Effect of Corneal Collagen Cross-Linking on Subsequent Corneal Fungal Infection in Rats

PURPOSE: The purpose of this study was to determine whether corneal collagen cross-linking (CXL) alters fungal susceptibility and increases the severity of keratitis through macrophage activation in rats. METHODS: Four weeks following CXL pretreatment, the corneal epithelium of adult rats was removed and inoculated with Candida albicans (C. albicans; CXL + inoculation group). The non-CXL-pretreated corneas were also inoculated with C. albicans (inoculation group). Clinical scoring and histopathological examination were performed to determine the severity of fungal keratitis. Immunofluorescence and confocal microscopy imaging were applied to determine the effects of CXL treatment on corneal local macrophage content. Real-time polymerase chain reaction (RT-PCR) and Western blots were used to evaluate mRNA and protein expression. Flow cytometry assays were performed to detect M1- and M2-type macrophages. RESULTS: CXL pretreatment (CXL + inoculation) resulted in higher infection success rate and more severe fungal keratitis than inoculation alone (inoculation group). On days 1, 3, and 7 following fungal infection, the increase in macrophage infiltration and IL-1β, MMP-9, and VEGFA expression was greater in the CXL + inoculation group than in the inoculation group. Number of M1- and M2-type macrophages, M1 to M2 ratio, M1-type macrophage genes, inducible nitric oxide synthase (iNOS), and tumor necrosis factor (TNFα) expression were higher in the CXL + inoculation group compared with the inoculation group. CONCLUSIONS: Our data demonstrate that CXL may increase the colonization of macrophages and activate more M1-type macrophages to increase fungal susceptibility and severity of keratitis. TRANSLATIONAL RELEVANCE: This study may aid long-term risk assessment and treatment of the complications of CXL.