14^th Annual

2022 Midwest Eye Research Symposium

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

MERS 2022 took place on July 29, 2022 at
the University of Iowa Medical Education and Research Facility

Keynote Speaker/Session Chairs

David J. Calkins, PhD - Keynote Address: There and Back Again: A Vision Scientist's Tale.
Dr. Calkins is the Denis M. O'Day Professor of Ophthalmology & Visual Sciences and the Assistant Vice President for Research of the Vanderbilt University Medical Center. He is also the Director of the Vanderbilt Vision Research Center, and the Vice-Chairman and Director for Research of the The Vanderbilt Eye Institute. He was awarded the Research to Prevent Blindness Senior Scientific Investigator and the Stein Innovation Award, was named an ARVO Gold Fellow, and serves in editorial positions for several journals.
His research focuses on the molecular mechanisms of neurodegeneration in the visual pathways in health, aging and disease.

Matthew J. Van Hook, PhD - Chair, Session I
Dr. Van Hook is an Associate Professor in the Department of Ophthalmology & Visual Sciences at the Truhlsen Eye Institute of the University of Nebraska Medical Center. He explores synaptic transmission between the neurons of the retina using patch-clamp electrophysiology, anatomical and histochemical techniques, calcium imaging, and two-photon microscopy.

Abbot Clark, PhD - Chair, Session II
Dr. Clark is the Regents Professor at the North Texas Eye Research Institute (NTERI). Although he has been involved in studies investigating all aspects of glaucoma, a main topic of his laboratory is the pathomechanism of the trabecular meshwork that leads to aqueous humor outflow disturbances in open angle glaucoma. He has published over 240 peer-reviewed scientific manuscripts and has given 140 invited national and international lectures.

Brian Clark, PhD - Chair, Session III
Dr. Clark is an Assistant Professor in the Department of Ophthalmology and Visual Sciences at Washington University, St. Louis. He has been studying how the vertebrate retina develops from a pool of multi-potent retinal progenitor cells (RPCs).

Matthew Harper, PhD - Chair, Session IV
Dr. Harper is an Associate Professor in the Department of Ophthalmology and Visual Sciences and the Interdisciplinary Graduate Program in Neuroscience at the University of Iowa. He research is focused on the visual consequences of blast-mediated traumatic brain injury (TBI).

Oral Presentations

Session I, Session Chair Matthew Van Hook, Ph.D.

Targeting Cholesterol Efflux Transporter 1 Mitigates Visual and Motor-Sensory Deficits In An Experimental Optic Neuritis Model

Anders, Jeffrey¹; Elwood, Benjamin¹; Godwin, Cheyanne^1,2; Kardon, Randy^1,3; Gramlich, Oliver W.^1,3
1Center for the Prevention and Treatment of Visual Loss, Iowa City VA Health Care; ²University of Iowa Carver College of Medicine, Iowa City, IA; ³Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA

Purpose: Acute optic neuritis is the initial symptom in 25-45% of newly diagnosed cases of Multiple Sclerosis (MS). Retinal nerve fiber layer (RNFL) thinning and impaired electrical function of the visual system is evident in MS patients as disease progresses and often serves to determine treatment success. The purpose of our study is to uncover new druggable molecular pathways for how systemic mesenchymal stem cell (MSC) therapy rescues the ocular phenotype in an experimental autoimmune encephalomyelitis (EAE) model.

Methods: EAE was induced in 36 female C57BL6 mice by immunization with MOG33-55, complete Freunds adjuvant and pertussis toxin. Half of the EAE mice received intraperitoneally delivered MSC (EAE+MSC). Pattern electroretinography (PERG) and RNFL thickness were measured 32 days after induction. Retinas were harvested and prepared for RNA sequencing and data was analyzed by ingenuity pathway analysis. Effects of the top candidate for augmentation, the cholesterol efflux transporter 1 (Abca1), were determined using systemic Gentisic Acid (GA) administration in a follow-up MOG-induced EAE study. Visual function was determined using PERG and visual acuity testing using optokinetic responses.

Results: EAE animals having received MSC show significantly lower motor-sensory impairment (area under the curve EAE:43±1.5 vs. EAE+SC: 30±1.4; p<0.0001) improvement in the PERG amplitude (EAE:16±4 vs. EAE+MSC:26±5 µV; p=0.0002), and diminished RNFL thinning (EAE:18±3µm vs. EAE+MSC:23±7µm; p=0.042). Significantly increased expression of gene clusters related to neurogenesis and cholesterol recycling was evident in MSC treated EAE mice (Nav3: +35%; p=0.0008 and Abca1: +49%; p=0.01). In a follow-up study, administration of GA to increase Abca1 expression likewise rescued PERG amplitude (EAE:16±4µV vs. EAE+GA: 20±4ms; p=0.03) and preserved visual acuity when compared to untreated EAE mice (EAE: 0.18±0.07c/d vs EAE+GA: 0.35±0.01c/d; p=0.0001).

Conclusions: Structural and functional analysis of the visual system is a viable tool to determine treatment success in EAE studies. We could identify that MSC positively influences expression of Nav3, an important factor for neurogenesis and neuroprotection, and cholesterol transport via Abca1. Augmentation of cholesterol efflux transporter 1 in EAE mice using GA significantly lessens motor-sensory deficits and promotes rehabilitation of the visual system. In summary, this study suggests that interventions to improve cholesterol recycling might be a viable add-on approach to promote rehabilitation in MS.

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Mechanism for Altered Dark-Adapted Electroretinogram Responses in DBA/2J Mice Includes Pupil Dilation Deficits

Bierlein, Elizabeth¹; Smith, Jennie¹; Van Hook, Matthew^1
1Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE

Purpose: The DBA/2J mouse is a model of pigmentary glaucoma, a form of open angle glaucoma. Because these mice develop increased intraocular pressure (IOP) at around seven months, these mice are used to understand the role of increased IOP on the visual system. Prior studies have noted that these D2 mice have deficits in the flash electroretinogram (ERG) responses, notably the A-wave and B-wave amplitude. The purpose of this study was to elucidate the origin of such differences in the D2 ERG waves.

Methods: We analyzed 9-month-old DBA/2J (D2) and DBA/2J-gpnmb+ (D2-control) for differences in scotopic ERG amplitudes and latencies, intraocular pressure, outer nuclear layer thickness, and pupil area.

Results: Results of the ERGs indicated deficits as D2 mice had lower A-wave amplitudes and longer implicit times. There was also a significant rightward shift in the intensity-response curve. While A-wave defects indicate photoreceptor dysfunction, there was no significant difference in the outer nuclear layer thicknesses. Additionally, the characteristic D2 IOP increase had a weak correlation with the ERG A-wave sensitivity. Following tropicamide eye drop administration, we found that D2 mice had abnormal pupillary shape and no dilation. The pupil size moderately correlated with the A-wave rightward sensitivity shift and was pharmacologically replicated with pilocarpine administration in C57Bl/6J mice. However, pilocarpine administration had no effect on the A-wave amplitudes seen in the D2 mice.

Conclusions: The data suggest that the smaller pupil size in the D2 mouse prevented the pupil from dilating. This contributed to the rightward shift of the intensity-response curve due to the smaller pupil size preventing light from reaching the photoreceptors. However, because the ERG A-wave amplitude differences in D2 mice could not be pharmacologically replicated by C57Bl/6J experiments, this likely results from dysfunctional photoreceptor responses.

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ATP-induced retinal damage in cone-dominant 13-lined ground squirrel throughout euthermia

Bowie, Owen¹; Follett, Hannah²; Yu, Ching²; Manfredonia, Nicole²; Guillaume, Chloe¹; Summerfelt, Phyllis¹; Merriman, Dana K.³; Carroll, Joseph¹
Departments of ¹Ophthalmology & Visual Sciences and ²Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI; ³Department of Biology, University of Wisconsin Oshkosh, Oshkosh, WI

Purpose: The 13-lined ground squirrel (13-LGS), a cone-dominant hibernator, has emerged as an accessible animal model for vision research. Induction of cone degeneration through exposure to cytotoxic chemicals has revealed variable effectiveness in the 13-LGS. Here, we sought to examine how the time of year (relative to hibernation emergence) influenced the degree of cone damage in 13-LGS following intravitreal injection of adenosine triphosphate (ATP).

Methods: Eighteen (9M,9F) 13-LGS in three experimental groups [early-season (N=6), mid-season (N=6), late-season (N=6)], underwent baseline imaging using scanning light ophthalmoscopy (SLO) and optical coherence tomography (OCT). Animals then received a 10µL intravitreal injection of 0.723M ATP, followed by OCT and SLO imaging at 1, 3, 10, and 21 days. Adaptive optics SLO (AOSLO) was performed among animals without retinal damage after follow-up. Retinal thickness and cone density measures were compared to values from wild-type controls (N=12).

Results: Five animals showed retinal damage following ATP injection, including 4/6 early-season, 0/6 mid-season, & 1/6 late-season (Fisher’s exact test, p= 0.065). A two-way ANOVA showed significant differences in axial length between early-season & mid/late season cohorts (p = 0.029; p = 0.035). All animals with retinal damage displayed lesions on SLO and disrupted retinal lamination on OCT. Follow-up imaging with AOSLO on mid-season and late-season animals without observed retinal damage showed no evidence of photoreceptor disruption at any of the 97 ROIs analyzed. In late-season animals, 518 of 528 eccentricity matched locations imaged by OCT were within the normative range (mean +/- 2SD) from the control group, consistent with no ATP-induced damage.

Conclusions: The 13-LGS retina may be more susceptible to retinal damage by intravitreal ATP injection during the early season. However, differences in both axial length and vitreous volume between groups may impact the effective dose. Future studies adjusting dose based on ocular biometry may help elucidate the impact of time of year on chemical response.

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Metabolic Deprivation Causes Retinal Ganglion Cells Loss in Human Retinal Organoids

Cheng, Lin^1,2; Merydith, Evylin G.^1,2; Scheetz, Todd¹; Kuehn, Markus H.^1,2
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; ²VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: Retinal ganglion cell (RGC) degeneration in glaucoma is likely due to multiple disease mechanisms acting both in concert and sequentially. Recent data from several laboratories indicate that disturbances in retinal energy metabolism are associated with RGC dysfunction and demise. Here, we are using human-induced pluripotent stem cell (iPSC) derived 3-D retinal cups to investigate the outcomes of glucose deprivation.

Methods: Retinal cups (organoids) were generated from human iPSC and maintained under normal culture conditions for 50 days. Organoids were then subjected to a gradual reduction of glucose concentration in the cell culture media. After five days of culture under low glucose (LC) conditions, organoids were harvested and subjected to single-cell RNA sequencing.

Results: Within 24 hours LC levels induced morphologic changes indicative of cell stress. Transcriptional profiling of 15,149 cells obtained from LC organoids and 17,369 cells from control organoids indicated that lack of glucose caused increased expression of genes related to stress responses, inflammation, and antigen presentation. The distribution of cell types within the organoids was similar in LC and control organoids, however, RGC were markedly less prevalent in LC (6.5% of all cells) than in control organoids (15%). Furthermore, LC organoids contained a significant proportion of cells (17.4%) that expressed high levels of cell stress and cell death-related genes and could not be categorized as a specific cell type. Such cells were nearly absent in control organoids (1.3%).

Conclusions: This model allows investigation of neuronal primary responses to metabolic challenges while removing the influence of retinal microglia on the one hand and environmental challenges, such as changes in oxygenation, on the other. Our findings indicate that RGC are much more susceptible to metabolic deprivation than other retinal neuronal cell types. These findings may provide a mechanistic basis for the concept that disturbances in cellular metabolism are a cause of RGC loss in glaucoma.

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Discovering the Molecular Pathogenesis of Glucocorticoid-induced Ocular Hypertension

Clark, Abbot^1
1North Texas Eye Research Institute, The University of North Texas, Fort Worth, TX

Purpose: Glucocorticoids (GCs) are the most frequently used anti-inflammatory and immunosuppressive agents because of their broad spectrum of action. However, a major side effect of prolonged GC therapy is the development of iatrogenic ocular hypertension and secondary open-angle glaucoma in susceptible individuals. Although most frequently occurring with ocular therapy, glucocorticoid-induced ocular hypertension (GC-OHT) can occur via numerous routes of administration. While only about 30-40% of the general population are "steroid responders", almost all POAG patients develop GC-OHT during GC therapy. The mechanism(s) responsible for differential steroid responsiveness is currently unknow; however, alternatively spliced GC receptor beta (GRβ) appears to play a role in "steroid resistant" individuals.

Methods: Mouse strains B6, B6.GR^dim (GR dimerization deficient), and D2.gpnnb+ were given weekly periocular injections of dexamethasone-acetate or vehicle, and IOPs were measured non-invasively using the TonoLab rebound tonometer. B6 mice receiving DEX-acetate also were treated with Ad5.GRβ or Ad5.null transducing viruses.

Results: We discovered mouse strain differences in the development of DEX-OHT. While B6 mice developed DEX-OHT, B6.GR^dim and D2.gpnmb+ mice did not. We also showed that GRβ gene therapy totally reversed DEX-OHT. We have dissected "TM rings" that contain only TM and sclera for RNAseq analyses.

Conclusions: Using the knowledge that B6 are DEX responders and D2 mice are non-responders, we are using the BXD panel of recombinant inbred mouse strains to map and identify the genes responsible for DEX-OHT. GR^dim mice lack the ability for GR transactivation; therefore, the GR transactivation pathway is responsible for the development of DEX-OHT in mice. RNAseq analyses of the TM from all these mice will enable us to determine the molecular pathways responsible for DEX-OHT.

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Using 'Calling Cards' to dial in cellular data during retinogenesis

Clark, Brian¹; Shiau, Fion¹; Ruzycki, Philip¹; Dougherty, Joseph¹; Mitra, Rob^2
1John F. Hardesty, MD Department of Ophthalmology and Visual Sciences, Washington University School of Medicine; ²Department of Genetics, Washington University School of Medicine

Purpose: Recent advances in single-cell genomics applied to the retina have elucidated the chromatin architecture and gene expression patterns governing retinal cell fate specification and cellular heterogeneity amongst retinal cell types. These studies have identified numerous transcription factors (TFs) expression patterns consistent with important functions in the regulation of cell fate specification. Despite this progress, we know little about where TFs bind during development to drive specification of individual retinal cell fates. In this talk, I will present proof-of-concept studies utilizing the 'Calling-Card' system to identify TF binding sites across retinal development within individual cells.

Methods: Transcription factor hyperPiggyBac (TF-hyPBase) fusion constructs were cloned using InFusion assembly including an L3 linker between protein coding sequences. Self-reporting tdTomato transposon constructs were utilized to report either genomic sites of Brd4 binding (hyPBase alone) or TF-hyPBase interaction. In vitro calling cards experiments were performed through transfection of NIH3T3 cells. In vivo calling card experiments were conducted through electroporation of retinal explants from post-natal day 0.5 (P0.5) mouse pups, cultured until P6 equivalence. Recovery of transposon integrations was performed on recovered RNA through established protocols, and sequenced on a NovaSeq S4 (GTAC@MGI) using 150bp paired-end reads. Sequence analysis was performed using custom scripts.

Results: Electroporation of pCAGIG-hyPBase and the self-reporting tdTomato transposon (SRT-tdTomato) constructs into P0.5 mouse retinal explants results in robust tdTomato expression and recoverable transposon insertions. Experiments assessing Brd4-binding within post-natal retinal explants from P0 to P6 correlates well with established Brd4 ChIP-seq datasets and accessible chromatin. Preliminary, in vitro, experiments assessing transcription factor binding sites are able to recover TF DNA-binding motifs.

Conclusions: The Calling-Card system displays robust activity within the developing retina, thereby establishing the Calling-Card system as a tractable model to further our understanding of the mechanisms by which cell fates are specified in the developing retina.

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Identification of clinically-relevant biomarkers in primary human trabecular meshwork cells exposed to TGF-β2 and mechanical strain

Dammak, Azza¹; Iqbal, Sana²; Rao, Vidhya¹; Kaja, Simon^1
1Dept. of Ophthalmology, Loyola University Chicago, IL ²Graduate Program in Pharmacology and Experimental Therapeutics, Loyola University Chicago, IL

Purpose: Glaucoma is the leading cause of irreversible blindness. The trabecular meshwork (TM) regulates intraocular pressure (IOP) through regulation of aqueous humor (AH). Elevated intraocular pressure (IOP) is a key pathological mechanism in primary open angle glaucoma, where fibrosis and age-related pathological changes prevent AH outflow, contributing to glaucoma pathophysiology. The purpose of this study was to quantify expression of clinically-relevant biomarkers in primary trabecular meshwork cells exposed to disease-relevant insults, transforming growth factor beta 2 (TGF-β2) and mechanical strain.

Methods: Primary human TM cells were cultured and seeded in collagen I-coated BioFlex® plates. Once confluent, cells were transferred to serum-free conditions and exposed to mechanical strain (10% static strain) in the presence of absence of recombinant TGF-β2 (5 ng/ml) for 48 h. The supernatant was collected and secreted proteins from TM cells were concentrated using centrifugal filter units (Amicon). Osteopontin (OPN) was quantified by ELISA (Millipore Sigma) according to the manufacturer’s protocol. Total RNA was extracted from cell lysates, and cDNA was prepared. Gene expression levels of osteopontin (SPP1), matrix metalloproteinase 9 (MMP9), tumor necrosis factor alpha (TNFA), vascular endothelial growth factor A (VEGFA), TGFB1 and TGB2 were quantified by quantitative PCR.

Results: Human TM cells secreted baseline levels of OPN, which increased ~5-fold in response to recombinant TGF-β2 treatment (n=3, P<0.01), which exposure to 10% static strain had no effect on secreted OPN levels (n=3, P=0.96). The combination of mechanical strain and TGF-β2 treatment resulted in an >350-fold increase in OPN secretion (n=3; P<0.01). qPCR experiments confirmed increased gene expression of OPN (SPP1) following TGF-β2 exposure. While TNFA expression was below the detection threshold in naďve TM cells, exposure to TGF-β2 significantly increased TNFA expression. Mechanical strain and TGF-β2 significantly increased MMP9 expression. Neither TGF-β2 nor mechanical strain altered VEGFA expression. Notably, mechanical strain significantly increased gene expression of both TGFB1 and T

Conclusions: TGF-β2 exposure to primary human TM cells resulted in significantly increased OPN levels, which were exacerbated by simultaneous exposure to mechanical strain. These data are consistent with previous findings in animal models. Our results suggest that mechanical strain exacerbates the fibrotic effects of recombinant TGF-β2, likely due to upregulation of TGFB1 and TGFB2 gene expression.

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Subretinal gene therapy treats retinal phenotypes in Bardet-Biedl Syndrome Type 10

Hsu, Ying¹; Bhattarai , Sajag¹; Thompson, Jacob¹; Mayer, Sara^1,3; Thomas, Jacintha¹; Mahoney, Angela¹; Datta, Poppy¹; Garrison, Janelle²; Searby, Charles C.²; Vandenberghe, Luk H.⁴; Seo, Seongjin¹; Sheffield, Val C.²; Drack, Arlene V.¹
Departments of ¹Ophthalmology and Visual Sciences, and ²Pediatrics, University of Iowa, Iowa City, IA; ³Interdisciplinary Graduate Program in Genetics, University of Iowa; ⁴Massachusetts Eye and Ear Infirmary, Grousbeck Gene Therapy Center, Harvard Medical School, Boston, MA

Purpose: Retinal degeneration is a cardinal feature of Bardet-Biedl Syndrome type 10 (BBS10). Patients experience gradual vision loss and become legally blind by their second or third decade of life. To prevent blindness in BBS10, we tested subretinal gene therapy in a mouse model of BBS10 and evaluated efficacy by measuring retinal function and photoreceptor survival in treated and untreated mice over a period of one year.

Methods: Mice homozygous for the knockout allele of the Bbs10 gene (Bbs10-/-) were subretinally injected with the AAV2/Anc80 viral vector containing a cassette encoding the mouse Bbs10 gene (AAV2/Anc80-CMV-mBbs10). Juvenile mice between 23-31 days of age were treated. In treated mice, one eye received the subretinal injection of the viral vector, and the contralateral eye was left untreated as an internal control. Electroretinography (ERG) was performed at 1, 2, 3, 5, 7, 9, 11-12 months after treatment to measure retinal function. Optical coherence tomography (OCT) was performed at 1, 3, 5, 7, 9, and 12 months after injection to determine photoreceptor survival. Histology and immunohistochemistry were performed at 10-12 months post injection to visualize retinal structures.

Results: Bbs10-/- mice have low cone function prior to notable degeneration. Cone-specific ERG tests show that gene therapy enabled recovery of cone function in Bbs10-/- mice. After subretinal treatment, a mixture of transduced and untransduced cells is present in the retina. Gradual loss of ERG amplitudes was observed in rod-specific ERG recordings in both injected and uninjected eyes, which could be explained by the loss of untransduced photoreceptors over time. After a few months, ERG amplitudes became stabilized in eyes injected with AAV2/Anc80-CMV-mBbs10 and non-recordable in uninjected eyes. Quantification of the photoreceptor nuclei layer in injected and uninjected eyes shows that subretinal gene therapy using AAV2/Anc80-CMV-mBbs10 drastically slows down retinal degeneration.

Conclusions: Subretinal gene therapy using AAV2/Anc80-CMV-mBbs10 enables cone photoreceptors, responsible for visual acuity, to regain electrical function after treatment in a mouse model of BBS10. Furthermore, subretinal gene therapy substantially slows the rate of retinal degeneration in treated Bbs10-/- mice. Results show that subretinal gene therapy for BBS10 has significant potential for clinical translation for the prevention of blindness in this disorder.

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CXCL13-CXCR5 signaling axis in the retina

Huang, Hu^1
1Department of Ophthalmology, University of Missouri, Columbia, MO

Purpose: CXCL13-CXCR5 signaling axis is vital for the formation and homeostasis of immune systems, and its dysregulation is implicated in the pathogenesis of cancers and immune disorders. However, its role in retinal immune regulation and disease is unknown. Therefore, this project aims to investigate the function and mechanism of this signaling axis in retinal immune regulation.

Methods: Global and conditional knockout mice of CXCL13 and CXCR5 were created and used. In-vivo fundus imaging, fluorescein angiography (FA), electroretinography (ERG), and histopathological assessments were employed for retinal structure and function. Primary retinal pigment epithelium (RPE), microglia, and Müller cells were cultivated for the in-vitro studies. CRISPR/Cas9 genome-editing approach is used to delete CXCR5 in RPE. The standard biochemical and molecular biology methods, such as western blots, immunohistochemistry, and qPCR, are exploited to examine the changes of critical proteins and marker genes.

Results: CXCR5 knockout causes retinal degeneration and age-related macular degeneration (RD/AMD)-like phenotypes in old mice, which are correlated with the (auto)immune dysregulation. CXCR5-deficient RPE cells are dysfunctional in mitochondrial metabolism, autophagic function, and epithelial-mesenchymal transition (EMT). CXCR5 deficiency leads to increased expression of inflammatory pathway genes, such as complement, toll-like receptor, and chemokine/cytokine in the retina and RPE. Oxidative stress downregulates the CXCR5 protein level in RPE, reversely correlated with the upregulation of C3a and NLRP3. CXCR5 knockdown by siRNA increases the levels of C3 and NLRP3 in RPE. CXCR5 protein structure is highly conserved across species. CXCR5 is expressed in retinal microglia and upregulated during infl

Conclusions: Our studies provide evidence that the CXCL13-CXCR5 signaling axis plays a vital role in regulating the immune functions of retinal cells. Its dysregulation contributes to the pathogenesis of immune-dysregulated retinal diseases, such as RD and AMD. The underlying mechanisms are to be further elucidated.

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Ocular Emergencies During the Coronavirus Disease "Safer-At-Home Order" in Wisconsin

Huang, Leslie¹; Maganti, Nenita²; van Landingham, Suzanne^2
1School of Medicine and Public Health; ²Department of Ophthalmology, University of Wisconsin-Madison, Madison, WI

Purpose: The COVID-19 pandemic posed many challenges for patients seeking care. Although hospital resources shifted towards intensive care units and inpatient wards, emergency ophthalmic care continued, as ocular trauma is an important source of morbidity. This study aims to compare the volume of ophthalmology consultations in the emergency room and inpatient settings at a single hospital in Wisconsin during the 2020 "Safer-At-Home" order compared to the same period in prior years.

Methods: This is a retrospective cohort study utilizing the electronic health record system comparing the volume of ophthalmology consultations performed between March 23 and May 26, 2020 to the same period in the four preceding years. Consultations were identified by searching institutional billing records and subdivided into location of consultation. Statistical analysis was performed using R. Chi-squared testing was used to compare categorical data and ANOVA to compare ages. Poisson models were used to compare the total number of consults and number of emergencies leading to surgical interventions in 2020 with prior years. A p-value less than 0.05 was considered statistically significant.

Results: One hundred and fifty-five consults were performed in 2020 (42 emergency department, 113 inpatient), compared to a mean of 214 over the four other years. The incidence rate ratio (IRR) of total consultations in 2020 was 0.74 (p=0.004) compared to previous years. When categorizing consultations by location, significantly fewer ED consultations (IRR=0.56, p=0.003) were performed, but not inpatient (IRR=0.84, p=0.17). The year 2017 had more than expected consultations overall (IRR=1.26, p=0.012) and in the inpatient setting (IRR=1.32, p=0.014), but not in the ED (IRR=1.32, p=0.014).

Conclusions: Our study demonstrated a 26% reduction in ophthalmology consultations at a major university hospital during the COVID-19-related "Safer at Home" order compared to the same period in the preceding four years. These findings were similar to those noted in other single center studies, and are the first to show a decrease in ophthalmology consultations during the lockdown order in the Midwestern United States.

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Function and Localization of Kv2.1/Kv8.2 channels in KCNV2 retinopathy

Inamdar, Shivangi¹; Laird, Joseph¹; Baker, Sheila^1
1Department of Biochemistry and Molecular Biology, University of Iowa, Iowa City, IA

Purpose: KCNV2 retinopathy is retinal disorder that is associated with mutations in KCNV2, that encodes for Kv8.2. Kv8.2 binds specifically with Kv2.1 to form a heterotetrameric voltage-gated potassium ion channel that is important for photoreceptor function. This channel is localized in the IS close to the OS boundary. In the absence of Kv8.2, Kv2.1 is still expressed but appears more punctate and is electrically inactive. These punctate appearances of Kv2.1 in other cells have been shown to function in the formation of ER-PM contact sites that play a role in exocytosis, transport, and regulation of calcium homeostasis. It is not known if Kv2.1 and Kv8.2 form ER-PM contact sites in photoreceptors and the goal of this study is to better define the membrane localization and function of this channel.

Methods: Kv8.2 KO mouse was generated using CRISPR technology. Loss of functional Kv8.2 protein was assessed by, western blotting, and immunohistochemistry. Retina structure and function was assessed by ERG and OCT. Kv2.1 creates ER-PM contact sites by recruiting VAPA so the photoreceptor expression of VAPA was assessed by immunohistochemistry and immunoprecipitation. To aid the study of Kv8.2 localization we generated a mouse model expressing Clover-tagged KCNV2.

Results: ERG recordings of Kv8.2 KO showed the characteristic phenotype of the human disease where rod activity is supernormally high while cone function is reduced. OCT measurements showed a slow but progressive decline in the ONL thickness but imaging of cone-arrestin labeled cones showed the cell loss was specific to rods. In the absence of Kv8.2, Kv2.1 still localizes to the inner segment near the OS-IS border, but distinct clusters also are formed. VAPA interacts with Kv2.1 but not Kv8.2 in IP experiments and immunohistochemistry demonstrated partial overlap between VAPA and Kv2.1.

Conclusions: Kv2.1 interacts with Kv8.2 in photoreceptors to create a channel essential for voltage regulation of the light response. In addition, a subpopulation of Kv2.1 can interact with VAPA and only a partial overlap along the OS-IS boundary suggests that ER-PM contact sites, additionally, may also be present in other cellular layers of the retina. In the absence of Kv8.2, Kv2.1 appeared more punctate which may explain two possible scenarios. The first scenario is that Kv2.1enhances the formation of ER-PM contact sites as a part of a mechanism to compensate for the altered ionic imbalance that occurs in the absence of Kv8.2. The other is that Kv2.1contained in the puncta might be the internalized pool of Kv2.1 destined for degradation.

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Analysis of Cav1.4 Dependent Cone Synaptic Development

Laird, Joseph¹; Kopel, Ariel²; Lankford, Colten³; Baker, Sheila^2
1Carver College of Medicine, University of Iowa, Iowa City, IA; ²Department of Biochemistry, University of Iowa; Iowa City, IA; ³University of Florida-Scripps

Purpose: The voltage-gated calcium channel, Cav1.4, mediates synaptic transmission and maintenance at the ribbon synapses of rods and cones. Mutations in the pore forming subunit, a1F, or the trans-synaptic accessory subunit, a2d4, resulting in congenital synaptic disorders such as, congenital stationary night blindness 2 (CSNB2), and cone-rod dystrophy synaptic development in a double-mutant mouse strain where all photoreceptors are cones.

Methods: We crossed conefull and Cav1.4 mutants to generate ‘Conefull: a1F KO’ and ‘Conefull: a2d4 KO’ to further investigate the role of Cav1.4 in the development and function specifically of cone synapses. As a measure of vision, a visually guided water maze was used. Retinal function was measured by electroretinogram (ERG), and retinal health was determined with Optical Coherence Tomography (OCT) and histology.

Results: Conefull: a1F KO could not pass the visually guided water maze whereas the Conefull: a2d4 KO could navigate the water maze as efficiently as the control group. The b-wave, indicative of synaptic transmission, was absent in Conefull: a1F KO and reduced in Conefull: a2d4 KO, and retinal thinning over 2-6 months of age progressed from 32-49% loss for Conefull: a1F KO but only 12-19% loss for Conefull: a2d4 KO. Histological analysis of Conefull: a1F KO demonstrated that photoreceptor loss begins as early as post-natal day 11.

Conclusions: In summary we have created a useful model for detailed analysis of Cav1.4 dependent cone synaptic development.

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Peripheral Trigeminal Nerve Blocks for Chronic Orbital Pain: An Extension Study

Lee, Grace¹; Pham, Chau²; Kardon, Randy^2,3; Shriver, Erin^2,3
1Carver College of Medicine, University of Iowa, Iowa City, IA; ²Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; ³Veteran’s Affairs Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: An initial retrospective study evaluating the therapeutic efficacy of peripheral trigeminal nerve blocks over a mean follow-up period of 2.4 years in 19 patients with chronic orbital pain demonstrated blocks can be successfully used to alleviate chronic orbital pain of wide-ranging etiologies and phenotypes. However, more subjects are needed to further characterize the phenotype of patients who are most likely to respond to treatment.

Methods: Twenty-nine patients who received peripheral trigeminal nerve blocks for chronic orbital pain from November 2016 to March 2022 were included. The injection method has been previously described in detail. Data reviewed included inciting factors, neuropathic symptoms of orbital pain, and injection composition (anesthetic alone versus anesthetic + dexamethasone). Primary outcomes assessed were overall treatment efficacy (no improvement, partial improvement, and complete improvement), injection response (no efficacy, moderate efficacy, and high efficacy), and duration of effectiveness (no effect, 0 – 2 weeks, 2 – 6 weeks, and > 6 weeks), based on patient-reported improvement in pain and quality of life post-injection.

Results: Twenty-nine patients who underwent a total of 154 injections for chronic orbital pain were included. During a mean follow-up period of 2.3 years (range 7 days – 5.5 years), 20 patients (69.0%) achieved at least partial improvement in pain. Of the 20 (69.0%) patients with baseline photophobia, 7 reported persisting photophobia at last follow-up; photophobia at last follow-up was not described for the remaining 13 patients. Of the 154 total injections, 123 (79.9%) received either moderate or high efficacy grades. Thirty-six (39.1%) of the 92 injections for which effect duration was recorded produced a response lasting greater than 6 weeks. There were no significant associations between dexamethasone inclusion and high efficacy injection response (p=0.4) or prolonged effect (p=0.8).

Conclusions: Modulation of trigeminal afferent nerve activity with peripheral trigeminal nerve blocks can help improve pain and quality of life in patients with chronic neuropathic orbital pain. However, their effects on photophobia may be limited. Moreover, long-term follow-up is needed to better assess their therapeutic durability. Physicians should continue shared decision making with patients to clarify personal values and improve their understanding of the therapeutic benefits and risks of the nerve blocks.

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A new diagnostic sign of carotid-cavernous fistula using laser speckle flowgraphy

Linton, Edward¹; Tedeschi, Thomas^1,2; Kardon, Randy^1,2
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; ²VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: Carotid-Cavernous Fistulas (CCF) may be difficult to diagnose without classic anterior ocular signs. Laser speckle flography (LSFG) is a non-invasive imaging technique that allows assessment of ocular blood flow with high enough temporal resolution to resolve changes in flow within a single cardiac cycle. We hypothesized that eyes affected by a carotid cavernous fistula would have disturbed arterial and/or venular flow detectable within the eye with LSFG.

Methods: Four patients with an indirect CCF underwent non-invasive measurement of retinal blood flow using Laser Speckle Flowgraphy. Blood flow parameters were compared to normal subjects (n=32). Choroidal and optic nerve tissue blood flow were quantified by measuring the Mean Blur Rate (MBR) in the fundus after masking out retinal vessels. Relative Flow Volume (RFV) waveforms over an average cardiac cycle were measured for retinal arteriole and venule companion pairs. Timing of peak venular blood flow velocity relative to the companion arteriole (venular delay) was expressed as a percent phase delay relative to the cardiac cycle duration (ΔATI). RFV range (max-min) in companion arterioles and venules was compared between each group.

Results: RFV Venular delay was significantly greater in eyes with CCF (10.8% ± 2.2) compared to unaffected fellow eyes (1.8% ± 0.2) or control eyes of normal subjects (mean 2.7% ± 0.3). This arteriole-venule delay disappeared after fistula thrombosis. In the presence of a CCF, there was a negative deflection of venule RFV during systole and the artery:vein ratio of RFV range was inverted. Choroid and optic nerve MBR was reduced in eyes with a unilateral CCF compared to fellow eyes.

Conclusions: A novel hemodynamic signature of carotid cavernous fistula, venular delay with expanded RFV range compared to the companion arteriole, was discovered in eyes with and without overt ocular signs, which immediately resolves after treatment. Our study is limited by sample size but changes in venous flow parameters within a given eye over time may be a sensitive biomarker for diagnosis and treatment response.

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Vascular injury precedes neuronal loss in a model of radiation retinopathy in mice

Liu, Emily¹; Tamplin, Michelle R.^1,3; Rosius, Jurnie¹; Tedeschi, Thomas S.²; Kardon, Randy H.^2,3; Grumbach, Isabella M.^1,3
Departments of ¹Internal Medicine and ²Ophthalmology and Visual Sciences, University of Iowa Iowa City, IA; ³VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: To determine the time course of vascular and neuronal injury and inflammation in the mouse retina after irradiation.

Methods: C57Bl/6J mice underwent cranial irradiation with 12 Gy. Retinal architecture, vascular density and leakage, and apoptosis were analyzed by histology and immunohistochemistry before or at 10 and 30 days, 8 and 12 months after irradiation. Retinal architecture was analyzed by hematoxylin and eosin staining. Vascular density and leakage were determined using lectin labeling (vasculature) and immunofluorescent staining for mouse IgG (leakage). Loss of nuclei and apoptosis were analyzed by immunofluorescent DAPI staining for nuclei and TUNEL staining for apoptosis.

Results: A significant loss of vasculature density was detected in the plexiform layers starting at 30 days after irradiation. Subtle perivascular leakage was present at 10 days, in particular in the outer plexiform layer. This corresponded to increased width of the outer plexiform layer. At 365 days after irradiation, the neuronal density was significantly reduced compared to baseline. Neuronal apoptosis was detected at 30 days and less prominently at 8 months.

Conclusions: Radiation results in vascular leakage at 10 days, followed by loss of vascular density, increased neuronal apoptosis leading to loss of neurons at 365 days

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The effect of age, sex, and vector on TGFβ2-induced ocular hypertension in C57BL/6J mice

Mao, Weiming¹; Sugali, Chenna Kesavulu¹; Peng, Michael¹; Margetts, Tyler¹; Rayana, Naga Pradeep¹; Dai, Jiannong^1
1Department of Ophthalmology, Indiana University School of Medicine

Purpose: The most important risk factor and causative factor of primary open angle glaucoma (POAG) is elevated intraocular pressure (IOP). Clinically, TGFβ2 is elevated in the aqueous humor of POAG eyes. Experimentally, TGFβ2 induces POAG-associated changes including extracellular matrix deposition, protein cross-linking, cross-linked actin networks in the trabecular meshwork (the tissue that regulates outflow resistance and IOP) as well as OHT in perfusion cultured human eyes and in vivo mouse eyes. Clark and colleagues first reported the use of adenoviral mediated overexpression of constitutively active TGFβ2 to induce OHT in mouse eyes. This approach has become an important research tool for trabecular meshwork research. Here, we determined the role of age, sex, and vector in this model.

Methods: Young (~3 months) and old (~5 months) male and female C57BL/6J mice were purchased from Jackson Laboratory. The mice were housed at the animal facility at Indiana University School of Medicine in reversed light cycle. Adenoviral vectors (Ad5-CMV) were purchased from Vector Biolabs. Lentiviruses were constructed using the pLVX-CMV or pLVX-EF1α vectors (Takara), produced and concentrated in the lab. Mouse IOP was measured at around 2pm under anesthesia using 2% isoflurane in a masked manner. Intravitreal or intracameral injection was performed using a 33-gauge needle under anesthesia using 2% isoflurane.

Results: Published studies showed that 2.5X10e7 PFU Ad5-CMV-TGFβ2 adenoviral vectors elevated IOP in female C57BL/6J mice. In this study, we found that 2.5X10e7 PFU adenoviral vector did not elevate IOP in 3- or 5-month old male C57BL/6J mice. In contrast, 5X10e7 PFU of the same viral vectors elevated IOP in both 3- and 5-month old male C57BL/6J mice. Also, 5-month old mice showed earlier OHT and higher IOP compared to 3-month old mice. We also intravitreally and intracamerally injected lentiviral viruses ovexpressing TGFβ2 using either the CMV or EF1α promoter in male and female mice. We did not observe significant IOP changes using either promoter or injection route at the dose of 8x10e5 PFU/eye. At the dose of 2x10e6 PFU/eye, intracameral injection of the lentiviral vector with the CMV promoter induced significant IOP elevation. However, not all mice showed IOP elevation.

Conclusions: Our data show that old mice respond better to Ad5-CMV-TGFβ2 viruses. Male mice require a higher dose compared to female mice. Also, lentiviral vectors can be used to induce OHT at high doses.

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Feline Trabecular Meshwork Cell Isolation and Culture using Magnetic Particles

Mathu, Virginia¹; Vogel, Kara¹; Torne, Odalys¹; Oikawa, Kazuya¹; Kiland, Julie A.¹; McDowell, Colleen M.¹; McLellan, Gillian J.^1
1University of Wisconsin-Madison, Madison, Wi

Purpose: The trabecular meshwork helps regulate intraocular pressure and is a target for glaucoma therapies. We have established a feline model of inherited glaucoma, but our studies are limited by lack of established methods for culturing feline trabecular meshwork cells (TMCs.) Dissection of trabecular meshwork tissue is complicated in cats by its location and presence of an angular aqueous plexus rather than a Schlemm’s canal. TMC growth from feline tissue explant cultures has been slow and often accompanied by fibroblast overgrowth. We therefore adapted a method originally developed for the culture of mouse TMCs that exploits the phagocytic properties of TMCs. Utilizing magnetic beads feline trabecular meshwork cells were cultured from normal and glaucomatous (LTBP2 mutant) subjects.

Methods: Anterior segment perilimbal tissue containing trabecular meshwork was dissected from 19 feline globes. The tissue was digested in collagenase A and cells were grown as a primary mixed culture then incubated for 2 days with media containing magnetic beads (2.9 uM, Spherotech Inc.) followed by magnetic separation of particle containing TMCs. The identity of the cells as TMCs was confirmed in accordance with consensus recommendations by immunocytochemistry for positive and negative markers for TM and adjacent tissue cell-types and by crossed-linked actin network (CLAN) formation and myocilin mRNA expression by microscopy and qPCR, respectively, following dexamethasone treatment (500 nM) relative to ethanol treated controls.

Results: Cells with morphology consistent with TMCs were isolated by this method. Dexamethasone treatment resulted in enhanced formation of CLANS, upregulation of myocilin mRNA expression in response to dexamethasone treatment. Cells were positively immunolabeled for fibronectin and were negative for cytokeratin, desmin, and Iba1, indicating that the cell cultures do not contain ciliary body epithelial cells, ciliary muscle cells, or macrophages.

Conclusions: Cultured feline TMCs share many properties with human TMCs in vitro, including protein and cell marker expression, and CLAN formation, and myocilin upregulation in response to Dexamethasone exposure. The cells isolated by this method can be used to investigate the differences between normal and glaucomatous trabecular meshwork cells, facilitating elucidation of the effects of LTBP2 mutation on TMCs in this translationally relevant model.

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Alzheimer’s Disease and the Visual System

McCool, Shaylah^1
1Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE

Purpose: Alzheimer’s disease (AD) is the most common neurodegenerative disorder and form of dementia worldwide. AD has been known to impact the visual system, but the mechanisms by which it does this are unknown. The 5xFAD mouse model is an AD model that rapidly develops an AD-like phenotype, specifically amyloid beta (Aβ) plaque formation at around 1.5 months of age. We use this AD model to study structural and functional deficits in neurons and synapses in the dorsal lateral geniculate nucleus (dLGN), the visual thalamus, during an Alzheimer’s disease state.

Methods: In this study, we analyzed amyloid beta density in the dLGN to validate our mouse model. Density of vGlut2 was considered a structural marker for retinal ganglion cell (RGC) axon terminals in the dLGN. Dendritic structure of thalamocortical (TC) neurons was determined via Sholl analysis. To determine functional differences in the retina, we analyzed electroretinogram (ERG) amplitudes, latencies, and oscillatory potentials.

Results: Analysis of Aβ density in the dLGN showed significant levels in the 5xFAD model compared to the C57 controls which had zero Aβ plaques present. vGlut2 immunohistochemistry indicated that RGC axon terminals are likely intact in the 5xFAD model. ERG results suggest no functional deficits in the retina, and Sholl analysis shows no structural changes in the dLGN between the C57 controls and 5xFAD mice.

Conclusions: Despite histopathological evidence of disease in the dLGN, there have not been any structural or functional deficits identified in the visual thalamus. mEPSC recordings of TC neurons may show deficits in synaptic transmission at retinogeniculate synapses, optomotor response (OMR) may give insight into the reflexive behavior of visual system function by testing visual acuity and contrast sensitivity, and identification of Aβ deposits in the retina or hippocampus may encourage future work.

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DAMPs and trabecular meshwork function

McDowell, Colleen¹; Mavlyutov, Timur¹; Mzyk, Philip¹; Myrah, Justin^1
1Department of Ophthalmology, University of Wisconsin-Madison; Madison, WI

Purpose: Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. The goal of this work is to characterize a novel mouse model to utilize in the dissection of the molecular pathology leading to elevated IOP and glaucoma. We investigated the role of a known damage associated molecular pattern (DAMP) and endogenous ligand of Toll-like receptor 4 (TLR4), fibronectin extra domain A isoform (FN-EDA), in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA+/+) that constitutively expresses FN containing the EDA isoform. FN-EDA is normally produced because of tissue damage and remodeling, leads to pathogenic TM damage, and is increased in the glaucomatous TM.

Methods: Eyes were processed for electron microscopy, polymerized in EPON, ultrathin sections (80 nm) were cut and placed on formvar coated slot grids, and poststained with uranyl acetate and lead citrate. Consecutive images of the entire TM area spanning from anterior to posterior parts of Schlemm’s canal (SC) were collected at 2500x and montaged into a single image. ECM accumulation, basement membrane thickness, and size/number of giant vacuoles quantified by ImageJ analysis. TLR4, FN-EDA, A20, and VE-cadherin expression in TM and Schlemm’s canal cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, outflow facility measured ex vivo using the iPerfusion system, and ON damage assessed by PPD stain.

Results: Ultrastructure analyses show the TM of B6.EDA+/+ mice have significantly increased accumulation of ECM between the TM beams with few empty spaces compared to control mice (P<0.05). SC basement membrane is thicker and more continuous in B6.EDA+/+ mice and there are significantly more smaller giant vacuoles compared to controls. No significant structural differences were detected in the TM of EDA null mice. The mRNA expression of TLR4, FN-EDA, VE-cadherin and A20 (a negative regulator of downstream TLR4 signaling) was increased in the TM of B6.EDA+/+ mice compared to control eyes (p<0.05). In B6.EDA+/+ mice, IOP was significantly higher (p<0.01), outflow facility was significantly decreased (p<0.05), and RGC and ON damage was detected at one year of age (p<0.001) compared to control eyes.

Conclusions: B6.EDA+/+ mice have increased accumulation of ECM in the TM, elevated IOP, decreased ouflow facility, and damage to RGC cell axons. These data implicate B6.EDA+/+ mice as a novel mouse model of glaucoma.

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Acute Macular Neuroretinopathy Secondary to Acute Myeloid Leukemia in a Pediatric Patient

Peesapati, Meghna¹; Kaehr, Mark²; Cipkala, Douglas^3
1Marian University College of Osteopathic Medicine, Indianapolis, IN; ²Associated Vitreoretinal and Uveitis Consultants, Indianapolis, IN; ³Ascension St. Vincent Hospital, Indianapolis, IN

Purpose: Retina complications are common in patients with acute myeloid leukemia (AML). To our knowledge, this is the first pediatric presentation of acute macular neuroretinopathy (AMN) as a sequela of AML. AMN is a rare posterior uveitis condition characterized by paracentral scotomas defined by red-brown petalloid or wedge-shaped lesions within the deeper vascular layers of the retina. This condition threatens acute vision loss most often in young adults with a flu-like prodrome but is often self-limited or improves after treatment of underlying conditions. The exact pathogenesis of this condition is unknown, but recent findings implicate the deep capillary plexus. The purpose of this case is to describe a novel mechanism of injury due to AML complications, resulting in the development of AMN.

Methods: Here we describe a 17-year-old male who presented with blurred vision in the right eye, to the left of his central vision for ten days, with associated floaters. PMH was significant for a recent diagnosis of AML. The patient had just completed the first of five chemotherapy rounds. He reported a blurred spot on the Amsler grid in the right eye with 20/16 visual acuity (20/20 in the left). Fundus exam revealed red-brown petalloid deep retinal change only in the right eye but was otherwise unremarkable in both eyes. Ocular examination did not reveal infection or hemorrhage. Macular OCT demonstrated disruption in the ellipsoid zone of the right eye. OCTa demonstrated possible dry rarefaction of the choroidal capillaries in the right eye. Fundal autofluorescence was unremarkable.

Results: This presentation was consistent with a diagnosis of acute macular neuroretinopathy. At one month follow-up, after the second round of chemotherapy, the patient’s condition was improved. He reported some wavy lines on the Amsler grid in the right eye. Fundus exam of the right eye revealed fading lesions in the deep retina. Macular OCT still demonstrated improvement in the disruption of the ellipsoid zone in the right eye. Overall, the patient demonstrated spontaneous improvement of the initial outer segment disruption and therefore did not require specific treatment. On imaging and clinical assessment, the patient’s retinal changes were still consistent with his initial diagnosis of AMN.

Conclusions: AMN due to AML has been reported in adults with ischemia of the deep capillary plexus due to anemia, thrombocytopenia, and/or leukocytosis, which may cause hyperviscosity leading to venous stasis, and thereby worsening local hypoxia. The development of AMN in this case is novel due to the patient’s age at presentation with AML and involvement of the choriocapillaris layer. This patient’s AMN could be secondary to a local hypoxic event due to his underlying leukemia or as a complication from his chemotherapy regimen. The case presented here highlights the characteristics of AML, including anemia, thrombocytopenia, and profound leukopenia, as potential causes for local hypoxia that caused the development of AMN. AML patients must therefore have routine preventative ophthalmic evaluations.

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Tyrosinase Hypomorphic Alleles and Visual Acuity in Patients with Albinism

Prokhoda, Polina¹; Woertz, Erica²; Curran, Erin¹; Costakos, Deborah¹; Carroll, Joseph^1
1Department of Ophthalmology & Visual Sciences,Medical College of Wisconsin; Milwaukee, WI; ²John A. Moran Eye Center, University of Utah, Salt Lake City, UT

Purpose: At least seven different genes can cause albinism when mutated. BCVA has been shown to vary between patients with different albinism subtypes, as well as number of pathogenic mutations. Hypomorphic alleles in the tyrosinase gene (TYR), specifically R402Q and S192Y, result in decreased enzymatic activity of tyrosinase. The purpose of this study was to examine the effect of hypomorphic alleles in TYR on best corrected visual acuity (BCVA) in patients with albinism.

Methods: We retrospectively analyzed BCVA and genetic data from 75 individuals with suspected albinism. We did not include individuals with retinal pathology, prematurity, or whose albinism subtype was unclear due to mutations in multiple genes. Patients were organized by subtype and number of mutations. For individuals with OCA1, 16 had a single pathogenic TYR mutation, while 25 had two or more pathogenic TYR mutations. For individuals with OCA2, 10 had a single pathogenic mutation and 17 had two pathogenic mutations. Seven individuals had OA1 caused by hemizygous mutations in GPR143. We further grouped individuals based on the number of TYR hypomorphic alleles that were identified. One-way ANOVA and Tukey’s multiple comparisons tests were performed to assess the difference in BCVA within groups.

Results: For patients with OCA1 who had two or more pathogenic mutations, we observed a significant difference in BCVA between the TYR hypomorphic allele groups. Post hoc testing revealed significant differences between zero versus two hypomorphic alleles (p=0.04) and one versus two hypomorphic alleles (p=0.001). For subjects with OCA1 who had one pathogenic mutation, there was no significant difference in BCVA between the TYR hypomorphic allele groups (p=0.56). For subjects with OCA2 with one or two pathogenic mutations, there was no significant difference in BCVA between the TYR hypomorphic allele groups (p=0.96 and p=0.22, respectively). For subjects with OA1, there was no significant difference in BCVA between the TYR hypomorphic allele groups (p=0.67).

Conclusions: In patients with OCA1 with two or more pathogenic mutations, the presence of hypomorphic alleles in TYR is associated with worse visual acuity, which could be due to further reduced tyrosinase function. While TYR hypomorphic alleles have been shown to alter foveal structure in individuals with normal vision, it remains to be seen if the hypomorphic allele-related changes in visual acuity described here are also associated with changes in foveal structure in albinism.

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Targeting polycystin-2 attenuates signatures of reactive astrocytes in primary optic nerve head astrocytes

Rao, Vidhya¹; Ghosh, Anita, K¹; Kaja, Simon^1
1Department of Ophthalmology, Loyola University, Chicago, IL

Purpose: Optic nerve head astrocytes (ONHA) are the primary cell type in the optic nerve head. In glaucomatous optic neuropathy, ONHA undergo reactive astrocytosis, the cellular mechanism responsible for optic nerve head remodeling that is characterized by changes to the extracellular matrix. Here we investigated the possible contribution of the intracellular calcium channel, polycysin-2 (TRPP2), to reactive astrocytosis.

Methods: Primary ONHA from adult Brown Norway rats were used for this study. Expression and functional properties of TRPP2 in ONHA were investigated by immunoblotting, immunocytochemistry and planar lipid bilayer electrophysiology. Reactive astrocytosis was induced by exposure to cyclic` mechanical strain (15%, 1 Hz, 16 h) on BioFlex® culture plates. siRNA and AAV2 encoding shRNA targeting TRPP2 were used to knock down polycystin-2 expression. Reactive astrocytosis was assessed by quantification of Reactive Oxygen Species (ROS) and effects on the actin cytoskeleton.

Results: Primary rat ONHA functionally express TRPP2, as determined by immunoblotting, immunocytochemistry and planar lipid bilayer single channel electrophysiology. Biophysical properties of TRPP2 channels were similar to those observed in other ocular cell types previously. Exposure to cyclic mechanical strain (15%, 1Hz, 16 h) resulted in a significant increase in Reactive Oxygen Species (ROS) as quantified by dichlorofluorescein fluorescence and reduced F-actin fiber length quantified by phalloidin labeling. Knock-down of TRPP2 prevented ROS generation and reactive astrocytosis-associated shortening of F-actin fibers.

Conclusions: Our data present the first evidence for functional expression of TRPP2 in ONHA. Partial TRPP2 knock-down attenuated signatures associated with reactive astrocytosis, identifying TRPP2 as a putative drug target for glaucoma.

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Neuritin 1: A therapeutic to promote human retinal ganglion cell survival in an ex-vivo pressurized model system

Sharma, Tasneem¹; Shahul Hameed, Shahna¹; Miller, Ryan¹; Bodi, Nicole^1
1Marilyn and Eugene Glick Eye Institute, Department of Ophthalmology, Indiana University School of Medicine, Indiana University - Purdue University Indianapolis, IN

Purpose: Glaucoma is a progressive neurodegenerative disease and a leading cause of irreversible blindness. Currently, the only targetable risk factor is elevated intraocular pressure (IOP). However, majority of patients continue to lose vision even after lowering IOP due to retinal ganglion cell (RGC) apoptosis and optic nerve atrophy. Thus, it is critical to target RGC loss to develop an effective glaucoma therapy. RGC degeneration is associated with trophic factor deprivation. Our previous studies have shown secreted human Neuritin1 can preserve RGCs after RGC axonal injury. Therefore, we propose to overcome RGC degeneration by over-expressing NRN1 using our novel ex-vivo perfused pressurized Translaminar Autonomous System (TAS) that can simulate pressure induced pathogenesis.

Methods: Human donor eyes were obtained according to Declaration of Helsinki. Quality of eyes was determined by gene expression and electroretinograms (ERG). The retinal integrity within the TAS model was tested by immunostaining of pre- and post-perfused retina. Expression of NRN1 was evaluated in control and glaucoma eyes. To recapitulate the in-vivo pressure stimulus in an ex-vivo environment, human posterior cups were cultured in TAS model at IOP: ICP conditions of 16:12 mmHg for 7 days with and without recombinant hNRN1 (200ng/mL). To assess RGC survival, we evaluated retinal, inflammatory, apoptotic and ECM markers through qPCR and western blotting. Retinal activity with and without hNRN1 was evaluated by ex-vivo ERG.

Results: Donor retinas had measurable retinal gene expression and electrical activity (N=5). Retinal staining was comparable between pre- and post-perfused retinas. Significant decrease of NRN1 expression in retina (N=6, p<0.05) and increased glycosylphosphatidylinositol-anchored NRN1 (N=4, p<0.05) in vitreous was observed in glaucoma versus control donors. In contrast to controls, we observed decreased gliosis (IBA-1, N=5, p<0.05), decreased apoptosis (CASP3, N=4, p<0.001) and increased neuronal survival (THY-1, N=5, p<0.01) with a significant reduction of ECM proteins COLIV and FN (N=3, p<0.05). We also identified measurable retinal activity with NRN1 treatment.

Conclusions: Our study identified that hNRN1 promotes RGC survival and increases retinal function under degenerative conditions. This study substantiates human NRN1 as a novel therapeutic target for glaucomatous degeneration.

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Resolution of retinal cyst cysts partially underlies beneficial effects observed after subretinal gene therapy in a mouse model of Juvenile X-linked Retinoschisis

Thompson, Jacob^1,2; Hsu, Ying^1,2; Gehrke, Ella J.^1,2; Mayer, Sara^1,2; Mahoney, Angela^1,2; Drack, Arlene V.^1,2
1Department of Ophthalmology and Visual Sciences; and ²Institute for Vision Research; University of Iowa, Iowa City, IA

Purpose: Juvenile X-Linked Retinoschisis (JXLR) is a vitreoretinal disorder and is the most common cause of macular degeneration in male children. JXLR is due to mutations in the RS1 gene on the X chromosome. Retinoschisin is a secreted protein encoded by RS1 implicated in cell adhesion and signaling. Clinical features include the splitting of retinal layers known as schisis, cyst formation, and an electronegative electroretinogram (ERG) b-wave. Previous studies have shown improved phenotype in Rs1 knockout mice (Rs1-KO) with intravitreal and subretinal gene therapy, but also with empty capsids. Improvements seen from empty capsids are unexplained. The purpose of this study is to investigate efficacy of subretinal gene therapy in Rs1-KO mice compared to buffer injection and natural history.

Methods: An AAV2/4-Ef1α-RS1 vector was delivered by 2 µL subretinal injection of 2E9 vg/mL to Rs1-KO mice (n=4). To control for the effect of the delivery method, 2 μL subretinal injection of buffer solution, 0.001% F68/PBS pH 7.4, was delivered Rs1-KO mouse retinas (n=5) and compared to eyes that received gene therapy and natural history in non-injected eyes. Injections occurred at postnatal day (P) 24-31. ERG, completed at 1 month post injection, and optical coherence to tomography (OCT), completed weekly to 1 month post injection, were chosen as endpoints to assess efficacy. OCT images were scored for severity of schisis and outer nuclear layer (ONL) thickness.

Results: OCT: Mice that received a subretinal injection of the gene therapy vector or the delivery buffer had thinner ONLs than non-injected mice at 1-week and 2-week post-injection, likely due to the effect of the subretinal injection itself. However, ONLs of non-injected and buffer injected mice thinned over time, and at 1-month post injection, mice that received the gene therapy vector had significantly thicker ONLs than mice that received the delivery buffer. Schisis severity was reduced in mice that received the gene therapy vector or the delivery buffer, compared to non-injected mice. ERG: Concomitant with the schisis severity results, mice injected with gene therapy vector or buffer had improved ERG amplitudes and b/a wave ratios compared to their non-injected counterparts.

Conclusions: Whereas preservation of ONL thickness was only observed in gene therapy treated mice, a transient reduction in schisis severity was observed in mice receiving gene therapy and mice receiving delivery buffer alone, along with improvements in ERG amplitudes. Though the cause is unknown, we speculate that the observed partial correction of ERG phenotypes is due to the short-term resolution of schisis. However, maintenance of ONL thickness is only observed in gene therapy treated mice indicating that resolving cysts does not lead to preservation of photoreceptors. This indicates that photoreceptor survival in JXLR requires RS1. Further studies are necessary to uncover whether gene therapy treatment leads to long-term preservation of ONL thickness and ERG metrics.

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Microglia in the dorsal lateral geniculate nucleus (dLGN) during retinal ganglion cell (RGC) degeneration.

Thompson, Jennifer¹; Smith, Jennie C.²; McCool, Shaylah³; VanHook, Matthew^2
1University of Nebraska Medical Center; ²Department of Ophthalmology and Visual Sciences, Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, NE; ³Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, United States. Department of Ophthalmology and Visual Sciences, Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, NE, United States. Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, United States.

Purpose: Although the majority of investigations concerning the response of microglia to glaucomatous neurodegeneration have been executed at the level of the retina, microglial responses and their interactions with the outputs of retinal ganglion cells (RGCs) in the dorsal lateral geniculate nucleus (dLGN) of the thalamus have not been previously described. The goals of this study were to characterize the response of dLGN microglia to ocular hypertension (OHT) in a mouse model of inherited glaucoma, and to determine the extent to which such features associate with elevations in intraocular pressure (IOP).

Methods: We performed longitudinal IOP measurements on DBA/2J mice, along with strain-matched controls, and collected tissue at 4, 9, and 12 months of age. We resolved microglia morphology by staining for ionized binding calcium adaptor molecule 1 (IBA1), then performed skeleton analysis on 50µm sections which contained central portions of the dLGN. We quantified RGC outputs by density measures of vesicular glutamate transporter 2 (vGlut2) puncta. In addition, we assessed the cleavage product of complement component 1 (C1q) by fluorescence intensity analysis. We analyzed between group differences using factorial ANOVAs and post-hoc Student’s T-tests. For IOP correlations, regressions were linearly fit.

Results: The average IOPs of D2 mice increased significantly from 4mo to 9mo, and from 9mo to 12mo. The average IOP of DBA/2JGpnmb+ mice (D2-Controls) did not differ with age and was significantly lower than D2 mice by 9 months. RGC axon terminals, identified as vGlut2 puncta, were similar for D2s and D2-Controls at 4 months, yet a significant reduction was present for D2s at each of the subsequent timepoints. Puncta densities for vGlut2 were negatively correlated with final IOP averages. In D2s, microglia (Iba1+) counts were higher at 9mo and 12mo than at 4mo, but this increase was not observed in controls. Microglia branching features (branch length per cell; endpoints) were negatively correlated with IOP. Deposition of C1q increased with age in D2 mice, and positively correlated with IOP.

Conclusions: Here, we show that increased IOP corresponds to changes in the morphology microglia of in the mature dLGN. We demonstrate that this immune response involves polarizing towards an activated phenotype, as evidenced by a retraction of processes and a reduction in contact points. Further, these changes become more pronounced as IOP increases and are concurrent with the loss of excitatory synaptic outputs. We show an inverse relationship between the intensity of C1q and the density of RGC outputs, suggesting that a selective tagging of synapses may be involved in loss of RGC output during RGC degeneration. Overall, our results suggest that, during chronic elevations in IOP, the loss of RGC synapses in the dLGN might be occurring by way of microgliosis and induction of the complement cascade.

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Neurons and synapses in the visual thalamus in mouse models of ocular hypertension

Van Hook, Matthew¹; Sladek, Asia¹; Zhang¹, Kevin (Yang)¹; Thompson, Jennifer¹; Bierlein, Elizabeth R.¹; Smith, Jennie C.^1
1Truhlsen Eye Institute, Dept. of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE

Purpose: Thalamocortical (TC) relay neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus receive convergent synaptic input from several retinal ganglion cells (RGCs) and convey those signals to the primary visual cortex for conscious vision. The goal of this study was to determine how TC neuron function and synaptic inputs from RGCs (retinogeniculate/RG synapses) are altered in mouse models of glaucoma.

Methods: We measured RG synaptic function in brain slices from male and female DBA/2J (D2) and DBA/2J-gpnmb+ (D2-control) mice at 6m, 9m, and 12m of age using optic tract stimulation in parasagittal brain slices while performing whole-cell voltage clamp recordings from post-synaptic TC neurons. In some experiments, intraocular pressure was experimentally elevated by injection of polystyrene microbeads into the anterior chamber. TC neurons were filled with neurobiotin during whole-cell recording in coronal slices. Anterograde RGC axon transport was measured as the fraction of dLGN area labeled by a unilateral intravitreal injection of cholera toxin-B (CTB). RGC synaptic terminal density weas quantified using immunofluorescence staining for vGlut2.

Results: There was also a reduction in the density of vGlut2 puncta at 9m and 12m and vGlut2 density was significantly correlated with strength of CTB labeling. There was a statistically significant reduction in the maximum synaptic current amplitude (EPSCmax) detected in slices from 12m D2 mice although the single-fiber EPSC (EPSCsf) was not different in amplitude between groups. The EPSCsf/EPSCmax ratio was higher in slices from 12m-old D2's indicative of reduced numbers of RGC inputs to each TC neuron. TC neurons from both D2 mice and mice with microbead-induced ocular hypertension showed increases in intrinsic excitability, firing more action potentials in response to depolarizing current. Finally, TC neurons in both models showed reduced dendritic complexity, as measured using Sholl analysis.

Conclusions: RGC synaptic loss is detectable in older DBA/2J mice and elevated eye pressure alters the structure and function of dLGN TC neurons. Central synapse dysfunction and altered TC neuronal excitability might contribute to glaucomatous vision loss.

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SARM1 ablation provides neuroprotection in experimental glaucoma

Wadkins, David^1,3; Chen, Nathan²; Elwood, Ben³; Gramlich, Oliver W.^1,3; Kuehn, Markus H.^1,3
1Department of Ophthalmology and Visual Sciences; and ²Carver College of Medicine, University of Iowa, Iowa City, IA; ³VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: Glaucoma is the leading cause of vision loss, impacting over 60 million people worldwide. Primary open angle glaucoma (POAG) is frequently accompanied by elevated intra-ocular pressure (IOP) which induces retinal ganglion cell (RGC) death and axonal degeneration. Current therapies primarily consist of IOP management; however, these therapies regularly fail to fully halt visual decline. Evaluating checkpoints in the retinal cell death pathways could lead to novel targets for pharmaceutical therapies to arrest vision decline. SARM1 is a gene that encodes an enzyme that is a sensor of metabolic stress and executioner of programmed neuronal cell and axon death. The purpose of this study was to determine if deactivation of SARM1 confers a neuroprotective effect in animal models of glaucoma.

Methods: Elevated IOP was induced in C57BL/6J-SARM1^em1Agsa/J (SARM1 KO) and control mice via injection of 3e8 pfa Ad5myoc virus into the anterior chamber of the eye (n=20/group). Elevated IOP was maintained for 4 months, and the optokinetic reflex (OKR) was measured monthly to functionally evaluate the loss of visual acuity. Pattern electroretinograms (pERG) were performed to assess electrophysiology of the retina and optic nerve. Retinas were then collected and stained with antibodies directed against RBPMS to evaluate RGC density. 8 images from the central region, mid periphery and outer periphery of each eye were acquired and these images were counted using an Image J macro. The counts were then normalized to cells per micron².

Results: Our data indicate that SARM1 ko mice exhibit robust increased RGC survival in all regions of the retina when compared to control animals. In the outer periphery SARM1 mice with elevated IOP lost on average 188.9 cells per mm², whereas control mice with the same IOP elevation lost 353.2 cells per mm² (p-value <0.01) in the mid peripheral and central retina SARM1 mice lost 214.7, and 250.4 cells/mm², respectively, whereas controls lost 451.6 and 491.8 cells/mm², respectively (p-values <0.01). Functional testing of visual acuity further supports the finding that loss of SARM1 is neuroprotective. Vision in SARM1 ko mice declined on average by 0.0356 cycles/degree, while vision in control mice decreased on average by 0.09722 cycles per degree (p-value 0.0494).

Conclusions: Taken together, our data indicate that lack of SARM1 reduces RGC loss and preserves visual function in a mouse model of chronic glaucoma. These findings suggest that SARM1 inhibition is a strong candidate for small molecule therapies to reduce visual decline in patients with glaucoma.

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Spatial Patterns of Retinal Structure in Glaucoma in a 2D Deep Learning Variational Autoencoder (VAE) Latent Space

Wang, Jui-Kai^1,3; Garvin, Mona^1,2,3; Kardon, Randy^1,3
Departments of ¹Ophthalmology and Visual Sciences, ²Electrical and Computer Engineering, University of Iowa, Iowa City, IA; ³VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: Glaucoma is a leading cause of irreversible blindness related to optic nerve damage and can result in both diffuse and regional patterns of retinal neuron loss due to damage to their axons at the optic nerve head. Standard quantitative estimations of glaucomatous progression use a global average and do not capture underlying spatial patterns. To simultaneously quantify and visualize the spatial patterns of neuron loss in glaucoma, we evaluated the feasibility of spatial modeling of macular ganglion cell plus inner plexiform layer (mGCIPL) thickness maps using a deep learning variational autoencoder (VAE). The proposed VAE model allows for the succinct representation (two latent variables in this study) and reconstruction of spatial structural thickness maps in optical coherence tomography

Methods: A VAE is designed by concatenating two simultaneously trained convolutional neural networks to provide a probabilistic model so that: 1) the encoder can learn how to efficiently decompose the input image into succinct digits – "latent variables," and 2) the decoder can learn how to correctly reconstruct the input image by only analyzing these succinct latent variables. In this work, the encoder and decoder have a symmetric structure of five concatenated convolutional layers with various channels followed by rectified linear units (ReLU). To enforce that the latent space is smooth, the sampling units introduced standard/normalized Gaussian distribution from the output of the encoder to constrain the distribution of the latent variables. Two latent variables were used in this study.

Results: To train the VAE model, we used 602/899 macular OCT scans from 25/25 normal/glaucoma subjects in the training stage. For the test set, 13/77 macular OCT scans from different 5/25 normal/glaucoma subjects were used for validation. Among all these 90 test eyes, the mean signed and root-mean-square (RMS) differences between the original and VAE-generated mGCIPL maps were 0.54 and 7.14 micrometers, respectively; the overall correlation coefficient was 0.86 (p<0.01).

Conclusions: Our results showed that the latent space tile montage contains meaningful spatial patterns of glaucomatous defects corresponding to the known superior and inferior arcuate patterns of nerve loss due to the nerve bundles being damaged at the optic disc head. The VAE model demonstrates a promising potential for glaucoma/normal case classification and for monitoring further glaucomatous progression in the clinic.

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Passive transfer of peripheral blood mononuclear cells from POAG patients causes retinal ganglion cell loss in NOD/scid recipient mice

Zeng, Huilan^1,2; Gramlich, Oliver^1,2; Wadkins, David^1,2; Kuehn, Markus H.^1,2
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; ²VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: We have previously shown that transfer of T-cells from glaucomatous mice to normal animals causes retinal ganglion cells (RGC) in recipients. Here we demonstrate that transfer of peripheral blood mononuclear cells (PBMC) from glaucoma patients causes RGC loss in humanized mice in accordance with ophthalmic metrics of the donating patient.

Methods: Blood samples were obtained from primary open angle glaucoma (POAG) patients without regard to disease state or rate of vision loss (n=20). In addition, samples were obtained from non-glaucomatous controls (n=10). Exclusion criteria were other retinal diseases (except AMD) autoimmune disease or neurodegenerative diseases. PBMC were isolated from these samples and transferred into immune deficient NOD/scid gamma (NSG) mice by intraperitoneal injection. Each mouse received PBMC from one donor only, creating an immunologic ‘avatar’ of the patient. After transfer mouse eyes received a microbead injection calibrated to cause mild, transient elevation of intraocular pressure (IOP). 50 days after PBMC transfer, RGC density was determined in retinal flat mounts using cresyl violet staining. Data

Results: Transfer of human PBMC did not cause significant side effects. However, retinas of mice having received PBMC from POAG donors displayed significantly fewer RGC than those having received PBMC from controls (255±64.8 RGC/field vs. 341±131 RGC/field, p=0.027) in the central area. Among donors with POAG, PBMC of those with more advanced disease caused less damage to the recipients’ RGC than PBMC with less advanced disease. Avatars of donors with a ganglion cell layer (GCL) thickness above the median displayed 197±60.9 RGC/field whereas those with GCL thickness below the median contained 282 ±75 RGC/field (p:0.017). Likewise, avatar mice of donors with visual field loss above the mediandisplayed 296±62 RGC/field, significantly more than those having received PBMC from donors with more advanced

Conclusions: These findings confirm that transfer of PBMC obtained from glaucoma patients causes more damage to RGC in NSG mice than those of controls. Our data also indicate that immune reactions may be more pronounced in POAG patents with moderate disease than in those with advanced disease.

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