Collagen structural changes in rat tarsus after crosslinking

Purpose: Surgery is the standard treatment for floppy eyelid syndrome, but crosslinking (CXL) tarsus has recently been proposed as an alternative. To the best of our knowledge, this study is the first to use second-harmonic generation (SHG) microscopy to examine tarsal collagen ex vivo before and after photo-activated crosslinking. To quantify crosslinking, this study examined fluorescence recovery after photobleaching (FRAP), which indirectly measures tissue stiffness. Methods: Upper eyelid tarsal plates were dissected from 21 Sprague–Dawley rats (total of 42 tarsal plates). Six normal plateswere sent for histopathology and SHG imaging; the remaining 36 were crosslinked with phosphate-buffered saline (PBS) alone or riboflavin in PBS (concentrations of 0.1%, 0.3%, and 0.5%). Tissues were irradiated with 365-nm ultraviolet A light (power, 0.45 mW/cm2) for 30 minutes and immediately underwent SHG microscopy. Stiffness was indirectly measured with FRAP using fluorescein isothiocyanate (FITC)–dextran. Results: SHG imaging of normal tarsus showed that the organization of collagen bundles is complex and varies greatly depending on location. After crosslinking with high-concentration riboflavin (0.5%), collagen fibers showed clear structural changes, becoming more densely packed and wavier compared to control. FRAP half-time to fluorescence recovery was significantly increased (P < 0.05), indirectly indicating increased tissue stiffness. No structural changes were observed after crosslinking with lower riboflavin concentrations of 0.1% and 0.3%. Conclusions: This is the first report of SHG microscopy used to image tarsus collagen before and after crosslinking. These results highlight collagen structural changes, with effects on tissue stiffness indirectly confirmed by FRAP. Translational Relevance: Collagen fibers in the tarsus may be a therapeutic target for crosslinking in order to treat symptomatic floppy eyelid syndrome.