Cellular Wound Healing Effects of Riboflavin-ultraviolet-A Collagen Cross-linking in Rabbit Corneas

Abstract

Purpose: : To investigate the wound healing processes that occur following riboflavin-UVA treatment in rabbit corneas.

Methods: : Nine 12 to 15-week-old female New Zealand white rabbits weighing 2.5-3.0 Kg each, were used in this study. Animals were divided into three groups. All animals received a 7 mm central epithelial debridement in the experimental eye. Six animals were then treated with 0.1% riboflavin solution (one drop every 5 minutes) in conjunction with UVA light (370 nm; irradiance, 3mW/cm2; dose, 5.4 J/cm2). Two control animals had one eye treated with UVA irradiation alone without riboflavin and 1 control animal had one eye treated with riboflavin without UVA. The treatment duration in each eye in each group was 30 minutes. The right eye of each rabbit served as an untreated control eye. All animals were euthanized 24 hours postoperatively and the corneoscleral rims were frozen in OCT. Corneal sections were evaluated with the TUNEL assay to detect stromal cell apoptosis and immunocytochemistry to detect the inflammatory marker CD11b expressed in monocytes and the alpha-smooth muscle actin (SMA) marker expressed in myofibroblasts. Cell counts were performed in randomly selected 400X fields bisecting the stromal surface

Results: : Rabbit corneas with riboflavin+UVA (26.3+2.5 SEM cells /400X field) had significantly more TUNEL+ cells in the central cornea compared to the rabbit corneas treated with UVA alone (2.3+0.6 cells/400X field). A single cornea treated with riboflavin alone (20.8 cells/400X field) had a similar level to treatment with riboflavin+UVA. CD11b+ monocyte influx was significantly less in the periphery of corneas treated with riboflavin+UVA (9.07+ 1.74 cells/400X field) than corneas treated with UVA alone (24.69+ 3.33 cells/400X field). A single control cornea treated with riboflavin alone also had low CD11b+ monocyte influx (3.5 cells/400X field). No SMA+ myofibroblasts were detected in any of the groups at this 24-hour after injury time point

Conclusions: : The riboflavin solution used in riboflavin-UV crosslinking appears to have a cytotoxic effect on keratocytes beyond the effects of UVA light exposure alone in corneas in which the epithelium is removed at 24 hours after surgery. On the other hand, less inflammatory cells are detected in the cornea at 24 hours after riboflavin+UVA treatment is performed compared to UVA alone. Additional time points out to 1 month (where haze is prominent in clinical riboflavin-UVA treatment) with additional control animals will be tested to further evaluate the corneal cellular effects of riboflavin-UVA treatment

Keywords: wound healing • cornea: stroma and keratocytes • apoptosis/cell death