Depth Resolved Differences After Corneal Crosslinking With and Without Epithelial Debridement Using Multimodal Imaging

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Purpose: : To quantitatively compare stromal collagen architecture and keratocyte viability between two methods of corneal crosslinking (CXL), one involving invasive epithelial debridement (standard riboflavin crosslinking) and the other by the modified method of riboflavin TRIS–ethylenediaminetetraacetic acid (riboflavin-TE) transepithelial crosslinking, which conserves epithelial layer, using multimodal imaging. Methods: : Corneas of fresh porcine globes were treated with standard or with riboflavin-TE crosslinking for 30 minutes under a 370-nm ultraviolet A (UVA) light source (3 mW/cm2). Depth resolved imaging by confocal and second harmonic generation (SHG) microscopy was performed after live/dead cell labeling. Evaluation of apoptotic keratocyte number and texture analysis based on surface roughness was performed on confocal and SHG image stacks, respectively. Results: : Surface texture and visual grading indicated significant alterations in stromal architecture following standard CXL compared with untreated controls, while riboflavin-TE CXL induced subtle alterations confined near the corneal surface. The standard CXL had a statistically lower (P < 0.05) average surface roughness (9.46 ± 2.8 AU), with no significant difference between controls (12.85 ± 2.96 AU) and riboflavin-TE CXL (14.34 ± 1.85 AU). Both CXL groups resulted in apoptotic keratocytes, differing significantly from controls, with 56.5% greater numbers of apoptotic cells in the standard CXL group than the TE-CXL group (P < 0.05). Conclusion: : Standard CXL produced significant architectural changes of the deeper stroma and caused increased collateral damage on the keratocytes in comparison to the riboflavin-TE CXL at all depths studied. Translational Relevance: : Multimodal imaging guided surface architectural analysis provided insights into the actual collagen changes that occur after CXL and may guide in real-time modifications of the CXL methods for effective therapeutic CXL.

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