Quantitative assessment of UVA-riboflavin corneal cross-linking using nonlinear optical microscopy

Corneal collagen cross-linking (CXL) by the use of riboflavin and ultraviolet-A light (UVA) is a promising and novel treatment for keratoconus and other ectatic disorders. Since CXL results in enhanced corneal stiffness, this study tested the hypothesis that CXL-induced stiffening would be proportional to the collagen autofluorescence intensity measured with nonlinear optical (NLO) microscopy.

Rabbit eyes (n = 50) were separated into five groups including: (1) epithelium intact; (2) epithelium removed; (3) epithelium removed and soaked in riboflavin, (4) epithelium removed and soaked in riboflavin, with 15 minutes of UVA exposure; and (5) epithelium removed and soaked in riboflavin, with 30 minutes of UVA exposure. Corneal stiffness was quantified by measuring the force required to displace the cornea 500 μm. Corneas were then fixed in paraformaldehyde and sectioned, and the collagen autofluorescence over the 400- to 450-nm spectrum was recorded.

There was no significant difference in corneal stiffness among the three control groups. Corneal stiffness was significantly and dose dependently increased after UVA (P < 0.0005). Autofluorescence was detected only within the anterior stroma of the UVA-treated groups, with no significant difference in the depth of autofluorescence between different UVA exposure levels. The signal intensity was also significantly increased with longer UVA exposure (P < 0.001). Comparing corneal stiffness with autofluorescence intensity revealed a significant correlation between these values (R(2) = 0.654; P < 0.0001).

The results of this study indicate a significant correlation between corneal stiffening and the intensity of collagen autofluorescence after CXL. This finding suggests that the efficacy of CXL in patients could be monitored by assessing collagen autofluorescence.