2023 AMAR - Trainee Poster Contest Handout

2023 Annual Meeting and Alumni Reunion

Annual Trainee Poster Contest

Trainee Poster Contest Floorplan

(The Newbury Hotel, Garden Ballroom)

Epidemic B8

Marta Stevanovic - Dual Molecular Diagnoses in Patients With Inherited Retinal Degenerations

Filippos Vingopoulos - Quantitative Contrast Sensitivity Correlates Better Than Visual Acuity With Geographic Atrophy Size in Advanced Dry AMD: A New Functional Endpoint

Melissa Yuan - Characteristics of Patients Presenting With Central Retinal Artery Occlusion to a Large Academic Center

Henry Zhou - Outcomes and Cost Analysis of Acute Commotio Retinae at a Tertiary Referral Center

Yadav Adhikari - Role of Polyploidy in Fuchs Endothelial Corneal Dystrophy

Hamid Alemi - Characterization of Corneal Fibrosis in Young versus Adult Mice Following Mechanical Injury

Shazia Ashraf - ATM-Mediated Prominent DNA Damage Response in Fuchs Endothelial Corneal Dystrophy

- Non-Human Primate Laser-Induced NAION Model for Assessing Neuro-Rejuvenating Gene Therapies

Neetu Kushwah - Myeloid Cell-Specific Deficiency of RORα Exacerbates Choroidal Neovascularization and Subretinal Inflammation in Mice

María Camila Lancheros Vega - Application of Enzyme-Loaded Microspheres to Overcome the Transplantation Barrier of the Inner Limiting Membrane

Volha Malechka - Modulation of Microglia by Co-Treatment With Soluble FasL and Annexin V Improves the Outcome After Donor RGC Transplantation

Meenakshi Maurya - Rod Photoreceptor-Specific REV-ERBα Deletion Accelerates Age-Related Synaptic Remodeling in the Mouse Retina

Riccardo Sangermano - In Vivo CRISPR-KO Screens for the Study of Photoreceptor Essential Genes

Madhura Shah - Epigenetic Reprogramming- A Novel Gene Therapy That Restores Vision Loss in a Nonhuman Primate Model of NAION

Sampath Vemula - Impaired Neuromuscular Junction Development in the Extraocular Muscles of Mice With Nystagmus

Candidate: Camille Andre

Poster #: A1

Genomic Epidemiology, Antimicrobial Resistance and Virulence Factors of Methicillin-Resistant S. aureus Isolates Causing Eye Infections.

Camille

André, Michael S. Gilmore, Paulo Bispo

Purpose: S. aureus is a major cause of eye infections. Methicillin-resistant S. aureus (MRSA) is commonly associated with multidrug-resistant (MDR) infections, resulting in treatment failure and poor visual outcome. To assess the emergence of high-risk MDR clones of MRSA causing eye infections, the repertoire of antimicrobial resistance (AMR) genes and virulence factors circulating among this population, we have sequenced the genomes of all MRSA isolates recovered from patients presenting with eye infections between January 2014 and December 2021 at the Massachusetts Eye and Ear and Sao Paulo hospital.

Methods: Whole Genome Sequencing was performed on 247 MRSA isolates causing eye infections using Illumina MiSeq. Molecular typing was performed by multilocus sequence typing. Comprehensive Antibiotic Resistance Database (CARD) and Pathogenwatch were used to identify genes and mutations that confer AMR. Routine antimicrobial susceptibility testing was performed by using the MicroScan WalkAway system. Virulence factor database (VFDB) was used to identify virulence genes that contribute to the pathogenicity of the bacteria.

Results: The population structure of MRSA isolates causing eye infections was dominated by lineages grouped within the clonal complex 5 (CC5) (50.6%) and CC8 (31.1%). Patterns of association of distinct genotypes have been noted with orbital abscess/cellulitis mainly caused by CC8 (60.0%) whereas keratitis were substantially enriched in the CC5 lineage (64.0%). 70% of MRSA CC5 isolates were MDR (resistance to ≥3 antimicrobial classes). In line with these observations, we found that the resistome of these CC5 strains was comprised of multiple acquired AMR genes that confer resistance to various clinically important antibiotics including aminoglycosides, macrolides, lincosamides, and streptograminB. Transference of some of these genes between strains is facilitated by horizontal gene transfer as they are found in mobile genetic elements such as transposons. In our CC5 population the mecA gene is integrated into a SCCmec cassette which also carries a full transposon (Tn554) containing the ermA and ant(9)-Ia genes, both present in 79.7% of our CC5 isolates. Ebh (adherence virulence factor) and 7 enterotoxin superantigens (seg, sem, sen, seo, seq, yent1 and yent2) were found to be significantly enriched among CC5 ocular isolates.

Conclusions: MRSA isolates causing eye infections mainly clustered within the CC8 and CC5, with patterns of disease. CC5 strains carried a variety of AMR genes that result in high levels of phenotypic resistance to first-line topical therapies. Specific virulence factors found in CC5 isolates may promote keratitis infections.

Candidate: Kanza Aziz

Poster #: A2

Genome-Wide Polygenic Risk Score for Primary Open Angle Glaucoma is Associated With More Severe Disease in a MultiEthnic Biobank

Kanza Aziz, Yan Zhao, Mohammad Eslami, Louis R. Pasquale, Tobias Elze, Ayellet V. Segre, David S. Friedman, Janey L. Wiggs, Nazlee Zebardast

Purpose: Primary open angle glaucoma (POAG) polygenic risk score (PRS) has been associated with increased risk of glaucoma, developing severe disease, maximum recorded IOP, younger age at diagnosis, and glaucoma progression. However, the utility of PRS in clinical practice is not well established. Here we aimed to evaluate the association between PRS and severity of POAG using data from a multi-ethnic clinical biobank.

Methods: A Lassosum penalized regression framework from 10238000 directly genotyped and imputed variants for UK Biobank samples (n=449186: 14171 cases, 394292 controls) was used to compute a genome-wide PRS. The calculated weights were used to compute PRS in 7689 participants with ocular data available in the Mass General Brigham Biobank. PRS were normalized within each genotype-inferred ancestral group. Participants were stratified into POAG cases and controls based on the presence of ICD codes 365.1, 365.8, 365.9, H40.1, H40.8, and H40.9. Data including intraocular pressure (IOP), cup-to-disc ratio (CDR), spherical equivalent (SE), optical coherence tomography derived macular ganglion cell complex (GCC) and optic nerve retinal nerve fiber layer (RNFL) global thicknesses were extracted. The worse measure from the worse eye was selected for each patient. Multivariable linear regression models were used to examine the association between ocular factors and PRS.

Results: Of the 7689 participants, 1077 (14%) were cases and 6612 (86%) were controls. The mean (SD) age was 67.5 (14.8) years, and 53% were female. The ancestral breakdown was 86.4% European, 6.1% Hispanic, 5.6% African, and 1.8% East/South Asian. The prevalence of POAG increased with each decile of polygenic risk and nearly tripled from 9.1% in the first decile to 24.2% in the 10th decile. After adjusting for age and sex, each SD increase in PRS was associated with 0.40 (CI:0.29,0.51; p<0.001) mmHg increase in IOP, 0.04 (CI:0.03,0.04; p<0.001) increase in CDR and -1.61 (CI: -2.84,-0.37; p=0.011) μm decrease in global RNFL thickness. There was no significant association between PRS and SE or global GCC thickness.

Conclusions: Our findings suggest that underlying POAG polygenic risk increases disease prevalence and severity, supporting the use of a POAG PRS to identify individuals at high risk of POAG and potentially risk stratify patients in our clinics.

Candidate: Nicole Belanger

Poster #: A3

An All-In-One Highly Multiplexed Diagnostic Assay for Rapid, Sensitive, and Comprehensive Detection of Ocular Pathogens

Purpose: Ocular infections are sight-threatening conditions that can lead to vision loss. Rapid identification of the etiologies plays a key role in early initiation of effective therapy to save vision. However, current diagnostic modalities are time consuming and lack sensitivity and inclusiveness. We present here a newly developed comprehensive ocular panel designed to improve diagnostic yields and provide a tool for rapid and sensitive pathogen detection.

Methods: A panel containing 46 pathogens and 2 resistance/virulence markers that are commonly detected in intraocular infections was developed. Genomic targets were scrutinized for stretches predicted to be specific for a particular species while being conserved across different strains. A set of primers for sample enrichment, and two 50mer NanoString compatible probes were then designed for each target. Probe-target hybrids were detected and quantified using the NanoString nCounter SPRINT Profiler. Diagnostic feasibility was assessed in two pilot clinical studies testing samples from infectious retinitis (n = 15), endophthalmitis (n = 12), and keratitis (n = 17) patients, for which the etiologies were confirmed by polymerase chain reaction (PCR) or culture.

Results: Analytical studies demonstrated highly sensitive detection of a broad spectrum of pathogens, including bacteria, viruses, and parasites, with limits of detection being as low as 2.5 femtograms per reaction. We also found excellent target specificity, with minimal cross-reactivity detected. The customdesigned NanoString ocular panel correctly identified the causative agent from all clinical specimens positive for a variety of pathogens.

Conclusions: This highly multiplexed panel for pathogen detection offers a sensitive, comprehensive, and uniform assay run directly on ocular samples that could significantly improve diagnostics of sightthreatening ocular infections.

Paulo Bispo, Nicole Belanger, Ashley Li, Renee Liu, Gayatri Susarla, Weilin Chan, Lawson Ung, James Chodosh, Michael S. Gilmore, Lucia Sobrin

Candidate: Madison Echavarri-Leet

Poster #: A4

Recovery From Amblyopia in Adulthood: A Meta-Analysis

Madison P. Echavarri-Leet, Hannah H. Resnick, Daniel A Bowen, Deborah Goss, Mark F. Bear, Eric D. Gaier

Purpose: Effectiveness of traditional amblyopia therapies is largely restricted to childhood, but recovery in adulthood is known to occur following removal or vision-limiting disease of the fellow eye (FE). Descriptions of this phenomenon are limited to case reports and small series, leaving knowledge gaps related to the incidence and clinical features of amblyopic eye (AE) recovery in adulthood. To address these gaps, we performed a meta-analysis of published cases. Regarding clinical features, we hypothesized that (1) FE retinal ganglion cell (RGC) injury is associated with better AE recovery, (2) strabismus blunts AE gains and (3) AE improvement is directly associated with the degree of FE vision loss.

Methods: A systematic review of 3 literature databases yielded 23 reports containing 109 original descriptions of patients ≥18 years with unilateral amblyopia and FE injury. Two meta-analyses were performed: (1) incidence: 3 case series (42 patients) reporting qualifying patients agnostic to recovery; (2) clinical features of recovery (hypotheses 1-3): 101 patients with any quantifiable AE best-corrected visual acuity (BCVA) improvement.

Results: Incidence: 32/42 (76%) and 29/42 (69%) adult amblyopic patients experienced ≥1 and ≥2 logMAR lines of AEBCVA improvement with FE disease.

Clinical Features: Among those with AE BCVA improvement, median change in AE BCVA was 5.0 (range 0.5–19.2) logMAR lines. Recovery occurred across amblyopia types and FE pathologies, contrary to hypotheses 1 and 2. FE disorders directly affecting FE RGCs demonstrated shorter latencies to AE BCVA recovery by 4 months (p<0.0124). Multivariate regression analysis revealed younger age, worse baseline AE BCVA, and worse nadir FE BCVA (supporting hypothesis 3) were independently associated with the greater gains in AE BCVA (p values<0.006).

Conclusions: The adult brain harbors the neuroplastic capacity necessary for clinically meaningful recovery. AE gains are greater with worse FE BCVA impariment and faster with disorders affecting RGCs. These results support the potential therapeutic utility of temporary retinal inactivation in adults with amblyopia.

Candidate: Kasem Seresikachorn

Poster #: A5

Evaluation of Polygenic Risk Score Prediction and Validation of Diagnostic Codes of Primary Open-Angle Glaucoma in Electronic Health Record (EHR)-Linked Biobanks

Joyce Kang, Jessica H. Tran, Kasem Seresirikachorn, Urvi Gupta, Elaine Han, Thi Ha Vy, Ghislain Rocheleau, Ron Do, Yan Zhao, Yuyang Luo, Ayellet V. Segrè, Janey Wiggs, Louis Pasquale, Nazlee Zebardast

Purpose: A polygenic risk score (PRS) may improve risk stratification for primary open-angle glaucoma (POAG). In constructing a PRS, case definitions are often based on diagnostic codes which may introduce misclassification. To assess the utility of International Classification of Disease (ICD)-codebased PRS construction and future usage, we validated ICD codes of POAG status and tested PRS performance among ICD-code-based and validated cases/controls in two electronic health record (EHR)-linked biobanks.

Methods: This retrospective cohort study of biobank participants included 42,286 Mount Sinai BioMe and 34,547 Mass General Brigham (MGB) Biobank participants.

We constructed the PRS using the Lassosum penalized regression framework from 10,238,000 directly genotyped and imputed variants for 449,186 UK Biobank samples (14,171 cases, 394,292 controls). The calculated weights were used to compute a POAG PRS for each site and normalized within each inferred ancestry group.

We reviewed records of ICD-identified POAG subjects and confirmed cases based on chart notes, optical coherence tomograms, and visual fields (VF). In a 5% random sample, controls were verified based on intraocular pressure and cup-disc ratio.

We compared the PRS area under curve (AUC) performance of manually reviewed vs. ICD-identified cases/controls.

Results: 650/1043 and 545/889 patients from BioMe and MGB with >1 POAG ICD code had their charts reviewed (Table 1).

For BioMe and MGB, respectively: positive predictive value was 56% vs. 52%; negative predictive value was 95% vs. 96%; sensitivity was 96% vs. 96%; specificity was 52% vs. 53%. Among cases confirmed manually, the most common ancestral groups represented were African (54.3%) and Hispanic (32.4%) in BioMe and European (80.5%) in MGB.

All AUCS between curves were >0.05 (Figure 1).

Conclusions: Our multi-site study demonstrates that case definitions using ICD codes to identify POAG cases and controls are highly sensitive and moderately specific. A PRS designed to identify POAG performed similarly using either manual clinical standard or ICD coding across most ancestries in two EHR-linked biobanks.

Candidate: Aaron Kaufman

Poster #: A6

Fuchs Endothelial Corneal Dystrophy Associations With Comorbid Systemic Disease, Lifestyle, and Nutritional Intake

Aaron R. Kaufman, Myriam S. Böhm, Francesca Kahale, Pia Leon, Viridiana Kocaba, Ula V. Jurkunas

Purpose: Fuchs endothelial corneal dystrophy (FECD) has multifactorial pathophysiology, with genetic and environmental factors. Systemic health contributions to FECD pathophysiology are incompletely characterized. This study aims to identify possible FECD associations with systemic comorbidities, lifestyle, and nutrition using a retrospective chart review and cross-sectional survey.

Methods: 50 FECD patients and 50 age/sex-matched control patients were enrolled. Inclusion criterion for the FECD group was FECD clinical diagnosis, and inclusion criteria for the control group were no FECD diagnosis and no guttae. Chart review examined demographics, FECD stage, medical history, and body mass index (BMI). Survey used a smoking & exercise questionnaire and a semiquantitative food frequency questionnaire (SFFQ). Statistical methods were Fisher exact text and Mann Whitney U test.

Results: Rates of cardiovascular diseases had greater occurrence in FECD compared to controls: hyperlipidemia (74% vs. 50%, p=0.023), atrial fibrillation (26% vs 8%, p=0.031), hypertension (68% vs 50%, p=0.103), coronary artery disease (20% vs 8%, p=0.148), and aortic stenosis (10% vs 2%, p=0.204). Diabetes had similar occurrence (10% vs 10%, p=1.000). No difference was observed for having ever smoked (56% vs 38%, p=0.109), but FECD patients had higher current daily smoking behavior (8.64 vs 4.92 packs/day, p=0.046), smoking duration (13.10 vs 8.52 yrs, p=0.048), and total smoking exposure (11.24 vs 6.12 pack- years, p=0.017). There were no differences in BMI (26.54 vs 26.17, p=0.879) or exercise activity (4.66 vs 4.56 hrs/wk, p=0.840). SFFQ computed levels for 231 nutritional items; differences in nutritional items included: sodium (2036.12mg vs 436.22mg, p=0.021), insulinogenic load (711 vs 667.79, p=0.077), and total fat (67.00g vs 71.00g, p=0.036).

Conclusions: Statistically significant differences between FECD and control patients were found for rates of cardiovascular diseases, smoking behavior, and sodium intake. Further investigation with a larger cohort may confirm these associations. Conclusions from the nutritional profile are limited by the single timepoint provided by a cross-sectional survey contrasting with the longitudinal nature of FECD pathogenesis. It might be prudent to counsel FECD patients about concomitant cardiovascular disease control, moderation of salt intake, and smoking cessation.

Candidate: Jonathan Lin

Poster #: A7

Aqueous Humor Neurofilament Light Chain is a Marker of Neurodegeneration in Glaucoma

Purpose: Neurofilament light chain (NfL) is a neuronal cytoskeletal protein that has been identified as a marker of neurodegeneration. Here, we investigated whether NfL in the aqueous humor (AH) can serve as a marker of glaucomatous neurodegeneration in a racially diverse North American population.

Methods: In this single-center, case-control study, we collected AH from 39 glaucoma patients and 10 patients without glaucoma undergoing ophthalmic surgery. AH NfL was quantified using the highly sensitive Single-Molecule Array (Simoa)® NF-light assay (Quanterix). Clinical information was obtained by reviewing the medical record.

Results: In a generalized linear model controlling for age and body mass index (BMI), AH NfL was significantly elevated in glaucoma patients (mean: 429 pg/ml; standard deviation [SD]: 1136 pg/ml) compared to patients without glaucoma (mean: 3.1 pg/ml; SD: 1.9 pg/mg): P=0.002. Higher AH NfL was associated with higher maximum intraocular pressure (IOP) (R=0.44, P=0.005), higher preoperative IOP (R=0.46, P=0.003), and more pre-operative glaucoma medications (Rs=0.61, P<0.001).

Conclusions: We provide evidence that AH NfL may have clinical utility as a marker of glaucomatous neurodegeneration. While further validation is necessary, our findings provide a foundation for longitudinal studies investigating the potential role of NfL in clinical practice.

Jonathan B. Lin, Kristen M. Pitts, Hani El Helwe, Cameron Neeson, Nathan E. Hall, Henisk Falah, Silas L. Wang, Kristine Lo, Christian Song, Milica A. Margeta, David Solá-Del Valle

Candidate: Anagha Lokhande

Poster #: A8

The Impact of Myopia on Regional Visual Field Loss and Progression in Glaucoma

Purpose: To investigate the impact of myopia on regional visual field (VF) loss and VF progression in glaucoma.

Methods: Reliable SITA Standard 24-2 VFs were selected from a large academic institution. Crosssectional linear regression analysis was performed to quantify the impact of myopia measured by spherical equivalent (SE) on regional VF loss. Associations for VF locations with p-values ≥ 0.05 were reset to zero for visualization. Logistic regression analysis was conducted by correlating SE with four different VF progression outcomes: (1) mean deviation (MD) progression: MD slope < 0 and p-value < 0.05; (2) VF index (VFI) progression: VFI slope< 0 and p-value < 0.05; (3) total deviation (TD) pointwise progression: at least three TD locations with TD slope ≤ -1 dB/year and p-value < 0.05; (4) MD fast progression: MD slope ≤ -1 dB/year and p-value < 0.05. All analyses were adjusted for patient demographics including age, sex, race, ethnicity, and language ability with additional adjustment for baseline MD for progression prediction. The sample consisted of 55,321 eyes from 55,321 patients (mean MD: -5.2 ± 6.3 dB; mean SE: 0.4 ±3.2 diopters; mean age: 57.2 ± 17.2 years; sex: female: 57.7%, male: 42.2% and unknown: 0.1%; race: White:69.7%, Asian: 6.2%, African American: 10.7%, and other or unknown: 13.4%; ethnicity: Hispanic: 6.9%, non-Hispanic: 84.4%, and unknown: 8.7%; primary language: English speakers: 87.5%, Spanish speakers: 4.0%, other languages speakers: 4.7%, and unknown: 3.8%) and 8,037 patients with at least five reliable 24-2 VFs over four years for longitudinal analyses.

Results: Cross-sectional analysis revealed that more negative SE values were associated with more negative TD values in the paracentral VF region. These results remained after MD adjustment. Longitudinal analysis revealed that with MD adjustment, three of the four measures of VF progression had a higher likelihood of deterioration with more negative SE: MD progression (odds ratio [OR] = 0.97, p = 0.008), VFI progression (OR = 0.97, p = 0.0001), and TD pointwise progression (OR = 0.97, p = 0.007). Similar results were seen without MD adjustment.

Conclusions: Eyes with myopia experience greater VF loss in the paracentral region. More severe baseline myopia in the context of glaucoma is associated with a higher likelihood of VF progression.

Anagha Lokhande, Yan Luo, Min Shi, Yu Tian, Tobias Elze, Louis R. Pasquale, Lucy Q. Shen, Nazlee Zebardast, David S. Friedman, Michael V. Boland, Mengyu Wang

Candidate: Christine Pang

Poster #: A9

The Impact of Demographics on Regional Visual Field Loss and Worsening in Glaucoma

Purpose: To elucidate the impact of self-reported demographics including gender, race, ethnicity and language ability on regional visual field (VF) loss and worsening in glaucoma on top of age.

Methods: We selected reliable SITA Standard 24-2 VFs from Massachusetts Eye and Ear Glaucoma Service. We performed multivariable linear regression analyses to assess the association of demographic parameters with regional VF loss and worsening measured with pointwise analyses. Longitudinal analyses were further adjusted for baseline mean deviation. Linear mixed modeling was applied to adjust for multiple samples from the patient and p-values were adjusted for multiple comparisons.

Results: We included 200,810 24-2 reliable VFs from 100,405 glaucoma patients (VF mean deviation: -3.9 ± 5.6 dB; age: 56.1 ± 17.8 years; gender: female: 42%; White: 70%, Asian: 6%, African American: 10% and other or unknown: 14%; Hispanic: 6%, non-Hispanics: 85% and unknown: 9%; English speakers: 80%, Spanish speakers: 4% and other languages speakers or unknown: 16%) for crosssectional analyses and 16,440 patients with at least 5 reliable 24-2 VFs over 4 years for longitudinal analyses. Males had worse VF loss at all locations (up to -0.8 dB) with the inferior hemifield being more affected. Compared with Whites, Asians had worse VF loss in the superior hemifield up to -0.4 dB and lesser inferior hemifield deficits. African Americans had worse VF loss in all locations than Whites with greater superior hemifield impairment up to -2.9 dB. Spanish and other language speakers had worse VF loss than English speakers globally up to -3.4 and -2.6 dB, respectively. In longitudinal analysis, males had greater VF loss worsening than females up to -0.07 dB/year in the inferior hemifield. Race and ethnicity appeared to be not relevant to rates of VF loss worsening. Spanish and other language speakers had more VF worsening than English speakers up to -0.12 dB/year with superior hemifield more affected.

Conclusions: Males had more inferior VF loss and worsening in this clinic-based population. African Americans were associated with more superior VF loss. Non-English speakers are associated with more superior VF loss and worsening. These findings can help clinicians monitor glaucoma based on patient demographics and suggest healthcare disparities.

Yueyin Pang, Melody Tang, Min Shi, Yu Tian, Yan Luo, Tobias Elze, Louis R. Pasquale, Nazlee Zebardast, David Friedman, Michael V. Boland, Lucy Q. Shen, Mengyu Wang

Candidate: Lyvia Zhang

Poster #: A10

Complications After Pediatric Penetrating Keratoplasty: An IRIS Registry Study

Lyvia J. Zhang, Isdin Oke, Tobias Elze, Joan W. Miller, Alice C. Lorch, Reza Dana, Thomas H. Dohlman

Purpose: Penetrating keratoplasty (PK) is effective in the management of corneal opacities; however, transplants in pediatric patients tend to have worse outcomes compared to adult patients, and there has been little attention paid to this in the literature. The purpose of this retrospective cohort study was to describe the postoperative complications in children who underwent PK using a large national electronic health record database.

Methods: Pediatric patients aged under 18 years in the Intelligent Research in Sight (IRIS) Registry who underwent PK between 01/2013 to 12/2020 were identified using Current Procedure Terminology codes. Postoperative complications were identified using International Classification of Diseases codes and categorized as either anterior segment (infectious keratitis, epithelial defect, neurotrophic keratitis etc.) or posterior segment (vitreous hemorrhage, retinal detachment, cystoid macular edema, etc.) complications. The frequency of each type of complication and the median time since PK are reported.

Results: Of the 544 pediatric patients (645 eyes) identified, 318 (58.5%) experienced postoperative complications, including 222 (70%) involving the anterior segment and 96 (30%) involving the posterior segment. The most common anterior segment complication was infectious keratitis (N=163, 30%) with a median onset time of 275 days (IQR) after PK. Vitreous hemorrhage (N=52, 9.6%) and retinal detachment (N=48, 8.8%) were the most common posterior segment complications. Complications with a median onset time greater than one year included neurotrophic keratitis, choroidal detachment, proliferative vitreoretinopathy, and epiretinal membrane.

Conclusions: Complications occurred more frequently in the anterior segment compared to the posterior segment. Postoperative complications threaten graft success as well as long-term visual outcomes, and this study illustrates the postoperative challenges faced in pediatric keratoplasty patients.

Candidate: Sandra Alhoyek

Poster #: B1

Longitudinal Assessment of Macular Thickness and Microvascular Changes in Children With Sickle Cell Disease

Sandra Hoyek,

Colin

Lemire, Omar Halawa,

Francisco

Altamirano-Lamarque, Efren Gonzalez, Nimesh A. Patel

Purpose: To longitudinally assess macular thickness and microvascular changes in children with sickle cell disease (SCD).

Methods: This is a retrospective consecutive series of 606 eyes of 303 children ≤18 years old with SCD who had an ophthalmic exam at Boston Children’s Hospital from January 1998 to August 2022. Qualitative and quantitative analyses of both optical coherence tomography (OCT) and OCT angiography (OCTA) images were performed. Main outcomes measures included total retinal thickness measured on macular OCT, as well as superficial capillary plexus (SCP) and deep capillary plexus (DCP) vessel density (VD), and foveal avascular zone (FAZ) area measured on 6 x 6 mm OCTA scans.

Results: There were 606 eyes of 303 children included. The breakdown by genotype was 280 (46%) HbSS, 154 (25%) sickle trait, 106 (18%) HbSC, 50 (8%) HbS thalassemia, and 16 (3%) unknown. OCT and OCTA images were acquired on 104 (17.2%) and 60 (9.9%) eyes at presentation and on 159 (26.2%) and 100 (16.5%) eyes at final visit. Overall, temporal retinal thinning was noted qualitatively in 35.6% of SCD patients at presentation and 40% at final visit. Of those patients with macular thinning, 94.6% and 90.5% had peripheral sickle cell retinopathy (SCR) at presentation and final visit. On quantitative OCT analysis, HbSS eyes had a lower retinal thickness in the fovea and temporal parafovea compared with HbSC, P < 0.05. Eyes with peripheral SCR had a larger FAZ at presentation compared to eyes without peripheral SCR (P = 0.004), a lower DCP VD at final visit in the inferior temporal macula (P = 0.03), and a higher DCP VD at final visit in the superior nasal macula (P = 0.01). At final visit, retinal thickness decreased at the fovea, inferior perifovea, and temporal perifovea compared to presentation, P < 0.05. In parallel, VD DCP in the superonasal quadrant increased at final visit, P = 0.03.

Conclusions: Macular retinal thinning was progressive and observed in eyes with and without peripheral SCR. Over time, there was a compensatory increase in DCP VD in the nasal macula on OCTA.

Candidate: Saghar Bagheri

Poster #: B2

Heidelberg Multispectral vs. Traditional Color Fundus Imaging for the Detection of Ocular Changes Due to Spaceflight

Saghar Bagheri, Jorge Nagel, Aditya Verma, Alex Huang, Srinivas Sadda, Sara Mason, Robert Gibson, William Tarver, Mary Van Baalen, Tyson Brunstetter

Purpose: To evaluate if multispectral imaging (= MultiColor imaging (MCI)) obtained with the Heidelberg SPECTRALIS® device is an effective replacement for traditional color fundus photographs as a diagnostic fundus screening tool onboard the International Space Station (ISS). Multispectral imaging significantly reduces crew time (approx. 115 minutes/session, 36 hours/year) while having the option to capture a larger field of view (55° vs. 35°).

Methods: 44 multispectral images, 14 of which were of normal eyes and 30 of abnormal eyes with pathologies resembling those that can occur due to spaceflight were graded and compared to corresponding color fundus pictures (ZeissVisupacFF450 plus) at UCLA and Doheny Eye Institute.

Results: Doheny Eye Institute grading concluded that multispectral imaging is more sensitive to traditional color fundus imaging for the detection of disc swelling, cotton wool spots, as well as retinal hemorrhage.

Conclusions: MCI can effectively detect all retinal and optic nerve findings detectable by CFP during spaceflight and is a suitable on-orbit replacement as a diagnostic screening tool. Additionally, dozens of crew hours are spared per year by utilizing MCI.

Candidate: Ines Lains

Poster #: B3

Plasma Metabolites Associated With Optical Coherence Tomography (OCT) Features of Age-related Macular Degeneration (AMD)

Ines Lains, Xikun Han, João Gil, Joana Providencia, Archana Nigalye, Rodrigo Alvarez, Vivian Paraskevi Douglas, Kevin Mendez, Raviv Katz, Gregory Tsougranis, Jinglun Li, Rachel S. Kelly, Ivana K. Kim, Jessica Lasky-Su, Rufino Silva, Joan W. Miller, Liming Liang, Demetrios Vavvas, John B. Miller, Deeba Husain

Purpose: The most widely used classifications of age-related macular degeneration (AMD) and its severity stages still rely on color fundus photographs (CFP). However, AMD has a wide phenotypic variability that remains poorly understood and is better characterized by optical coherence tomography (OCT). We and others have shown that patients with AMD have a distinct plasma metabolomic profile compared to controls. However, all studies to date have been performed solely based on CFP classifications. This study aimed to assess if plasma metabolomic profiles are associated with OCT features commonly seen in AMD.

Methods: Prospectively designed, cross sectional study. We included subjects with a diagnosis of AMD and a control group (> 50 years) from Boston, United States and Coimbra, Portugal. All participants were imaged with CFP, used for AMD staging (AREDS2 classification scheme), and with spectral-domain OCT (Spectralis, Heidelberg). OCT images were graded by two independent graders for the presence of characteristic AMD features, according to a predefined protocol. Fasting blood samples were collected for metabolomic profiling (using non-targeted high resolution mass spectrometry by Metabolon Inc). Analyses were conducted using logistic regression models including the worst eye of each patient (AREDS2 classification) and adjusting for confounding factors. Each cohort (US and Portugal) was analyzed separately and then results were combined by meta-analyses. False discovery rate (FDR) was used to account for multiple comparisons.

Results: We included data on 468 patients, 374 with AMD and 94 controls, and on 725 named endogenous metabolites. Meta-analysis identified significant associations (FDR < 0.05) between plasma metabolites and three OCT features: hyperreflective foci (n= 6), atrophy (n=6) and ellipsoid zone disruption (n=3). Most associations were seen with amino acids. No overlap was observed among metabolites and the OCT features they were statistically related to.

Conclusions: To our knowledge, we show for the first time that plasma metabolites have associations with specific OCT features seen in AMD. Our results support that the wide spectrum of presentations of AMD likely include different pathophysiologic mechanisms by identifying specific pathways associated with each OCT feature.

Candidate: Renee Liu

Poster #: B4

Clinical Outcomes for Intravenous Immunoglobulin Treatment in Autoimmune Retinopathy With Comparison to Natural History Controls

Renee Liu, Jacob T. Cox, Caroline L. Minkus, Ashley Li, Samuel Han, Lynn K. Stanwyck, Joseph F. Rizzo, Lucia Sobrin

Purpose: To compare clinical outcomes of autoimmune retinopathy (AIR) between patients treated with intravenous immunoglobulin (IVIg) and natural history counterparts.

Methods: Retrospective cohort study including all AIR patients followed for at least three months at the Massachusetts Eye and Ear Infirmary from 1988 to 2020 and treated with either IVIg or no immunomodulatory therapy. Descriptive statistics were used to characterize the study population. All visual acuity (VA), electroretinogram (ERG), optical coherence tomography (OCT), and Goldmann visual field (GVF) measurements were extracted via chart review. The change in these data from baseline to various timepoints (i.e., 6-, 12-, 24-months, final assessment) were calculated. Multivariate regression analyses were performed in Stata 13.1. The main outcome measure is the difference in VA progression between IVIg-treated patients and natural history controls.

Results: 21 patients met inclusion criteria. 9 were treated with IVIg and 12 were natural history patients. Among IVIg patients, 44% had significant VA improvement and 56% had stable VA at final assessment compared to initial presentation. In the natural history cohort, 67% had significant VA decline and 33% had stable VA. Assessing each eye individually, VA remained stable with a median change of 0.00 logMAR in the IVIg cohort but declined 0.26 logMAR in the natural history cohort (pvalue = 0.013). ERG analysis found White 0.5 Hz and White 30 Hz amplitudes were less likely to decline among IVIg patients compared to natural history controls (p -values = 0.041 and 0.013, respectively). Progression of OCT parameters and GVF area showed no significant difference between groups. Subset analysis of natural history patients found no significant difference in the progression of the aforementioned variables between paraneoplastic and non-paraneoplastic AIR.

Conclusions: IVIg therapy is significantly associated with preservation of VA and select ERG parameters in AIR patients when they are compared to natural history controls. This suggests IVIg may be an effective therapy for this difficult to treat, often devastating disease. Efforts to elucidate the natural history of AIR found no significant differences between the paraneoplastic and nonparaneoplastic subsets of AIR.

Candidate: Yan Luo

Poster #: B5

Assessing Retinal Layers' Association With Diabetes Using a Deep Learning Framework

Purpose: To quantify the association between retinal layer thickness and diabetes diagnosis with a deep learning approach.

Methods: From the population-based Leipzig Research Center for Civilization Diseases (LIFE) Adult Study, we included participants with macular Spectralis optical coherence tomography (OCT) scans and American Diabetes Association-stratified diabetes status. We exacted the retinal layer thickness maps (RLTMs) for the ten retinal layers segmented by the Heidelberg Engineering software, including retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), myoid zone (MZ), ellipsoid zone (EZ) and outer-photoreceptor segment (OS) combined (EZ+OS), interdigitation zone (IZ) and retinal pigment epithelium (RPE). We used each of the ten layers individually and ten layers together to predict diabetes diagnosis. We used the area under the receiver operating characteristic curve (AUC) to measure the association strength between each retinal layer and diabetes diagnosis. Participant-level separation was applied to divide our data into training and testing subsets with two-thirds and one-third of the total data, respectively.

Results: 17,715 OCT scans from 8,947 participants with information on diabetes status were included in this study. There were 4,901 participants with normal glucose tolerance (NGT), 2,786 with prediabetes, and 1,260 diabetes patients, respectively. Combining ten layers together, the AUCs to distinguish diabetes from NGT, diabetes from prediabetes, and prediabetes from NGT were 0.76, 0.64, and 0.65, respectively. The inner layers were generally more affected, including RNFL, GCL, IPL, INL, OPL, and ONL with AUCs ranging from 0.68 to 0.72 consistent with existing clinical knowledge. The top four layers that were most associated with diabetes diagnosis measured by AUCs were ONL, RNFL, RPE, IPL with AUCs of 0.72, 0.71, 0.69, and 0.69, respectively.

Conclusions: The deep learning approach was able to quantify the association between retinal layer thickness and diabetes status.

Yan Luo, Franziska G. Rauscher, Tobias Elze, Yu Tian, Min Shi, Saber Kazeminasab, Mohammad Eslami, Kerstin Wirkner, Thomas Peschel, Michael Stumvoll, Berend Isermann, Matthias Blueher, Markus Loeffler, Toralf Kirsten, Thomas Ebert, Mengyu Wang

Candidate: Joel Rämö

Poster #: B6

Overlap of Genetic Loci for Central Serous Chorioretinopathy With Age-Related Macular Degeneration

Joel Rämö, Erik Abner, Elon HC van Dijk, Xin Wang, Seung Hoan Choi, Tuomo Kiiskinen, Risto Kajanne, Juha Mehtonen, FinnGen, Priit Palta, Mitja Kurki, Lucia Sobrin, Suzanne Yzer, Steven A. Lubitz, Patrick T. Ellinor, Anneke den Hollander, Tõnu Esko, Mark J Daly, Joni A. Turunen, Aarno Palotie, Camiel JF Boon, Elizabeth J. Rossin

Purpose: Central serous chorioretinopathy (CSC) is a serous maculopathy of unknown etiology. Chronic CSC and age-related macular degeneration (AMD) have features that overlap and differ, and two of three previously reported CSC risk loci are also associated with AMD. Improved understanding of CSC genetics may unveil causal mechanisms in both diseases.

Methods: Using ICD code based criteria, we identified 552 CSC cases and 343,461 controls in the FinnGen study and 103 CSC cases and 178,573 controls in Estonian Biobank. We performed new genome-wide association studies (GWAS) and included previously reported results from 521 chronic CSC cases and 3,577 controls in a meta-analysis of 1,176 CSC cases and 526,787 controls. We assessed the expression of genes prioritized by the Polygenic Priority Score (PoPS) and nearest-gene methods in cultured choroidal endothelial cells and public ocular single cell RNA sequencing datasets. We evaluated polygenic scores (PGS) for CSC and AMD in FinnGen.

Results: We replicate two previously reported CSC risk loci (near CFH and GATA5) and report three novel loci (near CD34/46, NOTCH4 and PREX1). The CFH and NOTCH4 loci are associated with AMD but in the opposite direction. Prioritized genes are implicated in vascular hyperpermeability (PREX1) and development (NOTCH4 and CD34). Prioritized genes show increased expression in cultured choroidal endothelial cells (p = 0.0038) and are differentially expressed in choroidal vascular endothelial cells in single cell RNA sequencing data (average fold change 1.49–2.48 vs. other cell types, p < 7.1x10-20). A PGS for AMD is predictive of reduced CSC risk (OR = 0.76 per +1SD in PGS, p = 7.4x10-10); this association is mitigated after removing complement system loci (OR = 0.93 [0.851.01], p = 0.087).

Conclusions: We identify five genetic risk loci for CSC, highlighting a likely role for genes involved in choroidal vascular function and complement regulation. Polygenic AMD risk is negatively predictive of CSC and this genetic overlap is largely due to complement-related loci.

Candidate: Priya Shah

Poster #: B7

Choroidal Thickness Between Sex With Common Macular Diseases

Priya

Shah, Panisa Singhanetr, Isaac Bleicher, Elizabeth J. Rossin

Purpose: Central serous chorioretinopathy (CSCR) is a retinal disease indicated by serous detachment(s) of the neurosensory retina and may include detachment of retinal pigment epithelium (RPE). Age-related macular degeneration (AMD) is a macular disease characterized by the degeneration of photoreceptors and surrounding tissues, the addition of extracellular deposits, with the loss of central vision. Pattern dystrophy is characterized by pigment disposition in the retinal pigment epithelium (RPE) and is typically centered in the macula. Previous research reports in healthy populations, males have a thicker choroid than females. One study found that male patients with CSCR have a thicker choroid than females with CSCR and another found that in some types of AMD, like neovascular AMD, females have a thinner choroid than males.

Methods: From Massachusetts Eye and Ear (MEE), we gathered 574 patients with CSCR that were seen in the Retina clinic. Additionally, from a Greek data set, we gathered 91 pattern dystrophy patients. There were also 308 AMD patients from Greece and MEE that were used in the study. From here, any patients that received anti-VEGF injections, photodynamic therapy (PDT), had a choroidal neovascular membrane (CNVM), or where the choroid was obscured were excluded from the study. Finally, we used the left-over patients to measure their choroidal thicknesses using enhanced depth and spectral domain OCTs. Using R, we conducted a linear regression based on sex with a categorical predictor of age. Only the right eye was used for all bilateral disease patients.

Results: We identified 128 males (age = 46.67) and 56 females (mean age = 55.38) that met our inclusion criteria for CSCR. We identified 98 males (age = 80.99) and 70 females (mean age = 81.23) that met our inclusion criteria for AMD. And we also identified 55 males (age = 80.56) and 28 females (mean age = 79.54) that met our inclusion criteria for pattern dystrophy. In patients who had an initial diagnosis of unilateral CSCR (91 males, 40 females) and bilateral CSCR (37 males, 16 females), males had a thicker choroid than females (p= 0.042). The results also indicated that there is no statistically significant difference in choroidal thickness between AMD patients and pattern dystrophy patients based on sex.

Conclusions: These results illustrate a difference in choroidal thickness between males and females. While the etiology behind the sex imbalance in CSCR and pattern dystrophy is likely multifactorial, appreciating contributing factors will help to understand the etiology of both diagnoses better and a difference in baseline choroidal thickness may contribute. Further study will involve a larger sample size and a similar analysis in healthy individuals.

Candidate: Jared Sokol

Poster #: B8

Injection Drug Use-Associated Endogenous Endophthalmitis in the Modern Opioid Epidemic

Jared T. Sokol, David J. Krasinski, George N. Papaliodis, Eugene Lambert, Dean Eliott, Eric D. Gaier

Purpose: In the context of a historic opioid crisis, ophthalmologists can impact the care of persons with substance use disorders (SUDs) through the diagnosis and management of injection drug useassociated endogenous endophthalmitis (IDU-EE) and collaboration with an addiction consult service (ACS). We aimed to update SUD care and visual outcomes in the context of IDU-EE.

Methods: We reviewed cases of endophthalmitis from 2015-2021. Among patients with IDU-EE, the clinical features, treatment and outcomes of SUDs and IDU-EE were analyzed.

Results: Of 627 endophthalmitis cases, 62 subjects (9.9%; 65 eyes) with IDU-EE were included. Among 54 subjects who were inpatient, 37 (68.5%) were seen by the ACS. Of the 32 patients eligible for a new medication for opioid use disorder (MOUD) prescription, 13 (40.6%) were prescribed and accepted one (all seen by ACS).

Initial ophthalmic treatments were tap and inject (TAI) in 51 (78.5%) eyes and pars plana vitrectomy (PPV) in 9 (13.8%) eyes. Subsequent TAI was performed in 21 eyes (32.3%) and PPV in 30 eyes (46.2%). Intraocular cultures were positive in 34 (54.8%) eyes and systemic cultures were positive in 10 (16.1%) subjects. TAI was less likely to yield a positive culture (11/52) compared with PPV (26/39; p<0.0001). Median visual acuity (VA) improved from 1.3 (20/400 Snellen) to 0.48 logMAR (20/60) (p=0.0003). Retinal detachment occurred in 15 (23.1%) eyes. Fentanyl use increased the odds of poor (≤counting fingers) VA outcome (OR 5.0, 95% CI 1.3-22.8).

Conclusions: This is the largest case series of IDU-EE to date (Modjtahedi BS, et al. Ophthalmol Retina. 2017) and spans the current fentanyl crisis. Hospitalization and ACS collaboration resulted in a 4x increased likelihood of MOUD initiation. Initial PPV did not statistically improve visual outcomes over TAI but did facilitate pathogen identification. Fentanyl use conferred greater odds of poor visual outcomes.

Candidate: Marta Stevanovic

Poster #: B9

Dual Molecular Diagnoses in Patients With Inherited Retinal Degenerations

Marta Stevanovic, Kaitlin O’Connell, Priya Gupta, John S. Borchert, Jason Comander, Emily Place, Rachel M. Huckfeldt

Purpose: To highlight individuals with inherited retinal degenerations (IRD) whose likely underlying genetic explanation for retinal disease involves more than one gene. These findings may have prognostic implications, impact genetic counseling, and affect eligibility for gene-specific clinical trials.

Methods: Patients seen at Massachusetts Eye and Ear between 2020 and 2022 who had multiple potential genetic solutions for their clinical diagnosis were identified. Genetic testing was performed using commercial retinal dystrophy panels with variant classification following the American College of Medical Genetics criteria; only variants that were pathogenic (P) or likely pathogenic (LP) were included. The results of genetic testing were compared to clinical data, and family members were assessed when possible.

Results: Four patients were identified: two with macular dystrophies and two with retinitis pigmentosa (RP). Patient 1 (P1), who had a multigenerational family history of PRPH2-associated pattern dystrophy (PD), had anatomic disruption throughout the posterior pole with corresponding functional impact in contrast to the limited macular abnormalities in his asymptomatic affected parent. Genetic testing identified the familial PRPH2 variant (P) and two variants in ABCA4 (both P) in trans. Patient 2 (P2) had clinical findings of PD and was identified to have a PRPH2 variant (LP) and two ABCA4 variants (P, LP). There was no family history of IRD, and family members were not available for segregation analysis. Patient 3 (P3) had RP with two CNGA1 variants in trans (both P) as well as a RHO variant (P). There was no family history of RP. Patient 4 (P4), who had a diagnosis of Usher syndrome type 2, had two variants in USH2A (both P) and a PRPH2 variant (LP). There was no family history of retinal disease; familial assessments were pending.

Conclusions: This cohort demonstrates the value of broad genetic testing in explaining phenotypic variation within families (P1) and assessing familial risk even in the setting of presumed recessive disease (P4). These cases also illustrate the ambiguity introduced if segregation analysis cannot be performed (P2) and the challenges of understanding how coexisting variants in multiple genes impact clinical phenotype and prognosis even if phase is known (P3).

Candidate: Filippos Vingopoulos

Poster #: B10

Quantitative Contrast Sensitivity Correlates Better Than Visual Acuity With Geographic Atrophy Size in Advanced Dry AMD: A New Functional Endpoint

Filippos Vingopoulos, Marta Stevanovic, Peyman Razavi, Jack Tracy, Hanna Choi, Hannah Wescott, Deeba Husain, Leo A. Kim, Joan W. Miller, Demetrios G. Vavvas, John B. Miller

Purpose: Investigate the correlation between geographic atrophy (GA) size and quantitative contrast sensitivity function (qCSF) cross-sectionally and longitudinally.

Methods: 83 foveal involving GA eyes (defined as cRORA on OCT), 29 with more than one visit. Average follow up was 17.2 months (4-37). Same day qCSF contrast sensitivity (CS), OCT and FAF were performed. Regression models were used for associations between semiautomatic Heidelberg Regionfinder-measured total GA size on FAF, qCSF metrics and Snellen visual acuity (VA).

Results: Cross-sectionally (N=83), total GA was associated with CS thresholds at 1, 1.5, 3, 6, 12 (β=0.39, -0.33, -0.26, -0.29, -0.23, all p<0.05) but not with VA (β=0.08, p=0.47). Pearson correlations for GA size and qCSF outcomes (r=-0.35, -0.32, -0.31, -0.35, -0.23, all p<0.05) vs VA (r=0.14, p=0.23). Longitudinally (N=29), ΔGA size was associated with ΔCS threshold at 3cpd and ΔCA (β=-0.50, β=0.46, p<0.05) but not with ΔVA. (β= 0.03, p= 0.88).

Conclusions: Total GA size and ΔGA are associated with qCSF CS and ΔCS but not with VA or ΔVA. CS may be a better functional endpoint to measure treatment effect in AMD clinical trials.

Candidate: Melissa Yuan

Poster #: B11

Characteristics of Patients Presenting With Central Retinal Artery Occlusion to a Large Academic Center

Melissa Yuan, Sandra Hoyek, David N. Zacks, Joan W. Miller, Demetrios Vavvas, Nimesh A. Patel

Purpose: To characterize a large modern cohort of CRAO patients by describing presenting features as well as outcomes relating to OCT features, angiographic reperfusion and visual recovery.

Methods: This was a retrospective consecutive case series of 146 eyes of 145 patients initially presenting to a tertiary referral center between January 2017 and December 2021. ICD-10 codes for CRAO (H34.1) were used to identify subjects and records were manually reviewed. Fundus photographs, OCT, and fluorescein angiography images were analyzed. Primary outcomes included reperfusion, visual outcomes and development of neovascularization.

Results: The mean age at presentation was 69.4 ± 13.6 years. The average time to presentation was 1.6 ± 4.2 days, with 62.8% examined within 24 hours of vision loss. The average initial visual acuity (VA) in the affected eye was Snellen equivalent 20/5000, with a 90% rate of relative afferent pupillary defect. Only 7.6% had visible emboli. Cilioretinal artery sparing was observed in 19% of cases. Of the total tested, 32.6% had elevated inflammatory makers and 4 cases (2.7%) were due to biopsy confirmed giant cell arteritis. Etiology otherwise was 37% cryptogenic, 34.2% atheroembolic, 20% cardioembolic, 3.4% iatrogenic, and 1.4% associated with varicella zoster virus. Fluorescein angiography was performed in 22% of patients at presentation and showed delayed arteriovenous transit time (78%) and peripheral hypo or non-perfusion (37%). At final follow-up, the mean visual acuity was 20/3700, (p=0.13) with 35.2% having gained of 2 or more lines of vision. Twelve percent developed neovascularization. Out of 48 eyes (33%) imaged with fluorescein angiography at final follow-up, 1/3 showed reperfusion. In a multiple linear regression, VA at presentation (β=0.7, P<.001) was associated with a better visual outcome. Of 122 patients with available initial and final BCVA, 109 (89.3%) were 20/200 or worse at presentation. Of those 109 patients, 6 (5.5%) had a final vision of better than 20/200. Of those 6 patients, 3 (50%) had CRA sparing and 3 (50%) did not have CRA sparing. After CRAO, OCT initially shows macular thickening, then progressive thinning, largely due to inner retinal thinning as outer retinal thickness remains constant over time.

Conclusions: Those with acute CRAO often present within 24 hours of symptoms, with 1/3 having elevated inflammatory markers. In some cases, visual recovery and reperfusion are possible, both with and without the presence of a cilioretinal artery. Better visual acuity at presentation was associated with visual gain. OCT initially shows macular thickening, then progressive thinning, largely due to inner retinal thinning. This data can serve as baseline for future interventional trials.

Candidate: Henry Zhou

Poster #: B12

Outcomes and Cost Analysis of Acute Commotio Retinae at a Tertiary Referral Center

Purpose: To determine the utility in scheduled follow-up of patients with commotio retinae (CR) presenting emergently.

Methods: We reviewed 490 patients presenting to the MEEI ED from 1/1/2020–1/1/2022 for contusion or other injuries of the eye/orbit. 201 patients (41.0%) had CR on exam and were included for analysis.

Results: Mean visits to CR resolution was 2.27 ± 1.41 visits. 68 OCTs, 66 fundus photos, and 254 follow-up visits were performed prior to CR resolution, totaling $64,856. Dollars spent to detect one retinal tear on follow-up is $16,214. Loss to follow up rate is 29.3%. Four patients (2.0%) developed related retinal tears on follow-up, with one progressing to retinal detachment. Tears found on follow-up were preceded by worsening symptoms in 2/4 cases. Laser retinopexy was performed in 1/4 cases.

Conclusions: Patients with CR infrequently develop retinal complications and have a high rate of loss to follow up (no show). There is a significant cost and low utility in scheduled follow up for CR.

Henry W. Zhou, Sandra Hoyek, Shravan V. Savant, Scott R. Shuldiner, Grayson W. Armstrong, John B. Miller, Nimesh A. Patel

Candidate: Yadav Adhikari

Poster #: C1

Role of Polyploidy in Fuchs Endothelial Corneal Dystrophy

Yadav Adhikari, Neha Deshpande, Vicki Losick, Ula V. Jurkunas

Purpose: Fuchs endothelial corneal dystrophy (FECD) is an oxidative stress disorder associated with accelerated corneal endothelial cell (CEnCs) loss. It is the leading cause of corneal transplantation worldwide and is mainly characterized by loss of corneal endothelial cells and formation of guttae which compromise the transparency of the cornea. Although genetic and environmental factors have been shown to be implicated in FECD progression, the underlying molecular mechanisms leading to corneal endothelial cell loss and guttae formation in the cornea are yet to be understood. Using an ultraviolet light (UVA) irradiation based non-genetic mouse model we have previously shown that cellular senescence leading to fibrosis is involved in FECD. However, the potential implication of polyploidy in this process is poorly understood. Herein, we characterize the temporal evolution of polyploidy, senescence, and fibrosis processes in the corneal endothelium in response to UVA induced genotoxic stress.

Methods: Eight weeks old female C57BL/6J mice were irradiated with UVA light (500 Joules/cm2) on the right eye (OD) while non-irradiated left eyes (OS) served as controls. Corneas were harvested at 3-, 7-, 14- and 28- days post UVA irradiation. Nuclear Ploidy of corneal endothelial cells was evaluated using DAPI intensity. Development of senescence and fibrosis was evaluated by immunostaining against H3K9me3 and alpha SMA respectively.

Results: Increase in corneal endothelial nuclear ploidy was detected after UVA irradiation which was significantly higher at 3-days post UVA compared to control (p<0.001, One way ANOVA with Tukey;s HSD, N=3). Greater polyploidization was observed in central cornea compared to peripheral cornea at all time points evaluated after UVA irradiation (p<0.01, students t-test, N=3). Polyploidization was followed by onset of senescence (p<0.01) and fibrosis (p<0.01) which was maximum at 14-days post UVA irradiation (One way ANOVA with Tukey;s HSD, N=3).

Conclusions: We demonstrate that corneal endothelial cells polyploidize in response to UVA induced genotoxic stress. Polyploidization was followed by onset of senescence and fibrosis. Our study provides initial insights into understanding the role of polyploidy in determining the fibrotic processes in the corneal endothelium in the pathogenesis of FECD.

Candidate: Hamid Alemi

Poster #: C2

Characterization of Corneal Fibrosis in Young versus Adult Mice Following Mechanical Injury

Purpose: The presence of corneal scarring or fibrosis in children can have a significant adverse effect on visual and global development, making it important to understand the mechanisms of fibrosis in children. To date, relatively little is known regarding corneal fibrosis in the pediatric population, and so the purpose of this study was to characterize fibrosis in young versus adult mice using an established model of mechanical corneal injury.

Methods: Corneal injury was induced by the mechanical removal of the epithelium and anterior stroma in young (3-4 week old) and adult (9-10 week old) C57BL/6 mice using an Algerbrush II burr. Mice were then followed for two weeks. Mice were evaluated by slit-lamp examination to quantify corneal opacity using the Fantes scale and by anterior segment OCT to determine central corneal thickness (CCT). In Vivo Confocal Microscopy (IVCM) was used to evaluate corneal stromal keratocyte density. At day seven, corneas were collected to evaluate mRNA expression of alpha smooth muscle actin (αSMA) and type 1 collagen (COL1A1) by RT-PCR. At day 14, corneas were collected and evaluated for the density of α-SMA+ stromal cells (myofibroblasts) by immunostaining.

Results: At 14 days post-injury, young mice compared to adult mice showed: (i) an increased corneal opacity score (2.3±1.3 vs. 1.0±0.5, p=0.04); (ii) CCT (152.7±30. vs. 79.6±5.0μm, p=0.03); (iii) increased stromal keratocyte density (407±28 mm-2 vs. 271±23 mm-2, p=0.02); and (iv) an increase in α-SMA+ stromal cell density (353±32mm-2 vs. 147±33mm-2, p=0.01). At 7 days post-injury, young mice compared to adult mice showed higher corneal mRNA expression of both α-SMA (21.9 ± 2.77 vs. 9.0 ± 1.1 fold change relative to naive, p<0.01) and COL1A1 (8.5±0.6 vs. 0.8±0.2 fold change relative to naive, p<0.01). Of note, there was complete epithelial wound closure in both groups at the end of follow-up.

Conclusions: Our results demonstrate that following mechanical corneal injury, young mice show a relatively more robust pro-fibrotic response than adult mice, as evidenced by increased clinical corneal opacity, increased α-SMA+ stromal cell density and relatively higher expression of α-SMA and COL1A1.

Hamid Alemi, Katayoun Forouzanfar, Shudan Wang, Elsayed Elbasiony, Francesca Kahale, Shima Dehghani, Thomas H. Dohlman

Candidate: Shazia Ashraf

Poster #: C3

ATM-Mediated Prominent DNA Damage Response in Fuchs Endothelial Corneal Dystrophy

Shazia Ashraf, Neha Deshpande, Raymond Jeff Wong, Jeffrey Boakye, Mohit Nishitkumar Parekh, Ula V. Jurkunas

Purpose: Fuchs Endothelial Corneal Dystrophy (FECD) is an oxidative stress disorder leading to accelerated loss of corneal endothelial cells (CEnCs). Previous study has shown that ultraviolet-A (UVA) light causes DNA damage and leads to a greater FECD phenotype in female compared to male mice, underscoring greater prevalence of FECD in females. Ataxia Telangiectasia Mutated (ATM) is a key DNA damage response (DDR) kinase in post-mitotically arrested cells, which is activated and recruited to the damage site by Mre11-Nbs1-Rad50 (MRN) complex, leading to downstream activation of cell cycle checkpoints, DNA repair and/or apoptosis. In this study, we investigate the mechanism of ATM-mediated DDR activation in response to UVA leading to FECD.

Methods: To investigate DDR activation in response to UV irradiation in vitro, immortalized normal and FECD patient-derived cell lines were treated with UVA (10 J/cm2) + 4-OHE2 followed by recovery for 1 and 24 hours in serum-free media. Cells were lysed at 1-hr post-treatment to compare early DDR signaling between cell-lines. The phosphorylation status and levels of γH2AX (Ser139) and pATM (Ser1981) and its downstream effectors pCHK2 (Thr68), caspase-3 and caspase-9 were evaluated by western blotting. Cell-cycle distribution was measured 24-hrs post-treatment by flow cytometry. The effect of cell cycle reentry on DNA repair efficiency was determined by sorting the treated cells into G0/G1-G2/M with MoFLo and alterations in different DNA repair genes were quantified by qPCR. In vivo, cell cycle activation, DNA damage and senescence were tested by Ki67 staining in CEnCs using whole mount corneas (+/- UVA; 500 J/cm2) of wild-type (WT) and ATM-knockout mice.

Results: Normal CEnCs showed increased activation of pATM/ATM, and its effectors: pCHK2/CHK2, γH2AX/H2AX after treatment with UVA + 4-OHE2 compared to untreated CEnCs, indicating that ATM phosphorylation activated a DNA damage response, as seen in FECD cell lines in our previous data. Furthermore, UVA+4-OHE2-treated CEnCs were arrested in G2/M phase of the cell cycle and showed upregulation DNA repair genes (LIG3, NEIL2, TOP3A, XRCC1 and XPC) in G2/M phase compared to G0/G1, indicating that cell cycle arrest is critical for the DNA repair process to ensure. However, loss of NQO1, as seen in FECD, led to hyperphosphorylation of ATM with 2-fold increase in pATM/ATM compared to treated normal CEnCs; however, they showed a decrease in LIG3, indicating that a deficient DNA repair from oxidative stress. In vivo, UVA irradiation induced Ki67 staining and promoted cell cycle reentry in WT but not in ATM-knockout mice.

Conclusions: We demonstrate increased activation of ATM-mediated DDR in response to ROSinduced DNA damage in FECD. This study provides insights into understanding the role of DDR signaling cascade in determining the DNA repair pathways in FECD.

Candidate: Olufemi Folorunso

Poster #: C4

TIMP-2 Is a Major Metalloproteinase Inhibitor Secreted by Human Corneal Epithelial Cells

Olufemi Samuel Folorunso, Nishant Sinha, Wonkyung Cho, Sunil Chauhan

Purpose: Tissue inhibitors of metalloproteinases (TIMPs) modulate extracellular matrix remodelling for homeostasis, cell migration, proliferation, and wound healing. However, their roles in corneal injury are not well defined. Here, the expression of different TIMPs was characterised in the human corneal epithelial cells (HCLE) in a homeostatic and inflammatory milieu. Furthermore, we examined the topical application of TIMP-2 in the corneal wound healing assay.

Methods: Immortalized human corneal limbal epithelial cells (HCLE) were cultured in a monolayer until the confluence was more than 90%. Two inflammatory conditions were utilised in the in vitro experiments. Firstly, HCLE cells were stimulated with 100 ng/mL of recombinant human IL-1β (rhIL-1β), and secondly, a scratch wound was created on the cell monolayer. Both conditions were incubated overnight in 5% CO2 at 37oC. Lysates and supernatants of the cells were collected for RT-qPCR and antibody array to quantify the expression of injury-induced metalloproteinases (MMPs) and inflammatory cytokines. In the in vivo experiment, the corneal epithelium of C57BL/6 male mice was trephined (2 mm diameter) and mechanically removed with AlgerBrush II. The corneal injury was topically treated with recombinant TIMP-2 (rTIMP-2), and the wound healing was compared with control protein (mouse serum albumin)-treated mice. The rate of corneal re-epithelialization after the injury was monitored and quantified. Statistical analysis was performed assuming a significant difference at p<0.05 using 2-way ANOVA analysis.

Results: The HCLE cells express TIMP-1, -2, and -4 mRNA genes under inflammatory conditions; however, the TIMP-2 transcript level is higher following rhIL-1β stimulation or scratch-wound condition compared to untreated (p < 0.05). TIMP-1 and -2 are secreted HCLE following stimulatory conditions, but scratch wounds elevate the secretion of TIMP-2 protein more significantly than the control cells (p < 0.01). Scratch wounds treated with rTIMP-2 suppresses the expression of MMP-1, -3, -9, -10, -13, IL-8, and TNFα. Also, topical treatment of corneal injury in mice with recombinant TIMP-2 promotes wound healing and re-epithelialization of the injured cornea.

Conclusions: Human corneal epithelial cells secrete TIMP-1 and -2, but TIMP-2 is upregulated under inflammatory conditions and suppresses the expression of injury-induced MMPs and inflammatory cytokines. Topical treatment of corneal injury with recombinant TIMP-2 promotes wound healing and corneal re-epithelialization in mice, suggesting TIMP-2 could be a therapeutic target to promote wound healing and tissue remodelling.

Candidate: Katayoon Forouzanfar

Poster #: C5

Contribution of Regulatory T Cells to the Modulation of Corneal Neovascularization in a Diabetic Mouse Model

Purpose: Diabetic corneal changes are frequent but are often underdiagnosed complications in patients with diabetes mellitus (DM). Although corneal neovascularization (CNV) associated with DM is not commonly observed in the uninjured cornea; however, diabetics are more prone to develop CNV after surgery or inflammation. We have previously shown that naïve regulatory T cell (Treg) exert a potent anti-angiogenic effect on suture-induced inflamed CNV. Herein, we study this effect in the diabetic mouse model.

Methods: DM was induced by daily injection of streptozotocin (STZ) (50mg/kg) for 5 days in 8-weekold BALB/c mice. 8 weeks post-induction, CD4+CD25+ Treg from either naïve or diabetic mice were MACS-sorted and cocultured with a mouse vascular endothelial cell line (MS1 VEC) in triplicates for 4 hours in Matrigel. VEC tube formation was imaged by brightfield microscopy and both tube length and junctions were analyzed by image J. CNV was induced in diabetic and non-diabetic mice by placing a suture in cornea. Non-diabetic mice were treated with subconjunctival injection of either 1) saline, 2) Treg (5x105) from diabetic mice or 3) Treg from non-diabetic mice. In addition, diabetic mice were treated with 4) saline or 5) Treg from non-diabetic mice. On day 7, CNV was scored (0-4). The corneas were excised and immunostained with anti-CD31, imaged by confocal microscopy, and analyzed with Image J. Expression of anti-angiogenic factors in non-diabetic and diabetic Treg was evaluated by RTPCR.

Results: Diabetic mice had a higher CNV score (p<0.001) and CD31 positivity (P<0.001) vs. nondiabetic mice. Adoptive transfer of non-diabetic Treg to diabetic mice led to decreased CNV (p=0.001) while adoptive transfer of diabetic Treg to non-diabetic mice failed to reduce CNV. Compared to VECs alone, co-culture of VECs with non-diabetic Treg suppressed tube length (3161±346 vs. 5655±306, p=0.001) and the number of junctions (6.6±2 vs.24.6±4.2, p=0.015), while when co-cultured with diabetic Treg the changes were less significant for both tube length (4300±333, p=0.032) and number of junctions (12.25±2.6, p=0.050). Among all anti-angiogenic factors, PDL1 was significantly downregulated in diabetic Treg compared to non-diabetic Treg (p=0.005).

Conclusions: The diabetic state enhances CNV in response to inflammation and that correlates with the downregulation of antiangiogenic factors by Treg in diabetic mice.

Katayoon Forouzanfar, Shima Dehghani, Francesca Kahale, Seokjoo Lee, Akitomo Narimatsu, Aytan Musayeva, Hamid Alemi, Tomas Blanco, Reza Dana

Candidate: Francesca Kahale

Poster #: C6

Treatment with Neuropeptide α-Melanocyte Stimulating Hormone Suppresses Progression of Fuchs Dystrophy in a UV-A Induced Mouse Model

Francesca Kahale, Neha Deshpande, Hamid Alemi, Amirreza Naderi, Shudan Wang, Tomas Blanco, Thomas H. Dohlman, Jia Yin, Ula V. Jurkunas, Reza Dana

Purpose: Fuchs Endothelial Corneal Dystrophy (FECD) is characterized by a progressive loss of corneal endothelial cells (CEnC) leading to corneal edema and vision loss. Our previous work has shown the cyto-protective effect of early treatment with the neuropeptide alpha-Melanocyte Stimulating Hormone (α-MSH) in preventing the development of Fuchs dystrophy in a murine model of UV-A induced FECD. This study aims to investigate the suppressive effect of α-MSH on CEnC loss after the development of endothelial changes implicated in FECD.

Methods: 8 week-old female C57BL/6 mice (n=8/group) were irradiated with UV-A (500J/cm2). 2 weeks post-irradiation, CEnC were imaged using in vivo confocal microscopy. Cell density, %hexagonality, & coefficient of variation (CV) were quantified using Konan’s CELLCHEK. Development of early stages of the Fuchs phenotype (CEnC loss and morphological changes) was noted, and αMSH supplementation (1.67μg/g) was initiated in the treatment group and continued 3x/week for 10 weeks. At week 4, 8, and 12, CEnC were imaged and central corneal thickness (CCT) was measured via Optical Coherence Tomography. Cultured human CEnC were exposed to 150μM H2O2 and then incubated in medium containing 10 -6 M of α-MSH (treatment) vs. medium (control). Phosphorylated H2AX (γ-H2AX) staining was utilized to compared # of double stranded DNA breaks in treated vs. untreated groups.

Results: At 12 weeks post-irradiation, CEnC in the treated group compared to control had a significantly higher mean cell density (1252.4±83.3 vs. 1007±71.9, p<0.05), lower cell size variability (CV 44.7±2.6 vs. 57.5 ± 4.9, p<0.05) and higher % of cells retaining hexagonality (49.5±1.3 vs. 44.5±1.8, p<0.05). Corneal edema depicted by CCT was noted in untreated corneas starting at week 4, and progressively worsened. By week 12, CCT was significantly lower in the treated group compared to untreated (91.9±6.6 vs. 138.4 ±7.1, p=<0.001). In vitro, γ-H2AX staining was significantly lower in the group rescued with α-MSH compared to control (0.79±0.09 vs. 6.16±0.5 foci/cell, p<0.0001).

Conclusions: Treatment with α-MSH after the development of early UV-induced CEnC phenotypic changes, suppressed the progression and worsening of the FECD phenotype in this murine model. The preserved CEnC morphological and functional integrity maintained corneal thickness and prevented corneal decompensation.

Candidate: Liangju Kuang

Poster #: C7

Eye-Targeted Sustained Delivery of Anti-inflammatory Drugs to Injured Eyes

Liangju Kuang, Yimin Gu, Yuting Zheng, Yavuz Oz, Ann Yung, Amirreza Naderi, Reza Dana, Nasim Annabi

Purpose: Continuous and efficient delivery of anti-inflammatory therapeutics into inflamed ocular tissues can be challenging due to patient compliance as well as structural barriers of the eye. This study is aimed to develop a mucin-targeting eye drop capable of drug delivery in a sustained manner to improve the drugs’ therapeutic efficacy and reduce dose frequency.

Methods: Eye drops were formed by incorporating drug-loaded micelles (MCs) into a hyaluronic acidbased matrix solution. Drug-loaded MCs were formed from the self-assembly of block copolymers with an anti-inflammatory corticosteroid drug, loteprednol etabonate. The phenylboronic acid (PBA) group bearing the block copolymer polyethylene glycol-b-(N-(2-hydroxypropyl) methacrylamide-cooligolactate) was synthesized by radical polymerization and characterized by proton nuclear magnetic resonance (1H-NMR). The MCs were characterized by dynamic light scattering and transmission electron microscopy to determine particle size and polydispersity index, surface charge, and mucoadhesive properties. Drug loading efficacy was quantified by liquid chromatography. Eye drops were assessed for viscoelasticity by rheology and for wettability by measuring wetting areas on mucincoated glass slides.

Results: PBA conjugation efficacy ranged from 24.5% to 97%. The MCs were spherical with a diameter of 109.0 ± 9.2 nm and had a narrow size distribution with a polydispersity index of 0.094 ± 0.01. By increasing drug/polymer ratios, drug loading increased from 55.7 ± 10.0 to 589.2 ± 75.5 µg/mL with no effect on drug loading efficiency. The eye drops showed desirable shear thinning behavior and had a viscosity of 70 cps at a high shear rate (1000 s-1), pH of 7.4, and wetting areas of 27 ± 0.7 mm2 (20 µL). Increasing hyaluronic acid concentrations from 0.1% to 0.5% (w/v) significantly enhanced the viscosity and decreased wetting areas from 43 ± 0.9 to 27 ± 0.7mm2 (p<0.0001) while varying glycerin concentrations from 0.3% to 1% (w/v) had little impact on viscosity and wettability.

Conclusions: Drug-loaded MCs with optimal size, loading capacity, and mucoadhesive properties were successfully prepared and formulated as eye drops with suitable viscosity, pH, and wettability. Our targeting eye drops can potentially be used as a treatment for corneal inflammation with improved bioavailability and patient compliance.

Candidate: Amir Naderi

Poster #: C8

Effect of Neurokinin 1 Receptor Antagonist on Pain Response in Corneal Alkali Burn

Purpose: Patients with ocular chemical burns have been reported to experience significantly lower quality of life compared to other ocular diseases. Chronic ocular pain is a significant morbidity even in well-healed eyes or cases with only corneal injury. Substance P (SP) is a well-known modulator of pain and was shown to modulate nociception in murine cornea. The purpose of this study was to determine whether the inhibition of SP can reduce ocular pain following chemical injury to the cornea by local antagonism of SP’s preferred receptor, neurokinin 1 receptor (NK1R).

Methods: Corneal alkali burn was induced in C57BL/6 mice by means of a paper disc (2-mm diameter) soaked in 1 N NaOH for 10 seconds. 6 mice with 1:1 gender ratio were used for each experiment. SP levels in the cornea and trigeminal ganglion (TG) were measured by ELISA at 4, 7, 14, 21, and 28 days after alkali burn induction. Animals were randomized into two groups receiving L-733,060 (1μg/μl) or phosphate-buffered (PBS), topically twice a day for 4 weeks after alkali burn induction. On day 4, 7, 14, 21 and 28, eye wiping test (EWT) was performed to evaluate hyperalgesia after applying hypertonic saline (2M NaCl) as noxious stimulus. Following EWT, allodynia was assessed by quantifying palpebral ratio (PR) via an automated video analysis of the animals’ eye after instillation of a saline solution isoosmolar to tears (0.9 % NaCl) as neutral stimulus.

Results: The SP protein levels in the corneas at 4, 7, 14, 21, and 28 days after alkali burn were 4732 ± 812, 6360 ± 1044, 6268 ± 425, 6238 ± 1701, and 5572 ± 323 pg/mg, respectively. Compared with the SP level of naive corneas (4077 ± 241 pg/mg), there were significant increases in the SP levels of the corneas on days 7, 14, 21, and 28 after alkali burn (P < 0.001, P < 0.001, P < 0.001, P < 0.01, respectively). Eye wipe behavior was significantly higher in PBS group compared to normal mice at 4, 7, 14, 21, and 28 days after alkali burn. L-733,060 group showed significant decreases in the behavior compared to PBS group at day 4, 14, 21, 28 (P < 0.01, P < 0.01, P < 0.01, P < 0.05, respectively). PR was significantly lower in PBS group compared to normal mice at 4, 7, 14, 21, and 28 days after alkali burn. L-733,060 group showed significant increases in PR at day 4, 14, 21, 28 (P < 0.01, P < 0.01, P < 0.01, P < 0.05, respectively).

Conclusions: Our data demonstrate that NK1R antagonist reduces ocular pain response in a murine alkali burn model, suggesting SP blockade as a new therapeutic strategy in the management of ocular pain.

Amirreza Naderi, Hideaki Someya, Aytan Musayeva, Shudan Wang, Francesca Kahale, Akitomo Narimatsu, Yihe Chen, Reza Dana

Candidate: Sheyda Najafi

Poster #: C9

A Novel Murine Model of Neurotrophic Keratopathy (NK)

Sheyda Najafi, Zhirong Lin, Asmaa A. Zidan, Amirreza Naderi, Elsayed Elbasiony, Gabriel Corfas, Jia Yin

Purpose: Neurotrophic keratopathy (NK) is a degenerative disease characterized by impairment of corneal innervation. The growth factor Neuregulin 1 (NRG1) and its receptors (ErbB) are key regulators of axon-Schwann cell interactions. A transgenic line of mice (Line D) postnatally expresses a dominantnegative ErbB receptor (DN-ErbB4) in non-myelinating Schwann cells. These mice have shown progressive age-dependent peripheral sensory neuropathy in non-ocular studies. Herein we assess these mice as a model to study NK.

Methods: Initial assessment of Line D transgenic mice showed spontaneous corneal opacification, ulceration, and eventual perforation within 3 months of age in the homozygous. The heterozygous mice showed no gross corneal opacification or ulceration and were monitored clinically over time (1-8 months old). Cornea Corneal sensitivity, Fluorescein Staining (CFS), tear secretion, and eye wipe test were performed. Optical Coherence Tomography (OCT) and in vivo confocal microscopy (IVCM) were used to visualize ocular structures in vivo. To assess corneal innervation, immunohistochemistry of tubulin III was performed. The protein expression of the neuropeptides Alpha-Melanocyte-Stimulating Hormone (α-MSH), Calcitonin-Gene-Related Peptide (CGRP), and Substance P (SP) in the cornea was measured using ELISA. The mRNA expression of inflammatory and apoptotic markers in the trigeminal ganglia was done by real-time PCR. Data are presented as mean ± SEM.

Results: Transgenic mice show loss of sensory nerves in the cornea. Tubulin III staining of sub-basal plexus and the stroma of the central cornea in ErbB4+ heterozygous mouse shows progressive loss of innervation compared to wild-type mice. In vivo confocal imaging of the cornea of the ErbB4+ mice at 6 months age shows abnormal epithelial cells and fewer nerve fibers with smaller nerve trunks in stroma. Clinical assessment of the ErbB4+ mice showed significant decrease in the corneal sensitivity, tear secretion and eye wipe behavior and considerably higher CFS score in a progressive manner compared to the control mice (age= 1-8 months; n=12). However, the central corneal thickness measurement was not different between two groups. Molecular analysis of the expression of several neuropeptides in the cornea using ELISA shows decreased expression of CGRP, α-MSH, and Substance P in the transgenic mice. (age= 8 months; n=4, *p˂0.05). The mRNA expression of inflammatory cytokines as well as the apoptotic marker cleaved caspase-3 significantly increased in the Trigeminal ganglia of the transgenic mice compared to the control mice. (Age= 8 months; n=4, *p˂0.05).

Conclusions: Line D transgenic mice with ErbB4 mutations in non-myelinating Schwann cells demonstrate progressive loss of corneal innervation and sensitivity. In addition, they have ocular surface abnormalities, lower neuropeptide expression in the cornea, and more inflammation in the trigeminal ganglia. These mice may represent a novel murine model of neurotrophic keratopathy.

Candidate: Mohit Parekh

Poster #: C10

UVA Light and Estrogen Induce G2/M Cell Cycle Arrest Leading to Secretory Phenotype Seen in FECD

Mohit Parekh, Raymond Wong, Ula Jurkunas

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a female predominant disorder caused by genetic and environmental factors. It is characterised by loss of corneal endothelial cells (CEnCs) and extracellular matrix deposition in the form of guttae. Exposure to Ultraviolet-A (UVA) light has been shown to promote FECD characteristics. However, the understanding of how a combination of UVA and estrogen metabolite (4OHE2) lead to the formation of secretory phenotype (senescent associated secretory phenotype – SASP) that modulate the development of FECD is unknown. We therefore investigated whether UVA+4OHE2 cause DNA damage and cell cycle arrest resulting in the production of secretory factors that lead to the development of FECD.

Methods: Telomerase-immortalized CEnCs, were treated with UVA (25J/cm2) and recovered in the basal media containing 4OHE2 (20uM) at 3h and 24h. SASP was collected at the end of the treatment (4d). The cells were assessed for DNA damage (γH2AX), senescence (Sa-β-Gal, H3K9me3, and CDKN1A), EMT (TGFBIp), fibrosis (ACTA2), cell cycle checkpoint (cyclin B1), and compared to untreated cells by histochemical staining, immunostaining, western blotting and RT-PCR. Flow cytometry was used for cell cycle analysis and the cells from G0/G1 and G2/M phases were separately sorted and analysed. CEnCs were cultured for 24 hours with SASP and checked for senescence, EMT and fibrosis by histochemical staining and RT-PCR. Mann-Whitney and one-way Anova with post-hoc Bonferroni test was used to determine statistical difference. (*p<0.05, **p<0.01, ***p<0.001).

Results: Greater DNA damage (yH2AX+) was detected in the treated (14±3%) CEnCs compared to the controls (0.6±0.9%) (p<0.05); whereas p53 was expressed only in the treated cells. UVA+4OHE2 caused a shift from G0/G1 to G2/M arrest (53±14%) compared to untreated controls (19±2%) (p<0.001) and led to increased p21 nuclear staining (9±2%) compared to the controls (0.3±0.5%) (p<0.05). Cell sorting detected >2 fold upregulation of CDKN1A, SNAI1, TGFBIp, ACTA2, CLU, COL4A1 genes in G2/M vs G0/G1. SASP treated cells showed significant upregulation of SNAI1/2, TGFBIp, COL1A1, ACTA2 but not CDKN1A, indicating that acute SASP is insufficient to induce senescence.

Conclusions: UVA+estrogen metabolite (4OHE2) induces DNA damage leading to G2/M cell cycle arrest and early senescence. G2/M arrest plays a key role in the development of FECD as observed by upregulation of fibrotic and senescence markers in G2/M sorted cells. These cells secrete SASP, which in turn induces ECM deposition as observed in guttae formation. Females exposed to UVA can be more prone to developing FECD hence UVA protected eyewear is recommended.

Candidate: Vinay Kumar Pulimamidi

Poster #: C11

Mesenchymal Stem Cell-Derived Interleukin 11 Inhibits Effector T Cell Function

Vinay Kumar Pulimamidi, WonKyung Cho, Sharad K. Mittal, Sunil K. Chauhan

Purpose: Our group has previously shown that mesenchymal stem cells (MSCs) inhibit activation of innate immune cells, including neutrophils and macrophages. Here, we investigated the role and mechanisms by which MSCs regulate T cell function.

Methods: MSCs derived from human bone marrow (hMSCs) were phenotypically characterized for their expression of CD45- CD34- CD73+ CD90+. To co-culture MSCs with T cells, CD4+ CD25- T cells (purity: >95%) were magnetically sorted from human peripheral blood mononuclear cells. Expression and secretion of IL-11 by MSCs were assessed by qRT-PCR and ELISA, respectively. Presence of IL11 receptor was evaluated in activated CD4+ CD25- T cells by flow cytometry. Prior to co-culture, CD4+ CD25- T cells were stained with CFSE, and their proliferation was measured by flow cytometry using CFSE dilution. We co-cultured CD4+ CD25 T cells (stimulated with anti-CD3/CD28 beads at a 1:1 ratio) with MSCs at 1:1 ratio in the presence or absence of hIL-11 neutralizing antibody (20 µg/mL) for 24 hours (to initiate early activation) and 66 hours (to initiate proliferation). Early T- cell activation was assessed by evaluating the expression of CD40L and CD69 markers by flow cytometry.

Results: High levels of IL-11 were constitutively expressed by hMSCs at both mRNA and protein levels. IL-11 receptor was expressed by naive CD4+CD25- T cells, which increased by two folds upon CD3/CD28 stimulation. hMSCs significantly suppressed the activation of naïve T cells as evidenced by a reduction of approximately 70% in the expression of both CD69 (p=0.025) and CD40L (p=0.011). Following the neutralization of IL-11, MSC-mediated decrease in early T-cell activation was not observed. Additionally, CFSE dilution assay showed that hMSCs significantly suppress CD4+ CD25- T cell proliferation (p=0.006). However, this MSC-mediated suppression of T cell proliferation was reversed after neutralizing IL-11.

Conclusions: MSCs constitutively secrete high levels of IL-11, through which they suppress the activation and proliferation of CD4+ T cells.

Candidate: Jyoti Sharma

Poster #: C12

Minimally Invasive Keratoprosthesis (mi-KPro) for Vision Restoration After Severe Chemical Injury

Purpose: Approximately 1.5-2 million patients globally become corneal blind every year, of which a significant proportion is not amenable to standard corneal transplantation. To this end, artificial corneas have become the mainstay of treatment of severe corneal blindness. More than 20,000 eyes have been treated with the most successful FDA approved artificial corneas, the Boston Keratoprosthesis (B-KPro) and the Lucia. However, complications, such as, ocular hypertension, glaucoma, endophthalmitis and retroprosthetic membrane (RPM) formation often compromise long-term outcomes and cause terminal blindness. To overcome these limitations, we developed a minimally invasive KPro (mi-KPro) device, to minimize the aforementioned complications while being amenable for patients with severe ocular pathologies.

Methods: Corneal alkali and acid injury models were employed in rabbits, using 2N NaOH (n=12) or 2N HCl (n=12), respectively. One month after injury, animals underwent either mi-KPro surgery (n=12) or penetrating keratoplasty (PKP) (n=12). Animals were followed with anterior and posterior eye optical coherent tomography (OCT), photography, and intraocular pressure (IOP) with cannulation at baseline and monthly for 12 months. At 12 months, rabbits were euthanized and both the eyes and optic nerves were enucleated for histological analyses.

Results: Eye implanted with the mi-KPro had an excellent anatomic retention for 12 months and minimal complications as compared to standard keratoplasty that exhibited graft rejection within 3 months. None of the mi-KPro implanted rabbits developed ocular hypertension, while retinal thickness and optic nerve cup to disc ratio remained unchanged, as compared to baseline OCT measurements. In contrast, PKP control animals exhibited significant retinal thinning (n=6, p=0.004), and showed significant loss in optic nerve axons (n=6, p=0.01), as compared to KPro implanted eyes.

Conclusions: The mi-KPro achieves excellent anatomic retention for over a year in chemically inured eye and does not lead to post-surgical IOP elevation, glaucoma, PRM, or infection. This translates to preservation of the retina and optic nerve at 1 year after injury suggesting superiority in the treatment of chemically injured eyes, as compared to PKP or historical data from rabbit B-KPro surgeries. The preclinical safety and efficacy profile of the mi-KPro is exception and supportive of further evaluation in patients.

Jyoti Sharma, Thomas Dohlman, Chengxin Zhou, Peter York, Fengyang Lei, Jie Liu, Robert J. Woods, Claes H. Dohlman, James Chodosh, Roberto Pineda, Eleftherios I. Paschalis

Candidate: Nishant Sinha

Poster #: C13

IL-10 and TGFβ Induces ɑSMA Expression in Macrophages

Nishant R. Sinha, WonKyung Cho, Vinay Kumar Pulimamidi, Olufemi S. Folorunso, Lei Xi, Sunil K Chauhan

Purpose: Corneal fibrosis due to ocular trauma is a major cause of visual impairment worldwide. Studies in non-corneal tissue show macrophages have a role in fibrosis during the early stages of wound healing. Here, we investigated whether infiltrating macrophages contribute to fibrosis by differentiating into ɑSMA-positive cells.

Methods: C57BL/6 mice received a 2mm corneal injury using an AlgerBrush II to remove the epithelium and anterior stroma. Corneas were collected at day 6 for immunohistochemistry and stained with F4/80 (macrophage) and ɑSMA (myofibroblast-like cells). Human monocytic (THP-1) cell-derived macrophages were stimulated with or without 10 ng/mL IL-1β for 3 days. Next macrophages were divided into four treatment groups: 1) control, 2) 10 ng/mL IL-10, 3) 10 ng/mL TGFβ, and 4) 10 ng/mL IL-10 and 10 ng/mL TGFβ. ɑSMA expression was assessed with qRT-PCR and immunocytochemistry at day 6. Primary mouse macrophages were collected via peritoneal lavage and received the same treatment as human macrophage in group 4. Flow cytometry was used to quantify the number of F4/80 and ɑSMA double positive cells.

Results: qRT-PCR analysis of IL-1β stimulated human macrophages did not significantly increase ɑSMA expression following IL-10 only or TGFβ. However, IL-1β stimulated macrophages incubated with IL-10 and TGFβ had significantly increased ɑSMA compared to controls at day 3 (p<0.001) and day 6 (p<0.0001). Remarkably, resting macrophages co-treated with IL-10 and TGFβ had a two-fold increase in ɑSMA expression compared to controls; however, IL-1β stimulated macrophages had a greater increase compared to resting macrophages. ICC confirmed IL-1β simulated macrophages treated with IL-10 and TGFβ expressed ɑSMA. Flow cytometry showed mouse macrophages stimulated with IL-1β and stimulated with IL-10 and TGFβ had a significant increase in ɑSMA expression.

Conclusions: Infiltrating macrophages in the injured cornea transform into ɑSMA+ myofibroblast-like cells, which our data suggest depends on IL-10 and TGFβ in the milieu. Together these findings suggest that ɑSMA-positive macrophages may play role in corneal fibrosis.

Candidate: Shudan Wang

Poster #: C14

Corneal Myofibroblasts Suppress T Cell Immunity in Corneal Transplantation

Shudan Wang, Elsayed Elbasiony, Shima Dehghani, Hamid Alemi, Sunil Chauhan, Reza Dana, Thomas H. Dohlman

Purpose: To evaluate the suppressive effect of corneal myofibroblasts on effector T cells in corneal transplantation.

Methods: Allogeneic corneal transplantation (penetrating keratoplasty, PK) was performed in eight to ten week old male BALB/c mice. MK/T-1 corneal fibroblasts were cultured with transforming growth factor-beta-1 (TGF-β) for 48 hours to induce myofibroblasts. Allo-primed CD4+CD25- effector T cells were isolated from the cervical draining lymph nodes of PK mice 14 days post-op. Effector T cells were co-cultred with corneal myofibroblasts for 24 hours in the presence of CD3 and CD28. Effector T cell activation was evaluated by assessing the expression level of CD69 and CD40L using flow cytometry. The functional phenotype of effector T cells was assessed by measuring the intracellular cytokine staining level of IFN-γ by flow cytometry as well. To evaluate the effect of corneal myofibroblasts on T cell survival, we evaluated T cell apoptosis by Annexin V expression. Finally, the effect of corneal myofibroblasts on naïve effector T cell proliferation was assessed using a CFSE kit after co-culture of corneal myofibroblasts and effector T cells for 72 hours.

Results: We confirmed myofibroblsts formation by its morphology and higher expression of α-smooth muscle actin (α-SMA). Co-culture of allo-primed CD4+CD25- effector T cells with corneal myofibroblasts led to decreases in CD69, CD40L, and IFN-γ expression by T cells and an increase in T cell apoptosis. Myofibroblasts suppressed effector T cells proliferation compared to fibroblasts.

Conclusions: In addition to their role in fibrosis and extracellular matrix production, corneal myofibroblasts serve as immunoregulatory cells, suppressing CD4+CD25- effector T cell activation, function and proliferation and by promoting effector T cell apoptosis.

Candidate: Lei Xi

Poster #: C15

Ocular Surface Mast Cells Stabilize Inflammatory Blood Vessels

Lei Xi, WonKyung Cho, Elsayed Elbasiony, Aastha Singh, Sunil K. Chauhan

Purpose: Activated mast cells has been shown to promote ocular surface angiogenesis. Here we investigated whether mast cells delay the regression of corneal neovascularization.

Methods: Neovascularization was induced by placing a single figure-8 intrastromal suture in the cornea using 11-0 nylon. On day 14, the suture was removed upon establishment of mature blood vessels. Real-time vessel regression was followed by capturing slit-lamp pictures. To investigate the direct effect of mast cells on vessel regression, suture was placed in mast cell deficient cKitw-sh and their wildtype (WT) C57BL/6 controls. To evaluate the effect of pharmacological blockade of mast cells on the reversion of mature blood vessels, corneas were treated one day prior to suture removal with mast cell inhibitor, cromolyn (2% in PBS). Slit lamp images were analyzed using ImageJ software. Corneas were harvested on day of suture removal and 2 days post-suture removal and expression of VEGF-A.

Results: By 48 hours following suture removal, a significant 51.3% regression of blood vessels were observed in mast cell-deficient cKitw-sh mice, compared to 11.5% regression in WT controls (p=0.002). Near complete regression of corneal neovessels was observed in mast cell-deficient mice by day 7 post-suture removal compared to WT controls (p=0.004). Similarly, cromolyn-mediated suppression of mast cells accelerated the regression of inflammatory blood vessels, as demonstrated by complete regression of vessels by 24 hours post-suture removal, compared to PBS treatment. Furthermore, mRNA analysis showed a two-fold reduction in VEGF-A in the cromolyn-treated corneas, relative to PBS-treated groups (p=0.03).

Conclusions: Our data demonstrate that ocular surface mast cells delay the regression of pathological corneal blood vessels.

Candidate: Daniel Bowen

Poster #: D1

Using Visual Evoked Potentials to Assess Binocular Interactions in Mouse V1

Daniel A. Bowen, Daniel P. Montgomery, Mark F. Bear, Eric D. Gaier

Purpose: Interocular suppression is a quantifiable property and pathogenic driver of amblyopia. Despite the widely recognized importance, the underlying mechanisms serving interocular suppression are poorly understood. We aimed to deepen the understanding of interocular suppression and the effects of monocular deprivation in mouse visual cortex using 3 paradigms that employ visual evoked potentials (VEPs) with translational relevance.

Methods: Juvenile (P32) mice underwent chronic electrode implantation. Awake VEPs were elicited by phase-reversing sinusoidal grating stimuli (0.05-0.4 cycles/degree) presented on a 3D monitor with polarized lenses at P35. We quantified interocular suppression through changes in VEP magnitude imparted by unilateral intravitreal tetrodotoxin injection (experiment 1), and dichoptic presentations of phase-offset gratings (experiment 2) or binocularly rivalrous grating orientations (experiment 3).

Results: Retinal silencing via unilateral tetrodotoxin injection immediately augmented VEP magnitudes elicited through the uninjected eye by 1.5-2-fold on average, and the effect was spatial frequencydependent. Prior 2-week monocular deprivation (P21-P35) increased the degree of VEP magnitude augmentation, consistent with greater tonic suppression in the context of amblyopia. Dichoptic, phaseoffset gratings revealed a binocular balance point favoring the eye contralateral to the VEP recording hemisphere by a contrast ratio of ~2:1, reflective of the established contralateral bias of monocular VEP magnitudes. Preliminary data suggest that prior monocular deprivation shifted the binocular balance point in favor of the non-deprived eye. In the binocular rivalry paradigm, introduction of discordant (orthogonal) gratings reduced VEP magnitude in contrast- and spatial frequency-dependent manners. The effect on VEP magnitude was largely carried through reduction of the late, positive component of the VEP waveform. Prior monocular deprivation did not influence the effect of rivalry on VEP magnitude.

Conclusions: We have validated 3 distinct measures of interocular suppression in mice. Monocular deprivation shifts 2 of these measures in favor of the non-deprived eye, reflective of human psychophysical results in amblyopia. We plan to leverage these measures to dissect the mechanism of interocular suppression to optimize the treatment of amblyopia.

Candidate: Victor Correa

Poster #: D2

Transcriptomic Analysis of Retinal Pigment Epithelium Reveals Potential Injury Mechanisms Associated With Pentosan Polysulfate Treatment

Victor Correa, Dimitrios Ntentakis, Toshio Narimatsu, Demetrios G. Vavvas, Nikolaos E. Efstathiou

Purpose: Pentosan polysulfate sodium (PPS) is a sulfated polysaccharide used to relieve bladder pain and discomfort related to interstitial cystitis. Long-term exposure to PPS was linked to the pathogenesis of an insidious maculopathy, however The pathogenesis mechanisms of PPS maculopathy remains elusive Here we investigated the effects of PPS on retinal pigment epithelium (RPE) gene transcription to identify potential biochemical pathways that could be involved in PPS maculopathy development and progression.

Methods: RPE cell line (ARPE-19) was cultured until confluency, then treated with a high and non-toxic dose of PPS (1mg/ml) for 72h. RNA from treated cells and controls in triplicates were sequenced on Illumina platforms. The resulting data was gathered for bioinformatic analysis. Protein-protein interaction network was created using the STRING database. Functional and disease enrichment analysis was performed using the Gene Ontology (GO), KEGG, Reactome, Disease Ontology and DisGeNET pathway databases. Significance was established when adjusted p values (padj) <0.05.

Results: 486 genes were differentially expressed out of 10897 co-expressed genes between groups. Functional analysis showed a significant enrichment of extracellular matrix organization (padj=7.53e12), blood vessel morphogenesis (padj=7.53e-12), response to wounding (padj=4.18e-7), regulation of signaling receptor activity (padj=6.33e-9) and growth factor binding (padj=4.65e-7) pathways. Significantly enriched disease-associated pathways included retinal disease (padj=1.47e-6), inflammation (padj=1.46e-6), retinal vascular disease(padj=6.51e-5), retinal degeneration(padj=6.51e5), disease of capillaries (padj=2.6e-4) and age-related macular degeneration (padj=9.7e-4).

Conclusions: PPS treatment modified the transcriptomic profile of RPE cells, significantly altering pathways associated with extracellular matrix maintenance, vascular development, and growth factor binding and signaling. Disturbance of these pathways could contribute to the outer retinal injury observed clinically. These data provide new insights on the pathogenic mechanisms of PPS maculopathy.

Candidate: Egle Galdikaite-Braziene

Poster #: D3

Characterization of EYS in Human iPSC-Derived Retinal Organoids

Purpose: Mutations in the Eyes Shut Drosophila homolog (EYS) gene are one of the major causes of autosomal recessive retinitis pigmentosa (RP). Despite the high prevalence of this disease, little is known about the role of EYS in the retina and the molecular mechanism of the EYS-associated disease, which are necessary for the development of genetic therapies. The purpose of this project is to study EYS in the healthy human retina and human iPSC-derived retinal organoid (RO) model.

Methods: We differentiated wild type IMR90-4 human iPSC cell line to retinal organoids until day (d) 250 using 3D-2D-3D protocol (Cowan et.al., 2021) with alterations, including BMP-4 (Bone morphogenetic protein 4) spike at day 8. Samples for immunohistochemistry were collected at days 50, 70, 100, 130, 150, 170, 200, 230, 250. EYS expression was analyzed with RT-PCR. EYS localization was studied by immunohistochemistry and co-staining with photoreceptor and ciliary markers - Arrestin (ARR3), Rhodopsin (RHO), retinitis pigmentosa-1(RP1), Usherin (USH2A), Centrosomal protein 290 (CEP290) and Acetylated α-tubulin (AT) in mature retinal organoids and adult donor human eye. Human donor eye was obtained from Florida Lions Eye Bank and it was fixed with 2% paraformaldehyde for 4 h.

Results: Immunofluorescence analysis of the whole eye and retinal organoid sections showed EYS localization in both rod and cone photoreceptors as confirmed by colocalization with ARR3 and RHO. EYS expression in retinal organoids was observed at day 130 corresponding to the onset of outer segment formation and continue through later timepoints by immunohistochemistry. RT-PCR of EYS was confirmed at d170 to d250 retinal organoids. Co-staining of EYS and RP1 showed EYS expression at the base of the cilium in close proximity to the transition zone in adult human retina and retinal organoids. Colocalization analysis of EYS with USH2A, Acetylated α-Tubulin and CEP290 suggested possible EYS localization in the photoreceptor periciliary space.

Conclusions: Our study shows that EYS is expressed in rod and cone photoreceptors in human retina and retinal organoids. EYS localization was observed near the photoreceptor transition zone, possibly in the periciliary space. High resolution imaging techniques will need to be employed to determine the precise localization of EYS and its possible interacting partners.

Egle Galdikaite-Braziene, Julio Cesar Corral-Serrano, Cherrell Price, Marcela Garita-Hernandez, Kinga M. Bujakowska

Candidate: Cindy Hoppe

Poster #: D4

The Alternative Complement Pathway Triggers

Neuroinflammation, Glial Activation, and Retinal Ganglion Cell Death in a Microbead Induced Mouse Model of Glaucoma

Purpose: In glaucoma, glial activation and inflammation have been linked to retinal ganglion cell (RGC) death. The alternative complement pathway has recently been implicated in driving inflammation and propagating neurodegeneration in experimental models of spinal cord injury and brain injury, where specifically inhibiting the alternative pathway significantly reduced the extent of neurodegeneration. Herein we use complement factor B knockout mice (deficient in the alternative complement pathway) to determine the extent to which the alternative pathway contributes to glial activation, neuroinflammation, and RGC death in a microbead-induced mouse model of glaucoma.

Methods: Intracameral injection of magnetic microbeads (control:saline) was used to elevate the intraocular pressure (IOP) in complement factor B knockout (Fb-/- ) and wild type (WT) mice. At 2 weeks post-microbead injection, retinas were pooled (3-7 mice/grp) and processed for qPCR. At 5 weeks post-microbead injection, visual acuity was measured by optomotor reflex and RGC density and microglia activation (measurement of the longest process length) were assessed in retinal whole mounts stained with anti-Brn3a and anti-P2RY12 antibodies, respectively.

Results: IOP was increased equally in microbead-injected Fb-/- and WT mice. At 2 weeks postmicrobead injection, the induction of several inflammatory genes implicated in glaucoma (GFAP, Ccl2, Ccl3, Ccl4, Cxcl10, Il18, TNF, C3, C1q, NLRP3) were reduced by 2-fold or greater in microbead injected Fb-/- mice as compared to WT mice. At 5 weeks post-microbead injection, the Fb-/- mice showed significant preservation of visual acuity and RGC density as compared to WT mice. Moreover, microglia activation was significantly reduced in Fb-/- mice, as evidenced by the significant shortening of retinal microglia processes in microbead injected WT mice as compared to Fb-/- mice.

Conclusions: Our results demonstrate that knocking out the alternative complement pathway provides significant neuroprotection in a microbead-induced mouse model of glaucoma. Importantly, this neuroprotection coincides with inhibition of glial activation and neuroinflammation, indicating an important role for the alternative complement pathway in propagating glial activation and neuroinflammation in glaucoma.

Cindy Hoppe, Ryo Mukai, Nasrin Refaian, Amarachi Nwogu, Drenushe Krasniqi, Yinjie Guo, Maleeka Shrestha, Kip Connor, Meredith Gregory-Ksander

Candidate: Zhengping Hu

Poster #: D5

Endomucin Deletion Leads to Disorganized Choroidal Capillary Fenestration and Reduces Laser-Induced Choroidal Neovascularization in Mice

Zhengping Hu, Issahy Cano, Anton Lennikov, Melissa Wild, Urvi Gupta, Yin Shan Eric Ng, Patricia A. D’Amore

Purpose: The type I integral membrane glycoprotein endomucin (EMCN) is selectively expressed by capillary and venous endothelium. We have previously reported that knockdown of EMCN in vitro prevents VEGF165-induced VEGFR2 internalization and downstream activities including proliferation, migration, and tube formation. The goal of this study is to investigate the effect of EMCN deletion on the choriocapillaris as well as on angiogenesis in laser-induced model of choroidal neovascularization (CNV).

Methods: Global homozygous EMCN knock-out (EMCN-/-) mice were generated by crossing EMCNfloxed mice with ROSA26-Cre mice. Choroid, retina, kidney, lung, spleen, liver and thyroid were harvested and mRNA levels of EMCN were examined. Eyes from adult (4–6-month-old) EMCN-/- mice and EMCN+/+ control littermates were collected and fixed for transmission electron microscopy (TEM). For the laser-induced choroidal neovascularization (CNV) model, CNV was induced in EMCN+/+ and EMCN-/- mice (6-8 weeks-old) using 532 nm laser photocoagulation with an image-guided micron system. Mice were imaged in vivo using fluorescein angiography (FA) and optical coherence tomography (OCT) seven days after laser and then sacrificed. The lesion leakage was calculated by subtracting the integrated intensity of the earlier time point from the later time point. The retinal pigment epithelium/choroid was flat-mounted, stained with isolectin B4 (IB4), and imaged using Zeiss Axiscope. Expression of IB4, VEGF and microglial marker Iba1 were examined on flat mounts using an SP8 confocal microscope.

Results: The choroid had the highest level of EMCN mRNA level of the tissues examined (n=4). TEM revealed that the fenestrations of the choriocapillaris of EMCN-/- mice were disorganized and the endothelial cells were thickened compared to wildtype (n=3). Analysis of lesions leakage on FA revealed that EMCN-/- mice had significantly reduced leakage compared to EMCN+/+ mice (22.2 x 104 ± 2.2 x 104 pixels vs 41.06 x 104 ± 3.6 x 104 pixels, n=24, p<0.001, Student’s t-test). OCT examination showed that EMCN-/- mice had significantly smaller lesion area compared to EMCN+/+ mice (2.7 x 104 ± 841.2 um2 vs 3.5 x 104 ± 1119 um2, n=24, p<0.0001, Student’s t-test). Quantification of IB4-stained lesion area corroborated the OCT findings confirming that EMCN-/- mice had smaller lesions compared to EMCN+/+ mice (2.2 x 104 ± 1639 um2 vs 3.2 x 104 ± 2226 um2, n=23, p<0.001, Student’s t-test).

Conclusions: Consistent with our in vitro data, which indicate that EMCN is necessary for VEGFinduced VEGFR2 signaling, lack of EMCN in vivo leads to disorganized fenestrations in choroidal endothelium, structures known to be dependent on continuous signaling. In addition, the absence of EMCN interferes with angiogenesis and permeability, processes that are driven by VEGF signaling, pointing to EMCN as an alternative therapeutic target.

Candidate: Drenushe Krasniqi

Poster #: D6

Non-Human Primate Laser-Induced NAION Model for Assessing Neuro-Rejuvenating Gene Therapies

Drenushe Krasniqi, Madhura Shah, Meredith Gregory-Ksander, Sharon Rosenzweig-Lipson, Kasia Broniowska, Michel Wathier, Joan Mannick, Jennifer Cermak, Margarete Karg, Shintaro Shirahama, Nasrin Refaian, Yuancheng Lu, Mathew Lawrence, David Sinclair, Bruce Ksander, David Sinclair, Joseph F. Rizzo

Purpose: NAION (Non-Arteritic Ischemic Optic Neuropathy) is the most common ischemic optic neuropathy. The resulting non-reversable vision loss has no treatment and there is a 15% chance of a second NAION event in the contralateral eye. Notably, the pathophysiology is not completely understood. As a result, animal models are needed to advance understanding of NAION and to assess emerging treatments such as neuro-rejuvenating gene therapies. Bernstein et al first developed and tested a laser induced Non Human Primate (NHP) NAION model. The current study examined the variability in functional and histological damage to determine its utility in evaluating novel therapies.

Methods: Wild caught African green monkeys (n=8) of comparable ocular health had NAION induced on Day 0 in OS eye: IV rose Bengal (2.5 mg/kg; 0.1 ml/kg of 25 mg/ml) was administered intravenously, then 534 nm laser applied with contact lens at 100mW for 6 seconds to 500 μm spots in each ONH quadrant. OS and OD eyes were evaluated at baseline (Day -21) and weekly until study end point (Day 42) with slit lamp, fundus photography, pERG and VEP(VERIS), cSLO, and OCT (Heidelberg Spectralis HRA with HEYEX). At endpoint, eyes were enucleated and optic nerve sections stained with PPD for axon quantification; 8 mm punches of the whole mounted retinas were collected for IHC.

Results: Laser induced NAION triggered significant RNFL swelling on OCT at the ONH by Day 7 as compared to baseline. The lowest pERG responses (absolute N95 amplitude; V 10-6) compared to baseline were on Day 42 (6.9 +1.2 vs 2.6 + 0.33 V 10-6, P=0.0033, n=8). The macular ganglion cell thickness was also reduced on Day 42 as compared to baseline (228.0 +33.0 vs 354.1 + 7.0μm, P=0.0008) and untreated eyes (228.0 +33.0 vs 348.8 + 7.0μm, P=0.027) which coincided with a significant reduction in the number of healthy axons in the optic nerve and reduction in macular RNFL thickness.

Conclusions: NHP laser induced ONH damage is a robust and reproducible model of NAION. Interand intra-subject variability of functional and histological measurements achieved statistical significance using a reasonable sample size, demonstrating the model’s utility in studies evaluating novel therapies.

Candidate: Neetu Kushwah

Poster #: D7

Myeloid Cell-Specific Deficiency of RORα Exacerbates Choroidal Neovascularization and Subretinal Inflammation in Mice

Neetu Kushwah, Chi-Hsiu Liu, Kiran Bora, Meenakshi Maurya, Madeline Pavlovich, Ye Sun, Jing Chen

Purpose: Choroidal neovascularization (CNV) leads to visual decline in neovascular AMD, a leading cause of blindness in the elderly. Lipid-cholesterol metabolic pathway has been implicated in AMD pathology, precipitated by chronic non-resolving subretinal inflammation. Retinoic acid receptor-related orphan receptor alpha (RORα), a lipid-cholesterol sensing nuclear receptor has been implicated in neovascular AMD. Previously we found that systemic deficiency of RORα in Rorasg/sg mice worsened laser-induced CNV, with increased inflammation. Here, we investigated the role of myeloid cell-specific RORα deficiency during aging and in a laser-induced CNV model.

Methods: Aged systemic (Rorasg/sg) and myeloid specific RORα deficient (LysMCre/Rorafl/fl) mice were examined for fundus imaging with their respective wild type and flox controls. Immunohistochemistry was carried out in RPE/choroid flat mounts and cross sections to visualize microglia/macrophages. Young adult LysMCre/Rorafl/fl and flox control (Rorafl/fl) mice were characterized for laser-induced CNV. RPE/choroid flat mounts were stained with isolectin to examine CNV lesions at 7 days post laser. Mouse monocyte/macrophage cells (RAW 264.7) were treated with synthetic RORα inverse agonists (SR3335 and SR1001) to assess lipid uptake and lipogenic gene expression in vitro. Chromatin immunoprecipitation (ChIP) assay was performed with retinal lysate to identify RORα target genes.

Results: Aged Rorasg/sg and LysMCre/Rorafl/fl mice both showed significant increase in fundus lesions compared with their controls, with accumulation of lipid-enriched microglia/macrophages in the subretinal space. LysMCre/Rorafl/fl mice showed significant increase in CNV lesion size compared with flox controls. Moreover, RORα inhibition with synthetic inverse agonists enhanced macrophage lipid uptake of low-density lipoprotein in RAW 264.7 cells and substantially induced protein levels of PPARγ, a lipogenic gene. Direct binding of RORα to the promoter region of Pparg was confirmed with ChIP assay.

Conclusions: Our findings indicate that genetic deficiency of RORα in myeloid cells worsens subretinal inflammation during aging and exacerbates laser-induced CNV, in part by promoting macrophage lipid uptake and lipogenesis via PPARγ. Hence, RORα is a regulator of myeloid-mediated chronic subretinal inflammation and neovascularization in AMD.

Candidate: María Camila Lancheros Vega

Poster #: D8

Application of Enzyme-Loaded Microspheres to Overcome the Transplantation Barrier of the Inner Limiting Membrane

María Camila Lancheros Vega, Alba Aragón-Navas, Miriam Ana González-Cela-Casamayor, Irene Bravo-Osuna, Rocío Herrero-Vanrell, Karen Chang, Deepti Singh, Michael Young

Purpose: Cell therapy is a promising treatment for replacing the progressive loss of retinal ganglion cells (RGCs) with healthy RGCs intravitreally in diseases caused by neuron degeneration (e.g. Neurofibromatosis type I and glaucoma). However, the internal limiting membrane (ILM) represents a significant barrier hindering the success of cell-based therapies aimed at replacing retinal neurons. Several studies have shown that the majority of cells injected into the vitreous become located on the vitreal side of ILM and few actually reach the retina proper and therefore do not integrate into the RGC layer. The purpose of this study is to explore if the use of enzyme-loaded microspheres (MSs) to overcome the ILM without causing damage to the retina for improving RGC engraftment.

Methods: Microspheres were loaded with collagenase or pronase individually which were synthesized and sent from Dr. Herrero-Vanrell’s lab at the Complutense University of Madrid, Spain. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to test the degradation abilities of MSs in vitro. Culture plates were coated with Collagen Type IV (Col4) (10µg/mL) for 24 h. MSs loaded with either collagenase or pronase were added (0.1mg/mL) into the Col4-coated plates for 24 h. The remaining Col4 was visualized and assessed via immunofluorescence staining. To establish the degradation profile of the MSs, we measured Col4 for 14 days after an initial addition of MSs (0.1mg/mL) using ELISA. Additionally, adult C57BL/6J mice were intravitreal injected with either collagenase or pronase MSs (0.1mg/mL). Free collagenase (90U/mL & 30U/mL) and pronase (3U/mL) were used for comparison. After 5 days, mice were euthanized, and retinas were collected for cryosectioning. Retinal sections were stained with immunofluorescence against laminin and Col4 to check the integrity of the ILM after proteolytic enzyme degradation.

Results: Pronase-loaded MSs showed complete degradation compared to collagenase-loaded MSs, which showed partial degradation in the Col4-coated plates. Our ELISA profile showed maximum Col4 degradation rate in the first hour upon the addition of MSs and the degradation sustained up to 2 h. On top of that, our in vivo data showed decreased ILM thickness and integrity due to Col4 degradation with injection of collagenase loaded MSs. However, laminin remained intact within the ILM.

Conclusions: Taken together, our data indicate that collagenase loaded MSs could partially degrade collagen within the ILM. With further optimization, we can investigate the survival and integration rate of RGC after transplantation with the pretreatment of MSs. By understanding the interactions between the ILM and underlying retinal cells with MSs degradation, we hope to increase the efficiency of cell therapy for optic neuropathy in the future.

Candidate: Volha Malechka

Poster #: D9

Modulation of Microglia by Co-Treatment With Soluble FasL and Annexin V Improves the Outcome After Donor RGC Transplantation

Volha V. Malechka, Monichan Phay, Jonathan Soucy, John Dayron Rivera, Meredith S. Gregory-Ksander, Petr Baranov

Purpose: Stem cell-derived retinal ganglion cell (RGC) transplantation shows strong evidence of RGC integration into the retina in animal models. However, a major challenge of RGC replacement remains to be the number of RGCs that actually integrate and the variability in successful integration between recipients. The aim of this study was to investigate the role that innate immunity and host microglia play in this process and to improve the survival of donor RGCs by modulating microglia-RGC interaction.

Methods: RGC-deficient Brn3b-/- mice were used as hosts for RGC transplantation studies. The stemcell derived human RGCs were formulated, pretreated with soluble Fas Ligand (sFasL) and/or annexin V, and 1x104 of viable cells were injected into one eye of each mouse by subretinal delivery. Retinas were collected 3 days after transplantation and stained for donor RGCs (mCherry) and microglia/macrophages (Iba1). Structural integration of donor RGCs was evaluated in retinal flat mounts by confocal microscopy.

Results: Pretreatment of donor RGCs with sFasL alone, or both sFasL and annexin V, before subretinal delivery, significantly enhanced the donor RGC survival rate by 60% compared to control. In addition, microglia activation in the host retina was reduced by 40% in mice receiving donor RGCs pretreated with sFasL alone as compared to mice that received donor RGCs pretreated with both sFasL and annexin V.

Conclusions: Our findings confirm that sFasL and annexin V co-treatment creates a permissive environment for the survival of donor RGCs after transplantation through modulation of host microglia.

Candidate: Meenakshi Maurya

Poster #: D10

Rod Photoreceptor-Specific REV-ERBα Deletion Accelerates AgeRelated Synaptic Remodeling in the Mouse Retina

Purpose: Age-related retinal synaptic remodeling comprises neuronal function, as reported both in humansand mice. Alterations in outer retinal synapses were studied as a model to explore synaptic modification in aging. REV-ERBα (a nuclear receptor and transcription factor) regulates circadian rhythm and metabolism and is associated with synaptic strengthening in Alzheimer’s disease models. The present study explored the role of REV-ERBα in maintaining age-related synaptic integrity between rods and rod-bipolar cells using rod-specific REV-ERBα deficient mice.

Methods: Rod-specific conditional knockout (Rho-icre: Rev-erbα) mice with age-matched flox controls were generated. Genetic deletion of Rev-erbα was confirmed using RT-qPCR, Western blotting, and immunohistochemistry (IHC). Synapses were visualized in 12 months-old (12 MO) Rho-icre: Rev-erbα and flox controls by IHC with antibodies against synaptic ribbon protein Ribeye and rod bipolar cell marker PKC-α.The number and length of retracted photoreceptor synapses and outgrowth of bipolar dendrites were quantified (n=3 mice/group). LKB1, a downstream target of REV-ERBα, was quantified using immunoblotting(n=3 mice/group). The retinal visual function was assessed using scotopic ERG (12 MO, both male and female) and data were quantified (Mann-Whitney test, n=6 mice/group).

Results: REV-ERBα was localized in both rods and rod bipolar cells with IHC. In 12 MO Rho-icre: Reverbα retinas, the number, and length of rod synapse retraction were significantly increased compared with flox controls, accompanied by ectopic rod bipolar cell dendrite sprouts extending into the photoreceptor layer, similar to our previous observation in 11 MO systemic Rev-erbα mice. Western blot analysis revealed asignificant reduction in LKB-1 level in Rho-icre: Rev-erbα mice retina. Scotopic ERG analysis showed a significant reduction in the amplitude of rod-specific a- and b- waves, and in the sensitivity of rod b-wave only in 12 MO Rho-icre: Rev-erbα, compared to flox controls.

Conclusions: Our findings suggest that rod-specific knockout of Rev-erbα accelerates age-related outer retina synaptic remodeling and visual function decline, potentially through regulation of photoreceptor energy and metabolism via LKB1. These data suggest a rod-intrinsic role of REV-ERBα in maintaining photoreceptor synaptic integrity during aging.

Meenakshi Maurya, Chi-Hsiu Liu, Kiran Bora, Neetu Kushwah, Madeline C. Pavlovich, Zhongjie Fu, James D. Akula, Jing Chen

Candidate: Riccardo Sangermano

Poster #: D11

In Vivo CRISPR-KO Screens for the Study of Photoreceptor Essential Genes

Riccardo Sangermano, Egle Galdikaite-Braziene, Sudeep Mehrotra, Eric A. Pierce, Kinga M. Bujakowska

Purpose: CRISPR screens represent an effective tool to investigate the function of multiple genes simultaneously. Although these screens have been successfully applied on in vitro or ex vivo models, their use on mature photoreceptors has not been performed to date. Our study aims to develop an in vivo perturbation screen to study the function of retinal genes in mouse retina.

Methods: Cas9 transgenic mice were subretinally injected at post-natal day (PND) 3 with lentiviral particles (LVs) containing either fluorescent reporters or a 500-sgRNA library targeting 66 retinal genes. Twelve of them were considered positive controls as either previously associated with retinal degeneration and ciliopathy phenotypes (n=8) or implicated in other retina biology mechanisms (n=6). The library also contained 100 non-targeting sgRNAs serving as negative control. Integration of the LV library to the host photoreceptor cell genome allowed for subsequent sgRNA detection by PCR amplification of the sgRNA sequences from genomic DNA. Retinas injected with fluorescent markers were harvested at PND21 and PND90 and analyzed by flow cytometry to assess transduction efficiency. Retinas injected with the sgRNA library were harvested at PND90 and sgRNA dropout was analyzed by amplicon sequencing and bioinformatic software, including MAGeCK.

Results: To understand the number of genes that can be targeted in a screen, we first evaluated the optimal photoreceptor transduction efficiency by using four LV titers (4.8x1011 vp/ml, 2.4x1011 vp/ml, 1.2x1011 vp/ml, 6x1010 vp/ml, n=13/group). The average transduction efficiency of the highest titer was ~7%, however, it reduced to ~3% at PND90, possibly due to a toxic LV effect. The toxic effect has not been observed for the two lower titers, which reached transduction efficiency of ~3%, which allows for studying of ~160 genes with 6 targeting sqRNAs/gene in a pool of 4 retinas (768,000 transduced cells). Out of the 66 retinal genes perturbed, only eight positive control genes showed a statistically significant sgRNA dropout. Four of them were known disease genes (Rho, Nmnat1, Pde6b, Ttc21b) while the others were protein coding or microRNA genes essential for photoreceptor survival (Sarm1, Tsc2, Mir181a-1, Mir181b-1). The remaining positive controls of the perturbation assay (Aipl1, Cep290, Pde6a, Prpf8) didn’t reach statistical significance, possibly due to the poor efficacy of the gRNAs employed to ablate their expression.

Conclusions: Starting from the current knowledge in CRISPR screens, we have developed an in vivo CRISPR perturbation assay for the study of retinal photoreceptors in mice. This assay can be useful to study retinal biology in health and disease, to investigate the effects of ablation of novel uncharacterized genes and to study possible genetic modifiers of retinal phenotypes.

Candidate: Madhura Shah

Poster #: D12

Epigenetic Reprogramming- A Novel Gene Therapy That Restores Vision Loss in a Nonhuman Primate Model of NAION

Madhura Shah, Drenushe Krasniqi, Meredith Gregory-Ksander, Sharon Rosenzweig-Lipson, Kasia Broniowska, Michel Wathier, Joan Mannick, Jennifer Cermak, Margarete Karg, Shintaro Shirahama, Nasrin Refaian, Yuancheng Lu, Matthew Lawrence, David Sinclair, Joseph F. Rizzo, Bruce Ksander

Purpose: NAION (Non-Arteritic Ischemic Optic Neuropathy) is the most common ischemic optic neuropathy. The resulting non-reversable vision loss has no treatment and there is a 15% chance of a second NAION event in the contralateral eye. Notably, the pathophysiology is not completely understood. As a result, animal models are needed to advance understanding of NAION and to assess emerging treatments such as neuro-rejuvenating gene therapies. Bernstein et al first developed and tested a laser induced Non Human Primate (NHP) NAION model. The current study examined the variability in functional and histological damage to determine its utility in evaluating novel therapies.

Methods: Prescreened African green monkeys (n=20) had NAION induced on Day 0 by iv rose bengal followed by lasering the OS eye optic nerve head. Pre-treated NHP on Day -21 received an OS intravitreal (IVT) injection of doxycycline-inducible AAV2-OSK (5.7 x 1011 vg/eye; 1:1 ratio AAV2-TREOSK + AAV2-CMV-rtTAV16) (n=6), or vehicle (n=4). Post-treated NHP on Day +1 received an OS IVT injection of AAV2-OSK (n=6), or vehicle (n=4). Daily oral doxycycline was given until end point. Both eyes were examined at baseline and weekly intervals until Day +35 via slit lamp, fundus photography, pERG, VEP, cSLO, and OCT. At the endpoint, eyes were enucleated, optic nerve axons quantitated and retinal whole mounts stained to quantitate RGCs and Klf4.

Results: Consistent with NAION in humans, laser induced NAION damage significantly reduced pERG, OCT-measured RNFL thickness, and optic nerve axons when compared to baseline and untreated OD eyes (data in companion abstract). Pre-treated AAV2-OSK eyes displayed significantly elevated pERG responses (absolute N95 amplitude; V 10-6) as compared to vehicle treated eyes at the endpoint (4.4 +0.67 vs 2.6 +0.33 V 10-6, P=0.025, Day 35). After AAV2-OSK injection, it takes at least one week for OSK expression in RGCs. Post-treated eyes (treated after NAION induction) showed a pERG decline equivalent to controls at Day 7. Treated eyes showed a significant recovery by the endpoint (3.8 +0.29 vs 2.6 +0.33 V 10-6, P=0.021, Day 35).

Conclusions: Partial epigenetic reprogramming by AAV2-OSK gene therapy successfully restored visual function in a NHP model of NAION. These data support the strong clinical translational potential of this gene therapy for treating age-related retinal diseases.

Candidate: Sampath Vemula

Poster #: D13

Impaired Neuromuscular Junction Development in the Extraocular Muscles of Mice With Nystagmus

Sampath

Kumar Vemula, Tapiwa Muvavarirwa, Seoyoung A. Kim, Mary C. Whitman

Purpose: Infantile nystagmus syndrome (INS) is an eye movement disorder characterized by involuntary oscillations of the eyes starting in infancy. We recently showed decreased neuromuscular junction (NMJ) density as early as P10 in a mouse model of nystagmus (albino), as well as transient decreases in NMJ cross-sectional area. We now examine the development and maturation of the NMJs in three dimensions in albino EOMs.

Methods: We examined NMJ formation and maturation in EOMs of P3, P7, P10, P14, P21 and 12 weeks (wks) old albino and littermate wildtype (WT) mice. In whole-mount EOMs, we stained acetylcholine receptors (AChRs) with ɑ-bungarotoxin. NMJ development was assessed by measuring NMJ length, volume and morphology using Imaris software, and compared between WT and albino mice (n=2-6 animals per genotype for each time point). Data were analyzed using unpaired student’s ttest.

Results: In WT, NMJs start as plaque-like structures at P3, become increasingly perforated and irregular in shape at P7 and P14, and are fully perforated, pretzel-like structures by P21, which further increase in size to adulthood. In albinos, at P3 NMJ length, volume and % of plaques were similar to wildtype. But at P7, albinos had less perforated, thin and long NMJ plaques but smaller average volume (228.36±21.5 vs 249.73±7.43 µm3 in WT). At P14, these NMJ plaques developed into irregular shape, thin structures with smaller average volume (477.58±21.5 vs 553.09±14.25 µm3 in WT). At P21, albino NMJs were irregular in shape, with smaller average volume (647.98±38.99 vs 910.39±29.1 µm3 in WT). In adulthood, albino NMJs were short, with smaller average length (37.70±1.2 vs 46.93±0.49 µm in WT) but equal volume. There was a greater proportion with immature morphology (27.24±0.02% vs 10.21±1.31% in WT) in adult albino EOMs compared to WT.

Conclusions: In addition to a decreased density of NMJs in the EOMs of albino mice, the NMJs which are present are smaller and appear immature. Differences were noted as early as the second postnatal week, when albino mice EOMs show irregular NMJs. Altogether, the data suggest that irregular development of NMJs may contribute to the pathogenesis of INS in albino mice.