Elevation of hydrogen peroxide after spinal cord injury detected by using the Fenton reaction

Abstract

To reveal whether reactive oxygen species (ROS) play a role after spinal cord injury, we developed a unique method for assaying hydrogen peroxide (H2O2) and determined the time course of its concentration changes following impact injury to the rat spinal cord. Microdialysis was used to sample H2O2 in the extracellular space and the dialysates were collected into a vial containing salicylate and ferrous chloride (FeCl2). H2O2 collected in the vial was converted to hydroxyl radicals (*OH) by FeCl2 catalysis. 2,3- and 2,5-dihydroxybenzoic acid produced by reaction of *OH with salicylate in the collecting vial were measured by HPLC and calibrated to H2O2 concentrations. The postinjury levels of H2O2 were significantly increased (p = 0.02) for over 11 h. FeCl2 administered through the dialysis fiber catalyzes H2O2 conversion in the cord to *OH. This *OH does not reach the collecting vial due to its extremely short lifetime (nanoseconds). The reduced H2O2 levels in the vials validate the measurement of H2O2. The relatively long-lasting formation of H2O2 and superoxide reported herein and previously suggests that ROS may be important in secondary spinal cord damage and that removal of ROS may be a realistic treatment strategy for reducing injury caused by free radicals.