Transepithelial Riboflavin/Ultraviolet. A Corneal Cross-linking in Keratoconus: Morphologic Studies on Human Corneas

Abstract

To evaluate histologic and molecular changes in human keratoconic corneas after the procedure of transepithelial collagen cross-linking (CXL), without the removal of corneal epithelium. Experimental laboratory investigation. Thirty corneal buttons were examined, 18 of which were from patients affected by severe keratoconus and submitted to penetrating keratoplasty (PK). Among these, 8 were analyzed without any treatment, 4 were treated with transepithelial CXL 2 hours before PK, and 6 were treated with transepithelial CXL 3 months before PK. Twelve normal corneal buttons from healthy donors were used as controls. The corneal buttons were then evaluated by hematoxylin-eosin staining and by immunostaining with markers of epithelial junction proteins (ß-catenin and connexin 43), of stromal keratocytes (CD34), of apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] assay), and of collagen type I fibers. The analysis of epithelial markers showed a clear defective expression in keratoconic corneas before and soon after the transepithelial CXL treatment, returning to normal in corneas analyzed 3 months after transepithelial CXL. The analysis of stroma components indicated a loss of keratocytes in the upper stroma of keratoconic corneas and a trend toward a normal situation 3 months after transepithelial CXL; similarly, collagen fibers appeared disorganized in keratoconus, while their pattern appears to be close to normal 3 months after treatment. Histologic and immunohistochemical findings on human keratoconic corneas showed the presence of biochemical and morphologic alterations in the epithelium and the upper stroma that are significantly improved 3 months after transepithelial CXL. However, further studies are necessary to assess to what extent these results correlate with measurable biomechanical effects.