To assess the efficacy and safety of ascorbic acid in the treatment of keratoconus by increasing the number of "anchors" that bond collagen fibers together in human in vitro cornea using electron microscopy.

In this semi experimental study keratoconus cornea is divided into six equal parts after keratoplasty.Two doses of ascorbic acid (10-3 mg/ml and 10-4 mg/ml) in two time ranges of 4 hours and one week after the treatment are applied to analyze the deviations of cross-linking. A part of the cornea is considered as control sample (without ascorbic acid) for different times. Various parts are randomly assigned to different studied doses. Each section of cornea is evaluated using electron microscopy. Friedman and Wilcoxon tests are used for data analysis. The value of significance level was set at 0.05.

The average distance between collagen fibrils are measured after the treatment with two different doses of ascorbic acid for two different time ranges. These results showed that higher doses of ascorbic acid and longer treatment time led to lower distance between collagen fibrils of cornea (p <0.001). This implies the better strength of the cornea. Apoptosis and vacuolization were not observed in keratocytes by electron microscopy after treatment with ascorbic acid.

Results showed that ascorbic acid strengthens the cornea and decreases the distance between collagen fibrils (consequently increase cross-linking). Therefore, the efficacy of ascorbic acid is observed by more recovery through increasing its doses and passing time.