Glycosaminoglycans and collagen fibril distribution at various depths of the corneal stroma of normal and CXL treated rats

Corneal collagen cross-linking (CXL) is widely used to treat keratoconus and ecstatic corneal disorders. The present studies were carried out to investigate the distribution of glycosaminoglycans (GAGs) and collagen fibril (CF) at different depths of the normal and CXL treated corneal stroma of four week old rats 7 days after standard CXL application. Ten Wistar rats' corneas were used for the study. The epithelium of the cornea from the left eye of each rat was removed and treated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm² for 30 min). The cornea from the right eye was used as the control cornea. The cornea was removed from the eye and processed for transmission electron microscopy. A bottom mounted Quemesa camera was used to capture digital images and these images were analysed using iTEM software. In the control cornea, the GAGs area size was not significantly different in the anterior, middle, and posterior stroma. In the CXL treated rats the GAGs area size gradually increased from the anterior to the posterior stroma whereas the spacing between the GAGs gradually decreased. There were very large GAGs present in the posterior stroma of the CXL treated rats. When comparing the control and CXL cornea, the GAGs area in the CXL cornea was significantly higher and inter-GAGs-spacing was smaller than in the control cornea. In the control cornea, the collagen fibrils diameter was higher in the anterior stroma and lowest in the posterior stroma. In the CXL treated cornea, the CF diameter and the interfibrillar spacing gradually decreased from the anterior to the posterior stroma. On comparison between the control and the CXL treated cornea, the interfibrillar spacing was significantly smaller in the CXL treated cornea than the control cornea in the anterior, middle, and posterior stroma but there was no difference in the diameter. The CXL treatment significantly increased the GAGs area and decreased the inter-GAGs-spacing, and inter-CF-spacing. This could be due to the gradual decline in the availability of riboflavin, UVA, and oxygen in the middle and posterior stroma. Further studies are required to investigate the role of keratan sulphate and chondroitin sulphate by using monoclonal antibodies with immunogold technique.