Download Swelling of the Human Cornea Revealed by High

Cornea
Swelling of the Human Cornea Revealed by High-Speed,
Ultrahigh-Resolution Optical Coherence Tomography
Natalie Hutchings,1 Trefford L. Simpson,1 Chulho Hyun,2 Alireza A. Moayed,2
Sepideh Hariri,2 Luigina Sorbara,1 and Kostadinka Bizheva2,3
PURPOSE. To evaluate the change in thickness of the anterior,
stromal, and posterior corneal laminae in response to hypoxiainduced corneal swelling, by means of ultrahigh-resolution
optical coherence tomography (UHR-OCT).
METHODS. A UHR-OCT system, operating in the 1060-nm range,
was used to acquire in vivo cross-sectional images of human
cornea with a 3.2 ! 10-!m (axial ! lateral) resolution in
corneal tissue. Corneal edema was induced by inserting a thick,
positive-powered, soft contact lens, over which the eye was
closed and patched for 3 hours. Tomograms were acquired
from eight non– contact-lens wearers. Baseline images were
obtained before inducing corneal edema, immediately after
removal of the patch and the lens, and then every 15 minutes
for "2 hours. All images were postprocessed with a segmentation algorithm to identify the laminae visible in the image.
The apical thickness of the laminae (epithelium [EPI], epithelial-Bowman’s membrane [Ep-BM] complex, stroma, and endothelial-Descemet’s membrane [En-DM] complex) were determined at each time interval.
RESULTS. There was an interaction between time after removal
of the hypoxic stimulus and deswelling of the layers (RMANOVA; P # 0.001). The epithelial and stromal thickness
reduced significantly with time (P $ 0.001; P # 0.001, respectively), whereas the Ep-BM and En-DM complexes did not (P %
0.50). All layers except the En-DM complex exhibited a biphasic pattern of recovery.
CONCLUSIONS. UHR-OCT showed regional differences in swelling due to hypoxic provocation. On removal of the hypoxic
stimulus, the rate of recovery varied between layers, and all
layers except the En-DM complex exhibited a biphasic
recovery. (Invest Ophthalmol Vis Sci. 2010;51:4579 – 4584)
DOI:10.1167/iovs.09-4676
C
orneal metabolic function is reflected in corneal thickness,
since corneal deturgescence is tightly controlled, primarily for optical reasons,1 and as the hydration state changes, so
does corneal thickness.2 This thickness (and corneal epithelial
thickness), in addition to normal (diurnal) physiological varia-
tion,3 varies with the environment,4 –7 in systemic8 and ocular
disease,9 –12 after surgery,9,13 and with contact lens wear,14 –17
among other influences.
The cornea swells in hypoxic conditions and recovers when
normoxia returns.1,18,19 Edema of the cornea is a result of the
reduced availability of oxygen (primarily sourced at the epithelial surface), and the barrier function of the epithelium has
been shown to be disrupted with 1% hypoxia20 and due to
deprivation of oxygen after 1 hour of eye closure with a
low-Dk/t lens.21The endothelial barrier is the primary gatekeeper of edema, as the extent of edema is greater when the
endothelial barrier is removed than with removal of the epithelial barrier.22 The rate of recovery of hypoxia-induced
edema is thought to represent the endothelial pump function.23–25 In humans, extended eye closure during contact lens
wear is an effective model of hypoxia and has been used often
to model the normal physiological response of the cornea8,26
as well as in altered conditions in postsurgical27–29 and lenswearing24 patients.
It has been shown that the cornea swells regionally under
these conditions, with the greatest swelling occurring in the
anterior and posterior stroma.30 These measures were made
with clinical optical coherence tomography (OCT) systems
with limited axial resolution but having been previously proposed to be particularly useful in monitoring corneal physiological changes,31 particularly thickness and scatter. We therefore examined hypoxia-related corneal swelling and deswelling
by using ultrahigh-resolution optical coherence tomography
(UHR-OCT32). UHR-OCT has considerably higher spatial resolution than OCTs that have been used to examine corneal
swelling and deswelling, and thus enabled us to segment the
cornea into detailed, identifiable laminae.
The objectives of the study were to quantify regional laminar swelling after the hypoxic provocation of eye closure and
contact lens wear and to quantify deswelling after eye opening
and lens removal, to infer metabolic differences in these layers
using an UHR-OCT.
METHODS
From the 1School of Optometry and the Departments of 2Physics
and Astronomy and 3Electrical and Computer Engineering, University
of Waterloo, Waterloo, Ontario, Canada.
Supported by the Natural Sciences and Engineering Research
Council of Canada, Ontario Research and Development Challenge
Fund, and University of Waterloo.
Submitted for publication September 22, 2009; revised January 28
and March 19, 2010; accepted March 22, 2010.
Disclosure: N. Hutchings, None; T.L. Simpson, None; C. Hyun,
None; A.A. Moayed, None; S. Hariri, None; L. Sorbara, None; K.
Bizheva, None
Corresponding author: Natalie Hutchings, School of Optometry,
University of Waterloo, 200 University Avenue, Waterloo, ON, Canada
N2L 3G1; [email protected].
The imaging probe of the state-of-the-art, high-speed UHR-OCT system32 was modified to enable in vivo acquisition of two-dimensional,
cross-sectional images of the human cornea. Briefly, the device utilizes
a low coherence light source (Superlum, Ltd., Carrigtwohill, Ireland)
with a spectrum centered at 1020 nm and a spectral bandwidth of 110
nm used in combination with a linear array CCD camera (47-kHz line
rate; InGaAs; SUI Goodrich, Princeton, NJ), a detector array with
optimal efficiency for the source used. In corneal tissue, the system
provides 3.2-!m (axial) and 10-!m (lateral) resolution and a Rayleigh
range of "300 !m. The image-acquisition rate of 47,000 A-scans/s
corresponds to 38 frames/s. The optical power of the imaging beam
incident on the cornea was limited to 1.3 mW, which is well below the
maximum permissible exposure, as defined by the ANSI standard.33 At
this imaging power, the measured sensitivity of the UHR-OCT system
Investigative Ophthalmology & Visual Science, September 2010, Vol. 51, No. 9
Copyright © Association for Research in Vision and Ophthalmology
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Hutchings et al.
IOVS, September 2010, Vol. 51, No. 9
TABLE 1. Demographic and Refractive Data of the Study Sample
TABLE 2. Mean Thickness of the Layers
ID
Age
Sex
Refractive Error
Layer
Before Hypoxia
After Hypoxia
Average Change
1
26
F
2
27
F
3
22
M
Epithelium
Ep-BM
Stroma
En-DM
Total
58.40 ( 1.03
18.33 ( 0.46
519.22 ( 7.51
18.52 ( 0.95
613.91 ( 7.24
59.14 ( 1.26
19.37 ( 1.22
568.53 ( 8.13
18.60 ( 0.84
658.40 ( 7.71
0.74 ( 0.83
1.04 ( 0.36
49.31 ( 6.97
0.08 ( 0.63
51.20 ( 6.61
4
23
F
5
41
F
6
46
F
7
53
M
8
56
F
OD emmetropic
OS emmetropic
OD emmetropic
OS emmetropic
OD '8.25 DS
OS '7.25/'0.75 ! 044
OD '3.00 DS
OS '3.00 DS
OD '2.25/'0.50 ! 135
OS '2.50/'0.50 ! 055
OD '4.75/'1.25 ! 013
OS '4.75/'0.75 ! 180
OD &0.75/'0.50 ! 180
OS &1.00/'0.75 ! 180
OD &0.25/'0.75 ! 035
OS &2.50 DS
was 102 dB. In the absence of literature describing swelling of the
Bowman’s endothelial and Descemet’s layers, study sample size was
determined on the basis of power calculations of effect size,34 to show
corneal and epithelial swelling. Images were obtained from one eye of
eight non– contact-lens wearers (age range, 22–56 years). During image
acquisition, fixation was controlled by means of an external LED
positioned in front of the nonimaged eye. Summary data of the study
participants are shown in Table 1. Exclusion criteria comprised fulltime contact lens wear or any ocular disease in which delivery of the
hypoxic stimulus was contraindicated. All study participants provided
signed informed consent before entry into the study. The research
study was approved by the Office of Research Ethics, University of
Waterloo, and adhered to the tenets of the Declaration of Helsinki.
A series of two-dimensional tomograms (1000 ! 512 pixels corresponding to "5 ! 1-mm physical distance) were acquired from approximately the same location in the cornea in each volunteer. Initially, the condensing lens of the imaging probe was positioned
approximately level with the corneal apex. The central specular reflection was avoided just barely by shifting the position of the imaging
probe inferiorly by the smallest increment at which the optimum
contrast and image quality were visualized across the layers for the
imaged cornea. The imaging probe was translated downward (by no
more than "100 !m) with a mechanical stage while maintaining an
orthogonal position to the apical portion of the cornea and, therefore,
valid thickness measurements. The position of the probe was locked
for the duration of image acquisition.
Corneal edema was induced by inserting a thick, positive-powered,
low-Dk, FDA group IV hydrogel contact lens (CT $ 0.18 mm; &6.0 D)
onto the cornea, and the eye was then taped closed with surgical tape and
FIGURE 1. A representative two-dimensional UHR-OCT scan of the
human cornea obtained in the prehypoxic condition before (A) and
after (B) segmentation in a 27-year-old female participant. Four
distinct layers were defined: epithelium (EPI), Ep-BM, stroma (STR),
and En-DM.
Data are expressed as mean micrometers ( SE. Thickness was
derived from the absolute pixel distance, corrected for resolution, with
a tissue refractive index of 1.376.
covered with a gauze pad and an adhesive patch. The eye remained
patched for approximately 3 hours (group mean, 3 hours 19 minutes; SD,
18 minutes). The patch was removed, and a tomogram was obtained with
the lens in place. The first measurement without the lens was obtained at,
on average, 17 minutes after removal. Subsequent measures were obtained approximately every 15 minutes (measurement time points denoted in the figures by M01–M11) for approximately 3 hours.
The tissue boundaries were identified with a semiautomated segmentation algorithm.35 In the first step, the region of interest was manually
defined by identifying the front and back surface of the cornea. In the
second step, the segmentation algorithm sought four layers within this
region (Fig. 1). The definition was achieved by maximizing the fitting
criteria of a fourth-order polynomial function (based, initially, on the
characteristics of the curvature of the manually defined surfaces) across
the central 200 pixels of each two-dimensional scan. The search criteria
for the maxima followed a path that was perpendicular to the surface and
moved in a posterior direction from the front surface for the epithelium/
epithelial-Bowman’s membrane (Ep-BM) complex and (Ep-BM)/stroma
interfaces and moved in an anterior direction from the back surface for the
endothelial-Descemet’s membrane (En-DM) complex/stroma interface.
Segmented images were individually reviewed, and in cases in
which the segmentation algorithm had failed, the images were discarded. The apical thickness of the epithelium, Ep-BM complex,
stroma, and posterior En-DM complex were estimated as the average
difference between each segmented layer across the region of interest.
Repeatability of the thickness measures for each layer and for the total
corneal thickness was assessed by using the intraclass correlation
FIGURE 2. The overall change in thickness for the sample as a whole
from the time of removal of the contact lens. Each measurement
increment was separated by an average of 17 minutes.
UHR-OCT Study of Swelling of the Human Cornea
IOVS, September 2010, Vol. 51, No. 9
TABLE 3. Repeated-Measures ANOVA Summary Table for the Mean of
Differences in Thickness
Layer
Time
Layer!time
SS
df
F
P
1467.526
564.894
1417.447
4
10
40
2.145
5.032893
2.105595
0.1223
0.0001
0.0006
Data are derived from repeated-measures ANOVA. Layer and measurement time were taken as factors. Bold denotes significant differences.
coefficient (ICC) and correlation coefficient of concordance (CCC)36,37
between eight images obtained at two different acquisitions. ICCs for
all layers were %0.912 for images of the baseline and for images of the
hypoxic conditions. The corresponding CCCs ranged between 0.770
and 0.940 for the baseline condition and %0.933 for the hypoxic
condition.
Repeated-measures ANOVAs were used to examine the main effects of time and position on the outcome variable percentage of
swelling. In addition, post hoc tests (Tukey HSD) were used to determine pairwise differences between swelling measures after eye opening, and one-sample t-tests were used to examine whether swelling
differed from zero. For the En-DM measurements, there were three
outliers (each from different subjects) that were not used in the
analysis or reported in the figures.
RESULTS
The mean thicknesses of the identified layers are shown in
Table 2. Thicknesses were obtained by dividing the optical
thickness of each layer by the average refractive index of
corneal tissue: 1.376.
The overall corneal swelling and deswelling are shown in
Figure 2. Time was a statistically significant predictor, and the
interaction between time and position was also significant (both
P # 0.001). The RM-ANOVA results are shown in Table 3. Post
FIGURE 3. Swelling of each identified
layer after removal of the contact lens:
biphasic recoveries were shown by
the epithelium, stroma, and total cornea.
4581
hoc comparisons generally showed that there were significant
differences in swelling at time points separated by two steps,
except for the first (different from the next consecutive step) and
the last three (no difference across steps). One-sample t-tests
showed that swelling was not significantly different from 0 after
an interval of 100 minutes (as is also illustrated by the 95%
confidence intervals).
Regional Deswelling
The interaction indicated that the effect of time needed to be
determined for each layer. Significant effects of time on swelling
were found for the epithelium (P $ 0.011) and the stroma (P #
0.001), but not for the Ep-BM complex and the En-DM complex
(P $ 0.512, P $ 0.658, respectively).
The decline in thickness after removal of the lens showed a
two-phase recovery; an initial rapid phase of thickness change
followed by a phase with stabilized thickness.
Epithelial thickness had increased from baseline at the first
measure after lens removal and then decreased beyond baseline
values in recovery. The stroma and total corneal thickness increased from baseline and then decreased to baseline in recovery.
Figure 3 shows the group mean change in thickness for the Ep-BM
complex, stroma, and En-DM complex.
Rate of Deswelling
A bilinear function was fitted to the data to investigate the rate of
recovery for each structure and the time interval at which stabilization of the change in thickness occurred. The function was
fitted to the group average thickness over 15-minute intervals.
Table 4 shows the regression parameters. The thickness change
from baseline was different between structures, but the magnitude of change from the first measure to stabilization was similar
("10%). The rate of thinning before stabilization was different
between structures, with the anterior lamellae structures showing
thickness decreasing at a greater rate than the stroma. Although
the Ep-BM complex did not show a significant difference between
4582
Hutchings et al.
IOVS, September 2010, Vol. 51, No. 9
TABLE 4. Parameters for Bilinear Fit of the Recovery of Corneal Swelling Response over Time to Follow-up for the Layers Studied
Intercept, %
Slope, %/min
Time to break, min
Epithelium
Ep-BM Complex
Stroma
En-DM Complex
Total Thickness
&3.46
'0.172
44.4
&10.70
'0.187
41.6
&9.90
'0.090
98.1
0
0
—
&8.70
'0.085
98.0
All fits, P # 0.05. The En-DM did not show a change in thickness.
measures (RM ANOVA; F $ 0.27, df $ 10; P $ 0.623), it too
exhibited a biphasic pattern of recovery.
DISCUSSION
In this study, we identified separate layers of the cornea, including
fine layers anterior and posterior to the stroma. These layers were
nominally labeled the Ep-BM complex and the En-DM complex.
The thicknesses obtained in prehypoxia conditions (Table 1)
corresponded approximately to histology,38 reported measures of
the epithelium,39 total corneal thickness40 obtained with a lowresolution OCT, and epithelial thickness obtained with the confocal Rostock laser scanning microscope41 and a VHF ultrasound
system (Artemis; Ultralink, St. Petersburg, FL).42 Therefore, even
though the device (with its current sagittal scanning design) does
not unambiguously image individual cells, the physical thickness
of the layers derived by the backscattered light are very similar to
these histologically defined structural regions. The total thickness
of the cornea was found to be thicker than those obtained with
the confocal Rostock laser scanning microscope.41 This difference may have arisen as a function of the values used for the
refractive index. In this study, physical thickness of the cornea
was computed by using an average refractive index of 1.376,
although the layers undoubtedly have different refractive indices43,44 that are not known for the individual layers.
The corneal swelling response with lens wear and eye closure
averaged (across the group) "3.5% and "10% for the epithelium
and total cornea, respectively. This magnitude of swelling is
within the range found previously,23,45 and deswelling rates are
also similar to those reported for the epithelium and total corneal
thickness with in vivo optical methods.45As with the prehypoxia
condition, the boundaries of the layers in the posthypoxia condition were identified with a polynomial model that sought the
position of minimum intensity and maximum gradient in intensity. Three assumptions were made: First, the optical interfaces
occur at or near the physical (cellular) interfaces and correspond
with the position of minimum intensity and maximum gradient;
second, a polynomial approximation is a reasonable model to
determine the position of the interface across the examined area,
and local differences across the region are a minor factor in the
overall swelling response; and third, the optical and/or physical
characteristics of the boundaries are similar in the prehypoxia and
posthypoxia conditions. As a first approximation, these appear to
be reasonable assumptions because, as stated previously, the
thickness of the identified layers in the prehypoxia condition
corresponded reasonably with values obtained from histology
(which themselves have an inherent error due to the preparation
required for visualization of ex vivo samples) and, over the relatively small portion of the central cornea examined, adjacent
structures may be more likely to behave similarly than differently.
In normal configuration, light-scattering in the stroma is
thought to be primarily due to the nuclei of keratocytes46 and
contributes "1% of the total scattering observed, most being
observed at the anterior surface of the epithelium and the posterior endothelium.47 With edema, the extrafibrillar matrix in vitro
appears to increase in volume, resulting in a reduction of refractive index of the stroma,48 but this does not seem to represent the
total extent of increased scattering observed with swelling. If the
swelling is accompanied by injury, decreased crystallin expression as a function of the healing mechanism also appears to
contribute to increased light scattering.47 It is clear that the
dispersion of the source as it traverses the corneal structure can
be affected by several factors, even in the absence of injury. One
advantage of the UHR-OCT system used in this study is the illumination source used. Water dispersion has a null at 1060 nm and
changes slowly over the spectral range of the source and, as a
result, the axial resolution is preserved throughout the entire
thickness of the cornea. This advantage is not inherent to OCT
systems using illumination sources of "800 nm. It could be
argued that the imaging procedure adopted in the study by translation of the imaging probe inferiorly to avoid the strongest central specular reflection represents a shortcoming of the imaging
method. The specular reflex is an intrinsic complication of imaging the cornea and represents an artifact in the image that must be
managed. If the image is acquired with the strongest specular
reflection present, the contrast of the boundaries posterior to the
air– epithelium interface is reduced, and the data from the central
portion of the image become unusable. Reducing the power of
the illuminating beam to reduce the strength of the reflection
concurrently reduces the contrast of lateral portions of the image
and the deeper layers of the cornea to an unusable level. For these
reasons, a mechanical translation away from the position orthogonal to the central apex, while maintaining orthogonal alignment
with the apical region of the cornea, was selected as the preferable option. Although this may inevitably have introduced some
error into the measurement of thickness, averaging across the
central 200 pixels and across images would minimize the error to
some extent. In addition, the thicknesses obtained were in the
expected range and were repeatable. Thus, we conclude that the
technique, while perhaps not ideal, is valid.
The response to hypoxia was different among the various
layers. The En-DM layer did not show a significant difference in
pre- and posthypoxia conditions. The epithelium showed a
"3.5% increase in thickness from the prehypoxia condition and a
deswelling phase that stabilized to a thickness below baseline
values. This relative epithelial thinning has been reported in corneas recovering from hypoxia,49,50 and central epithelial thinning
has also been reported in a cohort of long-term soft contact lens
wearers.51 The finding may occur as a result of an autoregulation
effect leading to continuous extraction of water beyond the baseline levels,49 or it may be a mechanical effect associated with lens
wear. The stroma and total corneal thickness showed an increase
of "10% in thickness from the prehypoxia condition. Although
our experiment demonstrates that the proportional swelling response is different between layers we assume, but do not know,
that the relationship between the backscattered light in the preand posthypoxia conditions, in terms of the parameters used in
our model (minimum intensity and maximum gradient) may alter
in absolute terms, but remain consistent in relative terms.
The epithelium and endothelium are barriers that prevent
water from entering the stroma, to maintain the delicate balance
of hydration and transparency of the corneal structure. The transparency of the cornea appears to be a function of the precise
arrangement of the collagen fibrils in the stroma and the uniformity of the refractive index of the extrafibrillar matrix,47 with the
endothelial barrier acting as an active pump to maintain transpar-
IOVS, September 2010, Vol. 51, No. 9
UHR-OCT Study of Swelling of the Human Cornea
4583
a lack of systematic behavior for the group over time. A third and
related possibility is that the En-DM complex response is more
dynamic than the biphasic changes observed in other layers. Last,
central corneal thickness has been found to be related to the
endothelial cell density in young, normal subjects,58 and there is
a reduction in endothelial cell density with age.53 It may be that
the between-subject variability is due to different endothelial cell
densities across the various subjects’ ages in our sample, leading
to a variation in the endothelial pump capacity across the sample.
However, it has been reported that there is little relationship
between endothelial cell density and the permeability of the
endothelial barrier with age.54
In summary, using UHR-OCT and an automated segmentation
algorithm, we found regional differences in swelling induced by
hypoxia. On removal of the hypoxic stimulus, the rate of recovery
varied among layers, and all layers except the En-DM exhibited a
biphasic recovery.
Acknowledgments
FIGURE 4. Change in the En-DM complex after removal of the hypoxic
stimulus. Each subject’s data are identified by a separate line. Each
measurement (abscissa) was separated by approximately 15 minutes.
ency52 by drawing water into the aqueous.53 In the normal cornea, the anterior stroma has less water than the posterior stroma
and has been proposed to have a lower water-retention
capability.54 When the cornea swells, the posterior stroma has
been demonstrated to be more affected than the tightly packed
anterior stroma,48,55,56 perhaps also because of the proximity of
the endothelial pumps that are better able to evacuate water from
the posterior stroma and maintain stasis of the corneal water
content.54 It would therefore be expected that the anterior lamellae may swell less and/or show a more rapid rate of recovery than
the posterior lamellae. In this study, we identified a different rate
of recovery and return to a stabilized thickness between the
various layers identified. The epithelium and Ep-BM complex
showed minimal change in thickness from the baseline thickness
but showed different rates of recovery to a stabilized thickness
after removal of the hypoxic stimulus (epithelium, 4.7%/h; Ep-BM
complex 15.4%/h). The time to stabilization for both structures
was similar at approximately 40 minutes. Conversely, the stroma
and total corneal thickness took approximately twice the length
of time to reach a stabilized thickness ("98 minutes) with a rate
of recovery of 6.1%/h for the stroma and 5.3%/h for the total
cornea. These rates of recovery are similar to those found in the
epithelium and total cornea with a lower-resolution OCT.45
The En-DM complex did not exhibit a significant increase in
thickness between the pre- and posthypoxia conditions. Examination of the data for the group as a whole (Fig. 3) and for
individual subjects (Fig. 4) reveals a more variable response than
for the other layers identified. There are several possible explanations for this observation. First, as this layer is very thin and close
to the maximum resolution of the device, dispersion of the source
as it traverses the anterior tissues would have the greatest impact
at this layer. As a consequence, it may be expected that the
identification of the boundaries of the inner layer would be more
difficult to segment. The SD of the fit of the model within individuals was similar to that of other layers, suggesting that the high
group variability did not derive simply from technical issues related to the device or algorithm. Second, the dissimilarities in the
recovery profile between subjects could result from within- or
between-subject variations. It has been reported that both the
metabolic activity and the endothelial function are two factors
that contribute to corneal swelling.23,57 The interaction of these
and other factors in the recovery from edema may occur to
different extents between subjects and at different time points
after removal of the hypoxic stimulus for each subject, resulting in
The authors thank Justin Eichel, Akshaya K. Mishra, David Clausi, and
Paul Fieguth (Systems Design Engineering, University of Waterloo), for
development of the segmentation algorithm and Saad Shakeel and
Zainab Ramahi for assistance with the corneal data processing.
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