Decreased Riboflavin Impregnation Time Does Not Increase the Risk for Endothelial Phototoxicity During Corneal Cross-Linking

PURPOSE: To evaluate the riboflavin (RF) concentration and distribution in the corneal stroma and the risk for endothelial photodamage during corneal crosslinking (CXL) following 10- and 30-minute impregnation. METHODS: De-epithelialized rabbit corneas were subjected to impregnation for 10 and 30 minutes with different RF formulations. Human corneal endothelial cells (HCECs) were subjected to different RF concentrations and ultraviolet A (UVA) dosages. Assays included fluorescence imaging, absorption spectroscopy of corneal buttons and anterior chamber humor, and cell viability staining. RESULTS: After 10 and 30 minutes of impregnation, respectively, anterior chamber fluid showed an RF concentration of (1.6 ± 0.21)•10(−4)% and (5.4 ± 0.21)•10(−4)%, and trans-corneal absorption reported an average corneal RF concentration of 0.0266% and 0.0345%. This results in a decrease in endothelial RF concentration from 0.019% to 0.0056%, whereas endothelial UVA irradiance increases by 1.3-fold when changing from 30 to 10 minutes of impregnation. HCEC viability in cultures exposed to UVA illumination and RF concentrations as concluded for the endothelium after 10- and 30-minute impregnation was nonstatistically different at 51.0% ± 3.9 and 41.3 ± 5.0%, respectively. CONCLUSIONS: The risk for endothelial damage in CXL by RF/UVA treatment does not increase by shortened impregnation because the 30% increase in light intensity is accompanied by a 3.4-fold decrease of the RF concentration in the posterior stroma. This is substantiated by similar endothelial cell toxicity seen in vitro, which in fact appears to favor 10-minute impregnation. TRANSLATIONAL RELEVANCE: This study offers compelling arguments for (safely) shortening RF impregnation duration, reducing patients’ burden and costly operation room time.