Download ARVO 2016 Annual Meeting Abstracts 539 Glaucoma Thursday

ARVO 2016 Annual Meeting Abstracts
539 Glaucoma
Thursday, May 05, 2016 11:00 AM–12:45 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 6398–6429/D0118–D0149
Organizing Section: Physiology/Pharmacology
Program Number: 6398 Poster Board Number: D0118
Presentation Time: 11:00 AM–12:45 PM
In vivo evaluation of Schlemm’s canal in experimental glaucoma
monkeys
Byron H. Li, Shenouda Yacoub, Rad Daly, Sarah Webb,
Travis Jernigan, Terri Krause, Ganesh Prasanna, Dennis S. Rice.
Glaucoma Research, Novartis Institutes of Biomedical Research, Fort
Worth, TX.
Purpose: To determine the effect of pupillary changes on Schlemm’s
canal (SC) in normal and experimental glaucomatous monkey eyes
in vivo.
Methods: chronic ocular hypertension (OHT) was unilaterally
induced in 18 Cyno monkeys by laser trabecular photocoagulation.
The effect of 300 µg Pilocarpine, 600 µg Azopt®, or Phenylephrine
and Mydriacyl® on intraocular pressure (IOP) in both eyes
was evaluated in conscious animals using an applanation
pneumatonometer post a single topical ocular dose. The effect of
same treatments on pupil and SC area was evaluated in sedated
animals with Spectralis SD optical coherence tomography (OCT).
Pupil diameter (PD) was measured in OCT images. The size of SC
in a series of 49 OCT B-scan images obtained in the temporal limbal
region was measured by three masked readers using AMIRA (V.5.5)
image analysis software.
Results: Pilocarpine maximally decreased IOP by 23±3% at 1h postdose in OHT eyes and 14±3% in the fellow normal eyes. The baseline
IOP (±SEM) was 33±1 mmHg and 24±1 mmHg in OHT and normal
eyes, respectively. The pupil diameter in both eyes was decreased
similarly by about 70% compared to pre-dose reading (3.8±0.3 and
3.5±0.3 mm). The SC area was significantly increased by 218±122%
in OHT eyes and 122±57% in normal eyes. There was a positive
correlation between reduced PD and IOP reduction (r=0.889 in OHT
eye and r=0.670 in normal eye) but not with PD vs. SC and IOP vs.
SC. Azopt® maximally reduced IOP by 13±5% at 6h post-dose in
OHT eyes and by 4±2% in normal eyes. Azopt® did not significantly
alter pupil size or SC area. There was no correlation between IOP and
SC area. Maximal pupil dilation with mydriatic treatment increased
IOP by 9.0±9.3% in OHT eyes and 36.5±12.6% in normal eyes
at 1 hour post dose. The area of SC did not change in OHT eyes.
However, the area of SC significantly decreased (-34%) in normal
eyes at 1 hours post dose.
Conclusions: SC in the monkey eye can be noninvasively assessed
with OCT. The area of SC measured in the temporal quadrant is
passively changed along with pupillary responses to miotic or
mydriatic treatment. Pupil constriction induced A. greater change in
SC size and IOP than that following pupil relaxation and B. a greater
effect on SC size in OHT eyes compared to normal eyes. Pupil
relaxation induced greater changes on SC and IOP in normal eyes
than that of OHT eyes.
Commercial Relationships: Byron H. Li, None; Shenouda Yacoub,
None; Rad Daly, None; Sarah Webb, None; Travis Jernigan,
None; Terri Krause, None; Ganesh Prasanna, None;
Dennis S. Rice, None
Program Number: 6399 Poster Board Number: D0119
Presentation Time: 11:00 AM–12:45 PM
NEUROPROTECTIVE ACTIONS OF HYDROGEN SULFIDERELEASING COMPOUNDS AND CANNABINOIDS IN THE
BOVINE ISOLATED NEURAL RETINA
Leah Bush1, Jenaye Robinson1, Catherine A. Opere2, Sunny E. Ohia1,
Ya Fatou Njie-Mbye1. 1Pharmaceutical Sciences, Texas Southern
University, Houston, TX; 2Pharmacy Sciences, Creighton University,
Omaha, NE.
Purpose: There is evidence that hydrogen sulfide (H2S) and
endocannabinoids can protect the central nervous system against
oxidative stress and glutamate-induced excitotoxicity. In the present
study, we compared the neuroprotective actions of H2S (using
L-cysteine as a substrate and NaHS and GYY4137 as H2S-releasing
compounds) with that elicited by cannabinoids (meth-anandamide
and 2-arachidonyl glycerol (2-AG)) against H2O2-induced oxidative
stress in the isolated bovine retinae.
Methods: Isolated neural retinae were pretreated with L-cysteine (10
nM - 1 µM), NaHS (30 µM -100 µM) and GYY4137 (30 µM -100
µM), or meth-anandamide (1 nM -100 nM) and 2-AG (1 µM -10
µM) for 30 minutes prior to the application of the oxidative insult
with H2O2 (100 µM) for 10 minutes. Lipid peroxidation was assessed
by measuring the levels of 8-isoprostane in tissues via enzyme
immunoassay.
Results: In the presence of H2O2 (100 µM), there was a 20% increase
in 8-isoprostane levels in isolated retinal slices when compared to
untreated controls. Pretreatment of tissues with L-cysteine, NaHS,
GYY4137, meth-anadamide or 2-AG blocked the H2O2-induced
increase in 8-isoprostance levels. For instance, L-cysteine (10 nM),
NaHS (10 µM) and GYY4137 (100 µM) significantly (P<0.001)
reversed the H2O2 (100 µM)-induced elevation in 8-isoprostane
levels in the neural retina. Futhermore, meth-anandamide (1 nM) and
2-AG (3 µM) also completely prevented the H2O2 (100 µM)-induced
increase in the level of the lipid peroxidation product.
Conclusions: Both H2S-releasing compounds and cannabinoids can
protect the bovine isolated neural retina from oxidative stress.
Commercial Relationships: Leah Bush, None; Jenaye Robinson,
None; Catherine A. Opere, None; Sunny E. Ohia, None; Ya
Fatou Njie-Mbye, None
Support: NIH/NEI R15EY022215
Program Number: 6400 Poster Board Number: D0120
Presentation Time: 11:00 AM–12:45 PM
Delta Opioids Provide RGC Neuroprotection Via Adenylyl
Cyclase (AC) Superactivation
Shahid Husain, Sudha Singh. Ophthalmology, Medical University of
South Carolina, Charleston, SC.
Purpose:
This study was designed to determine the role of AC Superactivation
in delta opioid-receptors-mediated RGC neuroprotection during
glaucomatous injury.
Methods:
Brown Norway rats were used to elevate intraocular pressure (IOP)
by injecting 50 µL of 2M hypertonic saline into the circumferential
limbal veins. IOP was recorded as the average of 6-8 consecutive
measurements prior to surgery (baseline IOP) and weekly after
treatment, using a calibrated Tonolab tonometer. Animals were
treated with delta opioid-receptor agonist, SNC-121 (1 mg/kg; i.p)
daily for 7 days. Pattern electroretinograms (PERG), retinal ganglion
cells (RGCs) in flat mount, and axons were counted 4-6 week post
injury. The changes in the cAMP levels and phospho-cyclic AMPresponse element binding protein (p-CREB) were determined by
ELISA, Western blotting, and immunohistochemistry.
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ARVO 2016 Annual Meeting Abstracts
Results:
We measured the cAMP levels in the retina at 1, 7, 14, and 42 days,
post-glaucomatous injury in normal, glaucomatous, and SNC-121treated (1 mg/kg; 7 days, once daily) glaucomatous eyes. The level
of cAMP was decreased by 32% in the glaucomatous eyes, but
significantly increased in SNC-121-treated normal and glaucomatous
eyes. We found this data very interesting and highly unexpected as
opioids generally inhibit cAMP formation because they are linked to
Gi-proteins. As we expected, acute treatment by SNC-121 decreased
cAMP formation significantly at day 1. However, chronic treatment
with a δ-opioid agonist for 7 days increased the levels of cAMP,
which was further elevated significantly at the 14th day, while SNC121 treatment had been stopped at day 7. These data form a strong
rationale for the existence of “AC Superactivation” within the retina.
The p-CREB was also significantly reduced in glaucomatous eyes at
day 42. Glaucomatous injury caused significant (p<0.05) reduction
in pattern ERG (PERG), axonal, and RGC numbers at day 42, post
glaucomatous injury. PERG deficits, axonal, and RGC survival were
significantly improved by SNC-121 treatment.
Conclusions:
These data provide evidence that cAMP/CREB pathway plays a key
role in RGC neuroprotection during glaucomatous injury. Data also
provide novel information about the “AC Superactivation” in the
retina, which will open new avenues for RGC neuroprotection.
Commercial Relationships: Shahid Husain; Sudha Singh, None
Support: EY019081 (NIH) and an unrestricted grant to MUSC-SEI
from Research to Prevent Blindness, New York
Program Number: 6401 Poster Board Number: D0121
Presentation Time: 11:00 AM–12:45 PM
Elevated intraocular pressure increases melatonin levels in the
aqueous humour
Jesus J. Pintor1, Hanan Alkozi1, Juan Sanchez-Naves2, Maria
Jesus Perez de Lara1, Gonzalo Carracedo3, Begoña Fonseca1,
Alejandro Martinez Aguila1. 1Dep. Bioquimica, F de Optica UCM,
Madrid, Spain; 2Department of Ophthalmology, Institut Balear
Oftalmoligia, Palma de Mallorca, Spain; 3Dep. Optica II, F de Optica,
Madrid, Spain.
Purpose: To study the levels of melatonin in the aqueous humour of
normotensive and hypertensive IOP patients and to compare them in
an animal model of glaucoma.
Methods: 37 eyes of 37 patients who underwent cataract surgery
were included in the study and were divided into normotensive
patients, with IOP below 21 mmHg (n=23) and hypertensive patients,
with IOP > 21 mm Hg (n=14). Glaucomatous DBA/2J (n=6) and
control C57BL/6J (n=6) mice presenting 3 and 15 months of age for
each strain were also used. Human and mice aqueous humours were
aspirated using a 30-gauge Rycrof cannula on a tuberculin syringe
and further processed to quantify melatonin by HPLC analysis.
Results: Melatonin levels in normotensive patients (IOP below 21
mm Hg) presented values of 33.14 ± 11.61 ng/mL (n=23), while
hypertensive patients (IOP above 21 mm Hg) showed melatonin
concentrations of 95.87 ± 28.23 ng/mL (n=14) (p<0.039). Glaucoma
mice presented melatonin values of 0.44 ± 0.06 ng/mL (at 3 months
of age, before the pathology starts) which raised to 1.45 ± 0.18 ng/
mL (at 15 months of age, when the pathology is fully established and
IOP is maximum) (n= 6, p<0.001). Control mice did not significantly
modified melatonin concentrations between 3 and 15 months of age.
Conclusions: Glaucoma patients with high IOP present increased
concentrations of melatonin in their aqueous humour compared to
normotensive patients. This has been confirmed in a glaucomatous
animal model in which it has been possible to see a correlation
between the development of the pathology, with an increase in IOP,
and a concomitant elevation of melatonin in the aqueous humour.
Commercial Relationships: Jesus J. Pintor; Hanan Alkozi,
None; Juan Sanchez-Naves, None; Maria Jesus Perez de Lara,
None; Gonzalo Carracedo, None; Begoña Fonseca, None;
Alejandro Martinez Aguila, None
Support: SAF2013-44416-R, RETICS RD12/0034/0003 and
Universidad Complutense PR1/07-14890
Program Number: 6402 Poster Board Number: D0122
Presentation Time: 11:00 AM–12:45 PM
ROLE OF PROSTAGLANDINS AND ACETYLCHOLINE IN
THE CONTRACTILE ACTIONS OF HYDROGEN SULFIDERELEASING COMPOUNDS IN THE BOVINE ISOLATED
IRIS
Sunny E. Ohia1, Evelyn Ajelabi2, 1, Christina Anyikwa1, Christine Ly1,
Cherie Chua1, Cheryl Chua1, Jenaye Robinson1, Leah Bush1,
Catherine A. Opere2, Ya Fatou Njie-Mbye1. 1Pharmaceutical Sciences,
Texas Southern University, Houston, TX; 2Pharmacy Sciences,
Creighton University, Omaha, NE.
Purpose: Previously, we reported that hydrogen sulfide (H2S)releasing compounds can relax pre-contracted porcine isolated irides,
an effect that was dependent upon endogenous production of both
H2S and prostaglandins, and on the activity of KATP channels (Ohia et
al. Curr. Eye Res. 35: 402, 2010). In the present study, we investigate
the pharmacological actions of H2S-releasing compounds (NaHS
and L-cysteine) on basal tone in bovine isolated irides. Furthermore,
we studied role of prostaglandins and acetylcholine in the responses
elicited by the H2S-releasing compounds on this tissue.
Methods: Isolated bovine iris muscle strips were set up in an organ
baths containing oxygenated Krebs buffer solution maintained at
37°C and gassed with 95% O2: 5% CO2. The muscle strips were
set to a resting tension of 0.3 g and longitudinal isometric tension
was recorded via a Grass FT03 force-displacement transducer and
analyzed using PolyView Computer Software. Contractile responses
were elicited by the H2S releasing compounds in the absence
and presence cyclooxygenase (COX) inhibitors (flurbiprofen and
indomethacin) or the muscarinic receptor blocker, atropine.
Results:
NaHS (1 nM – 10 µM) and L-cysteine (100 nM - 1 mM) caused
a concentration-dependent contraction of isolated bovine irides
yielding EC50 values of 10 nM and 30 µM, respectively. Both
COX inhibitors, flurbiprofen (10 µM) and indomethacin (10 µM)
abolished the contractile actions of NaHS and L-cysteine on this
tissue. Likewise, atropine (1 nM – 10 nM) significantly (p < 0.001)
antagonized the contractile response to NaHS and L-cysteine.
Conclusions: We conclude that H2S-releasing compounds can elicit
contraction of isolated bovine irides, an effect that is dependent
upon the production of endogenous prostaglandins and the release of
acetylcholine.
Commercial Relationships: Sunny E. Ohia, None;
Evelyn Ajelabi, None; Christina Anyikwa, None;
Christine Ly, None; Cherie Chua, None; Cheryl Chua, None;
Jenaye Robinson, None; Leah Bush, None; Catherine A. Opere,
None; Ya Fatou Njie-Mbye, None
Support: NIH/NEI Grant R15EY022215-01
Program Number: 6403 Poster Board Number: D0123
Presentation Time: 11:00 AM–12:45 PM
The influence of TRPV4 ion channel blockade on lens water
content
Nicholas A. Delamere, Amritlal Mandal, Mohammad Shahidullah.
Physiology, University of Arizona, Tucson, AZ.
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Purpose: In previous studies we reported detection of TRPV4 ion
channels in porcine lens epithelium but not fibers. Activation of
TRPV4 has been implicated in a mechanism that activates Na,KATPase activity in the epithelium in response to swelling of the
lens fiber mass. Active transport by the epithelium plays a critical
role in ion homeostasis for the entire lens cell mass. Because water
homeostasis and ion homeostasis are linked, we tested the possible
impact of TRPV4 channel blockade on lens water content.
Methods: Na,K-ATPase activity was measured by quantifying
ouabain-sensitive ATP hydrolysis. Water content was measured by
drying the lens for 40h to completely remove water, then determining
the difference between wet and dry weight. Dried lenses were
digested in 30% nitric acid and sodium content was measured by
atomic absorption spectrophotometry.
Results: After 5 min, Na,K-ATPase activity was increased by
64.1±3.7% (n=6) in the epithelium removed from intact porcine
lenses subjected to a swelling challenge by deposit of 5 μl
hyperosmotic mannitol solution into the posterior fiber mass. Na,KATPase activity in the epithelium also was increased (93.0±5.0%;
n=6) in lenses exposed to a TRPV4 agonist GSK1016790A (10nM).
The TRPV4 antagonist HC067047 (10 μM) prevented the Na,KATPase responses. Porcine as well as rat lenses exposed to 10 μM
HC067047 in isosmotic Krebs solution for 90 min displayed a
significant net weight gain. HC-treated rat lenses showed an average
weight gain of 2.2±0.2 mg (n=4) compared to 0.2±.01 mg (n=11) in
control lenses. The sodium content of HC067047-treated rat lenses
was 72.2±10.4 (n=3) compared to 42.4±2.8 (n=9) μmoles/gm dry
weight in control lenses.
Conclusions: The findings are consistent with a role for TRPV4
in the mechanism that regulates Na,K-ATPase activity in the lens
epithelium. At steady state, intermittent TRPV4 activation may
contribute to fine tuning of Na,K-ATPase activity. On the basis
of these studies we suggest that Na,K-ATPase regulation, in turn,
influences lens water homeostasis.
Commercial Relationships: Nicholas A. Delamere;
Amritlal Mandal, None; Mohammad Shahidullah, None
Support: NIH EY009532.
facility C) and the uveoscleral pathway (outflow facility k). Steadystate IOP results from the balance between P/D of AH, and it solves
a non-linear algebraic equation. A sensitivity analysis (SA) is
performed to simulate healthy individuals (HI), OHT (C=0.3*Cref)
and the effect of IOP-lowering medications (reduced Δπ).
Results: The IOP frequency distribution, Fig1a, fits a right-skewed
Gaussian curve as in a population-based study on 12.000 individuals
(Carel 1984). Fig1c,d show the effect of a 25% Δπ reduction on HI
and OHT. The model predicts an average IOP reduction of 2.6mmHg
and 4.3mmHg, respectively. Fig2 shows the Sobol indexes for the
SA. The indexes quantify the relative importance of each parameter
in the variance of IOP. The results in Fig2a suggest that IOP is
strongly influenced by BP and Δπ and mildly influenced by the
level of L, C and EVP in HI. OHT patients, Fig2b, show a stronger
dependence on BP and Δπ and a weaker dependence on L, C and
EVP of IOP than HI.
Conclusions: The proposed model suggests that the outcomes of
IOP lowering treatments might depend on the initial IOP level of the
patient and on its individual clinical condition. The model identifies
the strong influence of BP and Δπ and the mild influence of L, C and
EVP on the level of IOP. Model predictions in synergy with clinical
and animal studies could help unravel the complex relationship
between the parameters involved and contribute to the formulation of
patient specific treatments.
Program Number: 6404 Poster Board Number: D0124
Presentation Time: 11:00 AM–12:45 PM
Mathematical modeling and statistical analysis of aqueous humor
flow towards individualized glaucoma treatment
Simone Cassani1, Giovanna Guidoboni1, Marcela Szopos2,
Christophe Prud’homme2, Riccardo Sacco4, Brent A. Siesky3,
Alon Harris3. 1Mathematics, Indiana University Purdue Univ,
Indianapolis, IN; 2University of Strasbourg, Strasbourg, France;
3
Ophthalmology, Indiana University, Indianapolis, IN; 4Mathematics,
Politecnico di Milano, Milano, Italy.
Purpose: Ocular hypertension (OHT), or elevated introcular pressure
(IOP), is a risk factor for vision loss. IOP results from the balance
between aqueous humor (AH) production and drainage (P/D) and is
influenced by several factors such as blood pressure (BP), episcleral
venous pressure (EVP) and blood/AH osmotic pressure difference
(Δπ). Such factors influence disease risks and treatment outcomes,
but are difficult to isolate experimentally. We use a theoretical
approach to: (1) quantify the relative influence of these factors
on IOP; (2) study the variability in the outcome of IOP-lowering
medications.
Methods: The mathematical model describes P/D of AH in analogy
with an electric circuit. AH production occurs via ultrafiltration
from the ciliary circulation and via the Δπ generated by ionic active
secretion, and is modulated by the total inflow facility (L). AH
drainage occurs via the trabecular meshwork pathway (outflow
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: Simone Cassani;
Giovanna Guidoboni, None; Marcela Szopos, None;
Christophe Prud'homme, None; Riccardo Sacco, None;
Brent A. Siesky, None; Alon Harris, Isama therapeutics (C),
Stemnion Inc. (C), Nano Retina (C), Oxymap (I), Biolight (C),
AdOM (C), Ono (C), Science Based Health (C), AdOM (I)
Support: This work has been partially supported by the NSF
DMS-1224195, NIH 1R21EY022101- 01A1, a grant from Research
to Prevent Blindness (RPB, NY, USA), an Indiana University
Collaborative Research Grant of the Office of the Vice President
for Research, the Chair Gutenberg funds of the Cercle Gutenberg
(France) and the Labex IRMIA (University of Strasbourg, France).
Program Number: 6405 Poster Board Number: D0125
Presentation Time: 11:00 AM–12:45 PM
Gap Junctions – their characterization and basis in porcine
ciliary epithelium
Ka Lok Li1, Sze Wan Shan1, Angela King Wah Cheng1,
Mortimer M. Civan2, 3, Chi-ho To1, Chi-wai Do1. 1School of
Optometry, The Hong Kong Polytechnic University, Hong Kong,
Hong Kong; 2Department of Physiology, University of Pennsylvania
Perelman School of Medicine, Philadelphia, PA; 3Department of
Medicine, University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA.
Purpose: Gap junctions provide a conduit between the intracellular
fluids of the pigmented (PE) and non-pigmented (NPE) cilary epithelial
cells, and are therefore critical in the secretion of the aqueous humor
by the ciliary epithelium. However, agreement has been incomplete
concerning the connexin (Cx) composition of the gap junctions. We have
now determined (1) the gene expression of Cxs in the porcine ciliary
epithelium, a favorable model for the human; and (2) the contribution of
the highest expressed Cx (Cx43) to secretion by this preparation.
Methods: Freshly-harvested porcine ciliary epithelial cells were
used. The mRNA and protein expressions of gap junctions were
assessed by reverse transcription polymerase chain reaction
(RT-PCR) and western blotting (WB), respectively. The relative
gene expressions of various connexins (Cx) were determined by
quantitative PCR (qPCR). The gap junction permeability of isolated
PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer.
Results: Using RT-PCR and WB, Cx43, Cx45, Cx50 and Cx60, but
not Cx26 and Cx40, were expressed in porcine ciliary epithelium.
Cx43 was the most abundant isoform, over 200-fold higher than other
Cxs. Knockdown of Cx43 by siRNA reduced mRNA and protein
expressions by 59±3% (n=4, p<0.001) and 71±13% (n=3, p<0.05),
respectively. The Cx43 knockdown significantly reduced dye transfer
from PE to NPE cells by ~30% (n=4).
Conclusions: Similar to other species, Cx43 was found to be the
major component of gap junctions in porcine ciliary epithelium.
Knockdown of Cx43 reduced gap junctional permeability, supporting
the functional significance of Cx43 in mediating fluid movement
across porcine ciliary epithelium.
Commercial Relationships: Ka Lok Li, None; Sze Wan Shan,
None; Angela King Wah Cheng, None; Mortimer M. Civan, None;
Chi-ho To, None; Chi-wai Do, None
Support: RGC/GRF: 5609/13M, 5607/12M, 151033/15M; PolyU
internal grants: G-YK88, A-SA27, G-YBBT; and Henry G Leong
Endowed Professorship fund
Program Number: 6406 Poster Board Number: D0126
Presentation Time: 11:00 AM–12:45 PM
Steady-state PERG after water drinking test in open angle
glaucoma and normal subjects
Giacomo Calzetti, Nicola Ungaro, Luigi Varano, Giulia Gennari,
Claudio Macaluso, Stefano A. Gandolfi. Eye Clinic, University of
Parma, Parma, Italy.
Purpose: To investigate the effect of water drinking test (WDT)
on steady-state pattern electroretinogram (PERG) in open angle
glaucoma (OAG) and in healthy subjects.
Our initial hypothesis was that WDT could affect negatively PERG in
glaucomatous patients.
Methods: Observational case-control study. 12 eyes of 8 OAG
patients scheduled for a routine WDT and PERG evaluation with a
visual field defect within stage 2 of the Glaucoma Staging System
2 (mean Standard Automated Perimetry-MD -1,62 ± 1,38 dB) and/
or glaucomatous alteration of macular ganglion cell layer + inner
plexiform layer thickness measured by Spectral Domain OCT, no
prior ocular surgery or laser and no ocular disease besides OAG
and 11 eyes of 6 age-matched normal controls (first-degree relatives
of glaucomatous subjects undergoing a full list of examinations
within a frame of a case-finding program) have been included. All
of the subjects underwent PERG and intraocular pressure (IOP - by
means of Goldmann applanation tonometry) recording before and 30
minutes after a WDT (1 liter of water in 10 minutes).
PERG amplitudes and IOP before and after water intake were
compared by means of Wilcoxon matched pairs test and paired
Student t test, respectively, in both glaucomatous and controls.
Changes in PERG amplitude and in IOP were correlated by means of
Spearman rank correlation test, in both groups.
Results: Mean baseline IOP values were 14,41 ± 2,53 mmHg in
the OAG cohort and 12,45 ± 2,29 mmHg in the control cohort.
After WDT mean IOP values were 19,25 ± 4,07 mmHg and 14,36
± 2,54 mmHg, with statistically significant increase in both groups
(P<0,0001 and P=0,0003, respectively).
PERG amplitude showed a trend towards increase after WDT only in
the OAG group (P=0,059 - median of amplitude values was 2,71 μV
at baseline and 3,78 μV after WDT).
A positive correlation between IOP changes and PERG amplitude
changes was found in glaucomatous patients (r =0,9183, P<0,0001),
while no correlation was found in healthy subjects.
Conclusions: Unexpectedly, we found a trend towards increase
of PERG amplitude after WDT in patients with OAG. The most
interesting finding is the positive correlation between changes in IOP
and in PERG amplitude after water intake found in patients with
OAG, but not in healthy subjects.
We hypothesize that a common unknown factor, possibly vascular
and linked to water intake, could cause both IOP and PERG
amplitude increase in patients with OAG.
Commercial Relationships: Giacomo Calzetti, None;
Nicola Ungaro; Luigi Varano, None; Giulia Gennari, None;
Claudio Macaluso, None; Stefano A. Gandolfi, None
Program Number: 6407 Poster Board Number: D0127
Presentation Time: 11:00 AM–12:45 PM
Brimonidine, like vancomycin, directly activates mast cells to
degranulate, releasing preformed mediators
Daniel Schwartz, Yoshihiro Fukuoka. Ophthalmology, Virginia
Commonwealth University, Richmond, VA.
Purpose: The effect of glaucoma medicines and their preservatives
on conjunctival mast cells is unclear. Alphagan (brimonidine 0.2%
and its preservative benzalkonium chloride (BAK)) is known to cause
local reactions, including follicular conjunctivitis. We examined
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ARVO 2016 Annual Meeting Abstracts
whether brimonidine, like vancomycin, directly degranulates mast
cells in vitro.
Methods: Degranulation was determined by β-hexosaminidase
release from mast cells exposed to various stimulants: alphagan,
alphagan-P, vancomycin (MRGPR-X2 positive control),
benzalkonium chloride, brimonidine, 22E7 (anti-FcεRI antibody
positive control) and Phosphate Buffer Solution (PBS, negative
control). Mast cells were dispersed from fresh surgical skin obtained
from the Cooperative Human Tissue Network, placed into culture
with Stem Cell Factor (100 ng/ml), removed after 6-12 weeks,
suspended at 1 x 10^6 cells/ml, preincubated at 37°C x 5 min,
stimulated for 15 min at 37°C, and stopped by adding two-volumes
of ice-cold Ca/Mg-free medium. Releasates were separated from
retentates by centrifugation. The pellet was combined with a
detergent (TX100) to extract β-hexosaminidase, and the activity
of released and retained enzyme was measured by cleavage of
pnitrophenyl-hexosamine; %degranulation being 100 x releasate/
(releasate + retentate). Viablity was determined by trypan blue
exclusion.
Results: Brimonidine (0.17%, n=6) reproducibly caused mast cell
degranulation without evidence of cell toxicity, %degranulation
ranging from 4.5 to 14.0. Alphagan (0.033% Brimonidine) and
alphagan P (0.025% Brimonidine) caused no detectable degranulation
(n=6). 22E7 consistently degranulated mast cells, %degranulation
ranging from 26.8 to 65.6. Vancomycin (8.3 μM to 83 μM), n=1
degranulated mast cells in a dose-dependent manner. There was no
spontaneous release to PBS alone in all experiments.
Conclusions: Our results show that brimonidine, at a pharmacologic
concentration of 0.17%, when applied topically to the eye, directly
stimulates mast cells to degranulate in vitro. The lower concentrations
of Brimonidine in Alphagan and Alphagan P did not do so. Whether
brimonidine concentrations applied to the eye remain high enough
after dilution with tears and diffusion through conjunctiva to where
mast cells reside is uncertain, but could result in conjunctival pruritis
in some patients.
Commercial Relationships: Daniel Schwartz, None;
Yoshihiro Fukuoka
Program Number: 6408 Poster Board Number: D0128
Presentation Time: 11:00 AM–12:45 PM
Robust extraction of the diversity of ganglion cell computation
via spatially correlated stimuli and nonlinear modeling
Hope Shi, Sarvenaz Memarzadeh, Joshua H. Singer, Daniel Butts.
Biology, University of Maryland, College park, MD.
Purpose: A prerequisite for understanding visual processing by
the retina is the ability to characterize the diverse responses of
retinal ganglion cells (GC) to light stimuli. Here, we designed a
spatiotemporal noise stimulus and applied a nonlinear modeling
approach to GC spike responses with the aim of gaining insight into
different forms of computation performed in parallel retinal pathways
terminating on individual GC types.
Methods: Spike responses of GCs to UV light in a whole-mount
in vitro preparation of ventral mouse retina were recorded on a
60-channel, perforated multi-electrode array mounted on an inverted
microscope. UV light was delivered through the objective by a
coupled and modified DLP projector. A spatially correlated noise
stimulus was generated by low-pass filtering random checkerboards,
which comprised shapes appropriate to map a variety of GC receptive
field features. GC receptive fields were derived from a nonlinear
input model (NIM), in which the transform from stimulus to response
via the integration of one or more excitatory and inhibitory subunits
(sensitive to different stimulus features). A separable form of the NIM
with parameters representing spatial and temporal tuning of multiple
subunits was developed, and parameters were determined using
maximum a posterior optimization.
Results: GCs were classified according to the structure of the
nonlinear model that described them. The use of the cloud stimulus
greatly enhanced characterization of receptive field surrounds and
also identified many more ON-OFF cells than were evident from
responses to full-field uniform contrast stimuli or the spike-triggered
average. Most categories of GC had nonlinear suppressive subunits
that often dominated the GC spatial surround relative to that
predicted by linear receptive fields.
Conclusions: Unlike commonly-used white noise stimuli, spatially
correlated stimuli drove surround responses of the GC robustly, and
NIM modeling provided significant insight into surround processing.
The NIM revealed large functional differences among receptive fields
of GCs that had similar responses to full-field stimuli. Due to the
correspondence between model subunits and physical retinal circuits,
our analyses will reveal mechanisms underlying GC function that
have been missed by standard linear-nonlinear modeling approaches.
Commercial Relationships: Hope Shi, None;
Sarvenaz Memarzadeh; Joshua H. Singer, None; Daniel Butts,
None
Support: University of Maryland, Biology Department internal grant
Program Number: 6409 Poster Board Number: D0129
Presentation Time: 11:00 AM–12:45 PM
Protection of visual function and neurodegeneration in a
mouse model of Leber’s hereditary optic neuropathy by drug
intervention
Alfred K. Yu, Lanying Song, Sandipan Datta, Gino Cortopassi.
University of California, Davis, Davis, CA.
Purpose: The pathophysiological mechanism of Leber’s Hereditary
Optic Neuropathy (LHON), which is caused by mutations in
mitochondrial complex I, is not well understood. Using the Ndufs4
knockout (KO) as a mouse model of LHON, we established
previously that mitochondrial complex I deficiency leads to an innate
immune and inflammatory wave that coincides with vision loss in
these mice as well as death of starburst amacrine cells and retinal
ganglion cells. We hypothesize that intervention with drugs that either
inhibit the inflammatory response or increase ATP synthesis will
preserve visual function and prevent neurodegeneration.
Methods: Ndufs4 knockout mice and littermate controls were treated
with vehicle or one of four drugs, multiple of which were protective
in a high-throughput screen of LHON cells, to be identified later due
to disclosure restrictions; R (8 mg/kg), Z (20 mg/kg), D (10 mg/kg),
or P (20 mg/kg). Doses were administered intraperitoneally daily for
14 days. Visual cliff testing was performed prior to dosing at P21 and
at the end of treatment at P35. Mice were then sacrificed and retinas
collected for qRT-PCR and flat mount immunostaining for ChAT
expression.
Results: All four drugs preserved visual function in the Ndufs4 KO
compared with Ndufs4 KO dosed with vehicle, which were unable
to detect the edge in the visual cliff test. Drugs R and P significantly
inhibited innate immune and inflammatory transcripts that were
elevated in the Ndufs4 KO, such as B2m, Cxcl10, Ccl5, Ccl12,
Aif1, Tlr2, and Tlr4, demonstrated by qRT-PCR. Drug R was able
to prevent neurodegeneration of starburst amacrine cells, which
was performed by counting ChAT-positive cells using confocal
microscopy.
Conclusions: An innate immune and inflammatory response and
starburst amacrine cell death appear to be early consequences of
complex I deficiency in the Ndufs4 KO mouse model of LHON,
and may be relevant to the pathomechanism of human LHON.
Drug screening has identified four drugs that rescue mitochondrial
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ARVO 2016 Annual Meeting Abstracts
blindness in the Ndufs4 context. R, Z, D, and P were shown to
preserve visual function in Ndufs4 KO mice, and could be considered
as therapeutic leads for LHON. The differential suppression of
inflammatory molecules of the different drugs suggests differential
mechanisms of protection.
Commercial Relationships: Alfred K. Yu, None; Lanying Song,
None; Sandipan Datta, None; Gino Cortopassi, None
Support: NEI Grant EY012245
Program Number: 6410 Poster Board Number: D0130
Presentation Time: 11:00 AM–12:45 PM
Effects of Nitric Oxide Donors on Intraocular Pressure in
Cynomolgus Monkeys
Shenouda Yacoub, Byron H. Li, Muneto Mogi, Dennis S. Rice,
Ganesh Prasanna. Novartis Institutes for BioMedical Research, Fort
Worth, TX.
Purpose: To determine the effects of various nitric oxide (NO)
donors on intraocular pressure (IOP) in experimental glaucomatous
monkey eyes following topical ocular dosing.
Methods: Chronic ocular hypertension (OHT) was induced in
the right eye of cynomolgus monkeys by argon laser trabecular
photocoagulation. The effect of various NO donors on IOP was
evaluated in conscious animals using a computerized applanation
pneumatonometer.
Results: Average baseline IOP in the hypertensive monkey eyes
was between 34 - 40 mmHg compared to the 24 – 30 mmHg in the
contralateral normotensive eye. A molsidomine derivative: SIN-1
(dosed at 300 mg) caused IOP lowering of 19 ± 3% at 3 hours and 18
± 4% at 6 hours post-dose in the hypertensive eye. In normotensive
monkey eyes, SIN-1 was not efficacious. Isosorbide 5-mononitrate,
an isosorbide caused IOP reduction of 14 ± 4% at 1 hour post-dose
but was not efficacious at 6 hours post-dose in the hypertensive
monkey eye. A dinitroglycerine derivative 1, 3 Dinitroglycerin (dosed
at 50 mg and 500 mg) caused an acute IOP reduction of 14% at 1
hour post-dose for both doses and was minimally efficacious at 6
hours (<8%) for both doses in the hypertensive monkey eye.
Conclusions: NO donors lower IOP in the hypertensive eyes of a
monkey model of glaucoma. Among the several classes of NO donors
tested, SIN-1 appeared to cause the most robust IOP reduction in the
monkey model of glaucoma compared to Isosorbide 5-mononitrate
and 1,3-Dinitroglycerin. Additional classes of NO donors are being
evaluated for their effects on IOP in this model of glaucoma.
Commercial Relationships: Shenouda Yacoub, Novartis;
Byron H. Li, Novartis; Muneto Mogi, Novartis; Dennis S. Rice,
Novartis; Ganesh Prasanna, Novartis
Program Number: 6411 Poster Board Number: D0131
Presentation Time: 11:00 AM–12:45 PM
Intravitreal delivery of chemically modified mRNA for
neuroprotection through Müller cell transfection
Joke Devoldere, Karen Peynshaert, Stefaan De Smedt,
Katrien Remaut. Laboratory of General Biochemistry & Physical
Pharmacy, Ghent University, Ghent, Belgium.
Purpose: Recent advances in the field of molecular biology have
revolutionized mRNA as a therapeutic. As chemical modifications to
in vitro transcribed mRNAs reduce its immunogenicity, prolonged
protein expression can be obtained, thus broadening the applicability
of mRNA in protein replacement therapies. This work aims to
evaluate the therapeutic potential of chemically modified mRNA as
a therapy for retinal neurodegeneration. In an experimental model
based on fresh bovine vitreous, we examined non-viral intra-vitreal
delivery of modified reporter mRNA to Müller cells via two lipidbased commercial carriers: messengerMAX and TransIT.
Methods: Human Müller glia cells (MIO-M1) were seeded in a
transwell system 5 days before transfection. Fresh bovine eyes were
obtained from a local abattoir. The vitreous was carefully removed
and applied on top of the insert. Lipid-based commercial carriers
with modified eGFP mRNA were either delivered to Müller cells via
serum-containing medium, injected into the intact vitreous or mixed
with enzymatically treated vitreous. Transfection efficiency was
quantified by flow cytometry. Cell viability was evaluated both with
flow cytometry and a standard MTT assay.
Results: For messengerMAX the percentage of GFP positive
cells reached up to 60% upon 24h incubation in serum-containing
medium. Transfection levels decreased when transfections were
performed in intact or enzymatically treated vitreous, indicating that
the vitreous is an important barrier for non-viral mRNA delivery
to the retina. Interestingly, although messengerMAX did not show
complete mRNA complexation with gel electrophoresis, it proved to
be superior over TransIT in all transfection media. However, toxicity
results indicated that the TransIT carrier possesses the most favorable
safety profile. Therefore, despite its lower transfection potential,
the low toxicity could make this carrier suitable in situations where
repeated transfections are required.
Conclusions: Our data demonstrate that non-viral lipid based carriers
show good potential to transfect Müller cells in vitro. Moreover,
chemical modification of the mRNA substantially increased GFP
expression in >80% of cultured Müller cells. However, to make these
mRNA therapeutics suitable for ocular targets, the development of
strategies to overcome the vitreal barrier will be crucial to implement
ocular mRNA therapy in the future.
Commercial Relationships: Joke Devoldere, None;
Karen Peynshaert, None; Stefaan De Smedt, None;
Katrien Remaut, None
Program Number: 6412 Poster Board Number: D0132
Presentation Time: 11:00 AM–12:45 PM
The effect of intravitreal injection of bevacizumab on the
intraocular pressure in mexican population
YOLANDA CHAVEZ ROMERO, Efrain Romo-Garcia,
Wilehaldo Quiñonez, SILVIA PAZ, ABEL RAMON,
PIMENTEL KARLA. Ophthalmology, CIDOCS, ZAPOPAN, Mexico.
Purpose: Purpose: The effect of intravitreal bevacizumab on the
intraocular pressure (IOP) has been studied and it has been reported
significant changes in the first 30 minutes with the dose of 0.05 ml,
with no significant changes in the first 24 hours after the injection.
In our study we measure the changes on IOP with the 0.1 ml dose
of intravitreal bevacizumab in the first 24 hours in a mexican
population.
Methods: Methods: We analyzed the records of all the subjects
that received an intravitreal injection of bevacizumab (0.1 ml) in
a period of 6 months in our hospital. We included all patients that
came back to the 24-hours IOP measurement. We excluded patients
with history of vitreoretinal surgery and silicon oil and patients with
previous glaucoma treatment. We measured the IOP 30 min before
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ARVO 2016 Annual Meeting Abstracts
the injection and 24 hours after the injection. The statistical analysis
was performed with the paired samples t-test.
Results: Results: a total of 60 patients, 31 male and 29 female, 132
applications. The mean of age was 65.7 years. The pre-application
IOP mean was 13.7±2.5 mmHg and the post-application IOP mean
was 13.8±2.5 mmHg, with no statistical difference (p=0.530). The
most common indications for the injections were Proloferative
Diabetic Retinopathy with clinically significant macular edema
(PDR + CSME) 38.2%, Non-proliferative Diabetic Retinopathy with
clinically significant macular edema (NPDR + CSME) 23.3% and
Age-related macular degeneration (ARMD) 20%.
Conclusions: Conclusions: The dose of 0.1 ml intravitreal injection
of bevacizumab showed safety in the changes of the intraocular
pressure in the period of 24 hours after the procedure.
Commercial Relationships: YOLANDA CHAVEZ ROMERO;
Efrain Romo-Garcia, None; wilehaldo quiñonez, None;
SILVIA PAZ, None; ABEL RAMON, None; PIMENTEL KARLA,
None
Program Number: 6413 Poster Board Number: D0133
Presentation Time: 11:00 AM–12:45 PM
ROLE OF BIOSYNTHETIC ENZYMES AND
PROSTAGLANDINS ON HYDROGEN SULFIDE LEVELS
IN PORCINE OCULAR ANTERIOR SEGMENT OUTFLOW
MODEL
Jenaye Robinson1, Leah Bush1, Olivia Nguyen1, Catherine A. Opere2,
Sunny E. Ohia1, Ya Fatou Njie-Mbye1. 1Pharmaceutical Sciences,
Texas Southern University, Houston, TX; 2Pharmacy Sciences,
Creighton University, Omaha, NE.
Purpose: In a previous study, we reported that H2S donors can
reduce intraocular pressure in normotensive rabbits presumably
by increasing aqueous humor (AH) outflow through the trabecular
meshwork. In the present study, we investigated the contribution
of endogenous H2S and the role of intramurally-generated
prostaglandins in the observed increase in AH outflow facility in an
ex vivo porcine ocular anterior segment model.
Methods: Porcine ocular anterior segment explants were perfused
with Dulbecco’s Modified Eagle’s Medium maintained at 37° C and
gassed with 5% CO2 and 95% air under an elevated pressure of 15
mmHg for four hours. Perfusates from the anterior segment explants
were collected and immediately assayed for H2S content using
the well-established Methylene Blue assay. In some experiments,
explants were perfused with an inhibitor of H2S biosynthesis
[aminooxyacetic acid (AOAA, 30-100 µM) and α-ketobutyric acid
(KBA, 1 mM)] or with cyclooxygenase (COX) inhibitors [flubiprofen
(30 µM and indomethacin (10 µM)]. The concentration of H2S
was also measured in the AH (as a positive control), and at normal
perfusion pressure of 7.35 mmHg in the absence of AOAA and COX
inhibitors.
Results: Elevating perfusion pressure from 7.35 to 15 mm Hg
significantly (p < 0.001) increased H2S concentrations from 0.4
± 0.1 to 67.6 ± 3.6 nM/µg protein. As a reference value, the H2S
concentration in the AH was 1.5 ± 0.2 nM/µg protein. In the presence
of AOAA (30 µM) or KBA (1 mM), the effects of elevated pressure
on H2S levels were significantly (p < 0.001) reduced from 67.6 ±
3.6 to 5.7 ± 0.3 nM/µg protein and 4.9 ± 0.8 nM/µg, respectively.
Likewise, flurbiprofen (30 µM) and indomethacin (10 µM) attenuated
the elevated pressure-induced increase in H2S levels.
Conclusions: We conclude that the elevated perfusion pressureinduced increase in H2S concentrations is dependent upon
endogenous biosynthesis of H2S and on intramurally-produced
prostaglandins in the porcine anterior segment explants.
Commercial Relationships: Jenaye Robinson, None; Leah Bush,
None; Olivia Nguyen, None; Catherine A. Opere, None;
Sunny E. Ohia, None; Ya Fatou Njie-Mbye, None
Support: NIH/ NEI RI5EY022215
Program Number: 6414 Poster Board Number: D0134
Presentation Time: 11:00 AM–12:45 PM
Pharmacokinetic Study of Vitreous and Aqueous Humors
Concentrations of Demethylvancomycin after Subconjunctival
Injection and Posterior Sub-tenon’s Continuous Infusion with a
Micro Pump in Vivo
Ding Lin1, 2, Yezhen Yang1, Yiqin Duan1, Xuetao Huang1. 1Aier School
of Ophthalmology,Central South University, Changsha, China;
2
Changsha Aier Eye Hospital, Changsha, China.
Purpose: How to deliver the drug with therapeutic local
concentrations to the area of infection remains a challenge, injections
by needles repeatedly may increase the risks of intraocular infection
and hemorrhage. This article compared the pharmacokinetics
of demethylvancomycin in aqueous humors and vitreous after
subconjunctival injection and Sub-tenon’s continuous infusion with
a micro-infusion pump in a rabbit model, explores the alternative of
using a Sub-tenon’s Continuous Micro-infusion for a long-term drug
release in vivo.
Methods: 40 adult New Zealand white rabbit randomly divided
into two groups. One group(N=20) received subconjunctival
injection of demethylvancomycin (3 mg in 0.3 ml) in their right
eyes, The other group(N=20) received the same concentration
of demethylvancomycin at a 1ml/h infusion velocity continuous
perfused into posterior sub-tenon in their right eyes. Four animals
were killed at each designated time points (1 hour, 3 hours,6 hours,12
hours, and 24 hours). The concentration of demethylvancomycin
in the aqueous humor and vitreous were determined by highperformance liquid chromatography(HPLC).
Results: The aqueous humor and vitreous regression equation is
Y=122952X+173.846 (R2=0.9998, P<0.05). The subconjunctival
injection group: demethylvancomycin concentration
were(1.35±0.472)μg/ml,(0.477±0.271)μg/mL in aqueous and vitreous
at 1 hour post-injection. Then the concentration achieve maximum at
3 hour post-injection and were (1.±4.427)μg/mL,(0.668±0.301)μg/
mL. After that the concentration showed a declining trend. The
continuous infusion group: demethylvancomycin concentration
were(1.678±0.716)μg/ml,(0.328±0.139)μg/mLin aqueous and
vitreous at 1 hour post-injection, then it achieve maximum at 3 hour
post-injection and were (3.21±0.621)μg/mL,(2.31±1.632)μg/mL,
then it decreased slightly and show a stable trend. No any ocular
complication was found throughout the studying duration.
Conclusions: In the subconjunctival injection group, the drug
concentrations in vitreous body and aqueous humors were lower
than minimal inhibitory contration (MIC) of the most gram-positive
bacteria after 3hours. In the sub-tenon’s continuous micro-infusion
group, the drug concentrations in vitreous body and aqueous was
greater than the MIC for more than 24 hours.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: Ding Lin, None; Yezhen Yang, None;
Yiqin Duan, None; Xuetao Huang, None
Program Number: 6415 Poster Board Number: D0135
Presentation Time: 11:00 AM–12:45 PM
A novel ex-vivo retina model for oxidative stress – chances for the
replacement of animal experiments
Sven Schnichels1, Sandra Kuehn2, Adelina Jashari2, Jose Hurst1,
Stephanie C. Joachim2. 1Ophthalmology, Centre for Ophthalmology
Tuebingen, Tuebingen, Germany; 2Ruhr-University Bochum,
Experimental Eye Research Institute, Bochum, Germany.
Purpose: Oxidative stress is a major player in several ophthalmic
diseases, including glaucoma, ischemia, AMD or diabetic
retinopathy. To investigate potential therapies we aimed to establish
an ex-vivo model with porcine eyes from the abattoir. On one hand,
hundreds of pigs are daily sacrificed for the food industry and these
eyes are simply discarded. On the other side, researchers kill many
animals to test for potential medical therapies. Not only saving the
lives of animals is a mayor advantage of using eyes form the abattoir,
additionally, the pig eye is much more similar to the human eye than
any rodent eye.
Methods: Organotypic cultures of porcine retina were cultivated and
treated with different doses of H2O2 (100, 300 & 500 µM) for 3h on
day 1. At day 3 and 8, retinas were cryo-conserved for histological
(n=6-8/group), Western blot (n=6/group) and qRT-PCR (n=6/
group) analysis. The apoptotic state of retinal ganglion cells (RGCs;
Brn3a+cleaved Caspase 3) and the activation of macroglia (GFAP;
vimentin) and microglia (Iba1; Fcγ) were analyzed. Further, the
expression of PGP9.5, GFAP, CD11b, HSP70, VEGF, INOS, TNFα,
IL1β & p21 were quantified via qRT-PCR.
Results: The amount of Brn3a+ RGCs was reduced in all H2O2
treated groups (p<0.05). In accordance, the amount of apoptotic
RGCs was higher compared to the control. A dose-dependent
increase of microglia was observed. In contrast neither histological
nor protein expression changes were found in regard to macroglia.
Quantitative RT-PCR confirmed these findings. Treatment with H2O2
had a negative effect on the expression of PGP9.5 after 3 (1.4 - 2-fold
reduction) and 8 days (1.4-fold reduction) of cultivation. At day 8,
a higher activation of CD11b (1.5-1.7-fold) was observed. After 3
days, a loss of IL1β mRNA (1.5-2.6-fold) and a rise of iNOS mRNA
(2-3-fold) was detected. However, this effect diminished at day 8.
HSP70 expression also increased by 2.3 fold after 3 days for all
concentrations.
Conclusions: A profound RGC death was observed after adding
H2O2. Also inflammatory markers showed an expected response.
An ex-vivo model for oxidative stress was therefore successfully
established. The use of this model offers high chances to reduce the
amount of animals used in retinal research. Moreover, the pig eye is
anatomically and molecular closer to the human eye, thus offering
higher chances of a successful prescreening of potential therapies.
Commercial Relationships: Sven Schnichels, None;
Sandra Kuehn, None; Adelina Jashari; Jose Hurst, None;
Stephanie C. Joachim, None
Support: SET Foundation (Germany)
Program Number: 6416 Poster Board Number: D0136
Presentation Time: 11:00 AM–12:45 PM
Simulating Distribution of Drugs Depending on logP Values in a
Rabbit Eye Model
Jihwang Lee1, Hye kyoung Hong2, Se Joon Woo2, Hyuncheol Kim1.
1
Chemical & Biomolecular Engineering, Sogang University, Seoul,
Korea (the Republic of); 2Ophthalmology, Seoul National University
Bundang Hospital, Seongnam, Korea (the Republic of).
Purpose: The objective of this study is estimating the distribution of
two drugs, which have different solubility in the vitreous, after the
intravitreal injection by simulating the 3-D mathematical model in a
rabbit eye.
Methods: Several ocular tissues including the vitreous were
isolated. The diffusion coefficients of two different drugs through
the isolated tissues were determined by using the Ussing chamber. A
3-dimensional finite element mathematical eye model were developed
and the convection and diffusion equations were applied to determine
the aqueous humor flow and drug distribution in the eye with the
experimentally determined diffusion coefficients.
Results: The average velocity of the aqueous humor through the
retina was 3.66×10-7 m/s which is comparable to the previously
reported data. The simulation study demonstrated that the
concentration of intravitreally administered more hydrophobic
drug (Brimonidine) was 3.6 and 4.4 fold higher than that of more
hydrophilic drug (Ganciclovir) at 6 and 12 hours in the vitreous,
respectively. The vitreal half-lives of Ganciclovir and Brimonidine
were determined to be 5 and 12 hours, indicating that the more
hydrophilic drug (Ganciclovir) were eliminated both anteriorly
and posteriorly more easily than the more hydrophobic drug
(Brimonidine).
Conclusions: To the best of our knowledge, it is the first study to
determine the diffusion coefficients for the two drugs, which show
different solubility in water, and compare the pharmacokinetics of
different solubility drugs with the 3-D mathematical eye mode. More
water-soluble drug in the vitreous showed less elimination rate and
longer half-life.
Commercial Relationships: Jihwang Lee, None; Hye
kyoung Hong, None; Se Joon Woo, None; Hyuncheol Kim, None
Program Number: 6417 Poster Board Number: D0137
Presentation Time: 11:00 AM–12:45 PM
Functional renin receptors in human Schlemm’s Canal cells
Jayter S. Paula1, 2, Kristin Perkumas2, Nicole E. Ashpole2,
W D. Stamer2. 1Department of Ophthalmology, Ribeirão Preto
Medical School, University of São Paulo, Ribeirão Preto, Brazil;
2
Department of Ophthalmology, Duke University School of
Medicine, Durham, NC.
Purpose: Intraocular pressure is set by resistance generation to
aqueous humor outflow in the juxtacanicular region where trabecular
meshwork (TM) and Schlemm’s canal (SC) cells interact. Elements
of the renin-angiotensin system (RAS) may contribute to outflow
resistance since modulation of some RAS enzymes decrease IOP in
experimental glaucoma. Our objective is to examine the expression
of the (pro)renin receptor (PRR) and evaluate the effects of renin on
signaling in and protein expression by cultured human SC cells.
Methods: RNA isolated from two human SC and three TM cells
strains at confluence was tested using RT-PCR for expression of
renin and PRR. In parallel, one SC cell strain was treated with
renin (10-6, 10-7, and 10-8M) in 1% FBS-low glucose DMEM for
48 h and fibronectin content in conditioned media was analyzed
by Western blot. Beta-catenin localization in cells was assessed by
immunofluorescence microscopy.
Results: PRR, but not renin, was expressed in both SC and TM
cells strains. Immunofluorescence analysis showed predominantly a
cytoplasmatic and plasma membrane distribution of beta-catenin in
untreated SC cells, while beta-catenin translocated to nucleus in 53%
of renin-treated cells. In a biphasic dose-dependent fashion, renin
increased fibronectin secretion from SC cells. Renin also appeared to
cause morphological alterations that needs to be characterized further.
Conclusions: Cultured human SC cells appear not to express renin,
but have a functional system for responding to renin in the aqueous
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ARVO 2016 Annual Meeting Abstracts
humor. Its role in resistance generation needs to be explored in future
studies.
Commercial Relationships: Jayter S. Paula, None;
Kristin Perkumas, None; Nicole E. Ashpole, None; W D. Stamer,
None
Support: São Paulo Research Foundation (FAPESP # 2014/26163-6)
Program Number: 6418 Poster Board Number: D0138
Presentation Time: 11:00 AM–12:45 PM
IOP-induced Changes in Schlemm’s Canal Dimensions and the
Relation to Shear Stress
Nicole E. Ashpole1, Dibyendu Mukherjee1, Guorong Li2,
Paolo Maccarini1, Joseph M. Sherwood3, C R. Ethier4,
Darryl R. Overby3, Sina Farsiu1, 2, W D. Stamer2, 1. 1Biomedical
Engineering, Duke University, Durham, NC; 2Ophthalmology, Duke
University, Durham, NC; 3Biomedical Engineering, Imperial College,
London, United Kingdom; 4Biomedical Engineering, Georgia
Institute of Technology, Atlanta, GA.
Purpose: In vascular endothelia including Schlemm’s canal (SC),
nitric oxide (NO) is produced by endothelial NO synthase (eNOS),
whose activity and abundance are regulated by shear stress. In
simplified elliptical models of SC, wall shear stress (WSS) at elevated
intraocular pressures (IOP) is calculated to be comparable to those in
large arteries. Using spectral-domain optical coherence tomography
(OCT) imaging of living mice, we create more realistic 3D models
of SC to estimate WSS and thus investigate the relationship between
IOP and shear stress.
Methods: IOP was controlled by intracameral cannulation of
anesthetized (ketamine/xylazine, intraperitoneal injection) C57BL/6
mice (6 months old), while simultaneously imaging iridocorneal
angle structures using OCT (Envisu R2200, 2 µm resolution,
Bioptogen, Inc). Incremental sagittal OCT sections along a 0.2 mm
length of SC were acquired at two IOPs (10 and 15 mmHg), to
create a stack of images (0.0085 mm distance between image slices).
Customized software (Matlab) was used to segment SC on each OCT
image, which was easily visualized at the two selected IOPs. A 3D
SC replica consisting of SC and 2 collector channels was built using
Aviso (FEI) and ANSYS software (ANSYS, Inc.).
Results: When changing IOP in living mice from 10 to 15 mmHg
the SC lumen imaged by OCT reduced by 43% on average from
an integrated volume of 13.8 ± 5.8 µm3 to 7.8 ± 4.8 µm3. This
corresponds to a decrease in SC height from 6.7 µm to 3.8 µm based
on previous elliptical models.
Conclusions: IOP elevation from 10 to 15 mmHg decreases SC
volume, corresponding to a pressure-dependent decrease in SC height
(inner-outer wall separation). Previous elliptical models suggest that
this change will lead to an increase in shear stress to levels capable
of increasing eNOS activity (~2 dynes/cm2). In conjunction with
computational fluid dynamics, our new 3D model of SC at both 10
and 15 mmHg will enable analysis of the fluid flow and estimation of
wall shear stress in Schlemm’s Canal.
Commercial Relationships: Nicole E. Ashpole;
Dibyendu Mukherjee, None; Guorong Li, None; Paolo Maccarini,
None; Joseph M. Sherwood, None; C R. Ethier, None;
Darryl R. Overby, None; Sina Farsiu, None; W D. Stamer, None
Support: National Institute of Health (Grant #EY017007 and
#EY022359)
Program Number: 6419 Poster Board Number: D0139
Presentation Time: 11:00 AM–12:45 PM
ETA receptor upregulation may be associated with ERK signaling
in a rat model of glaucoma
Nolan R. McGrady1, 2, Raghu R. Krishnamoorthy1, 2. 1UNT Health
Science Center, Fort Worth, TX; 2North Texas Eye Research Institute,
Fort Worth, TX.
Purpose: The endothelin system has been shown to play a causative
role in the neurodegenerative effects seen in animal models of
glaucoma. However, the mechanisms leading to neurodegeneration
need to be examined further. The goal of this study was to investigate
the endothelin signaling pathway to determine the contribution
of extracellular signal-regulated kinases 1 and 2 (ERK1/2) to
endothelin-mediated cell death.
Methods: Male retired breeder Brown Norway rats were subjected
to IOP elevation by the Morrison’s method and maintained for 2
and 4 weeks. Retinal sections obtained from the rats were subjected
to immunohistochemical analysis of ETA receptor expression. In
a separate set of experiments, western blots were performed on
transformed 661W cells transiently transfected with either the ETA
receptor or ETB receptor cDNA expression vector. Another set of
experiments was performed with stable clones overexpressing the
ETA receptor. The cells were grown on 100 mm dishes and treated
for 24 hr with 100nM endothelin-1 (ET-1) or endothelin-3 (ET-3).
Immunoblot analysis of levels of endothelin receptor and ERK1/2
phosphorylation was carried out.
Results: An increase in immunostaining for ETA receptors was
observed mainly in the inner plexiform layer and a modest increase
was also observed in the RGC layer which was significant at 4 weeks
of IOP elevation. Cell culture experiments showed an appreciable
upregulation of ETB receptors following overexpression of ETA
receptors and a reciprocal upregulation of ETA receptors following
overexpression of ETB receptors. A 1.8-fold increase in ERK1/2
phosphorylation was observed in stable clones overexpressing ETA
receptors, which was further elevated 2-3 fold after treating cells
with either endothelin-1 or endothelin-3, compared to empty vector
transfected cells.
Conclusions: While the two endothelin receptors may have distinct
functions, there is a significant overlap of the ETA and ETB receptor
mediated signal transduction pathways and there appears to be
some level of cross-talk between the two receptors. While there is
a substantial body of evidence for the pro-survival role of ERKs,
prolonged activation of ERK1/2 has been shown to be associated
with cell death. While the mechanisms are not completely clear, the
current study points to an association of ERK1/2 with cell death
following overexpression of ETA and ETB receptors.
Commercial Relationships: Nolan R. McGrady, None;
Raghu R. Krishnamoorthy, None
Support: NIH Grant EY019952
Program Number: 6420 Poster Board Number: D0140
Presentation Time: 11:00 AM–12:45 PM
BRINZOLAMIDE-TIMOLOL FIXED COMBINATION
AND TRAVOPROST AS MTMT IN PRIMARY OPEN
ANGLE GLAUCOMA: LONG TERM EFFICACY AND
TOLERABILITY
Teresa Rolle1, Laura Dallorto1, Gemma Caterina Rossi2,
Fiamma Campana3. 1Surgical sciences - Eye Clinic, University of
Torino, Torino, Italy; 2IRCCS Policlinico San Matteo, Pavia, Italy;
3
Ospedale Santa Croce e Carle, Cuneo, Italy.
Purpose: To evaluate long-term efficacy of brinzolamide-timolol
fixed combination and travoprost in terms of IOP values, tolerability
and visual impairment in POAG eyes.
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Methods: This was a multicenter, observational cohort, 12 month
study. Glaucomatous patients with different antiglaucomatous
therapies were switched to brinzolamide-timolol fixed combination
twice a day and travoprost 0.004 % once a day as maximum tolerate
medical therapy (MTMT). Complete ophthalmic examination
comprehensive of IOP, BCVA, SAP 24-2 SITA STANDARD, ocular
surface status (tear film break-up time and corneal staining) and
quality of life (OSDI) perception was performed at baseline (T0),
after 6 months (T1) and after 12 months (T2). Statistical analysis was
performed with Friedman test, statistical significance was considered
for p<0.05.
Results: 38 POAG patients (mean age 70.04±9.37ys, 12F/26M) were
enrolled in the study and treated with B+T FC TID and travoprost
0.004% once a day. 42.65% of the 68 eyes included in the study
were in therapy with two drugs and 57.35% received three antiglaucomatous drugs (88% were BAK-preserved drugs). The previous
therapy was changed for lack of efficacy and/or intolerance. B+T FC
TID and travoprost 0.004% were effective in reducing intraocular
pressure: the T1 mean IOP was 16% lower than the T0 mean IOP
(15.68 and 18.76 mmHg respectively, p <0.001). After 12 months
mean IOP value was 15.95 ± 2.76 mmHg (p<0.001). In table 1
BCVA, MD and PSD, BUT and corneal staining mean values are
illustrated. Ocular Surface Disease Index mean value was lower after
6 months and had a further decrease after 12 months (33.62 ± 10.74,
30.32 ±10.45, 28.48 ± 9.85 at T0,T1 and T2 respectively, p<0.001).
Only two patients discontinued the therapy (one for intolerance, the
other for failure to reach target IOP)
Conclusions: After the switch from a previous therapy to a
brinzolamide-timolol fixed combination and travoprost a significant
reduction in IOP was obtained with preservation of functional
parameters (BCVA, MD and PSD SAP values). Furthermore both
quality of life and ocular surface status statistically improved.
Brinzolamide-timolol fixed combination associated with travoprost is
therefore effective and safe for the ocular surface status.
Commercial Relationships: Teresa Rolle, None; Laura Dallorto,
None; Gemma Caterina Rossi, None; Fiamma Campana, None
Program Number: 6421 Poster Board Number: D0141
Presentation Time: 11:00 AM–12:45 PM
Non-linear changes in the pSTR and ERG responses of the rat
retina to visual stimulation for different levels of elevated IOP
Bingyao Tan1, Benjamin MacLellan1, Erik Mason1, Vivian Choh2,
Karen M. Joos3, Kostadinka K. Bizheva1, 2. 1Department of Physics
and Astronomy, University of Waterloo, Waterloo, ON, Canada;
2
School of Optometry and Vision Science, University of Waterloo,
Waterloo, ON, Canada; 3Vanderbilt Eye Institute, Ophthalmology and
Visual Sciences, Vanderbilt University, Nashville, TN.
Purpose: To evaluate the retinal response to visual stimulation at
different levels of elevated IOP.
Methods: One group of eleven-week old male Brown Norway
(n=6) rats were dark-adapted for at least 12 hours before isoflurane
anesthesia. The IOP of the right eye was raised from 10 mmHg to
70 mmHg in steps of 20 mmHg using a vascular loop anterior to
the equator of the eye. The left eye was left untouched and served
as control. Corneas were anesthetized and pupils were dilated
prior to collection of scotopic threshold responses (STRs) or fullfield Electroretinography (ERG) from both eyes simultaneously.
Measurements were conducted at baseline, all steps of elevated IOP
and at recovery, thirty minutes after loop removal.
Results: The pSTRs magnitude increased significantly from baseline
IOP = 10 mmHg to 30mmHg (p<0.0001), and decreased by ~20 %
for 50-mmHg (p=0.0002). As the IOP increased further to 60mmHg,
pSTR amplitude decreased significantly (p=0.0496) relative to
50mmHg and was on average 9.4µV lower than the baseline, though
not significantly (p =0.5903). The STR amplitude at 70-mmHg
was significantly lower than the baseline (p=0.0063) and recovered
to baseline within 30 min after the loop removal. In general,
significantly larger implicit pSTR times (p=0.0010) were measured
at higher IOP, however, there is no significant difference between the
baseline and recovery measurement (p=1.0000).
The ERG a-wave amplitude increased significantly (p<0.0001)
from baseline to 30 mmHg and remained relatively constant up to
60mmHg, then sharply decreased to baseline for IOP= 70 mmHg.
The ERG b-wave amplitude increased significantly from baseline and
peaked 30 mmHg, then progressively declined to baseline until ~55
mmHg. For IOP of 60 mmHg and 70-mmHg, the b-wave magnitude
was significantly lower than baseline (p = 0.0038 and p=0.0013,
respectively).
Conclusions: Results from this study suggest that acute increase of
the IOP can alter the normal retinal physiology, and provide better
understanding of the mechanism of early degeneration in retinal
ganglion cells induced by increased IOP.
STR recordings
Commercial Relationships: Bingyao Tan; Benjamin MacLellan,
None; Erik Mason, None; Vivian Choh, ARVO AP AMPC
member (S); Karen M. Joos, ARVO CME Committee Chair (S);
Kostadinka K. Bizheva, None
Support: Natural Sciences and Engineering Research Council
(NSERC) Discovery Grants, Joseph Ellis Family Glaucoma
Research Fund; William Black Glaucoma Research Fund; NIH
5P30EY008126­27 to Vanderbilt Vision Research Center; Unrestricted
Departmental Grant from Research to Prevent Blindness, Inc., N.Y.
to the Vanderbilt Eye Institute, Canadian Foundation for Innovation
Leaders Opportunity Fund
Program Number: 6422 Poster Board Number: D0142
Presentation Time: 11:00 AM–12:45 PM
Pathway of injectable PEA microfibers to clinical development:
preclinical safety evaluation in support of First-in-Man clinical
study
Madalina Natu Tavares, Mirian Gillissen, George Mihov. DSM
Biomedical, Geleen, Netherlands.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Purpose: Compliance with topical drugs for the treatments for ocular
hypertension and glaucoma is poor, and therefore sustained release
dosage forms are desired to advance the treatment of these diseases.
Subconjunctival administration of DSM’s polyesteramide (PEA)
microfiber drug delivery implants have the potential to deliver the
active compound over a multi-month period of time and degrade
safely in the ocular tissue (M. Kropp et al. 2014, J. Thies et al. 2014).
To advance DSM’s PEA microfiber into first-in-man studies, key
safety and tolerability attributes in GLP toxicology studies were
evaluated.
Methods: New Zealand white rabbits were used to evaluate safety
and tolerability in GLP toxicology studies for 12 months. Single dose,
triple dose, placebo and sham groups were included in the study.
Clinical ophthalmic examinations (slit-lamp biomicroscopy, indirect
ophthalmoscopy) and intraocular pressure measurements (IOP) were
performed and examinations utilized a modified McDonald-Shadduck
scoring system. Terminal investigations included histopathlogy
analysis, while in vivo microfiber degradation was evaluated at 4, 6, 9
and 12 months.
Results: Gross ocular observations found ocular swelling and
irritation in most study animals one day after test article implantation,
which resolved over the following days. These observations were
seen in all groups, including control and sham groups, indicating that
they were likely due to the implantation procedure and unrelated to
the test article. Clinical ophthalmic examinations similarly found
conjunctival congestion and swelling in the days immediately
following test article implantation in most study animals, a finding
that resolved at later time points. Later findings of occasional
conjunctival congestion, swelling, and discharge were scattered, not
systematically correlated with any group, and generally resolved
quickly. No adverse events were observed over the course of the
study suggesting both the microfiber and its degradation products are
well tolerated. No adverse changes in IOP were observed during the
first 6 months of the study.
Conclusions: Subconjunctival administration of DSM’s
polyesteramide (PEA) microfibers resulted in safety and tolerability
observations which were mild and transient.
Conjunctival swelling in single dose group
Conjunctival swelling in triple dose group
Commercial Relationships: Madalina Natu Tavares, DSM
Biomedical; Mirian Gillissen, DSM Biomedical; George Mihov,
DSM Biomedical
Program Number: 6423 Poster Board Number: D0143
Presentation Time: 11:00 AM–12:45 PM
Correlation among parameters of aqueous humor dynamics and
biometrics in healthy Chinese adults
Shan Fan1, 2, Tao Guo2, 3, Sruthi Sampathkumar4, Fang Wang2,
Carol B. Toris1, 4. 1Ophthalmology, University of Nebraska Medical
Center, Omaha, NE; 2Ophthalmology, Tenth People’s Hospital of
Tongji University, Shanghai, China; 3No. 9 People’s Hospital of
Shanghai Jiaotong University School of Medicine, Shanghai, China;
4
Case Western Reserve University, Cleveland, OH.
Purpose: This study investigates correlations among and between
parameters of aqueous humor dynamics and biometric measurements
in young and old healthy Chinese adults.
Methods: This prospective, single center study of healthy Chinese
volunteers was divided into young (between 20 and 30 years of age,
n=32), and old (50 years of age and older, n=36) groups. Aqueous
humor dynamics assessments were intraocular pressure (IOP),
aqueous flow (Fa), episcleral venous pressure (EVP), outflow facility
(C) and uveoscleral outflow (Fu). Biometric measurements included
anterior chamber depth (ACD), anterior chamber volume (ACV),
axial length and central cornea thickness (CCT). Parameters were
analyzed using GraphPad Prism, and SPSS19- linear mixed effects
model. Averaged values from both eyes of each volunteer were used
to assess associations between two parameters by Pearson correlation
analysis. Data are represented as mean ± SD. Statistical significance
was set at p < 0.05.
Results: Irrespective of age, a positive linear correlation was
present between IOP and EVP [r (66)=0.43, p<0.001], Fa and Fu [r
(62)=0.35, p=0.005], Fa and C [r (65)=0.28, p=0.02], EVP and Fu [r
(56)=0.32, p=0.01] and Fa and ACV [r (65)=0.32, p=0.008]. Negative
linear correlation occurred between Fu and C [r (56)= -0.6, p<0.001].
In the old group, correlations occurred between CCT and Fa [r (34) =
-0.36, p = 0.03] and ACV and C [r (34)=0.4, p=0.02] but not in young
heathy individuals.
Conclusions: The interplay among parameters of aqueous humor
dynamics suggests the possible presence of autoregulatory
mechanisms in the eye required for IOP maintenance. With higher
EVP, IOP increases. When trabecular outflow facility is low, outflow
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ARVO 2016 Annual Meeting Abstracts
favors the uveoscleral path of lesser resistance. The more aqueous
humor produced, the more drains through the uveoscleral outflow
pathway and the higher the outflow facility. With aging, correlations
among parameters of AHD and ocular biometrics increase
underlining the importance of ocular biometrics in the study of
aqueous humor dynamics and aging.
Commercial Relationships: Shan Fan, None; Tao Guo, None;
Sruthi Sampathkumar, None; Fang Wang, None; Carol B. Toris,
None
Support: Shanghai municipal commission of health and family plan
fund (20124100); RPB
Program Number: 6424 Poster Board Number: D0144
Presentation Time: 11:00 AM–12:45 PM
Pressure-independent outflow in ex vivo mouse eyes
Michael Madekurozwa, Joseph M. Sherwood, Ester Reina-Torres,
Jacques A. Bertrand, Darryl R. Overby. Bioengineering, Imperial
College London, London, United Kingdom.
Purpose: Mice are commonly used for studies of aqueous humor
dynamics, but it has been reported that up to 80% of outflow is
pressure-independent, which would typically be attributed to nontrabecular routes. Such high non-trabecular outflow would invalidate
the mouse as a model for human outflow, which is primarily
trabecular. This study aims to directly measure pressure-independent
outflow at zero pressure, Q0, in ex vivo mouse eyes. As the flowpressure relationship is often assumed to be linear, we also examined
whether non-linear behaviour could contribute to the appearance of
pressure-independent outflow.
Methods: 8 pairs of enucleated eyes from C57BL/6 mice aged 9-16
weeks were perfused with PBS+5.5mM glucose using the iPerfusion
system. Pressure steps of 0, 3, 6, 9, 12, 15, and 18 mmHg were
applied while measuring intraocular pressure (P) and the flow rate
(Q) into the eye. The contralateral eye received the same pressure
regimen with an artificial pressure-independent inflow of 120 nl/min
infused with a syringe pump. This was chosen to mimic reported in
vivo rates of aqueous production minus pressure-independent outflow.
To examine the non-linearity of the Q-P relationship, 66 additional
unpaired eyes were perfused at 4-20 mmHg. Q-P data were fit by
linear (Q = C P + Q0) and non-linear (Q =Cr (P / Pr)βP + Q0) models
where C is the facility and Cr the facility at Pr = 8 mmHg. β is an
index of non-linearity.
Results: Non-zero values of β (0.75 ± 0.63; mean ± 2SD, n=66)
confirmed that the Q-P relationship was non-linear. For the artificial
inflow cases, Q0 was not significantly different from zero when
accounting for the resolution of the flow meter (-6 ± 2 nl/min, n=8),
and Q0 was not significantly different from 120 nl/min with infusion
(-115 ± 8 nl/min, n=8). There was a strong correlation between the
non-linearity parameter β and Q0 predicted by the linear model
(p<10-6, n=66), suggesting that non-linearity in the Q-P relationship
introduces an artificial pressure-independent outflow.
Conclusions: There is negligible pressure-independent outflow in
enucleated eyes from young C57BL/6 mice. Non-linearity in the
Q-P relationship contributes to the artificial appearance of pressureindependent outflow when analysed using a linear model. The
form of the Q-P relationship thus influences interpretation of the
mechanism and pathway of outflow, and assumptions regarding
linearity or constancy of outflow facility should be more carefully
evaluated.
Commercial Relationships: Michael Madekurozwa;
Joseph M. Sherwood, None; Ester Reina-Torres, None;
Jacques A. Bertrand, None; Darryl R. Overby, None
Support: We thank the donors of National Glaucoma Research, a
program of the BrightFocus foundation
Program Number: 6425 Poster Board Number: D0145
Presentation Time: 11:00 AM–12:45 PM
Membrane Cholesterol Differentially Regulates TRPV4 DrugChannel Efficacy and Osmotic-Evoked Swelling in Müller
Astroglia
Anthony Iuso1, 2, Andrew Jo2, Oleg Yarishkin2, Alen Delic2,
David Krizaj1, 2. 1Neuroscience, University of Utah, Salt Lake City,
UT; 2Ophthalmology, University of Utah, Salt Lake City, UT.
Purpose: The polymodal TRPV4 cation channel is a promiscuous
cellular sensor capable of integrating various stimuli, such as
temperature, osmolality and tensile forces from the extracellular
milieu. However, how these diverse stimuli regulate channel gating
and what role, if any, the lipid bilayer has in the regulation of
gating, has been a long-standing question in the field. To address
it, we studied how TRPV4 activation in retinal neurons, Müller
glia and heterologous HEK293 overexpressors – induced through
pharmacological agents, hypotonicity and heat – is impacted
following the sequestration of membrane cholesterol with methyl-βcyclodextrin (mβCD).
Methods: Membranous cholesterol was removed from TRPV4-OE
HEK293 cells and dissociated retinal cells via incubation with 10mM
mβCD or filipin (4 ug/ml). In a subset of experiments, the cholesterol
content was replenished by incubating cells with cholesterol-loadedmβCD (1:10mM). Optical Ca2+ imaging with ratiometric indicator
Fura-2 (10μM) was used to measure [Ca2+]i. The extent of cell
swelling due to osmotic challenge was determined by changes in
fluorescence resulting from intracellular volume changes.
Results: Lowering membrane sterol content was sufficient to
attenuate agonist (GSK1016790A) and hypotonicity-evoked
[Ca2+]i elevations in TRPV4-OE HEK293 cells and Müller
cells, whereas responses to heat were unaffected. Ca2+ signaling
deficits were rescued by cholesterol restoration delivered through
cholesterol:mβCD complex substitution, arguing against nonspecific
consequences of depletion. In the absence of membrane cholesterol,
TRPV4 antagonist (HC067047) lost its effectiveness as an inhibitor
of hypotonically evoked, TRPV4-mediated [Ca2+]i responses, which
however were blocked by the nonspecific Ca2+ channel pore blocker,
gadolinium. Cholesterol-depleted Müller cells exhibited a reduced
capacity for swelling, slow swelling kinetics and an absence of
regulatory volume decrease.
Conclusions: Our findings suggest that TRPV4 activation requires
the formation of local, cholesterol-enriched, lipid microdomains
that are likely to regulate the energy barrier for conformational
switches effected by pharmacological agents and hypo-osmotic
stretch. We conclude that cholesterol saturation and the membrane
lipid environment are important regulators of retinal-glial TRPV4
signaling, calcium homeostasis and volume regulation.
Commercial Relationships: Anthony Iuso, None; Andrew Jo,
None; Oleg Yarishkin, None; Alen Delic, None; David Krizaj,
None
Support: NIH T32EY024234
Program Number: 6426 Poster Board Number: D0146
Presentation Time: 11:00 AM–12:45 PM
Real-time Visualization of AR-13324 Effects on Schlemm’s Canal
and Scleral Vessels in Living Mice
Guorong Li1, Dibyendu Mukherjee1, Iris Navarro1, Sina Farsiu1,
Pedro Gonzalez1, Casey Kopczynski2, W D. Stamer1. 1Department
of Ophthalmology, Duke Eye Center, Durham, NC; 2Aerie
Pharmaceuticals, Inc., Durham, NC.
Purpose: The goals of this study were to (i) monitor the effects of
a topical rho kinase inhibitor (AR-13324) on conventional outflow
tissues using spectral domain-optical coherence tomography (SD-
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ARVO 2016 Annual Meeting Abstracts
OCT) at controlled pressure levels in living mouse eyes and (ii) test
new software to quantify changes.
Methods: Two strains of young mice (C57 and CD1, 2-5 months old)
were given AR-13324 or placebo topically. Schlemm’s Canal (SC)
was imaged by OCT while intraocular pressure (IOP) was held at
10, 15 or 30 mmHg. New software was developed to quantitatively
assess changes in cross sectional area of SC and scleral vessels.
Effects of AR-13324 on fluorescent tracer deposition in anterior
segment flat mounts, IOP and outflow facility were also determined.
Results: AR-13324 significantly lowered IOP by 5.1±0.5 mmHg,
corresponding to an increase in outflow facility of 166% ± 33% in
C57 mice. Fluorescence due to tracer deposition in outflow tissues
was increased by 2.9-fold in the presence of AR-13324. When
holding IOP at 10 mmHg, the average area of SC was 381.8±51.5
µm2, dilating by 132% ± 14%, upon AR-13324 treatment. At elevated
IOPs, AR-13324 prevented SC collapse. Moreover, AR-13324
increased the relative speckle variance intensity and total area of
scleral vessels conducting aqueous humor by 173% ± 29% and 193%
± 33% respectively. Reproducibility of the software to quantify
SC area was 91.7 % ± 7.4 % and 99.7 % ± 4.4 % (inter- and intraobserver). Similar results were observed in CD1 mice.
Conclusions: IOP-lowering of AR-13324 in living mice works
predominantly by increasing perfusion of conventional outflow
tissues. The changes of SC lumen and flow pattern in scleral vessels
can be effectively monitored by OCT and discriminated by our
newly developed software. This study in living animals is a first step
towards the development of OCT technology to monitor glaucoma
drug treatment responses in humans.
Commercial Relationships: Guorong Li, None;
Dibyendu Mukherjee, None; Iris Navarro, None; Sina Farsiu,
None; Pedro Gonzalez; Casey Kopczynski, Aerie Pharmaceuticals,
Inc; W D. Stamer, Aerie Pharmaceuticals, Inc. (F), Aerie
Pharmaceuticals, Inc (C)
Support: BrightFocus Foudation
Program Number: 6427 Poster Board Number: D0147
Presentation Time: 11:00 AM–12:45 PM
Aging changes in aqueous humor dynamics and ocular biometrics
of SPARC null mice
Sruthi Sampathkumar, Yuxi Zheng, Min Hyung Kang,
Douglas J. Rhee, Carol B. Toris. Ophthalmology & Visual Sciences,
Case Western Reserve University, Cleveland, OH.
Purpose: SPARC (secreted protein, acidic, cysteine rich) is a
matricellular protein involved in regulation of extracellular matrix,
cell adhesion, migration and collagen I deposition. Previous studies
show that SPARC null (KO) mice exhibit lower intraocular pressure
(IOP) and a more uniform outflow than wild-type (WT) mice. This
study investigated the effects of SPARC deletion on aqueous humor
dynamics (AHD) in aging mice.
Methods: Studied were SPARC WT and KO mice of 3 age groups
(a, 4-8 weeks; b, 3-5 months; c, 20-28 months). AHD included
IOP by rebound tonometer, aqueous flow (Fa) by a modified
fluorophotometer and outflow facility (C) by a multi-level constant
pressure perfusion method. Anterior segment optical coherence
tomography (AS-OCT) was used to measure central corneal thickness
(CCT) and anterior chamber depth (ACD). AS-OCT images were
segmented semi-automatically to determine the volumes of the
cornea (Kv) and anterior chamber (ACv). Unpaired t-tests and
ANOVA were used to compare differences among strains and age
groups respectively.
Results: Refer Table 1 for a complete description of results. Key
findings include; 1. Lower IOP in young KO mice compared to WT
counterparts. This difference is lost with aging, 2. Thicker central
corneas in young KO mice, 3. Absence of aging associated reduction
in Fa in KO mice.
Conclusions: SPARC deletion abrogates the normal aging changes
seen in WT mice, namely a decrease in aqueous flow, increase in
outflow facility, AC deepening and an increase in CCT that maintains
IOP unchanged in the WT animals. Absence of these aging changes
along with cataract development in all KO mice eventually fails to
keep IOP lower than in WT mice. In KO mice, an increase in ACv
with no accompanying changes in ACD indicates that the peripheral
AC might be widening secondary to loss of lens volume from
leaky mature cataracts. The lower IOP of young SPARC KO mice
cannot be explained by outflow facility or by corneal changes. Since
collagen I is a major structural component of the cornea and sclera,
biomechanical factors might affect measured IOP. These findings
support the conclusion that SPARC plays a role in normal physiologic
changes that maintains IOP with aging.
Table 1. Statistical significance (p<0.05) is indicated as * for
comparisons between WT and KO mice, 1; a and b, 2; a and c, and 3; b
and c. Results are represented as mean±SD (n).
Commercial Relationships: Sruthi Sampathkumar, None;
Yuxi Zheng, None; Min Hyung Kang, None; Douglas J. Rhee,
None; Carol B. Toris, None
Support: Research to Prevent Blindness
Program Number: 6428 Poster Board Number: D0148
Presentation Time: 11:00 AM–12:45 PM
The effects of isoflurane and ketamine:xylazine on the scotopic
threshold response (STR) in albino rats with elevated intraocular
pressure (IOP)
Akshay Gurdita1, Karen M. Joos3, Bingyao Tan2, Yunwei Feng1,
Kostadinka K. Bizheva1, 2, Daphne L. McCulloch1, Vivian Choh1.
1
School of Optometry and Vision Science, University of Waterloo,
Waterloo, ON, Canada; 2Physics and Astronomy, University
of Waterloo, Waterloo, ON, Canada; 3Vanderbilt Eye Institute,
Ophthalmology and Visual Sciences, Vanderbilt University,
Nashville, TN.
Purpose: To compare the scotopic threshold response (STR) of
acute, moderately-elevated intraocular pressure (IOP) in the retinas
of Sprague Dawley rats under isoflurane or ketamine:xylazine (kx)
anesthesia.
Methods: Two group of rats, isoflurane anesthetized (n=9) and kx
anesthetized (n=6) underwent acute IOP elevation to 35 mmHg using
a vascular loop around the treated eye anterior to the equator, for one
hour, while the other eye served as a control. Binocular STRs were
recorded prior to loop wear, 45 minutes into the IOP elevation, and
45 minutes after removal of the loop.
Results: The positive STR (pSTR) amplitude was greater with kx
than with isoflurane and increased during IOP elevation with both
anesthetics. For isoflurane anaesthetized rats, peak pSTRs (mean
± S.D.: 61.2 ± 39.9 μV) were greater than those before loop wear
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ARVO 2016 Annual Meeting Abstracts
(6.4 ± 6.4 μV; p<0.0001) and those after loop removal (10.1 ±7.5
μV; p<0.0001) with no significant difference in pSTR between
the pre- and post-wear conditions (p=1.000). For control eyes,
pSTR amplitudes did not change over the course of the experiment
(p=1.000 for all comparisons). In kx-anesthetized animals, pSTR
amplitudes during loop wear (158.5 ± 81.7 μV) were also greater
than those before loop wear (58.3 ± 37.9 μV, p<0.0001). However,
post-loop pSTR amplitudes remained elevated and were not
significantly different from those during loop wear (126.4 ± 62.2 μV,
p=0.1347) and were also larger than pre-loop values (p=0.0007). The
control eye for the kx anesthetized rats also changed over the course
of the experiment, with post loop pSTR amplitudes larger than those
prior to loop wear (compare 115.5 ± 55.3 μV vs 58.4 ± 34.6 μV,
respectively; p=0.0028).
Conclusions: IOP elevation to 35 mmHg is associated with an
increase in the pSTR response irrespective of the anaesthetic
used. Additionally, although kx anesthesia resulted in larger pSTR
amplitudes, the anaesthetic was associated with a significant systemic
increase in the control eye pSTR amplitude. Thus, the anesthesia
used may be important in electrophysiological studies that depend on
consistent control eye values over the time course of the experiment.
Commercial Relationships: Akshay Gurdita; Karen M. Joos,
ARVO CME Committee Chair (S); Bingyao Tan, None;
Yunwei Feng, None; Kostadinka K. Bizheva, None;
Daphne L. McCulloch, None; Vivian Choh, ARVO AP AMPC
member (S)
Support: Natural Sciences and Engineering Research Council
(NSERC) Discovery Grants, University of Waterloo CIHR
Research Incentive Fund; University of Waterloo Propel Centre
Disease Prevention Initiative Seed Grant; Joseph Ellis Family
Glaucoma Research Fund; William Black Glaucoma Research
Fund; NIH 5P30EY008126-27 to Vanderbilt Vision Research
Center; Unrestricted Departmental Grant from Research to Prevent
Blindness, Inc., N.Y. to the Vanderbilt Eye Institute
Program Number: 6429 Poster Board Number: D0149
Presentation Time: 11:00 AM–12:45 PM
Retrospective evaluation of visual fields and pattern-ERG
in glaucoma worsening patients treated with forskolin,
homotaurine, and L-carnosine
Caterina Gagliano1, 2, Antonio Longo2, Maurizio G. Uva2,
Roberta Amato1, Teresio Avitabile2, Ganluca Scuderi3,
Giulia Malaguarnera1. 1Ophthamology, NEST (Neurovisual Science
Technology), Catania, Italy; 2Ophthalmology, University of Catania,
Catania, Italy; 3Ophthalmology, University La Sapienza Roma,
Roma, Italy.
Purpose: Development of glaucoma neuroprotective treatment
is therefore a pressing unmet medical need. We carried out an
observational retrospective clinical study to analyze the synergic
neuroprotective effects of forskolin, homotaurine and L-carnosine
(Gangliolife®) in glaucoma worsening patients.
Methods: We retrospectively recruited 25 patients with Primary
Open Angle Glaucoma (POAG) who last year had a progressive
worsening of visual field (Humphrey field analyzer SITA standard
threshold test 24-2) and IOP < 18 mmHg. Evaluation of the P-ERG
with Retimax® with the ISCEV parameters was performed. The
endpoint were:
- Stabilization of the visual field indices (SITA Standard 24-2,
Glaucoma Staging System GSS-2) after a period of 24 months.
- Amplitude increase> 2:00 uV (P50-N95 wave) in pattern
electroretinogram (P-ERG) after a period of 24 months.
Results: Significant MD average increase (beta 0:07, p = 0.038) was
observed (costant trend see fig. 1).
In the first six months, the value of MD remains fairly stable
(differences 12:18, p = 0.690). At 9 months you will notice a
significant increase from baseline that is maintained to 12 months and
at 18 months the value of MD decrease again. The change between
baseline and 24 months is of borderline significance (1.55, p = 0.058).
PSD significant mean decrease (beta -0.07, p <0.001) was noted
(Fig.2). Unlike the pre period in which the value of the PSD tended
to increase, during the study the PSD decreases significantly already
at 6 months and continues to decrease up to 18 months. At 24 months
there seems to be a slight increase (but the value is still significantly
lower than the baseline).
PERG avarage increase (0.08 - p <0.001) was observed (Fig. 3). The
increase is significant already at 6 months (+1.23, p = 0.001). The
value of the P-ERG continues to grow up to 18 months. At 24 months
we observed a decrease, but the value is still significantly higher than
baseline.
Conclusions: We showed that a combined treatment with forskolin,
homotaurine, and L-carnosine affords neuroprotection in glaucoma
worsening patients. Steady state in visual field parameters (MD, PSD)
and significant increase of P50-N95 wave (P-ERG) were the clinic
data collected. All that reported confirm the synergic neuroprotective
effect on RGC survival recently observed in vivo experimental
model.
Commercial Relationships: Caterina Gagliano; Antonio Longo,
None; Maurizio G. Uva, None; Roberta Amato, None; Teresio Avitabile, None; Ganluca Scuderi, None; Giulia Malaguarnera, None
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