Changes in Rat Scleral Collagen Structure Induced by UVA–Riboflavin Crosslinking at Various Tissue Depths in Whole Globe Versus Scleral Patch

PURPOSE: To investigate structural changes in scleral collagen fibers at various tissue depths before and after photosensitized crosslinking (CXL) both isolated scleral patch versus whole globe using second-harmonic generation (SHG) imaging. METHODS: Scleral tissues were harvested from Sprague–Dawley rats and separated into three groups: untreated sclera (control), full-thickness scleral patch for CXL (Free Scleral CXL group), and sclera in intact globe for CXL (Globe CXL group). The CXL groups were soaked in 0.1% riboflavin and irradiated with 365 nm ultraviolet-A light (power, 0.45 mW/cm(2)) for 30 minutes. SHG images were acquired every 5 µm between 10 and 60 µm from the outer scleral surface. Collagen fiber waviness was calculated as the ratio of the total length of a traced fiber and the length of a straight path between the fiber ends. RESULTS: In the Free Scleral CXL group, collagen waviness was significantly increased compared to the control group at 35 to 50 µm (P < 0.05). In the Globe CXL group, collagen waviness was decreased compared to control at all depths with statistical significance (P < 0.05) achieved from 10 to 45 µm. CONCLUSIONS: Depending upon its initial state (i.e., free scleral patch versus mechanically loaded intact globe under pressure), collagen may experience different structural changes after CXL. In addition, the extent of the CXL effects may vary at different depths away from the surface. TRANSLATIONAL RELEVANCE: Understanding the CXL effects on collagen structure may be important in optimizing the scleral crosslinking protocol for future clinical applications such as preventing myopic progression.