The crosslinking effect of photorefractive ablation with riboflavin evaluated with transmission electron microscopy

Abstract

Transmission electron microscopy is one of the key methods in studying the pathogenesis of collagen crosslinking. Aim. To evaluate the effect of crosslinking induced by photorefractive ablation of the cornea with riboflavin using transmission electron microscopy. Material and methods. This experimental in vivo study was performed on 16 eyes of 16 Chinchilla rabbits weighing from 2.5 to 3.5 kg, aged from 1 to 1.5 years. Animals were divided into control and experimental groups, including 8 animals for each of the transepithelial photorefractive keratoectomy (TransFRK) methods, with and without prior saturation of the stroma with a 0.25% isotonic riboflavin solution., The epithelium and outer and deep layers of the stroma were studied in the projection of surgery at the light-optical level. Based on data of the transmission electron microscopy, collagen fibers and bundles, and their concentration per unit area, and the cellular component of corneal stroma were evaluated. Results. The rabbit cornea studied at the light-optical level upon completion of epithelialization maintained a normal morphology of the flat multi-layered non-squamous epithelium with almost complete recovery of the layer stratification. In post-ablation samples with the prior saturation of stroma with riboflavin solution, isolated vacuoles were observed in cells of the basal layer along with emergence of pseudostratified epithelium. According to results of the complex morphological evaluation, corneas of the experimental group (with riboflavin pre-saturation of the stroma) showed changes corresponding to the stroma crosslinking with formation of stable cross-links of collagen fibers. In the cornea of the control group (without riboflavin saturation of the stroma), the stromal structure completely recovered after the laser treatment without any signs of stromal crosslinking. Conclusion. As detected by transmission electron microscopy, the stable cross-links in collagen structures of the corneal stroma induced by excimer laser ablation with riboflavin confirm the presence of the crosslinking effect in using the given method of the photorefractive
keratoectomy.