Effect of Green Light-Mediated Corneal Cross-Linking on the Delivery of Rose Bengal and Vancomycin in Staphylococcus aureus-Infected Eyes.

Rose Bengal (RB)-green light (GL) cross-linking (RGX), a phototherapy used to treat infectious keratitis, activates RB to generate reactive oxygen species (ROS), promoting bacterial killing and corneal strengthening through collagen cross-linking. Despite its promise, the way GL exposure alters RB behavior within the infected cornea remains poorly understood. This study investigates RB distribution and fluorescence changes following RGX in methicillin-susceptible Staphylococcus aureus-infected bovine corneas with denuded epithelium. Ex vivo RGX treatment resulted in a pronounced, infection-dependent loss of RB fluorescence. Infected corneas exposed to GL showed a 70-85% reduction in RB fluorescence across corneal tissues and aqueous humor, compared with non-illuminated RB-treated controls. Consistent with these findings, fluorescent RB was reduced from 63 to 19% dose/g of stroma with GL in infected eyes. In contrast, RGX-treated uninfected eyes showed increased RB fluorescence in the endothelium and aqueous humor, while stromal RB levels were similar to those in infected and uninfected eyes without RGX. RGX treatment did not affect corneal stromal integrity in infected or uninfected eyes, as assessed by biomechanical piercing forces and vancomycin distribution in the corneal stroma and endothelium. Vancomycin delivery ranged from 50 to 63% dose/g of stroma. To determine whether RB fluorescence loss could be reproduced under in vitro conditions, complementary studies were performed. GL exposure alone, or with live or heat-killed bacteria, did not result in RB fluorescence loss. However, replication of these experiments in the presence of bovine corneal stromal extract (BCSE) resulted in a decline in RB fluorescence, analogous to that observed in infected RGX-treated ex vivo eyes. This effect was attenuated by the addition of ROS scavengers, L-histidine, D-mannitol, and vitamin E, targeting Type I and Type II photochemical pathways. These findings suggest that ROS generated from GL-activated RB drive the infection- and tissue-dependent loss of RB fluorescence, without affecting corneal piercing force and vancomycin delivery to the corneal stroma or endothelium.