2022 AMAR - Program

June 24, 2022 Harvard

For a complete program, including the abstracts for the trainee poster contest, visit https://joom.ag/SCed or scan the QR code to the right with your smartphone or tablet.

Annual Meeting and Alumni Reunion Co-chairs

Joan W. Miller, MD

Alumni Reunion Co-chair, David Glendenning Cogan Professor of Ophthalmology, and Chair, Department of Ophthalmology, Harvard Medical School; Chief of Ophthalmology, Mass. Eye and Ear and Mass General Hospital; Ophthalmologist-in-Chief, Brigham and Women’s Hospital

Dr. Miller earned her medical degree and received her ophthalmology residency training from Harvard Medical School (HMS), and then completed a vitreoretinal fellowship at Mass. Eye and Ear, joining the faculty in 1991. She was the first female physician to achieve the rank of Professor of Ophthalmology at HMS, and the first woman to chair the Department of Ophthalmology at HMS. Dr. Miller is also the first woman appointed as Chief of Ophthalmology at both Mass. Eye and Ear and Massachusetts General Hospital.

Dr. Miller’s clinical research interests focus on retinal disorders, including age-related macular degeneration (AMD) and diabetic retinopathy. Over the last two decades, she and her colleagues at Mass. Eye and Ear/HMS pioneered the development of photodynamic therapy using verteporfin (Visudyne®), the first approved pharmacological therapy able to reduce and slow vision loss in patients with AMD. The group also identified the key role of vascular endothelial growth factor (VEGF) in ocular neovascularization, leading to the development of anti-VEGF therapies now administered to millions of people annually around the world.

Joseph F. Rizzo III, MD

Dr. Miller’s current investigations include the genetics of AMD, strategies for early intervention in AMD, and neuroprotective therapies for retinal diseases.

An internationally recognized expert in the field of retina, Dr. Miller has published nearly 220 peer-reviewed papers, 95 book chapters, review articles, and editorials. She is a member of the National Academy of Medicine and the Academia Ophthalmologica lnternationalis. Among her numerous honors, Dr. Miller delivered the 2012 Edward Jackson Lecture for the American Academy of Ophthalmology (AAO), and was a co-recipient of the 2014 António Champalimaud Vision Award, the highest distinction in ophthalmology and visual science. In 2015, Dr. Miller became the first woman to receive the Mildred Weisenfeld Award for Excellence in Ophthalmology from ARVO; in 2018, she became the first woman awarded the Charles L. Schepens Award from AAO. Recently, Dr. Miller was awarded the 2018 Lucien Howe Medal from the American Ophthalmological Society, and the 2018 Gertrude D. Pyron Award from the American Society of Retinal Specialists.

Alumni Reunion Co-chair, David Glendenning Cogan Professor of Ophthalmology in the Field of Neuro-Ophthalmology and Director of Ophthalmology Alumni, Harvard Medical School; Director, Neuro-Ophthalmology Service and Co-Director, Mobility Enhancement and Vision Rehabilitation Center of Excellence, Mass. Eye and Ear

Dr. Rizzo is a graduate of Louisiana State University and Louisiana State University Medical School, where he received the “Dean’s Award” in recognition of outstanding leadership and performance. He completed an internship in adult medicine at the University of California at Los Angeles Medical Center, followed by a neurology residency at Tufts University - New England Medical Center, and an ophthalmology residency at Boston University. He then performed a clinical fellowship in Neuro-Ophthalmology under Dr. Simmons Lessell at Mass. Eye and Ear. He is board certified in both ophthalmology and neurology.

Dr. Rizzo joined Mass. Eye and Ear in 1986 and received a five-year Physician Training Award from the National Institutes of Health. The laboratory training was under the supervision of Dr. Richard Masland. In 1988, Dr. Rizzo initiated, and has since served as Co-director, to the Retinal Implant Project, a joint effort of MIT, Mass. Eye and Ear, Boston VA, and Cornell University to de-

velop a retinal prosthesis to restore some vision to the blind. In addition to an active clinical practice, Dr. Rizzo has served as Director of the Neuro-Ophthalmology Service at Mass. Eye and Ear and Director of Alumni, Harvard Department of Ophthalmology. He also founded two companies, Bionic Eye Technologies and Visus Technology, which are developing devices to assist the visually-impaired.

Each year, Dr. Rizzo supervises and teaches three clinical fellows and eight residents in the basic evaluation and management of neuro-ophthalmic disorders. As Director of the Neuro-Ophthalmology Service, he also designs and provides oversight for the fellowship program in Neuro-Ophthalmology. For more than 25 years, he directed the Neuro-Ophthalmology section of the Lancaster Course in Ophthalmology, which is the oldest and largest educational course that is designed for residents-in-training.

Joseph B. Ciolino, MD

Alumni Reunion Co-chair, Associate Professor of Ophthalmology, Associate Director of Ophthalmology Alumni, and the Henry Freeman Allen Cornea Scholar, Harvard Medical School, Mass. Eye and Ear

Dr. Ciolino earned his medical degree from Georgetown University School of Medicine and subsequently pursued an ophthalmology residency at Albany Medical College, where he served as Chief Resident in his final year. Returning to Boston for a two-year fellowship in the Cornea, Refractive Surgery and External Disease Service, he received the nationally recognized Claes Dohlman Fellowship Award. He then joined Harvard Medical School's full-time faculty at Mass. Eye and Ear under the K12 Harvard-Vision Clinical Scientist Development Program, a highly competitive institutional program funded by the National Institutes of Health.

Working with collaborators at Boston Children's Hospital and Massachusetts Institute of Technology, Dr. Ciolino has developed a drug-eluting contact lens capable

Gena Heidary, MD, PhD

of delivering various pharmaceutical agents, such as antibiotics, antifungals, and glaucoma medication. These drug-eluting lenses have the potential to improve surgical outcomes for patients with keratoprosthesis by both protecting the ocular surface of the eye and by preventing post-operative infections, and they may one day replace eye drops.

In addition to his clinical practice and his research program, Dr. Ciolino also supervises medical students and participates in training residents and fellows. He is a named inventor on a U.S. patent application for a contact lens drug delivery device, and his research innovations have multiple direct therapeutic applications that may ultimately benefit a broad and diverse patient population.

Annual Meeting Co-chair, Associate Professor of Ophthalmology, Harvard Medical School and Director of the Pediatric Neuro-Ophthalmology Service, Boston Children’s Hospital

Dr. Heidary earned her MD, PhD in Neuroscience from the University of Pennsylvania School of Medicine. She completed her ophthalmology residency training at Harvard Medical School. She is dually fellowship trained, having completed a fellowship in pediatric ophthalmology and adult strabismus at Boston Children’s Hospital under the mentorship of Dr. David Hunter and a second fellowship in Neuro-Ophthalmology at Mass. Eye and Ear under the mentorship of Drs. Joseph Rizzo and Simmons Lessell.

As the Director of the Pediatric Neuro-Ophthalmology Service at Boston Children’s Hospital, Dr. Heidary’s clinical work focuses on pediatric neuro-ophthalmology and

Rachel Huckfeldt, MD, PhD

the management of pediatric and adult strabismus. Her research focuses on improving the management and treatment of patients with visually threatening, pediatric neuro-ophthalmic disease and complex strabismus. Specific efforts have focused on the development of a method to non-invasively assess elevated intracranial pressure using otoacoustic emissions in patients with idiopathic intracranial hypertension or pseudotumor cerebri. Additionally, Dr. Heidary has been working toward the development of a novel system to accurately assess visual field dysfunction in pediatric patients using saccadic vector optokinetic perimetry (SVOP). With this technique, eye-tracking technology is used to evaluate pediatric patients for visual field loss.

Annual Meeting Co-chair, Assistant Professor of Ophthalmology, Harvard Medical School, Mass. Eye and Ear

Dr. Huckfeldt earned her MD and PhD in Neuroscience from Washington University in St Louis. After completing her ophthalmology residency at Harvard Medical School, she conducted postdoctoral research focused on novel therapeutics for retinal dystrophies in the lab of Dr. Jean Bennett at the University of Pennsylvania. She subsequently completed clinical fellowships in medical retina (University of Iowa) and inherited retinal disorders (Mass Eye and Ear) with the latter under the guidance of Dr. Eric Pierce with support from a Foundation Fighting Blindness Clinical/Research Fellowship Award.

Dr. Huckfeldt’s research focuses on improving the visual outcomes of patients with inherited retinal

disorders (IRDs). She is the site principal investigator for multiple first-in-human clinical trials of genetic therapies for IRDs, and she also leads Mass Eye and Ear’s participation in clinical studies conducted by the Foundation Fighting Blindness Consortium. Dr. Huckfeldt was recently selected to serve as Co-chair for this multi-institution research group. Her own research program, which encompasses topics including retinitis pigmentosa-associated cystoid macular edema, is supported by a Career Development Award from the Foundation Fighting Blindness as well as the 2021 Iraty Award. Dr. Huckfeldt is also the director of the Inherited Retinal Degenerations clinical fellowship at Mass Eye and Ear.

Friday, June 24

The Liberty, Ballroom - 215 Charles Street, Boston, MA

7:30–8:00am Registration & Continental Breakfast - Liberty Ballroom

8:00–8:13am

8:00–8:13am

8:13–8:24am

Welcome & Opening Remarks

Gena Heidary, MD, PhD (2008, 2010) - Annual Meeting Co-chair, Associate Professor of Ophthalmology, HMS

Rachel Huckfeldt, MD, PhD (2013, 2016) - Annual Meeting Co-chair, Assistant Professor of Ophthalmology, HMS

Faculty Session II - Speaker Introductions

Gena Heidary, MD, PhD (2008, 2010) - Annual Meeting Co-chair, Associate Professor of Ophthalmology, HMS

Rachel Huckfeldt, MD, PhD (2013, 2016) - Annual Meeting Co-chair, Assistant Professor of Ophthalmology, HMS

8:24–8:35am

8:35–8:46am

8:46–8:57m

8:57–9:08am

9:08–9:19am

9:19–9:30am

Caught Between a Rock and a Hard Place - Pediatric Optic Nerve Head Drusen

Ryan A. Gise, MD (2020)

Instructor in Ophthalmology, Harvard Medical School

Mass. Eye and Ear

The Role of Galectin-3 in the Etiology and Treatment of Glaucoma

David A. Sola-Del Valle, MD (2015)

Assistant Professor of Ophthalmology, Harvard Medical School Mass. Eye and Ear

Updates and Subtleties in the Histopathologic Diagnosis of Orbital Inflammatory Disease - Anna M. Stagner, MD (2016)

Assistant Professor of Ophthalmology, Harvard Medical School

Mass. Eye and Ear

Developing Deep Learning Models for Data Cleaning in Glaucoma

Mengyu Wang, PhD (2017)

Assistant Professor of Ophthalmology, Harvard Medical School

Schepens Eye Research Institute of Mass. Eye and Ear

Transnasal Endoscopic Orbital Surgery

Suzanne K. Freitag, MD (2001)

Associate Professor of Ophthalmology, Harvard Medical School

Mass. Eye and Ear

Molecular Mechanisms of Proliferative Vitreoretinopathy

Leo A. Kim, PhD, MD

Associate Professor of Ophthalmology, Harvard Medical School

Mass. Eye and Ear

Rosebud: Challenges and Opportunities in the Indian Health Service

Silas L. Wang, MD

Instructor in Ophthalmology, Harvard Medical School Mass. Eye and Ear

9:30–10:10am Break - Outdoor Courtyard

Faculty Session II - Speaker Introductions

10:10–10:17am

10:17–10:28am

Gena Heidary, MD, PhD (2008, 2010) - Annual Meeting Co-chair, Associate Professor of Ophthalmology, HMS

Rachel Huckfeldt, MD, PhD (2013, 2016) - Annual Meeting Co-chair, Assistant Professor of Ophthalmology, HMS

The Tilt You See and The Tilt You Don’t: Orbital and Ocular Strategies to Enhance Binocular Fusion - Linda R. Dagi, MD (1989)

Associate Professor of Ophthalmology, Harvard Medical School Mass. Eye and Ear

Friday, June 24 (Continued)

10:28–10:39am

10:39–10:50am

10:50–10:55am

10:55–11:00am

11:0011:20am

11:2011:25am

11:25–11:55am 11:55–12:00pm

Targeted Therapy for Inherited Retinal Disorders

Qin Liu, PhD, MD

Assistant Professor of Ophthalmology, Harvard Medical School Mass. Eye and Ear

Better (Academic) Living Through Computers

Michael V. Boland, MD, PhD

Associate Professor of Ophthalmology, Harvard Medical School Mass. Eye and Ear

Harvard Ophathlmology Mentoring Awards

David Hunter, MD, PhD (1991) - Professor of Ophthalmology and Vice Chair for Promotions and Reappointments, Harvard Medical School; Richard M. Robb Chair in Ophthalmology and Ophthalmologist-in-Chief, Boston Children's Hospital

Summer Scholars Program - Alumni Introduction

James Chodosh, MD, MPH

Edith Ives Cogan Professor of Ophthalmology, Harvard Medical School Mass. Eye and Ear

Department Overview

Joan W. Miller, MD (1989, 1991) - Alumni Reunion Co-chair, David G. Cogan Professor of Ophthalmology, and Chair of Ophthalmology, Harvard Medical School; Chief of Ophthalmology, Mass. Eye and Ear and Mass General Hospital; and Ophthalmologist-in-Chief, Brigham and Women’s Hospital

Perspective on Mariana D. Mead & Introduction to the 2022 Mariana D. Mead Lecturer

Joseph F. Rizzo III, MD (1986) - David Glendenning Cogan Professor of Ophthalmology in the field of NeuroOphthalmology, Director of Alumni, and Co-director, Mobility Enhancement and Vision Rehabilitation Center of Excellence, Harvard Medical School; Director, Neuro-Ophthalmology Service, Mass. Eye and Ear

The 2022 Mariana D. Mead Lecture

The Elena Barraquer Foundation. A Global Challenge to Solve Avoidable Blindness #NoMoreCataracts

Elena Barraquer, MD (1987)

ophthalmologist and Assistant Medical Director at the Barraquer Centre CEO of the Barraquer Foundation

Presentation of the Mariana D. Mead Bowl - Presented by Joan W. Miller, MD

12:00–1:00pm Boxed Lunch - Outdoor Courtyard

Alumni Reunion Welcome

1:00–1:05pm

1:05–1:17pm

1:17–1:29pm

Joseph F. Rizzo III, MD (1986) - Alumni Reunion Co-chair, Director of Alumni, Harvard Medical School

Joseph B. Ciolino, MD (2009) - Alumni Reunion Co-chair, Associate Director of Alumni, Harvard Medical School

The Future of Big Data and Real World Evidence

Durga Borkar, MD (2017)

Assistant Professor of Ophthalmology Duke University School of Medicine

Teleretinal Screening for Diabetic Retinopathy: NYEE Experience

Meenakashi Gupta, MD (2012)

Assistant Professor, Ophthalmology

New York Eye and Ear Infirmary of Mount Sinai

Friday, June 24 (Continued)

1:29–1:41pm

1:41–1:58pm

1:58–2:15pm

2:15–2:35pm

Mimickers of Idiopathic Intracranial Hypertension

Marc J. Dinkin, MD (2007)

Associate Professor of Ophthalmology

Weill Cornell Medicine

Building a Surgical Curriculum for Ophthalmology Residents

Shahzad I. Mian, MD (2002)

Professor of Ophthalmology and Visual Sciences and Vice Chair for Clinical Sciences and Learning. University of Michigan Medical School, Kellogg Eye Institute

Mentoring: Why Should It Matter to You and Your Career

Quan Dong Nguyen, MD, MSc (1997, 1999)

Professor of Ophthalmology at the Byers Eye Institute

Stanford University School of Medicine

Alumni Speaker Panel

2:35–3:15pm Break - 4th Floor

3:15–3:32pm

3:32–3:37pm

3:37–3:42pm

3:42–4:17pm

Greetings from Tokyo!

Annabelle A. Okada, MD (1992)

Professor of Ophthalmology

Kyorin University School of Medicine in Tokyo, Japan

Alumni Spotlight: Michael V. Drake, MD (1982)

Joseph F. Rizzo III, MD (1986) - Alumni Reunion Co-chair, Director of Alumni, Harvard Medical School

Introduction of Distinguished Research Achievement Awardee

Eric A. Pierce, MD, PhD (1994) - William F. Chatlos Professor of Ophthalmology and Director, Ocular Genomics Institute, Harvard Medical School; Director, Berman-Gund Laboratory for the Study of Retinal Degenerations, Mass Eye and Ear

4:17–4:22pm

4:22–4:57pm

4:57–5:00pm

Distinguished Research Achievement Lecture: Neuroprotective Approaches for the Treatment of Photoreceptor and Retinal Ganglion Cell Degeneration

Donald J. Zack, MD, PhD (1988)

Professor of Ophthalmology and Co-Director, Stem Cells and Ocular Regenerative Medicine (STORM) Johns Hopkins University

Introduction of Distinguished Clinical Achievement Awardee

Joan W. Miller, MD (1989, 1991) - Alumni Reunion Co-chair, David G. Cogan Professor of Ophthalmology, and Chair of Ophthalmology, Harvard Medical School; Chief of Ophthalmology, Mass. Eye and Ear and Mass General Hospital; and Ophthalmologist-in-Chief, Brigham and Women’s Hospital

Distinguished Clinical Achievement Lecture: Peering Beyond the Blind Spot Seeking Authentic Risk Factors: A Case Study - The Ocular Hypertension Treatment Study

Eve J. Higginbotham, SM, MD, ML (1984)

Professor of Ophthalmology and Vice Dean for Penn Medicine Office of Inclusion, Diversity, and Equity Perelman School of Medicine at the University of Pennsylvania

Closing Remarks

Joseph F. Rizzo III, MD (1986) - Alumni Reunion Co-chair, Director of Alumni, Harvard Medical School

Joseph B. Ciolino, MD (2009) - Alumni Reunion Co-chair, Associate Director of Alumni, Harvard Medical School

The Newbury Hotel - One Newbury Street, Boston, MA

6:00–7:00pm Trainee Poster Contest & Reception - Garden Room

7:00–9:00pm Gala Dinner - Assembly Ballroom

ACCREDITATION

In support of improving patient care, Boston Children’s Hospital is jointly accredited by the Accreditation Council for Continuing Medical Education (ACCME), the Accreditation Council for Pharmacy Education (ACPE), and the American Nurses Credentialing Center (ANCC), to provide continuing education for the healthcare team.

Boston Children’s Hospital designates this live activity for a maximum of 7.5 AMA PRA Category 1 Credits™. Physicians should claim only credit commensurate with the extent of their participation in this activity.

Fees associated with AMA PRA Category 1 Credits™ have been provided by the HMS Department of Ophthalmology.

CME AND CE CERTIFICATES

IMPORTANT: Be sure to sign in each day on the sign-in sheets at registration, if you wish to apply for CME or CE. For Optometric CE, pick up your certificate at registration at the end of each day. For CME, the Boston Children’s CMED will send an email after the course with an survey link to all participants. Each participant will be asked to attest to the hours that he or she participated in the activity. Once the survey is complete, CME attendees can download their CME certificates. If you have any questions or concerns, contact: cmedepartment@childrens.harvard.edu.

COMPLETE PROGRAM

For a complete program, including the abstracts for the trainee poster contest, visit https://joom.ag/SCed or scan the QR code to the right with your smartphone or tablet.

Contest Floorplan

Trainee Poster

(The Newbury hotel, Garden Ballroom)

BASIC & TRANSLATIONAL RESEARCH

Monica Akula - Altered Transcription Kinetics Drives Save or Abort Decision for Retinal Cell Fate in Retinitis Pigmentosa: Reset with Modifier Gene Therapy

Hamid Alemi - Efficacy of Alpha-Melanocyte Stimulating Hormone (α-MSH) in Suppressing Corneal Edema Following Acute Injury

Camille Andre - Characterization of the Resistome in Gram-Positive Bacteria Causing Keratitis

Kiran Bora - Genetic Deficiency of RORα Leads to Retinal Bipolar Cell Dysfunction in Aging Mice

Neha Deshpande - Deregulated DNA Repair in Fuchs Endothelial Corneal Dystrophy

Nikolaos Efstathiou - Expression of Peripherin 2 (PRPH2) in Retinal Pigment Epithelial Cells

Tessa Fitch - Resveratrol Protects RPE Against TNFα-Induced Inflammation in Age Related Macular Degeneration

Yilin Guan - Ocular Surface Mast Cells Promote Nerve Damage and Trigeminal Ganglion Inflammation Following Corneal Injury

Cindy Hoppe -Targeting the Alternative Complement Pathway as a Neuroprotective Therapy in Glaucoma and Optic Nerve Injury

Zhengping Hu - Endomucin Deletion Delays Retinal Vascular Development and Inhibits Neovascularization in Oxygen-Induced Retinopathy

Mohammad Mirazul Islam - Chemical Crosslinker-free Pro-regenerative Corneal Implants

- Cyto-protective Effect of Alpha-Melanocyte-Stimulating Hormone on UV-A Induced Fuchs Endothelial Corneal Dystrophy

Margarete Karg - Microglia Prevent the Loss of Visual Function in the Aging Retina by Maintaining the Health of Retinal Pigment Epithelial Cells

Changrim Lee - Conjunctival Goblet Cells Stimulated with an Allergic Mediator Histamine Secrete Extracellular Vesicles that Exhibit Paracrine Secretagogue Activity

Seokjoo Lee - CD11b+Gr-1+MDSC Prevent IL-6-Induced Treg Dysfunction Partially Through Secretion of IL-10

Meenakshi Maurya - REV-ERBα Deficiency Accelerates Age-Related Synaptic Remodeling in Mouse Retina

Amirreza Naderi - The Effect of Neurokinin-1 Antagonism in Treating the Allergic Red Eye

Mohit Parekh - Effect of ROCK Inhibitor on Cell Migration in Fuchs Endothelial Corneal Dystrophy

Suelen Scarpa de Mello - Blocking the Emergence of Resistance to Last Line Antibiotic, Daptomycin

Jonathan Soucy - Chemokine-directed Migration Improves the Structural Integration of Human Stem Cellderived Retinal Neurons

Feng Tian - Core Transcription Programs Controlling Injury-Induced Neurodegeneration of Retinal Ganglion Cells

BASIC & TRANSLATIONAL RESEARCH

Amrita Saha Vadher - Potential Role of High Mobility Group Box 1 in Formation of Corneal Subepithelial Infiltrates in Adenovirus Keratitis

Asmaa Zidan - Calcitonin Gene-related Peptide Promotes the Healing of Corneal Epithelial Stromal Injury

CLINICAL RESEARCH

Inas Aboobakar - Genetic Risk Scores of Mitochondrial TXNRD2 and ME3 Variants are Associated with Specific Primary Open-angle Glaucoma Phenotypes

Atitaya Apivatthakakul - Prevalence of Ocular Inflammatory Conditions in Patients with Small Fiber Neuropathy in a Large Healthcare System Database

Bleicher- Outcomes of Far Posterior Open Globe Injuries: The Case for Zone 4

Henisk Falah - Comparative Outcomes of Phacoemulsification Combined with Hydrus or Kahook Dual Blade

Jacoba - Automated Machine Learning Models for Diabetic Retinopathy Screening Using Handheld Fundus Cameras in a Low-resource Community Screening Program

Li - High Positive Predictive Value of Fluorescein Angiography Contiguous, Perinerve

Yangjiani Li - Age-controlled Correlation Between Oral Glucose Tolerance Test (OGTT) and Thicknesses of Ten Retinal Layers in Non-diabetes

Coherence Tomography Angiography and Visual Outcomes in RAO

Luis Martinez-Velazquez - Predicting Visual Outcomes in Central Retinal Vein Occlusion (CRVO) with Vascular Metrics and Non-Perfusion Area (NPA) Measured by Wide-Field Swept-Source OCT-Angiography (WF SS-OCTA)

Kristen Pitts - APOE and Galectin-3, Markers of Activated Microglia, are Elevated in the Aqueous Humor of Glaucoma Patients

Chhavi Saini - Restoration of Vision in Severe, Cicatricial, Ocular Surface Disease with the Boston Keratoprosthesis Type II

Aastha Singh - Mesenchymal Stem Cells-derived Interleukin-11 Inhibits Activation and Proliferation of T cells

Karen Wai - Recovery of Vision in Open Globe Injury Patients with Initial No Light Perception Vision

Shudan Wang - Neurotrophic Keratopathy-Associated Eye Pain and Quality-of-Life-Related Parameters in Patients with Ocular Graft-Versus-Host Disease

Category: Basic and Translational Research

Candidate: Monica Akula

Poster #: B1

Altered Transcription Kinetics Drives Save or Abort Decision for Retinal Cell Fate in Retinitis Pigmentosa: Reset with Modifier Gene Therapy

M. Akula, P. Kadam, S. Bhambra, O. Dube, M. Alejandro, S. Li, Z. Love, K. Holton, M. DeAngelis, N.B. Haider

Purpose: Retinitis pigmentosa (RP) is a large group of genetically heterogeneous disorders that result in severe vision loss. Our prior and recent in silico and in vivo analysis revealed that Nr2e3 regulates several key biological networks that are critical to maintaining homeostasis in the retina. In this study we determine the expression profiles of the key retinal transcription regulators such as Nr2e3, Nr1d1, Nrl, Crx, Rora and Thrb in multiple RP models before and after treatment with AAV-Nr2e3 therapy.

Methods: RNA was isolated from retinas of several RP models including Nr2e3rd7/rd7, Pde6βrd1, Rho-/-, RhoP23H, RhoP347S and Cep290rd16. Gene expression profiling of key regulator genes was carried out by RNA-Seq (n=3) and quantitative real-time PCR (n≥7). Controls included B6 (normal) retinas, and untreated retinas were compared to AAV-Nr2e3 treated retinas.

Results: Monogenic diseases such as RP have been studied for the loss of single genes. However, our studies revealed that there is significant downregulation in expression of key retina transcription factors in several models of RP. This shift in turn caused misregulation of key homeostasis gene networks as disease progressed in each model, and this mutational load of the system likely contributes to disease. AAV-Nr2e3 therapy attenuated retinal degeneration in each of these models and resulted in increased expression of key retinal transcription factors and a reset of retinal homeostasis.

Conclusions: This is the first report evaluating the impact of transcriptome kinetics on retinal degeneration using RNA-seq and remodeling of the retina transcriptome following modifier gene therapy. The primary mutation causes a shift of transcription kinetics, forcing the retina to make a save or abort decision early in disease, leading to progressive photoreceptor degeneration. This combinatorial mutational load including the primary mutation and key retinal transcription factor modulation determines phenotypic outcome of each disease. This study further shows the profound impact of AAV-Nr2e3 modifier gene therapy in attenuating retinal disease in a mutation agnostic manner.

Category: Basic and Translational Research

Candidate: Hamid Alemi

Poster #: B2

Efficacy of Alpha-Melanocyte Stimulating Hormone (α-MSH) in Suppressing Corneal Edema Following Acute Injury

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

Purpose: Corneal endothelial cells (CEnC) have minimal regenerative capacity and insults to the endothelium may lead to persistent corneal edema. Following ocular injury, the concomitant decrease in density and function of both corneal nerves and CEnC suggests that nerves have a trophic role for CEnC. Herein, we investigate the efficacy of the neuropeptide α-MSH in preventing corneal edema following injury and explore the underlying mechanisms.

Methods: Corneal injury was induced by transcorneal freezing in BALB/c mice (n=8/group) by placing a -72oC 2 mm-diameter steel rod on the center of the cornea for 10 seconds. Mice received 10µL subconjunctival injection of α-MSH (10-4M) or PBS (control) immediately post-injury and twice weekly for eight weeks. The following measures were tracked clinically: Central Corneal Thickness (CCT) by optical coherence tomography, corneal opacity by slit lamp, and CEnC density by in vivo confocal microscopy. Corneal endothelial tissue was evaluated for apoptosis (TUNEL assay), proliferation(5-ethynyl-2’-deoxyuridine [EdU] incorporation), and functional differentiation (Na+/K+-ATPase mRNA and protein expression). Immortalized human CEnC (HCEnC-T21) were cultured with/without α-MSH (10-7M) for 24hrs and a scratch assay and live microscopy were used to evaluate wound healing, while CEnC proliferation was measured using the PrestoBlue® assay.

Results: In the control group, transcorneal freezing led to a 0.61-fold increase in CCT (vs. baseline:116±6, P<0.001) and a 0.38-fold reduction in CEnC density (vs. baseline:2954±67, P<0.001) 8wks post-injury. α-MSH restored CCT to baseline values (117±12µm, P=0.004) and increased CEnC density by 0.2-fold (P<0.001). Compared to control, α-MSH treatment led to a 0.61-fold reduction in apoptotic CEnC frequency and a 0.4-fold increase in the number of newly generated (EdU+) CEnC 48hrs post-injury. Supplementing HCEnC culture with α-MSH led to a 0.25-fold increase in wound healing rate and a 0.62-fold increase in CEnC proliferation (P=0.03). While control-treated mice experienced a 0.3-fold and 0.63-fold decrease in CEnC mRNA and protein expression of Na+/K+-ATPase (P=0.001 and P=0.004, respectively), α-MSH restored CEnC Na+/K+ATPase expression with a 2.3-fold increase in mRNA (P=0.044) and a 1.85-fold increase in protein levels (P<0.001) compared to the control group.

Conclusions: Our results demonstrate that following injury, treatment with α-MSH prevents corneal edema through its cytoprotective function and additionally promotes the differentiation and proliferation of newlyformed CEnC.

Category: Basic and Translational Research

Candidate: Camille Andre

Poster #: B3

Characterization of the Resistome in Gram-Positive Bacteria Causing Keratitis

Camille André, Michael S. Gilmore, Paulo J. M. Bispo

Purpose: Antimicrobial resistance (AMR) in microbial keratitis is a concerning issue that can result in treatment failure and poor visual outcome. The aim of this study was to characterize phenotypically and genomically the AMR patterns of gram-positive bacteria (GPB) isolated from keratitis at Massachusetts Eye and Ear from 2014 to 2017.

Methods: Whole Genome Sequencing was performed on 161 GPB keratitis isolates using Illumina HiSeq. Molecular typing was performed by MLST. CARD algorithm was used to identify genes and mutations that confer AMR. Minimum inhibitory concentrations were determined by broth microdilution.

Results: Staphylococcus aureus was the most common pathogen (53.4%) and its population structure was dominated by lineages grouped within the clonal complex 5 (32.6%), which includes epidemic MRSA strains commonly associated with multidrug-resistant (MDR) infections. Compared with methicillin-susceptible S. aureus, resistance to other antibiotics was more prevalent among MRSA isolates, with rates of resistance higher than 70% for fluoroquinolones (FQ) and azithromycin. The newest FQ (besifloxacin and moxifloxacin) had lower MIC90 compared with the earlier ones. More than 30% of coagulase-negative Staphylococcus were resistant to FQ and half of them were resistant to azithromycin (52.9%). We found between 2 and 4 mutations in the quinolone resistance-determining regions (QRDR) of gyrA and parC genes for staphylococci isolates (21.7% of S. epidermidis and 25.0% of S. aureus) that were associated with high FQ MICs. 52.1% of S. epidermidis and 22.1% of S. aureus were MDR. CARD analysis revealed that 26.7% of S. aureus co-harbored ant(9)-Ia and ermA genes that confer resistance to aminoglycosides, and to macrolides, lincosamides and streptogramin B, localized in a Tn554 transposon, whereas macrolide resistance in S. epidermidis was frequently associated with a mphC/msrA genotype. S. epidermidis were also positive for the dfrC (82.6%) and tetK genes (21.7%) which confers resistance to diaminopyrimidine and tetracycline respectively. Among S. pneumoniae isolates, resistance rates were less than 10% to all antibiotics tested except for ofloxacin (14.4%), azithromycin (30.8%) and penicillin (30.8%). These isolates often carried macrolide resistance genes (ermB and mefA/msrD) that are integrated into mobile genetic elements

Conclusions: We provided an overview of current rates of resistance to antibiotics commonly used to treat keratitis caused by GPB and a characterization of the diversity of the associated molecular mechanisms that confer AMR.

Category: Basic and Translational Research

Candidate: Kiran Bora

Poster #: B4

Genetic Deficiency of RORα Leads to Retinal Bipolar Cell Dysfunction in Aging Mice

Kiran Bora†, Chi-Hsiu Liu†, Felix Yemanyi, Alexandra K. Blomfield, Meenakshi Maurya, Ye Sun, James D. Akula, Jing Chen*

Purpose: Retinoic acid receptor-related orphan receptor alpha (RORα) is a lipid-sensing nuclear receptor transcription factor expressed in many retinal cell types including photoreceptors, ganglion cells and bipolar cells. It plays a crucial role in regulating expression of target genes involved in diverse physiological processes in the eye, such as those in lens and photoreceptor development. Genetic variations of RORα have been linked to development of age-related macular degeneration. In this study, we investigated the role of RORα in retinal bipolar cell function during aging using mice with spontaneous RORα deletion mutation (RORαsg/sg).

Methods: RORαsg/sg and age-matched wild-type (WT) mice were assessed at various time points during aging for functional and phenotypical analysis. The distribution of RORα in retina was analyzed using single cell RNA sequencing (scRNA seq) database and its localization was monitored by immunohistochemistry, along with evaluation of alterations in bipolar cell morphology using PKCα, a rod bipolar cell selective marker. Further, visual function was assessed by scotopic full-field electroretinography (ERG), and retinal expression of genes involved in signal transmission through bipolar cells was determined in RORαsg/sg and WT mice.

Results: RORα expression was found in retinal bipolar cells in scRNA seq analysis and its localization was confirmed in retinal sections. RORasg/sg mice showed substantial impairment of visual function with significant attenuation (P=0.0002) of b-wave ERG amplitude at 5 month, with greater reduction (P=0.04) at 12 month, suggestive of bipolar dysfunction. However, ERG a-waves were comparatively normal. RORasg/sg mice revealed significant degeneration and progressive loss of bipolar cells upon aging with notable loss of rod bipolar cells at 5 months, followed by more conspicuous loss and severely disrupted morphology upon aging (12 months) with respect to WT. Furthermore, RORasg/sg retinal tissues revealed significant dysregulation of genes involved in glutamate and calcium signaling.

Conclusions: These findings suggest that RORα deficiency results in progressive, age-related rod bipolar cell degeneration with associated visual dysfunction, indicating a crucial role of RORα in regulating rod bipolar cell integrity and function in aging eyes.

Category: Basic and Translational Research

Candidate: Neha Deshpande

Poster #: B5

Deregulated

DNA Repair in Fuchs Endothelial Corneal Dystrophy

Neha Deshpande, Shazia Ashraf, Geetha Melangath, Shivakumar Vasanth, Marianne Price, Francis Price Jr., Ula V. Jurkunas

Purpose: Fuchs endothelial corneal dystrophy (FECD) is an age-related disorder involving corneal endothelial (CE) degeneration resulting from lifelong exposure to oxidative stress. We previously reported heightened accumulation of nuclear (nDNA) and mitochondrial (mtDNA) damage to be a hallmark of FECD. This finding was recapitulated in the ultraviolet-A (UVA) light-induced FECD mouse model which showed early stage mtDNA and late stage nDNA damage to contribute to degenerative CE phenotype. In this study, we explore impaired DNA repair pathways that may drive the build-up of deleterious DNA lesions in FECD.

Methods: Differential expression profiles of DNA repair genes were analyzed by real-time PCR arrays in which 84 genes and five housekeeping genes were tested. Total RNA was extracted from Descemet’s membrane-CE stripped from age-matched normal donors (n=4) and FECD specimens (n=8). cDNA was subjected to real-time PCR analysis on Human DNA Repair RT Profiler plates. Change in mRNA expression of <0.5 or >2.0-fold in FECD relative to normal was set as the cutoff value for down- or upregulation. Total protein was extracted from CE stripped from 7-9 weeks old C57BL/6 mice irradiated with 1000 J/cm2 UVA at 1 day and western blotting was carried out to assess DNA repair protein levels.

Results: FECD specimens differentially expressed (p<0.05) 6 nucleotide excision repair (NER), 7 base excision repair (BER), 3 double strand break (DSB) repair, 2 mismatch repair (MMR) and 1 unclassified pathway gene compared to normal donors. Both MMR genes belonged to the upregulated group. A majority of the deregulated genes predominantly belonged to NER and BER pathways (8 out of 11 downregulated, and 5 out of 8 upregulated genes). NER gene XPC and BER gene lig3 were both downregulated (0.20-fold; p<0.005 and 0.32-fold; p<0.005) in FECD specimens and were also reduced (0.3-fold; p<0.05 and 0.4-fold; p<0.05) in the FECD mouse CE model 1 day post UVA irradiation, correlating to the early time-point mtDNA damage shown earlier.

Conclusions: Our findings indicate that of the 4 major pathways involved in DNA repair; NER, BER and DSB pathways feature in the sets of both up and down regulated genes, while MMR pathway features only in the set of upregulated genes in FECD. Deficient NER and BER pathways may trigger the accumulation of unresolved DNA damage in FECD. Downregulation of XPC and lig3 may contribute to the early mtDNA damage observed in the UVA-mouse model of FECD.

Category: Basic and Translational Research

Candidate: Nikolaos Efstathiou

Poster #: B6

Expression of Peripherin 2 (PRPH2) in Retinal Pigment Epithelial Cells

Nikolaos E. Efstathiou, Victor S.M.C. Correa, Toshio Narimatsu, Dimitrios P. Ntentakis, Yuki Morizane, Constantine D. Georgakopoulos, Demetrios G. Vavvas

Purpose: Peripherin2(PRPH2),encoded by the PRPH2 gene, also known as Retinal Degeneration Slow(RDS),is a transmembrane protein found in the outer segment of both rod and cones photoreceptor cells.Complete loss of PRPH2 prevents photoreceptor outer segment formation.Up to date there have been identified more than 190 mutations in the PRPH2 gene causing a heterogenous set of retinal diseases characterized by various phenotypes.In cases like Central Areolar Choroidal Dystrophy caused by the R172W mutation of the PRPH2 gene the lesion observed is well circumscribed and affects predominantly in the macular area including the retinal pigment epithelium.That prompt us to examine if PRPH2 is expressed in RPE

Methods: Here we use human fetal primary (hfpRPE), ARPE-19 and human induced pluripotent stem cell (iPSC)-derived RPE cells to examine the expression of PRPH2 at RNA level. We use agarose gel and Sanger DNA sequencing to verify our results. In addition, we examine the protein levels of PRPH2 by Western Blot in hfpRPE and ARPE-19 cells. Finally, we used human retinal tissue isolated from retinal disease-free donors to examine the PRPH2 protein level by WB.

Results: We found that PRPH2 is expressed in low quantities in RNA level of hfpRPE, ARPE-19 and iPSCderived RPE cells. PRPH2 wasn’t detectable by WB in hfpRPE and ARPE-19 cells. PRPH2 was detected by WB in Retina and RPE/Choroid in human donor samples. PRPH2 protein levels were higher in macular versus periphery RPE/choroid isolated from human donor eyes.

Conclusions: PRPH2 is expressed in low amounts in retinal pigment epithelial cells. There is higher expression of PRPH2 in macular compared to periphery retinal pigment epithelial cells.

Category: Basic and Translational Research

Candidate: Tessa Fitch

Poster #: B7

Resveratrol Protects RPE Against TNFα-Induced Inflammation in Age Related Macular Degeneration

Tessa C Fitch, Scott Frank, Leo A. Kim, Daisy Y. Shu

Purpose: Age-related macular degeneration (AMD) is a leading cause of irreversible blindness and vision loss worldwide. Damage to retinal pigment epithelial cells (RPE) from inflammatory cytokines plays a key role in the pathogenesis of AMD. This study examined the effects of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine that is heavily involved in AMD, on RPE health and function. Resveratrol is a natural organic compound known to have very potent anti-inflammatory effects. In this study, we investigated the capacity of resveratrol to block TNFα-driven inflammation and metabolic dysfunction in RPE.

Methods: Matured H-RPE cells were treated with TNFα (10 ng/ml) and/or treated with resveratrol (50 μM) for 24 hours to 5 days. Glycolytic and oxidative (OXPHOS) metabolic profiles were determined with the Seahorse XFe96 Bioanalyzer. Gene expression of metabolic markers was assessed using qPCR. IL-6 secretion was quantified by using the Human IL-6 ELISA Kit. The EVOS m700 microscope was used for imaging morphology and fluorescence. Data and statistical analysis were performed using GraphPad Prism, Excel, ImageJ, and BioRender.

Results: Resveratrol suppressed TNFα-induced upregulation of IL-6, IL-8, TLR2, and MCP-1. Maximal respiration, spare respiratory capacity, and glycolysis levels were enhanced with resveratrol on the Seahorse XFe96. Co-treatment with resveratrol and TNFα further enhances oxygen consumption rate. Cells treated with TNFα showed an elongated morphology with loss of the cuboidal cobblestone morphology. Concurrent treatment with resveratrol rescued morphology with cells appearing similar to control. Cells with resveratrol alone maintained the regular morphology. Resveratrol suppressed TNFα-induced upregulation of cytoplasmic reactive oxygen species (ROS) down to basal levels on the CellROX assay.

Conclusions: RPE cells are profoundly affected by the proinflammatory effects of TNFα. TNFα not only disrupts the structural epithelial morphology of H-RPE cells, it also causes dysfunction of metabolic pathways and mitochondria. Treatment with the natural organic compound, resveratrol, efficiently blocks TNFα-induced proinflammatory activation, bioenergetic reprogramming and ROS upregulation of RPE. These results reveal a critical interplay between inflammation, metabolic dysfunction and oxidative stress in RPE, identifying resveratrol as a potent drug to treat AMD.

Category: Basic and Translational Research

Candidate: Yilin Guan

Poster #: B8

Ocular Surface Mast Cells Promote Nerve Damage and Trigeminal Ganglion Inflammation Following Corneal Injury

Yilin Guan, WonKyung Cho, Elsayed Elbasiony, Aastha Singh, Sharad Mittal, Sunil Chauhan

Purpose: Nerve damage following injury has been associated with impaired wound healing. We have previously shown that corneal injury leads to increased activation of ocular surface mast cells. Here, we investigated whether mast cells interact with corneal nerve and contribute to nerve degeneration and neuroinflammation.

Methods: Corneal injury was induced by mechanical removal of the epithelium (3 mm) and one-third of the anterior stroma in C57BL/6 mice using an Algebrush II. To evaluate the proximity of mast cells and damaged nerves, corneas were harvested 6 hours post-injury and stained with β-tubulin III (corneal nerves), and avidin (mast cells), for immunohistochemistry (IHC) analysis. Trigeminal ganglions (TGs) were harvested post-injury and lysates were prepared to measure levels of tryptase (mast cell activation marker), CD11b and Substance P (SubP), using colorimetric assay and PCR analysis. To assess direct interaction between mast cells and inflamed corneal nerves, primary TGs were co-cultured with bone marrow-derived mast cells for 24h. Brightfield images were captured, and neurite length was quantified using ImageJ software. TGs harvested from co-cultures were assessed for the expression of nerve activation marker SubP and CGRP. To assess the in vivo effect of mast cell activation on nerve damage, injured corneas were treated with mast cell inhibitor cromolyn (2% in PBS) and hyperactivation of nerves were measured using eye wipe test.

Results: IHC analysis demonstrated that ocular surface mast cells infiltrated into the injured cornea in close proximity to the damaged corneal nerves. Corneal injury resulted in a significant activation of TG mast cells and inflammation, as indicated by increased levels of tryptase (p=0.003), CD11b (p<0.001) and SubP (p=0.009). Co-culturing of TGs with mast cells resulted in an approximate 13-fold upregulation in CGRP (p<0.001), and ~1.5-fold increase in SubP (p=0.008), compared to TGs cultured alone. Moreover, mast cells resulted in significant neuronal degeneration, as indicated by 50% decrease in neurite length compared to control TG cultures (p<0.001). Pharmacological inhibition of ocular surface mast cell activation resulted in a significant decline in TG inflammation and hyperalgesia, as shown by decreased eye wipes (p=0.005).

Conclusions: Ocular surface mast cells exacerbate nerve degeneration and promote inflammation in the trigeminal ganglion.

Category: Basic and Translational Research

Candidate: Cindy Hoppe

Poster #: B9

Targeting the Alternative Complement

Pathway as a Neuroprotective Therapy in Glaucoma and Optic Nerve Injury

Hoppe, Cindy; Guo, Yinjie; Shrestha, Maleeka; Verma, Bhupender; Connor, Kip; Gregory-Ksander, Meredith S.

Purpose: Accumulating evidence from both human and animal models of glaucoma implicates the classical complement pathway as an important mediator of neuroinflammation and glaucomatous neurodegeneration. However, the alternative complement pathway mediates amplification of the complement pathway and has recently been implicated in driving inflammation and propagating injury in experimental models of spinal cord injury and brain injury, where specifically inhibiting the alternative pathway significantly reduced the extent of neurodegeneration. Herein we examine whether specifically inhibiting the alternative pathway is neuroprotective in experimental models of glaucoma and optic nerve injury.

Methods: Intracameral injection of magnetic microbeads (control: saline) was used to elevate the intraocular pressure (IOP) in complement factor B knockout mice (Fb-/-, deficient in the alternative complement pathway), complement component 3 knockout mice (C3-/-, deficient in all three complement pathways), and wild type (WT) mice. The optic nerve was crushed (ONC) approximately 2mm behind the globe with jeweler’s forceps for 10 seconds. Visual acuity was measured by optomotor reflex (OMR). RGC density was quantified in retina whole mounts stained with a RGC-specific anti-Brn3a antibody. The nerve fiber layer (NFL) thickness was analyzed by spectral domain coherence tomography.

Results: IOP was increased equally in microbead injected Fb-/-, C3-/- and WT mice as compared to saline controls. At day 35 post-microbead injection, visual acuity was significantly reduced in WT mice (0.37 ± 0.03 cyc/deg to 0.29 ± 0.04 cyc/deg, p<0.001) while no significant loss was observed in Fb-/- mice (0.39 ± 0.03 cyc/deg to 0.36 ± 0.03 cyc/deg, p>0.05) or C3-/- mice (0.35 ± 0.03 cyc/deg to 0.34 ± 0.04 cyc/deg, p>0.05). A significant thinning of the NFL and significant reduction in RGC density was also detected in microbead injected WT mice, but not Fb-/- mice. Similar results were observed following ONC, with Fb-/- mice displaying a significant increase in RGC survival at two weeks post ONC when compared to WT mice.

Conclusions: Our data shows that specifically inhibiting the alternative complement pathway is neuroprotective in mouse models of glaucoma and ONC, revealing the importance of the alternative pathway in the pathogenesis of glaucoma and optic nerve injury and introducing a potential new neuroprotective treatment.

Category: Basic and Translational Research

Candidate: Zhengping Hu

Poster #: B10

Endomucin Deletion Delays Retinal Vascular Development and Inhibits Neovascularization in Oxygen-Induced Retinopathy

Zhengping Hu, Issahy Cano, Magali Saint-Geniez, Yin Shan Eric Ng, Patricia A. D’Amore

Purpose: Endomucin (EMCN) is a type I integral membrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that EMCN knockdown in vitro significantly inhibits VEGF165-induced VEGFR2 internalization and downstream activities including proliferation, migration and tube formation. The goal of this study is to characterize the role of EMCN in normal retinal vascular development and in pathologic neovascularization using the EMCN knock-out mice.

Methods: Homozygous EMCN knock-out (EMCN-/-) mice were obtained by crossing EMCN-floxed mice with the ROSA26-Cre strain. Eyes from adult (8-16 weeks) EMCN-/- mice and EMCN+/+ control littermates were collected and retinas and RPE/choroids complex were dissected for RNA extraction, and qPCR to determine gene expression. Eyes from P5 mice and adult mice (12-16 weeks) were collected for retinal flat mounts; retinal vasculature was stained with Isolectin-B4 (IB4). For the oxygen-induced retinopathy (OIR) model, P7 mice were housed in 75% oxygen for five consecutive days and returned to room air at P12, eyes at P12 and P17 were collected and the retinal vasculature stained with IB4 and the avascular and neovascular areas were quantified using photoshop.

Results: EMCN mRNA in both retinas and RPE/choroids from the EMCN-/- mice was undetectable (n>4, p<0.0001), compared to EMCN+/+ mice by qPCR. The area of retinal vascularization at P5 was significantly lower in the EMCN-/- pups compared to EMCN+/+ controls (0.14±0.01 vs 0.2±0.013, p<0.0001, n>10 for both groups). Adult retinal vascular density remained lower in the EMCN-/- mice compared to EMCN+/+ mice (0.135 0.015± vs 0.154±0.017, p<0.05, n= 13). In the OIR model, the avascular area generated by high oxygen exposure at P12 was similar in the EMCN-/- and EMCN+/+ mice (24.57±1.4% vs 23.18±1.0%, p=0.9, n>6). However, pathological neovascularization at P17 was significantly reduced in the EMCN-/- mice compared to EMCN+/+ (8.98 ± 2.9% vs 11.98±1.4%, p<0.05, n>10) while the avascular area at P17 was comparable between the EMCN-/- and controls (9.9± 1.5% vs 8.85±1.0%, p>0.5, n>10).

Conclusions: Genetic ablation of the EMCN gene reduces retinal vascularization under normal and pathological conditions. As a critical regulator of retinal angiogenesis, EMCN represents a novel therapeutic target for ocular diseases characterized by pathological blood vessel growth.

Category: Basic and Translational Research

Candidate: Mohammad Mirazul Islam

Poster #: B11

Chemical Crosslinker-free Pro-regenerative Corneal Implants

Mohammad Mirazul

Islam,

Alexandru Chivu,

Dina

B. AbuSamra, Amrita Saha, Sumit Chowdhuri, Bapan Pramanik, Claes H. Dohlman, Debapratim Das, Pablo Argüeso, Jaya Rajaiya, Hirak K Patra, James Chodosh

Purpose: Transplantation of the cornea is a standard procedure for the treatment of corneal blindness, but there is a severe scarcity of donor corneas. The development of an artificial cornea could help to quell the demand. Collagen-based artificial corneas, in which the collagen has been chemically crosslinked, have shown promise in human trials. However, most crosslinking agents are cytotoxic. In the absence of crosslinking, collagen implants are mechanically weak and susceptible to enzymatic degradation. In this work, we successfully fabricated transparent hydrogels as corneal substitutes from collagen, without using any crosslinkers or modifying the native collagen structure.

Methods: We developed a pyrene conjugated dipeptide amphiphile PyKC consisting of lysine and cysteine for supramolecular gelation where collagen molecules are intertwined inside the PyKC network without any alteration of the collagen. Hydrogels of 10% and 15% collagen (as controls), 10% collagen-1% PyKC, 10% collagen-2% PyKC, 15% collagen-1% PyKC, and 15% collagen-2% PyKC, are referred to as Coll10, Coll15, Coll10-PyKC1, Coll10-PyKC2, Coll15-PyKC1 and Coll15-PyKC2, respectively. We measured physicochemical properties, biocompatibility, immunogenicity and antiviral property of the hydrogels. One-way ANOVA with Tukey post hoc test was performed to compare between groups.

Results: Light transmission studies showed the hydrogels are transparent in visible light and block UV light. The hydrogels were mechanically comparable with controls (Coll10 and Coll15-PyKC2, p=0.0555), enzymatically stable, and the tolerance for sutures was increased compared to controls (Coll10 and Coll15PyKC2, p=0.0128). The hydrogels also supported the growth and function of corneal epithelial, stromal, and endothelial cells. In vitro immune response studies showed that the hydrogels suppressed the inflammatory differentiation of human monocyte-derived dendritic cells. The hydrogels also restricted adenovirus propagation (Coll15 and Coll15-PyKC1, p=0.0007).

Conclusions: Our newly developed crosslinker-free fabrication strategy may dramatically impact the development of corneal implants. The hydrogels can also be modified to encapsulate cells, drugs, growth factors, and/or antibodies for therapeutic use.

Category: Basic and Translational Research

Candidate: Francesca Kahale

Poster #: B12

Cyto-protective Effect of Alpha-Melanocyte-Stimulating Hormone on UV-A Induced Fuchs Endothelial Corneal Dystrophy

Francesca Kahale; Neha Deshpande; Hamid Alemi; Amir Reza 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) from accumulated exposure to oxidative stress. FECD is a leading cause of corneal transplantation worldwide with no definitive pharmacologic treatment available. UV radiation, specifically UV-A has been shown to induce DNA damage by means of free radical generation, contributing to FECD pathogenesis. We have previously demonstrated the protective function of the neuropeptide alpha-Melanocyte Stimulating Hormone (α-MSH) against oxidative stress on ex vivo corneas. This study aims to investigate the in vivo protective effect of α-MSH in a mouse model of UV-A irradiation ind

Methods: 8-week-old female C57BL/6 mice (n=6/group) were irradiated with UV-A for 16 minutes, delivering a dose of 500J/cm2. Mice received an intraperitoneal injection of 0.01mL/g of α-MSH (10-4 M) immediately following irradiation and thrice weekly for 4 weeks. CEnC density, hexagonality, and coefficient of variation (CV) were evaluated by in vivo confocal microscopy pre-irradiation and at day 1, 14 and 28 post-irradiation. Image analysis was done via the KONAN CellChek® software. Corneal edema was evaluated by measuring central corneal thickness by optical coherence tomography.

Results: Corneal endothelial cells visualized by in vivo confocal microscopy pre-UV exposure at days 14 and 28. At day 28, mean cell density was significantly higher in the treated (1970.9 ± 285 cells/mm2) compared to the untreated group (1137.5 ± 398 cells/mm2), p=0.0005. Polymegathism evaluated by coefficient of variation was also significantly lower in the treated group at 37.2 ± 5.5 compared to untreated at 64.4+20.3 (p=0.005). Percentage of cells retaining hexagonality was significantly higher in the treatment group (54.3 ±3.4 %) compared to untreated (45.1 ±6.8 %), p=0.006. Central corneal thickness measured by optical coherence tomography demonstrates difference in CCT starting at D14 between treated and untreated groups. By day 28, corneal edema is significantly higher in the untreated group (114.8 ±16.5μm ) compared to treated group (93.4 ±5.4μm), p=0.0074.

Conclusions: Treatment with α-MSH leads to a decrease in UV-A induced CEnC cell loss and preserves cellular morphology. The conferred endothelial cyto-protection prevents corneal decompensation and edema.

Category: Basic and Translational Research

Candidate: Margarete Karg

Poster #: B13

Microglia Prevent the Loss of Visual Function in the Aging Retina by Maintaining the Health of Retinal Pigment Epithelial Cells

Margarete M. Karg, Drenushe Krasniqi, May Moorefield, Emma Hoffmann, Daisy Y. Shu, Hannah Philipose, Bruce R. Ksander, Magali Saint-Geniez

Purpose: Aging leads to the progressive deterioration of visual function that coincides with increasing cellular dysfunction in retinal neurons and retinal pigment epithelial (RPE) cells. Interestingly, aging also coincides with the migration of microglia from the inner to the outer retina where they reside in the subretinal space between photoreceptor outer segments and the apical surface of RPE. Healthy young microglia act as the immune sentinels of the retina, and thus are protective and maintain retinal homeostasis. However, age alters cellular functions and the role of aging subretinal microglia is not known.

Methods: Here we investigated whether aged microglia residing in the subretinal space are either preventing or promoting loss of visual function. Microglia were depleted in 33-month-old C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) which is expressed by microglia and is required for their proliferation, differentiation, and survival. As a negative control, age-matched mice received a control diet. Following microglia depletion, the optomotor reflex (OMR) was used to assess visual acuity and contrast sensitivity and electroretinogram (ERG) assessed retinal function. Morphological analysis was determined by immunohistochemical staining and TEM.To distinguish microglia from infiltrating macrophages, retinas from old mice were stained with P2RY12, a microglia-specific marker.

Results: The vast majority of cells residing between the photoreceptor outer segments and RPE layer in old mice were P2RY12+ microglia. These cells displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets similar to lipofuscin. PLX5622 treatment depleted up to 90% of the microglia. Microglia preserved visual function, and in the absence of microglia, there was a significant loss of contrast sensitivity and ERG c-wave, as compared with untreated controls. Loss of microglia coincided with a loss of RPE cells and increased RPE swelling indicating that microglia support RPE in aged animals. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, thereby compensating for the known age-related decline of RPE phagocytosis.

Conclusions: We conclude that microglia in aging mice migrate to the subretinal space of the outer retina to support and maintain RPE cell functions that preserves vision.

Category: Basic and Translational Research

Candidate: Changrim Lee

Poster #: B14

Conjunctival Goblet Cells Stimulated with an Allergic Mediator

Histamine Secrete Extracellular Vesicles that Exhibit Paracrine Secretagogue Activity

Changirm Lee, Darlene A. Dartt

Purpose: Goblet cell functions, especially secretion of mucins, are relatively well studied owing to their protective role on the ocular surface; however, little attention is given to the other secretory products, such as extracellular vesicles (EVs). The purpose of this study is to examine the existence and the function of goblet cell secreted EVs and test whether EVs produced by inflammatory stimuli will cause distinct actions compared to those produced by healthy (baseline) cells on recipient goblet cells.

Methods: First, serum-starved primary human conjunctival goblet cells (HCGCs, grown from conjunctiva explants) were incubated for 4 h with and without the allergic mediator histamine (His, 10-3 ~ 10-5 M) to induce inflammation or to indicate normal cells, respectively. EVs isolated from each treatment were denoted as EVsH (Histamine) and EVs-B (Basal), respectively. Second, a nanoparticle tracking analyzer and western blotting were used to analyze the size and the molecular identity of the isolated EV particles. Third, to examine the secretagogue activity of EVs, freshly prepared recipient HCGCs were treated for 4 h with either EVs-H or EVsB diluted to 1, 10, 100, and 1000 ng/mL in Hank’s balanced salt solution (HBSS). The amount of secreted high molecular weight glycoproteins (HMWP) in the cell culture media during this 4 h period was measured using an enzyme-linked lectin assay (ELLA). Fourth, to test the paracrine and endocrine actions of HCGC EVs, fluorescent Ca2+ indicator Fura-2 loaded HCGCs and primary human corneal epithelial cells were spiked with EVs-H or EVs-B and observed for changes in intracellular Ca2+ concentration (delta[Ca2+]i).

Results: HCGCs stimulated with His secreted a 2-fold higher amount of high molecular weight glycoproteins (HMWP) as well as a 7-fold higher number of cell-derived nano-scale particles (EVs-H) into the media compared to the control group, which confirmed that His stimulation increases both mucin secretion and EV release in primary HCGCs. The mean diameter of isolated EVs-H and EVs-B was 204.4 and 170.9 nm, respectively. Isolated EVs were enriched with CD9, ALIX, and CD81 but negative for GM130 (Golgi marker) and calnexin (ER marker), showing comparable biophysical and molecular characteristics to the standard set forth by the international community (MISEV 2018). When 10 ng/mL of EVs-H were incubated with recipient HCGCs, HMWP secretion was increased by 14-fold compared to HBSS treated HCGCs (negative cntl). This increase was also 6-fold and 4-fold higher compared to the 10 ng/mL EVs-B treated HCGCs and 10-5 M His treated HCGCs (positive cntl), respectively. An increase of 2~3-fold was observed at other EV concentrations (1, 100, and 1,000 ng/mL); however, all were lower than the activity seen in 10 ng/mL EVs-H. EVs-H also induced an increase in intracellular Ca2+ concentration ([Ca2+]i) not only in HCGCs but also in primary human corneal epithelial cells. In HCGCs, 100 ng/mL EVs-H increased [Ca2+]i by 7-fold compared to mock treatment (negative cntl) and this increase was comparable to 10-5 M His (positive cntl) treatment. In corneal epithelial cells, only 10 and 100 ng/mL EVs-H showed positive delta[Ca2+]i while all other doses showed negative values for delta[Ca2+]i upon addition. EVs-H’s ability to mobilize intracellular Ca2+ in both HCGCs and corneal epithelial cells suggests that they are capable of inducing intracellular signaling in paracrine and endocrine manner on the ocular surface.

Conclusions: We conclude that EVs secreted from inflammatory mediator stimulated HCGCs (EVs-H) have differential secretagogue activity and intracellular Ca2+ mobilization capacity when compared to EVs secreted

from the basal HCGCs (EVs-B). Despite the indispensable role of HCGC EVs in regulating goblet cell secretion and intracellular signaling at the extracellular level, their contributions to the ocular surface homeostasis have been almost neglected until today. The results presented here warrant in-depth investigation into their molecular properties, mechanism of action, and their contributions to the overall ocular surface health.

Category: Basic and Translational Research

Candidate: Seokjoo Lee

Poster #: B15

CD11b+Gr-1+MDSC Prevent IL-6-Induced Treg Dysfunction Partially Through Secretion of IL-10

Seokjoo Lee, Francesca Kahale, Aytan Musayeva, Shudan Wang, Tomas Blanco, Thomas H. Dohlman, Sunil K. Chauhan, Yihe Chen, Reza Dana

Purpose: Regulatory FoxP3+ T cells (Tregs) are critical in maintaining corneal allograft tolerance. The highly vascularized inflammatory microenvironment in corneal transplantation adversely affects the suppressive function of Tregs. Emerging evidence indicates the functional crosstalk between CD11b+Gr-1+ myeloidderived suppressor cells (MDSCs) and Tregs, but little is known about the impact of CD11b+Gr-1+ MDSCs on the rescue of inflammation-induced Treg dysregulation. The purpose of this study is to determine the rescue effect of CD11b+Gr-1+ MDSCs on IL-6-mediated Treg dysregulation, and to further elucidate the underlying mechanism by which this rescue occurs.

Methods: BALB/c bone marrow cells were cultured in complete RPMI medium in the presence of IL-6 (20ng/ml) and GM-CSF (20ng/ml) for 4 days to differentiate MDSCs. CD11b+Gr-1+ MDSCs and CD11b+ cells were obtained using CD11b+Gr-1+ MDSC MACS kits. Tregs were isolated from BALB/c splenocytes using CD4+CD25+ MACS kits. Tregs (2.5x105) were co-cultured either with CD11b+Gr-1+ MDSCs or with CD11b+ cells at a ratio of 1:1 in the absence or presence of IL-6 (40ng/ml) for 48 hours. IL-10 knockdown (KD) CD11b+Gr-1+ MDSCs were generated by IL-10 siRNA (10nM) transfection for 6 hours prior to co-culture with Tregs. FoxP3 expression and IL-10 secretion of Tregs were analyzed by flow cytometry, immunoblotting, and real-time PCR.

Results: Flow cytometric quantification showed Tregs cultured with IL-6 had significantly reduced expression of FoxP3 (12.5±0.3% vs. baseline 15.97±0.6%, p=0.0318) and modestly decreased secretion of IL-10 (5.16±1.2% vs. baseline 10.7±1.9%, p=0.0495) as compared to culture without IL-6. Addition of CD11b+ cells to culture partially preserved FoxP3 expression (18.2±0.3%) and IL-10 production (32.63±12.3%) by Tregs in the presence of IL-6. In contrast, addition of CD11b+Gr-1+ MDSCs to culture led to significantly higher FoxP3 (32.93±1.9%, p=0.0035) and IL-10 (60.03±13.3%, p=0.0238) expression by Tregs than addition of CD11b+ cells. Furthermore, Tregs co-cultured with IL-10 KD CD11b+Gr-1+ MDSCs exhibited significantly reduced FoxP3+ Tregs (23±1.8%) and IL-10-secreting FoxP3+ Tregs (55.93±4.4%) as compared to those co-cultured with WT CD11b+Gr-1+ MDSCs (32.3±2.4% and 82.53±3.4%, respectively, p=0.0403 and p=0.0015, respectively).

Conclusions: Our data demonstrate that in vitro generated MDSCs are effective in preventing inflammatory cytokine-induced impairment of Treg function, which can be explored as a novel therapeutic approach in ocular autoimmune and alloimmune disorders, including high risk corneal transplantation.

Category: Basic and Translational Research

Candidate: Meenakshi Maurya

Poster #: B16

REV-ERBα Deficiency Accelerates Age-Related Synaptic Remodeling in Mouse Retina

Meenakshi Maurya*, Chi-Hsiu Liu*, Shuo Huang, Felix Yemanyi, Kiran Bora, Alexandra K. Blomfield, Jing Chen

Purpose: Synaptic remodeling occurs during aging in both humans and mice, leading to neuronal dysfunction. Age-related alterations in the outer retinal synapses have been investigated as a model system to study agedependent neuronal and synaptic modifications such as in AMD. The role of REV-ERBα has been reported in synaptic strengthening in mice model of Alzheimer’s disease. REV-ERBα, a nuclear receptor and transcription factor, regulates multiple biological processes including circadian rhythm, metabolism and neuronal function. The present study aimed to explore whether REV-ERBα regulates age-related retinal synaptic alterations using REV-ERBα knockout (KO) mice as a model.

Methods: 5- and 11- month-old wild-type (WT) and systemic REV-ERBα KO mice were used. Retinal cross sections were immunostained with rod bipolar cell marker Protein kinase C (PKC-α) and pre-synaptic protein synaptophysin antibody to assess synaptic alterations. The number and length of ectopic rod bipolar cell dendrite sprouts were quantified. REV-ERBα localization was analyzed with REV-ERBα antibody immunostaining in WT retinas and with β-Gal reporter staining in REV-ERBα KO retinas. Expression of REVERBα in retinal cells was assessed using an online single cell RNA sequencing (scRNA seq) database. RTqPCR was performed in WT and REV-ERBα KO retinas to evaluate expression of genes involved in cellular energy homeostasis and axonal growth.

Results: REV-ERBα was localized in both outer and inner nuclear layer with REV-ERBα immunostaining. scRNA seq analysis also indicated expression of REV-ERBα in both rod photoreceptors and bipolar cells. The present study showed an accelerated retraction of rod axons accompanied with increase in both the number and length of ectopic rod bipolar cell dendrite sprouts extending into the photoreceptor layer by synaptophysin and PKC-α co-localization in REV-ERBα KO retinas that were significantly increased compared to WT in 5and 11-months old mice. REV-ERBα KO retinas exhibited significantly decreased expression of multiple genes involved in cellular energy and metabolic regulation.

Conclusions: Our findings suggest that genetic loss of REV-ERBα accelerates age-related synaptic remodeling in the outer retina, possibly through modulating expression of genes controlling cellular energy and metabolism to mediate axonal retraction and dendrite growth.

Category: Basic and Translational Research

Candidate: Amirreza Naderi

Poster #: B17

The Effect of Neurokinin-1 Antagonism in Treating the Allergic Red Eye

Amirreza Naderi1, 2; Shudan Wang; Lingjia Liu; Tomas Blanco ; Xia, Yutong; Chen, Yihe; Reza Dana

Purpose: Allergic conjunctivitis and various conditions can cause conjunctival vessel dilation, leading to ocular redness (OR). Using an animal model of allergic OR, we evaluated the therapeutic efficacy of topical blockade of substance P (SP), which preferentially binds to neurokinin-1receptor (NK1R) , in treating red eye.

Methods: Allergic OR was induced in guinea pigs with topical 1.5 mg/mL histamine. NK1R antagonist, L703,606, was applied 10 minutes before or after OR induction as pre-induction or post-induction treatment, respectively. Their eyes (N= 12/group) were imaged by a slit lamp every 30 seconds in the first 2 minutes, and at 6, 10, 20, 30, 40, 50, and 60 minutes. The redness was analyzed via pixel analysis to calculate the ocular redness index (ORI), a score between 0 to 100. After 60 minutes, tear fluid was collected to quantify SP levels by ELSIA (N= 6/group). Expression levels of IL-4, IL-5, IL-13 and TNF-α, cytokines associated with allergic response, were evaluated in conjunctiva via PCR (N= 6/group). Lastly, conjunctiva were harvested and stained with H&E to quantify the size of blood vessels and eosinophil and neutrophil infiltration. Toluidine blue staining was used to enumerate mast cells (N= 6/group).

Results: With histamine induction, the ORI peaked at 10 minutes (maximum ORI: 17.04±0.99). Topical treatment of L-703,606 significantly (P<0.05) reduced ORI (pre-induction: 7.38±0.71; post-induction: 10.91±0.59).Compared to naive guinea pigs, histamine increased the conjunctival blood vessel diameter nearly three times (naive: 10.15±1.41; histamine:30.66±4.12). Both pre- and post-induction treatment decreased blood vessels diameter (pre-induction: 16.84±2.21; post-induction: 23.69±2.36) but only pre-induction had significant (P<0.05) change. The tear concentration of SP was 71.64±19.16 pg/mL in the naive group, and 270.60±38.04 pg/mL in histamine-induced group. Pre-induction treatment significantly (P<0.05) decreased SP concentrations to 154.10±9.72 pg/mL and post-induction treatment lowered SP levels to 191.10±13.52 pg/mL. The numbers of eosinophils and neutrophils were 8.50±2.69 and 4.67±2.91 respectively in the conjunctiva of the naive group. Histamine increased the eosinophil count to 46.00±2.57 and neutrophils to 31.47±2.33. Postinduction treatment reduced eosinophils to 35.50±1.66 and neutrophils to 21.87±1.97, while pre-induction treatment significantly (P<0.01) decreased eosinophils to 26.00±2.92 and neutrophils to 14.53±2.31. Finally, application of histamine significantly increased the expression levels of IL-4, IL-5, IL-13, and TNF-alpha by 3-6 times compared to the normal control. Pre-induction treatment with L-703,606 effectively suppressed the upregulation of all cytokines to nearly normal levels, and post-induction treatment significantly reduced the expression levels of these cytokines by ~50%.

Conclusions: Topical administration of the NK1R antagonist effectively reduces allergy-induced ocular redness by decreasing SP release in the tear fluid, allergy-associated cytokines in the conjunctiva and leukocyte infiltration.

Category: Basic and Translational Research

Candidate: Mohit Parekh

Poster #: B18

Effect of ROCK Inhibitor on Cell Migration in Fuchs Endothelial Corneal

Dystrophy

Mohit Parekh, Annie Miall, Neha Deshpande, Ula V Jurkunas

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a progressive loss of corneal endothelial cells (CECs) that are post-mitotically arrested with limited proliferative capacity. Therefore, wound healing is mainly achieved through cell enlargement and migration. Inhibition of Rho-kinase, a key regulator of cytoskeletal reorganization has been shown to promote cellular migration. The purpose of this study was therefore to investigate the effect of a novel Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (K-115) in promoting CEC cell migration on Descemet’s membrane in FECD ex vivo.

Methods: Normal and FECD Descemet’s membrane-CEC tissues were obtained from cadaveric corneas or patients undergoing Descemet’s membrane endothelial keratoplasty (DMEK) surgeries. The tissues were preserved in Optisol-GS and after washing in PBS were stained with Hoechst 33342 for 30 sec and attached to the plastic plates by air drying for 3 min with the endothelial cell side facing up. The tissues were treated with 1uM of K-115 drug and monitored using live cell imaging microscope (Leica DMi8) for 5 hours. Controls were treated with Chen’s media without the drug. The images from three biological and three technical replicates were collected per group, and the velocity and displacement of the cells were analyzed using the TrackMate plugin in ImageJ. Mann-Whitney and one-way Anova tests were used for statistical analysis with p<0.05 being statistically significantly different.

Results: Mean velocity (pixels/hour SD) of CECs without the drug was 0.45 0.11 in normal and 0.64 0.21 in FECD tissues; and increased to 0.65 0.20 (p<0.05) and 0.82 0.39 (p<0.001) with K-115, respectively. Mean displacement (pixels/hour SD) of the cells from the normal and FECD tissues without the drug was 4.33 2.19 and 6.63 5.8, which increased to 13.49 10.32 (p<0.001) and 15.02 13.10 (p<0.001) respectively with K-115. Although the fold change in mean displacement between normal and FECD cells did not change significantly following the treatment with K-115, a significantly higher mean velocity (p<0.01) was observed in FECD compared to normal cells.

Conclusions: FECD cells migrate faster on the Descemet’s membrane following the treatment with K-115 compared to the normal cells, which further provides a promising therapeutic approach for the treatment of FECD using ripasudil after Descemetorhexis without endothelial keratoplasty (DWEK).

Category: Basic and Translational Research

Candidate: Suelen Scarpa de Mello

Poster #: B19

Blocking the Emergence of Resistance to Last Line Antibiotic, Daptomycin

Suelen Scarpa de Mello, Michael S. Gilmore

Purpose: Multidrug resistant Enterococcus faecium is a leading cause of postsurgical infections of the eye and other anatomical sites. Daptomycin (DAP) is a last-line drug for treating vancomycin-resistant E. faecium infections but is compromised by the emergence of resistant mutants during protracted therapy. We discovered that defects in the gene encoding a glycosyltransferase involved in biosynthesis of the cell wall component, lipoteichoic acid, render E. faecium hypersusceptible to DAP. Our work has focused on determining the role of this gene in cell wall biosynthesis and determining whether it is dominant to mutations that otherwise are capable of conferring DAP resistance to the organism.

Methods: We first generated a deletion of the glycosyltransferase lafB gene, to generate a lafB-defective E. faecium strain incapable of reversion by back mutation. Minimum Inhibitory Concentration (MIC) assays were determined for daptomycin and other antibiotics. We next introduced mutated genes known to confer increased daptomycin resistance, specifically mutant forms of liaR and cls synthetase. Finally, we attempted to evolve daptomycin resistant derivatives of the lafB-deleted strain (along with wild type controls).

Results: The deletion was successfully constructed and as predicted was found to be hypersusceptible to daptomycin and select other antibiotics. Although mutations in liaR and cls synthetase genes led to an increase daptomycin resistance in a wild type E. faecium cell, they did not confer resistance to the lafB deleted mutant, supporting the prospect that lafB mutation is dominant to known daptomycin resistance mechanisms. Whereas from the evolved strains– as expected – strains exhibiting over 100-fold resistance to daptomycin were readily obtained when beginning with wild type E. faecium controls, little change in resistance was observed for the otherwise identical lafB-deleted strain.

Conclusions: These results support the notion that daptomycin resistant mutations will not emerge in an E. faecium strain in which the LafB glycosyltransferase is inhibited. We are now launching a small molecule screen for LafB inhibitors to identify compounds that sensitize E. faecium to daptomycin and prevent emergence of resistance.

Category: Basic and Translational Research

Candidate: Jonathan Soucy

Poster #: B20

Chemokine-directed Migration Improves the Structural Integration of Human Stem Cell-derived Retinal Neurons

Jonathan

Soucy, Petr Baranov

Purpose: Cell transplantation has been proposed to restore retinal ganglion cell (RGC) populations lost in glaucoma and other optic neuropathies. While donor RGCs can survive in host retinas following transplantation, cell survival does not equate to vision restoration, and poor integration remains a significant challenge for successful RGC replacement. The inner limiting membrane (ILM) has recently been identified as a major barrier to donor cell migration into the ganglion cell layer (GCL) following intravitreal delivery. Therefore, to overcome this limitation, we delivered donor RGC subretinally, then directed their migration towards the GCL by engineering chemokine gradients across the retina.

Methods: To identify our lead candidate for RGC recruitment, we studied the migration of human RGC in response to different chemokine gradients in vitro. For this and other experiments, RGCs were differentiated from pluripotent stem cells by using retinal organoid cultures. After identifying SDF1 as the most potent chemokine for RGC recruitment, we transplanted human RGCs subretinally and delivered recombinant SDF1 protein intravitreally to establish a chemokine gradient across the retina. To investigate the mechanisms of chemoattraction and the modes of donor RGC migration, we applied small-molecule inhibitors to donor RGCs prior to transplantation for 1) the CXCR4 receptor – AMD3100, 2) somal translocation (ST) – CK666, and 3) multipolar migration (MP) – roscovitine. Three days post-transplantation, retinas were stained for donor and host neurons and evaluated by tracking the position of each donor RGC in 3D reconstructions of retinal flat mounts.

Results: Treatment with SDF1 increased donor RGC integration into the GCL from 16 ± 11% to 44 ± 11%. We confirmed the direct effect of SDF1 on donor cells by inhibiting in response to SDF1 by blocking SDF1-CXCR4 binding in donor cells with AMD3100 (17 ± 5.9%). Further, inhibiting MP migration significantly limits the percentage of donor RGCs that migrate to the GCL in response to SDF1 (No inhibition: 45 ± 15%; MP inhibition: 20 ± 3.7%), whereas inhibiting ST does not affect donor RGC migration (ST inhibition: 47 ± 12%) –indicating donor RGCs migrate via MP in vivo. The inhibition of migration did not affect survival, and each migration pattern was confirmed with an in vitro time-lapse study.

Conclusions: Altogether, our studies confirm that donor cell behavior is controllable by modulating the tissue microenvironment and that SDF1 gradients across the host retina improve the structural integration of donor RGCs. Furthermore, our results show that MP migration is the primary mode of migration for surviving donor human RGCs in the retina and that SDF1 acts through the CXCR4 receptor – suggesting the possibility that RGCs selected for these features before transplantation will improve donor cell integration.

Category: Basic and Translational Research

Candidate: Feng Tian

Poster #: B21

Core Transcription Programs Controlling Injury-Induced Neurodegeneration of Retinal Ganglion Cells

Feng Tian, Yuyan Cheng, Songlin Zhou, Qianbin Wang, Aboozar Monavarfeshani, Kun Gao, Weiqian Jiang, Riki Kawaguchi, Qing Wang, Mingjun Tang, Ryan Donahue, Huyan Meng, Yu Zhang, Anne Jacobi, Wenjun Yan, Jiani Yin, Xinyi Cai, Zhiyun Yang, Shane Hegarty, Joa

Purpose: Regulatory programs governing neuronal death and regenerative failure in neurodegenerative diseases remain poorly understood. In adult mice, optic nerve crush (ONC) injury by severing retinal ganglion cell (RGCs) axons, results in massive RGC death and regenerative failure. We aim to dissect the transcirptional programs that regulate RGC survival and axon regeneration in nonbiased approaches.

Methods: In this study, we conducted multiple orthogonal, but complementary, functional genomic analyses, starting with a comprehensive, in vivo CRISPR screen of all known mammalian TFs. In parallel, we performed a RNA-seq coupled to an ATAC-seq analysis to define transcriptional networks and their drivers in intact and injured RGCs. Remarkably these different approaches converged on an overlapping set of TFs as critical regulators of neuronal injury responses, highlighting a core transcription program that mediates injury-induced neuronal degenerative outcomes.

Results: Through two orthogonal genome-wide strategies, in vivo CRISPR screening and combined assays of chromatin accessibility (ATAC-seq) and gene expression (RNA-seq), we identified several TFs that play key roles in injury-induced neuronal loss. We then focused on a set of four TFs – ATF3, ATF4, C/EBPγ and CHOP identified through these independent approaches. We showed that their activation and complementary action play key roles in the early engagement of different degeneration-regulatory pathways in injured RGCs. Manipulation of these TFs also led to significant neuroprotection in a newly modified glaucoma model.

Conclusions: Our results reveal core transcription programs that transform an initial axonal insult into a degenerative process and suggest novel strategies for treating neurodegenerative diseases.

Category: Basic and Translational Research

Candidate: Amrita Saha Vadher

Poster #: B22

Potential Role of High Mobility Group Box 1 in Formation of Corneal Subepithelial Infiltrates in Adenovirus Keratitis

Amrita Saha, Mohammad Mirazul Islam, Ashrafali Mohamed Ismail, Xiaohong Zhou, James Chodosh, Jaya Rajaiya

Purpose: Ocular surface infection by human adenovirus species D (HAdV-D) causes epidemic keratoconjunctivitis (EKC). The most significant long-term complication of EKC is subepithelial infiltrate (SEI) formation, occurring in about one-third of cases. However, the mechanism of SEI formation after adenoviral infection of the cornea remains uncertain. High-mobility group box protein 1 (HMGB1) is an important late inflammatory mediator; extracellular HMGB1 variably regulates inflammatory responses and leukocyte infiltration. In this study, we aimed to define a potential role for HMGB1 in progression to SEI formation.

Methods: Primary human corneal epithelial (PHCE) cells, hTERT-immortalized human corneal epithelial (THE) cells, and primary human corneal fibroblasts (HCFs) were infected with HAdV-D37 or HAdV-C5 at MOI = 5, and cell supernatants were collected through 48 hours post infection. Mass spectrometry (LC-MS/MS) analysis was performed on cell lysates. Immunoblotting was performed on infected cell supernatants, and on cytoplasmic and nuclear cellular fractions, using acetylated HMGB1 antibody. ELISA for secreted HMGB1 was conducted on cell-free supernatants, and HMGB1 gene expression was studied using realtime qPCR. Confocal microscopy was performed to visualize HMGB1 translocation. Cytokine expression by HCFs treated with recombinant HMGB1 was analyzed using human cytokine protein arrays.

Results: HAdV-D37 infection resulted in HMGB1 translocation from the nucleus to the cytoplasm and then to the extracellular space in THE cells, but not in HCF. HAdV-C5, a virus not associated with EKC, did not induce secretion of HMGB1 from any of the cell types tested. Finally, treatment of HCF with recombinant HMGB1 triggered expression of pro-inflammatory mediators.

Conclusions: Our data suggest that HMGB1 is specifically released by adenovirus infected corneal epithelial cells andalso provides insights into possible mechanism of corneal SEI formation in EKC. HMGB1 expressed by adenovirus infected corneal epithelial cells could induce underlying stromal cells to express proinflammatory mediators, leading indirectly to the development of SEI. HMGB1 may be a viable therapeutic target for preventing the corneal stromal complications of EKC.

Category: Basic and Translational Research

Candidate: Asmaa Zidan

Poster #: B23

Calcitonin Gene-related Peptide Promotes the Healing of Corneal Epithelial Stromal Injury

Asmaa A. Zidan, Shuyan Zhu, Jia Yin

Purpose: Calcitonin gene-related peptide (CGRP) is an anti-inflammatory neuropeptide abundantly expressed by corneal nerves. Its effects on the corneal wound healing, inflammation, and opacity have not been studied before. In this study, we aim to examine the effects of CGRP on corneal epithelial stromal injury in mice.

Methods: Corneal epithelial stromal injury was induced in C57BL/6 mice by removing the epithelium and anterior portion of the stroma using a hand-held Alger brush II. Following injury, mice were topically treated either with CGRP or PBS control three time daily for 14 days. Corneal epithelial healing, stromal opacity, and thickness were examined using slit lamp photography and OCT. Corneal endothelial cells integrity, alphasmooth muscle actin (αSMA) and TGFβ-1 expression were assessed by ZO-1, αSMA, TGFβ-1 immunostaining, respectively. Leukocyte infiltration was determined using flow cytometry.

Results: CGRP accelerated epithelial wound healing and led to significantly reduced corneal opacity after injury. CGRP decreased CD45+ cell infiltration into the cornea from 10.2±1.2% to 6.1±0.3% (P =0.03) at day 5. At day 14, CGRP reduced corneal edema compared to PBS treatment (central corneal thickness 159.7±29.2µm in PBS group vs 85.2±1µm in CGRP group, P=0.03) and preserved corneal endothelial cell integrity.

Conclusions: CGRP promotes healing of corneal epithelial stromal injury by accelerating epithelial wound closure and preventing corneal opacification and edema. Potential mechanisms include inhibiting TGFβ-1 signaling and the production of αSMA, reducing leukocyte infiltration, and preserving corneal endothelial cells.

Category: Clinical Research

Candidate: Inas Aboobakar

Poster #: C1

Genetic Risk Scores of Mitochondrial TXNRD2 and ME3 Variants are Associated with Specific Primary Open-angle Glaucoma Phenotypes

Inas F. Aboobakar, Tyler G. Kinzy, Baojian Fan, Louis R. Pasquale, Jessica N. Cooke-Bailey, Janey L. Wiggs, NEIGHBORHOOD CONSORTIUM

Purpose: Genetic variants in regions that include the mitochondrial genes TXNRD2 and ME3 are associated with primary open-angle glaucoma (POAG) in genome-wide association studies (GWAS). To assess their clinical impact, we investigated whether genetic risk scores (GRSs) of TXNRD2 and ME3 variants are associated with specific glaucoma phenotypes.

Methods: 2617 POAG cases and 2634 controls from the NEIGHBORHOOD consortium were studied. All POAG-associated single nucleotide polymorphisms (SNPs) in the TXNRD2 and ME3 loci were identified using GWAS data (p<0.05). Of these, 20 TXNRD2 and 24 ME3 SNPs were selected after adjusting for linkage disequilibrium. The correlation between SNP effect size and gene expression levels was investigated using the Gene-Tissue Expression (GTEx) database. GRSs were constructed for each individual using the unweighted sum of TXNRD2, ME3, and TXNRD2+ME3 combined risk alleles. Age and gender-adjusted odds ratios (ORs) for POAG diagnosis were calculated per decile for each GRS. Additionally, the clinical features of POAG cases in the top 1, 5, and 10% of each GRS were compared to the bottom 1, 5, and 10%, respectively.

Results: Increased SNP effect size strongly correlated with higher TXNRD2 and lower ME3 expression levels (r=0.72 and -0.97, respectively). Individuals in decile 10 of TXNRD2+ME3 GRS had the highest odds of POAG diagnosis (OR=1.79, 95% CI 1.39-2.30, p<0.001). Individuals in the top 1% of TXNRD2 GRS had higher mean maximal IOP compared to the bottom 1% (19.9 mmHg vs 15.6 mmHg, p=0.01). Individuals in the top 1% of ME3 and TXNRD2+ME3 GRS had higher prevalence of paracentral field loss compared to the bottom 1% (72.7-89% vs 14.3-33%, p=0.02 and 0.01, respectively).

Conclusions: Individuals with higher GRSs have higher odds of POAG diagnosis, with a greater effect seen for TXNRD2+ME3 combined compared to either gene alone. Moreover, POAG patients with higher GRSs have higher IOP and greater prevalence of paracentral field loss. These data suggest that TXNRD2 and ME3 functionally contribute to POAG development and disease severity, potentially through a mechanism involving NADPH levels and mitochondrial dysfunction.

Category: Clinical Research

Candidate: Atitaya Apivatthakakul

Poster #: C2

Prevalence of Ocular Inflammatory Conditions in Patients with Small Fiber Neuropathy in a Large Healthcare System Database

Atitaya Apivatthakakul, Daniel A. Brill, Lucia Sobrin

Purpose: The purpose of this study is to determine the prevalence of ocular inflammatory diseases in SFN patients.

Methods: The Mass General Brigham health care system database was queried for cases defined as SFN patients with an eye exam and a diagnosis code for an ocular inflammatory disease (episcleritis, scleritis, and neurotrophic keratopathy). The diagnoses were verified by chart review. Patients with co-existing autoimmune/infectious diseases that are also associated with ocular inflammation were excluded. Prevalence of ocular inflammation was calculated, and clinical characteristics were determined.Controls were identified from the Mass General Brigham database with the criteria of patients without the diagnosis of SFN who had at least one eye examination by an eye doctor and no diagnosis for ocular inflammatory conditions.

Results: Results:Of 5,609 SFN patients identified, 857 had an eye examination. Of these, 42 patients had an ocular inflammatory disease, yielding a prevalence of 4.9%. 31 (83.8%) had a history of eye pain. 33 (78.6%) patients were female, and 37 (88.1%) had bilateral ocular disease. Mean age of ocular diagnosis was 55.7 years (range 22-81 years). Eight (19.0%) patients had episcleritis, ten (23.8%) had scleritis, and eight had neurotrophic keratopathy. Seventeen patients had at least two ocular inflammatory conditions. We identified 1,310,601 controls. 3,195 had scleritis, 2,884 had episcleritis and 210 had neurotrophic keratopathy.Ocular inflammatory conditions were significantly more common in SFN patients compared to non-SFN patients. P values all <.0001 for scleritis, episcleritis and neurotrophic keratopathy.

Conclusions: Conclusions:Ocular inflammatory conditions, including episcleritis/scleritis, are present at a higher rate in SFN patients than in the general population

Category: Clinical Research

Candidate: Isaac Bleicher

Poster #: C3

Outcomes of Far Posterior Open Globe Injuries: The Case for Zone 4

Isaac Bleicher, Laurel Tainsh, Eric D. Gaier, Grayson Armstrong

Purpose: Open globe injuries (OGIs) are categorized by zone of injury, with Zone 3 (Z3) defined as ≥5mm posterior to the corneal limbus. Heterogeneity in outcomes of Z3 OGIs precludes precise prognostication and surgical management decisions. We hypothesized that Z3 OGIs with far posterior wounds would have worse visual and anatomic outcomes.

Methods: We performed a retrospective review of Z3 OGIs with at least 30 days of follow-up treated at a tertiary care center over a 10-year period. Far posterior OGIs (pZ3) were defined as those with wounds extending ≥10mm posterior from the corneal limbus. Secondary surgeries, visual outcomes, and anatomic outcomes were compared between pZ3 and anterior Z3 (aZ3) eyes at presentation, the 3 months and the latest available timepoints after injury. Chi-square and Mann-Whitney U tests were used to compare pZ3 and aZ3 outcomes.

Results: Of 300 eyes, 258 met inclusion criteria. Of these, 62% were classified as pZ3. At final follow-up, pZ3 eyes were more likely to be no light perception (NLP) (pZ3: 49%, aZ3: 33%), hand motion (HM) or worse (pZ3: 75%, aZ3: 57%) or <20/400 Snellen acuity (pZ3: 84%, aZ3: 65%) (p ≤0.02). The effect of zonal group on HM and <20/400 acuity persisted even when excluding patients presenting NLP (p ≤0.05). These relationships were also seen at 3 month follow-up for NLP, HM, and 20/400 visual acuities (all p < 0.01) Compared to presentation, pZ3 eyes were less likely to have acuity improve (pZ3: 33%, aZ3: 44%) and more likely to have worsened acuity (pZ3: 19%, aZ3: 11%) (p<0.02). pZ3 eyes were more likely to become phthisical or be eviscerated/enucleated (pZ3: 56%, aZ3 40%, p<0.02). pZ3 and aZ3 eyes underwent means (±SD) of 1.5±1.6 and 1.4±1.5 secondary procedures, respectively (p>0.76).

Conclusions: Eyes with OGIs extending ≥10 mm posterior to the corneal limbus have poorer visual and anatomic outcomes compared to those limited to the more anterior Z3. While the potential for recovery in posterior OGIs necessitates careful assessment and emergent repair in all cases, further zonal categorization within zone 3 injuries may help improve prognostic precision and refine surgical approaches.

Category: Clinical Research

Candidate: Mohammad Eslami

Poster #: C4

Evaluation of Deep Learning Visual Field Prediction Models for Clinical Relevance

Mohammad Eslami, Miao Zhang, Julia Kim, Dolly Chang, Yangjiani Li, Saber Kazeminasab, Mojtaba Fazli, Vishal sharma, Michael Boland, Nazlee Zebardast, Mengyu Wang, Tobias Elze

Purpose: Deep learning methods have recently been used for predicting future visual fields (VFs) using baseline or longitudinal VFs. In clinical practice, glaucomatous VF loss progression is a comparatively rare event. It is of particular clinical relevance if these prediction models can accurately identify patients with disease progression to aid clinicians in avoiding vision loss. Here, we evaluate two previously described models for potential biases in over- or underestimating VF changes over time.

Methods: We consider two recent studies, namely Wen et al. (MWen) to predict VF sensitivity and Park et al. (MPark) to predict total deviations. All reliable (false negatives/positives≤30%, fixation losses≤30%) Humphrey 24-2 VFs from Mass. Eye and Ear glaucoma services from 1999 to 2020 were included. We re-implemented the methods and made them available to other investigators. As in the original studies, pointwise mean absolute error (PMAE) was used to measure model prediction accuracy. A 5-fold cross-validation scheme was utilized, and the models are additionally compared against a no-change model, i.e. the baseline VF for MWen and the last-observed VF for MPark were used as the predicted VF. The evaluation dataset included 54,373 samples from 7,472 people for MWen and 24,430 samples from 1,809 people for MPark, depending on the method's needs.

Results: The PMAE results (%95 CI, MWen:2.21-2.24, MPark:2.56-2.61) are close to the original papers. Also, the scatterplots w.r.t. mean of sensitivity for MWen and mean of deviation for MPark are considered to show predicted vs truth values. This shows that both approaches produce satisfactory outcomes well close to y=x (predicted = truth). But, for further investigation, more scatterplots were examined by showing the prediction's errors vs. actual changes. With this investigation, both methods exhibit a large error in projecting worsening cases and were not even close to the hypothetical unbiased model.

Conclusions: Our evaluation of the two VF prediction models confirms the low PMAEs reported in the original studies. However, both models underpredicted the worsening of VF loss over time. As detecting the progression of VF loss is a major motivation to obtain clinical VFs, we suggest explicitly considering this aspect in future model evaluations as well as the data characteristics.

Category: Clinical Research

Candidate: Henisk Falah

Poster #: C5

Comparative Outcomes of Phacoemulsification Combined with Hydrus or Kahook Dual Blade

Blake Oberfeld, BS; Nathan Eli Hall, MS; Henisk Falah David Solá-Del Valle, MD

Purpose: To report the relative efficacy of combining phacoemulsification with Schlemm canal microstent (Phaco/Hydrus) or dual blade trabecular excision (Phaco/KDB).

Methods: Phaco/Hydrus or Phaco/KDB procedures from January 2016 to July 2021 were included. Data were collected as available for 36 months postoperatively. Primary outcomes were IOP and medication burden evaluated by Generalized Estimating Equations (GEE). Two Kaplan-Meier Estimates (KM) assessed survival without additional glaucoma procedure or medication while maintaining: (1) IOP ≤21 mmHg and ≥20% IOP reduction or (2) IOP ≤ preoperatively designated goal.

Results: Mean preoperative IOP was 17.70 mmHg ± 4.91 (SD) on 0.28 ± 0.86 medications in the Phaco/Hydrus cohort (N=69) and 15.92 ± 4.34 mmHg on 0.19 ± 0.70 medications in the Phaco/KDB cohort (N=62). At 12 months, mean IOP was reduced to 14.98 ± 2.77 mmHg on 0.12 ± 0.60 medications after Phaco/Hydrus and 13.52 ± 4.13 mmHg on 0.04 ± 0.19 medications after Phaco/KDB. GEE models of IOP (p<0.001) and medication burden (p=0.05) had significant patterns of reduction across all timepoints. There was no difference in the reduction in IOP (p=0.94) or medication burden (p=0.95) when evaluated between procedures. Phaco/Hydrus and Phaco/KDB were not different in survival by KM1 (p=0.72) or KM2 (p=0.11).

Conclusions: Both Phaco/Hydrus and Phaco/KDB resulted in significantly reduced IOP and medication burden for more than 12 months. Phaco/Hydrus and Phaco/KDB confer similar

Category: Clinical Research

Candidate: Cris Jacoba

Poster #: C6

Automated Machine Learning Models for Diabetic Retinopathy

Screening Using Handheld Fundus Cameras in a Low-resource Community Screening Program

Cris Martin P. Jacoba, Duy Doan, Lizzie A. Aquino, Joseph Paolo Y. Silva, Claude M. Salva, Recivall P. Salongccay, Glenn P. Alog, Kexin Zhang, Kaye Locaylocay, Aileen V. Saunar, Jennifer K. Sun, Tunde Peto, Lloyd P. Aiello, Paolo S. Silva

Purpose: Evaluating diabetic retinopathy (DR) severity from retinal images is one of the most common uses of artificial intelligence (AI) in medicine. However, commercially available algorithms are expensive, not universally generalizable, and are limited to specific retinal cameras. Automated machine learning (AutoML) allows the development of code-free models that decrease the barrier to using AI for clinical needs. This is useful in low resource settings where the financial incentive of commercial ventures is low, but the clinical need is high. This study used Google AutoML Vision to train models based on locally sourced data from community programs in the Philippines to detect referable DR.

Methods: AutoML Vision (Google Cloud) models were generated based on previously acquired 5-field (macula centered, disc centered, superior, inferior, temporal macula) retinal images (17,237 images, 3447 eyes) from the Philippine DR screening program (DRSP). Each individual image was labelled based on the International DR and diabetic macular edema (DME) classification and performed by four certified graders from a centralized reading center (RC), with secondary adjudication done by a senior retina specialist. Images for the initial model were split 8-1-1 for training, optimization and testing to detect referable DR [(refDR), defined as moderate nonproliferative DR or worse or any level of DME]. The AutoML platform provided model evaluation metrics by showing the area under the precision-recall curve (AUPRC), and the confusion matrix. Internal validation of the autoML model was performed using a holdout image set that used a 2-field imaging protocol (disc and macula-centered, 225 eyes) using the same device in the same population, evaluated by the same RC. External validation was performed using a high-quality published image set (macula-centered, 207 eyes) using tabletop retinal cameras, which showed a different patient population. This dataset is publicly available from the Eye Picture Archive Communication System (EyePACS) Kaggle, USA. Sensitivity, specificity, positive predictive value, negative predictive (SN, SP, PPV, NPV), accuracy and F1 score for refDR were calculated.

Results: In the training set, refDR was present in 17.3%, non-refDR 82.7%. The model’s AUPRC was 0.995 with a precision and recall of 97% using a score threshold of 0.5. In the internal validation set, refDR was present in 39.1%, non-refDR 60.9%. The SN, SP, PPV, NPV, accuracy and F1 scores were 0.96 (95% CI: 0.88-0.99), 0.98 (95% CI: 0.94-0.99), 0.96 (95% CI: 0.88-0.99), 0.98 (95% CI: 0.94-0.99), 0.97 and 0.96, respectively. Some false positive eyes had other pathologies that warranted referral. In the external validation set, refDR was present in 42.5%, non-refDR 57.5%. The model performed well with the high-quality external validation set, with SN, SP, PPV, NPV, accuracy and F1 scores at 1.0.

Conclusions: This study demonstrates the accuracy and feasibility of low-cost autoML models for identifying refDR developed for a DRSP using handheld retinal imaging in a low-resource setting community program. The performance approaches and potentially outperforms published diagnostic accuracy metrics of commercial models used for DRSP. These data emphasize the use of local data in the development and optimization of machine learning models to potentially improve performance in the populations that they will be used. Furthermore, the use of autoML may increase access to machine learning models adapted for specific programs that are guided by clinicians to rapidly address disparities in patient care.

Category: Clinical Research

Candidate: Ashley Li

Poster #: C7

High Positive Predictive Value of Fluorescein Angiography

Contiguous, Perinerve Retinal Vascular Leakage Pattern for Birdshot Chorioretinopathy

Ashley Li, Atitaya Apivatthakakul, Lucia Sobrin

Purpose: Birdshot chorioretinopathy (BSCR) is a posterior uveitis with ovoid yellow-white choroidal lesions. If untreated, patients’ vision declines, so early diagnosis is critical. While indocyanine green angiography (ICGA) is used to identify subtle lesions, ICGA is not always available. Fluorescein angiography (FA) is more common and a contiguous, perinerve retinal vascular leakage pattern has been described in BSCR patients. The purpose of this study is to determine the sensitivity and positive predictive value (PPV) of a contiguous, perinerve retinal vascular leakage FA pattern for BSCR diagnosis.

Methods: Mass General Brigham patients with a FA Common Procedural Terminology code and mention of BSCR in the free text of notes were identified. BSCR diagnosis required HLA-A29 positivity and typical lesions on fundus photos and/or ICGA hypofluorescent spots. Chart review was performed to confirm BSCR vs. other uveitis/retinal vasculitis diagnosis and symptom duration. The first FA was analyzed for a contiguous, perinerve pattern of leakage by two readers. A perinerve leakage pattern was defined as leakage primarily around the optic nerve and along the larger arcade vessels (Fig 1). We compared the rates of this FA pattern in BSCR vs. other posterior uveitis/retinal vasculitis patients using the chi-square test and determined the sensitivity and PPV of this FA pattern for BSCR diagnosis. All statistical analyses were performed in STATA.

Results: 54 BSCR patients and 42 patients with other posterior uveitis and/or retinal vasculitis were identified (Table 1). A perinerve FA pattern was more common in BSCR patients vs. patients without BSCR (53.7% vs. 4.76%, p=3.63 X 10-7). The sensitivity and specificity of the FA pattern were 53.7% and 95.2%, respectively. The PPV was 93.5%. BSCR patients with an FA perinerve pattern had a shorter time from symptom onset to FA vs. BSCR patients without the pattern (217 vs. 1510 days, p=.0015, t-test).

Conclusions: An FA contiguous, perinerve retinal vascular leakage pattern is a useful tool to identify potential BSCR for further imaging and serological testing. This pattern is more common when the interval between symptom onset and FA is shorter.

Category: Clinical Research

Candidate: Yangjiani Li

Poster #: C8

Age-controlled Correlation Between Oral Glucose Tolerance Test (OGTT) and Thicknesses of Ten Retinal Layers in Non-diabetes and Prediabetes Participants from macular optical coherence tomography (OCT) volume scans

Yangjiani Li, Franziska G. Rauscher, Mengyu Wang, Mohammad Eslami, Jennifer K. Sun, Konstantina Sampani, Kerstin Wirkner, Markus Loeffler, Joachim Thiery, Anke Tönjes, Thomas Ebert, Christoph Engel, Tobias Elze

Purpose: People with prediabetes are at higher risk of developing diabetes. We investigate the age-controlled relationship between glucose parameters and ten retinal layer thicknesses in optical coherence tomography (OCT) macular volume scans of non-diabetic and prediabetic participants with/without glaucoma in a large study. The fasting, 30-min, and 2-h plasma glucose (FPG, 30-min PG, and 2-h PG) during an oral glucose tolerance test (OGTT) were employed.

Methods: All eyes of non-diabetic and prediabetic participants with available OGTT and OCT scans from the population-based, sex- and age-stratified LIFE-Adult study (age range: 20-80) were included. Macular volume scans (97 horizontal B-scans with 512 A-scans each) from Spectralis OCT were automatically segmented into ten layers (Figure 1) after exclusion of unreliable B-scans (quality < 20 dB). Pointwise partial Pearson correlation during OGTT adjusted for age was calculated. The analysis was repeated excluding participants with self-reported glaucoma diagnosis or medication.

Results: Figure 2 visualizes the age-controlled correlation of glucose level and A-scans in each layer from 5532 eyes of 2787 participants, including 128 participants with glaucoma. For FPG, a predominantly negative correlation (red) with layer thicknesses of RNFL, ONL, and MZ and a positive correlation (blue) with EZ and OS were observed (Figure 2A). Similar correlation patterns were found for 30-min PG with layer thicknesses of RNFL, MZ, and EZ, with a slightly increasing negative correlation with layer thicknesses of GCL and IPL (Figure 2B). For 2-h PG, there is an apparent negative correlation with layer thicknesses of GCL, IPL, and MZ (Figure 2C). The correlation patterns remained unchanged when participants with glaucoma were excluded.

Conclusions: There were specific correlation patterns between retinal layer thicknesses and measures during OGTT. With the prolongation of the time post-glucose intake, higher glucose level was more clearly related to thinner GCL and IPL and firmly associated with thinner MZ, while the correlation with RNFL, ONL, EZ, and OS became less pronounced. Macular layer thicknesses could be an early indication of the retinal structural change prior to diabetes and in this cohort did not seem to be impacted by comorbidity of glaucoma.

Category: Clinical Research

Candidate: Jonathan Lin

Poster #: C9

Systemic Complement Activation in Non-Exudative Age-Related Macular Degeneration

Jonathan B. Lin, Stylianos Serghiou, Joan W. Miller, and Demetrios G. Vavvas

Purpose: Although complement inhibition has emerged as a possible therapeutic strategy for certain forms of advanced age-related macular degeneration (AMD), it is unknown what portions of the complement pathway are dysregulated in AMD or when this dysregulation occurs relative to AMD stage; in this meta-analysis, we tested the hypothesis that complement dysregulation may occur during early/intermediate but not advanced non-exudative AMD.

Methods: Using PubMed, Google Scholar, and Embase, we identified articles from database inception to October 11, 2020 that reported systemic complement activation profiles in patients with early/intermediate nonexudative AMD or geographic atrophy (GA) and non-AMD controls. We meta-analyzed the data by generating multi-level random-effects models using restricted maximum likelihood estimation. Standard errors were adjusted using the Knapp-Hartung correction. Risk of bias was assessed using a modified Newcastle-Ottawa score.

Results: The seven meta-analyzed studies included 710 independent participants (mean per study: 101; standard deviation: 35) with 23 effect sizes. Compared with non-AMD controls, patients with early/intermediate non-exudative AMD (N=246) had significantly higher systemic complement activation as quantified by the levels of complement proteins generated by common final pathway activation (standardized mean difference [SMD]=0.52 [95% confidence interval (CI): 0.19 to 0.86]) and significantly lower systemic complement inhibition (SMD=-0.57 [95% CI: -1.07 to -0.07]). In contrast, there were no statistically significant differences in systemic levels of complement common final pathway activation products (SMD=0.34 [95% CI: -0.05 to 0.74]) or complement inhibition (SMD=-0.02 [95% CI: -0.29 to 0.24]) in patients with GA (N=178) versus non-AMD controls.

Conclusions: We provide evidence that systemic complement over-activation is a feature of early/intermediate non-exudative AMD; no such evidence was identified in patients with advanced nonexudative AMD, suggesting that complement inhibition may have improved efficacy for patients with early disease rather than for patients who have already developed GA as has been studied in multiple clinical trials to date.

Category: Clinical Research

Candidate: Yifan Lu

Poster #: C10

Quantitative Wide-field Swept-source Optical Coherence Tomography

Angiography and Visual Outcomes in RAO

Yifan Lu, Ying Cui, Ying Zhu, Edward S. Lu, Rebecca Zeng, Rohan Bajaj, Raviv Katz, Rongrong Le, Jay C. Wang, John B. Miller

Purpose: Retinal artery occlusion (RAO) is an ophthalmic emergency that can lead to poor visual outcome and is associated with an increased risk of cerebral stroke and cardiovascular events. Fluorescein angiography (FA) is the traditional diagnostic tool for RAO; however, wide-field swept-source optical coherence tomography angiography (WF SS-OCTA), as a nascent imaging technology, is able to provide quick and non-invasive angiographic information with a wide field of view. In this study, we looked for associations between OCT-A vascular metrics and visual acuity in patients with prior diagnosis of RAO.

Methods: Patients with diagnoses of central retinal artery occlusion (CRAO) or branched retinal artery occlusion (BRAO) were included. A 6mm x 6mm Angio and a 15mm x 15mm AngioPlex Montage OCT-A image were obtained for both eyes in each patient using the Zeiss Plex Elite 9000 WF SS-OCTA device. Each 6mm x 6mm image was divided into nine Early Treatment Diabetic Retinopathy Study (ETDRS) subfields. The average measurement of the central foveal subfield, inner ring, and outer ring was calculated for each parameter. Non-perfusion area (NPA) was manually measured using 15mm x 15mm Montage images. A linear regression model was utilized to identify a correlation between the imaging metrics and visual acuity. A P-value less than 0.05 was considered to be statistically significant.

Results: Twenty-five subjects were included in the study. For RAO eyes, there was a statistically significant negative correlation between vision and retinal thickness as well as superficial capillary plexus vessel density (SCP VD). A negative correlation was found between vision and deep capillary plexus vessel density (DCP VD) without statistical significance. There was a positive correlation between vision and choroidal thickness as well as choroidal volume without statistical significance. No statistically significant correlation was found between vision and the above metrics in contralateral eyes. For NPA measurements, no significant correlation was found between vision and NPA.

Conclusions: This is the first study to investigate the utility of WF SS-OCTA in RAO and to demonstrate correlations between various retinal vascular imaging metrics and visual outcomes. Further investigations should explore the associations between these imaging findings and cardiovascular risk as RAO patients are at elevated risk for symptomatic stroke. The results of this study provide a basis to understand the structural changes involved in visual outcomes in RAO. Furthermore, they may help guide management of RAO and prevention of cerebral stroke and cardiovascular accidents in patients with RAO.

Category: Clinical Research

Candidate: Luis Martinez-Velazquez

Poster #: C11

Predicting Visual Outcomes in Central Retinal Vein Occlusion (CRVO) with Vascular Metrics and Non-Perfusion Area (NPA) Measured by Wide-Field Swept-Source OCT-Angiography (WF SS-OCTA)

Luis A. Martinez-Velazquez1,2, Grace Baldwin1,2, Itika Garg1,2, Ying Cui1,3, Raviv Katz1, Rebecca Zeng1,2, Margaret Duich1, Diane N. Sayah1, Jade Moon1,2, Fillipos Vingopolous1,2, Hannah E. Wescott1,2, Thomas Koch1,2, Nimesh A. Patel2, David M. Wu2, John

Purpose: To quantify vascular metrics following central retinal vein occlusion (CRVO) using wide-field swept source OCTA (WF-SS-OCTA) and evaluate for its associations with visual acuity (VA). To determine the possible clinical application of WF-SS-OCTA in monitoring patients that suffered from a CRVO for the development of worsening ischemia.

Methods: A cross-sectional observational study was conducted from March 2019 to December 2021. WF SSOCTA (PLEX® Elite 9000, Carl Zeiss Meditec Inc.) 3x3, 6x6, and 12x12-mm fovea-centered angiograms were obtained. The ARI Network was used to quantify vessel density (VD), vessel skeletonized density (VSD), and foveal avascular zone (FAZ) area, circularity, and perimeter. Non-perfusion area (NPA) was calculated using ImageJ. Mixed effects multiple linear regression analyses and receiver operating characteristics (ROC) curve analyses were performed.

Results: The cohort included 156 eyes, with 76 CRVO eyes, including 12 ischemic CRVO, and 80 fellow eyes. All the OCTA vascular metrics, except FAZ area, were found to have significant associations with VA on regression analyses controlling for age, sex, hypertension, hyperlipidemia, and diabetes mellitus. Using stepwise bidirectional elimination, a regression model accounting for age (β=0.01, p=0.008), BMI (β=-0.01, p=0.030), FAZ perimeter (β=0.26, p=0.001), FAZ area (β=-1.20, p=0.003), and NPA (β=0.01, p<0.001) on 12x12-mm images was best for predicting VA<20/50. On ROC analysis, a multivariate regression model using NPA to predict VA>20/50 performed best (area under the curve =0.91).

Conclusions: SS-OCTA can be used to robustly detect microvascular changes in eyes following CRVO, and these changes are strongly associated with the resulting visual acuity. ROC analysis found that out of the OCT-A metrics, NPA was more strongly associated with predicting visual acuity worse than 20/50, supporting that NPA is a strong correlate for ischemia. Our data also supports that vascular metrics from 12x12 angiograms can be used to statistically separate ischemic versus non-ischemic RVOs, supporting the role for SS-OCTA in monitoring and prognosticating patients following CRVO.

Category: Clinical Research

Candidate: Kristen Pitts

Poster #: C12

APOE and Galectin-3, Markers of Activated Microglia, are Elevated in the Aqueous Humor of Glaucoma Patients

Kristen Pitts, Cameron Neeson, Nathan Hall, Henisk Falah, Milica Margeta, David Solá-Del Valle

Purpose: Microglial activation has emerged as a critical event contributing to retinal ganglion cell loss in glaucoma. Apolipoprotein E (APOE), the major apolipoprotein in the brain, and Galectin-3 (Gal-3), a secreted carbohydrate-binding lectin, have been identified as molecules critical for microglial cytotoxicity in mouse models of glaucoma. In this study, we aim to examine the role of APOE and Gal-3 in human disease by analyzing their concentrations in aqueous humor (AH) collected from glaucoma patients and healthy controls.

Methods: AH was collected from 71 patients at the start of surgery, including 57 glaucoma patients undergoing various ophthalmic procedures and 14 control patients undergoing routine cataract surgery. APOE and Gal-3 levels were quantified by enzyme-linked immunosorbent assay and total protein concentration by bicinchoninic acid (BCA) assay. Patients’ medical records were carefully reviewed to collect relevant preoperative data. Descriptive statistics, Pearson’s correlation coefficient, and multivariate linear regression analyses were conducted to characterize associations between clinical and demographic variables and APOE and Gal-3 levels.

Results: APOE and Gal-3 levels were significantly elevated in the AH of glaucoma patients (1.64 +/- 1.45 μg/mL and 2.33 +/- 2.09 ng/mL, respectively) compared to controls (0.40 +/- 0.19 μg/mL, 0.92 +/- 0.81 ng/mL) [p<0.001; p=0.004]. APOE and Gal-3 levels were moderately positively correlated across the entire cohort (r=0.65, p<0.001). No association was observed between APOE and total protein or Gal-3 and total protein, indicating that APOE and Gal-3 levels were not increased in glaucomatous AH due to nonspecific protein accumulation (p>0.3). Multivariate linear regression analyses revealed significant associations between Gal-3 and maximum recorded intraocular pressure measurement (p=0.009), and between APOE and number of past ophthalmic surgeries (p=0.031).

Conclusions: Our findings demonstrate that APOE and Gal-3 are increased in the AH of glaucoma patients and significantly correlate with clinical measures of glaucoma severity. Thus, these molecules could serve as biomarkers to identify patients who may benefit from microglia-based neuroprotective glaucoma therapies.

Category: Clinical Research

Candidate: Chhavi Saini

Poster #: C13

Restoration of Vision in Severe, Cicatricial, Ocular Surface Disease with the Boston Keratoprosthesis Type II

Chhavi Saini, Teresa C. Chen, Lucy H. Young, Demetrios G. Vavvas, Mark Vangel, George N. Papaliodis, Shizuo Mukai, Angela V. Turalba, Douglas J. Rhee, David M. Wu, Dean Eliott, John B. Miller, Brian J. Song, Lucy Q. Shen, Louis R. Pasquale, James Chodosh

Purpose: The Boston Keratoprosthesis type II (BK2) device may be the only viable option to salvage vision in patients with severe cicatrizing ocular surface disease. The prior BK2 model was modified to the present day (click-on) model in 2009. We assessed clinical outcomes of the current click-on BK2 model.

Methods: Study Design and Study Participants: This was a retrospective cohort study including all BK2 surgeries at Massachusetts Eye and Ear (MEE) from June 2009 to March 2021. One eye per participant was included. For patients with both eyes eligible, the eye with maximum follow-up was considered for univariate and time-to-event analysesDevice Design and Assembly: The BK2 click-on model consists of a front plate made of clear polymethyl methacrylate plastic and a back plate made of titanium. The front plate is placed face down on the adhesive patch followed by the donor cornea, and then the slitted “click-on” titanium back plate. The back plate is secured in place with gentle pressure by the assembly tool

Results: 56 eyes of 53 patients with a mean follow-up of 45.8 months were included.Visual Outcome: Visual acuity improved from LogMAR 2.2±0.5 preoperatively to 1.5±1.2 at final follow-up. 89.3% saw ≥20/200 at some point postoperatively. Of the eyes with a follow-up of more than 5 years, 50.0% retained a visual acuity of ≥20/200 at their final follow-up.Complications: The most common complications were de-novo or worsening glaucoma (41.1%), choroidal effusions (30.3%), retinal detachment (25.0%) and end-stage glaucoma (25.0%). Preoperative systemic immunosuppressive therapy was associated with retention of ≥20/200 acuity.

Conclusions: At 5 years postoperatively, approximately 50% of eyes with the BK2 retained a BCVA better than preoperative BCVA. For patients with severe cicatricial corneal blindness, implantation of a BK2 offers the potential for long term visual function but can also be associated with vision threatening complications.

Category: Clinical Research

Candidate: Aastha Singh

Poster #: C14

Mesenchymal

Stem

Cells-derived Interleukin-11 Inhibits Activation and Proliferation of T cells

Aastha Singh, Sharad Mittal, WonKyung Cho, Yilin Guan, Elsayed Elbasiony, Sunil Chauhan

Purpose: Uncontrolled T cell activation can result pathological conditions including autoimmunity and graft rejections. Our group has previously shown that mesenchymal stem cells (MSCs) inhibit activation of innate immune cells including neutrophils and macrophages. Here, we investigate whether MSCs regulate T cell responses by secreting interleukin-11 (IL-11).

Methods: Human bone-marrow derived MSCs (hMSCs) were purchased and phenotypically characterized for their expression of CD45- CD34- CD73+ CD90+. CD4+ CD25- T cells (purity: >95%) were magnetically sorted from human peripheral blood mononuclear cells for the MSC-T cell co-culture assays. Expression and secretion of IL-11 by hMSCs were evaluated using real-time PCR and ELISA, respectively. Expression of IL-11 receptor was confirmed in activated CD4+ CD25- T cells using flow cytometry. CD4+ CD25- T cells stimulated with anti-CD3/CD28 beads (1:1 ratio) were co-cultured with MSCs at 1:1 ratio for 24 hours (for early activation) and 66 hours (for proliferation) in the presence and absence of hIL-11 neutralizing antibody (20 µg/ml). Early T cell activation was assessed by evaluating expression of CD40L and CD69 (Median Fluorescence intensity; MFI) using flow cytometry. For proliferation, CD4+ CD25- T cells were stained with CFSE prior to co-culture and their proliferation was quantified by measuring CFSE dilution via flow cytometry.

Results: hMSCs constitutively express high levels of IL-11 at both mRNA and protein levels. Naïve CD4+ CD25- T cells showed the expression of IL-11 receptor, which was upregulated by 2-fold following CD3/CD28 stimulation. hMSCs significantly suppressed early activation of naive T cells, as indicated by an approximate 70% reduction in expression of both CD69 (p=0.025) and CD40L (p=0.011). This MSC mediated reduction in early T cell activation was not observed following the neutralization of IL-11. Furthermore, our CFSE dilution assay demonstrated that hMSCs significantly prevented the proliferation of CD4+ CD25 T cells (p=0.006); however, this MSC-mediated suppression of proliferation was abrogated following IL-11 neutralization.

Conclusions: IL-11 secretion by human mesenchymal stem cells is critical for inhibition of early T cell activation and proliferation.

Category: Clinical Research

Candidate: Karen Wai

Poster #: C15

Recovery of Vision in Open Globe Injury Patients with Initial No Light Perception Vision

Karen M. Wai, Noha A. Sherif, Kevin Makhoul, Racquel Bitar, Marisa G. Tieger MD, Grayson W. Armstrong MD MPH

Purpose: The recovery of vision in open globe injuries (OGI) presenting with no light perception (NLP) vision is poorly understood, and patients presenting with NLP vision often undergo primary enucleation or evisceration to mitigate the risk of sympathetic ophthalmia. We conducted a retrospective chart review to identify patient characteristics, OGI features, and surgical events that may predict the recovery of any vision in patients initially presenting with NLP and inform clinical considerations for management.

Methods: The Massachusetts Eye and Ear (MEE) OGI Database was used to identify patients with NLP vision at presentation from January 1999 to March 2022 with at least one week of follow up. The medical records of these patients were analyzed to identify demographic characteristics; zone of injury; time from injury to surgical repair; presence of retinal detachment, vitreous hemorrhage (VH), intraocular foreign body (IOFB), vitrectomy, hyphema, amongst 17 other features; and visual acuity (VA) at last follow-up appointment. Logistic regression was used to analyze the relationship between regained vision at most recent follow-up visit and the above dependent variables (Python 3.10.1).

Results: A total of 152 OGI cases with NLP at presentation that met inclusion criteria were identified. Of these, the majority were zone 3 injuries (68.4%). The average time of OGI to repair was 0.85 days, and the average length of follow up in our study was 3.5 years. Of patients with initial NLP vision with OGI included in our study, 29 cases (19.1%) regained better than NLP vision at most recent follow-up. Regained vision ranged from VAs of 20/100 (3.4%) to LP (58.62%). Vitrectomy was positively associated with regained vision (B=3.1, p<0.001) while zone 3 injury was negatively associated with regained vision (B=-1.94, p=0.05). Previous intraocular surgery approached statistical significance for a negative association with regained vision (B=-1.36, p=0.08)

Conclusions: While OGIs cause severe ocular morbidity, a significant proportion of OGIs with initial NLP vision regained some vision during follow-up care. Vitrectomy was positively associated with return of vision, while zone 3 injuries were negatively associated. These findings argue for primary closure of OGIs given potential for return of vision rather than primary enucleation, even in the presence of NLP vision at presentation.

Category: Clinical Research

Candidate: Shudan Wang

Poster #: C16

Neurotrophic Keratopathy-Associated Eye Pain and Quality-of-LifeRelated Parameters in Patients with Ocular Graft-Versus-Host Disease

Shudan Wang, Rohan Bir Singh, Erdem Yuksel, Aytan Musayeva, Thomas H. Dohlman, Reza Dana

Purpose: Neurotrophic keratopathy (NK) is a degenerative disorder of the cornea characterized by decreased sensory innervation, epitheliopathy, and impaired epithelial healing. In this study, we performed an assessment of ocular pain and quality-of-life-related parameters in ocular graft-versus-host disease (oGVHD) patients with and without NK.

Methods: We included 213 oGVHD patients in this retrospective study, including 29 patients with NK assessed by Cochet-Bonnet esthesiometer. We evaluated their records for ocular pain assessment survey (OPAS) scores and clinical parameters including corneal sensation, corneal fluorescein staining (CFS) score, Schirmer test, tear break-up time (TBUT), and ocular surface disease index (OSDI) score.

Results: In the cohort, NK was diagnosed by significantly lower corneal sensation scores. We observed a significantly higher CFS score and shorter TBUT in patients with NK than patients without NK. The patients with NK reported a significantly higher ocular pain intensity than patients without NK (average eye pain intensity in the past 24h: 1.1±1.9 vs. 2.0±2.8; p =0.03). The patients with NK more commonly reported burning sensation (0.2 ± 0.3 vs. 0.3 ± 0.4; p = 0.021) and sensitivity to light (0.2 ± 0.3 vs. 0.3 ± 0.4; p = 0.049) compared to patients without NK. Ocular pain had negative correlation with TBUT (r=-0.34, p=0.11) among NK patients.

Conclusions: The clinical parameters are worse in oGVHD patients with NK and they experience a higher intensity of ocular pain and have a worse quality of life, compared to oGVHD patients without NK.

Alumni Giving Society

OF HAR V ARD OPHTHALMOL OG Y @ MA SS . E YE AND EAR

We extend our grateful thanks to the current Society members (as of 5/28/2019)

CHAMPION Gifts of $25,000 or more

CHAMPION Gifts of $25,000 or more

Glenville A. March, Jr., MD

Robert J. D'Amato, MD, PhD and Janey L. Wiggs, MD, PhD

(as of 09/30/2021)

Joan W. Miller, MD

Francois C. Delori, PhD

C. Stephen Foster, MD

VISIONARY Gifts of $10,000 - $24,999

VISIONARY Gifts of $10,000 - $24,999

Kathryn A. Colby, MD, PhD

Herbert Giller, MD

Anthony P. Adamis, MD

Mark S. Borchert, MD

Jack V. Greiner, OD, DO, PhD

INNOVATOR Gifts of $5,000 - $9,999

Jack V. Greiner, OD, DO, PhD

David G. Hunter, MD, PhD

David G. Hunter, MD, PhD

Nalini A. Madiwale, MD

J. Van de Velde, MD, PhD

Frederick A. Jakobiec, MD, DSc.* Michael J. Price, MD, FRCSC

Samir A. Melki, MD, PhD

Nicholas J. Volpe, MD

John H. Niffenegger, MD and Arysol Soltero Niffenegger, MD

Nicholas J. Volpe, MD

Ann M. Bajart, MD, FACS

INNOVATOR Gifts of $5,000 - $9,999

Michael J. Bradbury, MD

Mark B. Abelson, MD

Dean M. Cestari, MD

Nathalie Azar, MD and Dimitri Azar, MD

Dean M. Cestari, MD

C. Douglas Evans, MD

Reza Dana, MD, MPH, MSc

PIONEER Gifts of $2,500 - $4,999

PIONEER Gifts of $2,500 - $4,999

Mark W. Balles, MD

Ann M. Bajart, MD, FACS

Patricia A. D’Amore, PhD, MBA

Sheila Borboli-Gerogiannis, MD

Matthew F. Gardiner, MD

Matthew F. Gardiner, MD

Aubrey Gilbert, MD, PhD

Ralph H. Hinckley, MD

Rosa Y. Kim, MD

Deborah Pavan Langston, MD, FACS

FRIEND Gifts of $1,000 - $2,499

Kenneth B. Graham, MD

Melanie Ryan Graham, MD

Michael S. Gilmore, PhD

Steven R. Hamilton, MD

John W. Gittinger, Jr., MD

Susan M. Pepin, MD

Evangelos S. Gragoudas, MD

Steven R. Hamilton, MD

Michael S. Gilmore, PhD

John I. Loewenstein, MD

Scott H. Greenstein, MD

Zhonghui Katie Luo, MD, PhD

Cynthia L. Grosskreutz, MD, PhD

Michael F. Marmor, MD

Byron S. Lingeman, MD

Shizuo Mukai, MD

Eric A. Pierce, MD, PhD

Joseph F. Rizzo, III, MD

FRIEND Gifts of $1,000 - $2,499

Anthony P. Adamis, MD

Anonymous

James V. Aquavella, MD

Christopher M. Andreoli, MD

Mary Beth Aronow, MD

Hasan M. Bahrani, MD

Charles K. Beyer-Machule, MD

Charles K. Beyer-Machule, MD

Nancy Canter Weiner, MD

Nancy Canter Weiner, MD and Mark J. Weiner, MD

Stanley Chang, MD

Kenneth K. Chang, MD

Cynthia S. Chiu, MD, FACS

James Chodosh, MD, MPH and Jaya Rajaiya, PhD

Thomas G. Chu, MD, PhD

Paul R. Cotran, MD

Thomas G. Chu, MD, PhD

Anthony B. Daniels, MD, MSc

Steven H. Cobb, MD

Jason I. Comander, MD, PhD

Deborah S. Eappen, MD

Paul R. Cotran, MD

Evangelos S. Gragoudas, MD

Marc J. Dinkin, MD

Kevin E. Houston, OD

Michael W. Gaynon, MD

Rachel M. Huckfeldt, MD, PhD

David Z. Greenseid, MD

B. Thomas Hutchinson, MD*

*Deceased

John A. Irvine, MD

Ula V. Jurkunas, MD

Scott H. Greenstein, MD

David M. Hinkle, MD

Ivana K. Kim, MD

Robert A. Petersen, MD

Michael J. Price, MD, FRCSC

Subhransu K. Ray, MD

Leo A. Kim, MD, PhD and Nahyoung Grace Lee, MD

Joseph F. Rizzo III, MD

George N. Papaliodis, MD

Michael F. Marmor, MD

Shizuo Mukai, MD

Michael P. Rubin, MD

Philip M. Skidd, MD

Eric A. Sieck, MD

Philip M. Skidd, MD

Harold A. Woodcome, Jr., MD

Frans J. Van de Velde, MD, PhD

Eric A. Pierce, MD, PhD

Roberto Pineda II, MD

Anthony B. Nesburn, MD

Michael B. Raizman, MD

David E. Newman-Toker, MD, PhD

Rosa Y. Kim, MD

Dorothy J. Horns, MD

Deeba Husain, MD

Leo A. Kim, MD, PhD

John A. Irvine, MD

Sahar Kohanim, MD

Cameron G. Javid, MD

Ernest W. Kornmehl, MD

Ernest W. Kornmehl, MD

David W. Lamberts, MD

Magdalena G. Krzystolik, MD

Peter R. Laibson, MD

Charles Leahy, OD

Mark A. Latina, MD

Nahyoung Grace Lee, MD

Charles D. Leahy, OD

Daniel R. Lefebvre, MD

Daniel R. Lefebvre, MD

Michael M. Lin, MD

Robert M. Mallery, MD

Dennis M. Marcus, MD

Anthony B. Nesburn, MD

Joan W. Miller, MD

Nancy J. Newman, MD

Sandra R. Montezuma, MD

Victor L. Perez, MD

Dale C. Oates, MD

Richard M. Robb, MD

Michael W. Pennett, MD

Felix N. Sabates, Sr., MD

Victor L. Perez, MD

Alfredo A. Sadun, MD, PhD

Richard M. Robb, MD

Aaron M. Savar, MD

Felix N. Sabates, Sr., MD

Jose-Alain Sahel, MD

Ankoor S. Shah, MD, PhD

Richard J. Simmons, MD

Nurhan Torun, MD

Kazuo Tsubota, MD

Erich C. Strauss, MD

Judith E. A. Warner, MD

Daniel J. Townsend, MD

Raymond Wee, MD

Mark J. Weiner, MD

Luk H. Vandenberghe, PhD

Stuart R. Winthrop, MD

Demetrios Vavvas, MD, PhD

Richard B. Weber, MD

Natalie Wolkow, MD, PhD

Michael K. Yoon, MD

Mark J. Weiner, MD

Frans