Longitudinal study of Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) effects on rabbit corneas

Abstract

Purpose : Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel treatment for progressive infectious keratitis in which a photosensitizer is used to induce corneal crosslinking and the formation of antimicrobial reactive oxygen species (ROS). However, the depth and duration of its effect have not been completely elucidated. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes in the acute phase and at 5 weeks post-treatment, as well as characterize its crosslinking effect and clearance of RB dye from the cornea in an in vivo rabbit model.

Methods : Rose Bengal solution (0.1% RB in 0.9% NaCl) was applied to the right cornea of rabbits for 30 minutes and irradiated using 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 was performed on frozen corneal sections of treated and control eyes. A second group (N=4) was followed for 5 weeks post-treatment with AS-OCT with blue fluorescence. On day 34 post initial treatment, one of the rabbits was re-exposed to green light to evaluate RB dye re-activation. TUNEL assay was performed on corneal cryosections of the second group of rabbits on day 35.

Results : Confocal imaging demonstrated a superficial corneal penetration of Rose Bengal with associated cellular apoptosis evident in up to 1/3 of the anterior stromal thickness on frozen sections after 24 hours of treatment. On long term follow up, rabbit corneas demonstrated complete reepithelization by postoperative day 7 and corneas remained optically clear. AS-OCT revealed crosslinking demarcation line after 7 days of treatment in all rabbits at a mean depth of 140.9 +/- 6.93 μm. RB and collagen crosslinking fluorescence remained evident in all treated eyes after 5 weeks of treatment. RB retention in the corneal stroma was corroborated by fluorescence confocal microscopy on frozen sections. Nonetheless, there was no evidence of a sustained cytotoxic effect via TUNEL at this time point, nor was there detectable TUNEL assay positivity following the second green light exposure.

Conclusions : Our results demonstrate there is no long-term toxicity following RB-PDAT. Even though the RB dye is retained in the corneal stroma after one month of treatment, oxidative stress was only produced during the acute phase of RB-PDAT.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.