Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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. 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 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. 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: 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 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 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 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 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 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 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/ to access the versions of record. 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 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 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. 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 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 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 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- 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 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 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 (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 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.