Excimer laser-induced hydroxyl radical formation and keratocyte death in vitro

PURPOSE. To characterize the type of reactive oxygen species (ROS) produced by excimer photoablation of aqueous solutions and to show the effects of ROS and antioxidants on corneal stromal cells in vitro. METHODS. Electron spin-resonance spectroscopy was performed using the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for the detection of the superoxide anion and the hydroxyl radical in an acellular DMPO solution irradiated with the excimer laser. Hydroxyl radicals were produced by the Fenton reaction in vitro by the mixture of hydrogen peroxide and ferrous iron (Fe²⁺), and the effects on cultured corneal fibroblasts were observed by fluorescent microscopy using the cell death marker, propidium iodide (PI) and TdT-mediated dUTP nick-end labeling (TUNEL). RESULTS. Excimer photoablation of a 1% DMPO solution produced a species-specific spin-trapping adduct for the hydroxyl radical (·OH), but not for the superoxide anion or other unidentified free radical. The signals were inhibited dose dependently by the hydroxyl radical scavenger dimethylsulfoxide (DMSO) and an L-ascorbic acid analogue, EPCK-1. The production of ·OH in the supernatant of cultured rabbit corneal fibroblasts by the Fenton reaction caused an increase in PI (+) and TUNEL (+) cells by 90 minutes, which was significantly inhibited by the addition of DMSO. CONCLUSIONS. Hydroxyl radicals may be partly responsible for stromal fibroblast cell apoptosis after excimer photoablation.