Regional and Anisotropic Changes in Stromal Diffusivity After Corneal CXL With Riboflavin and UVA Light
Narrative Responses:
Purpose
The purpose of this study is to investigate regional and anisotropic changes in stromal diffusivity after crosslinking (CXL). We hypothesize that regional changes, observed with fluorescence recovery after photobleaching (FRAP), will be a sensitive measurement of crosslinking effect, and anisotropic changes will provide insight into the mechanism.
Methods
Standard, epithelium-off corneal CXL was performed on fresh, intact porcine eyes and human corneas, while controls received riboflavin without irradiation. FRAP was performed at different stromal depths, and nasal-temporal (X) and superior-inferior (Y) diffusivities were calculated with a custom program. FRAP was also performed in wells of riboflavin solution before and after UVA-irradiation to control for its effect on solute diffusivity in free solution. To evaluate the effect of crosslinking on diffusivity in the anterior-posterior (Z) direction, an electrical method was employed to calculate overall conductivity, dependent on small ion diffusivity.
Results
Normalized XY diffusivities of crosslinked and control porcine corneas were significantly increased (P=0.005, ANOVA) in the anterior cornea. This difference decreased as stromal depth increased (R=-0.87, Pearson’s). However, in the Z direction, conductivity decreased (P<0.0001, ANOVA) in porcine corneas, indicating a corresponding decrease in diffusivity in the Z-direction. UVA irradiation had no effect on riboflavin diffusivity in free solution.
Conclusion
The effect of CXL is more pronounced in the anterior stroma. We present a non-destructive ex vivo method for quantifying and comparing crosslinking effect with respect to stromal depth. The observed anisotropic change in diffusivity has implications for the CXL mechanism and ophthalmic drug delivery.