Protection of Corneal Limbus from Riboflavin Prevents Epithelial Stem Cell Loss after Collagen Cross-Linking

PURPOSE: To investigate whether the protection of corneal limbus from riboflavin exposure during collagen cross-linking (CXL) prevents limbal epithelial stem cell (LESC) loss. METHODS: Ten New Zealand white rabbits received an epithelium-off CXL using an accelerated protocol. Seven days before procedure, 5-bromo-2-deoxyuridine (BrdU) was intraperitoneally injected. During procedure, riboflavin was applied to the corneal surface within a 9 mm diameter retention ring in 5 rabbits, thereby preventing the limbus from riboflavin exposure. In other 5 rabbits, riboflavin was instilled every 2 min, allowing the spillover to the limbus. One day after UVA irradiation, corneas were subjected to histological and molecular assays. RESULTS: There were no differences in corneal thickness and epithelial healing between the groups. The numbers of BrdU-labelled and p63(+) limbal epithelial cells were markedly reduced in the group without a ring, but significantly increased when a ring was used. Robust expression of CK3/12 was observed in the limbal epithelium in the group with a ring. The mRNA levels of ABCG2, FGF2, IL-1β, and IL-6 were significantly increased in the corneas with a ring. CONCLUSIONS: Protection of limbus from riboflavin during CXL was effective in preserving LESCs. However, inflammation was increased in the cornea treated with riboflavin using a ring.