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2018 Agenda and Abstracts | < Previous Next >

2018 OMIG Abstract

Cellular and Molecular Assessment of Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) on Corneal Endothelium and Limbal Stem Cell Niche

Andrea Naranjo, MD1,2, Daniel Pelaez, PhD2, Esdras Arrieta, MD1, Enrique Salero-Coca, PhD2,
Jaime D. Martinez, MD1,2, Alfonso L. Sabater, MD, PhD2, Guillermo Amescua1, MD2,
Jean-Marie Parel, PhD, FARVO1,2,
1Ophthalmic Biophysics Center and 2Anne Bates Leach Eye Hospital, Bascom Palmer Eye Hospital, University of Miami Miller School of Medicine, Miami, Fl


Purpose: Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis in which a photosensitizer is used to induce corneal crosslinking and the formation of antimicrobial reactive oxygen species (ROS). The purpose of this study was to evaluate the effect of RB-PDAT on endothelium and limbal stem cell niche, as well as the distribution of keratocyte death following treatment.

Methods: Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 minutes and then irradiated by a custom-made green LED light source (525nm, 6mW/cm2) for 15 minutes (5.4J/cm2). Three rabbits were sacrificed and enucleated after 24 hours. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. Limbal stem cells of both eyes were isolated and cultured to perform MTT viability and proliferation, as well as scratch wound healing assays under time-lapse microscopy.

Results: TUNEL assay on whole mounts showed no endothelial cell death following treatment. Superficial Rose Bengal penetration with associated cellular apoptosis in up to 1/3 of stromal thickness was evidenced on frozen sections. Immunohistochemistry on frozen sections of limbal stem cells displayed no structural difference between the treated and non-treated eyes. There was no difference in limbal stem cell proliferation rates between cells isolated from control or treated eyes and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes after 24 hours.

Conclusion: Even though penetration of the Rose Bengal dye has been shown to be superficial, our data suggests that oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on limbal stem cell viability or function.

Disclosure: N

Support: Edward D. and Janet K. Robson Foundation, Florida Lions Eye Bank and the Beauty of Sight Foundation, Drs. KR Olsen, ME Hildebrant, Raksha Urs and Aaron Furtado; NIH Center Grant P30EY14801, Research to Prevent Blindness, Henri and Flore Lesieur Foundation (JMP).


2018 Agenda and Abstracts | < Previous Next >