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

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.