Rose Bengal Photodynamic Antimicrobial Therapy: A Pilot Safety Study.

PURPOSE: To evaluate the in vivo corneal changes after Rose Bengal photodynamic antimicrobial therapy (RB-PDAT) treatment in New Zealand White rabbits. METHODS: Sixteen rabbits were divided into 5 groups. All groups underwent deepithelialization of an 8 mm diameter area in the central cornea. Group 1: balanced salt solution drops only, group 2: 0.2% RB only, group 3: green light exposure (525 nm, 5.4 J/cm2) only, group 4: 0.1% RB-PDAT, and group 5: 0.1% RB-PDAT. All rabbits were followed clinically. Group 5 rabbits were followed using anterior segment optical coherence tomography (AS-OCT) and clinically. On day 35 after initial treatment, 1 rabbit from group 5 was re-exposed to green light (5.4 J/cm2) to evaluate reactivation of the remaining RB dye, and terminal deoxynucleotyl transferase-mediated UTP-biotin-nick-end labeling assay was performed on corneal cryosections. RESULTS: Complete reepithelization was observed, and corneas remained clear after treatment in all groups. In group 5, AS-OCT revealed a cross-linking demarcation line. AS-OCT showed RB fluorescence and collagen cross-linking in all treated eyes of group 5 animals after 5 weeks of treatment. Photobleached RB retention in the corneal stroma was corroborated by fluorescence confocal microscopy on frozen sections. There was no evidence of a sustained cytotoxic effect through terminal deoxynucleotyl transferase-mediated UTP-biotin-nick-end labeling at 5 weeks. CONCLUSIONS: RB-PDAT with 0.1% RB is a safe procedure. There was no difference clinically and on histopathology compared with control groups. In eyes where RB dye is retained in the corneal stroma after 1 month of treatment, oxidative stress is not evidenced at long term.

Cellular and molecular assessment of rose bengal photodynamic antimicrobial therapy on keratocytes, corneal endothelium and limbal stem cell niche.

Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 min and then irradiated by a custom-made green LED light source (525 nm, 6 mW/cm2) for 15 min (5.4 J/cm2). Three rabbits were sacrificed and enucleated after 24 h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, 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 LSC viability or function.