Identification of Treatment Protocols for Effective Cross-Linking of the Peripheral Cornea: An Experimental Study

INTRODUCTION: This study aimed to test and evaluate modified corneal cross-linking (CXL) protocols regarding improved treatment effects on the peripheral cornea in terms of tissue stability and cellular response. METHODS: Peripheral CXL (pCXL) was performed within a ring of 9–11 mm of 36 human donor corneas with variations in applied energy (5.4, 7.2, and 10 J/cm(2)) at 9 mW/cm(2) irradiance. Each energy level was additionally modulated regarding the oxygen level surrounding the cornea during treatment (21%; 100%). Stress–strain tests with endpoints at 12% strain and collagenase A-assisted digestions to complete digestion were performed to evaluate the rigidity and resistance of treated and control tissue. Further, corneas were processed histologically via TUNEL assay and H&E staining to demonstrate the effects on stromal cells during treatment under varying CXL conditions. RESULTS: Increases in energy dosage achieved significant increases in resistance to stress in all variations except when comparing protocols A and B under normoxic conditions. Supplemental oxygen significantly increased rigidity in protocols B (p < 0.01) and C (p = 0.018). Hyperoxic conditions significantly increased resistance to digestion in all protocols. The number of DNA strand breaks in TUNEL assay staining showed significant increases in all increases in energy as well as with oxygen supplementation. CONCLUSIONS: Increases in energy and supplemental oxygen improved the effect of CXL, though endothelial safety could not be verified with confidence in high-fluence CXL with supplemental oxygen. Results suggest that CXL protocols using 7.2 J/cm(2) with 100% O(2) or 10 J/cm(2) without supplemental oxygen prove most effective without anticipated risk of endothelial damage.