11^th Annual

2018 Midwest Eye Research Symposium

Sponsored by the Iowa City VA Center for the Prevention and Treatment of Visual Loss

MERS 2018 took place on August 3^rd, 2018 at
The University of Iowa Medical Education and Research Facility

Keynote Speaker/Session Chairs

Vittorio Porciatti, DSc, FARVO - Keynote Speaker
Dr. Porciatti is the James L. Knight Professor of Ophthalmology and the Director and Vice Chairman of Research at the Bascom Palmer Eye Institute. Dr. Porciatti has pioneered non-invasive electrophysiologic and imaging techniques in human and mouse models to investigate how retinal ganglion cells become dysfunctional, how and when they become susceptible to stress, and how their function may be improved. He has authored over 160 well-regarded manuscripts, is a member of several professional societies and serves on review panels of NIH, other funding agencies,and scientific journals.

Denise Inman, Ph.D. - Chair, Session I
Dr. Inman is an Assistant Professor in the Department of Pharmaceutical Sciences at Northeast Ohio Medical University. She has had a longtime interest in the role of retinal glia in glaucoma. Most recently she has been investigating mitochondrial function and retinal ganglion cell metabolic vulnerability in the disease.

Daniel M Lipinski, M.Sc. D. Phil. - Chair, Session II
Dr. Lipinski is an Assistant Professor in the Department of Ophthalmology and Visual Sciences at the Medical College of Wisconsin, Milwaukee. He is also the director of the Ocular Gene Therapy Laboratory at the Medical College of Wisconsin. The laboratory is developing gene-based treatments to prevent cone photoreceptor cell death in RP, achieve effective vascular choroidal gene delivery, and the design of viral vectors with increased loading capacity to deliver large genes.

Oliver W. Gramlich, Ph.D. - Chair, Session III
Dr. Gramlich is an Investigator at the VA Center and a member of the University of Iowa Department of Ophthalmology and Visual Sciences. His research is focused on the biology and causes of optic neuropathies. Particular research interests of Dr. Gramlich's are the pathobiology, early detection, and treatment of multiple sclerosis.

Chondroid metaplasia of an eyelid cyst: an unusual occurrence

Chandana, Papudesu MD; Meghana, Agni MD; Bradley, Lemke MD; Shilpa, G. Reddy MD; Potter, Heather D, MD
University of Wisconsin-Madison Department of Ophthalmology and Visual Sciences

Purpose: A 76-year-old woman presented with an oblong skin-colored pedunculated growth on her right temporal upper eyelid. The lesion had been present for the past two years and was affecting her vision. Pathologic examination of the excised tissue revealed areas of keratinizing stratified squamous epithelium with an epidermoid cyst in the dermis. The cystic lumen was filled with loose eosinophilic debris. Extruding from one side of the cyst was a mass of mature hyaline cartilage with chondrocytes in lacunae confirmed with Masson Trichrome stain. This represents chondroid metaplasia likely secondary to epidermoid cyst rupture. There is limited literature on chondroid metaplasia of the skin especially of the eyelid.

Methods:

Results:

Conclusion:

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Visuo-retinal effects of chemotherapy in hematological malignancy

Anderson, David E.^{1, 2} Kedar, Kedar^{1, 3, 4} Bhatt, Vijaya R.^{2, 5} Holstein, Sarah A.^{2, 5} Lunning, Matthew^{2, 5} Rizzo, Matthew ^1
1 Department of Neurological Sciences University of Nebraska Medical Center (UNMC); ² Fred & Pamela Buffett Cancer Center UNMC; .³ Department of Ophthalmology UNMC; ⁴ Truhlsen Eye Institute UNMC; ⁵Division of Oncology & Hematology Department of Internal Medicine UNMC

Purpose: Multiple studies have demonstrated chemotherapy-related cognitive impairment (CRCI) in hematological malignancy (HM) patients. Putative mechanisms include inflammation-mediated white matter (WM) and gray matter (GM) degradation. Our pilot study evaluated the visuo-retinal correlates for cerebral changes seen in CRCI.

Methods: We studied 9 HM patients receiving chemotherapy (CTX+ group) 2 HM patients receiving supportive care only (CTX- group) and 14 healthy comparisons (HC). We measured contrast sensitivity (CS) using 5% ETDRS charts at 4m and retinal nerve fiber layer (RNFL) and ganglion cell/inner plexiform layer (GCL-IPL) thickness using optic disc and macular cube protocols of Cirrus OCT. Prospective assessments occurred at baseline (T1) 1-month (one CTX cycle; T2) and 3-months (three CTX cycles; T3). Data were analyzed in SAS using PROC FREQ and MIXED procedures.

Results: Contrast Sensitivity: Mean ±SD (logMAR values; T1; T2; T3): CTX+ OD: (0.34 ±0.15; 0.38 ±0.16; 0.39 ±0.05); OS: (0.37 ±0.13; 0.39 ±0.11; 0.39 ±0.04); CTX- OD: (0.39 ±0.18; 0.34 ±0.17; 0.40 ±0.21); OS: (0.55 ±0.32; 0.50 ±0.36; 0.49 ±0.29); HC OD: (0.40 ±0.09; 0.39 ±0.09; 0.36 ±0.10); OS: (0.35 ±0.11; 0.35 ±0.11; 0.32 ±0.10). Trend for mild CS worsening across visits in CTX+ group did not reach statistical significance. Global RNFL thickness (mm; T1; T2; T3) Mean ±SD: CTX+ OD: (93.6 ±17.8; 98.4 ±14.0; 88.5 ±10.8); OS: (93.2 ±15.9; 97.7 ±14.7; 91.3 ±6.5); CTX- OD: (88.9 ±11.8; 91.1 ±7.9; 90.7 ±81); OS: (85.0 ±10.4; 87.3 ±10.8; 89.3 ±9.2); HC OD: (87.1 ±9.1; 86.6 ±9.4; 88.1 ±8.4); OS: (84.6 ±9.9; 82.8 ±11.0; 82.9 ±10.8). A trend for RNFL thickening at T2 followed by thinning at T3 in CTX+ group did not reach statistical significance. In CTX+ group 86% showed RNFL thickening from T1 to T2 and 80% from T2 to T3 (c2=4.9 p=.09). GCL-IPL thickness showed no change across visits or groups. Modeling CS T1-T2 changes showed significant effects of patient group (F=6.8 p=.01) and T2-T3 RNFL thickness changes (F=9.4 p=.01). CS decrease for Ctx+ group (t=3.64 p=.003) was greater than Ctx- (t=3.57 p=.004) and HC (t=2.68 p=.02).

Conclusion: HM patients receiving chemotherapy showed small changes in CS and RNFL thickness. Initial acute thickening and subsequent thinning of RNFL may reflect inflammation-induced axonal swelling followed by degeneration. Our pilot study was not adequately powered but these results support a larger study to evaluate these biomarkers.

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Inhibitory and excitatory inputs to alpha retinal ganglion cells are unaffected in a mouse model of ocular hypertension

Bhandari, Bhandari¹ Smith, Jennie ² Van Hook, Matthew J. ³
Truhlsen Eye Institute

Purpose: Optic nerve damage and loss of retinal ganglion cells (RGCs) are hallmarks of glaucoma. Elevated intraocular pressure (ocular hypertension OHT) plays a significant role in optic nerve damage and RGC loss. The goal of this study is to evaluate the effects of early OHT on the excitatory and inhibitory synaptic inputs and the dendritic structure of On-sustained alpha RGCs (On-aRGCs) in the mouse retina.

Methods: Bilateral injection of 10 micron polystyrene beads was used to induce OHT in a mouse line expressing GFP in melanopsin expressing ganglion cells (Opn4Cre/+ ; Z/EG). Both Saline injected and uninjected animals were used as controls. Intraocular pressure was measured using a Tonolab tonometer and experiments were carried out 6-8 weeks post-injection. Large GFP positive presumptive On-aRGCs (soma >15 microns) were targeted for patch clamp recording in flat-mounted retinas from dark-adapted animals. The cesium-based pipette solution was supplemented with 100 microM CF 568 to enable 2-photon imaging of RGC dendrites following whole-cell recording. To characterize RGC physiology we measured the spontaneous spiking frequency light-evoked spiking (500ms flash of 385nm LED) and excitatory and inhibitory current responses. RGC images were traced using the Simple Neurite Tracer plugin in ImageJ and the total dendritic length and soma size were measured. Sholl analysis was performed to measure dendritic complexity.

Results: In cell-attached recordings we observed a significant reduction in the spontaneous spike frequency from 23.78 ± 2.435 (n=15) to 11.12 ± 1.436 (n=14; p<0.05) in ocular hypertensive mice. No significant changes were observed in light-evoked spiking. In voltage-clamp we found that OHT had no effect on either the excitatory or inhibitory currents across a 5 log-unit range of stimulus intensities. Significant reduction in total dendritic length from 6229 ± 416.1 (n=12) to 4501 ± 283.4 (n=9; p<0.05) was observed in the OHT mice while there were no significant changes to the soma diameter total number of branch points or dendritic field diameter.

Conclusion: The major conclusion of this study is that although OHT alters On-sus aRGC dendrites and spontaneous spiking cone-driven excitatory and inhibitory synaptic inputs are unaffected. Thus changes in RGC spiking behavior are likely to be solely the result of homeostatic adjustment of RGC excitability.

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RNA binding proteins in eye development: rbm24a regulates sox2 and leads to microphthalmia

Brastrom, Lindy¹, Dash, Soma² Scott, C. Anthony¹, Spatz, Lillian³, Lachke, Salil², Slusarski Diane^1
1Department of Biology, University of Iowa, Iowa City, Iowa ²Department of Biological Sciences, University of Delaware, Newark, Delaware ³Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana

Purpose: Developmental eye disorders such as microphthalmia affect roughly 1 in every 7000 live births. Nearly 20% of these cases are due to a mutation in the proliferative gene sox2. While transcriptional control of sox2 is well understood the mechanisms for its post-transcriptional control have not been addressed. Our preliminary data has found the RNA binding protein rbm24a regulates sox2. The purpose of our study is to determine the cellular mechanism underlying the microphthalmia phenotype and identify additional RNA targets of rbm24a.

Methods: We use gene knockdown and CRISPR genome editing to reduce rbm24a in zebrafish. We characterize developmental defects evaluate predicted targets by qPCR and perform behavioral studies to assess visual acuity.

Results: Knockdown and somatic CRISPR mutation of rbm24a leads to microphthalmia. We find that knockdown of rbm24a leads to a 70% decrease in sox2 expression. Using the vision startle response with software developed in our lab we show rbm24a morphants are able to detect stark changes in light. However rbm24a deficient embryos are unable to follow a sinusoidal pattern suggesting a subtle defect in vision or a defect in visual processing.

Conclusions: Our study highlights a novel mechanism for rbm24a in the post-transcriptional regulation of sox2. Additionally this work demonstrates the usefulness of the zebrafish as a model for visual function studies.

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CX43 knockout hiPSC fail to generate normal optic cups

Cheng, Lin¹, Kuehn, Markus^1,2
1Department of Ophthalmology and Visual Science, The University of Iowa, Iowa City, IA ²Center for Prevention and Treatment of Visual Loss, Iowa City VA Healthcare System

Purpose: Connexin43 (CX43) is a major protein that forms gap junction channels in embryonic stem cells. Mutations in the gene Gap Junction Protein Alpha 1 (GJA1) expressing CX43 were identified in a families with oculodentodigital dysplasia (ODDD) syndrome. Symptoms include microdontia caries enamel hypoplasia thin nose syndactyly and eye abnormalities such as microphthalmos iris atrophy and glaucoma. We established a model of ODDD using human induced pluripotent stem cells (hiPSC)-derived optic cups. These may be useful to investigate if microphthalmos can be rescued by small molecules or gene therapy.

Methods: GJA1 was disrupted using CRISPR/Cas9. The established lines were characterized by immunofluorescence and qPCR. The GJA1 knockout cell lines and WT hiPSC were differentiated into retinal organoids. Expression levels of neural makers and retinal markers were identified by qPCR and immonostaining.

Results: Using CRISPR/Cas9 genome editing we successfully obtained multiple hiPSC GJA1 knockout clones. To prevent potential batch variation between different hiPSC clones we used at least two GJA1 knockout clones for further characterization. GJA1^-/- hiPSC remained undifferentiated and morphologically indistinguishable from wild-type (WT) hiPSC. They remained typical hiPSC morphology and had high expression level of pluripotency markers such as OCT4 SOX2 and NANOG. GJA1^-/- hiPSCs had no defects in self-renewal and pluripotency state in primed states. However after 26 days differentiation GJA1^-/- hiPSC failed to generate thick neuroepithelium in retinal organoids culture which resulted in smaller retinal cups and thin neural retina. At day26 the neural identity marker PAX6 was significantly lower than in the WT.

Conclusions: GJA1 is not required for pluripotency in hiPSC in primed states but is required for hiPSC to develop into normal eye cups. Future investigations could be 1) characterize the laminar distribution of retina organoids between GJA1^-/- hiPSC and WT especially in the retinal ganglion cells and photoreceptors; 2) find the regulating pathways why GJA1^-/- hiPSC fail to differentiate to default neural ectoderm fate; 3) see if we can rescue the phenotype by blocking the pathway with small molecules or gene therapy.

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The Effect of Sleep Position Preference on Periorbital Symmetry

Dabin Choi B.S.¹ Kenten D. Kingsbury C.N.A.² Audrey C. Ko M.D.³ Keith D. Carter M.D. F.A.C.S.³ and Erin M. Shriver M.D. F.A.C.S.^3
1Carver College of Medicine, University of Iowa, Iowa City, Iowa ²University of Iowa, Iowa City, Iowa ³Department of Ophthalmology and Visual Sciences, University of Iowa Health Care, Iowa City, Iowa

Purpose: Periorbital structures such as eyebrow and eyelid play key roles in visual function and aesthetic appearance. Sleep position preference has been shown to have a significant correlation with facial aging and symmetry in previous studies. The effect of sleep position on the eyelids and eyebrow position has not been evaluated. Knowledge of the effect of sleep position on these characteristics is crucial to understanding patients' baseline eyelid and eyebrow characteristics surgical planning and maximizing post-operative results. This study investigates the relationship between patients€™ sleep position preference and the degree of eyebrow ptosis eyelid ptosis and upper eyelid dermatochalasis.

Methods: A prospective randomized study of consecutive patients at the University of Iowa Department of Ophthalmology and Visual Sciences was conducted in compliance with the Institutional Review Board. Eligibility criteria included the absence of any periocular-altering trauma surgery or disease process. Study participants were given a questionnaire to ascertain their sleep position preference. Standardized digital photographs of patients were obtained and Image J software was used to obtain positional measurements for the eyebrow and upper and lower eyelid positions. The image measurements assessed were the distance from the pupil centroid to the upper eyelid margin (MRD1; marginal reflex distance 1) the pupil centroid to the lower eyelid margin (MRD2; marginal reflex distance 2) the upper eyelid margin to the visible upper eyelid skin fold (TPS; tarsal platform show) and the pupil centroid to the superior limit of the eyebrow (BP; brow position). These results were then compared to the patient reported sleep position preference to determine correlation.

Results: A total of 86 patients who satisfied the selection criteria were evaluated of which 32 patients preferred right side 25 patients preferred left side and 15 patients preferred sleeping on both sides. 14 patients who preferred supine or prone sleep position were not included in the analysis. Patients with right or left sleep side preference demonstrated significantly lower MRD1 image measurements on the side which they customarily sleep. No significant differences were seen in other image measurements among patients with strong sleep side preference. Among patients who sleep on both sides there were also no significant differences between right and left periorbital image measurements.

Conclusion: These results suggest that patients with a predominant sleep side preference demonstrate a significant ipsilateral increase in upper eyelid ptosis. There were no differences noted in eyebrow position amount of upper eyelid dermatochalasis and lower eyelid position.

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Examining axial length in congenital achromatopsia (ACHM)

Connolly, Sarah; Patterson, Emily J.; Heffernan, Elizabeth; Warr, Emma; Carroll, Joseph
Froedtert and the Medical College of Wisconsin Eye Institute

Purpose: Subjects with congenital ACHM characterized by reduced/absent cone function nystagmus and photophobia and a lack of color vision are generally reported to be hyperopic. However robust data regarding axial length in adults and in the developing eye is lacking. Here we examine axial length in adults and children both with normal vision and ACHM across multiple visits.

Methods: Axial length was measured in both eyes using the Zeiss IOL Master in 302 normal trichromats and 119 subjects with genetically-confirmed ACHM ranging in age from 5 to 84 years. Six trichromats and 31 ACHM subjects under 18 years of age had repeated measurements acquired over 0.5 to 5.6 year period. An unpaired t-test was conducted for statistical analysis using GraphPad Prism.

Results: Mean ± SD axial length for adults (>18 years) was not significantly different between control subjects (24.39 ± 1.25 mm n = 271) and ACHM subjects (24.42 ± 1.97 mm n = 73) p=0.8699. Likewise analysis of axial length data over multiple visits for subjects under 18 years of age revealed no significant difference in eye growth p = 0.727: the mean slope was 0.145 ± 0.097 in control subjects and 0.142 ± 0.099 in ACHM subjects consistent with previous estimates of axial elongation rates in children.

Conclusion: As anticipated axial length in all subjects under 18 years of age increased more dramatically in younger years and growth began to slow down with age. However despite a disruption in cone structure/function in ACHM axial length and eye growth do not appear to be affected.

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Effective arrest of retinal degeneration depends on the timing of gene restoration in BBS17 mouse model

Datta, Poppy; Hendrickson, Brandon; Seo, Seongjin.
Department of Ophthalmology and Visual Sciences University of Iowa College of Medicine; Institute for Vision Research Iowa City IA 52242.

Purpose: Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy with blindness being a cardinal feature. The current study is aimed to examine the presence of a 'limited time-window' for effective gene augmentation therapy to rescue vision loss and prevent retinal degeneration in BBS using the LZTFL1 (aka BBS17) mouse model.

Methods: We used an Lztfl1 mutant mouse line in which the gene trap cassette suppressing Lztfl1 expression can be eliminated using tamoxifen-inducible FLP recombinase; i.e. mutant alleles are converted to normal by tamoxifen injections. Tamoxifen was injected to three age groups (i.e. at postnatal days 21 at postnatal days 45 and at postnatal days 90). The injected animals were followed at 1 month 3 months and 6 months post-injection to assess the effect of gene restoration on disease phenotypes. Immunoblotting was used to assess the restoration of LZTFL1 and other photoreceptor specific proteins' level. Immunohistochemistry and electroretinography (ERG) were used to analyze photoreceptor specific polarized protein distribution and retinal visual function respectively.

Results: Restoration of Lztfl1 expression initiated in younger mice (i.e. at P21) significantly preserved photoreceptor cells' morphology as well as visual function compared to rescue initiated at older age (i.e. at P45) as observed in 1 month and 3 months post-injection follow-ups. Interestingly however protein mislocalization phenotype was only partially rescued even in younger rescued animals after 3 months of recovery time. In older animals (i.e. rescued at P45) restoration of Lztfl1 expression failed to preserve photoreceptors and retinal function essentially undistinguishable from un-rescued mutant animals.

Conclusion: Our study suggests that effective and sustained prevention of retinal degeneration and vision loss in BBS by gene therapy may depend on the age of individuals or the status of photoreceptors. The gene restoration clearly showed notable and sustained rescue effects when initiated at younger age compared to older animals. This further implies the presence of 'limited time-window' for successful gene therapy to preserve vision in BBS17 patients.

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The BAX activation mechanism of Bax deficient mice is deactivated several months after optic nerve injury

Donahue, Ryan; Grosser, Josh; Nickells, Robert
University of Wisconsin - Madison

Purpose: When the Bax gene is knocked out of retinal ganglion cells they survive indefinitely after acute or chronic injury. This makes BAX a promising target in the search for a therapeutic to prevent neurodegeneration. In this study Bax^+/- mice were used to test the hypothesis that lowering the quantity of BAX in retinal ganglion cells would delay the onset of apoptosis following and acute optic nerve injury.

Methods: Optic nerve crush was performed on Bax^+/- mice and then used immunofluorescence qPCR and western blotting to monitor the status of the BAX activation mechanism in the months following the crush injury. The apoptotic susceptibility of injured cells was directly tested by introducing exogenous GFP-BAX into Bax^-/- retinal ganglion cells after injury using an AAV2 virus.

Results: After crush it took 12 weeks for a significant percentage of retinal ganglion cells to undergo apoptosis. Quantitative measures demonstrated that this was not due to a deficiency in the BAX activation mechanism of Bax^+/- retinal ganglion cells. Intriguingly we found that cells deactivated their Bax activation mechanism between 7 and 12 weeks after crush and that by 12 weeks after crush damaged cells no longer displayed increased susceptibility to BAX activation relative to their naïve counterparts. At this same time glial activation subsided in the retina and the signaling of the pro-apoptotic JNK pathway abated.

Conclusions: These findings demonstrate that lowering the quantity of BAX in neurons is neuroprotective to them after an acute injury. Furthermore damaged retinal ganglion cells appear to enter a quiescent state months after injury where they are no longer susceptible to apoptosis. This implies that therapeutically lowering the quantity of BAX in neurons may help them survive a temporary pathologic environment until other therapeutic interventions can restore a more benign homeostasis.

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Changes in the retina and optic nerve in a mouse model of blast-induced traumatic brain injury

Dutca, Laura; Castonguay, William Hedberg-Buenz, Adam; Bielecki, Michael; Boehme, Nicholas; Herlein, Judith; Pellack, Danielle; Harper, Matthew; Anderson, Michael
¹Center for Prevention and Treatment of Visual Loss, Iowa City VA Healthcare System;
Departments of ²Ophthalmology and Visual Science, ²Psychiatry, ³Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA

Purpose: Detection management and treatment of symptoms relating to blast-induced traumatic brain injury (TBI) have become increasingly relevant to Veteran’s healthcare. Our recent work in a mouse model of blast-induced TBI suggested that retinal ganglion cells (RGCs) may be particularly prone to damage associated with over-pressure waves. The purpose of this study was to characterize cellular and tissue changes in the retina and optic nerve in this model.

Methods: Mice were exposed to an overpressure wave (20 PSI) directed to the head using a custom-built blast chamber. In vivo retinal structure was characterized using optical coherence tomography (OCT). RGCs were quantified on whole mounted retinas immunostained with antibodies recognizing BRN3A, a marker for RGCs. Whole mounted retinas stained with hematoxylin and eosin (H&E) were used to determine the total number of cells in the RGC layer and total retinal area. Cross sections of optic nerves were stained with paraphenylenediamine (PPD) to assess integrity of myelin and axons and cresyl violet to analyze cell infiltration.

Results: Decreased thickness of RGC complex layer was observed by OCT at all time-points analyzed. A significant loss in RGCs positive for BRN3A was detected at 1, 5, 16, and 99 weeks post-injury. The H&E staining showed a decrease in total cell density in the RGC layer at 5 and 16 weeks post-injury while retinal area stayed constant. Optic nerves stained with PPD and cresyl violet at 5 weeks post-injury showed a decrease in number of myelinated axons and an increase in number of glial cells. At 16 weeks post-injury myelinated axons appear relatively normal while increased number of glial cells persisted. The poster by Hedberg et al. presents a more in-depth analysis of the optic nerves.

Conclusions: Over-pressure wave exposure induced significant loss of BRN3a positive RGCs as early as 1 week after injury. At 5 weeks post-injury significant RGC loss in retina and loss of myelinated axons with increased glial presence in optic nerve were observed. Similarly loss of RGCs and myelinated axons appears to occur early after injury (5 weeks) with no further loss at chronic time points. In optic nerves increase in glia persists to 16 weeks post-injury while axon number is partially recovered. A better understanding of changes in the visual system and the time-line of these changes after blast exposure will help in the development of improved clinical testing and treatment.

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Human Pluripotent Stem Cell Derived Retinal Ganglion Cells Display Extensive Neurite Outgrowth in Response to Intrinsic and Extrinsic Signals

Fligor, Clarisse¹; Campbell, Peter²; Langer, Kirstin¹; Ren, Yuan¹; Guido, William³; Suter, Daniel⁴; Meyer Jason^1
1IUPUI; ²University of Louisville; ³University of Louisville; ⁴Purdue University

Purpose: Retinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain and as such the loss of RGCs often leads to blindness. Human pluripotent stem cells (hPSCs) can be differentiated into RGCs providing an unlimited source of cells for translational strategies such as disease modeling cell replacement and drug screening. During development intrinsic and extrinsic cues such as neuronal activity extracellular matrix composition and growth factor signaling are beneficial for survival proper guidance and innervation of RGC axons. However many previous studies of RGC outgrowth have been based on animal models with little emphasis on the response of human RGCs to these factors. Therefore the development of an in vitro model utilizing hPSC-derived RGCs would allow for the precise study of factors that may enhance axonal outgrowth and encourage synaptogenesis of RGCs with their target tissue.

Methods: Efforts have focused on the development of effective assays to test the ability of hPSC-derived RGCs to extend axons in response to a variety of cues as well as display target specificity. In order to better identify those guidance receptors expressed specifically within hPSC-derived RGCs the transcriptional profiles of individual cells were analyzed via quantitative RT-PCR. Subsequently the ability of both extrinsic and intrinsic factors to enhance RGC neurite outgrowth was analyzed. Finally to determine target specificity aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN) a primary post-synaptic target of RGCs.

Results: Results demonstrated that hPSC-derived RGCs cells possessed receptors essential in influencing outgrowth as well as target specificity including those for Netrins Slits and Semaphorins. Enriched populations of RGCs exhibited significant outgrowth within the first 24 hours which was significantly enhanced in the presence of certain neurotrophic factors such as BDNF and Netrin-1. Furthermore when co-cultured with LGN explants RGCs displayed a responsiveness to LGN signaling with the neurites projecting towards LGN explants extending over the longest distances.

Conclusion: Overall these results will facilitate the replacement of RGCs following their loss due to disease and degeneration as extensive axonal outgrowth will be critical for the development of personalized transplant therapies for optic neuropathies. The success of these strategies depend on the ability of RGCs to extend lengthy neurites which integrate with appropriate target tissue. Additionally once this axonal pathfinding is accomplished these axons must also be able to form functional synaptic connections.

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Preservation of Retinal Ganglion Cell Density in Immune Compromised Mice

Godwin, Cheyanne^{1, 2}; Gramlich, Oliver^{1, 2}; Steele, Moriah¹; Kuehn, Markus^{1, 2, 3
1}Department of Ophthalmology and Visual Sciences University of Iowa Iowa City IA USA; ²VA Center for the Prevention and Treatment of Visual Loss Iowa City IA USA; ³Institute for Vision Research University of Iowa Iowa City IA

Purpose: We have previously shown the presence of T cells in the retina and demonstrated that elevating intraocular pressure (IOP) leads to loss of retinal ganglion cells (RGC) in normal C57BL/6J (B6) mice. We elevated IOP in B6 and B6.129S7-Rag1^tm1Mom/J (Rag) mice which are deficient in mature B and T cells. The purpose of this study was to determine if the immune system contributes to RGC loss with elevated IOP.

Methods: IOP elevation was induced unilaterally in B6 and Rag mice by injecting an adenoviral vector expressing a mutant version of myocilin into the anterior chamber of one eye. 16 weeks after transfection mice were sacrificed and their retinas were fixed in 4% PFA for 2 hours. Retinal ganglion cell density was analyzed by staining retinas for the presence of Brn-3a and imaged in the periphery, mid-periphery, and central regions of the retina. RGC counts of the treated eye were compared to the contralateral eye and to controls having received an intraocular injection of an empty adenoviral vector.

Results: Elevated IOP was induced in approximately 75% of mice. The IOP profiles for B6 and Rag mice were not significantly different. Pressure increased by 2 weeks post transfection and remained elevated for the 16 weeks mice were monitored. 16 weeks post infection a significant decrease in RGC density in the treated versus untreated eye in B6 mice but not in Rag was noted. A loss of RGC in the untreated contralateral eye of B6 when compared to eyes treated with an empty viral vector was also noted.

Conclusion: The significant loss of RGC in treated verses untreated eyes in B6 mice but not Rag mice may be attributed to the lack of mature B and T cells in Rag mice. The loss in RGC in the untreated contralateral eyes in B6 mice indicates the possibility of a systemic process leading to RGC death even in the absence of elevated pressure. Combined these results indicate the presence of a pressure independent loss of RGCs mediated by the immune system.

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The Effect of Eyelid Surgery on the Ocular Surface Tear Composition and Meibomian Gland Morphology and Function

Gorrepati, Pavane L. BA; Carter, Keith MD FACS; Ko, Audrey C. MD; Shriver, Erin M. MD FACS
University of Iowa

Purpose: It is known that eyelid surgery directly affects blink dynamics and the Meibomian glands but little is known regarding the effect of eyelid surgery on dry eye markers tear film composition and Meibomian gland morphology. To date the most studied eyelid surgery is upper eyelid blepharoplasty which has been demonstrated to result in dry eye symptoms in 11-27% of patients postoperatively. The incidence of dry eye is greater in patients who undergo concurrent upper and lower eyelid surgery and those who undergo resection of the orbicularis muscle in addition to skin resection. While the dry eye symptoms post-blepharoplasty are established the literature is lacking on the procedure's effect on objective measures of dry eye such as tear meniscus and Meibomian gland morphology.

Methods: A prospective study evaluating all patients undergoing elective eyelid procedures with the University of Iowa Oculoplastics Division was conducted in compliance with the Institutional Review Board. The procedures include blepharoplasty ptosis repair ectropion repair eyelid reconstruction after Mohs excision of malignancy etc. Preoperative and postoperative measurements of tear meniscus imaging of Meibomian glands and ocular redness were collected using the Oculus Keratograph 5M. A subjective dry eye score using the Ocular Surface Disease Index (OSDI) was also collected. All patients were evaluated preoperatively and at one-week postoperatively and will be evaluated at two months and six months postoperatively.

Results: To date seven patients were included in this analysis of patients preoperatively and one-week postoperatively. No significant differences were noted between the preoperative and 1-week postoperative OSDI scores (Table 1) and OSDI dry eye categorization (Table 2). There were no significant differences noted between preoperative and one-week postoperative tear meniscus measurements. There was a significant difference in the left bulbar redness between preoperative and one-week postoperative scores (Table 4). There was also a significant difference in the redness categorization for the right eye when comparing preoperative measurements to one-week postoperative measurements (Table 5).

Conclusion: These findings suggest that eyelid surgery does not significantly impact dry eye conditions at one-week postoperatively. There is a significant difference in the ocular redness at one-week postoperatively which would be expected in the immediate postoperative period. Future directions include continuing to recruit patients and following-up with recruited patients at two months and six months postoperatively.

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Structural and Functional Evaluation of the Visual System in a Mouse Model of Multiple Sclerosis

Gramlich, Oliver W.^{1, 2}; Brown, Alexander J.^{1, 2}; Kardon, Randy H.^{1, 2
1}Center for the Prevention and Treatment of Visual Loss, Iowa City VA Health Care, Iowa City, IA
²Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA

Purpose: Optic neuritis is an associated pathology of Multiple Sclerosis (MS), and is often the first symptom of the disease. The purpose of our study is to determine changes in Pattern electroretinography (PERG) could be used as early and sensitive biomarkers to predict magnitude of optic nerve degeneration in an experimental autoimmune encephalomyelitis (EAE) model.

Methods: EAE was induced in female C57BL6 mice by immunization with MOG33-55, complete Freunds adjuvant and pertussis toxin. One EAE group received treatment with prednisolone and sham-injected (sham) mice served as controls. Clinical progression was recorded according to a 5-point EAE scoring scheme. PERG was recorded at baseline, 14 days after immunization and prior to death at day 35. Retinal nerve fiber layer (RNFL) thickness was determined by optical coherence tomography.

Results: Motor-sensory deficits were apparent 10 days after induction. EAE animals treated with prednisolone showed decreased clinically severity. A delayed PERG conduction speed in EAE mice was noticed 14 days after induction, even in prednisolone-treated animals, and latency did not recover at later time points. Amplitudes are not affected after 14 days but after 35 days, EAE mice demonstrate a significantly decreased amplitude (15 ±8µV) when compared to the sham group (21 ±6µV, P=0.03) and a notable decrease in prednisolone-treated EAE mice (18 ±2µV). Likewise RNFL thickness was significantly decreased in EAE mice in the naso-temporal axis (EAE: 19 ±3µm, sham: 23 ±1µm; P=0.001) while prednisolone–treated EAE mice show RNFL preservation (24 ±3µm).

Conclusions: Herein we describe that decreased optic nerve function correspond to the timeline of motor-sensory and structural deficits in the EAE model. Our data also suggest that the impairment of visual function relates to RNFL thinning and RGC loss. Clinical translation of these results could help predict MS relapses and rehabilitation in MS patients.

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HDAC3 Ablation in DBA/2J Mice is Mildly Protective Against Retinal Ganglion Cell Apoptosis

Grosser, Joshua; Schmitt, Heather M.; Nickells, Robert W.
University of Wisconsin-Madison

Purpose: Previous work in our lab has shown that Histone Deacetylase 3 (HDAC3) is important for retinal ganglion cell (RGC) apoptosis. HDAC3 inhibition using an HDAC3-selective inhibitor RGFP966 or conditional knockout (cKO) of Hdac3 in mice attenuated RGC loss in mouse models of acute optic nerve damage. The current study expands on the previous studies by using a chronic model of optic nerve injury the DBA/2J mouse to assess the effect of inhibiting Hdac3 in glaucoma.

Methods: In the first experiment DBA/2JRgos1BALB mice were injected intraperitoneally with either 2mg/kg RGFP966 or vehicle every three days from 6 to 10 months of age. Intraocular pressures (IOPs) were measured at 4, 6, and 10 months of age and mice were euthanized at 10 months of age. In the second experiment Hdac3fl/fl was back-crossed onto a DBA/2J background and mutations in the Gpnmb and Tyrp1 genes (known to cause anterior segment dysgenesis) were verified using PCR. Hdac3 was conditionally knocked out following an injection of 1 μL AAV2-Pgk-Cre virus at four months of age. IOPs were measured weekly and all mice were euthanized at 10 months of age. DBA/2J-Hdac3+/fl mice served as a 4 month uninjected control group. RGCs were visualized by staining for BRN3A. Optic nerves were cross sectioned stained for βIII-Tubulin and damage was scored. Both optic nerves and retinal whole mounts were counter stained with DAPI.

Results: Mice treated with RGFP966 had higher total cell and RGC counts than the vehicle-treated control after 10 months. Both groups experienced significant total cell and RGC loss compared to the 4-month control and treatment with RGFP966 did not prevent optic nerve degeneration relative to vehicle treated animals. We compared AAV2-Pgk-Cre injected DBA/2J-Hdac3fl/fl and DBA/2J-Hdac3^+/+ mice and found that average IOPs increased from approximately 12 to 20 mmHg over the course of fifteen weeks and remained elevated until death at 25 weeks post-injection. Average maximum IOPs did not statistically differ between Hdac3fl/fl and Hdac3^+/+ cohorts. RGC counts were modestly but significantly higher in conditional knockout mice while total cell counts and optic nerve scores did not statistically differ from the age-matched control.

Conclusion: Conditionally knocking out Hdac3 helped preserve RGCs in a chronic model of glaucoma and use of the HDAC3 selective inhibitor RGFP966 resulted in a modest but significant decrease in RGC death. HDAC3 inhibition is not a viable method of lessening optic nerve degeneration.

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670nm Photobiomodulation Modulates Levels of Glutathione and VEGF in a Cell Model of Diabetic Retinopathy

Hall, Alexandria; Fisher, Hannah; Senthikumar, Gopika; Abroe, Betsy; Liedhegner, Elizabeth; Eells, Janis
¹University of Wisconsin-Milwaukee

Purpose: Diabetic Retinopathy (DR) is a common and debilitating complication of diabetes mellitus resulting in vision loss and blindness. Hyperglycemia caused by diabetes disrupts normal cellular mechanisms in the retina leading to oxidative stress inflammation and the growth of abnormal blood vessels (angiogenesis). Excess oxidative stress results in the production of reactive oxygen species (ROS). Increased ROS occurs due to diminished levels of antioxidants including glutathione and activates vascular endothelial growth factor (VEGF) responsible for angiogenesis. Increased expression of VEGF contributes to the overgrowth of blood vessels in the eye which eventually rupture and cause blindness. Current treatment for DR includes intraocular injections to inhibit VEGF which have limited efficacy and are associated with adverse effects. An alternative non-invasive therapy using far-red light (photobiomodulation PBM) may be an improvement over current therapy. PBM is a unique therapeutic treatment which uses light in the 630-1000 nm range of the light spectrum and has shown efficacy in experimental and clinical studies of retinal disease including DR. We hypothesize that 670nm PBM will ameliorate oxidative stress and decrease expression of VEGF in a cell model of DR.

Methods: Our experiments were performed in cultured retinal Müller glial cells. Müller cells span 70% of the retina and have been shown to be the primary source of inflammation in DR. Müller glial cells were cultured in normal (5 mM) or high glucose (25 mM) medium to mimic normal and diabetic conditions. Cells will be treated for 3 or 4 days with 670nm irradiation for 180 seconds at a power intensity of 25mW/cm² (4.5 Joules/cm²) or with no light (sham). Reduced/oxidized glutathione concentrations and levels of VEGF expression were measured.

Results: Müller cells grown in diabetic conditions expressed increased levels of VEGF and decreased levels of glutathione consistent with DR pathogenesis. Treatment of cells with 670nm light was found to reduce levels of VEGF and oxidative stress similar to those levels seen in normal glucose conditions.

Conclusions: Data from these experiments support our hypothesis and provide insight to the mechanisms by which PBM mediate DR. This study suggests that treatment with 670nm PBM ameliorates oxidative stress, by increasing levels of antioxidants, and decreases VEGF expression, the main angiogenic factor seen in DR pathogenesis. Here, we provide further evidence that PBM may be a beneficial therapeutic approach for the treatment of DR.

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Optic nerve damage in a mouse model of blast-induced traumatic brain injury

Hedberg-Buenz, Adam; Dutca, Laura; Harper, Matthew; Anderson, Michael
Center for the Prevention and Treatment of Visual Loss VA Healthcare System Iowa City IA; The Departments of Molecular Physiology and Biophysics Ophthalmology and Visual Science The University of Iowa Iowa City IA

Purpose: Traumatic brain injury (TBI) is a leading cause of death and injury in the United States. Over 20% of current Service members have experienced some form of TBI many arising from exposure to blasts. Thus treating symptoms related to TBI are increasingly relevant to Veteran's healthcare. Using a mouse model of blast-induced TBI our research indicates that retinal ganglion cells (RGCs) and their axons in the optic nerve are susceptible to damage by blast. The purpose of this study was to identify and characterize optic nerve pathology in this model. For a more in-depth analysis of damage to RGCs using this model please attend the presentation by Dutca et. al.

Methods: C57BL/6J mice were exposed to an overpressure wave in a custom-built blast chamber. Analyses of the RGC complex layer were performed using in vivo optical coherence tomography. Optic nerve damage was evaluated using dyes and antibodies targeting markers of myelination axon structure and glial activity.

Results: In blast-injured mice thickness of the RGC complex was decreased compared to sham controls. Histopathology revealed decreased myelination and increased immunoreactivity for markers specific to oligodendrocytes microglia and astrocytes in blast-injured optic nerves compared to controls. Immunoreactivity to markers of axon structure were decreased and appeared disordered in blast-injured nerves compared to controls.

Conclusion: In this model exposure to blast causes optic nerve damage including defects to axon myelination and structure and increased glial activity. A better understanding of pathological changes occurring in the visual system following exposure to blast will aid in the development of improved clinical detection and treatment of TBI.

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Setting up connections in the inner retina and the role of LRRTM4

Hoon, Mrinalini¹; Siddiqui, Tabrez²; Rieke Fred³; Craig, Ann Marie⁴; Wong, Rachel⁵; Sinha, Raunak^6
1Dept. of Ophthalmology and Visual Science University of Wisconsin-Madison ²Dept. of Physiology and Pathophysiology University of Manitoba ³Dept. of Physiology and Biophysics University of Washington ⁴Dept. of Psychiatry University of British Columbia ⁵Dept. of Biological Structure University of Washington ⁶Dept. of Neuroscience University of Wisconsin-Madison

Purpose: The retina lines the back of the eye and converts light photons to electrical impulses which are transmitted to the brain for perception. Retinal neurons communicate via specialized junctions called "synapses". Understanding molecules regulating synapse assembly and circuit formation is essential for identifying retinal substrates and molecular targets of disease and degeneration conditions. This study investigates the role of a synaptic cell-adhesion protein (Leucine rich repeat transmembrane protein 4 or LRRTM4) for the establishment and function of the retinal synapses.

Methods: To determine the localization of LRRTM4 in the retina, an isoform-specific antibody was utilized. We used a knockout (KO) approach to investigate the role of LRRTM4 for the assembly and function of retinal synapses. LRRTM4 KO retinas were characterized with cell- and synapse-specific markers by immunohistochemical methods 3D reconstructions of synaptic ultrastructure was performed by serial block-face electron-microscopy and synapse function was determined by electrophysiology (whole-cell patch clamp recordings).

Results: LRRTM4 was localized to synapses of the dim-light retinal circuit. Rod bipolar cells (RBCs) of this circuit enrich LRRTM4 at their axon terminals. By double labeling with synaptic markers LRRTM4 was localized to inhibitory GABAergic postsynapses of RBC terminals. Eliminating LRRTM4 from the retina resulted in impaired GABA receptor clustering and inhibitory feedback responses at RBC terminals as revealed by correlating synapse structure and function. At the ultrastructural level RBC output connections at ribbon synapses were perturbed.

Conclusions: Our results reveal a role for LRRTM4 in the assembly and function of feedback inhibitory synapses of the dim-light retinal circuit. Unlike its role for organizing excitatory glutamatergic synapses in the rest of the brain, in the retina LRRTM4 uniquely organizes and establishes inhibitory synaptic connections.

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Development of an animal model to investigate Cone Dystrophy with Supernormal Rod Responses

Inamdar, Shivangi; Mahoney, Angela; Laird, Joseph; Bhattarai, Sajag; Drack, Arlene; Baker, Sheila
Department of Biochemistry, University of Iowa

Purpose: Cone Dystrophy with Supernormal Rod Response (CDSRR) is an autosomal recessive disorder that results in cone dystrophy accompanied by unusual rod signaling. CDSRR patients present with reduced visual acuity photophobia color vision abnormalities nyctalopia and eventually vision loss. CDSRR is caused by mutations in the KCNV2 gene which encodes a voltage-gated potassium channel named Kv8.2. Kv8.2 functions to regulate membrane potential and filter photoresponses. The majority of CDSRR associated information comes from human patients and while in vitro studies have provided valuable insights into the possible mechanism of this disease there is no animal model for in vivo studies of the disease. Our goal is to create a knockout mouse line that phenocopies the human disease.

Methods: In this work we developed a Kv8.2 knockout mouse (KO) model using CRISPR/Cas technology. Guide RNAs were designed to target exon 1 of KCNV2 in order to generate frameshifting indels. Validation of the Kv8.2 KO line was done using genomic sequencing reverse-transcriptase digital droplet PCR and western blotting. Retinal function was measured using the standard ISCEV protocol for rodent electroretinography (ERG).

Results: The founder KO animal obtained from the UI Genome Editing core had a 101 bp deletion in KCNV2. Expression levels were reduced by 50%. Western blotting confirmed Kv8.2 protein was not detectable while the expression of its binding partner Kv2.1 was unchanged compared to WT littermates. The ERG a-wave was unchanged indicating intact phototransduction. The scotopic b-wave indicative of rod signaling was delayed but of normal amplitude. Flicker responses were decreased indicating decreased cone function. The oscillatory potentials were absent suggesting that the disruptions in photoreceptor signaling severely compromised inner retina signaling.

Conclusion: The preliminary characterization of the Kv8.2 KO mouse indicates we have generated a useful model for investigating the mechanisms of CDSRR.

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Axonal Metabolic Rescue in Glaucoma

Inman, Denise¹; Harun-Or-Rashid Muhammad^1
1Northeast Ohio Medical University

Purpose: Axon degeneration can arise from metabolic stress potentially a result of mitochondrial dysfunction or lack of appropriate substrate input. We investigated whether the metabolic vulnerability observed during optic neuropathy in the DBA/2J (D2) model of glaucoma is due to dysfunctional mitochondria or impaired substrate delivery to axons the latter based on our observation of significantly decreased glucose and monocarboxylate transporters in D2 optic nerve (ON) human ON and mice subjected to acute glaucoma injury.

Methods: We placed both sexes of D2 mice destined to develop glaucoma and mice of a control strain the DBA/2J-Gpnmb+ on a ketogenic diet to encourage mitochondrial function.

Results: Eight weeks of the diet generated mitochondria improved energy availability by reversing monocarboxylate transporter decline reduced glial hypertrophy protected retinal ganglion cells and their axons from degeneration and maintained physiological signaling to the brain.

Conclusions: These results suggest that energy compromise and subsequent axon degeneration in the D2 is due to low substrate availability secondary to transporter loss.

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Investigating synaptic disease in an all-cone mouse retina

Kopel, Ariel; Laird, Joseph; Baker, Sheila
Department of Biochemistry, University of Iowa

Purpose: Perturbations in the synaptic calcium channel Cav1.4 or its associated subunits and regulatory proteins leads to synaptic degeneration and either a progressive or stationary retinal disease. Previous studies in mice have found that rods synapses are more affected than cone synapses. The purpose of this study was to establish a series of mouse lines for the investigation for Cav1.4 mutations specifically in an all-cone mouse retina.

Methods: We obtained the NRL knockout (KO); RPE65 R91W knockin line where all photoreceptors are S-cones and the decreased activity of the RPE65 mutant reduces the occurrence of confounding neural rosettes. From here on this line is referred to as 'conefull' . Conefull mice were bred to the following Cav1.4 mutants: alpha1 (a1) KO or alpha 2 delta 4 (a2d-4) KO. Histology and optical coherence tomography (OCT) was used to investigate retina health.

Results: Conefull and conefull:a2d-4 KO lines were investigated conefull:a1 lines are still in production. At three weeks old the conefull and conefull:a2d-4 KO retina appeared largely normal with the notable exception that the conefull line has a reduced density of photoreceptors compared to wildtype animals and they are all cones as evident from the chromatin organization of the nuclei. OCT images were collected at two four and six months of age. The outer nuclear layer in conefull mice exhibited a slight thinning of 2.73 ± 0.02 µm from two to six months of age. This slow degeneration was accelerated in the conefull:a2d-4 KO mice to 5.93 ± 0.62 µm over this time interval.

Conclusion: Investigating loss of the Cav1.4 subunit a2d-4 in an all cone mouse retina revealed that this subunit is needed to prevent cone degeneration. Since mutations in human a2d-4 cause cone-rod dystrophy this animal model will be useful for further investigations into the differences between rod and cone synapses in congenital synaptic diseases.

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Poster # 10

The Effect of Image Size on OCTA Retinal Vasculature Metrics

Kraker Jessica; Linderman Rachel; Cava Jenna; Omoba Bisola; Schmidt Taly; Chui Toco; Rosen Richard; Carroll Joseph
Medical College of Wisconsin

Purpose: To investigate the effect of scan size on commonly used metrics describing the foveal avascular zone (FAZ) as measured by optical coherence tomography angiography (OCT-A).

Methods: Nineteen subjects with normal retinal morphology had their right eyes imaged using OCT-A. Ten scans each of three nominal scan sizes (3x3mm 6x6mm 8x8mm) were acquired per subject (30 scans total per subject). For each scan size the 10 scans were ranked according to image quality and the five highest ranked were registered and averaged to generate a single image for each scan size for each subject (3 images per subject 57 images total). The images were then cropped and scaled to the same size before the FAZ was using a semi-automatic algorithm. FAZ area FAZ acircularity and parafoveal intercapillary area (PICA) were analyzed.

Results: No significant differences were found when comparing FAZ area (0.262 ± 0.130 mm2; 0.260 ± 0.129 mm2; 0.270 ± 0.127 mm2; repeated ANOVA) or FAZ acircularity (1.253 ± 0.130; 1.233 ± 0.128; 1.225 ± 0.095; repeated ANOVA) for the 3mm 6mm and 8mm scans respectively. However significant differences (p < 0.001; repeated ANOVA) were found in PICA metrics of 8x8mm scans (6445.7 ± 1561.1 µm 2) when compared to 3x3mm (3036.9 ± 713.15 µm 2) and 6x6mm (3339.0 ± 717.78 µm 2) sized scans.

Conclusion: While scan size appears to have minimal impact on FAZ metrics it can affect other retinal vasculature metrics. When selecting OCTA scan size clinicians and scientists should be sure to choose one that is appropriately suited for their metric(s) of interest.

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Induction of rod synaptogenesis in adult mice

Laird, Joseph G.¹ Gardner, Sarah¹ Kopel, Ariel¹ Joiner, Mei-ling² Lee, Amy² Baker, Sheila¹
University of Iowa; Departments of ¹ Biochemistry and ²Molecular Physiology & Biophysics

Purpose: Cav1.4 is a photoreceptor specific voltage-gated calcium channel clustered at the presynaptic active zone or 'ribbon' ™ of photoreceptors. Ca2+ influx via Cav1.4 is essential for communication across the first visual synapse; additionally Cav1.4 has been found to be required for synaptic development. Loss of Cav1.4 or altering the activity of the channel can result in either a stationary (CSNB2) or progressive (CORDX3) disease. In mouse models of these rare diseases there is always gross malformation and degeneration of the synapse. We expect efforts to gain mechanistic insight into the regulation of photoreceptor synaptogenesis will be a boon to ongoing studies seeking to cure blindness through cell replacement which will often require not only new photoreceptors but new wiring to the rest of the retina. The purpose of this study was to develop a rescue strategy for investigating the ability of Cav1.4 to trigger synaptogenesis in both immature and mature mouse retinas.

Methods: We used in vivo electroporation to transiently express Cav1.4 or a tamoxifen-inducible Cav1.4 in a subset of Cav1.4 knock out mouse rods and assayed for rescue of synaptogenesis using morphological markers and a behavioral assay for vision.

Results: This approach allowed for rescue of Cav1.4 expression synaptic development and limited vision in Cav1.4 knock out mice independent of the age at which Cav1.4 expression was restored.

Conclusion: The observation that synaptogenesis can be triggered by adding back Cav1.4 to either immature or mature photoreceptors indicates that these synapses retain a sufficient degree of plasticity to support future restorative therapies.

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Astrocytes Regulate the Developmental Timeline of Retinal Ganglion Cells Differentiated from Human Pluripotent Stem Cells

Langer, Kirstin; Vij, Ridhima; Ohlemacher, Sarah; Sridhar, Akshayalakshmi; Feder, Elyse; Baucum, Anthony; Cummins, Theodore; Meyer, Jason
¹Indiana University Purdue University, Indianapolis

Purpose: Human pluripotent stem cells (hPSCs) are capable of generating any cell type of the body including those cells of the retina. As numerous disorders of the retina lead to loss of vision hPSCs are often considered attractive tools for applications in disease modeling and regenerative medicine. However traditional retinal differentiation protocols require extensive periods of time to generate retinal cells and often result in retinal cells constituting a fraction of the overall differentiated population. Hence efforts were undertaken to enhance both the efficiency and timing of retinal differentiation from hPSCs. BMP4 a member of the TGF-ß family prominently known for its role in bone formation also has many vital roles in the development of other tissues of the body including the retina.

Methods: hPSCs were differentiated to a retinal lineage including the formation of three-dimensional retinal organoids following established protocols. To enhance retinal differentiation BMP4 was added to cultures for five days at early stages of retinal differentiation and hPSCs were then allowed to further differentiate into a variety of retinal cell types. Analyses of retinal differentiation efficiency and timing were accomplished by immunocytochemistry and quantitative RT-PCR.

Results: In this study the temporally-specific use of BMP4 was found to result in a higher yield of retinal organoids including a greater percentage of retinal organoids expressing CHX10 following BMP4 exposure. Additionally BMP4 accelerated retinal differentiation from hPSCs with the expression of specific retinal-associated genes occurring earlier than controls including CHX10 and BRN3.

Conclusions: These studies demonstrate the ability of BMP4 to act as a modulator to boost and refine retinal differentiation from hPSCs with these results facilitating the use of hPSC-derived retinal organoids in modeling retinal diseases and for high throughput analyses for pharmacological development.

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A role for 14-3-3 in the downregulation of HCN1

Lankford, Colten; Evans, Amy; Inamdar, Shivangi; Laird, Joseph; Houtman, John; Baker, Sheila
Department of Biochemistry, University of Iowa

Purpose: Hyperpolarization activated cyclic nucleotide-gated 1 (HCN1) channels are expressed throughout the central nervous system and primarily act to regulate neuronal excitability. HCN1 is also present in the retina where its function has been best characterized in rod and cone photoreceptors. Here HCN1 assumes a different function acting as the fast reset to return photoreceptors from their light activated hyperpolarized state back to their depolarized dark state. In the absence of HCN1 light activated signaling is prolonged resulting in saturation of the downstream neural network at mid-range light intensities and a reduction in temporal resolution. HCN1 has been shown to be dynamically regulated with multiple signaling inputs altering its activity. Unlike the other HCN family members HCN1 shows little modulation by cyclic-nucleotide binding and instead is modulated based on the density of channel at the cell surface; however the mechanisms by which this modulation is achieved are poorly understood. Here we focus on the novel interaction between HCN1 and the phosphoserine/phosphothreonine binding 14-3-3 family of proteins.

Methods: Co-immunoprecipitation and isothermal titration calorimetry assays were used to demonstrate an interaction between HCN1 and 14-3-3. Semi-quantitative western blotting was used to assess the relative expression level of HCN1 mutants in a cell culture model while SNAP-tag technology was used to perform pulse-chase experiments to assess the half-life of HCN1 in this model.

Results: Our results demonstrate that 14-3-3 interacts with HCN1 in both the retina and in a human cell lines and that three predicted sites 14-3-3 sites all act as 14-3-3 ligands in vitro. Mutations at these sites appear to stabilize HCN1 as we observe elevated expression levels over the wild-type channel when expressed in human cell lines. Further we have observed a shift in the half-life with one of these mutants exhibiting a slowed degradation rate compared to the wild-type channel.

Conclusion: Our data suggests that HCN1 is a 14-3-3 client protein and that loss of this interaction stabilizes HCN1 channels. This suggests that 14-3-3 binding is involved in the degradation of HCN1 channels.

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Accelerated and improved retinal differentiation of human pluripotent stem cells via BMP4 signaling

Lavekar, Sailee; Fligor, Clarisse; Shields, Priya; Langer, Kirstin; Meyer, Jason
Indiana University, Purdue University, Indianapolis

Purpose: Human pluripotent stem cells (hPSCs) are capable of generating any cell type of the body including those cells of the retina. As numerous disorders of the retina lead to loss of vision hPSCs are often considered attractive tools for applications in disease modeling and regenerative medicine. However traditional retinal differentiation protocols require extensive periods of time to generate retinal cells and often result in retinal cells constituting a fraction of the overall differentiated population. Hence efforts were undertaken to enhance both the efficiency and timing of retinal differentiation from hPSCs. BMP4 a member of the TGF-β family prominently known for its role in bone formation also has many vital roles in the development of other tissues of the body including the retina.

Methods: hPSCs were differentiated to a retinal lineage including the formation of three-dimensional retinal organoids following established protocols. To enhance retinal differentiation BMP4 was added to cultures for five days at early stages of retinal differentiation and hPSCs were then allowed to further differentiate into a variety of retinal cell types. Analyses of retinal differentiation efficiency and timing were accomplished by immunocytochemistry and quantitative RT-PCR.

Results: In this study the temporally-specific use of BMP4 was found to result in a higher yield of retinal organoids including a greater percentage of retinal organoids expressing CHX10 following BMP4 exposure. Additionally BMP4 accelerated retinal differentiation from hPSCs with the expression of specific retinal-associated genes occurring earlier than controls including CHX10 and BRN3.

Conclusion: These studies demonstrate the ability of BMP4 to act as a modulator to boost and refine retinal differentiation from hPSCs with these results facilitating the use of hPSC-derived retinal organoids in modeling retinal diseases and for high throughput analyses for pharmacological development.

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Development of an inducible gene therapy for the treatment of age-related macular degeneration (AMD)

Lipinski, Daniel
Department of Ophthalmology, Medical College of Wisconsin

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the developed world. In 10-15% of patients AMD progresses to an exudative stage characterized by chorodial neovascularization (CNV) and loss of central vision. As the development of CNV is triggered largely through extracellular vascular endothelial growth factor (VEGF) signalling current treatment strategies focus on the repetitive intravitreal administration of soluble VEGF inhibitors such as Eylea (Aflibercept). Although anti-VEGF agents have proven to be effective at preventing CNV formation repetitive bolus dosing is highly invasive and has been linked to accelerated retinal pigment epithelium (RPE) and photoreceptor atrophy. Herein we demonstrate that incorporation of a riboswitch element within the recombinant adeno-associated virus (rAAV) expression cassette allows for precise control of Aflibercept dosing in vivo that correlates with lesion severity in a mouse model of laser CNV.

Methods: Six-week-old C57BL/6J mice were intravitreally injected with PBS or 5.0x1010vg of rAAV2/2[MAX] packaging either a constitutively active (smCBA-Eylea) or a tetracycline-inducible (smCBA-Eylea-1x-TC45) Eylea construct. smCBA-Eylea-1x-TC45 injected mice were divided into two groups with mice either receiving standard diet (ON-state; high levels of Eylea expression) or tetracycline diet (OFF-state; reduced Eylea expression). CNV was induced using an 810nm diode laser six weeks following injection. Seven days following laser injury CNV formation was evaluated using fluorescein angiography with leakage independently graded by three blinded individuals. Finally eyes were harvested and the intraocular concentration of Eylea determined by ELISA.

Results: Constitutive expression of Eylea (smCBA-Eylea) significantly reduced the severity of CNV lesions compared to PBS sham injected eyes (p<0.0001). Moreover smCBA-Eylea-1x-TC45 injected mice receiving standard diet (Eylea expression ‘ON’) also showed significantly reduced number of clinically significant ‘2B’ lesions compared to smCBA-Eylea-1x-TC45 injected mice receiving tetracycline diet (Eylea expression ‘OFF’) (p=0.0008). Tetracycline mediated activation of the TC45 riboswitch resulted in a significant decrease in Eylea concentration in smCBA-Eylea-1x-TC45 injected mice (p<0.05).

Conclusions: Over-expression of Elyea in the mouse eye following rAAV-mediated delivery significantly reduced the severity of CNV lesions observed following laser injury. Incorporation of a riboswitch element within the expression cassette allowed the intraocular concentration of Elyea to be modulated simply through oral administration of an activating ligand. Increasing the intraocular concentration of Elyea through activation of the riboswitch element resulted in a significant and reproducible reduction in lesion severity. When developed further this technology may allow for personalized dosing of an anti-VEGF agent following a single intravitreal administration of rAAV redefining the current treatment paradigm and substantially reducing invasiveness by eliminating the need for repetitive monthly bolus injections.

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Diversity in synaptic function of retinal inputs to the SCN revealed by optogenetics

Stothert, Andrew R. and Van Hook, Matthew J.
¹Truhlsen Eye Institute, University of Nebraska Medical Center

Purpose: The suprachiasmatic nucleus (SCN) is a bilateral nucleus of the hypothalamus responsible for controlling the circadian clock. A subset of retinal output neurons termed intrinsically photosensitive retinal ganglion cells (ipRGCs) innervate the SCN via the retinohypothalamic tract (RHT) and mediate photic entrainment of the circadian clock. The features of synaptic transmission along the RHT in both health and disease are largely unknown. Therefore we set out to characterize excitatory synapses made by ipRGCs in the SCN and determine whether changes in the retina during ocular disease affects retinal signaling to the SCN.

Methods: We generated an optogenetic reporter mouse line in which ipRGCs express the lightsensitive channel Channelrhodopsin-2 (ChR2). Then we performed whole cell voltage clamp recordings in acute coronal SCN slices measuring LED-evoked synaptic currents. Paired pulse stimulation (intervals of 0.2 to 60 s) was used to determine short-term synaptic plasticity and 4Hz pulse train recordings to determine vesicle release probability (Pr). Frequency and amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs) were measured to examine presynaptic neurotransmitter release and postsynaptic receptor regulation.

Results: Currents evoked by 470 nm LED stimulation reversed near 0 mV and were completely blocked by CNQX. Immunohistochemistry revealed complete loss of ChR2 staining in the SCN following bilateral enucleation. Analysis of EPSCs revealed high variability in ipRGC synaptic function at the SCN; at the 200 ms paired pulse interval we found an average paired pulse ratio (PPR EPSC2/EPSC1) of 0.72 ±0.22 with a wide range of values from 0.35 to 1.03 (n=8). Analysis of EPSC amplitudes evoked by a 4 Hz pulse train revealed variability in Pr (low of 0.16 to high of 0.72 mean of 0.43 ± 0.19 n=7). mEPSCs measurements comparing control mice (n=5 mice 10 cells) to ocular hypertension (anterior chamber microbead injection OHT; n=1 mouse 2 cells) suggested that OHT causes an decreased in mEPSC amplitude (16.09 ±9.1 pA to 4.99 ±0.48 pA) and an increase in frequency (7.99 ±8.2 Hz compared to 23.03 ±4.9 Hz).

Conclusions: Initial analysis suggests presence of AMPA-mediated synaptic currents while ipRGCs express ChR2 and synapse directly on SCN neurons. Variability in PPR and Pr suggests ipRGC input strength to the SCN is the summed activity of all inputs to the SCN and might be required to encode irradiance across a wide range of intensities for circadian photoentrainment. mEPSC analysis suggests OHT causes a downregulation of post-synaptic AMPARs and an increase in presynaptic Pr. With these results we aim to gain a clearer understanding of retinohypothalamic synaptic function allowing us to better understand early stages in the progression of ocular disorders.

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Exome-based Investigation of the Genetic Basis of Human Pigmentary Glaucoma

van der Heide, Carly^{1, 2, 3} Goar, Wes^{1, 2} Alward, Wallace^{1, 2} Roos, Ben^{1, 2} Meyer, Kacie (3) Kwon Young^{1, 2} Anderson, Michael^{1, 2, 3} Scheetz, Todd^{1, 2} Fingert, John^{1, 2
1} Department of Ophthalmology and Visual Sciences Carver College of Medicine University of Iowa Iowa City IA; ²Institute for Vision Research University of Iowa Iowa City IA; ³Department of Molecular Physiology and Biophysics Carver College of Medicine University of Iowa Iowa City IA

Purpose: Pigmentary glaucoma (PG) is a secondary form of glaucoma that affects young adults and develops from a common predisposing condition pigment dispersion syndrome (PDS) in which iris pigment is released in the eye. Dispersed pigment accumulates within the trabecular meshwork and ultimately leads to elevated intraocular pressure optic nerve damage and permanent vision loss. Multiple lines of evidence suggest that PDS has a genetic basis but no causative mutations have been identified in humans. The goal of this study is to screen a cohort of PDS patients for mutations within candidate genes that are known to cause pigment dispersion in mice.

Methods: DNA was collected from PDS patients (n=210) and controls (n=362) who underwent complete eye examinations including slit lamp examination gonioscopy and ophthalmoscopy. Patients were included in the study if they exhibited at least two of four principal clinical features of PDS: iris transillumination defects a Krukenberg spindle a Scheie stripe and moderate to heavy pigmentation of the trabecular meshwork. Patients and controls were evaluated by whole exome sequencing and tested for rare (MAF < 2.5%) loss-of-function variations within five genes known to cause pigment dispersion in mice (TYRP1, GPNMB, LYST, DCT, and MITF).

Results: Four loss-of-function mutations within the five PDS candidate genes were detected. Three were rare nonsense mutations in TYRP1 LYST and DCT and were found only in the PDS cohort. The fourth loss-of-function mutation was a 14-base pair deletion (frameshift leading to premature termination) in GPNMB detected in one of the control patients. No loss of function mutations were detected in MITF.

Conclusion: In the largest genetic screen of PDS patients to date we identified three rare loss-of-function mutations within genes known to cause pigment dispersion in mice. The mutations we detected in TYRP1 LYST and DCT are novel candidates for a genetic cause of some cases of PDS in humans. We are currently using in vitro assays and mouse models to explore whether these mutations could cause pigment dispersion by damaging the pigmented cells of the iris.

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Ocular hypertension alters spiking behavior of neurons in the visual thalamus

Van Hook, Matthew J.
University of Nebraska Medical Center, Omaha, NE

Purpose: Elevated eye pressure (ocular hypertension OHT) causes optic nerve (ON) injury that leads to eventual dysfunction and degeneration of retinal ganglion cells in glaucoma. The goal of this study is to determine how early OHT affects the function of thalamocortical relay neurons (TC neurons) in the dorsal lateral geniculate nucleus (dLGN) a major brain nucleus receiving input from retinal ganglion cells.

Methods: Bilateral anterior chamber injection of 10-micron polystyrene microbeads was used to induce OHT in mice expressing the optogenetic reporter channelrhodopsin in a subset of dLGN-projecting retinal ganglion cells. Eye pressure was monitored with a TonoLab tonometer and experiments were performed at ~5-6 weeks following injection prior to detectable RGC loss. Control animals received saline injections. TC neurons were targeted for whole-cell patch clamp recordings in coronal brain slices. Synaptic currents were evoked by stimulation of ChR2. Input resistance (Rin) was measured with hyperpolarizing current injection while spiking was evoked by depolarizing current. The spike adaptation index (SAI) was quantified as the ratio of the 2nd spike instantaneous frequency to the steady-state spike frequency. Data are mean+SD and statistical significance was tested using a t-test (p<0.05 threshold).

Results: Using optogenetic activation of RGC axons we found that OHT triggered changes in short-term synaptic plasticity consistent with increased synaptic vesicle release probability. In current-clamp we found that TC cells were slightly depolarized relative to controls (control: -77.6+3.5 mV n = 24; OHT: -73.9+3.0 mV n = 19) and that Rin was slightly elevated in OHT cells (OHT: 442+99 MOhm; control: 372+72 MOhm). Depolarization with 500-ms current injections revealed that OHT TC cells fired more action potentials than controls (160 pA; control: 19.5+6.1; OHT: 24.3+5.9). This was reflected in a higher steady-state spike frequency in OHT (48+11 Hz) relative to controls (39+11 Hz). However the initial frequency of firing was reduced from 136+20 Hz to 111+23 Hz indicating a change in the overall pattern of TC neuron spiking behavior. Consistent with this the SAI was increased in OHT (0.47+0.18) relative to controls (0.31+0.12). Hinting at changes in Ca2+-activated currents responsible for spike frequency adaptation the amplitude of the slow after hyperpolarization (AHP) following a current step was reduced from -2.9+1.0 mV in controls to -2.2+1.2 mV in OHT.

Conclusions: These findings show that OHT alters the excitability of neurons receiving input from retinal ganglion cells in the visual thalamus before RGC loss. Changes are likely to be a homeostatic response to altered retinal inputs and might contribute to later pathology. The mechanisms of these changes and their influence on responses to RGC synaptic inputs have yet to be determined.

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Functional Characterization of the Bardet-Biedl Syndrome1 M390R Mutation in Zebrafish

Westfall, Trudi A. ¹; Scott, C. Anthony¹; Baye, Lisa M. ^{1, 2}; Sheffield, Val C. ³; Slusarski, Diane C. ¹
Trudi A. Westfall¹ C. Anthony Scott¹ Lisa M. Baye^{1, 2} Val C. Sheffield3 Diane C. Slusarski^{1 1} Department of Biology, University of Iowa Iowa City IA ² Augustana University Sioux Falls SD. ³ Department of Pediatrics, University of Iowa Carver College of Medicine

Purpose: Dysfunctional cilia underlie a number of human genetic conditions such as Bardet-Biedl syndrome (BBS) and affect multiple organs causing blindness heart disease infertility obesity and/or diabetes. BBS proteins form a complex the BBSome which mediates protein trafficking. BBS1 is the most commonly mutated gene in BBS patients and a single missense mutation causing a methionine to arginine substitution at position 390 (M390R) accounts for 80% of all BBS1 mutations. The purpose of the current study is to characterize the cellular mechanism by which the mutant BBS1 protein causes disease.

Methods: We generated several zebrafish alleles with CRISPR/Cas9 and performed rescue experiments with either BBS1 or BBS1 M390R RNA in knockdown zebrafish. We analyzed the ciliated Kupffer's Vesicle intracellular transport and visual function. We also evaluated subcellular localization and protein binding.

Results: Bbs1 homozygous mutants show reduced cilia delayed cellular transport and visual function defects. BBS1 RNA is sufficient to rescue all knockdown defects while BBS1M390R mRNA had differing abilities depending on the tissue type. Tagged expressed zebrafish BBS1 protein is found at the centrioles while BBS1M390R is destabilized and fails to localize to the centrosome. We also find that BBS1M390R maintains interaction with some components of the BBSome complex but not with BBS4.

Conclusion: We conclude that the underlying disease mechanism of BBS1M390R stems from reduced interaction with BBS4 and mis-localization. This leads to a global loss of BBS1 protein stability which likely impacts other BBSome protein complex members.

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Assessing ganglion cell layer topography in human albinism using optical coherence tomography

Woertz, Erica N.¹; Omoba, Bisola²; Chiu, Stephanie J. ³; Farsiu, Sina ³; Carroll, Joseph^{1, 4
1}Department of Cell Biology Neurobiology & Anatomy Medical College of Wisconsin; ²Medical College of Wisconsin; ³Department of Ophthalmology Duke University; ⁴Department of Ophthalmology & Visual Sciences Medical College of Wisconsin

Purpose: The purpose of this study was to characterize ganglion cell layer (GCL) topography in subjects with albinism and normal individuals.

Methods: Optical coherence tomography (OCT) scans were acquired in 14 subjects with albinism (8 M, 6 F, ages 9-44) and 17 normal subjects (8 M 9 F ages 12-33). Horizontal and vertical 6- or 7-mm B-scans were acquired at the fovea (or incipient fovea) followed by registration and averaging of 28-30 frames from each scan using ImageJ and DeMotion strip registration software. The Duke OCT Retinal Analysis Program was used to automatically segment the combined GCL-inner plexiform layer (IPL) and total retinal thickness followed by by program-assisted manual segmentation of the boundary between the GCL and IPL. Image segmentation coordinates were then used to calculate layer thickness and area under the curve (AUC) within 2.7 mm of the (incipient) fovea. Nasal-temporal (N-T) and superior-inferior (S-I) asymmetry was calculated as a ratio of AUC in each quadrant then compared using a two-tailed t-test.

Results: In control subjects the GCL comprised 0-100% of the combined GCL-IPL thickness while in subjects with albinism the GCL comprised 13.6-72.4% (0.2-2.7 mm from fovea). When AUC in all four quadrants was summed neither GCL nor IPL AUC was significantly different in controls from that in albinism. Both groups showed N-T asymmetry in the GCL consistent with histologic studies showing greater ganglion cell density nasally than temporally. Notably this asymmetry was significantly greater in albinism than in controls (p<0.01) and showed 1.62-fold variation across subjects with albinism.

Conclusion: Subjects with albinism have increased N-T asymmetry in the GCL compared to control subjects suggesting alterations in GCL topography. Further the variation in GCL contribution to the combined GCL-IPL layer seen in all subjects illustrates the need to delineate the GCL from the IPL when evaluating inner retinal layers in any population as measuring the combined GCL-IPL obscures each layers' independent contribution.

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The Effect of Visual Aids in Preoperative Patient Counseling for Oculoplastic Surgery

Yom, Kelly H.; Kingsbury, Kenten D.; Carter, Keith D.; Shriver, Erin M.; Ko, Audrey C.
University of Iowa

Purpose: In preoperative patient counseling the effect of exemplary immediate postoperative photographs demonstrating common events such as bruising swelling and asymmetry has not been previously evaluated. Therefore this study investigates the effect of such visual supplementation on patient education regarding the healing process after oculoplastic surgery. This study also determines if there are significant differences if counseling is performed by the medical team in person versus prerecorded video.

Methods: A prospective randomized controlled trial was conducted in compliance with the Institutional Review Board. 250 consecutive surgical patients seeking oculoplastic eyelid surgery at a single institution were randomized to an 'oral only' or 'oral and visual' patient education group. The 'oral only' group received routine preoperative oral counseling regarding what to expect after the operation. The 'oral and visual' group received routine oral counseling supplemented with exemplary photographs demonstrating common postoperative findings,such as swelling bruising and asymmetry, either via the medical team in person or prerecorded video. Patient-reported emotions and expectations of postoperative findings were assessed by validated survey instruments (the State-Trait Anxiety Inventory and Surgical Fear Questionnaire). Postoperative surveys assessing the same emotional parameters were administered during routine follow up visits at postoperative week one and month two.

Results: 59 patients were included in this preliminary analysis. 19 received oral only education 18 received oral education accompanied by photographs and 22 received oral education via video. No significant differences were noted in patient demographics between groups (Table 1). Preoperatively there were no significant differences in patient-reported feelings of fear anxiety or preparedness (Table 2). There were also no significant differences in patients' expectations of post-operative adverse events. At the one week postoperative visit there were no significant differences in patient feelings of anxiety preparedness or satisfaction nor were there significant differences in patient-reported adverse events (Table 3). The triage call rate was also similar amongst all three groups.

Conclusion: Visual supplementation during patient counseling does not significantly impact triage call rates or patient expectations anxiety or satisfaction regarding their surgery and postoperative healing process compared to standard oral counseling alone. Moreover patient education via video is equivalent to counseling in person by the medical team. Thus implementation of video counseling may help to standardize the education given to patients and increase efficiency without affecting the patient experience.

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Oxidative Stress and Vascular Damage in a Mouse Model of Diabetic Retinopathy

Zhu, Jiyao¹; Price, Tulin²; Wang, Shoujian³; Suscha, Andrew³; Shah, Gul²; Sheibani, Nader³; Ogilvie, Judith M.¹
Department of Biology¹ and Department of Biochemistry and Molecular Biology² Saint Louis University; Department of Ophthalmology and Visual Sciences ³ University of Wisconsin-Madison

Purpose: Diabetic retinopathy (DR) can be considered as both a neurodegenerative and a vascular disease. The complex biochemical and molecular interactions underlying DR are not yet fully understood. However oxidative stress is recognized to play a major role in damaging the diabetic retina given its high-energy demands. The gradual onset and development of symptoms in animal models of DR have slowed research efforts to elucidate these processes. Here we use the streptozotocin (STZ) injected thrombospondin-1 knockout (TSP1^-/-) mouse model which displays early onset vasculopathies. Our current goal is to use this robust model of progressive retinal neurovascular degeneration to determine the time course of markers of hyperglycemia-induced oxidative stress with respect to microvascular pathologies seeking to gain a better understanding of how oxidative damage affects retinal neurovasculature.

Methods: To perform a time course study retinas from TSP1^-/- mice injected with either STZ or vehicle were harvested at three time points: 4, 8, and 12 weeks after onset of diabetes. After retinas were extracted they were either subject to measurements for reduced glutathione (GSH) and superoxide dismutase (SOD) using commercial kits or fixed in paraformaldehyde followed by trypsin digest to analyze the retinal vasculature.

Results: Both GSH and SOD levels were decreased in STZ TSP1^-/- mice with significantly less GSH by 8 weeks and significantly less SOD by 12 weeks. Similarly a significant loss of pericytes as indicated by the endothelial cell/pericyte ratio was observed at 12 weeks.

Conclusion: We have confirmed that decreased GSH an indicator of oxidative stress precedes vascular pathology in STZ TSP1^-/- diabetic mouse retinas. Future studies will include neurodegenerative markers to reveal the temporal sequence of microvascular and neurological pathologies.

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