforming growth factor-ß1 (TGF-ß1)
synthesized by migrating periocular mesenchymal cells on ocular surface tissue morphogenesis
during development. Methods: Using tetracycline-inducible (Tet-Off) system, a Kera-tTA
(keratocan promoter-tetracycline transcription activator) minigene containing 3.2 kb genomic
DNA fragment 5’-flanking sequence, exon 1 and part of intron 1of Kera was used to prepare a
Kera-tTA transgenic mouse line expressing tTA by migrating periocular mesenchymal cells
during development. The Kera-tTA mice were mated with tetO-TGFβ1 transgenic mice to
produce Kera-tTA/tetO-TGFβ1 bitransgenic mice. The experimental mice were fed normal
diet, and doxycycline in water and chow to examine the effects of excess TGF-ß1 on ocular
surface tissue morphogenesis by histology and immunofluorescence staining of keratins
at postnatal days 12, 14, 3 weeks and 3 months. Results: Excess TGF-ß1 synthesized by
mesenchymal cells resulted in malformation of eyelids. Histological examination revealed
absent or underdeveloped Meibomian glands. Hyperkeratosis, angiogenesis and inflammation
were also observed in central cornea in bitransgenic mice, and immunohistochemistry revealed
absence of K12 and sporadic expression of K14. These phenotypes observed in bitransgenic
mice were alleviated by feeding the experimental animals with doxycycline.Conclusions:TGFß1 overexpression causes eyelid abnormalities e.g., missing Meibomian glands and may also
perturb corneal type epithelium differentiation signified by K12 expression in Kera-tTA/tetOTGFβ1 bitransgenic mice. It remains unknown, however, whether the abnormality of corneal
epithelial defect is caused directly by excess TGF-β1 or secondary to the abnormal eyelid.
CR: S.H. Yi, None; K. Terai, None; Y. Hayashi, None; W.W. Kao, None.
Support: NIH EY13755, EY14207; RPB; OLERF
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2174-2180 / B943-B949
265. Corneal Development Organizing Section: CO
2180 - B949
Comparison of Corneal Thickness Between Dogs With Retinal Dystrophy Due to a
Null Mutation in Cyclic GMP Phosphodiesterase Alpha (PDE6A) and Phenotypically
Normal Heterozygous Carriers
D.M. Eifler1, F.M. Montiani-Ferreria2, F.F. Cardoso3, C.A. Johnson1, S.M. PetersenJones1. 1Dept. Small Animal Clinical Sciences, Michigan State University, East Lansing,
MI; 2Dept Veterinary Medicine, Federal University of Paraná, Curitiba-PR, Brazil;
Embrapa Pecuária Sul, Bagé- RS, Brazil.
Purpose: To compare the corneal thickness of dogs with a retinal dystrophy model of retinitis pigmentosa due
to a null mutation in PDE6A with closely related phenotypically normal dogs heterozygous for the mutation.
Methods: The central corneal thickness was measured by ultrasonic pachymetry in a colony
of Cardigan Welsh Corgi dogs with a mutation in PDE6A that results in rod dysfunction and
retinal degeneration. Measurements were performed in 28 dogs from 2 - 40 weeks of age (11
homozygous for the PDE6A mutation and 17 phenotypically normal dogs heterozygous for the
mutation). In some dogs measurements were repeated at different ages. The average corneal
thickness between the right and left eyes was not significantly different, therefore these
measurements were averaged to obtain a mean corneal thickness for each animal at each age
tested. Statistical analyses were performed on corneal thickness by repeated measures ANOVA
as a preliminary analysis that determined the significance of disease status, eye, gender, and time
effects. Segmented nonlinear least square regression was used to capture the two phases observed
in the corneal development plotted against age. The first phase, corresponding to a decrease in
thickness after neyelid opening, was modeled by a quadratic polynomial function on age, and
the second phase corresponding to the corneal growth was modeled by a logistic growth curve.
Results: Homozygous PDE6A mutant dogs had a mean corneal thickness of 0.565 +/- 0.010 mm,
which was significantly greater than that of heterozygous carriers (0.533 +/- 0.009 mm). There was no
significant difference between males and females and no interaction between gender and disease status.
The corneal thickness of the maturing eye of homozygous mutant dogs fitted a different prediction
equation model than that of the carriers, the difference appearing to be greater in the younger dogs.
Conclusions: This study showed that dogs with a retinal dystrophy due to a null mutation in the
PDE6A gene had a significantly greater corneal thickness than closely related heterozygous carriers
of the mutation. This difference could result from effects on ocular growth due to the abnormal retinal
function of mutant dogs or might be due to the effects of a gene linked to the PDE6A locus.
CR: D.M. Eifler, None; F.M. Montiani-Ferreria, None; F.F. Cardoso, None; C.A. Johnson,
None; S.M. Petersen-Jones, None.
Support: NIH Grant EY14160. Midwest Eye-Banks. MSU Companion Animal Funds
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2181-2195 / B950-B964
266. Corneal Stroma and Keratocytes I Organizing Section: CO Contributing Section: VI
2181 - B950
2182 - B951
Morphology and Histochemistry of Bowman’s Layer in Selected Vertebrates
D.A. Samuelson1, K.C. Ganesh1, S.M. Miles1, A.Z. Zivotofsky2, D.Zivotofsky3, P.A. Lewis1.
Small Animal Clinical Sciences, University of Florida, Gainesville, FL; 2Brain Science
Center, Bar Ilan University, Ramat Gan, Israel; 3Private Practice, Jerusalem, Israel.
Purpose: Our understanding of the Bowman’s layer in the vertebrate eye is limited to that
described in primates and birds. Recently, we have discovered similar structures in the
giraffe (Giraffa camelopardalis) and two spp of whales (Tursiops truncatus, dolphin, and
Globiocephela macrorhynchus, pilot whale). Knowledge of Bowman’s layer especially
with regard to the role that this structure plays in the corneas of these species is limited. To
that end, we have begun to investigate the morphology and histochemistry of Bowman’s
layer in eyes of the giraffe, the two mentioned whale spp, the chicken and monkey.
Methods: Formalin-preserved specimens from anterior eyes of two or more individuals of the
adult giraffe, bottle-nosed dolphin, pilot whale, domestic chicken and macaque monkey were
embedded and sectioned sagittally and treated with a variety of stains that included: H&E,
Masson’s trichrome, PAS, elastin stain, heparan sulfate proteoglycan antibody, and a battery
of lectins (asparagus pea lectin, wheat germ agglutinin, Concanavalin agglutinin, Dolichos
biflorus lectin, Bandeiraea simplicifolia agglutinin). Results: The thickness of Bowman’s
layer was 4-8 microns in the giraffe, monkey and chicken and 16-22 microns in the whale spp.
The construction of Bowman’s layer appeared nearly identical among the species as viewed
by sections stained with H&E and trichrome, having been homogenous and acellular. In the
monkey, chicken and giraffe the staining intensity was similar to that of the adjacent stroma,
having differed only in the whale spp. Concomitantly, there was an absence of staining for
elastic fibers in Bowman’s layer of the land-based animals, having been present in both marine
mammals. Bowman’s layers of all spp. possessed PAS-positive anterior margins. Among the
lectins, wheat germ agglutinin reacted positively only with Bowman’s layer of the two whale
spp. The rest of the lectins reacted weakly or inconclusively with Bowman’s layer of all spp.
Conclusions: Bowman’s layer of the land-based spp. share similarities in size, morphology
and histochemistry, differing substantially from that of marine mammals, which may reflect a
variation of roles that this structure plays. Further analysis, especially with regard to collagen
composition and ultrastructural appearance, is warranted.
CR: D.A. Samuelson, None; K.C. Ganesh, None; S.M. Miles, None; A.Z. Zivotofsky,
None; D. Zivotofsky, None; P.A. Lewis, None.
Support: None.
Light Scattering Calculations From TEM of Healed Penetrating Corneal Wounds
R.L. McCally1,2, R.Grebe2, A.DeLaCruz2, W.R. Green2, D.E. Freund1. 1Johns Hopkins Univ
Applied Phyics Lab, Laurel, MD; 2Wilmer Eye Institute, Baltimore, MD.
Purpose: Calculate the light scattering from structures depicted in TEM of aged (semi)
transparent scars resulting from penetrating wounds in rabbit cornea and compare it with
light scattering measurements made on the fresh tissue from which three scattering groups
(moderate, intermediate and high) were identified. [McCally and Bonney-Ray, IOVS 43, Eabstract 1710 (2002)] Methods: 2 mm diameter penetrating wounds were made in the central
cornea of rabbits in Dr. Charles Cintron’s laboratory and allowed to heal for periods of 3.5 to 4.5
years, at which time the rabbits were sacrificed and the corneas excised. Corneal transmissivity,
T, was measured immediately and the tissue was prepared for TEM. TEM from corneas in
the moderate and intermediate scattering groups having regions with an organized lamellar
structure were analyzed to determine fibril positions and radii. Scattering was calculated using
the direct summation of fields method. Results: Stromal lamellae in the moderate scattering
group are mildly irregular at all depths. Calculations revealed high transparency (T>90% from
400 to 700 nm) and scattering characteristic of the short-ranged order in the fibril positions
found in normal cornea - findings consistent with slit-lamp observations showing some highly
transparent regions in these scars. Lamellar structure in the intermediate transparency group
is highly irregular and is absent in some areas where fibrils often appear tangled. There are
also many voids (“lakes”). Calculated transmissivity is lower (80%<T<93% from 400 to
700 nm) and scattering is characteristic of that expected from regions containing lakes - a
finding consistent with the scattering measurements. Conclusions: Structural features in
TEM are consistent with the scattering categories identified from scattering measurements.
Not unexpectedly, transmissivity calculated from the regions having an organized lamellar
structure is higher than that measured from the entire corneal depth, but is consistent with
the scattering groups identified from measurements.
CR: R.L. McCally, None; R. Grebe, None; A. DeLaCruz, None; W.R. Green, None; D.
E. Freund, None.
Support: NIH Grant EY12165
2183 - B952
2184 - B953
The Effect of Swelling on the Lamellar Arrangement of the Corneal Stroma
K.M. Meek, S.Hayes, C.Boote. Optometry & Vision Sciences, Cardiff University, Cardiff,
United Kingdom.
Purpose: To investigate how the prefer red orientations of the collagen
lamellae in the human corneal stroma change when the tissue swells in vitro.
Methods:Synchrotron x-ray scattering was used to map, at 0.4mm resolution,
the preferred orientation of lamellae across the human cornea and then to remap after the cornea had been swollen in distilled water for about 1 hour.
Results: The unswollen cornea displayed the usual preferred arrangement of lamellae (inferiorsuperior/nasal-temporal) at the centre and the diagonal anchoring lamellae in the periphery
(Aghamohammadzadeh et al., Structure 12, 249-256, 2004). After one hour, the cornea was
visibly swollen and cloudy (H=11.6). However, the vertical and horizontal preferential orientation
of the lamellae was still evident, and the distribution of both preferentially aligned and total
fibrillar mass (projected onto the two-dimensional plane of the cornea) showed little change.
Conclusions: When the human cornea swells, the angles between adjacent lamellae are not
significantly changed.
CR: K.M. Meek, None; S. Hayes, None; C. Boote, None.
Support: MRC Grant G0001033
Role of Keratocytes in Corneal Transparency as Assessed by Classical Light Scattering
D.W. Hahn1, K.Kim1, J.V. Jester2. 1Mechanical & Aerospace Eng, University of Florida,
Gainesville, FL; 2Ophthalmology, University of California at Irvine, Irvine, CA.
Purpose: Recent studies suggest that increased light scattering from corneal keratocytes
contributes to the loss of corneal transparency and the development of haze, for example
following excimer laser corneal ablation. Perturbations in the refractive index of keratocytes
have been proposed as a possible biophysical mechanism underlying this phenomenon.
To better understand the role of keratocytes in the development of corneal haze, we have
modeled cells using classical light scattering theory. Methods: Keratocytes in a transparent
corneal matrix were modeled using two classical light scattering theories, namely the solution
for homogenous spheres of arbitrary size, and the solution for arbitrarily-sized coated
spheres such that the core material and outer shell may have different optical properties. A
parametric study was performed to assess the degree to which differential scattering crosssections were altered when the refractive indices of the various components were perturbed.
Baseline calculations were matched to properties anticipated for normal corneal structures.
Results: The calculated scattering coefficients for small angle (forward) scattering were
found to vary significantly for modest changes in the refractive index. For coated spheres,
changes of only 10% in the refractive index of a 200-nm thick outer shell increased the forward
scattering by more than an order of magnitude. Similar effects were observed for changes
in the refractive index of the solid sphere. In addition, resonance behavior was observed
at select forward scattering angles, resulting in 100-fold enhancements of light scattering.
Conclusions: The present analyses reveal that modest changes in the optical properties of
corneal keratocytes can significantly alter the scattering characteristics. In particular, changes
in only the outer thin shell, representing the cell wall, may induce these changes. Overall,
these results support the hypothesis that small changes in the refractive index of keratocytes
may underlie the development of corneal haze after injury.
CR: D.W. Hahn, None; K. Kim, None; J.V. Jester, None.
Support: Supported in part by NEI grant EY13215 (JVJ) and Senior Scientist Award from
2185 - B954
2186 - B955
A New Technique for Mechanically Characterising Hydrogels for Tissue Engineering
K.Y. Then1, M.Ahearne2, K.K. Liu2, Y.Yang2, E.Siamantouras2, P.J. McDonnell1, S.Shah1,
S.Rauz3, A.El Haj2. 1Corneal and External Diseases, Birmingham and Midland Eye Centre,
Birmingham, United Kingdom; 2Institute for Science and Technology in Medicine,
Keele University, Stoke-on-Trent, United Kingdom; 3Academic Unit of Ophthalmology,
Division of Immunity and Infection, The University of Birmingham, Birmingham, United
Purpose: One of the main difficulties in growing corneal tissue in-vitro is to
replicate the physical characteristics of the cornea. This experiment looked into
how the mechanical characteristic of a corneal construct can be monitored in vitro.
Methods: Alginate and agarose are hydrogels that have similar mechanical properties to
collagen gel and both have been used for engineering tissue. A novel ball loading indentation
system has been set up. It consists of a computerised long-focal CCD microscope, image
analyser and clamping system. The circular corneal equivalent is clamped around its outer
diameter and a ball of known weight and size is placed on top of it causing a deformation. The
CCD microscope allows the capture of a side-view deformation profile. A theoretical model
was derived to correlate quantitatively the viscoelasticity to the time-dependent deformation
profile of the stromal equivalent. The force required to cause a deformation to occur was
measured by the force transducer. The elastic modulus of the hydrogel can be calculated
from the deformation. Viscosity values can be calculated from the creep or relaxation curves
Results: Alginate exhibits highly viscoelastic deformation and the determined viscosity decreased
with the increase of alginate concentration. The presence of cells appears to initially weaken hydrogel.
By day 3, it appears that the mechanical strength of the gel improves in the presence of the gel.
Conclusions: This technique is capable of characterising the mechanical strength of the
corneal equivalent construct. It can also be used to study the affect of cyclic strain on the
cells in a 3D matrix.
CR: K.Y. Then, None; M. Ahearne, None; K.K. Liu, None; Y. Yang, None; E. Siamantouras,
None; P.J. McDonnell, None; S. Shah, None; S. Rauz, None; A. El Haj, None.
Support: North Staffordshire R&D Consortium
Promotion by Latanoprost of Collagen Gel Contraction Mediated by Human Corneal
R.Yanai, Y.Liu, Y.Lu, S.Hirano, T.Sagara, T.-I.Chikama, T.Nishida. Biomolec Recognition
& Ophthal, Yamaguchi Univ School of Med, Ube City, Japan.
Purpose: Measurement of intraocular pressure depends on the shape and thickness of
the cornea. The possible effects of the antiglaucoma drugs latanoprost, timolol maleate,
and pilocarpine on the contraction of corneal tissue were examined with the use of threedimensional cultures of human corneal fibroblasts. The effects of these drugs on collagen
degradation as well as their potential cytotoxicity were also examined with this system.
Methods:Human corneal fibroblasts (CFs) were cultured in three-dimensional gels of
type I collagen and in the presence of medium containing various concentrations (0.01
to 1000 µM) of latanoprost, timolol maleate, or pilocarpine. Collagen gel contraction
was evaluated by daily measurement of the gel diameter for 3 or 4 days. The extent of
collagen degradation was determined by measurement of the amount of hydroxyproline
generated by acid-heat hydrolysis of the culture supernatants. The release of lactate
dehydrogenase from the cells was determined as an index of drug cytotoxicity.
Results: Latanoprost promoted CF-mediated collagen gel contraction in a dose- and
time-dependent manner, whereas timolol maleate and pilocarpine did not exhibit such
an effect. Latanoprost and timolol maleate, but not pilocarpine, showed a cytotoxic effect
at the highest concentration (1000 µM) tested. Neither latanoprost, timolol maleate, nor
pilocarpine affected collagen degradation by human CFs at the concentrations examined.
Conclusions: Among the antiglaucoma drugs investigated, only latanoprost promoted
CF-mediated collagen gel contraction. Latanoprost may thus affect corneal shape through
this effect on CFs.
CR: R. Yanai, None; Y. Liu, None; Y. Lu, None; S. Hirano, None; T. Sagara, None; T.
Chikama, None; T. Nishida, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2181-2195 / B950-B964
266. Corneal Stroma and Keratocytes I Organizing Section: CO Contributing Section: VI
2187 - B956
2188 - B957
Corneal Epithelial Cells and Stromal Fibroblasts but Not Stromal Myofibroblasts
Efficiently Convert Plasminogen to Angiostatin
S.S. Twining1, D.J. Warejcka1, A.M. Bernstein2. 1Department of Biochemistry, Medical
College of Wisconsin, Milwaukee, WI; 2Department of Ophthalmology, Mount Sinai
School of Medicine, New York, NY.
Purpose: Controlling neovascularization in the cornea is essential to maintain corneal
transparency. This is especially important during wound healing when factors that induce
angiogenesis are present, such as FGF-2 and extracellular proteases. Proteolysis of the kringle
region of plasminogen generates angiostatins, molecules that control angiogenesis in the cornea
during wound healing. Epithelial, stromal and endothelial cells of human corneas synthesize
plasminogen. Angiostatins are found in extracts of the three layers of the cornea and in serumfree conditioned medium from human corneal organ culture. The purpose of this study was to
compare the ability of different types of corneal cells to convert plasminogen to angiostatins.
Methods: Primary human corneal epithelial cells were cultured in keratinocyte medium.
Primary human corneal stromal cells were cultuered in serum-free media with supplements
and were converted to fibroblasts with FGF-2 or to myofibroblasts with TGF-β. Human
plasminogen (33nM) was added to cultured cells and medium was collected at 24, 48 and 72
hrs. Medium proteins were separated under non-reducing conditions and identified on Western
blots. The angiostatins were isolated using an ε-aminocaproic acid-Sepharose column. FGF2 was used to stimulate human umbilical vascular endothelial cell (HUVEC) proliferation.
Results: Confluent cultures of epithelial cells converted the plasminogen to angiostatin by
24 hrs while stromal fibroblasts converted the added plasminogen to angiostatin by 48 hrs.
Even at 72 hrs, stromal myofibroblasts converted very little plasminogen to angiostatin. The
epithelial and stromal fibroblast products were very similar. The initial cleavage removed
the N-terminal peptide and secondary cleavages formed products that contained kringles
1-4 plus part of kringle 5 (52 and 49 kDa), kringles 1-4 (45 and 41 kDa) and kringles 1-3
(33 kDa). Over the 72 hr period, the larger polypeptides were further cleaved. Purified
angiostatin cleavage products were able to inhibit FGF-2 induced proliferation of HUVECs.
Conclusions: These results suggest that the motile cells of the cornea, the epithelial cells
and stromal fibroblasts in culture can produce angiostatins from plasminogen, while the less
mobile myofibroblasts do not have the proteases to carry out this conversion.
CR: S.S. Twining, None; D.J. Warejcka, None; A.M. Bernstein, None.
Support: EY012731 (SST), F32EY07049 (AMB)
Purpose: Cornea has a unique structure and unique extracellular components which are essential for
tissue integrity and transparency. Cells used for corneal tissue engineering must have the ability to
secret such a cornea-specific matrix. Keratocytes are the major cellular constituents of corneal stroma,
but in vitro human keratocytes change into fibroblasts losing their unique phenotype. Multipotent
adult progenitor cells (MAPC) have recently been identified in a number of tissues. These cells are
considered candidates for cellular therapy and tissue engineering. In this study, we investigated
whether corneal stromal derived stem cells (CSSC) and adipose derived stem cells (ADSC) can
adopt the keratocyte phenotype and secret unique components of the corneal stromal extracellular
matrix. Methods: CSSCs were isolated by cell sorting using Hoechst 33342 dye exclusion and
ADSCs were isolated by collagenase digestion and differential centrifugation. Each cell type was
cultured and passaged in medium containing 2% fetal bovine serum (FBS) with growth factors. For
keratocyte differentiation, the stem cells were cultured in defined medium using Advance MEM
(In Vitrogen) with addition of horse serum or of bovine corneal extract (Exp Eye Res 2003 77:273).
Cells were cultured either as pellet or in fibrin gels. Keratocan and keratan sulfate were detected
by immunostaining, immunoblotting, and RT-PCR. Pellets and fibrin gel were fixed, sectioned,
and stained for keratocan and keratan sulfate. Results: Stem cells from human corneal stroma and
from human subcutaneous adipose tissue can express keratocan and keratan sulfate after culture in
defined medium. After 14 days in pellet culture, both cells types secret an amorphous connective
tissue containing keratocan and keratan sulfate. In fibrin gels, the cells also secret keratocan and
keratan sulfate, and exhibit extensive intracellular connection via extended cellular processes
similar to those observed in the corneal stroma in vivo. Conclusions: Stem cells from human corneal
stroma and from adipose tissue can differentiate into cells showing the characteristic morphology
and connective tissue secretion of human keratocytes. Ability to expand cultures of human stem
cells and transform them into functional keratocytes may represent a key step development of a
bioengineered corneal bioprostheses.
CR: Y. Du, None; M.L. Funderburgh, None; M.M. Mann, None; J.L. Funderburgh, None.
Support: NIH Grants EY09368, 30-EY08098, Research to Prevent Blindness, Eye and Ear Foundation
of Pgh.
2189 - B958
2190 - B959
2191 - B960
2192 - B961
Comparison of the Water-Soluble Proteomes From Keratocytes and Serum-Cultured
Corneal Fibroblasts
H.Karring1, T.Ostergaard1, I.B. Thogersen1, G.K. Klintworth2, J.J. Enghild1, T.MollerPedersen3. 1Department of Molecular Biology, University of Aarhus, Aarhus, Denmark;
Departments of Pathology and Ophthalmology, Duke University Medical Center,
Durham, NC; 3Department of Ophthalmology, Aarhus University Hospital, Aarhus,
Purpose: To compare the water-soluble protein expression profiles of freshly-isolated
keratocytes versus serum-cultured corneal fibroblasts to obtain a better understanding
of the biochemical processes regulating corneal transparency and haze development.
Methods: Keratocytes were isolated from porcine corneas by collagenase digestion for 12
hours. Serum-cultured corneal fibroblasts were obtained using explant technique and grown
to third passage in 10% FBS. Both phenotypes were lysed by sonication and the water-soluble
proteomes were compared using 2D gel electrophoresis and analysed by mass spectrometry.
Results: The proteome analysis revealed distinct differences in the protein expression
profiles of keratocytes and serum-cultured corneal fibroblasts. The “phenotypespecific” proteins are involved in many cellular processes including cell metabolism
and protection from oxidative stress. Specifically, the expression of the enzymecrystallins, aldehyde dehydrogenase 3A1 and transketolase, are dramatically
reduced in serum-cultured corneal fibroblasts, as verified by immunoblotting.
Conclusions: This proteomic study reveals distinct differences in the protein expression
profiles of keratocytes versus the serum-cultured fibroblast phenotype. Together with enzymecrystallins, these “phenotype-specific” proteins may be involved in the regulation of corneal
cellular transparency.
CR: H. Karring, None; T. Ostergaard, None; I.B. Thogersen, None; G.K. Klintworth,
None; J.J. Enghild, None; T. Moller-Pedersen, None.
Support: NIH Grant RO1-EY12712 and The Danish Medical Research Council.
A Novel Phagocytosis Uptake Mode of Herpes Simplex Virus-1 Involves Receptor
Localization Within the Phagosome
C.Clement1A, V.Tiwari1A, P.M. Scanlan1A, T.Valyi-Nagy1B, B.Y. J. T. Yue1A, D.Shukla1A.
Ophthalmology and Visual Sciences, BPathology, 1University Illinois-Chicago, Chicago,
Purpose: To investigate the mechanism of entry of herpes simplex virus 1 (HSV-1) into human
corneal fibroblasts. Methods: This study was designed to assess alternate mechanism(s) of entry
of HSV-1 using primary cultures of fibroblasts from the stroma of the human cornea, to further
understand HSV-1 physiopathology of the stroma. We examined electron micrographs of a purified
recombinant strain of wild type HSV-1 (KOS gL86) entry into Chinese hamster ovary (CHO) cells
transfected with alphaherpesvirus entry receptor, nectin-1 (or HVEM) and identified a typical
phagocytosis uptake mode. We adopted an assay in which the virus as the phagocytosis effector was
evaluated. Cytochalasin D and lysosomotropic agents were used to interfere with plasma membrane
projections and low pH, to block HSV-1 uptake. Furthermore the role of pH in virus entry was
assessed by a virus-free cell fusion luciferase assay. Heparan sulfate (HS) mediated phagocytosis
engulfment in response to HSV-1 attachment was evaluated by a spinoculation technique in which
the low efficiency of virus entry in HS deficient CHO-745 cells was compensated for by centrifugal
force. To colocalize the receptor and phagosome we used nectin-1-EGFP, a fluorescent homologue
and Texas-red tagged transferrin in cell systems infected with virus and consequently showed virus
trafficking within infected cells by a green fluorescent tagged virus and a marker for early phagosome.
Results: Our results show that HSV-1 entry is via a unique phagocytosis uptake mode and this route
is characteristic of human corneal fibroblasts. However, within the corneal tissue there was variation
of entry as related control primary trabecular meshwork cells appeared to aid attachment and fusion
at the plasma membrane as previously reported. Electron micrographs showed cellular projections
engulfing intact viral particles. Interestingly, we colocalized receptor and phagosome and showed
virus presence within these vesicles suggesting that they are essential for tethering virion envelope
to phagosomal membrane. Furthermore, low pH favored enhanced fusion of the virion envelope with
the phagosomal membrane. Conclusions: We have demonstrated a unique HSV-1 entry mechanism
relevant in human corneal fibroblasts. The engulfment of intact virions and release of capsids within
the cell suggest a complex immune response to HSV-1 once it infects the corneal stroma, perhaps
contributing to the severity of stromal keratitis.
CR: C. Clement, None; V. Tiwari, None; P.M. Scanlan, None; T. Valyi-Nagy, None; B.Y.J.T.
Yue, None; D. Shukla, None.
Support: Career Development Award from Research to Prevent Blindness to (DS)
Adult Stem Cells Secrete Corneal Specific Matrix Components
Y.Du, M.L. Funderburgh, M.M. Mann, J.L. Funderburgh. UPMC Eye Center,
Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Department of
Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA.
The Effect of Atmospheric Composition and Hydrostatic Pressure on Keratocyte
Viability: Preliminary Experiments
I.Jalbert1, R.Augusteyn1, F.Stapleton1,2. 1Vision Cooperative Research Centre, Sydney,
Australia; 2School of Optometry and Vision Science, The University of New South Wales,
Sydney, Australia.
Purpose: The aim of this study was to investigate the short-term effects
of anoxia and increased pressure on keratocyte viability in tissue culture.
Methods: Primary cultures of bovine and porcine keratocytes were exposed to anoxia
using a humidified chamber filled with 100% nitrogen. Hydrostatic heads of tissue culture
medium equivalent to pressures of 400 and 1,000 Pa were also established. Negative controls
were exposed to air and positive controls to D-sorbitol (Sigma-Aldrich, Sydney, Australia).
Test conditions were applied for six hours and keratocyte viability assessed immediately
afterwards using vital staining (Hoeschst 33258, annexin V-EGFP, propidium iodide).
Results: No viable keratocytes could be detected following six hours of anoxia. The percentage
of viable cells reduced from 95% during normal atmospheric conditions to 58% and 41%
after 6 hours exposure to 400 and 1,000 Pa of hydrostatic pressure, respectively. Raised
hydrostatic pressures induced a combination of necrosis and apoptosis in keratocyte cultures.
Conclusions: Oxygen deprivation and physiologically relevant levels of pressures are able to
directly impact keratocyte survival in primary culture. These findings may underpin some of
the recent clinical observations described in the stroma of soft contact lens wearers.
CR: I. Jalbert, None; R. Augusteyn, None; F. Stapleton, None.
Support: None.
ALDH3A1 Protects Against UVB-Induced Inhibition of the Proteasome
T.B. Estey, N.Lassen, V.Vasiliou. Department Pharmaceutical Sciences, University of
Colorado Health Sciences Center, Denver, CO.
Purpose: ALDH3A1 is one of the most abundantly expressed proteins in the mammalian
cornea, representing up to 40% of the total water-soluble fraction in some species. Though
the precise biological role of ALDH3A1 in the cornea remains to be elucidated, it is clear
that ALDH3A1 plays a dynamic role in protecting the cornea against oxidative stress. We
have recently demonstrated that cells transfected with human ALDH3A1 were resistant to
UV- and 4-hydroxynonenal-induced oxidative damage. In this study, we sought to further
characterize the effects of UV radiation by evaluating the activity of the proteasome, which
is susceptible to UV-induced inhibition. We hypothesize that ALDH3A1 may prevent
UV-induced proteasome damage. Methods: A rabbit keratinocyte cell line (TRK43) was
transfected with human ALDH3A1. Control TRK43 cells (empty vector) and ALDH3A1transfected cells were exposed to UVB-light at doses of 0.05 and 0.25 J/cm 2. After 24 hr
post-exposure incubation, the cells were lysed and assayed for proteosome activity using a
substrate for chymotrypsin-like activity (LLVY-AMC). Identical samples were prepared with
a proteosome inhibitor (MG 132) to verify the specificity of the assay. In addition, Western
blot analysis was used to detect 4-HNE-adducted proteins. Results: In the TRK43 vector
cells, we observed that the chymotrypsin-like activity of the proteasome decreased by nearly
60% after both 0.05 and 0.25 J/cm 2 UVB doses. Cells transfected with ALDH3A1, however,
demonstrated no loss in proteasome activity. We did not observe an accumulation of 4-HNEadduct proteins 24 hours post-exposure in either vector or ALDH3A1-transfected TRK43 cells.
Conclusions: In this report, we describe for the first time that ALDH3A1 protects the
proteasome against UV-induced inhibition. These findings support the multifunctional role
of ALDH3A1 in the protection of the cornea against oxidative stress.
CR: T.B. Estey, None; N. Lassen, None; V. Vasiliou, None.
Support: NIH Grant EY11490
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Monday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 2181-2195 / B950-B964
266. Corneal Stroma and Keratocytes I Organizing Section: CO Contributing Section: VI
2193 - B962
Glucose Permeability of Human, Bovine, and Porcine Corneas in vitro
J.Noolandi1A, D.Myung1A,1B, K.Derr1A, P.Huie1A, C.Ta1A. AOphthalmology, BChemical
Engineering, 1Stanford University, Stanford, CA.
Purpose: To measure the glucose diffusion coefficients of human, bovine,
and porcine corneas in vitro. The results of this study would serve as a guide
in the development of nutrient-permeable materials for cor neal implants.
Methods: Diffusion of glucose across human (n = 8), bovine (n = 7), and porcine corneas (n =
8) was measured using a modified blind well chamber apparatus (Boyden chamber). Dialysis
membranes (MWCO 14000) (n = 7) and non-porous mylar membranes (n = 7) were used as
positive and negative controls, respectively. Corneal and polymer samples were sandwiched
between an upper chamber filled with deionized water and a lower chamber filled with a
circulating glucose solution maintained at a fixed concentration of 10 mg/mL. Motorized
stirring bars were used to mix the solution in the upper chamber in order to prevent boundary
layering of glucose. The glucose concentrations of the upper chambers were measured at
30 minute intervals with a FreeStyleTM glucose meter. Diffusion coefficients of glucose for
each of the samples were calculated from the glucose flux data using Fick’s law of diffusion.
Results: The diffusion coefficient of glucose is highest for human corneas (3.0 ± 0.2 x
10 -6 cm 2/s) followed by porcine corneas (1.8 ± 0.6 x 10 -6 cm 2/s) and bovine corneas
(1.6 ± 0.1 x 10 -6 cm 2/s) (P < 0.05). The diffusion coefficients of all tested corneas were
significantly higher (P < 0.05) than that of dialysis membrane (3.4 ± 0.2 x 10 -7 cm 2/s).
Conclusions: The glucose diffusion coefficients of human, bovine, and porcine corneas in
vitro is on the order of 10 -6 cm 2/s. Human corneas have the highest permeability to glucose,
followed by porcine and bovine corneas. The results of this study provide an index by which
polymers can be evaluated for their potential as corneal implant materials based on their
permeability to glucose.
CR: J. Noolandi, VISX, Inc. P; D. Myung, VISX, Inc. P; K. Derr, VISX, Inc. F; P. Huie,
VISX, Inc. F; C. Ta, VISX, Inc. P.
Support: VISX, Incorporated; Stanford Bio-X Program
2194 - B963
Highly Reflective Cells in the Corneal Stroma Visualized by in vivo Confocal
M.J. Quadrado1A, M.Popper1A,2, A.M. Morgado1B,1C, J.N. Murta1A, J.A. Van Best1A, L.J.
Muller3. AIBILI Centre of Ophthalmology, BIBILI Dept. of Instrumentation, CDept.
of Physics, 1University of Coimbra, Coimbra, Portugal; 21st Dept. of Ophthalmology,
Semmelweis University, Budapest, Hungary; 3The Netherlands Ophthalmic Research
Institute (NORI), Amsterdam, The Netherlands.
Purpose: To study the highly reflective cells with a tail (HRC) in more detail in the
corneal stroma of diabetic and control patients with in vivo confocal microscopy (IVCM).
Methods: We analyzed eight type 2 diabetic patients, four with level 20 ETDRS (age 53y
to 69y) and four with level 35 (age 59y to 64y) and eight healthy controls (age 35y to 74y).
The number of HRC found in the stroma was counted using registered images obtained by
IVCM (Tomey Confoscan P4). Length and width of HRC and keratocyte nuclei was measured.
Results: Structures consisting of a highly reflective head and a vague tail (straight or undulated)
were found mainly in anterior and mid-stroma of patients and controls. They were often
observed near the stromal nerves and were rare in posterior stroma. The average number of
HRC was 52.5±9.3 and 40.0±14.0 in level 20 and 35 diabetic patients, respectively. There were
significantly less present in healthy controls, 6.8±2.1 (p<0.001). In level 35 patients more HRC
were found in the posterior stroma, usually associated with a higher tortuosity of the adjacent
nerves. In general, stromal nerves are not located in the posterior stroma of the central cornea.
The average length and width of a HRC is 19.5±5.7 µm and 10.4±4.4 µm, respectively,
and those of a keratocyte nucleus are 27.2±2.5 µm and 9.5±3.5 µm, respectively.
There is a significant difference in length (p<0.001) but not in width (p= 0.16).
Undulated st r uct u res with dimensions similar to large st romal ner ves
(width: 8 to 11 µm) were also obser ved in the diabetic patients.
Conclusions: HRC is a new kind of structure found in high densities in the corneal stroma
of diabetic patients but their identity and their origin are yet unknown. The undulated large
stromal structures in diabetic patients might represent altered straight stromal nerves as
observed in controls.
CR: M.J. Quadrado, None; M. Popper, None; A.M. Morgado, None; J.N. Murta, None; J.
A. Van Best, None; L.J. Muller, None.
Support: FCT SFRH/BD/13710/2003
2195 - B964
Analysis of Glucose Diffusion Across the Acufocus Corneal Inlay Using a Finite
Element Method
T.A. Silvestrini1, P.M. Pinsky2, B.Christie1. 1Research and Development, AcuFocus, Irvine,
CA; 2Department of Computational Mechanics, Stanford University, Palo Alto, CA.
Purpose: To estimate the steady state glucose concentration profile in a cornea implanted
with the AcuFocus Corneal Inlay (ACI), a device designed to restore near vision.
Methods: The finite element method was used to model the steady-state Fickian diffusive
transport of extracellular glucose across a cornea implanted with a thin, microperforated,
semi-opaque membrane designed to treat presbyopia. As glucose diffuses across the
cornea from the endothelium to the epithelium, it is partially consumed by keratocytes at
rates that depend on the level of extracellular glucose. Based on published experimental
data, a model for glucose consumption is incorporated by the addition of a nonlinear
sink term in the diffusion equation. The aqueous provides the corneal glucose source
and it is assumed that the glucose concentration is uniform and constant at the stromaDescemet’s membrane interface. At the epithelium, experimental evidence indicates that
glucose consumption by the epithelial cells is proportional to the extracellular glucose
concentration and this is incorporated through a boundary condition that relates glucose
normal flux to the extracellular glucose concentration. The non linear finite element
solves for the glucose concentration over a representative region of the cornea taking into
account the full three-dimensional details of the microperforations in the implanted device.
Results: Steady state glucose concentration profiles determined for ACIs positioned
at depths of 5, 65, and 120 microns in the cornea showed a drop in the glucose level at
the corneal epithelium of between 5% and 40%, depending on the spacing of the
micro perforation pores. The model provides a detailed view of extracellular glucose
transport in the vicinity of the pore holes in the implanted device and can provide
useful guidance in the creation of optimal designs for the implant microperforations.
Conclusions: The dimensions and permeability of the ACI appear to allow good nutritional
flow of glucose throughout the cornea. The model provides a complete understanding of how
the design variables of the ACI affect glucose levels in the cornea.
CR: T.A. Silvestrini, AcuFocus E; P.M. Pinsky, AcuFocus C; B. Christie, AcuFocus E.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 8:30 AM - 10:15 AM Ballroom BCD Paper Session Program Number Range: 2301-2307
310. Corneal Wound Healing II Organizing Section: CO
2301 - 8:30AM
ATP and UTP Mediated Epithelial Injury Response Is Associated With Transactivation
of EGF Receptors
V.Trinkaus-Randall1A, L.Yang1B, I.Weinger1B, V.Klepeis1C. AOphthalmology and
Biochemistry, BBiochemistry, CPathology, 1Boston University Med School, Boston, MA.
Purpose:We have shown that injury to corneal epithelial cells releases nucleotides and that a calcium
wave is propagated to neighboring cells. Furthermore we have elucidated which P2Y receptors
are expressed. Our goal is to determine which P2Y receptors play a role in the wound response and
elucidate the cross-talk that occurs with the EGF pathway to generate cellular signaling cascades.
Methods: Experiments were performed on both primary corneal epithelial cells and
HCE-Ts. Calcium signaling was monitored using live cell confocal imaging with a
perfusion system. Localization of the receptors was assessed using confocal microscopy.
Cell migration was determined using scratch assays and modified Boyden chambers.
Expression and phosphorylation of PLCgamma1 and EGFR were performed using
western blot analysis. Assays were performed to determine levels of diacylglycerol.
Results: We have demonstrated that there is a dose dependent response to the nucleotides
where ATP and UTP show saturation at lower concentrations than ADP or UDP. To
determine which are involved in the wound response, cells were pre-treated and then
injured. When cells were pretreated with ATP or UTP the injury induced calcium wave
was inhibited indicating desensitization of receptors. Desensitization of receptors was not
detected when cells were pretreated with ADP and UDP, as the injury induced calcium
wave was present. In addition, only pretreatment with ATP and UTP decreased the EGF
induced Ca2+ response. Migration induced by either ATP or UTP was significantly greater
than that induced by ADP or UDP and the latter were enhanced by EGF. Furthermore
the integrin peptide, RGD, diminished trinucleotide induced migration. Cross-talk was
exhibited when ATP attenuated the ability of EGF to activate phospholipase C gamma1. In
adition, while ATP and UTP transactivated ErbB1 and ErbB2, the trinucleotides decreased
the ability of EGF to induce autophosphorylation of the EGFR. Other experiments using
CRM 197 and TIMP showed that specific parts of the signaling pathway could be inhibited.
Conclusions: These results indicate that the injury response is mediated by P2Y2 and
P2Y4 receptors and we hypothesize that cross-talk may occur via a triple-membrane pass
CR: V. Trinkaus-Randall, None; L. Yang, None; I. Weinger, None; V. Klepeis, None.
Support: NIH Grant EY06000, Mass Lions
2303 - 9:00AM
2302 - 8:45AM
Connexin 43 (Cx43) Serine Residue Phosphorylation (pser) in the Corneal Epithelium
During Homeostasis and Wound Repair
L.M. Carrington1,2, J.Cai1,2, P.Martin3, J.Albon1,2, M.Berry4, W.J. Armitage4, M.Boulton1,2.
School of Optometry & Vision Sciences, Cardiff University, Cardiff, United Kingdom;
Cardiff Institute of Tissue Engineering & Repair (CITER), Cardiff, United Kingdom;
Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, United
Kingdom; 4Department of Clinical Science, Bristol University, Bristol, United Kingdom.
Purpose: This study aims to characterise the phosphorylation state of Cx43 during
corneal epithelial homeostasis, and the changes that occur during wound repair.
Methods: Bovine corneas were maintained in organ culture for up to 7 days, cryopreserved
and sectioned. Immunofluorescence detection of 5 antibodies i) Cx43 (phosphorylation-state
independent), ii) Cx43npcterm, iii) Cx43pser255, iv)Cx43pser262, v) Cx43pser279/282 was
performed using Alexafluor conjugates and analysed. Positive controls were bovine heart and liver.
Results: In the unwounded cornea, Cx 43 was immunolocalised to a single basal layer of cells
in the limbus, with both intensity and number of cell layers staining increasing with proximity
to the centre. Cx43npcterm, Cx43pser255 and Cx43pser262 showed similar staining patterns to
the phosphorylation-independent antibody, increasing toward the central epithelium, although
Cx43pser 255 showed additional strong nuclear staining in the more superficial cell layers.
Cx43pser 279/282 staining was absent. Following wounding, Cx43 was lost at the leading edge,
and Cx43pser262 staining decreased, co-ordinated with an increase in nuclear pser255. Transient
location of a) Cx43pser279/282 was seen in the basal cells within the wound-site immediately
following closure, and b) Cx43pser262 was observed, associated with sites of proliferation.
C-terminus phosphorylation was lost during migration over the cut edge of the wound
Conclusions: Cx43 appears to be phosphorylated at serine residues 255 and 262 on the
carboxyl tail, but not at 279/282 or any of the c-terminus cluster serines during homeostasis.
During repair phospho-isoform changes may regulate both proliferation and differentiation
of corneal cells.
CR: L.M. Carrington, None; J. Cai, None; P. Martin, None; J. Albon, None; M. Berry,
None; W.J. Armitage, None; M. Boulton, None.
Support: Guide Dogs for the Blind
2304 - 9:15AM
Rapid Induction of Hyaluronan Synthesis and Hyaluronan Synthase in Corneal
Wound Healing
J.L. Funderburgh1, R.Tammi2, Y.Du1, N.Guo1, M.M. Mann1, D.Kanter1, M.L. Funderburgh1.
UPMC Eye Center, Ophthalmology and Visual Science Research Center, Eye and Ear
Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine,
Pittsburgh, PA; 2Department of Anatomy, University of Kuopio, Kuopio, Finland.
Urokinase Pathway Regulation in Corneal Fibroblasts and Myofibroblasts
A.M. Bernstein1, S.S. Twining2, K.A. Vaughan2, S.K. Masur1. 1Ophthalmology, Mount Sinai
Sch of Med, New York, NY; 2Biochemistry, Medical College of Wisconsin, Milwaukee,
2305 - 9:30AM
2306 - 9:45AM
Purpose: Corneal wounds deposit non-transparent stromal matrix containing abnormal proteoglycans.
Hyaluronan (HA), a high molecular weight glycosaminoglycan not present in the normal stroma,
is frequently observed in corneas with various pathological conditions. HA is highly bioactive,
involved in cell migration and proliferation as well as in mediation of inflammatory response. The
presence of HA in wounds suggests an active participation in the cellular events of scar formation.
HA is synthesized in mammalian tissues by three different hyaluronan synthase (HAS) enzymes.
This study was initiated to determine the time course during which HA appears in the corneal stroma
after wounding, and to identify the cells and HAS isoforms involved in HA stromal biosynthesis.
Methods: The temporal appearance of HA in mouse corneal scalpel wounds was examined by
staining with biotin labelled hyaluronan binding protein (bHABP). HA in corneal extracts was
quantified using fluorophore assisted-carbohydrate electrophoresis (FACE). Cellular HAS enzyme
expression was detected by immunostaining with peptide antibodies to HAS1 and 2. HAS gene
expression in cultured keratocytes was determined using real time RT-PCR. Results: Unwounded
and 1 hr wounded corneas showed no HA by staining with bHABP but by 3 hr a diffuse stain was
detected throughout the wounded cornea. Intensity of the staining increased up to 48 hr becoming
granular. Staining decreased after 2 weeks but was clearly detected in the healed corneas for up to
3 months. HA in the early wounds was confirmed by FACE analysis. Immunohistochemistry of the
wounded region showed strong expression of HAS2 protein on cells near the wound within 24 hr.
Many of these cells were also CD45 positive. HAS2 mRNA in cultured keratocytes was markedly
upregulated within 1-3 hr of serum stimulation. Conclusions: Accumulation of HA in the cornea
represents the most rapid and pervasive change in stomal extracellular matrix yet observed in response
to wounding. Staining and RT-PCR suggest that HAS2 enzyme is induced in stromal keratocytes
in response to wounding, however inflammatory cells may also participate. The rapid, abundant,
and persistent presence of HA in the wounded tissue is likely an important factor in several of the
cellular healing processes including cell migration cell division and localization of inflammatory
cells in the healing tissue.
CR: J.L. Funderburgh, None; R. Tammi, None; Y. Du, None; N. Guo, None; M.M. Mann,
None; D. Kanter, None; M.L. Funderburgh, None. Support: NIH EY09368,30-EY08098, Research
to Prevent Blindness, Eye and Ear Foundation of PGH
TGFβ Induced Myofibroblast Differentiation in Rabbit Corneal Keratocytes Requires
p38 but Not ERK1/2 Signaling
J.V. Jester1, J.Huang2. 1Ophthalmology, University of California, Irvine, Orange, CA;
Ophthalmology, University of Texas Southwestern Medical Center, Dalls, TX.
Purpose: Previous studies show that inhibition of PDGF or integrin signaling blocks TGFβ induced
myofibroblast differentiation without affecting TGFβ receptor mediated SMAD signaling. The purpose
of this study was to identify possible alternative signal transduction pathways that become activated
during myofibroblast differentiation which lead to expression of alpha smooth muscle actin (α-SMA).
Methods: Primary rabbit corneal keratocytes and serum starved immortalized human
corneal fibroblasts were treated with 1 ng/ml TGFβ in the presence or absence of inhibitors
to PI-3Kinase (LY294002), ERK1/2 (PD98059) and p38 kinase (SB203580). Myofibroblast
differentiation was then assessed by immunocytochemistry and western blotting for α-SMA.
Cell proliferation was assessed by Ki67 staining and cell counting. Immunopreciptation
and western blotting using antibodies specific for p38 assessed phosphorylation of p38.
Results: Inhibition of ERK1/2 using PD98059 at 40 uM completely blocked cell cycle entry
induced by TGFβ after 3 days in culture but had no effect on cell spreading or expression of αSMA. Treatment of keratocytes with LY294002 showed a dose dependent inhibition of α-SMA
expression and cell cycle entry. LY294002 also showed a dose dependent reduction in the number
of keratocytes while treatment with LY294002 alone showed no effect suggesting that inhibition of
PI-3K induces apoptosis of activated and differentiating corneal keratocytes and acted downstream
of myofibroblast differentiation. Keratocytes treated with SB203580, however, showed a dose
dependent decrease in expression of α-SMA that was completely inhibited by 30 uM without
affecting cell proliferation. Furthermore, treatment of TGFβ-modified myofibroblasts with SB203480
significantly reversed myofibroblast differentiation. Immunoprecipitation experiments showed
phosphorylation of p38 beginning at 2 hours after TGFβ treatment that was inhibited by SB203580.
Conclusions: These data indicate that myofibroblast differentiation involves a p38 mediated signal
transduction cascade. Furthermore, cell proliferation is not required for myofibroblast differentiation.
Overall these finding suggest new approaches to controlling myofibroblast differentiation in vivo.
CR: J.V. Jester, None; J. Huang, None.
Support: NIH Grant EY07348; Senior Scientist Award and Unrestricted Grant from RPB, Inc.
Purpose: Upon corneal wounding, the normally quiescent cells in the stroma differentiate into
fibroblasts, which secrete proteases that degrade and remodel the extracellular matrix as they migrate. The
urokinase-type plasminogen activator (uPA) is an extracellular serine protease expressed in the stroma of
wounded corneas. When uPA’s precursor, pro-uPA, binds to its cell-surface receptor, uPAR, it is cleaved
into active uPA which in turn cleaves plasminogen into the broad-spectrum protease, plasmin. Plasmin
remodels extracellular matrix, activating latent growth factors and stimulating cell migration. Using an
in vitro wounding model that reproduces the transition from keratocyte to fibroblast to myofibroblast,
we have investigated differences in the uPA/uPAR pathway that correspond with these stromal
phenotypes. Methods: Human corneal fibroblasts were seeded onto collagen and cultured for three
days in DMEM/F12 with supplements plus FGF-2/heparin for fibroblasts or TGFβ1 for myofibroblasts.
uPAR cleavage and PAI-1 expression were detected by western blot. uPA activity was evaluated by
zymography and a colorimetric activity assay. Results: We previously demonstrated that uPA/uPAR
expression is upregulated in fibroblasts and that the binding of uPA to uPAR promotes uPAR’s
association with the actin cytoskeleton through the integrin, αvβ3 (IOVS, 45:2967, 2004). We now
report that FGF-2/heparin stimulated an increase in cell-associated uPA activity and a decrease in
secreted pro-uPA. In contrast, myofibroblasts significantly down-regulated the uPA/uPAR pathway
by 1) promoting the cleavage of uPAR’s domain 1 into a form that can no longer bind to pro-uPA, 2)
increased pro-uPA secretion and decreased cell-associated uPA activity (possibly because cleaved
uPAR can not bind uPA) and 3) increased secretion of PAI-1 (uPA/uPAR inhibitor). The TGFβ1
induced uPAR cleavage was blocked by 1% FBS or FGF-2/heparin treatment in serum-free media but
not by a serine protease inhibitor (aprotinin), or metalloprotease inhibitor (GM6001).Conclusions:
Together our data support a model in which fibroblasts are able to migrate into a wound using the
uPA/uPAR/αvβ3/vitronectin pathway. Myofibroblast differentiation correlates with inhibition of the
uPA/uPAR pathway through cleavage of uPAR into a non-uPA binding form. PAI-1, by promoting
the endocytosis of uPA/uPAR, further down-regulates uPA/uPAR activity. By this mechanism,
myofibroblasts may become non-motile and fulfill their role in promoting wound closure.
CR: A.M. Bernstein, None; S.S. Twining, None; K.A. Vaughan, None; S.K. Masur, None.
Support: NIH Grant F32 EY07049, NIH Grant R01 EY09414, NEI Core Grant, Research to Prevent
Differential Regulation of the Dynamic Sub-Cellular Mechanical Activity of Corneal
Fibroblasts by Rho and Rac
W.Petroll, L.Ma, A.Kim, L.Ly, M.Vishwanath. Department of Ophthalmology, UT
Southwestern Medical Center, Dallas, TX.
Purpose: The small GTPases Rac and Rho play a central role in regulating spreading and contraction,
respectively, of a variety of cell types on 2-D substrates. The goal of this study was to determine the
morphological and sub-cellular mechanical effects of Rho and Rac on corneal fibroblasts inside 3-D
matrices. Methods: Human corneal fibroblasts were plated at low density inside 100 μm thick fibrillar
collagen matrices and cultured for 1 to 3 days in serum free (S-) media. Time-lapse imaging was
then performed at 1-2 minute intervals using Nomarski DIC. After 1 hour, perfusion was switched
to S- media containing either 1 mM LPA (which activates Rho), 10 ng/ml PDGF (which activates
Rac), or the Rho-kinase (ROCK) inhibitor Y-27632 (10 μM) for 1 hour. Perfusion media was then
changed to either LPA + Y-27632, or PDGF + Y-27632. In other experiments, time-lapse imaging was
performed following microinjection of constitutively active Rac (L61Rac, 800 μg/ml) or wild type
Rac (as a control). Changes in cell morphology and extracellular matrix (ECM) deformation were
measured using MetaMorph. Results: Cells produced little or no ECM deformation in S- media.
Addition of LPA (n=6 cells) induced retraction of cell processes and contraction along the cell body,
as indicated by a decrease in cell length (-12.1 + 7.0%, p <0.05) and cell area (-11.8 + 12.2%). Force
generation was greatest along the cell body, as indicated by the location of maximum compressive
ECM strain (-14.7 + 7.9%, p < 0.05). These effects were reversed after adding Y-27632. In contrast to
LPA, stimulation with PDGF (n=7 cells) induced rapid cell spreading, as indicated by an increase in
cell length (30.8 + 34.1%, p = 0.054), cell area (45.9 + 27.1%, p<0.05), and the number of pseudopodial
processes (10.4 + 2.6 vs. 5.7 + 3.7 processes per cell, p<0.05). Forces were more transient than those
observed after LPA treatment, and the pattern of matrix strain was more complex. However, in all
cases maximal ECM compression was located at the leading edge of extending pseudopodia (-11.0 +
5.3%, p<0.05). A similar response was induced by microinjection of L61Rac (n= 3 cells). Following
ROCK inhibition with Y-27632, PDGF induced a similar, but even more localized response at the
tips of extending pseudopodia (n=6 cells). Conclusions: Taken together, the data suggest that during
Rac-induced cell spreading, transient forces are generated at the ends of extending pseudopodia via
a ROCK-independent mechanism. In contrast, during Rho-induced cell contraction, more sustained
forces are generated along the cell body via a ROCK-dependent mechanism.
CR: W. Petroll, None; L. Ma, None; A. Kim, None; L. Ly, None; M. Vishwanath, None.
Support: NIH Grant EY13322, and a Lew R. Wasserman Merit Award (WP) and Departmental
Grant from RPB
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 8:30 AM - 10:15 AM Ballroom BCD Paper Session Program Number Range: 2301-2307
310. Corneal Wound Healing II Organizing Section: CO
2307 - 10:00AM
Lysophospholipase D and Elevated Lysophosphatidyl Choline as Sources of Elevated
Aqueous Humor LPA Following Corneal Injury
M.A. Watsky1, W.Hatsuyama2, T.Tsutsumi3, K.Fukuzawa2, A.Tokumura2. 1Physiology,
University of Tennessee Health Science Center, Memphis, TN; 2Institute of Health
Biosciences, University of Tokushima Graduate School, Tokushima, Japan;
Pharmaceutical Sciences, Kyusyu University of Health and Welfare, Nobeoka, Japan.
Purpose: The cornea is bathed by the aqueous humor, which contains various growth factors that may
participate in the healing process following corneal injury. We previously found that lysophosphatidic
acid (LPA), an important growth factor present in animal sera, is accumulated in rabbit aqueous
humor following corneal freeze wounds. In the current study, we examined the possibility that
soluble lysophospholipase D (lysoPLD), an enzyme that generates LPA from lysophosphatidylcholine
(LPC), is relevant to this injury-induced increase in aqueous humor LPA concentration.
Methods: Rabbit corneas were freeze wounded, and aqueous humor samples were collected 24 h following
injury. LysoPLD activity was measured by the enzymatic determination of choline concentration
following incubation of aqueous humor samples with exogenous LPC. Lysophospholipids present in
aqueous humor were analyzed by liquid chromatography-electrospray ionization mass spectrometry.
Results: Aqueous humor samples from both control and injured corneas showed a high, but similar
degree of lysoPLD activity. LysoPLD enzymatic properties, such as sensitivity to inhibition by
bivalent ion chelators (EDTA and o-phenanthroline), metal-ion requirement, potentiation by
Co2+, and pattern of relative activities for various lysophospholipids, resembled those of
lysoPLD found in human plasma, which was recently identified by us as autotaxin (a member
of the ecto-nucleotide pyrophosphatase/phosphodiesterase-I family). Autotaxin was originally
identified as a tumor cell-motility stimulating protein found in conditioned medium of
human melanoma cells. Mass spectrometry experiments determined that levels of four major
molecular species of LPC, predominant substrates of lysoPLD in aqueous humor, were 513 times higher in the aqueous humor samples from injured eyes than those in control eyes.
Conclusions: These results suggest that lysoPLD is constitutively released into rabbit aqueous
humor, with no injury-induced increase in release following freeze-wounding. They also show that
wound-induced increases in LPA are likely due to an increase in the aqueous humor concentration
of several LPC species, which are being catalyzed to form LPA by the constitutively active lysoPLD.
An elevated aqueous humor LPA concentration likely plays a role in corneal wound healing.
CR: M.A. Watsky, None; W. Hatsuyama, None; T. Tsutsumi, None; K. Fukuzawa, None; A.
Tokumura, None.
Support: Ministry of Education, Culture, Science, Sports and Technology of Japan (A.T.)
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 11:15 AM - 1:00 PM Ballroom BCD Symposia Program Number/Board # Range: 2356-2361
323. Innervation of the Anterior Segment in Health and Disease - Minisymposium Organizing Section: CO
2356 - 11:15AM
2357 - 11:32AM
2358 - 11:50AM
2359 - 12:07PM
2360 - 12:25PM
2361 - 12:42PM
Corneal Nerve Structure
L.J. Muller. Ocular Signal Transduction, Netherlands Ophthalmic R I, Amsterdam, The
Corneal nerves are important to maintain the integrity of the ocular surface. Their distribution
in the plexus between basal epithelial cells and Bowman’s layer is highly specific in humans
and differs from that in other mammals (e.g. rats, rabbits and dogs). One of the consequences
of dissection of these nerves by refractive surgery is dry eyes. In the past years our research
has focused on the subbasal nerves in humans. After a summary of our current knowledge,
new data will be presented on the organization of the stromal and subbasal nerves in the
human cornea. This knowledge might give us clues on how to influence the process of nerve
repair after damage.
CR: L.J. Muller, None.
Support: None.
Neural Regulation of Tear Production
D.Dartt. Schepens Eye Research Institute, Boston, MA.
The tear film protects the eye from the continuously changing external environment. To
respond tear secretion must occur rapidly, in a coordinated fashion and thus uses a neural
reflex arc. The afferent limb of this pathway is the sensory nerves of the cornea and conjunctiva
that activate the efferent parasympathetic and sympathetic nerves that innervate the glands
and epithelia that secrete tears. These tissues include: conjunctival goblet cells that secrete
gel-forming mucins of the inner mucous layer of the tear film; lacrimal gland, conjunctiva,
and cornea that secrete the electrolytes, water and proteins in the middle aqueous layer; and
meibomian glands that secrete the outer lipid layer. Stimulation of parasympathetic nerves
releases the neurotransmitters acetylcholine (Ach) and VIP that induce mucin secretion by
conjunctival goblet cells, as well as protein, electrolyte, and water secretion by the lacrimal
gland. Stimulation of sympathetic nerves releases norepinephrine that via beta-adrenergic
receptors stimulates water and electrolyte secretion by conjunctival and corneal epithelia and
via alpha-adrenergic receptors produces protein secretion by the lacrimal gland. Although the
meibomian gland is innervated, lipid secretion appears not to be subject to neural control. To
stimulate secretion Ach and VIP activate their canonical signaling pathways, although there
are some unique aspects to the lacrimal gland and goblet cells. Activation of alpha-adrenergic
receptors in the lacrimal gland stimulates a pathway specific to this gland. Damage to the
neural reflex arc can occur in ocular surface diseases, aging, and LASIK surgery and can
play a major role in ocular surface pathogenesis.
CR: D. Dartt, None.
Support: None.
Effect of Disease on Nerve Function
T.Tervo. Helsinki Univ Eye Hospital, University of Helsinki, Helsinki, Finland.
The importance of corneal innervation for the maintenance of a healthy ocular
surface, stability and quality of tear film, optical properties of the eye, or normal
corneal wound healing has been known since early 1800`s but has recently received
increasing interest partially because of expanding corneal refractive surgery.
Certain congenital conditions such as Riley-Day syndrome, Men II b syndrome, congenital
hypaesthesia, infectious diseases such corneal zoster, severe or repeated HSV infections,
some bacterial ulcers or Acantamoeba infections may induce corneal sensory damage
and ocular surface disease (OSD). The same may result from iatrogenic conditions
such as anterior segment ischaemia related to retinal buckling surgery, corneal lamellar
surgery, transplantations, refractive surgery, orbital or intracranial surgery. Additionally
a number of systemic diseases of which diabetes is the most important as well as certain
neurological conditions may affect the trigeminal pathway to the cornea. Such conditions
need to be clarified and corneal sensitivity tested, when examining patients with OSD.
Recent studies have also revealed that corneal nerves may be reversibly compromised by a
disease such as recurrent erosion or conditions such as refractive surgery. We also postulate
that in some conditions the sensations in the cornea may be altered simulating dry eye
symptoms or corneas may show hyperaesthesia and minor pain following refractive surgery.
Developing corneal imaging techniques combined with a non-contact esthesiometers capable of
evaluating multiple sensory modalities will help us to understand these conditions better.
CR: T. Tervo , None.
Support: None.
Neural Mechanisms of Sensation in Normal and Injured Corneas
C.Belmonte. Instituto de Neurociencias de Alicante, Universidad Miguel HernandezCSIC, San Juan de Alicante, Spain.
The cornea is innervated by the peripheral axons of three main types of sensory neurons
located in the trigeminal ganglion: Mechano-nociceptive neurons that respond only to
injurious mechanical forces, polymodal neurons that are additionally excited by chemical
irritants and noxious heat, and cold-sensitive neurons that respond to moderate decreases of
corneal temperature Excitation with a gas esthesiometer (Belmonte et al. 1999, IOVS 40:513)
of mechano- and polymodal corneal neurons evokes sensations of irritation and pain while
sensations of innocuous cooling are evoked by small temperature reductions. Combined
stimulation of the various populations of corneal sensory endings leads to sensations of
distinct qualities. Corneal inflammation and/or injury of corneal nerves induce short- and long
term changes in responsiveness of corneal neurons. These include sensitization, development
of aberrant spontaneous activity and altered responses to natural stimuli. Altered response
of corneal nerves may explain reduced sensitivity and disesthesias developed by patients
following ocular surgery.
CR: C. Belmonte , None.
Support: None.
Effects of PRK, LASIK, and Topical Glaucoma Therapy on Corneal Nerve
W.Bourne. Department of Ophthalmology, Mayo Medical School, Rochester, MN.
Corneal nerve fiber bundles appear in confocal images of the cornea as bright linear objects
that extend across the field of view, and sometimes show texture and branch. The number,
density and orientation of corneal nerves can easily be measured by using standardized
sampling techniques. Changes in innervation of the cornea can be examined after surgical
procedures and treatment of the cornea. For example, we examined corneal nerves before and
after the refractive surgical procedures photorefractive keratectomy (PRK) and laser-assisted
in situ keratomileusis (LASIK). Immediately after these procedures most subbasal nerves
disappear. Subbasal nerves return to near pre-operative densities by 3-5 years, although they
return somewhat faster after PRK than after LASIK. We also examined subbasal nerves in
ocular hypertensive patients enrolled at the Mayo Clinic site of the Ocular Hypertensive
Treatment Study. Subbasal nerve density was 39% lower in 19 patients who received topical
drug therapy for their ocular hypertension for 7 to 9 years as compared with subbasal nerve
density in 10 patients who did not receive therapy (p<0.007). Sampling methods will be
discussed as well as possible artifacts that can change visibility of nerves in longitudinal
studies, artifacts such as diminished corneal clarity, increased scattered light, and changes
in operating parameters of the microscope and camera.
CR: W. Bourne , None.
Support: None.
Role of Innervation of the Cornea and Development of a New Mode of Treatment for
Neurotrophic Keratopathy
T.Nishida. Dept Biomolec Recog & Ophthal, Yamaguchi Univ Sch of Medicine, Ube City,
The maintenance of a clear cornea requires innervation by the trigeminal nerve. The loss
of such innervation can result in various types of corneal epithelial disorders, including
superficial punctate keratopathy, persistent epithelial defects, and, occasionally, corneal
ulceration. We recently found that substance P, a sensory neurotransmitter present in the
cornea, sensitizes corneal epithelial cells to the migration-promoting effects of insulin-like
growth factor-1 (IGF-1), fibronectin, and interleukin-6. These observations suggested that
the innervated cornea is responsive to changes in the concentrations of humoral factors
encountered in vivo, but that loss of innervation might render corneal epithelial cells insensitive
to the biological actions of such humoral factors. On the basis of results of both in vitro and in
vivo experiments, we have been treating the corneal lesions of individuals with neurotrophic
keratopathy by the administration of eyedrops containing a substance P-derived peptide
(FGLM-amide) and IGF-1.
CR: T. Nishida , Santen Pharmaceutical F, P.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Ballroom BCD Paper Session Program Number Range: 2418-2424
332. Lacrimal Gland, Tear Film and Dry Eye Organizing Section: CO
2418 - 3:00PM
Loss of Muscarinic, Parasympathetic Activation Induces Long-Term Changes in
Proinflammatory and Immune Response Gene Expression in the Rat Lacrimal Gland
D.Nguyen1, T.L. Nhan1, H.Toshida2, L.P. Pedroza1, R.W. Beuerman1. 1Ophthalmology, LSU
Eye Center, New Orleans, LA; 2Juntendo University School of Medicine, Tokyo, Japan.
Purpose: To examine long-term loss of parasympathetic activation of secretion on the
expression of proinflammatory mediators and the immune status in the rat lacrimal gland.
Methods: Rats underwent unilateral pre-ganglionic parasympathetic sectioning of the greater
superficial petrosal nerve and were sacrificed after 2.5 months. The structure of the LG acini was
examined by light microscopy. The LG from the contralateral (Ctla) and parasympathectomized
(Px) side was used for real-time RT-PCR (n=3). All reactions were done in triplicate using the
iCycler IQ real-time detection system. Mean cycle threshold (MCt) for the genes in the Ctla
and Px LG were normalized to 18S rRNA MCt values. Relative fold change was calculated for
each of the genes using the 2-ΔΔCT method and compared to the expression level after 7 days.
Results: Light microscopy showed an increase in the number of seromucous granules and
apoptotic cells in the Px LG. Expression of caspase 1, NFκB p105, and MMP-12 remained
elevated (greater than 2-fold) in the Px LG after 2.5 months. Expression of the MHC class
II antigen RT1.B (HLA-DQB orthologue) and RT1.D (HLA-DRA orthologue), increased
from 2.0- and 2.4-fold after 7 days, to 7.6- and 6.3-fold after 2.5 months, respectively. In
addition, expression of Psmb8, a proteasome subunit RC1 involved in peptide processing,
increased from 1.4-fold after 7 days, to 4.6-fold after 2.5 months. Conversely, expression
of the ER-resident protein, protein disulphide isomerase, which showed a 2-fold decreased
after 7 days, was not altered after 2.5 months. The MCt of the 18S rRNA (Mean + SEM)
was not significantly different between Ctla (11.70 + 0.00) and Px LGs (11.63 + 0.04).
Conclusions: Removal of parasympathetic control of secretion leads to a chronic and persistent
upregulation of the immune response in the lacrimal gland. These changes correlate with
age-associated changes in the LG.
CR: D. Nguyen, None; T.L. Nhan, None; H. Toshida, None; L.P. Pedroza, None; R.W.
Beuerman, None.
Support: EY12416, RPB, EY002377, BRIN
2420 - 3:30PM
2419 - 3:15PM
Antibody Array Analysis of the Distribution of Angiogenic Modulators, Growth
Factors, MMPs and TH-1/TH-2 Cytokines in Normal and Pathological Tears
R.A. Sack, L.Conradi, S.Sathe, T.Hawasly, A.R. Beaton. Biological Sciences, SUNY
College of Optometry, New York, NY.
Purpose: We recently utilized stationary phase antibody arrays (AA) to characterize the
distribution of 80 low abundance proteins in pooled open and closed (OTF and CTF) eye
tears (IOVS in press). In this study, we further increased the signal-to-noise ratio of the AA
thereby allowing the differential analysis of low abundance proteins in individual normal
and pathological tears. Methods: AA were constructed with the aims of reducing cross
talk between secondary antibodies and enhancing the sensitivity of detection. Arrays were
calibrated using recombinant standards achieving sensitivities in many instances in the low
femtogram range. Parallel analyses were carried out using commercial micro-well plate
formatted AA of many proteins employing a protocol minimizing confounding tear matrix
effects. Arrays were used to characterize OTF and CTF from normals and individuals with
active chronic allergic diseases (with and without overt ocular surface tissue involvement),
systemic diseases and acute unilateral allergic conjunctivitis. Results: Analysis reveals
that the protein profiles of tears recovered from normal individuals and those with active
chronic atopic reactions were strikingly different with the pathological samples exhibiting
exceptionally high levels of many TH-1/2 cytokines, MMPs, unique growth factors such as
FGFb and HB-EGF that are not detected in normal tear fluid. The latter factors could drive
mucin secretion. These differences were far more pronounced in CTF samples. This also
proved true in tear samples recovered from individuals with chronic sinusitis without overt
symptoms of ocular surface tissue involvement. In contrast, only marginal differences were
observed in the distribution of cytokines in tear fluid obtained from normals and individuals
with acute allergic conjunctivitis. Conclusions: AA can be constructed that are sufficiently
sensitive to allow the differential screening of normal and pathological tears. These arrays
demonstrate marked differences in the distribution of a wide range of bioactive proteins in
tear samples from normals and individuals with active chronic allergic diseases.
CR: R.A. Sack, None; L. Conradi, None; S. Sathe, None; T. Hawasly, None; A.R. Beaton,
Support: CIBA Vision
2421 - 3:45PM
SELDI-TOF-MS ProteinChip Array Profiling of Tears From Dry-Eye Patients:
Diagnostic Applications of New Protein Biomarkers
C.A. Kramann, N.Pfeiffer, F.Grus. Department of Ophthalmology, Johannes GutenbergUniversity, Mainz, Germany.
Purpose:. Protein and peptides in tears play an important role in ocular surface diseases. In
previous studies we demonstrated changes in the electrophoretic protein profiles of dry-eye
patients. The aim of this work was to determine the usefulness of SELDI-TOF-MS (Surface
Enhanced Laser Desorption/Ionisation Time-of-Flight Mass-Spectrometry) ProteinChip®
Array technology for the automated analysis of proteins and peptides in tear fluid.
Methods: Dry-eye patients (DRY, n=88) and healthy subjects (CTRL, n=71) were examined.
Their tear proteins were analyzed using SELDI-TOF-MS ProteinChip Arrays with three
different chromatographic surfaces (CM10 cation exchange, Q10 anion exchange, and H50
reverse phase) prepared by means of a laboratory liquid handling robotic workstation. The
data were analyzed by multivariate statistical techniques and artificial neural networks and
the most important biomarkers purified and then identified by tandem mass spectrometry.
Results: Complex patterns of tear proteins and peptides were detected. The different
chromatographic surfaces revealed the selective enrichment of proteins such as lipocalin
and lysozyme. Analysis of discriminance demonstrated highly significant changes in the
protein profiles in dry-eye patients (P<0.001). Using a seven peptide multimarker-panel,
an artificial neural network could differentiate between dry-eye and healthy individuals
with a specificity and sensitivity of 90%. The identification of biomarkers revealed an
increase of inflammatory markers in dry-eye patients and a decrease of some proteins
which might have protective functions, such as members of the proline rich protein family.
Conclusions:. The SELDI-TOF-MS technology seems to be ideally suitable for the massscreening of peptides and proteins in tears. This highly sensitive approach dramatically
reduces the analysis time and provides protein profiles with great mass accuracy. In this
study, new protein biomarkers for dry eye could been demonstrated. Thus, it might become
a very useful tool in the search for potential biomarkers for diagnosis and new therapeutics
in ocular diseases such as dry-eye.
CR: C.A. Kramann, None; N. Pfeiffer, None; F. Grus, None.
Support: None.
Tear Lipocalin (TL) Scavenges Lipids From the Corneal Surface
B.J. Glasgow, O.K. Gasymov, A.R. Abduragimov, T.N. Yusifov. Pathology and
Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA.
2422 - 4:00PM
2423 - 4:15PM
Slow Cycling Cells at the Corneal Limbus and Mucocutaneous Junctional Epithelium
in Rabbit Models of Dry Eye
H.Toshida1, A.Murakami1, D.H. Nguyen2, R.W. Beuerman2,3. 1Ophthalmology, Juntendo
University School of Medicine, Tokyo, Japan; 2Ophthalmology, Louisiana University
School of Medicine, New Orleans, LA; 3Singapore Eye Research Institute, Singapore,
Purpose: To compare and evaluate the changes in the density slow cycling cells at the
corneal limbus and mucocutaneous junctional epithelium (MCJ) in dry eye rabbits.
Two types of dry eye models were used; one was acute section of the parasympathetic
pre-ganglionic, the other by extended wear of soft contact lenses (SCLs).
Methods: Two rabbit dry eye models were used for this study: 1) unilateral sectioning of the
greater superficial petrosal nerve, the preganglionic input to the pterygopalatine ganglion,
2) bilateral removal of the nictitating membranes followed by extended SCL wear (2 week
Pure TM, Seed, Tokyo, Japan) by eyelid suturing or simple eyelid suturing on one side. The
control side received tarsorrhaphy. Clinical observations and Schirmer’s tear tests (STT) were
performed prior to and during the experiment in both groups of rabbits. BrdU (50mg/kg) was
injected (i.p) for 14 days after surgery. At 2 months after final injection of BrdU was given, the
corneal limbus and lower eyelid were removed and sectioned. A fluorescence method was used
to detect BrdU-incorporating cells in the limbus and MCJ. The number of BrdU-labeled cells
was automatically counted using the Scion image software (Scion Corporation, MD, USA).
Results: The STT values in both the denervated (5.3±0.4mm) and extended SCL wear
(12.4±2mm) models decreased compared to the levels prior to surgery (16.2±2.4mm,
16.5±3.2mm each). The density of BrdU-incorporating cells in the corneal limbus in
denervated eyes increased (49.6±2.4cells/0.5mm) compared to the contra-lateral eyes
(22.3±2.9cells/0.5mm). The same pattern is seen in the MCJ. In contrast, no observed changes
in the density of BrdU-incorporating cells were found in the extended wear SCL model.
Conclusions: These results show that parasympathetic nerve is involved in maintaining the
density of slow cycling cells in the corneal limbus and MCJ.
CR: H. Toshida, None; A. Murakami, None; D.H. Nguyen, None; R.W. Beuerman,
Support: None.
Purpose: The cornea is coated with membrane spanning and secreted mucins that create a
hydrophilic surface. Apical shedding of squamous epithelium and ectodomain shedding of mucins
as well as minor trauma may leave areas of corneal epithelium exposed (Dartt DA, Exp Eye Res
2004;78:173-185, Gipson IK, Exp Eye Res 2004;78:379-388). Lipid contamination of these areas
could create an unwettable surface. TL the principle lipid binding protein in tears, has been shown
to scavenge lipids from hydrophobic surfaces (IOVS 1999;40:3100-7). The hypothesis that TL can
remove contaminating fatty acids and phospholipids from the human corneal surface was tested.
Methods: TL was purified from pooled human tear samples by size exclusion and ion exchange
chromatographies (ibid). Tears without TL were reconstituted from eluted fractions of Sephadex
G-100 (ibid). SDS PAGE confirmed the resultant mixture was depleted of TL. Fresh and formalinfixed human eyes, were obtained from exenteration specimens. Fluorescent analogs, 5.3 mM, of
fatty acid (16-(9-anthroyloxy)palmitic acid, 16AP) or phospholipid (NBDC6 HPC), Molecular
Probes, were applied uniformly to the corneal epithelial surface. Excess lipid was removed by
gentle rinsing in buffer (10mM sodium phosphate, 100 mM NaCl, pH 7.4). The corneas, in quartz
cuvettes were overlayed with solutions of buffer, tears, TL (70 μM), or tears depleted of TL. Entry
of 16AP bound to protein in solution was monitored by fluorescence, λ em=450nm, (λ ex=361nm),
until saturation was achieved. For NBDC 6HPC, λ ex=420nm, λ em =524 nm. Tears used in the
experiments were fractionated by size exclusion to determine the component of tears associated
with fluorescence. Histopathology was performed to verify the presence of corneal epithelium.
Results: 16AP and NBDC6HPC demonstrate enhanced fluorescence and a blue shift of the emission
peak when protein is bound. Significant enhancement of fluorescence for 16AP and NBDC6HPC
was evident in solutions incubated with whole tears and purified TL but not with tears depleted of
TL for fixed corneas. Similar results were obtained for both lipids in fresh human corneas. After the
experiment, size exclusion fractions of tears showed that the fluorescence component co-eluted with TL.
Conclusions: Lipids may stick to the cornea surface in which mucins are depleted. These data
provide evidence that TL, a potent binding protein, scavenges lipids from the human corneal
surface and delivers them into aqueous phase of tears. TL may have an important role in removing
lipids from the cornea surface where mucins are depleted to maintain the wettability and integrity
of the ocular surface.
CR: B.J. Glasgow, None; O.K. Gasymov, None; A.R. Abduragimov, None; T.N. Yusifov,
Support: EY 11224, EY00331, an unrestricted grant from Research to Prevent Blindness
Anti-Inflammatory Therapy Preserves Corneal Barrier Function in Experimental
Murine Dry Eye
C.S. De Paiva, R.M. Corrales, A.L. Villarreal, W.Farley, D.-Q.Li, M.E. Stern, S.C.
Pflugfelder. Ophthalmology, Baylor College Medicine, Houston, TX.
Purpose: To investigate the effects of anti-inflammatory therapy on tight junction (TJ)
protein expression and barrier function of corneal epithelia in experimental murine dry eye.
Methods: C57BL6 mice aged 6-8 weeks were treated with systemic scopolamine and exposure
to an air draft for 5 days to induce experimental dry eye (EDE), with or without topical therapy,
1% methylprednisolone, 0.025% doxycycline or balanced salt solution (BSS) 4 times per day.
Untreated (UT) mice were used as controls. The expression of involucrin, zonula occludens (ZO1) and occludin were evaluated by laser scanning confocal microscopy (whole mount corneas)
and immunofluorescent staining (tissue sections). Epithelial proliferation was evaluated by BrdU
incorporation. Oregon green dextran (OGD, 70kDa) was used to measure corneal permeability.
Results: EDE significantly increased expression of involucrin, compared to UT and
treatment groups. EDE also induced marked changes in occludin and ZO-1 expression,
with increased cytoplasmic and decreased membrane staining in apical cells of the corneal
epithelia. Extensive desquamation of apical cells was observed in the EDE group. Corneal
permeability to OGD increased in EDE, with punctate and confluent dye staining, mimicking
human corneal disease. BrdU incorporation increased in corneal epithelial cells of EDE.
Treatment of EDE with methylprednisolone or doxycycline preserved the TJ network,
decreased expression of involucrin, reduced BrdU incorporation and corneal permeability
to OGD, indicating preserved corneal barrier function. BSS treatment showed no protective
effect, with TJ expression, BrdU incorporation and corneal permeability similar to EDE.
Conclusions: EDE causes altered expression of TJ proteins in the corneal epithelium that is
accompanied by increased expression of a cornified envelope protein, involucrin. Treatment
of EDE with methylprednisolone or doxycycline preserved corneal barrier function.
CR: C.S. De Paiva, None; R.M. Corrales, None; A.L. Villarreal, None; W. Farley, None; D.
Li, None; M.E. Stern, Allergan Inc, Irvine, California E; S.C. Pflugfelder, None.
Support: NIH Grants EY11915-05(SCP), T32EY07001-29(CSP);Allergan, RPB,Oshman
Foundation,William Stamps Farish
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Ballroom BCD Paper Session Program Number Range: 2418-2424
332. Lacrimal Gland, Tear Film and Dry Eye Organizing Section: CO
2424 - 4:30PM
Exposure to a Dry Environment Induces Strain-Specific Dry Eye in Mice
S.Barabino1,2, S.Rashid1, L.Shen1, L.Chen1, M.Rolando2, R.Dana1. 1Laboratory of
Immunology, Schepens Eye Research Institute, and Massachusetts Eye and Ear Infirmary,
Department of Ophthalmology, Harvard Medical School, Boston, MA; 2Department of
Neurosciences, Ophthalmology, and Genetics, University of Genoa, Genoa, Italy.
Purpose: Most current dry eye animal models have a single causative mechanism, and do not take
into consideration the influence of environmental conditions on tear secretion and ocular surface
signs. The purpose of the present study was to test the hypothesis that strains with specifically
biased T cell responses develop differential signs of dry eye when exposed to a low humidity setting.
Methods: Eight to12-week-old BALB/c (T helper-2 biased) and C57BL/6 (T helper-1 biased)
mice were placed in our controlled environment chamber (CEC) where relative humidity (RH),
temperature (T), and air flow (AF) are regulated and monitored. Mice were exposed to specific
environmental controlled conditions (RH = 21.7 ± 5.2%, AF = 15 l/min, T = 21-23°C) for 3 and
7 days. Control mice were kept in a normal environment (RH = 50-70%, no AF, T = 21-23°C)
for the same duration. Aqueous tear production by means of the cotton thread test, corneal
fluorescein staining (score 0-15), goblet cell density in the superior and inferior conjunctiva,
and corneal epithelial thickness in histologic sections were measured by a masked observer.
Results: No statistically significant differences between the groups were found at baseline.
Statistically significant decreases in tear secretion were seen after exposure to the CEC
environment. Mean cotton thread wetting was respectively 1.8, 1.4, and 0.9 mm for
control, BALB/c, and C57BL/6 at day 3, and 1.7, 0.9, and 0.4 mm for control, BALB/c,
and C57BL/6 mice at day 7. C57BL/6 mice showed reduced tear secretion as compared
to BALB/c at each time point (P<.005, t test). BALB/c and C57BL/6 mice showed a
significant increase in corneal fluorescein staining and epithelial thickness at day 3 and
7 as compared to controls. Goblet cell density was significantly decreased in the superior
and inferior conjunctiva in the BALB/c at day 7, and even earlier (day 3) in C57BL/6 mice.
Conclusions: This study indicates that exposure of non-pharmacologically modified normal
mice to a low humidity environment in the CEC can lead to significant alterations in tear
secretion, goblet cell density, and acquisition of dry eye-related ocular surface signs which
appear to be strain-specific, and more significant in T helper-1 biased C57BL/6 mice.
CR: S. Barabino, None; S. Rashid, None; L. Shen, None; L. Chen, None; M. Rolando,
None; R. Dana, None.
Support: Allergan Inc.; Vistakon, Johnson & Johnson
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2590-2610 / B143-B163
342. Corneal Molecular Biology Organizing Section: CO
2590 - B143
Induction of Gelatinase B (MMP-9) Promoter Activity by Platelet-Activating Factor
in Human Corneal Epithelial Cells Requires Multiple Transcription Factor Binding
Sites Including NFκB (-600) and Sp1 (-558)
F.Taheri1A, H.E. P. Bazan1B. ABioch & Mol Biol, BOphthalmology/Neuroscience,
LSUHSC, New Orleans, LA.
Purpose: MMP-9 is an important matrix metalloproteinase involved in remodeling of corneal
epithelium during wound healing. Expression of the gene encoding MMP-9 is elevated after corneal
injuries in epithelial cells. One important inducer is platelet activating factor (PAF), an inflammatory
mediator that accumulates in corneal epithelium following injuries. Here, we studied the promoter
region of human MMP-9 gene to determine the induction efficiency of PAF-stimulated expression
of MMP-9 and the role of different transcription factors in the responsiveness of the promoter.
Methods: Human corneal epithelial cells (HCEC) were transfected using FuGene 6 reagent
with luciferase reporter constructs fused to the wild type (-2172/+54) or 5` deleted fragments
of the human 92-kDa gelatinase B promoter (including -1511, -1112, -670, -460, -324, and -8).
Transiently transfected cells were either left untreated or stimulated with 100nM PAF for 1-3
days and the expression of luciferase was tested. HCEC were stimulated by PAF and nuclear
extracts were examined by electrophoretic mobility shift assay (EMSA) for the DNA binding
activity of NFκB, Sp1, AP-1, and AP-2. Transiently transfected HCEC with an NFκB-d2EGFP
plasmid were also stimulated with PAF and monitored up to 12 hours for the expression of GFP.
Results: Expression of luciferase in transiently transfected HCEC was highly elevated by the presence
of the segment spanning -324 to -8, which carries the TATA and GT boxes, and some transcription
factor binding sites including AP-1. Another increase in the expression of the gene was observed
when the segment spanning -1112 to -670 was present. PAF treatment of transiently transfected cells
showed that the region spanning -670 to -460 is very crucial in the induction of promoter activity by
PAF. We also found an increase in the binding activity of NFκB, Sp1 and AP-2 by EMSA. Expression
of NFκB driven destabilized green fluorescent protein (NFκB-d2EGFP) was also observed in HCEC
within 8-12 hours after stimulation with 100nM PAF, indicating the activation of NFκB in these cells.
Conclusions: Induction of the promoter activity by PAF was found to be dependent on a segment
spanning -670 to -460, which contains several transcription factor binding sites including NFκB (600), Sp1 (-558), PEA3 (-540) and AP-1 (-533). Upregulation of NFκB and Sp1 DNA binding activity
and d2EGFP over-expression by NFκB motif confirm the contribution of these two transcription
factors in PAF-mediated induction of MMP-9 in HCEC.
CR: F. Taheri, None; H.E.P. Bazan, None.
Support: NIH Grant EY04928
2592 - B145
2591 - B144
Modeling Corneal Cell Kinetics: Implications for Gene Transfer
M.I. Rosenblatt1, C.M. Nimigean2, D.T. Azar1. 1Massachusetts Eye & Ear Infirmary
and Schepens Eye Research Institute, Harvard Medical School, Boston, MA; 2Dept. of
Physiology, University of California-Davis, Davis, CA.
Purpose: To develop a novel modeling scheme to characterize the theoretical kinetics
of corneal cell layer maintenance and regeneration, allowing predictions of gene transfer
behavior. Methods: For modeling, a cell layer was divided into three potential compartments.
Compartment A contained stem cells capable of dividing or moving to compartment B or
C. Compartment B represented dividing cells capable of either replicating or moving to
compartment C. Compartment C contained cells which could not replicate, but which could be
lost. Models contained rate constants for replication and transitions between compartments.
The model for corneal epithelium contained all three compartments (A, stem cells; B, basal
cells; and C, superficial cells) as well as rate constants for division and movement. For stromal
cells, only a B compartment was modeled with a rate constant for cell division. The endothelial
layer was modeled using a C compartment with a rate constant for cell loss. Gene transfer with
retroviral vectors (stable infection of dividing cells only), lentiviral vectors (stable infection of
both dividing and non-dividing cells), or adenoviral vectors (transient expression in all cells)
was modeled for each cell layer. For adenoviral vectors a rate constant for vector degradation
was added. Results: Epithelium: For retroviral infection, the initial percentage of expression
in A was lower than in B. Over time the percentage of infection within B approached the initial
percentage of A infection. Initial lentiviral infection was higher than for retrovirus. Again,
the B compartment expression would over time approach the percentage of that seen in the
A and overall expression would remain higher. For adenoviral infection all compartments
would initially have high levels of expression, but the levels would eventually be zero.
Stroma: The rate of infection would remain constant for retroviral and lentiviral infection, although
the initial rate would be higher for the lentiviral vectors. Adenoviral infection would steadily decline.
Endothelium: Retroviral infection was not possible. Lentiviral infection demonstrated a
constant percentage of infected cells, albeit a steady decline in total cells infected. Adenoviral
infection steadily declined. Conclusions: The variable mechanisms for regeneration and
maintenance of the corneal cell layers pose challenges in choosing a gene transfer system.
Using theoretical modeling, however, may provide guidance in choosing the appropriate vector
system to alter specific corneal processes. CR: M.I. Rosenblatt, None; C.M. Nimigean,
None; D.T. Azar, None. Support: EY10101 and Joint Clinical Research Center Fellowship
2593 - B146
Pnn-Dependent Modulation of E-Cadherin Gene Expression in Corneal Epithelial
S.P. Sugrue, R.Alpatov, J.-H.Joo, G.C. Munguba, M.R. Jackson. Anatomy & Cell Bio
- POB 100235, University of Florida, Gainesville, FL.
Regulation of Gene Expression During the Transition From Human Corneal Epithelial
Cell Stem Cell to Differentiated Cell: A Role for AP1 Transcription Factors
R.L. Eckert1A,1B, G.Adhikary1A, F.Bone1A, R.Gopalakrishnan1A, J.Lass1C, J.Crish1A.
Physiology & Biophysics, BBiochemistry, COphthalmology, 1Case School of Medicine,
Cleveland, OH.
Purpose: Division of corneal epithelial stem cells gives rise to transient amplifying cells
that ultimately differentiate to form the multilayered corneal epithelium. In the present study
we use the involucrin gene as a model to study the regulation of gene expression during this
progression. Human involucrin (hINV) is a structural protein that is absent from the limbal
corneal epithelial stem cells, but is expressed in transient amplifying and differentiated cells.
Methods: Involucrin gene expression and promoter activity were monitored in primary
cultures of normal human corneal epithelial cells and in hINV promoter transgenic mice.
Cells were transfected with hINV promoter-luciferase reporter constructs and cell extracts
were prepared and monitored for luciferase activity as an index of promoter activation.
Transcription factor binding was monitored using nuclear extracts and gel mobility
shift analysis. Parallel studies utilized hINV promoter transgenic mice to monitor the
effects of transcription factor binding site-specific mutations on gene expression in vivo.
Results: Our studies identify an activator protein one (AP1) DNA binding site in the hINV gene
upstream regulatory region that is essential for baseline and stimulus-associated hINV promoter
activity. Mutation of this site, AP1-5, results in a lose of hINV promoter activity. In vivo studies,
using hINV promoter transgenic mice, reveal that targeted mutation of AP1-5 causes a complete
cessation of hINV gene expression in the corneal epithelium, confirming that this site is essential
for in vivo expression. Gel mobility supershift analysis reveals interaction of the AP1 factors,
Fra-1, Fra-2 and Jun-B, with the AP1-5 element. In addition, expression of dominant-negative
c-jun inhibits AP1 site-dependent hINV expression. Moreover, treatment with TPA, a protein
kinase c activator, increases hINV promoter activity, a response that correlates with increased
nuclear AP1 factor level and AP1 factor binding to the hINV gene AP1-5 response element.
Conclusions: These findings provide definitive evidence that AP1 factors are required for
activation of gene expression during the transition form corneal stem cell to differentiated
CR: R.L. Eckert, None; G. Adhikary, None; F. Bone, None; R. Gopalakrishnan, None; J.
Lass, None; J. Crish, None.
Support: None.
2594 - B147
2595 - B148
Purpose: Previously, we have shown that pinin (Pnn) is involved in the regulation of epithelial cell
adhesion. Pnn and its associated proteins of the SR-family, which are involved in mRNA splicing
events, co-localize and interact in nuclear speckles. Interestingly, Pnn can also up regulate Ecadherin gene activity through its binding to the co-repressor CtBP, a multifunctional transcriptional
regulator, which targets E-cadherin promoter. Transcriptional co-repressors bridge basal transcription
machinery to enzymatic complexes capable of maintaining the silenced chromatin state within the
gene regulatory regions. We are now addressing the mechanism by which Pnn may impact both
transcription and mRNA processing. Methods: Pnn expression was knocked-down in HCET cells
by expressing a short hairpin RNAi from pU6-vector. In vivo interaction studies from transiently
transfected cells were carried out using anti-Flag or anti-myc affinity agarose. Pol II/Pnn complex
was immuno-isolated from HeLa nuclear extracts using anti-Pol II. Chromatin immunoprecipitation
(ChIP) were performed using the following antibodies: anti-HA, anti-acetylated histone H3, antipolymerase 2, anti-dimethylated histone H3 at lysine9 (H3K9). For the PCR reactions primers
spanning E-cadherin promoter region or GAPDH promoter region were utilized. Results: Knocking
down Pnn expression with shRNA results in a down regulation of E-cadherin and loss of epithelial
cell adhesion. In addition, the effect of Pnn-RNAi could be rescued by transfection of Pnn vectors
carrying conserved substitutions within the RNAi target. These results are consistent with Pnn’s
role in regulating epithelial cell-cell adhesion. ChIP analyses revealed that Pnn is associated with
E-cadherin promoter but not GAPDH promoter. In addition, Pnn expressing cells exhibited enhanced
acetylation of H4, reduction in dimethylation at H3K9, and increased presence of Pol II at the Ecadherin promoter. Conclusions: These data suggest that expression of Pnn is linked to increase in
cell-cell adhesion and elevation in E-cadherin. Furthermore, the presence of Pnn at the E-cadherin
promoter region may promote transcriptionally favorable local chromatin state along with the
recruitment of transcriptional machinery associated with activation of the epithelial gene expression.
Thus, Pnn may play a fundamental role in transcriptional regulation in addition to its previously
reported role in RNA splicing, adding Pnn to the growing list of proteins that functionally bridge
transcription and mRNA processing. (Supported by NIH grant EY07883)
CR: S.P. Sugrue, None; R. Alpatov, None; J. Joo, None; G.C. Munguba, None; M.R. Jackson,
Support: NIH grant EY07883
Keratocan-Cre Transgenic Mouse: A New Model for Neural-Crest Cell Lineage
Analysis During Embryonic Development
D.Y. Weng1, C.Y. Liu2, W.W. Y. Kao1. 1Ophthalmology, University of Cincinnati, Cincinnati,
OH; 2Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of
Medicine, Miami, FL.
Purpose: We have made use of a novel mouse strain carrying Cre whose expression
is controlled by a tissue-specific promoter (kerotocan) which can be used to
determine the fates of periocular mesenchymal cells during embryonic development.
Methods: To create a transgenic mouse line, a gene construct containing keratocan
promoter and Cre recombinase minigene was microinjected into the fertilized
mouse eggs. We then crossed these mice to Z/EG (EGFP) and ROSA26-LacZ
reporter strains to reveal the fates of Cre expressing cells in situ. The Cre activity
was assessed by the detection of green fluorescence and the β-galactosidase activity
of bi-transgenic Kera-Cre/ZEG and Kera-Cre/Rosa26-LacZ mice, respectively.
Results: Twenty two independent transgenic mouse lines were obtained. Seven founders did
not transmit the transgene to its offspring. Four mouse lines did not express the Cre activity.
The rest of mouse lines were examined further. We detected strong lacZ expression in corneal
stroma of the Kera-Cre/Rosa26-LacZ bi-transgenic mice and green fluorescence in the corneal
stroma of the Kera-Cre/ Z/EG bi-transgenic mice. However, we also observed that the lack of
the β-galactosidase and green fluorescence in the corneal endothelium and limbal region of
the corneal stroma. Interesting, through whole body survey, LacZ expression was detected
not only in the corneal stroma, but also in the CA1 and CA2 region of the hippocampus
Conclusions: Kera-Cre transgenic mice are a new genetic tool for the analyses of neural-crest
cell lineage and tissue-specific gene targeting.
CR: D.Y. Weng, None; C.Y. Liu, None; W.W.Y. Kao, None.
Support: NIH grant EY14207; EY12486; RPB; OLERF
Over-Expression of Pax6 Retards Cell Cycle Progression and Reduces Cell Migration
Rate in Corneal Epithelial Cells
J.Ouyang1,2, S.-Y.Cheng1, L.-K.Yeh1, C.-Y.Liu1, M.E. Fini1. 1Bascom Palmer Eye Institute,
University of Miami School of Medicine, Miami, FL; 2Physiology/Biophysics, University
of Miami School of Medicine, Miami, FL.
Purpose: Transcription factor Pax6 resides at the top of a regulatory hierarchy controlling formation
of the eye during embryological development. Pax6 is also expressed in several tissues of the adult
eye. The goal of this study was to learn more about the role of Pax6 in the corneal epithelium.
Methods: Pax6 dosage is critical for proper formation of the eye. Thus to study its role in corneal
epithelial cells, we increased its expression level. A rabbit corneal epithelial cell line, SIRC, was
used to establish stable transformant clones carrying a mouse wild-type Pax6 full-length coding
region or a mutant Pax6, lacking the c-terminal transactivation domain. The full-length mPax6 or
mutant mPax6 transgene was regulated using a doxycycline-based (tet-on) inducible gene expression
system. Expression of Pax6 protein or truncated Pax6 protein was induced by adding doxycycline to
the culture medium. A cell growth curve, cell cycle profile, cell proliferation index, cell migration,
and apoptosis was studied. In all experiments, induced and non-induced tetO-Pax6 or tetO-delta286
transformed SIRC cells were compared. Results: Pax6 over-expression has been reported to retard
or inhibit the cell cycle in lens cells and neurons. We found that over-expression of Pax6 protein also
retards the SIRC cell cycle. Consistent with this, retinoblastoma tumor suppression protein (pRB) was
up-regulated as judged by immuno-precipitation analysis. In contrast, truncated Pax6 protein without
the transactivation domain did not affect the cell cycle. Pax6 over-expression induced apoptosis in a
small fraction of cells. Western blotting experiments suggested that this effect was mediated via a
caspase-3 independent pathway. In addition, we previously reported that Pax6 levels and DNA binding
activity increase at the epithelial front migrating to heal a corneal wound, and migration is faster in
“small eye” mice with decreased Pax6 dosage. Consistent with this, over-expression of wild-type
but not mutant Pax6 inhibited the rate of SIRC cell migration. Conclusions: These results suggest
that normal dosage of Pax6 protein plays a pivotal role in the maintenance of corneal epithelial cell
function. The feature of cell cycle retardation in Pax6 over-expressing cells is consistent with the
notion that Pax6 may control the rate of epithelial turnover and repair and contribute to the slow
cycling of stem cells. The effects on cell migration are consistent with a proposed role in controlling
corneal re-epithelialization.
CR: J. Ouyang, None; S. Cheng, None; L. Yeh, None; C. Liu, None; M.E. Fini, None.
Support: EY014801,EY12486,Walter G. Ross Foundation, RPB
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2590-2610 / B143-B163
342. Corneal Molecular Biology Organizing Section: CO
2596 - B149
Repression of the Alpha6 Integrin Gene Promoter by the ECM Component Laminin
M.Gaudreault, S.Leclerc, S.L. Guérin. Oncology and Molecular Endocrinology Research
Center and Ophthalmology Research Unit, CHUL Research Center, Sainte-Foy, PQ,
Purpose: Injury to the corneal epithelium induces a massive secretion of fibronectin (FN)
within the first few hours following the eye damage. During the wounding process, FN
secretion is progressively turned off while secretion of laminin (LM) increases. These
extracellular matrix components are recognized by membrane-bound receptors that belong
to the integrin family. Both the α6β1 and α6β4 integrins recognize LM as their ligand. The
promoter from the α6 gene has been recently cloned and shown to bear multiple binding
sites for members of the Sp1 family of transcription factors. In this study, we characterized
the regulatory influence exerted by Sp1 and Sp3 on both the promoter and 5’ flanking region
of the α6 gene when primary cultures of rabbit corneal epithelial cells (RCECs) are grown
in the presence of LM. Methods: Recombinant plasmids bearing the CAT reporter gene
fused to various segments from the α6 promoter were transfected into both RCECs grown
on untreated or LM-coated culture plates, or into Sp1-deficient Drosophila Schneider cells.
Expression and DNA binding of Sp1/Sp3 was monitored by Western blot and electrophoretic
mobility shift assays (EMSAs), respectively. DNA target sites for Sp1/Sp3 in the α6 promoter
and 5’ flanking sequence were identified by DNaseI footprinting analyses. Experiments
in northern blot and RT-PCR were conducted to evaluate α6 mRNA expression in RCECs
grown on LM-coated culture plates. Results: Transfections conducted into Drosophila cells
provided evidence that Sp1 and Sp3 act in an additive manner on the activity driven by the α6
promoter. Two Sp1/Sp3 target sites were identified in the α6 promoter by DNaseI footprinting
analyses. Transfections conducted into RCECs grown on LM-coated culture plates resulted
in a dose-dependent repression in the activity directed by the α6 promoter that also vary in
strength in a cell density-dependent manner. Most of all, expression of α6 mRNA diminished
dramatically when RCECs are grown on LM-coated plates. Repression of the α6 promoter
activity was suggested to result from the proteolytic cleavage of Sp1/Sp3 when cells are grown
on LM, as revealed by both EMSA and Western blot analyses. Conclusions: Taken together,
these results suggest that the signal transduced by the binding of LM to its integrin receptor
subunit α6, triggers the activation of one (or more) unknown protease(s) that control the level
to which Sp1/Sp3 are to be expressed in RCECs. CR: M. Gaudreault, None; S. Leclerc,
None; S. L. Guérin, None. Support: FRSQ Network in Vision Research
2598 - B151
2597 - B150
The Role of Tetracyclines in Healing of Canine Refractory Ulcers
H.L. Chandler1, C.M. H. Colitz1, W.W. Miller2, D.F. Kusewitt1. 1Veterinary Biosciences,
The Ohio State University, Columbus, OH; 2Animal Ophthalmology Clinic, Memphis, TN.
Purpose: Re-epithelialization in refractory corneal ulcers does not occur properly. Normal
corneal re-epithelialization resembles the process of epithelial-mesenchymal transition
(EMT), the transformation of epithelial cells into more fibroblastic cells. EMT is initiated by
growth factors like transforming growth factor-ß (TGF-ß) that stimulate production of Slug.
Slug modulates changes in cell adhesion and motility leading to EMT and is required for
normal corneal epithelial cell (CEC) migration. Tetracyclines enhance TGF-ß expression. We
hypothesize that tetracycline stimulation of TGF-ß activity leads to increased expression of
Slug and, ultimately, to the EMT-like changes required for successful corneal wound healing.
Methods: Canine refractory corneal ulcers were examined to determine levels of expression
of Slug and its putative target genes. Western blotting and real-time RT-PCR were used to
examine the expression of Slug protein and mRNA compared to normal corneas. Also, CEC
were wounded in vitro and treated with oxytetracycline. Wound closure was monitored
and RT-PCR performed to determine Slug expression. Cultured CEC were incubated
with oxytetracycline and TGF-ß blocking antibody, and cell migration was monitored.
Results: There was decreased expression of Slug protein and mRNA in refractory ulcers
compared to normal corneas. Oxytetracycline increased both Slug expression and the rate
of wound closure in cultured CEC compared to untreated controls. Blocking the TGFß pathway decreased the rate of cell migration in CEC treated with oxytetracycline.
Conclusions: Refractory ulcers have even lower Slug expression than normal corneal
epithelium, suggesting reduced stimulation of cell migration. In vitro treatment of wounded
CEC with oxytetracycline enhanced wound closure and Slug expression. Blocking the TGF-ß
pathway decreased the rate of cell migration in tetracycline-treated CEC, suggesting that
tetracycline may use this pathway to stimulate wound closure. Treatment of refractory ulcers
with tetracycline may increase the expression of Slug and its targets, thus allowing normal
re-epithelialization to occur.
CR: H.L. Chandler, None; C.M.H. Colitz, None; W.W. Miller, None; D.F. Kusewitt,
Support: American Animal Hospital Foundation
2599 - B152
The Notch Receptors and Their Ligands in Human Corneal Epithelium
A.Ma, B.Zhao, J.Cai, M.E. Boulton, J.Albon. Optometry & Vision Science, Cardiff
University, Cardiff, United Kingdom.
Purpose: To investigate the expression and role of Notch receptors and their ligands in the
human corneal epithelium. Methods: Human corneal epithelial cells were cultured from donor
human corneas (n=60). Following RNA extraction from epithelial cells, RT-PCR was performed
to determine gene expression of the Notch receptors (N1-4) and their ligands (Delta 1, 3, 4 and
Jagged 1, 2). Western blotting and indirect immunofluorescence was then used to confirm protein
expression of the genes identified in corneal epithelial cells in vivo and ex vivo respectively.
Following serum starvation (0.2% serum for 30 minutes), γ-secretase inhibitor at a
concentration of 25μM or 50 μM was added to cultured corneal epithelial cells in triplicate
for 4 hours. The consequent changes in expression of Notch1 and Notch2, the proliferation
marker: Ki67 and the differentiation marker: Cytokeratin3 were evaluated by Western blotting.
Results: Notch 1, 2, Delta 1 and Jagged 1 but not Notch 3, 4, Delta 3, 4 or Jagged
2 genes and proteins were identified in human corneal epithelial cells. Notch 1
and 2 were immunolocalised throughout the corneal epithelium in all suprabasal
cell layers, but absent from the basal layer. In contrast, Delta 1 and Jagged 1
appeared to be expressed in cell borders in all cell layers of the corneal epithelium.
γ-Secretase inhibition of cultured epithelial cells resulted in a significant
decrease in Notch1 and 2 expression (p<0.01), accompanied by a reduced
level of Ki67 (p<0.01) and an increase in Cytokeratin3 expression (p<0.01).
Conclusions: Notch 1, 2 and their ligands Delta1, Jagged 1, are likely to play a pivotal
role in corneal epithelial cell homeostasis. Downregulation of Notch 1 and 2 in corneal
epithelial cells is associated with decreased proliferative activity and promotion of epithelial
CR: A. Ma, None; B. Zhao, None; J. Cai, None; M.E. Boulton, None; J. Albon, None.
Support: ORS
Metabolic and Molecular Signaling Pathways of Retinoids on Human Ocular Surface
H.Nezzar1A,2, F.Chiambaretta1A,2, G.Marceau1B,2, B.Dastugue1B, D.Rigal1A,2, V.Sapin1B,2.
Department of Ophthalmology, BDepartment of Biochemistry, 1University Hospital,
Clermont-Ferrand, France; 2Faculty of Medicine, ARDEMO, Clermont-Ferrand, France.
Purpose: The integrity of the human ocular surface has an absolute requirement of vitamin
A and its active derivatives, the retinoic acids. These retinoids regulate transcriptional levels
of target genes through the activation of ligand-dependant nuclear receptors: Retinoic Acid
Receptors and Retinoid X Receptors. Partial characterization of these receptors have been
realised in rabbit, mouse and human cornea, but systematic tissue and cellular expression of
the 3 RARs and RXRs had to be completed at the human ocular surface. The first objective
of this work was to define their expression patterns in term of genes and proteins for total
human conjunctiva, cornea and the major cell types present constituting human ocular surface.
The second objective was to demonstrate the presence of differents enzymes transforming
vitamin A to retinoic acid and the activity of this retinoids pathway in the ocular surface.
Methods: Total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial
cells, corneal keratocytes, corneal endothelial cells and conjunctival epithelial cell and
submitted to RT-PCR experiments in order to determine the expression patterns of the RARs
and RXRs using specific primers. Immunological staining (histo- and cellular chemistry)
experiments were performed to better define the RARs and RXRs protein localization in
ocular surface at tissular and cellular levels. The expression patterns of retinal deshydrogenase
(RALDHs) and alcohol deshydrogénase (ALDHs) was also determined in corneal epithelial
cells. The metabolic pathway is tested by targeted disruption of different enzyme step who
transforms retinol to active derivatives. Results: RAR alpha, gamma and RXR alpha are
expressed in cornea, conjunctiva and all of their constitutive cells, checked in our study.
RXR gamma was never detected in cornea and conjunctiva. RXR beta was only present in
primary cultures of corneal keratinocytes, which don’t express RAR beta in contrast of the
other corneal and conjunctival cells.ALDH3, ALDH4, RALDH1, RALDH 3 are expressed
in ocular surface. Conclusions: For the first time, we established an exhaustive description
of RARs and RXRs, ALDHs and RALDHs expression patterns, in the human ocular surface.
Our results demonstrated at the human ocular surface, the presence of all the actors of retinoic
acid signalling pathway and their activation.
CR: H. Nezzar, None; F. Chiambaretta, None; G. Marceau, None; B. Dastugue, None; D.
Rigal, None; V. Sapin, None. Support: JE 2447 ARDEMO MRT
2600 - B153
2601 - B154
Multi-Drug Resistance Proteins Are Expressed in Human Cornea
U.Becker1A, C.Ehrhardt1A,2, U.F. Schaefer1A, K.W. Ruprecht1B, C.M. Lehr1A.
Biopharmaceutics & Pharm. Tech., BUniversity Eye Hospital, Institute of Orthoptics,
Saarland University, Saarbruecken, Germany; 2School of Pharmacy, University of Dublin,
Trinity College, Dublin, Ireland.
Purpose:To study the expression of proteins with relevance to drug absorption such as Pglycoprotein (MDR1), multi drug resistance protein 1 (MRP1), multi drug resistance protein
2 (MRP2), lung resistance-related protein (LRP) and breast cancer resistance protein (BCRP)
in human corneal epithelium. Introduction: The eye is considered a good target for local
therapy, but absorption of certain drugs can be amended by multi-drug resistance (MDR).
MDR has evolved as a problem in tumor therapy and intestinal drug delivery. Recently, the
presence of MRP1 in rabbit corneal epithelial cell culture has been reported (Dey et al.), but
few data is available on MDR expression and function in human corneal tissue. In this study,
we determined the presence of MDR-related proteins in native human corneal epithelium.
Methods: RT-PCR and western blotting of lysate gained from human corneas were performed.
In addition, functionality of MDR1 was assessed by bi-directional transport studies across
excised human cornea using a substrate, rhodamine 123 (Rh123), with and without presence
of an MDR1-inhibitor, verapamil. Fluorescein-sodium (FLU) permeability was studied as a
passively diffusing marker. Results: By RT-PCR the presence of the MDR-related proteins
was assessed on the genomic level. Western blot studies gave similar information on the
protein level. Rh123 showed a significant net secretion in transport studies (i.e., basolateral
to apical transport) across intact cornea, which was regulated by verapamil. There was no
significant directionality in FLU-transport, exhibiting permeability values in the range
found in commonly used cell culture models. Conclusions: The presence of MDR-proteins
in intact human cornea could be verified, giving new criteria for the evaluation of cell
culture models representing the corneal epithelium. Acknowledgements: Ms. Gross and Dr.
Eschmann (Ursapharm) are thanked for their support of this work. Drs. Baldes and Daum
are thanked for their help in molecular biology, the staff of the Lions Corneabank, Homburg
for their co-operation.
CR: U. Becker, Ursapharm Arzneimittel GmbH&Co KG, Saarbruecken, Germany F; C.
Ehrhardt, None; U.F. Schaefer, None; K.W. Ruprecht, None; C.M. Lehr, None.
Support: Ursapharm Arzneimittel GmbH & Co KG, Saarbruecken, Germany
Influence of the Fibronectin-Mediated Activation of the MAPK Pathway on Poly(ADPRibose) Polymerase-1 (PARP-1) Gene Expression During Corneal Wound Healing
K.Zaniolo, S.Leclerc, M.Audette, S.L. Guérin. Oncology and Molecular Endocrinology,
CHUL Research Ctr, Sainte-Foy, PQ, Canada.
Purpose:Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that catalyzes
the transfer of ADP-ribose units onto PARP-1 itself and other nuclear proteins involved in
chromatin architecture and DNA metabolism. It is therefore involved in several important
functions such as DNA repair, DNA transcription, carcinogenesis and apoptosis. The activity
directed by the PARP promoter is mainly dictated through its recognition by the transcription
factors Sp1 and Sp3. Recently, we demonstrated that fibronectin (FN), a component from the
extracellular matrix that is required for proper corneal epithelial wound healing, exerted a
positive regulatory influence on the α5 promoter in primary cultured rabbit corneal epithelial
cells (RCECs). This FN-mediated increase in α5 promoter activity has been shown to be
mediated by the activation of the MAPK pathway. Because both PARP and α5 genes are
regulated by Sp family members, we examined whether activation of this signaling pathway
would alter PARP1 expression in manner similar to that observed for the α5 integrin gene
promoter. Methods:Expression of PARP-1, Sp1/Sp3, Erk1/Erk2 and phospho-Erk1/Erk2 in
RCECs grown in the presence of either (or both) FN or the MAPK inhibitor PD98059 was
monitored by Western blot. Electrophoretic mobility shift assays (EMSAs) were conducted to
assess the extent of both Sp1 and Sp3 DNA binding in nuclear extracts from RCECs grown in
the presence of FN and/or PD98059. Recombinant constructs bearing the PARP-1 promoter
fused to the CAT reporter gene were transfected into RCECs under similar culture conditions.
Results:Expression of endogenous PARP-1, Sp1 and Sp3 increased when RCECs were grown
on FN-coated culture plates. This also translated into a similar increase in the ability of both
Sp1 and Sp3 to bind their high affinity target site in DNA. In agreement with these results,
the activity directed by the PARP-1 promoter increased in the presence of FN. Inhibition
of the MAPK pathway by PD98059 abolished these properties. Conclusions:Expression
of PARP-1, Sp1 and Sp3 is likely stimulated by the presence of FN during corneal wound
healing in vitro. Binding of FN to its integrin receptor α5β1 is also thought to alter the level
of Sp1 phosphorylation by activated Erk1/Erk2 through activation of the MAPK pathway.
PARP may therefore contribute to gene transcription during the highly proliferative phase
that characterizes corneal wound healing. CR: K. Zaniolo, None; S. Leclerc, None; M.
Audette, None; S.L. Guérin, None. Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2590-2610 / B143-B163
342. Corneal Molecular Biology Organizing Section: CO
2602 - B155
2603 - B156
Expression Pattern of EphrinB Ligand and EphB Receptor and Cell-Cell Interaction
in Corneal Epithelial and Keratocyte Cell Lines
T.-Y.Chung, F.H. Casanova, T.Sakimoto, J.A. Javier, E.Albe, J.H. Chang, D.T. Azar.
Department of Ophthalmology, Mass Eye and Ear Infirmary, Schepens Eye Research
Institute, Harvard Medical School, Boston, MA.
Gene Expression of Matrix and Cell Adhesion Molecules in PAF-Treated Corneal
X.Chen, P.Ottino, J.He, H.E. P. Bazan. Ophthalmology and Neuroscience Center,
LSUHSC, New Orleans, LA.
Purpose: Recent studies from our laboratory demonstrated that the inflammatory
mediator platelet-activating factor (PAF) induces, by a receptor-mediated mechanism,
the expression of selective matrix metalloproteinases (MMPs) in corneal myofibroblasts.
(Ottino et al, IOVS, in press) Since myofibroblasts are key components of contraction
of collagen and wound healing, in this study we compared the gene-expression
profiles of matrix and adhesion molecules of rabbit corneal keratocytes and
myofibroblasts and the effect of PAF on the expression of these genes in myofibroblasts.
Methods: A human extracellular matrix and adhesion molecules gene array containing
96 genes (Superarray) was used. Corneal keratocytes were isolated from rabbit corneas.
Myofibroblasts were obtained from corneal fibroblast subcultured at low cell density in
DMEM-F12 with 2%-5% PPHS. The cells were identified by morphology and a-smooth
muscle actin expression. Myofibroblasts were serum-starved overnight and treated with
or without 100 nM PAF for 4 h and RNA was isolated. Changes in gene expression of
more than three fold were considered significant. Real-time PCR was employed to
confirm the cDNA array results and to identify the fold increases of gene expression.
Results: cDNA expression array analysis comparing keratocytes with myofibroblasts showed
that the cell adhesion molecule MICA and the MMPs -10,- 13 and -24 were down-regulated
in myofibroblasts, while the extracellular protein caveolin-1 and the integrin alpha 3 were
up-regulated. Stimulation of myofibroblasts with PAF induced up-regulation of the cell
adhesion molecule contactin -1, and the integrin beta 1 (ITGB1), the extracellular matrix
proteins collagen type 1 and fibrinogen beta, the MMPs -11 and -20, in addition to those already
described. Gene expression of ITGB1 increased 3.5 fold at 8 hours as assayed by real-time. PCR.
Conclusions: Changes in the expression of cell-matrix adhesion molecules between keratocytes
and myofibroblasts could reveal differences in function between these two cell phenotypes.
The up-regulation of specific extracellular matrix and cell adhesion genes, such as ITGB1,
suggests a role of the inflammatory mediator PAF in cell-matrix adhesion.
CR: X. Chen, None; P. Ottino, None; J. He, None; H.E.P. Bazan, None.
Support: NIH grant EY04928
2604 - B157
2605 - B158
2606 - B159
2607 - B160
Purpose: Eph rinB ligands and EphB receptors are involved in blood vessel
differentiation and nerve pathfinding. Our purpose was to investigate the expression
pattern of ephrinB and EphB in mouse corneal epithelial and keratocyte cell lines.
Methods: Excised corneas were immortalized and subcloned to generate wild type mouse corneal
epithelial and keratocyte cell lines, which were characterized using immunohistochemistry. Indirect
immunofluorescent assays with antibodies against ephrinB ligands (ephrinB1, B2 or B3) and EphB
receptors (EphB1, B2, B3, B4 or B6) were performed on the corneal epithelial and keratocyte cell
lines as well as mouse neuroblastoma (CCL-131) and endothelioma (CRL-2299) cell lines, which
served as controls. Further immunolocalization of ephrinB2 and EphB4 was performed in vivo and in
epithelial and keratocyte cell lines seeded at low (1×104 cells/ml) and high (5×10 4 cells/ml) densities.
Results: Positive staining for ephrinB2, B3 and EphB1, B2, B3 and B4 was observed in corneal
epithelial cells (with EphB4 showing highest levels of expression and ephrinB3, EphB1 and
EphB2 showing moderate expression levels). Positive staining for ephrinB1, B2 and B3 and
EphB1, B4 and B6 was observed in keratocytes (with ephrinB1 and EphB4 showing highest level
of expression and ephrinB2 and EphB1 showing moderate expression levels). Neuroblastoma
cell line showed intense immunolocalization of ephrinB1, B2 and EphB1 and B4. Endothelioma
cell line showed intense immunolocalization of ephrinB1, EphB1 and EphB4. EphB4 and
ephrinB2 showed diffuse membrane immunolocalization in keratocytes seeded at low density,
while EphB4 showed focal areas of intense immunolocalization in keratocytes seeded at high
density. Corneal epithelial cells seeded at low and high densities showed diffuse membrane
immunolocalization of ephrinB2 and EphB4. Mouse corneal epithelium showed expression
of both ephrinB2 and EphB4 (with EphB4 showing higher expression than ephrinB2).
Conclusions: Certain members of the ephrinB and EphB families are exclusively expressed in corneal
epithelial cells (EphB2 and EphB3) and keratocytes (ephrinB1 and EphB6). Despite similarities in
the expression of other ephrinB and EphB family members in corneal epithelial cells and keratocytes
(ephrinB2, B3 and EphB1 and B4), they respond differently to changes in cell seeding densities.
The differences in the response to cell-cell interactions may be important in the regulation of
angiogenesis in the cornea.
CR: T. Chung, None; F.H. Casanova, None; T. Sakimoto, None; J.A. Javier, None; E. Albe,
None; J.H. Chang, None; D.T. Azar, None. Support: None.
LASIK Surgical Smoke Analysis by MALDI Indicates Presence of Polypeptides
B.Woo1A, A.D. Baribeau2, C.A. Carroll1B, S.T. Weintraub1B, R.D. Glickman1A.
Ophthalmology, BBiochemistry, 1University of Texas Health Science Center at San
Antonio, San Antonio, TX; 2LASIK Eye Institute, San Antonio, TX.
Purpose: LASIK is the most widely performed refractive surgical procedure. Surgical
smoke inhalation is a health concern to refractive surgeons and researchers. The present
theory of laser photochemical ablation suggests that only small terminal degradation
products would be present in the plume. Preliminary data from amino acid analysis
and gel electrophoresis indicated the presence of proteinaceous material of up to 5
kDa molecular weight. (Glickman et al, Proc. SPIE v. 4951:124-32, 2003). Peptides of
this size are sufficient to provoke allergenic reactions, and thus pose allergeneic risk to
those exposed to those exposed to surgical smoke produced during LASIK. The purpose
of this study was to refine the analysis of biomolecules in the excimer induced plume.
Methods: A Mastel clean room smoke evacuation unit was used with a LADARVISION
excimer laser. The Mastel filters were used to trap the plume particles produced
during LASIK procedures. Unused filters were used as a control. The PTFE filter
elements were removed from the plastic housings in a negative pressure fume hood and
stored at -70C until analysis. Material trapped on the filter was eluted with successive
washes of 50% and 90% acetonitrile (ACN) with 1% TFA. The extracted material was
concentrated by evaporation of the ACN prior to analysis with MALDI mass spectroscopy.
Results: Previous analysis indicated that amino acids were present as oligopeptides. This
was consistent with MALDI analysis which indicated randomly fragmented polypeptides
in the range of 600 to 2000 m/z were present only on the patient-exposed filters. The
randomly fragmented nature of the peptides precluded identification of the parent protein(s).
Conclusions: Results to date suggest that polypeptides are present in the LASIK plume
with masses consistent with the range identified in our previous analysis. The presence of
these large molecules is not predicted by the current theory of excimer laser photochemical
ablation. We hypothesize that local thermal reactions may cause fragmentation and ejection of
polypeptides. Regardless of the mechanism of fragmentation, the present findings underscore
the biohazardous nature of the LASIK plume and the need for efficient smoke evacuation
during these procedures.
CR: B. Woo, None; A.D. Baribeau, None; C.A. Carroll, None; S.T. Weintraub, None; R.
D. Glickman, None. Support: RMG Research Fund and Research to Prevent Blindness
Exogenous Gene Delivery and Expresion in Human Corneal Stroma Cells Using an
Adenoviral Vector
G.Amescua, S.Pfahler, E.C. Carlson, V.L. Perez. Ophthalmic Research, Cole Eye Inst/
CCF, Cleveland, OH.
Pur pose: To develop a mi nimally i nvasive and ef f icient means of
delivering and expressing a gene of interest in the human corneal stroma.
Methods: Utilizing the technique of intrastromal injection using 1cc syringe and a
30G needle, 5 x 109 pfu of adenoviral vector expressing an EGFP reporter gene driven
by a CMV promoter in a volume of 50 μl was delivered to the corneal stroma of human
corneal buttons from the Cleveland Eye Bank. The corneal buttons were then cultured
in an incubator at 37 °C and 5%CO2 in RPMI media for a total of 14 days. Fluorecent
stereo-micrograph were taken every 24 hrs, using an appropriate filter for EGFP. At 7 days
corneal buttons were removed from cultures, DAPI stained and confocal microscopy was
performed to generate 3D reconstructions from z-stack images in 1 micron increments.
Results:No positive EGFP expression could be detected by fluorescent stereomicroscopy
at 24 hrs post-injection; however, expression of the EGFP reporter gene was initially
observed at 48 hrs post-injection in 50-60 percent of the total corneal area. The
area of expression began to diminish at day 9. By day 14, the corneal area with
positive EGFP expression was 19.6 percent. Three-dimensional confocal analysis
demonstrated the presence of EGFP was exclusively localized to the stroma
Conclusions: Ex vivo transfection of human corneal buttons can be achieved with intrastromal
injection of adenoviral vector. This ability of intrastromal injection to deliver an adenoviral
construct, may prove useful as a tool to deliver exogenous genes of interest to the corneal
CR: G. Amescua, None; S. Pfahler, None; E.C. Carlson, None; V.L. Perez, None.
Support: K08EY014912-01
Corneal Fibroblasts Support Osteoclast-Like Cell Differentiation of Macrophages
A.Sharma1,2, R.R. Mohan2, S.Sinha2, S.E. Wilson2. 1Current address: Pharmacutical
Sciences, Punjabi university, Patiala, India; 2Cole Eye Institute, The Cleveland Clinic
Foundation, Cleveland, OH.
Purpose: Osteoblasts and corneal fibroblasts both express membrane-bound receptor
activator of NFkB (RANK) ligand and macrophage colony stimulating factor (mCSF),
which confer on osteoblasts the capacity to modulate macrophages to differentiate into
osteoclasts. The aim of the present study was to investigate the capacity of corneal
fibroblasts to support osteoclast-like differentiation from macrophages in vitro.
Methods: Corneal fibroblasts were isolated from C57BL/6 mice by dispase and collagenase
digestion. For macrophage cell culture, mouse bone marrow obtained from tibia and femur
was dispersed in alpha MEM medium and incubated at 37°C for 24 hours. Non-adherent
cells were collected, layered over Ficoll and centrifuged. The interphase thus obtained was
collected, washed, and diluted with alpha MEM containing 10 ng/ml of mCSF and plated at
50,000 cell/well in 4-chamber culture plates. Three days later, 50,000 first passage corneal
fibroblasts were added to the macrophage culture. These co-cultured cells were incubated
together. Cells were stained for osteoclast marker tartrate-resistant acid phosphatase (TRAP)
using a commercially available kit (SIGMA). The effects of osteoprotegerin (soluble decoy
RANKL receptor antagonist) (100 ng/ml) and/or dexamethasone (100 nM) in these co-cultures
were also evaluated. Dexamethasone has been shown to down regulate OPG expression in
fibroblast cells. Soluble RANKL added to macrophage cultures served as positive controls.
Results: Cells staining positive for TRAP were generated after 15 days of co-culture
of corneal fibroblasts and macrophages, suggesting formation of osteoclast-like cells.
No TRAP staining was noted in corneal fibroblasts or macrophages that were cultured
separately to serve as negative controls. Addition of OPG, to the fibroblast-macrophage
co-culture significantly decreased the number of TRAP-positive cells. Supplementing
co-cultures with dexamethasone increased TRAP-positive cells several fold.
Conclusions: Corneal fibroblasts are competent to induce the formation of osteoclast-like cells
from macrophages in vitro. This interaction is likely mediated by RANKL, since formation
of the osteoclast-like cells is blocked by OPG and augmented by dexamethasone.
CR: A. Sharma, None; R.R. Mohan, None; S. Sinha, None; S.E. Wilson, None.
Support: EY10056
Sphere Initiating Cells in the Mouse Corneal Stroma Exhibit Multipotency in Addition
to the Keratocyte Phenotype in vitro
S.Yoshida1A, S.Shimmura1B, Y.Matsuzaki2A, J.Shimazaki1B, H.Okano2A, K.Tsubota2B,1B.
Cornea Center, BOphthalmology, 1Tokyo Dental College, Ichikawa, Japan; APhysiology,
Ophthalmology, 2Keio University School of Medicine, Tokyo, Japan.
Purpose: To analyze the self-renewal capacity and multipotency of sphere initiating
cells in the mouse cornea. Methods: Dissociated mouse corneal stromal cells were
cultured in serum-free DMEM/F12 medium supplemented with EGF and bFGF to
initiate spheres. Methylcellulose culture method was used to assess self-renewality of
sphere-forming cells, which were analyzed by the Hoechst 33342 dye exclusion assay
to isolate cells with side-population (SP) phenotype. Cells from P6 to P10 spheres
were subcultured onto plastic dishes or type I collagen gels for phenotype analysis.
Expression of keratocan, aldh, CD34, cytokeratin K12 (K12), Pax6, and musashi-1 was
analyzed by RT-PCR. Immunocytochemistry was done against sphere cells subcultured
in condition medium to express α-SMA (myofibroblast), βIII-tublin and MAP2,(neuron),
and O4 (glia cell). Oil-red staining was done to identify lipid producing adipocytes.
Results: Primary keratocytes formed spheres, which were cultured for over 12 passages.
A high fraction of these cells demonstrated the SP phenotype by FACS. Subculture using a
methylcellulose sustrate showed regeneration of spheres from single cells. Suspended sphere
cells expressed vimentin, keratocan, CD34 and lumican, but were negative for K12 and Pax6.
Sphere cells subcultured on plastic showed dendritic morphology characteristic of keratocytes,
and maintained keratocan, Aldh and CD34 expression in serum-free medium. Sphere cells
subcultured with serum became fibroblastic, and expressed α-SMA when stimulated by TGFβ. The neural progenitor marker musashi-1 was detected in spheres, and dissociated sphere
cells subcultured in condition medium up-regulated the neural and glial markers βIII-tublin,
MAP2, O4, and GFAP. Lipid producing adipocytes were also observed by oil-red staining.
Conclusions: Stromal spheres cells contain self-renewing keratocyte progenitors with
intact keratocyte phenotype and the ability to differentiate into neurons, glial cells and
CR: S. Yoshida, None; S. Shimmura, None; Y. Matsuzaki, None; J. Shimazaki, None; H.
Okano, None; K. Tsubota, None.
Support: None.
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2590-2610 / B143-B163
342. Corneal Molecular Biology Organizing Section: CO
2608 - B161
In vivo Identification of the Cis-Regulatory Elements of the Murine Keratocan (mKera)
H.Yi1, E.Carlson2, J.Ouyang1, V.Perez2, Y.-F.Lee1, W.-Y.Kao3, C.-Y.Liu1. 1Ophthalmology,
University Miami, Miami, FL; 2Ophthalmology, Cleveland Clinic Foundation, Cleveland,
OH; 3Ophthalmology, University of Cincinnati, Cincinnati, OH.
Purpose: To identify the elements of the mKerapr3.2-bpA promoter responsible
for cor nea-specif ic gene expression using deletion mutant analysis i n vivo.
Methods: The entire intron 1 sequence (1.5 kb) of mKera was inserted into mKerapr3.2-EGFP-bpA
to generate mKerapr3.2-int-EGFP-bpA. A series of deletion mutant mKera promoters (-2.8kb, -2.0 kb,
-1.1 kb, -0.8 kb, and -0.3 kb) or internal deletion of the intron 1 (0.5 kb) were generated by restriction
enzyme digestion and re-ligation in the mKerapr3.2-int-EGFP-bpA vector. A 0.5 kb fragment of the
intron 1 was inserted into the 3’ to the bpA of the mKerapr3.2-EGFP-bpA to generated mKerapr3.2EGFP-bpA-int. Deletion mKera promoters and control plasmids were delivered in vivo to murine
corneal stroma and conjunctiva by intrastromal and conjunctival injections. In vivo expression was
determined by measuring EGFP using a stereomicroscope at different time points. After 24 hours
eye were enucleated and analyzed further using fluorescent stereo and confocal microscopies.
Results: In vivo transfection of corneas with the mKerapr3.2-int-EGFP-bpA promoter resulted
in stronger in vivo expression. In contrast, the mKerapr3.2-EGFP-bpA construct resulted in poor
EGFP reporter gene expression in corneal stroma cells. Interestingly, the mKerapr3.2-EGFPbpA-int showed compatible EGFP expression to that of the mKerapr3.2-int-EGFP-bpA, indicating
that the 0.5 kb intron 1 fragment contains a putative enhancer. In vivo deletion mutant analysis
showed that deletion from -3.2 kb to -2.8 kb reduced promoter activity by ~60% and further
deletion to -1.1 kb resulted in the loss of promoter activity. However, deletions to -0.8 kb and -0.3
kb restored the promoter activity back to 40 % and 90 %, respectively, as compared to that of the
-3.2 kb promoter. All of the mKera promoters tested showed cornea-specific EGFP expression
Conclusions: These results show the ability of in vivo promoter analysis to identify cis-regulatory
elements in mKera between -1.1 and -0.8 kb which serve as silencers that can be overcome by enhancers
between -3.2 kb to -1.1 kb. Furthermore, we have identified mKera intron 1 as being crucial for its
promoter activity in corneal stromal cells and the -0.3 kb mKera promoter region being sufficient
to drive cornea-specific expression.
CR: H. Yi, None; E. Carlson, None; J. Ouyang, None; V. Perez, None; Y. Lee, None; W.
Kao, None; C. Liu, None.
Support: NIH RO1EY12486, EY11845, EY 13755, K08EY014912-01, RPB, and Knights
Templar Foundation
2609 - B162
Variation of Krüppel-Like Factors Levels in Mouse Corneal Model of Angiogenesis
F.Chiambaretta1A, F.De Graeve2, L.Feraille3, H.Nezzar1A, G.Marceau1B, B.Dastugue1B,
D.Rigal1A, P.Elena3, V.Sapin1B. AOphthalmology, BBiochemistry, 1Clermont Ferrand
Hospital, Clermont Ferrand, France; 2Ophthalmology, Clermont Ferrand University,
Clermont Ferrand, France; 3Ophthalmology, Iris Pharma, La gaude, France.
Purpose: The Krüppel-like factors (KLFs) nuclear transcription factors family regulate
numerous cell processes in various human tissues as digestive tract, placenta and lung.
Accumulating publications supports an important role for these factors in vascular biology.
Neovascularization remains a severely disabling condition, resulting in loss of the immunologic
privilege of the cornea and in visual impairment. Given the fact that we recently demonstrated
that the KLFs were expressed in human ocular surface, we sought to explore KLFs implication
in corneal angiogenesis. The first step was to evaluate the mRNA levels of key genes in
angiogenesis in a sutured-induced mouse corneal model of neovascularization. Then we
determine mRNA levels of KLFs (fifteen members) in angiogenesis versus normal mouse
cornea. Materials and Methods: Eight mice used for suture-induced CNV experiments (Right
eye versus Left eye control) were between 8 and 12 weeks of age and were treated in accordance
with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. In
order to evaluate the mRNA levels of key genes in angiogenesis and KLFs, total RNA was
isolated from cornea (eight days after suture-induced CNV). Relative quantitative RT-PCR
was performed with key genes in angiogenesis-specific primers (VEGFA, VEGFR2, FGF2,
FGF2R, TGFb1, TGFbR, endoglin, Angiopoietin, Tie2, PDGF, PDGFR, Thrombospondin,
TNFAIP2, Akt3, uPA, MMP19, PAI-1, PECAM), KLFs-specific primers and primer sets for
GAPDH RNA used as an internal standard. In order to check the localization of PECAM
proteins, immunohistochemistry experiments were realized on normal and angiogenesis
cornea. Results: The mRNA levels of key genes demonstrate their classical variation in this
late stage of angiogenesis. The immunohistochemistry confirm the corneal neovasularization
process. We find significant increase of KLF2, KLF7, KLF9, KLF10 and significant decrease
of KLF4, KLF5, and KLF14. Conclusions: In conclusion, the present study demonstrates for
the first time, the potential implications of some KLFs in corneal neovascularization, using
an established mouse corneal angiogenesis model.
CR: F. Chiambaretta, None; F. De Graeve, None; L. Feraille, None; H. Nezzar, None; G.
Marceau, None; B. Dastugue, None; D. Rigal, None; P. Elena, None; V. Sapin, None.
Support: None.
2610 - B163
Molecular Analysis of the Zebra Fish Lumican (zLum) and Keratocan (zKera) Genes
L.K. Yeh1,2, J.Ouyang3, C.-Y.Liu3, W.-Y.Kao4, I.-J.Wang5. 1Ophthalmology, Chang Gung
Mem Hosp, Taoyun Hsien, Taiwan Republic of China; 2Ophthalmology, Bascom Palmer
Eye Institute/university of miami, Miami, FL; 3Ophthalmology, Bascom Palmer Eye
Institute/University of Miami, Miami, FL; 4Ophthalmology, University of Cincinnati,
Cincinnati, OH; 5Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
Republic of China.
P u r p o s e : To e s t a b l i s h a n e w ve r t e b r a t e m o d e l s y s t e m f o r
st udy i ng molecu la r genet ics of t he cor neal d isea ses i n zebr af ish.
Methods:In the present study, we isolated and identified the zebrafish corneal keratan sulfate
proteoglycan genes, zLum and zKera. Human lumican and keratocan sequences were used
to blast search zebrafish homologues. Oligo-primers specific to the zLum and zKera were
synthesized for polymerase chain reaction (PCR). The zLum and zKera full-length genomic DNA
and cDNA were isolated by PCR of genomic DNA and RT-PCR of total eye RNA, respectively.
The structures of the zLum and zKera gene were determined by Southern blot hybridization,
sequencing, and DNA analysis tools provided by DNASIS MAX™. Northern blotting and in
situ hybridizations were carried out to determine the expression patterns of zLum and zKera.
Results:Genomic southern blotting analysis showed that zebrafish genome contains single
copy of zLum and zKera genes. In zebrafish genome, the zLum and zKera genes are 11 kb
apart where zLum is located 5’upstream to the zKera. The zLum and zKera span 3.0 kb and
2.2 kb of the zebrafish genome, respectively. Both genes consist of 3 exons and two introns,
and do not have a conventional TATA box of transcription initiation in the 5’-flanking regions.
zLum and zKera mRNA encode 344 and 341 amino acids as verified by cDNA sequence.
The zebrafish lumican and keratocan are 50% and 56% identical in amino acid sequence to
their counter parts of human proteins. Northern hybridization revealed that zLum mRNA
was detected in many tissues including eye, eyeless head, and headless body, while zKera
mRNA was only found in the eye. In situ hybridization confirmed that zKera mRNA was
restricted to corneal stroma, while zLum mRNA was present in corneal and scleral stroma.
Conclusions: These results demonstrate that lumican and keratocan gene structure and
expression patterns are highly conserved in zebrafish and human. Thus, zebrafish may serve
as a model to studying corneal biology and diseases.
CR: L.K. Yeh, None; J. Ouyang, None; C. Liu, None; W. Kao, None; I. Wang, None.
Support: NIH RO1EY12486,EY11845,EY13755,RPB
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2611-2650 / B164-B203
343. Corneal Inflammation and Keratitis Organizing Section: CO Contributing Section: CL
2611 - B164
Best Classification of Acanthamoeba Keratitis
H.Shiota, M.Inoue, Y.Kubo, Y.Hayashi, H.Eguchi, T.Naitou. Ophthalmology/Vis Neurosci,
Univ Tokushima, Tokushima, Japan.
Purpose:Acanthamoeba keratitis is one of the most difficult ocular infections to treat. It is
often misdiagnosed as herpetic keratitis or keratomycosis. Its clinical stages have not been
properly classified yet. Therefore, the classification of Acanthamoeba keratitis was tried.
Methods:Eight eyes of Acanthamoeba keratitis were carefully observed by
slit-lamp along with the treatment. The classification of the disease was done
by the clinical progression of our cases with due consideraton of literature.
Results:Our 8 eyes showed common features in their clinical progression, starting
from punctate keratitis or corneal erosion, forming ring ulcer or infiltrate and finally
resulting in corneal opacity. One eye showed nodular scleritis with corneal ulcer.
Conclusions:We propose the classification of Acanthamoeba keratitis into 5 stages: early(stage
1), growing(stage 2), established (stage 3), regression(stage 4a), perforated(stage 4b) and
cicatrical(stage 5). Also it should be born in mind that the disease can start healing at any
stage with proper medication and in rare occasions nodular scleriris is complicated.
CR: H. Shiota, None; M. Inoue, None; Y. Kubo, None; Y. Hayashi, None; H. Eguchi,
None; T. Naitou, None.
Support: None.
2613 - B166
2612 - B165
Heidelberg Retina Tomograph II Findings of Acanthamoeba Keratitis
N.Fayol, T.Bourcier, B.Dupas, V.Borderie, C.Chaumeil, P.Larricart, C.Baudouin,
L.Laroche. Ophthalmology 5, Quinze Vingts Natl Ctr Ophth, Paris, France.
Purpose: Acanthamoeba Keratitis (AK) is a painful, potentially blinding condition that, like any
infectious keratitis, benefits from early detection and treatment. Heidelberg Retina Tomograph II
(HRTII) (Heidelberg engineering, Dossenheim, Germany) examination was performed with cornea
module in one patient with AK to provide images detailing characteristic findings of the disease.
Methods: A 34-year-old woman presented with clinical signs and symptoms
of AK. HRTII with cornea module was performed. Pictures were taken from
all corneal structures of the central and peripheral cornea and compared to
those of healthy subjects. The patient underwent laboratory investigations.
Results: HRTII examination with cornea module revealed numerous 20 to 26 μm diameter
high-contrast round particles within the corneal epithelium and anterior stroma, resembling
acanthamoeba cysts. Morphologic details of these cyst-like particles suggested the presence
of a double wall. Stellate cells as well as ovoid irregular objects, possibly inflammatory
cells, trophozoites, altered cysts, or activated keratocytes, were also present in the area
of stromal infiltrates. An irregularly thickened stromal nerve was seen, consistent with
radial keratoneuritis. Confirmation of AK was obtained by cytological examination
of corneal smears which revealed the presence of numerous acanthamoeba cysts.
Conclusions: HRTII cornea module provides non-invasive, high-contrast, in vivo images of
the cornea. It can be helpful in the diagnosis of AK by identifying acanthamoeba cyst-like
structures in the cornea. This technique has also potential uses in monitoring the efficiency
of anti-infective treatment.
CR: N. Fayol, None; T. Bourcier, None; B. Dupas, None; V. Borderie, None; C. Chaumeil,
None; P. Larricart, None; C. Baudouin, None; L. Laroche, None.
Support: None.
2614 - B167
Confocal Microscopy in Fungal and Acanthamoebal Keratitis: Sensitivity and Utility
in Guiding Treatment
E.T. Aliprandis, D.Miller, E.Alfonso. Ophthalmology, Bascom Palmer Eye Institute,
Miami, FL.
Purpose: To evaluate the sensitivity of confocal microscopy in diagnosing
fungal and acanthamoebal keratitis at the Bascom Palmer Eye Institute.
Methods: In vivo confocal microscopy recordings of patients with positive
fungal or acanthamoebal corneal cultures from January 2002 to November 2004
were reviewed for the presence of hyphal elements or acanthamoebal cysts.
Sensitivity was determined by comparison of confocal findings to culture results.
Results: Fungal hyphal elements were present in 50% (2 out of 4) patients with culture
proven filamentous fungal keratitis who had confocal microscopy done. Acanthamoeba
cysts were clearly identified on confocal microscopy in 50% (5 out of 10) of patients
with culture proven acanthamoeba keratitis who had confocal microscopy done.
Two of these patients had serial confocal examinations during treatment, and the
number of cysts declined with each examination during the treatment course.
Conclusions: Confocal microscopy may be helpful in the early diagnosis of fungal and
acanthamoebal keratitis as well as in guiding length of treatment. Limitations to the technique
include improper alignment with the specific area of corneal pathology, dense corneal
opacification, and decreased sensitivity in early infections with lower microbial density.
CR: E.T. Aliprandis, None; D. Miller, None; E. Alfonso, None.
Support: None.
Acanthamoeba Keratitis and Confocal Microscopy: New Horizons
D.D. Freitas1, E.M. Nakano1, K.Nakano2, M.Oliveira2, W.Portellinha2. 1Ophthalmology,
Fed Univ Sao Paulo, Sao Paulo, Brazil; 2Ophthalmology, Excimer Laser Santa Cruz, Sao
Paulo, Brazil.
Purpose: Based on confocal microscopic images obtained with Nidek ConfoScan
2.0 System in corneas with clinical suspect of AK that had the diagnosis confirmed by
either cytological and/or histological analysis, this study intends to establish an in vivo
pattern of presentation and behavior of Acanthamoeba, once such pattern is lost in any
diagnosis procedure which involves corneal scrapping. More over, authors venture
on a encystment and excystment process proposal based on in vivo observations.
Methods: Sixteen eyes with clinical diagnosis of Acanthamoeba keratitis ( AK) underwent
confocal microscopy evaluation. All procedures were performed by one single observer and images
were analysed by several experts in the field. Statistical analyses were applied in the Results:
Hyper -ref lective round, double-walled structures, measuring from 10 to 30µm
were observed in all cases of confirmed AK. In one case a rare amoeboid body
was seen and believed to be an Acanthamoeba trophozoite. Images suggesting
different stages of encystment and excystment of the trophozoite were seen.
Conclusions: Acanthamoeba keratitis findings in confocal microscopy are not well established
in literature, therefore this study may contribute for the enhancement of new features on this
serious ocular infection.
CR: D.D. Freitas, None; E.M. Nakano, None; K. Nakano, None; M. Oliveira, None; W.
Portellinha, None.
Support: None.
2615 - B168
2616 - B169
Dacryoadenitis Associated With Acanthamoeba Keratitis
M.Tomita1, S.Shimmura1, T.Sumi1, K.Tsubota2, J.Shimazaki1. 1Ophthalmology, Tokyo
Dental College, Ichikawa, Japan; 2Ophthalmology, Keio University, Tokyo, Japan.
Purpose: Acanthamoeba keratitis is known to cause various clinical findings
including severe anterior and posterior scleritis, anterior uveitis, cataract, glaucoma,
lid edema, and reactive pseudoptosis. The purpose of this study is to report a
series of patients with dacryoadenitis associated with acanthamoeba keratitis.
Methods: We investigated all cases of acanthamoeba keratitis (18 cases, 19 eyes) diagnosed
and treated at Tokyo Dental College Ichikawa General Hospital, Japan, between May 1994 and
November 2004. We recorded the incidence and clinical findings of dacryoadenitis diagnosed
by computed tomography (CT), magnetic resonance imaging (MRI) and histopathology.
Results: Six eyes of 6 cases (32%) presented with dacryoadenitis simultaneously with
acanthamoeba keratitis. Dacryoadenitis was diagnosed by histopathological findings and CT
in 1 case, MRI in 3 cases, and clinical signs of lacrimal gland swelling in another 2 cases.
Histopathological examination of 1 case revealed moderate infiltration of lymphocytes and
plasma cells in the lacrimal gland compatible with dacryoadenitis. No acanthamoeba organisms
were found in the lacrimal gland. Standard protocol for acanthamoeba keratitis was done without
particular treatment for dacryoadenitis in all cases. Lacrimal gland swelling improved in
conjunction with symptoms of keratitis, however, 1 case required blepharoplasty for residual ptosis.
Conclusions: Dacryoadenitis is a clinical finding associated with acanthamoeba keratitis.
CR: M. Tomita, None; S. Shimmura, None; T. Sumi, None; K. Tsubota, None; J.
Shimazaki, None.
Support: None.
Acanthamoeba Keratitis With Herpes Simplex Keratitis Is a Separate Clinical Entity
T.John1A,2, C.Bouchard1A, J.I. Perlman3, C.Thomas1B, R.Bala4. AOphthalmology, BPathology,
Loyola University at Chicago, Maywood, IL; 2Chicago Cornea Research Center, Tinley
Park, IL; 3Ophthalmology, Edward Hines, Jr., Veterans Administration Hospital, Hines,
IL; 4Pathology, Little Company of Mary Hospital, Evergreen Park, IL.
Purpose. To describe the combination of Acanthamoeba keratitis (AK) and herpes simplex
keratitis (HSK) as a separate clinical entity. All cases were diagnosed after a therapeutic
penetrating keratoplasty (TPK). Methods: Case 1: A 28-year-old woman with HSK and anterior
uveitis developed a corneal ring infiltrate. Corneal culture for acanthamoeba was negative. The
prolonged clinical course and failed medical therapy necessitated TPK. Case 2: A 68-year-old
man who underwent PK for HSK and acromonium fungal keratitis developed a ring infiltrate
in the previously clear graft. Corneal biopsy was performed for acanthamoeba. Case 3: A 16year-old boy with HSK and stromal keratitis unresponsive to medical treatment underwent a
TPK. Confocal microscopy for AK was negative. Results: Clinical presentation is significant
for absence of pain, mild conjunctival injection, negative culture and confocal microscopy, and
prolonged clinical course. All cases were diagnosed after TPK, with histopathology revealing
numerous stromal acanthamoeba cysts. Case 1: 5 years postoperatively there has been no
recurrence of AK and the corrected vision is 20/40. Case 2: 6 years postoperatively there has
been no recurrence of AK. Case 3: 2 months postoperatively there has been no recurrence
of AK. Conclusions: The combination of AK and HSK is a separate clinical entity with
atypical presentation and prolonged clinical course that necessitated surgical intervention.
Supported by the Richard A. Perritt Charitable Foundation
CR: T. John, None; C. Bouchard, None; J.I. Perlman, None; C. Thomas, None; R. Bala,
Support: Richard A. Perritt Charitable Foundation
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2611-2650 / B164-B203
343. Corneal Inflammation and Keratitis Organizing Section: CO Contributing Section: CL
2617 - B170
Prevention of Ocular Herpes Using a Replication Defective Herpes Simplex Virus
S.S. Tuli1A, D.C. Bloom1B, A.S. Lewin1B, J.Liu1B, G.S. Schultz1C. AOphthalmology, BMolecular
Genetics and Microbiology, CObstetrics and Gynecology, 1University Florida, Gainesville,
Herpes simplex keratitis is the most common infectious cause of corneal blindness
in developed nations. A large percentage of the population is infected with herpes
simplex and, once infected, the virus remains latent in the nerve ganglia lifelong. It
undergoes periodic reactivation resulting in corneal infection and scarring. Prevention
of the initial infection by vaccination would be ideal but has been very elusive to date.
Purpose: To evaluate the ability of a replication defective herpes simplex virus to prevent wild
type herpes infection of the cornea and to determine the possible mechanisms of protection.
Methods: A replication defective herpes simplex type-1 (HSV-1) virus was injected by the
intrastromal route into one cornea of New Zealand white rabbits. Four months later, 2 x 105
pfu of a wild type HSV-1 containing a lacZ reporter was topically applied to both corneas of
the rabbits after scarification and blood was drawn for serology. Four days later, conjunctival
swabs were taken and the corneas were harvested and stained for β-galactoside activity.
Results: The corneas previously injected with the vector showed a
dramatic decrease in the number and extent of epithelial dendrites. In
some cases, there was a complete absence of dendrites (bottom image).
The contra lateral corneas, on the other hand, had extensive dendrites
(top image). The dendrites were quantified using Sigma Scan Pro
software. The difference in area infected was statistically significant
by the paired t-test (P = 0.01). The serum antibody titre in these rabbits
was very low (1:5 - 1:10 versus 1:100 for rabbits infected with the
wild type virus) indicating a predominantly local immune response.
Conclusions: A replication defective herpes simplex virus confers local
immunity to the cornea and has the potential to ‘vaccinate’ the cornea
against herpes simplex infections. It is also a useful tool to study the
pathophysiology of herpes infection and latency.
CR: S.S. Tuli, None; D.C. Bloom, None; A.S. Lewin, None; J. Liu,
None; G.S. Schultz, None.
Support: None.
2619 - B172
2618 - B171
Herpetic Stromal Keratitis: Virus Entry Into Corneal Fibroblasts Is Mediated by 3O-Sulfated Heparan Sulfate
D.Shukla1A, C.Clement1A, V.Tiwari1A, P.Scanlan1A, B.Y. J. T. Yue1A, T.Valyi-Nagy1B.
Ophthalmology/Visual Sciences, BPathology, 1Univ Illinois-Chicago, Chicago, IL.
Purpose: To identify the receptor used for Herpes Simplex Virus-1 entry
into cor neal f ibroblasts cultured from the stroma of the human cor nea.
Methods: First, to verify the natural susceptibility of cultured corneal fibroblasts (CF) in vitro,
the cells were exposed to β-galactosidase-expressing and enhanced green fluorescent proteinexpressing HSV-1 virions. Deconvolution and confocal microscopy were used to monitor entry
into live cells. Immunohistochemistry and reverse transcriptase-PCR were used to examine entry
receptors in CF. Disaccharide analysis of surface heparan sulfate isolated from CF was performed
to verify the expression of 3-O-sulfated heparan sulfate. Spinoculation, antibody blocking
and viral glycoprotein D (gD) binding assays were performed to verify receptor usage by CF.
Results: A three dimensional model of HSV-1 entry into CF was generated by use of
deconvolution and confocal microscopy. Expression of HSV-1 gD rendered resistance to HSV-1
entry suggesting an important role for gD receptors. Expression of HVEM and 3-O sulfated
heparan sulfate (3-OS HS), but not nectin-1, was detected by reverse transcriptase-PCR.
Absence of nectin-1 was also evident from immunohistochemistry and resistance of cultured
CF to bovineherpesvirus-1, which uses nectin-1 for entry. Demonstrating the significance of
HVEM in entry, anti-HVEM antibody partially blocked HSV-1 entry. This effect was more
pronounced when combined with heparinase treatment; suggesting an important role for HS.
Finally, using a spinoculation technique, it was found that heparinase treatment of cultured CF
blocked entry at fusion step, implicating 3-OS HS as a mediator of membrane fusion. Presence
of 3-OS HS on CF was also verified by disaccharide analysis with its gD binding ability
established by an immunoprecipitation assay. Involvement of an alternate receptor other than
HVEM or nectin-1 was also suggested by blocking of HSV-1 entry by an anti-gD monoclonal
antibody DL6, which blocks entry mediated by 3-OS HS but not via nectin-1 or HVEM.
Conclusions: We provide molecular and biochemical evidence that HSV-1 entry into primary
cultures of human corneal fibroblasts is mediated by HVEM and 3-OS HS but not nectin-1.
CR: D. Shukla, None; C. Clement, None; V. Tiwari, None; P. Scanlan, None; B.Y.J.T.
Yue, None; T. Valyi-Nagy, None.
Support: RPB Career Award
2620 - B173
Archipelago Keratitis: A Herpes Mediated Epithelial Cell Activation Syndrome?
T.Hoang-Xuan1, N.Alfonsi1, L.Racine1, G.Sultan2, C.Baudouin2, E.E. Gabison1.
Ophthalmology, Fondation Rothschild, Paris, France; 2Ophthalmology, CHNO des XV/
XX, Paris, France.
Purpose: To report 5 cases of Archipelago keratitis, a distinct entity of herpetic epithelial
keratitis and to propose a hypothesis regarding the physiopathogenesis of this syndrome.
Methods: Case report with clinical examination, corneal scraping for Herpes PCR and in
vivo HRT2 confocal microscopy.Results: We describe a form of herpetic keratitis consisting
of a limbal infiltrate associated with epithelial erosions which have a linear centripetal
disposition. All the patients had a past history of herpetic keratitis. The evolution over 3
weeks was marked by the appearance of nummular subepithelial opacities. Corneal scraping
performed in 3 cases revealed positive PCR for herpes simplex in two cases (HSV1, HSV2).
Confocal in vivo microscopy (HRT2 with corneal unit ) revealed the presence of ballooned
epithelial cells and subepithelial infiltrates of inflammatory cells. All the patients healed
with oral valacyclovir treatment.Conclusions:We believe that during the evolution of a
limbal recurrence of herpetic disease, inflammatory mediators may in some cases promote
the migration of limbal epithelial cells towards the center of the cornea, hence facilitating
the dissemination of the viral infection.
CR: T. Hoang-Xuan, None; N. Alfonsi, None; L. Racine, None; G. Sultan, None; C.
Baudouin, None; E.E. Gabison, None.
Support: None.
Latanoprost Associated Noninfectious Dendritiform Keratitis
K.K. Chang1, J.C. Affeldt2. 1Department of Ophthalmology, Loma Linda University
School of Medicine, Loma Linda, CA; 2Ocular Surface Center, Doheny Eye Institute, Los
Angeles, CA.
Purpose: Latanoprost has been reported to promote recurrences of herpetic
keratitis. We wish to document for the first time the clinical characteristics
of a non-her petic, dend r itifor m keratitis related to Lat anoprost use.
Methods: Case Report.
Results: A middle aged woman with open-angle glaucoma with no previous history of ocular
herpes simplex developed a bilateral branching epitheliopathy after starting Latanoprost
therapy. The lesion was manifested by an opaque whitish elevated epithelial cell congregation,
which formed rough branching dendritic figures without terminal bulbs. The lesion was
limited to the inferocentral cornea, with the long axis oriented at 180 degrees. The lesion
stained with fluorescein, and was broadly surrounded by a dense field of punctate keratopathy.
Additionally, average central corneal sensation (Cochet-Bonnet esthesiometer) was intact,
reflecting a non-neurotrophic environment. Following a negative viral culture, treatment was
unsuccessfully attempted with oral acyclovir and aggressive lubrication in the form of punctal
occlusion. Discontinuation of Latanoprost resulted in prompt resolution of the epitheliopathy.
Conclusions: Not all keratopathies associated with Latanoprost use are of herpetic origin.
Latanoprost associated noninfectious dendritiform keratitis appears to represent a variant of
vortex keratopathy (cornea verticillata), which can be easily confused with dendritic herpes
simplex or zoster keratitis. It can be distinguished clinically from its infectious counterparts
however by its distinctive presentation including lesion color, location, orientation, and presence
of surrounding punctate keratopathy field; as well as its rapid response to discontinuation
of Latanoprost. This observation may help clarify the current controversy concerning the
relationship of Latanoprost to apparent infectious herpetic keratitis.
CR: K.K. Chang, None; J.C. Affeldt, None.
Support: None.
2621 - B174
2622 - B175
Expression of SLPI in Ocular Host Defense Against S. Aureus Endophthalmitis and
Herpes Simplex Virus Keratitis
V.E. Reviglio1, A.Olmedo1, M.Falco1, J.D. Luna1, R.Sambuelli1, A.Berra2, C.P. Juarez1.
Cornea & External Eye Diseases, Fundacion VER & Catholic University, Cordoba,
Argentina; 2Cornea, Pathology Dep. Buenos Aires University, Buenos Aires, Argentina.
Purpose: Secretory leukocyte protease inhibitor (SLPI) is an antimicrobial
protein. Staphylococcus and herpes simplex virus are leading causes of potentially
blinding microbial disease. In this study we investigated whether Staphylococcus
aureus or HSV infection could induce expression of SLPI in ocular tissues.
Methods: An HSV keratitis mouse model (right eye) was developed in forthy
BALB/c mice using KOS strains. Endophthalmitis was induced in forthy Lewis
rats by intravitreal injection (right eye) of colony-forming units of viable S. aureus.
Twenty-four hours post-infection, the animals were sacrificed. Corneal & retinal
tissues were excised and processed for histopathology, immunohistochemical
staining and Western blot assay studies to determine the site of SLPI production.
Results: Our initial studies established that SLPI is expressed abundantly in the cornea after HSV1 infection. Also, SLPI was detected in endophthalmitis vitreous samples at increased levels. The
SLPI expression in retinal tissue was primarily associated with leukocytic inflammatory infiltration.
Conclusions: Eyes infected with S. aureus or HSV demonstrated an intense expression
of SLPI compared with normal control eyes. These results suggest that SLPI may have an
antimicrobial function, promoting up-regulation of local eye innate immunity.
CR: V.E. Reviglio, None; A. Olmedo, None; M. Falco, None; J.D. Luna, None; R.
Sambuelli, None; A. Berra, None; C.P. Juarez, None.
Support: None.
Sensitivity of Filamentous Fungi Isolated From Fungal Keratitis to Amphotericin B, Natamycin,
Caspofungin, Itraconazole, Voriconazole, and Posaconazole
B.L. Shapiro1, P.Lalitha2, A.W. Fothergill3, J.Ruiz3, M.Srinivasan2, N.V. Prajna2, J.Chidambaram1,
Y.Pan1, S.McLeod1, T.M. Lietman1. 1Proctor Foundation, UCSF, San Francisco, CA; 2Aravind Eye
Care System, Madurai, India; 3Department of Pathology, University of Texas Health Sciences Center
at San Antonio, San Antonio, TX.
Purpose: Fungal corneal ulcers are notoriously difficult to manage, and physicians currently choose
antifungal agents empirically. A range of new antifungal agents has become available that has not been widely
applied in ocular disease. The non-ocular infectious disease literature has suggested that susceptibility
testing of fungal isolates can provide clinically relevant information. This study aims to characterize the
susceptibility of six antifungal agents against filamentous fungi cultured from scrapings of fungal keratitis.
Methods: Corneal isolates from 98 consecutive cases of culture-proven fungal keratitis presenting
to the Aravind Eye Hospital were collected. Fungi were identified and tested for susceptibility
to the following antifungal agents: Amphotericin B, Natamycin, Caspofungin, Itraconazole,
Voriconazole, and Posaconazole. The fungal isolates were analyzed for susceptibility to antifungal
agents by conducting Minimal Inhibitory Concentration (MIC) testing performed using the
macrobroth dilution technique according to NCCLS guidelines set forth in NCCLS M38-A. MIC50
and MIC90 values for Fusarium spp., Aspergillus spp., and all species together were estimated.
Results: We identified the fungal genus in 95 of the 98 specimens; the other 3 were unidentifiable
or contaminated. Thirty-nine isolates were identified as Fusarium spp. and 43 as Aspergillus spp.
Table 1: Minimal inhibitory concentration of antifungal agents to filamentous fungi
Fusarium spp.
All Corneal
Natamycin Caspofungin Itraconazole Voriconazole Posaconazole MIC50 MIC90 MIC50 MIC90 MIC50 MIC90
Results are broken down by species.
Conclusions: No single agent was uniformly most effective for all filamentous species tested. In vitro,
Posaconazole had the lowest MICs against Aspergillus spp., while Voriconazole gave the lowest MICs
against Fusarium spp.. Future studies may determine how in vitro susceptibilities correlate with in
vivo clinical results.
CR: B.L. Shapiro, None; P. Lalitha, None; A.W. Fothergill, None; J. Ruiz, None; M. Srinivasan,
None; N.V. Prajna, None; J. Chidambaram, None; Y. Pan, None; S. McLeod, None; T.M. Lietman,
Support: That Man May See (TMMS), Alta California Foundation
Copyright 2005 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
Commercial Relationships are noted at the end of each abstract by “None” or with codes.
Tuesday, May 3, 3:00 PM - 4:45 PM Hall B/C Poster Session Program Number/Board # Range: 2611-2650 / B164-B203
343. Corneal Inflammation and Keratitis Organizing Section: CO Contributing Section: CL
2623 - B176
2624 - B177
Comparative Efficacy of Topical Voriconazole With Amphotericin B and Fluconazole in
the Treatment of Candida Albicans Keratitis in an Animal Model
P.J. Botelho1, C.Zhang1, I.C. Kuo1, J.D. Dick2, T.P. O’Brien1. 1Cornea, Refractive, Ext
Disease, Wilmer Eye Institute, Baltimore, MD; 2The Johns Hopkins University, Dept of
Microbiology, Baltimore, MD.
Purpose: To determine the efficacy of topical Voriconazole with Amphotericin B and
Fluconazole in the treatment of experimental Candida Albicans keratitis in an animal model.
Methods: The concentrations of the antifungal agents used in this study were based on
published recommended levels recommended for parenteral use. Fungal keratitis models were
created by injecting into the central cornea of the right eye a 100 ul suspension of Candida
Albicans containing 1 X 10 ^6 organisms (colony forming units). Forty four rabbits were
randomly divided into four groups and each group was treated hourly for 24 hours with
topical voriconazole (10mg/mL), amphotericin B (3mg/mL), fluconazole (2mg/mL), and
balanced salt solution (control). Prior to the administration of the last dose, the presence of
conjunctival hyperemia and discharge was noted. One hour after administrating the last dose,
the animals were sacrificed and a uniform corneal button excised with an 8.5 mm sterile
trephine. The corneal buttons were then homogenized, serially diluted and plated on trypticase
soy agar. Fungal colony counts were performed after 72 hours incubation at 37 degrees C.
Results: Using one-way nonparametric analysis of variance(ANOVA) statistical analysis, fungal
colony counts for the initial homogenized samples revealed that only the voriconazole group had
a statistically significant reduction in CFU compared to the control group (P 0.02). Comparison
of serially dilution 1x10-1 through 1x10-3 demonstrated that the voriconazole, amphotericin
B and fluconazole groups all yielded a statistically significant reduction in candid albicans
CFU when compared to the control group. Although the average CFU counts were lower for
voriconazole than for amphotericin B and fluconazole for each level of dilution, the differences
were not statistically significant. The amphotericin B group demonstrated a significant increase
in conjunctival hyperemia and mucus discharge compared to the other three groups.Conclusions:
Topical voriconazole was found to be as efficacious in the treatment of candida albicans keratitis
as the gold standard, amphotericin B with potentially less ocular toxicity. Results suggest that
voriconazole is a potential treatment agent against candida albicans keratitis.
CR: P.J. Botelho, None; C. Zhang, None; I.C. Kuo, None; J.D. Dick, None; T.P. O’Brien,
Support: None.
Can MRSA (Methicillin Resistant Staphylococcus aureus) Keratitis Be Successfully
E.M. Happ, R.P. Kowalski, F.S. Mah. Ophthalmology, University of Pittsburgh,
Pittsburgh, PA.
Purpose: The purpose of this study is to compare the clinical outcomes of infectious keratitis
by methicillin resistant Staphylococcus aureus (MRSA) to methicillin sensitive Staphlococcus
aureas (MSSA) and to report the susceptibility of these pathogens to potential therapeutic options.
Methods: A retrospective analysis of the seven year period starting August 1996 and ending August
2003 of all keratitis patients culture positive for Staphylococcus aureus (20 cases of MRSA; 14
cases of MSSA) from our clinic was completed. Age and gender were compared. Resolution of
infectious keratitis was calculated by time to re-epithelization, and time to infiltrate resolution.
Susceptibilities of the Staphylococcus aureus isolates to various antibiotics was completed.
Results: All 34 keratitis cases resolved without adverse events. There were no statistical
differences in age or gender. The time range for re-epithelization for MRSA infections was
1-88 days, and 3-55 days for MSSA keratitis. The median/mean time for infiltrate resolution
for MRSA was 19/39 days and 19/27 days for MSSA. There were no statistical differences
between MRSA and MSSA (p>0.05). Comparing MICs for MRSA isolates, moxifloxacin had
statistically significant lower MICs than the other currently available topical fluoroquinolones
(p=0.00001). Among all antimicrobial agents, vancomycin, bacitracin and cefazolin had
the best coverage of Staph aureus keratitis isolates in terms of percent susceptibility.
Conclusions: Methicillin-resistant Staphylococcus aureus (MRSA) causing acute bacterial
keratitis can be successfully eradicated. Clinical response along with in vitro antibiotic
susceptibility testing should be used as guides in the management of infectious keratitis
until the ophthalmic community can determine better predictors for the successful treatment
of bacterial keratits.
CR: E.M. Happ, None; R.P. Kowalski, Alcon Labs, Inc. F, R; Allergan, Inc. F; Novactyl
Pharma, Inc. F; F.S. Mah, Alcon Labs, Inc. F, R; Allergan, Inc. F; Novactyl Pharma, Inc F.
Support: None.
2625 - B178
2626 - B179
What Is the Best Antibiotic for the Treatment of Group B Streptococcus Keratitis?
J.M. Kurilec1A, G.W. Zaidman1A, J.N. Kruh1B, M.E. Aguero-Rosenfeld1C. AOphthalmology,
New York Medical College, Valhalla, NY; CPathology, 1New York Medical College,
Valhalla, NY.
Purpose: To compare the eff icacy of several commonly used topical
opht h a l m ic a nt ibiot ic pr e pa r at ion s i n t he t r e at me nt of G roup B
Streptococcus (GBS) in a New Zealand white (NZW) rabbit keratits model.
Methods: The choice of topical antibiotics investigated was based on in-vitro testing which
demonstrated sensitivity of GBS as follows - fortified cephazolin 50mg/ml > moxifloxacin
0.5% > gatifloxacin 0.3% > ciprofloxacin 0.3% > fortified vancomycin 50mg/ml and resistance
to sulfacetamide 10% and balanced salt solution (BSS). Fourteen rabbit corneas received
intrastromal injections with 1x103 colony-forming units of GBS in 20μl tryptic soy broth.
Following an incubation period of 7 hours each rabbit was treated, around the clock, for one week
with one of the following topical ophthalmic preparations - moxifloxacin 0.5%, gatifloxacin
0.3%, fortified vancomycin 50mg/ml, fortified cephazolin 50mg/ml, and BSS as a control. The
eyes were treated every hour for the first 24 hours, then every 2 hours for the next 4 days, and
finally every 6 hours for the remainder of the study. Daily examinations were performed. On
day 7 all rabbits were examined under an operating microscope, euthanized and enucleated.
One representative eye from each treatment group underwent histologic examination.
Results: By day 7 the rabbits treated with fortified cefazolin and moxifloxacin clinically
demonstrated near complete resolution of the infection. The rabbits treated with
gatifloxacin were improving but still had visible infiltrates. Corneal ulcers and a severe
keratitis developed in the rabbits treated with fortified vancomycin. The control rabbits
developed hypopyons and severe corneal neovascularization. Overall efficacy appeared
to be fortified cephazolin = moxifloxacin > gatifloxacin > fortified vancomycin > BSS.
Conclusions: We have developed a working NZW rabbit model for GBS keratitis. Of the
antibiotics tested, fortified cefazolin and moxifloxacin (instead of fortified vancomycin) were
the most effective for topical treatment of GBS keratitis.
CR: J.M. Kurilec, None; G.W. Zaidman, None; J.N. Kruh, None; M.E. Aguero-Rosenfeld,
Support: None.
2627 - B180
Response of Microbial Keratitis to Fourth Generation Fluoroquinolones
A.F. Koreishi1A, E.Aliprandis1A, D.Miller1B, E.Alfonso1A, S.Yoo1A. ACornea, BMicrobiology,
Bascom Palmer Eye Inst, Miami, FL.
Purpose: To assess the outcomes of culture-positive microbial keratitis treated with the
commercially available fourth generation fluoroquinolones, Moxifloxacin and Gatifloxacin.
Methods: Positive culture results for patients treated with fourth generation fluoroquinolones
were analyzed for pathogens and susceptibility profiles. Charts corresponding to these culture
results were reviewed to assess clinical outcomes and specifically in-vivo antibiotic response.
Results: Of the culture-positive isolates, 47% were non-bacterial (fungi, acanthamoeba,
nocardia), while 53% were gram positive and negative bacteria. General bacterial susceptibility
was 94% for moxifloxacin and 92% for gatifloxacin. In patients pre-treated with fourth
generation fluoroquinolones, S. aureus and S. epidermidis were the most common gram
positive pathogens and P. aeruginosa was the most frequent gram negative isolate.
Conclusions: The majority of culture-positive microbial keratitis pre-treated with the new
fourth generation fluoroquinolones was bacterial; however, a large number of isolates was nonbacterial. The microbiology isolates that showed in-vitro resistance to the fourth generation
fluoroquinolones were analyzed and cross-referenced to clinical outcomes.
CR: A.F. Koreishi, None; E. Aliprandis, None; D.