Corneal Enzymatic Resistance Following High-Fluence Accelerated Corneal Cross-Linking Using Rose Bengal And Green Light Ex Vivo

Rose Bengal (RB) generates reactive oxygen species (ROS) when photoactivated with green light at 522 nm. In the cornea, ROS kill pathogens and cross-link stromal molecules. This both strengthens the cornea and increases resistance to enzymatic digestion by steric hindrance – many collagen cleavage sites become hidden. In infectious keratitis, reduced digestion equals less melting and smaller scar sizes. We evaluated the digestion-inhibiting effects of high-fluence RB CXL protocols.

Laboratory investigation. ELZA Institute, Dietikon, Switzerland and Dept. of Ophthalmology, University of Antwerp, Antwerp, Belgium.

This ex vivo study aimed to evaluate whether high-fluence RB CXL protocols increase resistance to enzymatic digestion relative to non-treated control corneas. Porcine corneas (n=86) were assigned to 4 groups. Groups 1 and 2 served as controls (abrasion only and abrasion/RB soaking, respectively). Group 3 received CXL treatment using a 10 J/cm² high-fluence cross-linking protocol (9′15″ @ 18 mW/cm²), whereas Group 4 was treated with an accelerated 15 J/cm² high-fluence protocol (8′20″ @ 30 mW/cm²). All irradiations were performed using a custom-built device and 0.1% rose Bengal solution. All corneas were digested in 0.3% collagenase A solution and time to complete dissolution was observed.

Both high-fluence cross-linking protocols significantly increased resistance to digestion. The mean times to complete digestion in Groups 1 through 4 were: 21.36 ± 3.25 h, 31.21 ± 3.84 h, 33.7 ± 2.38 h, and 34.39 ± 1.85 h, respectively. Corneas in Groups 2–4 displayed significantly higher (p<0.0001 for all comparisons) digestion resistance than corneas in the non-irradiated, no RB control group, Group 1. Corneas in Groups 3 and 4 also displayed significantly increased digestion resistance when compared to Group 2 (RB, no irradiation control) corneas (0.0392 and 0.0036, respectively). There was no significant difference between the two high-fluence groups 3 and 4.

Photoactivated chromophore for keratitis-CXL (PACK-CXL) with Rose Bengal and green light has been successfully used for the treatment of infectious keratitis. Interestingly, unlike its pathogen-killing effect, RB does not need direct, focused irradiation with 543 nm light to inhibit collagenase digestion of the cornea, although this effect is enhanced with 543 nm light irradiation. This may suggest that part of this enzymatic digestion inhibition effect is achieved through an alternative mechanism, which may be independent of photoactivation