The Effect of Dietary Nitrate Supplementation on Retinal

The eﬀect of dietary nitrate supplementation on retinal vessel responses in young healthy subjects

Posterboard#: A0120

Abstract Number: 5725 - A0120

AuthorBlock: Naim Terai1, Fabian Helbig1, Lisa Ramm1, Richard P. Stodtmeister1, Lutz E. Pillunat1 1Ophthalmology, University of Dresden, Dresden, Germany;

DisclosureBlock: Naim Terai, None; Fabian Helbig, None; Lisa Ramm, None; Richard P. Stodtmeister, None; Lutz E. Pillunat, None;

Purpose In peripheral arterial disease, dietary nitrate supplementation enhances patients exercise performance via an increase in nitric oxide (NO) bioavailability and an improvement of tissue oxygenation. Therefore, the aim of the present study was to investigate whether an oral intake of inorganic nitrate in form of beetroot juice may have an impact on retinal vessel responses in young healthy subjects as a sign of an increased NO bioavailability in the endothelium of retinal vessels.Methods In a prospective, placebo-controlled randomized study, retinal vessel response was measured in 62 healthy subjects before and two hours after oral intake of either beetroot juice (group I, 200 ml) or apple juice (group II, 200 ml) as a placebo. Static and dynamic vessel analysis was performed with the Dynamic Vessel Analyzer (DVA, Imedos Systems UG). Intraocular pressure (IOP), systemic blood pressure and blood glucose level were obtained before and after oral intake of both juices.Results Mean age was 25.4 ± 2.3 years and 25.1 ± 2.8 years in group I and II with no statistical significant difference between both groups (p = 0.665). Systemic blood pressure, blood glucose level and IOP did not change significantly after supplementation of beetroot juice or apple juice. Dilatation responses of the arterioles and the venules (+ 3.84 ± 2.3 % and + 4.57 ± 2.96 %) did not change significantly after intake of beetroot juice (+ 3.76 ± 1.88 % and + 4.66 ± 2.26 %, p = 0.624 and p = 0.752). Also, the constriction responses of the arterioles (- 1.08 ± 1.18 %) did not change significantly after beetroot juice intake (- 0.990 ± 1.40 %, p = 0.310). Static vessel analysis revealed non-significant changes of the central retinal artery equivalent (197.42 ± 23.5 µm to 195.88 ± 15.1 µm, p = 0.477) and the central vein equivalent (221.19 ± 29.4 µm to 218.02 ± 17.18 µm, p = 0.848).Conclusions In the present study retinal vessel responses, as measured by the DVA, were not affected by dietary nitrate supplementation in young healthy subjects. It might be hypothesized that an unaffected endothelial function as seen in young subjects may not show any further improvement of vessel responses after additional nitrate intake. However, further studies should elucidate in how far patients with an endothelial dysfunction (e.g. glaucoma) may benefit from an nitrate supplementation.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Retinal venous pressure at fixed airway pressure levels.

Posterboard#: A0121

Abstract Number: 5726 - A0121

AuthorBlock: Richard P. Stodtmeister1, Soﬁe Heimann1, Naim Terai1, Lutz E. Pillunat1 1Ophthalmology, TU Dresden, Dresden, Germany;

DisclosureBlock: Richard P. Stodtmeister, None; Soﬁe Heimann, None; Naim Terai, None; Lutz E. Pillunat, None;

Purpose A weak correlation between airway pressure (AirP) and retinal venous pressure (RVP) increase was observed in a previous study in which the subjects themselves determined the AirP during the Valsalva maneuver (VM). We hypothesize that a closer correlation may be seen when the RVP is measured at fixed AirP levels.Methods 43 healthy volunteers (table 1) were investigated. Instruments used: RVP by contact lens dynamometry (CLD). IOP by dynamic contour tonometry. AirP by aneroid manometer. Blood pressure by auscultation and aneroid manometer. Examination procedure: IOP, pupil dilation, IOP, BP, assessment of the spontaneous venous pulsation (SVP) of the central retinal vein by direct ophthalmoscopy, RVP measurement in absent SVP, measurement of IOP and RVP during enhanced AirP at 4 fixed levels of AirP: 10, 20, 30 and 40 mmHg. At each pressure level 4 CLD readings were taken and the median was calculated. The sequence of the AirP levels was randomized.Results Table 2 shows the RVP, the difference RVP- IOP and the IOP before and during the VM at the fixed pressure levels. AirP before the VM is zero by definition. The values of the 4 single CLD readings maximally differed by 0.0(0.0;1.8)mmHg) [Median(Q1;Q3)] at AirP 0 mmHg. This difference rose to 3.3(1.9;5.0)mmHg at AirP 40mmHg. The RVP increase was steepest from AirP 0 mmHg to AirP 10 mmHg. Then the RVP increase flattened. RVP and AirP showed a good correlation (Spearmans ρ=0.64, p<0.05). The range of the RVP values was wide. The increase of the IOP with increasing AirP was clearly smaller than that of the RVP and was maximally 1.9 mmHg at an AirP of 40Conclusions AirP and RVP showed a good correlation. This finding is in contrast to our earlier results and may be due to the wider range of the AirP in the present study. The increase in IOP was small in comparison to results in literature. This may be explained by the RVP and IOP measurement already 3 seconds after the onset of the VM. The scatter of the 4 single CLD readings increased with the AirP during the VM which may be caused by the agitation of the subjects due to the effort in enhancing the AirP. However, the scatter was too small to cause the wide range of the RVP. It can be assumed that the variability of the venous anatomy proximal of the jugular vein may be responsible for the scatter of the RVP during the VM. Also, the interindividual variability of the compliance of these vessels may have increased this effect.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Clinical circulation research in ophthalmology is mainly concerned with the arterial side of the circulation. The primary reason may be seen in the fact that there was no method of measuring the venous pressure in the eye until recently. Therefore, the assumption was made, that the introcular venous pressure equals the intraocular pressure whose measurement is clinical routine. Twentyfive years ago, however, it was demonstrated that it is possible to measure the retinal venous pressure. Six years ago we could show that the retinal venous pressure may be higher than the intraocular pressure in about one third of glaucoma patients. Thus the assumption: “intraocular venous pressure equals introcular pressure” has to be modified: we have to discriminate between the retinal venous pressure and the choroidal venous pressure. In the study presented here we show that the retinal venous presssure goes up much higher than the intraocular pressure when the airway pressure is enhanced like in trumpet playing. It may be concluded from our results that the retinal circulation is much more influenced by enhancing the airway pressure than assumed until now. Characteristics of Retinal Blood Flow, Velocity and Vessel Diameter of eyes with Branch Retinal Vein Occlusion as revealed by Doppler Optical Coherence Tomography Flowmeter

Posterboard#: A0122

Abstract Number: 5727 - A0122

AuthorBlock: Kengo Takahashi1, YS Song1, Kenji Sogawa1, Takafumi Yoshioka1, Tomofumi Tani1, Takanari Wada1, Masahiro Akiba2, Jun Sakai2, Syunsuke Nakamura2, Kana MINAMIDE2, Satoshi Ishiko1, Yasuo Yanagi1,3, Akitoshi Yoshida1 1Asahikawa Medical University, Asahikawa, HOKKAIDO, Japan; 2R&D division, Topcon Corporation, , Japan; 3Singapore National Eye Centre, , Singapore;

DisclosureBlock: Kengo Takahashi, None; YS Song, None; Kenji Sogawa, None; Takafumi Yoshioka, None; Tomofumi Tani, None; Takanari Wada, None; Masahiro Akiba, Topcon, R&D division Code E (Employment), Jun Sakai, Topcon, R&D division Code E (Employment), Syunsuke Nakamura, Topcon, R&D division Code E (Employment), Kana MINAMIDE, Topcon, R&D division Code E (Employment), Satoshi Ishiko, None; Yasuo Yanagi, None; Akitoshi Yoshida, Topcon, R&D division Code P (Patent)

Purpose Doppler optical coherence tomography flowmeter (DOCT flowmeter) instantaneously (< 3sec) analyses rheological properties of retinal blood vessels, including measurement of absolute retinal blood flow (RBF), blood velocity and vessel diameter. Herewith, we report the characteristics of RBF, blood velocity and vessel diameter on the occluded and the non-occluded vein in eyes with branch retinal vein occlusion (BRVO) and the vein in healthy eyes as measured by DOCT flowmeter.Methods Twenty patients with unilateral BRVO were enrolled. Using DOCT flowmeter, RBF, blood velocity, and diameter were measured from 3 veins, i.e., 1. an occlude vein, 2. a non-occluded vein in BRVO eyes and 3. a vein in the fellow eye (non-affected vein), in each patient. Paired t-test was employed to compare the parameters of DOCT flowmeter and Pearson chi-square test were used to test the association between RBF and vessel diameter. Bonferroni corrected P < 0.05 was considered statistically significant.Results Mean patient age was 65.2 ± 9.8 yrs. Mean systolic and diastolic blood pressure were 141.6 ± 19.8 mmHg and 80.1 ± 11.9 mmHg, respectively. The RBF in the occluded veins (4.7 ± 3.5 µL/min) was lower than that in the non-occluded veins (11.0 ± 5.0 µL/min) and that of the fellow eyes (8.9 ± 3.4 µL/min) (p < 0.01). There were no significant differences in the blood velocity among the three types of veins. (p > 0.1). The diameter in the occluded vein (96.1 ± 25.5 µm) was smaller than the one in the non-occluded (130.7 ± 24.1 µm) and the healthy veins (123.3 ± 15.0 µm) (p< 0.01). Additionally, the RBF was correlated with the diameter in the occluded vein (p< 0.05, r=0.46).Conclusions DOCT, for the first time in BRVO patients, disclosed that the RBF of the occluded veins was significantly lower than the other veins and the RBF in the occluded vein was correlated with the vessel diameter, suggesting that RBF reduction in the occluded veins of BRVO depends on vasoconstriction of vessels but not on decrease in blood velocity.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. The role of Acid-sensing ion channel 1a in a mouse model of ischemic retinopathy.

Posterboard#: A0134

Abstract Number: 5739 - A0134

AuthorBlock: Ayumi Ouchi1,4, Edith Aguilar1, Kyle Vincent Marra1, Min Qiang2, Yang Guang2, Richard Lerner3,2, Martin Friedlander1 1Molecular Medicine, The Scripps Research Institute, La Jolla, California, United States; 2Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, , China; 3Chemistry, The Scripps Research Institute, La Jolla, California, United States; 4Ophthalmology, Juntendo University School of Medicine, , Japan;

DisclosureBlock: Ayumi Ouchi, None; Edith Aguilar, None; Kyle Vincent Marra, None; Min Qiang, None; Yang Guang, None; Richard Lerner, None; Martin Friedlander, None;

Purpose Acid-sensing ion channel 1a (ASIC1a) has a critical role in neuronal cell death under acidic conditions such as ischemia or metabolic stress. This study examined the role of ASIC1a in a mouse model of ischemic retinopathy and acidosis-induced retinal cell death in vitro.Methods Oxygen-induced retinopathy (OIR) was induced on C57BL/6J mice via exposure to 75% oxygen from postnatal day 7 (P7) to P12 and subsequent transfer to room air. ASIC1a-blocking combinatorial antibody ASC06-IgG1 developed through the screening of human monoclonal antibody libraries (Qiang M et al. PNAS 2018) was injected intravitreally to OIR eyes. The area of vaso-obliteration (VO), neovascularization (NV) and the number of GFAP positive astrocytes were quantified in ASC06-IgG1 or control antibody treated OIR retina at various time points. We also evaluated retinal astrocytes to determine the mechanism of action of ASC06-IgG1. Primary human retinal astrocytes (HRA) were cultured in pH7.4 or pH5.5 medium with addition of ASC06-IgG1 or an isotype control. Astrocyte viability and apoptosis were measured by CCK-8 assay after 24 hours and flow cytometry for PI and Annexin V staining after 6 hours, respectively.Results In OIR, P12 injection of ASIC1a-blocking antibody ASC06-IgG1 significantly decreased the percentage retinal area of VO and NV at P17 by 55% and 41%, respectively, relative contralateral eyes treated with isotype control (p<0.0001). When injected into P7 eyes, ASC06-IgG1 significantly decreased the area of VO in OIR retina by 34% at P10 and 28% at P14. At the same time, the reduction in the number of astrocytes in the VO area was inhibited by ASC06-IgG1 treatment compared to control during hyper- and hypoxic phases (P10; Control: 67% ASC06-IgG1: 5%, P14; Control: 64% ASC06- IgG1: 38% p<0.0001). ASC06-IgG1 treated HRA showed 24% reduction in acidosis-induced apoptosis, and the viability of HRA with ASC06-IgG1 treatment under pH5.5 acidic condition increased by 20% in comparison to isotype control treated HRA (p<0.05, p<0.0001).Conclusions Our data suggest that blocking ASIC1a prevents hyperoxia-induced vessel degeneration and hypoxia- derived retinal neovascularization. Blocking ASIC1a may preserve astrocytes during hyper- and hypoxic phases in OIR, thus providing protection from acid-induced astrocyte cell death.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Theoretical predictions of oxygenation in a heterogeneous vascular network of the retina

Posterboard#: A0123

Abstract Number: 5728 - A0123

AuthorBlock: Lucas Rowe1, Alon Harris1, Brendan C. Fry3, Alice Chandra Verticchio Vercellin4,5, Brent A. Siesky1, Julia Arciero2 1Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, United States; 2Mathematical Sciences, IUPUI, Indianapolis, Indiana, United States; 3Mathematical and Computer Sciences, Metropolitan State University of Denver, Denver, Colorado, United States; 4Ophthalmology, University of Pavia, Pavia, Italy; 5IRCCS - Fondazione Bietti, Rome, Italy;

DisclosureBlock: Lucas Rowe, None; Alon Harris, CIPLA Code C (Consultant) , AdOM Code C (Consultant) , AdOM Code I (Personal Financial Interest) , Oxymap Code I (Personal Financial Interest) , Shire Code C (Consultant) , Brendan C. Fry, None; Alice Chandra Verticchio Vercellin, None; Brent A. Siesky, None; Julia Arciero, None;

Purpose This study uses theoretical modeling to predict blood and tissue oxygenation in a heterogeneous vascular network of the mouse retina for different levels of tissue oxygen demand.Methods Confocal microscopy images of the mouse retina have revealed a very complex and non-uniform distribution of blood vessels supplying the retina. Here, a realistic model of oxygen transport is adopted which explicitly represents the interactions among vessels and tissue in a vascular network with non-uniform geometry. By the conservation of mass, oxygen diffusion in tissue is equated to oxygen consumption in tissue, which is assumed to follow Michaelis-Menten kinetics. Vessels are modeled as discrete oxygen sources, and the tissue regions are considered as oxygen sinks. The resulting oxygen concentration at any tissue point is calculated by summing the oxygen fields produced by each of the surrounding blood vessels.Results Figure 1 (Panel A) shows the model-predicted contour map of the oxygenation of the arteriolar network and surrounding tissue under well-oxygenated conditions (incoming arterial saturation to all branches is 96%). In Panel B, the inflow saturation in one of the six arteriolar branches is reduced to

33%. As a result of this simulated oxygen impairment, average tissue PO2 in the entire network decreased from 67.2 mmHg to 61.7 mmHg. Importantly, the minimum tissue PO2 dropped from 18.7 mmHg to 8.1 mmHg. Thus, tissue oxygenation in one branch of the retina is predicted to decrease by nearly 56% following a 66% reduction in arterial blood saturation.Conclusions By incorporating a realistic model of oxygen transport within the multi-layer and heterogeneous geometry of the retina, this model allows, for the ﬁrst time, more accurate predictions of retinal oxygenation in response to changes in oxygen demand, arterial saturation, viscosity, or hematocrit.

The results of this study highlight the critical point that the average value of PO2 in the retina is a poor indicator of network oxygenation. Many terminal arteriolar vessels will have abnormally low PO2 levels despite reasonable average values in the overall network, which can lead to areas at risk of hypoxia—an eﬀect that would not be observed in a non-heterogeneous description of the network.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your ﬁndings, not the study itself and the associated details.

A rapid technique to quantify retinal oxidative stress and the protection provided by novel nitroxide-based antioxidant / anti-inﬂammatory compounds

Posterboard#: A0135

Abstract Number: 5740 - A0135

AuthorBlock: Nigel L. Barnett2,3, Steven E. Bottle1, Jason Tong3, Komba Thomas1, Cassie L. Rayner3 1Queensland University of Technology, Brisbane, Queensland, Australia; 2Faculty of Health Sciences & Medicine, Bond University, Gold Coast, Queensland, Australia; 3Queensland Eye Institute , South Brisbane, Queensland, Australia;

DisclosureBlock: Nigel L. Barnett, None; Steven E. Bottle, None; Jason Tong, None; Komba Thomas, None; Cassie L. Rayner, None;

Purpose To develop a rapid screening technique to evaluate the capacity of novel nitroxide-based antioxidant compounds to reduce oxidative stress in cultured retinal cells, and to analyse the neuroprotection offered by a candidate compound in an in vivo model of retinal injury.Methods Cultured 661W mouse photoreceptor cells were incubated (45 min) with a novel mitochondrially- targeted fluorescent probe (methyl ester tetraethylrhodamine nitroxide, ME-TRN) that is reversibly responsive to cellular redox status. Oxidative stress was induced with antimycin (AMC, 1 µM) following incubation (30 min) with a nitroxide (5,6-dicarboxy-1,1,3,3-tetraethylisoindolin-2-yloxyl, DCTEIO; 2KT109A or 1KT141D) or lutein antioxidant. ME-TRN fluorescence was quantified by flow cytometry. The effects of DCTEIO were then assessed in vivo. Unilateral retinal ischemia-reperfusion (I/R) was induced in anaesthetized rats by increasing intraocular pressure (110mmHg for 1hr). DCTEIO (20 mg/kg i.p.) was administered 1 hour prior and 1 hour after I/R. An additional intravitreal injection (2µl, final conc. 100 µM) was given 30 mins into the reperfusion phase. Animals recovered for 8 days prior to assessment of retinal function by scotopic flash electroretinography (ERG).Results Untreated 661W cells converted the probe from a non-fluorescent (oxidized) state to a fluorescent (reduced) state. Oxidative stress decreased mean fluorescence intensity by 50%. Antioxidant data were normalized to the maximal effect of AMC and expressed as the % amelioration of the AMC- induced change in mean fluorescence. Lutein (10 µM) significantly blunted the effects of AMC by 82±23%, p=0.0002, n=8, ANOVA; DCTEIO (nitroxide antioxidant, 500 µM) by 62±13%, p=0.0002, n=5; 2KT109A (indomethacin-nitroxide hybrid, 10 µM) by 97±12%, p=0.0003, n=5, and 1KT141D (aspirin-nitroxide hybrid, 100 µM) by 92±10%, p=0.0004, n=5. A significant reduction in ERG a-wave amplitude was induced by ischemia-reperfusion, which was ameliorated by DCTEIO (I/R 265±48 µV, I/R+DCTEIO 451±43 µV, p=0.0011, n=6).Conclusions Flow cytometry with a reversible fluorescent probe for oxidative stress is an effective tool to assess antioxidant compounds in retinal cells. Novel nitroxide antioxidant compounds significantly ameliorated the effects of oxidative stress on 661W cells and may provide an effective neuroprotective strategy in vivo.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Measurement of flicker induced hyperemia in the retina and optic nerve head by Laser Speckle Flowgraphy

Posterboard#: A0124

Abstract Number: 5729 - A0124

AuthorBlock: Doreen Schmidl1, Klemens Fondi1, Ahmed Bata1, Nikolaus Luft2,3, Katarzyna Witkowska1, René M. Werkmeister4, Leopold Schmetterer1,5, Gerhard Garhofer1 1Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria; 2Department of Ophthalmology, Kepler University Hospital, Linz, Austria; 3University Eye Hospital, Ludwig-Maximilians- University , Munich, Germany; 4Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria; 5Singapore Eye Research Institute, , Singapore;

DisclosureBlock: Doreen Schmidl, None; Klemens Fondi, None; Ahmed Bata, None; Nikolaus Luft, None; Katarzyna Witkowska, None; René M. Werkmeister, None; Leopold Schmetterer, None; Gerhard Garhofer, None;

Purpose Ocular blood flow regulation during stimulation with flicker light has been extensively investigated using a variety of different techniques. The aim of the present study was to evaluate whether Laser speckle flowgraphy (LSFG), a new commercially available technique for measuring ocular blood flow, is capable to assess flicker-induced hyperemia in the retina and optic nerve head (ONH). Methods Twenty healthy subjects (mean age 25±7 years) participated in this cross-sectional study. Retinal and ONH blood flow was measured before and during stimulation with flicker light. Mean blur rate (MBR) in the ONH (MBRONH) and relative flow volume (RFV) in retinal arteries (RFVART) and veins (RFVVEIN) were assessed using LSFG. Results

Stimulation with flicker light induced a significant increase in MBRONH by +17.5%±6.6% (p<0.01), as well as in RFVART by +23.8±10.0% (p<0.05) and in RFVVEIN +23.1%±11.0 (p<0.05). When comparing retinal sectors, the observed response was higher in nasal RFV compared to temporal RFV in arteries (nasal: +28.9%±20.0%; temporal: +20.4%±17.6%, p<0.05) and also in veins (nasal: +28.3%±19.6%; temporal +17.8%±18.9%, p<0.05). Conclusions As expected, flicker stimulation led to a significant increase in retinal and ONH blood flow. These results indicate that LSFG is an appropriate method to study flicker-induced hyperemia in the retina and ONH and may be applied as a non-invasive, easy to use tool to assess neurovascular coupling in the future.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Delayed trans-scleral electrical stimulation preserved the survival of retinal ganglion cells after ischemia-reperfusion injury in mice model

Posterboard#: A0136

Abstract Number: 5741 - A0136

AuthorBlock: LIN Youhong1, Vincent Wing Hong Lee1, Kin Chiu1,2 1Ophthalmology, HKU, Hong Kong, China; 2State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong, Hong Kong;

DisclosureBlock: LIN Youhong, None; Vincent Wing Hong Lee, None; Kin Chiu, None;

Purpose Trans-corneal electrical stimulation (TcES) has shown neuroprotective effects on retina under stress. Compares to TcES, the pads of trans-scleral electrical stimulation (TsES) were placed on the sclera without touching cornea, will not damage the cornea even when applied repeatedly. In this study, we studied the effect of TsES on retinal ganglion cells (RGCs) with 24 hours delayed application in a mouse model of retinal ischemia-reperfusion injury.Methods Adult C57BL/6J mice were divided into 4 groups: A) normal; B) sham; C) pad; D) TsES group. Retinal ischemia was induced by cannulation of the anterior chamber with a 33G needle connected to a sterile fortified balanced salt solution (BSS Plus) bag. The water level of BSS bag was set at 170 cmH2O above the eye level to create a consistent hydrostatic pressure for 60 minutes. And intraocular pressure (IOP) was monitored during the ischemia procedure. At 24 hours after the reperfusion, a pair of tailor-made gold pads were placed at the medial and lateral canthus of the mouse eye. In the TsES group, the parameters are frequency at 20 Hz, power at 100 microA for 30 mins, while no ES was applied in the pad group. Retina samples were collected at 7 days after the ischemic injury. Morphological changes of retinal neurons were analyzed in cross sections and flat mounted retinas.Results Under general anesthesia, IOP measured by Tonolab was 84±4.6 mmHg during ischemic insult.60- minute acute retinal ischemia followed by reperfusion for 7 days could induce nearly 60% of RGC loss. The survival rate of RGC in the TsES group was about 10% more than that in the pad control group.Conclusions Twenty-four hours delayed TsES has neuroprotective effect on ischemia-reperfusion induced RGC loss. This treatment window is very important to patients with such ischemia insult or acute glaucoma.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Real-time evaluation of systemic-ocular microcirculation using laser speckle flowgraphy in white rabbits

Posterboard#: A0125

Abstract Number: 5730 - A0125

AuthorBlock: Tetsuya Komatsu1, Tomoaki Shiba1, Yoshinobu Nagasawa2, Megumi Aimoto2, Kiyoshi Sakuma2, Tatsuo Chiba2, Xin Cao2, Akira Takahara2, Tadashi Matsumoto1, Yuichi Hori1 1Opthalmology, Toho university, Ota city, Tokyo, Japan; 2Pharmacology, Toho university, , Japan;

DisclosureBlock: Tetsuya Komatsu, None; Tomoaki Shiba, None; Yoshinobu Nagasawa, None; Megumi Aimoto, None; Kiyoshi Sakuma, None; Tatsuo Chiba, None; Xin Cao, None; Akira Takahara, None; Tadashi Matsumoto, None; Yuichi Hori, HOYA Code F (Financial Support), Senju Code F (Financial Support), KOWA Code F (Financial Support), Menicon Code F (Financial Support)

Purpose To establish systemic-ocular microcirculation using laser speckle flowgraphy (LSFG) in white rabbits and investigate the circulatory response to a systemic adrenaline load.Methods We used six normal adult male New Zealand white rabbits (16 weeks of age, 2.8–3.3 kg). After intramuscular administration of xylazine (5 mg/kg) and ketamine (35 mg/kg), the rabbits were intubated and administered vaporized isoflurane (1.5-2.0%, room air 2 L/min). Surgical indwelling needles were inserted into the right and left auricular veins; these were used for infusion and drug administration. Heparinized arterial cannulas were placed in the right brachial and right rectal arteries for measurement of blood pressure while the blood flow rates of the right femoral artery and the left common carotid artery were measured using laser Doppler to determine blood flow volume in each artery. Mean blur rate (MBR) of the retinal vessels and choroid area were measured using LSFG- rabbit™ (Softcare, Fukuoka, Japan), developed for animals. Continuous intravenous adrenaline (0.2 mL/kg /min; 100-1000 ng/kg) was administered for 10 minutes, and changes in each index were observed for 30 minutes after the start of administration.Results The MBR increased in the retinal vessels and choroid area in a dose-dependent manner with the adrenaline load. The Δ mean blood pressure showed a significant correlation (R = 0.37 to 0.77) with each area of ΔMBR. The Δ carotid blood flow volume showed a significant negative correlation with Δ retinal blood vessel MBR at 100-300 ng/kg dose and the Δ femoral arterial blood flow volume showed a significant positive correlation with Δ choroid area of MBR at each dose. Conclusions We successfully demonstraited a real-time evaluation system for systemic-ocular microcirculation using LSFG in white rabbits. A systemic adrenaline load caused an increase in MBR, associated with elevation in blood pressure.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Longitudinal Measurements of Changes in Retinal Blood Flow in Feline Retinal Vein Occlusion Model Measured by Doppler Optical Coherence Tomography and Optical Coherence Tomography Angiography

Posterboard#: A0126

Abstract Number: 5731 - A0126

AuthorBlock: Takanari Wada1, YS Song1, Tsuneaki Omae1, Kenji Sogawa1, Takafumi Yoshioka1, Seigo Nakabayashi1, Kengo Takahashi1, Tomofumi Tani1, Akihiro Ishibazawa1, Satoshi Ishiko1, Yasuo Yanagi1,2, Akitoshi Yoshida1 1Asahikawa Medical University, Asahikawa, Hokkaido, Japan; 2Singapore National Eye Centre, , Singapore;

DisclosureBlock: Takanari Wada, None; YS Song, None; Tsuneaki Omae, None; Kenji Sogawa, None; Takafumi Yoshioka, None; Seigo Nakabayashi, None; Kengo Takahashi, None; Tomofumi Tani, None; Akihiro Ishibazawa, None; Satoshi Ishiko, None; Yasuo Yanagi, None; Akitoshi Yoshida, None;

Purpose We established retinal vein occlusion (RVO) model using argon laser and observed longitudinal changes in retinal blood flow (RBF) and microvasculature using Doppler optical coherence tomography flowmeter (DOCT flowmeter)and optical coherence tomographyangiography (OCTA).Methods Branch RVO (BRVO) was induced by Argon green laserphotocoagulation (PC) in 6 eyes of 6 cats.After successful occlusion of the retinal vein was confirmed with OCTA, RBFwas measured from a first-order retinal artery and vein by DOCT flowmeter, and microvascular changes around the occluded vessels (12x12mm and 3x3mm) was assessed by OCTA before (at baseline) and right after PC and 1, 4, 7, 14 days thereafter. Systemic and ocular parameters such as mean blood pressure (MABP), heart rate (HR), percutaneous oxygen saturation (SpO2), intra ocular pressure (IOP), ocular perfusion pressure (OPP) were monitored during the measurements. One-way analysis of variance (ANOVA) was used for repeated measurements, followed by post hoc comparisons with the Dunnett procedure.Results There were no significant differences in any systemic and ocular parameters before and after PC. Arterial RBF (aRBF) increased significantly on day 1 (160.6 ±21.1% vs baseline, P < 0.05) and decreased under the baseline level after day 1 through day 14. Venous RBF (vRBF) decreased right after PC (17.4 ±23.5 % vs baseline, P < 0.05) and then gradually increased afterwards, but did not return to the baseline level. OCTA disclosed dilatation of retinal venules immediately after PC to day 1. Collateral vessels began to form on day 4 and became matured on day 7. The collateral vessels were pruned on day 14, and formed as mature as normal retinal venule diameters.Conclusions Arterial RBF increased within 1 day after inducing RVO. However, both arterial and venous RBF ended up decreasing during the observation period. Venules gradually expanded to form collateral vessels after PC, suggesting that collateral vessels originate from existing capillary networks but not by angiogenesis. The feline RVO model, combined with retinal flow analysis, has possibilities to elucidate the pathogenesis of RVO.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Regulation of retinal blood flow in response to an experimental increase in intraocular pressure

Posterboard#: A0127

Abstract Number: 5732 - A0127

AuthorBlock: Kornelia Schutzenberger1, Stefan Puchner1, Laurin Ginner1, Doreen Schmidl2, Gerold C. Aschinger1, Gerhard Garhofer2, Rainer A. Leitgeb1, Leopold Schmetterer1,3, Rene Marcel Werkmeister1 1Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria; 2Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria; 3Singapore Eye Research Institute, Singapore National Eye Centre, Singapore, Singapore;

DisclosureBlock: Kornelia Schutzenberger, None; Stefan Puchner, None; Laurin Ginner, None; Doreen Schmidl, None; Gerold C. Aschinger, None; Gerhard Garhofer, None; Rainer A. Leitgeb, None; Leopold Schmetterer, None; Rene Marcel Werkmeister, None;

Purpose There is evidence from several studies that retinal blood flow (RBF) is autoregulated in response to changes in ocular perfusion pressure (OPP). The present study set out to investigate RBF autoregulation during an experimental increase in intraocular pressure (IOP) using a custom-built Doppler optical coherence tomography (OCT) system.Methods Healthy male and female subjects were included in this open study. Retinal blood flow was measured at baseline using Doppler OCT. Thereafter, IOP was stepwise increased by a suction cup. Retinal blood flow was assessed at each step and systemic hemodynamics were evaluated every minute. After a resting period of 30 minutes, application of the suction cup was repeated and IOP was measured at each level of suction using applanation tonometry. OPP was calculated as OPP = 2/3 MAP-IOP.Results Fifteen healthy subjects aged between 23 and 32 years participated in the present study. Application of the suction cup induced a significant increase in IOP and a decrease in OPP (p < 0.01). During the stepwise increase in IOP, the decrease in RBF was less pronounced than the decrease in OPP (maximum decrease in OPP: -94.1 ± 8.8% , maximum decrease in RBF: -59.4 ± 20.8% from baseline, respectively).Conclusions Experimental increase in IOP induced a significant decrease in RBF. This decrease was less pronounced than the decrease in OPP, indicating some degree of retinal autoregulation. This is in line with previous studies using different techniques for assessment of retinal blood flow. Therefore, Doppler OCT seems to be a valuable technique for investigation of RBF autoregulation in humans.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details.

Raspberry Pi controlled ﬂicker stimulation in dynamic retinal vessel analysis

Posterboard#: A0128

Abstract Number: 5733 - A0128

AuthorBlock: Dietmar Link1, Sascha Klee1 1Biomedical Engineering & Informatics, Technische Universitaet Ilmenau, Ilmenau, Germany;

DisclosureBlock: Dietmar Link, None; Sascha Klee, None;

Purpose In recent years, flood-illumination fundus cameras have been increasingly equipped with LEDs for retinal illumination and stimulation. Segmented LED light sources have been introduced to realize spatial adaptation to reducing stray light effects in order to improve fundus image quality. We present a Raspberry Pi controlled 24-fold segmented surface-mount LED light source for fundus cameras. To test its performance and reliability we performed a comparative study. The new setup was tested by means of dynamic retinal vessel analysis against the Gold standard.Methods We studied 15 young volunteers (9f, 6m, 24.8±1.89 years) all free of ocular or systemic disease. We examined four vessel segments: one superior temporal artery and vein (STa/STv), one inferior temporal artery and vein (ITa/ITv), as well. For statistical analysis, the relative maximum vaso- dilatation value was used. The light source was connected to a mydriatic fundus camera. The Raspberry Pi was programmed to run the standard flicker protocol (50s baseline, 3 cycles of 20s flicker stimulation and 80s constant illumination). The light source pattern was annular (all segments on). Each volunteer was measured two times. One measurement with the Gold standard and one with the new setup, in a random order. To allow sufficient recovery of retinal function the study incorporated a rest period of 10 minutes between the two measurements. Comparison of the two setups were demonstrated and analyzed using the Bland-Altman method. To value the differences among the group means we performed a paired t-test. The Shapiro-Wilk test was used for testing on normal distribution.Results The comparison analysis according to Bland-Altman resulted in the following mean differences (MD) and Limits of Agreement (LoA) (Gold standard vs. new setup). STa: MD=-0.300%, LoA=-2.57%/+1.97. ITa: MD=0.810%, LoA=-1.65%/+3.27. STv: MD=0.291%, LoA=-0.989%/+1.57. ITv: MD=0.521%, LoA=-1.436%/+2.48. The t-test p-values were 0.432 (STa), 0.085 (ITa), 0.189 (STv), and 0.082 (ITv). p≥0.05 means no significant difference.Conclusions We successfully performed dynamic retinal vessel analysis in humans controlling a miniaturized surface-mount device LED light source by means of a Raspberry Pi. The differences in the maximum dilatation values of the new setup compared to the Gold standard were not statistically significant. The new setup can be used for dynamic retinal vessel analysis.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. The effect of hyperoxia and hypercapnia on retinal vascular blood flow in healthy adults

Posterboard#: A0137

Abstract Number: 5742 - A0137

AuthorBlock: Keiko Yamada1,2, Brendan Seto1, Christopher Llerena1, Christopher Hsu1, Chie Sotozono2, Takatoshi Maeno3, Jorge G. Arroyo1 1Beth Israel Deaconess Medical Center, Massachusetts, United States; 2Ophthalmology, Kyoto Prefectural University of Medicine, , Japan; 3Ophthalmology, Toho University Sakura Medical Center, , Japan;

DisclosureBlock: Keiko Yamada, None; Brendan Seto, None; Christopher Llerena, None; Christopher Hsu, None; Chie Sotozono, None; Takatoshi Maeno, None; Jorge G. Arroyo, None;

Purpose We used two non-invasive imaging devices including spectral domain optical coherence tomography (SD-OC) and laser-speckle flowgraphy (LSFG) to measure retinal arteriolar vasoreactivity using both normobaric hyperoxia and hypercapnia.Methods Imaging consisted of optic nerve head photography under the tracking mode of SD-OCT (Heidelberg Spectoralis, Heidelberg Engineering, Germany) and retinal vascular imaging using the LSFG (LSFG- NAVI, SoftCare, Fukuoka, Japan). Subject initially sat for 15 minutes before taking baseline retinal images. They then inhaled CO2 rich air for 3 minutes before and during follow-up retinal images. After breathing room air for 15 minutes, subjects inhaled 12 L/min of Oxygen for 15 minutes using a facemask before taking a final set of images. In order to include both usual exhalation and inspiration periods, each measurement was performed three times while maintaining a respiratory rate constant at approximately one breath every 5 seconds.Results This study studied eighteen retinal arteries (RA) of 2 male and 1 female non-smoking healthy volunteers (mean age: 47±9 years old). The mean RA diameters at baseline was 132.69±20.28 μm and did not vary significantly by subject (F = 1.25, p = 0.31). The changes in diameter from baseline to hypercapnia (mCO2 / Air = 1.08, p < 0.05) and baseline to hyperoxia (mO2 / Air = 0.912, p < 0.05) were statistically significant.Conclusions Hypercapnia and hyperoxia had an opposite effect on retinal vasoreactivity. This change in vessel diameter may have an effect on vessel blood flow and oxygenation. Further research should examine the difference between retinal arterial dilation and constriction in patients with a variety of retinal conditions.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Comparison of Retinal Blood Velocity Measurements using Non-invasive Retinal Imagers

Posterboard#: A0129

Abstract Number: 5734 - A0129

AuthorBlock: Kyoung-A Cho1, Edmund Arthur2, Yici Jing1, Amit Guruprasad1, Seyed Hamed Haghshenas1, Abhishek Rege1, Delia Cabrera DeBuc2 1Vasoptic Medical Inc., Baltimore, Maryland, United States; 2Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, Florida, United States;

DisclosureBlock: Kyoung-A Cho, Vasoptic Medical Inc. Code E (Employment), Edmund Arthur, Vasoptic Medical Inc. - Grant Code F (Financial Support), Yici Jing, Vasoptic Medical Inc. Code E (Employment), Amit Guruprasad, Vasoptic Medical Inc. Code E (Employment), Seyed Hamed Haghshenas, Vasoptic Medical Inc. Code E (Employment), Abhishek Rege, Vasoptic Medical Inc. Code I (Personal Financial Interest), Vasoptic Medical Inc. Code P (Patent), Vasoptic Medical Inc. Code F (Financial Support), Vasoptic Medical Inc. Code E (Employment), Delia Cabrera DeBuc, Vasoptic Medical Inc.- Grant Code F (Financial Support)

Purpose To compare retinal blood flow velocity (BFV) measurements between the retinal function imager (RFI) and the XyCAM ID, which uses laser speckle contrast imaging, in an in-vitro blood flow model and healthy subjects.Methods A custom in-vitro model was designed with features comparable to the normal human eye. The back surface contained a tube connected to a calibrated syringe pump controlling the flow of human whole blood. The imagers simultaneously captured the model set at a range of known velocities. Ten eyes from ten healthy subjects were also imaged using both devices, and vessels were chosen from the optic disk and macula using reference fundus images (Figure 1). BFVs were computed from the in- vitro setup, arterioles and venules in the RFI using backscattered light from erythrocyte movement by tracing these vessels. The average, standard deviation (SD), and coefficient of variation (CV) of the BFVs from the RFI-generated data were computed. All vessels with CV>0.45 were considered unreliable and excluded from the analysis. BFV from the model and corresponding vessels were computed from the XyCAM ID by analyzing the blurring of laser speckles to obtain BFV over 6 seconds of acquisition. BFV values were averaged across all pixels in each selected vessel segment and the mean BFV of a cardiac cycle was used for analysis. Pearson product moment correlation assessed the association between the two devices.Results Significant association was found for BFV in the in-vitro setup between the two devices (Figure 2), r=0.80, p<0.001. In human subjects, the overall mean BFV across 63 vessels was 3.11±0.99 mm/s for the RFI and 9.35±1.28 a.u. for the XyCAM ID. The average CV was 30.5±10.6% for RFI measurements and 4.4±4.0% for the XyCAM ID measurements. The BFVs of the arterioles (3.14±0.91 mm/s, 9.36±0.93 a.u.) and venules (3.08±1.08 mm/s, 9.34±1.56 a.u.) were not significantly different, p>0.05 for the RFI and XyCAM ID respectively.Conclusions Significant correlations were found between the two non-invasive retinal BFV measurement techniques for the in-vitro set up; however, due to the large CV, comparison of clinical data between the two devices was infeasible. The XyCAM ID, with a low CV of BFV measurements, may find application in research and eventually clinical diagnostics of retinal pathophysiology.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Noninvasive High-resolution Measurement of Retinal Blood Velocity and Flow

Posterboard#: A0130

Abstract Number: 5735 - A0130

AuthorBlock: Cherilyn Mae Acorda Palochak1,2, Hee Eun Lee1, Stephen A. Burns3, Amani A. Fawzi1 1Ophthalmology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States; 2Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States; 3School of Optometry, Indiana University, Bloomington, Indiana, United States;

DisclosureBlock: Cherilyn Mae Acorda Palochak, None; Hee Eun Lee, None; Stephen A. Burns, None; Amani A. Fawzi, None;

Purpose We used Adaptive Optics Scanning Laser Ophthalmoscopy (AOSLO) to noninvasively image red blood cell flow through small caliber retinal vasculature and validate its utility in accurately quantifying retinal vessel size, blood velocity, and flow.Methods We used AOSLO with split-detection (Boston Micromachines, Cambridge, MA) to perform XT imaging, which involved scanning a line repeatedly across a target vessel at 15kHz. The horizontal axis within an XT scan represents retinal scan position. The vertical axis represents time. Erythrocytes passing along the scan line produce diagonal streaks, representing erythrocyte flowing across the scan over time. Two vertical intensity gaps were used to measure vessel diameter, taking into account angle α (Fig 1). Arteries and veins were identified by the direction of the diagonal streaks, either away from or towards the optic disc, respectively. Velocity was calculated using vessel geometry. Large variations in the velocity of a target vessel, representing pulsatility related to the cardiac cycle (max and min values), were averaged to estimate average velocity. Flow was calculated by combining the areal cross section with velocity. Two graders analyzed and validated the results.Results Twelve healthy subjects were imaged, with a total of 13 eyes, 19 arteries, 9 veins, and 50 targets. Measured arterial diameter ranged from 16-95µm. Velocity and flow ranged from 4-29mm/sec and 0.06-12µL/min, respectively. Measured veins ranged from 15-47µm with velocity and flow ranges of 3-15mm/sec and 0.05-1.4µL/min, respectively. Blood flow varied with diameter by a power of 3.16 (Fig 2). Ocular pulse was observed in arteries and veins, with average velocity fluctuations of 5 and 2.4µm/sec, respectively. In one patient, we measured the flow in arterial branches before and after a bifurcation. Calculated time-averaged blood flow rate before the bifurcation was 5.94µL/min and the sum of the daughter vessels after was 5.95µL/min, consistent with physical expectations.Conclusions Our results are consistent with previous studies that demonstrate the linear relationship of blood velocity and diameter, and exponential variation of flow with diameter. Ocular pulse was observed by velocity fluctuations in vessel cross sections, consistent with the cardiac cycle. These results further validate AOSLO measurements and the potential to detect early vascular flow changes associated with retinal disease.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details.

Dilatation of the retinal capillary plexuses in branch retinal vein occlusion

Posterboard#: A0131

Abstract Number: 5736 - A0131

AuthorBlock: Christopher Patrick Long1, Mathieu Bakhoum1, Christopher Brian Toomey1, Dirk-Uwe G. Bartsch1, Eric Nudleman1, Michael Henry Goldbaum1, K Bailey Freund3, David Sarraf2, William R. Freeman1 1University of California, San Diego, San Diego, California, United States; 2University of California Los Angeles, California, United States; 3Vitreous Retina Macula Consultants NY, New York, United States;

DisclosureBlock: Christopher Patrick Long, None; Mathieu Bakhoum, None; Christopher Brian Toomey, None; Dirk-Uwe G. Bartsch, None; Eric Nudleman, None; Michael Henry Goldbaum, None; K Bailey Freund, None; David Sarraf, None; William R. Freeman, None;

Purpose The retinal capillary system in the macula is arranged into three distinct layers, the superficial, intermediate and deep capillary plexus (SCP, ICP and DCP). Yet, the exact path of blood flow has been a matter of conjecture. A parallel organization implies independent arterial supply and venous drainage occurring at each layer. An in-series system implies arterial inflow to the SCP which then feeds into the ICP and then DCP where venous outflow originates. By utilizing optical coherence tomography angiography (OCTA) which is depth-resolved, we sought to examine whether the DCP is preferentially dilated in eyes with venous occlusion.Methods This was a retrospective case series that included patients presenting with branch retinal vein occlusion (BRVO). All patients had OCTA imaging. Exclusion criteria included poor quality OCTA imaging and any underlying macular pathology. En face OCTA was performed using the Angiovue (Optovue, Fremont, California, USA). For each eye, a 3x3 mm macular scan was obtained. Automated OCTA segmentation was performed and accurate segmentation was confirmed for each case. Capillary diameter was measured using diameterJ (open source plugin for NIH ImageJ, Hotaling et al. Biomaterials 2015; 61:327-38). Individual segments in the SCP images were obtained and analyzed after excluding large vessels. Changes in capillary diameter within the affected hemisphere were established by comparing them to the capillary diameter in the normal hemisphere within the same eye. The same measurements were repeated in the contralateral eyes that served as negative controls.Results Six out of 10 patients (2 male and 4 female, mean age of 67 years), met the inclusion criteria. SCP diameter in the affected hemisphere increased by 4.2% (-4.1% to 8.6%), vs a decrease of 1.7% (-5.3% to 3.7%) in the contralateral eye, p = 0.03. DCP diameter in the affected region increased by 9.4% (-0.03% to 18.8%), vs 1.3% (-1.8% to 2.8%) in the contralateral eye, p = 0.02. The overall change in DCP diameter was greater than that of the SCP (9.4% vs 4.2%, p = 0.11).Conclusions In BRVO, both the SCP and DCP are dilated. Capillary dilatation was more pronounced in the DCP, albeit statistically insignificant. While this may suggest an in-series arrangement of the retinal vascular plexuses, independent autoregulation of each plexus may account for their relative susceptibility to dilatation.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Blood flows from arteries to veins. The intermediate structures, called capillaries, allow oxygen diffusion from the blood to the neighboring tissue. In the retina, capillaries are arranged into distinct layers. Here we utilize high resolution imaging that in patients with obstruction in the venous system

the capillaries are dilated. Xanthohumol protects corneal epithelial cells against oxidative stress in vitro

Posterboard#: A0138

Abstract Number: 5743 - A0138

AuthorBlock: Samatha Ankireddy1, Harsh Nilesh Hariani2, Karoline Anna Orloﬀ1, Avinash Kolli3, Jenni J. Hakkarainen4, Anita K. Ghosh2,5, Simon Kaja3,5 1Department of Molecular Pharmacology and Therapeutics, Loyola University Medical Center, Maywood, Illinois, United States; 2Graduate Program in Neuroscience, Loyola University Medical Center, Maywood, Illinois, United States; 3Department of Ophthalmology, Loyola University Medical Center, Maywood, Illinois, United States; 4Research and Development, Experimentica Ltd., Kuopio, Finland; 5Research and Development, eyeNOS Inc., Oak Park, Illinois, United States;

DisclosureBlock: Samatha Ankireddy, None; Harsh Nilesh Hariani, None; Karoline Anna Orloff, None; Avinash Kolli, None; Jenni J. Hakkarainen, Experimentica Ltd. Code F (Financial Support), Experimentica Ltd. Code I (Personal Financial Interest), Experimentica Ltd. Code E (Employment), Experimentica Ltd. Code R (Recipient), Experimentica Ltd. Code S (Non-remunerative), Anita K. Ghosh, K&P Scientific LLC Code R (Recipient), eyeNOS Inc. Code F (Financial Support), eyeNOS Inc. Code I (Personal Financial Interest), eyeNOS Inc. Code P (Patent), eyeNOS Inc. Code R (Recipient), eyeNOS Inc. Code S (Non-remunerative), Simon Kaja, K&P Scientific LLC Code F (Financial Support), K&P Scientific LLC Code I (Personal Financial Interest), K&P Scientific LLC Code P (Patent), K&P Scientific LLC Code S (Non-remunerative), Experimentica Ltd. Code F (Financial Support), Experimentica Ltd. Code I (Personal Financial Interest), Experimentica Ltd. Code R (Recipient), Experimentica Ltd. Code S (Non-remunerative)

Purpose Xanthohumol (Xn) is a natural compound found in the hops plant (Humulus Lupulus) and recognized as a potent antioxidant. The purpose of this study was to determine the cytoprotective effects of Xn against oxidative stress in human corneal epithelial cells in vitro.Methods Human Corneal Epithelial cells (HCE-T; Riken; Japan) were exposed to concentrations from 1 nM to 100 μM Xanthohumol and cell viability and proliferation were assessed using the lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake assays in order to establish the in vitro toxicity profile. We next exposed HCE-T to chemically- induced exogenously-applied oxidative stress by treating cells with tert-butyl hydroperoxide (tBHP) for 6 hr. Cells were pretreated for 24 hr with Xanthohumol concentrations from 0.1 μM to 5 μM Xanthohumol, vehicle (0.01% DMSO) or remained untreated. Cytoprotective effects were assessed using MTT and LDH assays. Analysis of calcein-AM uptake and kinetics of the fluorescence were used to determine effects of Xn on P-glycoprotein 1, using cyclosporine A as a positive control.Results Xn did not show any cytotoxicity up to 10 μM as determined by LDH and MTT assays. In cytoprotection experiments, pre-treatment with Xn resulted in a statistically significant dose- dependent protection of HCE-T cells from tBHP-induced oxidative stress compared with vehicle- treated cells. The EC50 of tBHP shifted from 15.2 ± 0.5 μM (untreated, n = 4) to 33.3 ± 3.4 μM (5 μM, n = 4, P < 0.01) in the MTT assay and, similarly, in the LDH assay (13.4 ± 0.4 μM vs.100.0 ± 11.7 μM; n = 4; P < 0.01). Changes in expression levels of components of the endogenous antioxidant system, including the intracellular target of Xn, Nrf2, in response to tBHP are currently being investigated.Conclusions Xn exerts potent dose-dependent cytoprotective effects against oxidative stress in HCE-T cells. Our in vitro findings support testing Xn in preclinical models for DED, either as monotherapy or in combination with established anti-inflammatory treatment modalities.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Cellular response of human meningothelial cells to oxidative stress

Posterboard#: A0139

Abstract Number: 5744 - A0139

AuthorBlock: Xiaorong Xin1 1Qinghai Red Cross Hospital, Xining, China;

DisclosureBlock: Xiaorong Xin, None;

Purpose Meningothelial cells (MECs) are fundamental cells of the sheaths covering the brain and optic nerve, but their exact roles remain unclear.The present study was designed to investigate the inﬂuence of hydrogen peroxide (H2O2) induced-oxidative stress on cellular functional changes of MECs.Methods

Cell viability was measured after MECs were exposed to diﬀerent concentrations of H2O2 (0, 25, 50, 100, 150, 250 µM) for 12h, 24h, 36h and 48h respectively. The intracellular reactive oxygen species

(ROS) level was determined using the DCFH-DA probe following cells were treated with 150 µM H2O2 for 24h and 48h respectively. Proteomic changes in H2O2-triggered stress were comparatively analyzed using a two-dimensional gel electrophoresis (2DE) and mass-spectrometry- (MS-) based proteomic approach.Results Our study showed that cell viability of MECs was inhibited after cells were exposed to oxidative agents. Cells treated with H2O2 for 24h exhibited an elevation of ROS activity compared with control cells(p <0.05). ROS level was significantly increased in H2O2-exposure group for 48h compared with the untreated cells (p<0.001). 95 protein spots with more than 2 fold difference were detected in two dimensional electrophoresis (2DE) gels and 10 proteins were identified through TOF/MS analysis. These differential proteins are involved in cellular functions such as modulation of energy metabolism, mitochondrial regulation, and cell cycle control.Conclusions MECs are sensitive to oxidative stress through inhibition of cell viability , accumulation of intercellular ROS and alteration of protein expression. Any impairment of MECs function probably disturbs the integrity of the brain/optic nerve-CSF barrier. These findings maybe enhance our understanding of the role of MECs in the pathology of brain and optic nerve disorders.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. The effects of Nrf2 activator in a rodent model of anterior ischemic optic neuropathy (rAION)

Posterboard#: A0140

Abstract Number: 5745 - A0140

AuthorBlock: Shun-Ping Huang1,2, Jia-Ying Chien3, Yu-Yau Chou1 1Molecular Biology and Human Genetics, Tzu Chi University, , Taiwan; 2Opthalmology, Taichung Tzu Chi Hospital, Taichung, Taiwan; 3Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan;

DisclosureBlock: Shun-Ping Huang, None; Jia-Ying Chien, None; Yu-Yau Chou, None;

Purpose To investigate the effects of Nrf2 activator on retinal ganglion cell survival and its molecular mechanisms in a rodent model of anterior ischemic optic neuropathy (rAION)Methods AION induction was achieved using laser-coupled photoactivation of optic disc after intravenously injection of rose Bengal through tail vein6. The male Wistar rats were divided into three experimental groups: (1) sham, (2) AION induction and subcutaneously (SC) injected with PBS, (3) AION induction and SC injected with RTA402 (20mg/Kg in 0.2ml PBS) for 3 consecutive days. Animals were sacrificed at different time post infarct. Evaluation methods include TUENL assay and retrograde labeling FluoroGold (FG) of retinal ganglion cells (RGCs), immunohistochemical studies of ED1 in optic nerve (ON). Western blot analysis for pro-surviving signaling. Visual function evaluated by flash visual evoked potential (FVEP). Results RTA402 treatment significantly reduced optic nerve edema in the acute phase of rAION. The RGC survival were significantly improved in AION induction with RTA402-treated rats compared with those of AION induction with PBS-treated rats (79.6% vs 35.95% survival in the central and 85.9% vs 40.4% in mid-peripheral retina). For FVEP assessment, RTA402 treatment significantly restored the visual function after AION induction. Furthermore, less ED1 positive cells were accumulated in RTA402- treated ON indicate the anti-inflammatory effect of RTA402 at the ON after infarct. Conclusions Our results demonstrated that the rescue effects of RTA402 in rAION model work through the dual actions of anti-apoptosis and anti-inflammationLayman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Characterization of Vascular Cell Receptors that Regulate Blood Perfusion of the Mouse Retina

Posterboard#: A0132

Abstract Number: 5737 - A0132

AuthorBlock: Alejandro Luis Acosta2,1, Armando L. Garcia2,1, Diogo Felipe Muller2,1, Maria Jesus Chaves2,1, Manuel N. Tapia2,1, Daniel Pelaez2,1, Sanjoy K. Bhattacharya2,1, Luis E. Vazquez2,1 1Ophthalmology , University of Miami, Miller School of Medicine, Miami, Florida, United States; 2Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida, United States;

DisclosureBlock: Alejandro Luis Acosta, None; Armando L. Garcia, None; Diogo Felipe Muller, None; Maria Jesus Chaves, None; Manuel N. Tapia, None; Daniel Pelaez, None; Sanjoy K. Bhattacharya, None; Luis E. Vazquez, None;

Purpose To identify the vascular cell signaling pathways that regulate blood perfusion of the retina.Methods SMA-GFP transgenic (a.k.a. “OTO2-10”) mice, which express GFP under the acta2 promoter, were acquired from the National Eye Institute. Their retinas were dissected and dissociated with papain into single cell suspensions. VSMCs were purified by the collection of GFP positive cells with the use of a fluorescence activated cell sorter (FACS). Western blots (WB) from the sorted GFP positive cells were performed to confirm the purification of vascular cells, and liquid chromatography-mass spectrometry (LC-MS) was used to identify cell signaling components that regulate contractility. Immunohistochemistry of retinal flat mounts was used to validate key targets identified by our exploratory LC-MS profile.Results Confocal microscopy of SMA-GFP retinal flat mounts found GFP expression to be exclusive to the vascular tree. WB of GFP positive cells showed enrichment of alpha-smooth muscle actin (α-SMA), smooth muscle-myosin heavy chain (SM-MHC), neuron-glial antigen 2 (NG2), and depletion of neurofilament and glial fibrillary acidic protein (GFAP), consistent with the purification of VSMCs and pericytes via FACS. LC-MS yielded a total of 6,175 proteins within three replicates of SMA-GFP mice with 100 high confidence proteins (FDR < 0.01), 18 medium confidence proteins (FDR 0.01 - 0.05), and 6057 low confidence proteins (FDR > 0.05). LC-MS identified proteins that potentially regulate cell contractility include: cell surface receptors (such as glutamate and acetylcholine receptors), generators of intracellular calcium, cyclic AMP, ATP, and nitric oxide (second messengers), and effector proteins of contractility (such as myosin light chain kinase, myosin light chain phosphatase, and Rho-kinase). The localization of key regulators of contractility within the retinal vasculature is shown with flat mount immunohistochemistry.Conclusions Neurovascular coupling and regulation of blood flow in the retina remain poorly understood. We used mass spectrometry to identify VSMC cell surface receptors and downstream signaling pathways that regulate vascular tone to further understand neurovascular coupling in the mouse retinaVSMC receptors and signaling pathways that regulate contractility play a key role in vascular resistance in the retina. Our findings shed light on the molecular regulation of ocular perfusion.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details. Determination of absolute diastolic erythrocyte velocities in peripapillary arterioles and venuoles of human subjects using erythrocyte mediated angiography

Posterboard#: A0133

Abstract Number: 5738 - A0133

AuthorBlock: Victoria Chen1, Breanna Tracey1, Christopher Le1, Corinne Renner1, Jiaqi Li1, Lakyn Mayo1, Joby Tsai1, Michael Ou1, Sachin Kalarn1, Lily Im1, Mona Kaleem1, Osamah Saeedi1 1Department of Ophthalmology and Visual Sciences, University of Maryland School of Medicine, Baltimore, Maryland, United States;

DisclosureBlock: Victoria Chen, None; Breanna Tracey, None; Christopher Le, None; Corinne Renner, None; Jiaqi Li, None; Lakyn Mayo, None; Joby Tsai, None; Michael Ou, None; Sachin Kalarn, None; Lily Im, None; Mona Kaleem, None; Osamah Saeedi, Heidelberg Engineering Code F (Financial Support), Vasoptic Medical Inc Code F (Financial Support)

Purpose Erythrocyte-mediated angiography (EMA) is a novel technique that permits direct visualization of indocyanine green (ICG)-labeled erythrocyte ghost cells in vivo. This allows for the measurement of individual erythrocyte velocities and observation of erythrocyte dynamics. We performed a cross- sectional human study to measure erythrocyte velocity in the peripapillary retinal microvasculature using EMA.Methods A Heidelberg Retinal Angiograph 2 (Heidelberg Engineering, Germany) was used to acquire 12-20 second angiograms of 17 participants undergoing EMA at 24.6 frames per second. After image registration with a semi-automated MATLAB script, a certified grader measured distances traveled by individual ICG labelled erythrocyte ghosts between frames in peripapillary non-branching vessels ranging from 30 to 65 microns in diameter. Distance traveled was measured in pixels and then converted to microns using patients’ corneal curvature from keratometry values and angiogram scaling from the Heidelberg imaging platform. Time was measured in frames and converted to seconds using the frame rate conversion (24.6 frames per second). Average diastolic velocity for a vessel was defined as the average of all tracked erythrocyte velocities at and below the 50th percentile in a single angiogram.Results 100 angiograms of 53 vessels from 17 individuals (8 females and 9 males) were analyzed. The average age of participants was 57.4 ± 5.9 years. The average diastolic venous velocity was 4.6 ± 1.0 mm/s. The average diastolic arterial velocity was 5.2 ± 1.8 mm/s. We observed a statistically significant difference between average diastolic venous and arterial velocities was observed (p=0.03).Conclusions Using erythrocyte mediated angiography, the average diastolic erythrocyte velocity in this sample was determined to be 4.6 ± 1.0 mm/s in human retinal venules and 5.2 ± 1.8 mm/s in arterioles. Further development and use of EMA may improve our understanding of ocular disease.Layman Abstract (optional): Provide a 50-200 word description of your work that non-scientists can understand. Describe the big picture and the implications of your findings, not the study itself and the associated details.