Evaluation of effects of riboflavin and/or ultraviolet-A on survival of rat limbal epithelial stem cells in ex-vivo.

Abstract

To investigate the effects of riboflavin and/or ultraviolet-A (UV-A) irradiation on the cell viability of ex-vivo-cultured rat limbal stem cells (LSCs). LSCs of male Wistar rats (N = 12 eyes) were cultured, and immunofluorescence staining was performed to evaluate them. After characterization, these cells were assigned to four groups of control (C), a group that was exposed to UV-A radiation (UV), a group that was treated with riboflavin (R), and a group that cotreated with both UV-A and riboflavin (UV+R). To determine the cell viability of LSCs, these cells were subjected to MTT assay on days 1, 3, and 7 after exposure to UV-A and/or riboflavin. The duration of exposure to UV-A and riboflavin was similar to levels used during the conventional corneal collagen cross-linking procedure. Compared with the viable cells in the control group, there was a significant decrease (P < 0.0001) in the number of LSCs in the UV group during all study days. In the R group, the level of viable LSCs was as same as the level of viable LSCs in the C group. Combined treatment with UV-A plus riboflavin significantly decreased the survival of LSCs on days 1 and 3 (P < 0.0001, P < 0.001, respectively) compared with the control group. Interestingly, in the UV+R group, the photosensitizing effect of riboflavin significantly decreased the cytotoxic effect of UV irradiation 7 days after exposure. These results suggest that the administered UV energy in the presence or absence of riboflavin can damage LSCs. Likewise, riboflavin could decrease the toxic effect of UVA on LSCs.