Application of in vivo esr spectroscopy to measurement of cerebrovascular ROS generation in stroke

Abstract

Reactive oxygen species (ROS) are believed to be very important in ischemic nerve cell death. Nitroxyl radicals react with O2- in the presence of reducing agents and with OH, and hence suppress lipid peroxidation. These reactions were reported to reduce the electron spin resonance (ESR) signals for the nitroxyl radical, and we proposed the utilization of nitroxyl radicals as spin probes for in vivo ESR spectroscopy to determine ROS generation in vivo. This study used an in vivo ESR spectroscopy / spin probe technique to measure directly the generation of reactive oxygen species (ROS) in the brain after cerebral ischemia-reperfusion. Transient middle cerebral artery occlusion (MCAO) was induced in rats by inserting a nylon thread into the internal carotid artery for 1 h. The in vivo generation of ROS and its location in the brain were analyzed from the enhanced ESR signal decay data of three intra-arterially injected spin probes with different membrane permeabilities. The ESR signal decay of carbamoyl-PROXYL having intermediate permeability was significantly enhanced 30 min after reperfusion following MCAO, while no enhancement was observed with the other probes or in the control group. The enhanced in vivo signal decay was significantly suppressed by superoxide dismutase (SOD). Neither catalase nor 100 mM DMTU (OH scavenger) suppressed the enhanced signal decay rate. None of the inhibitors had any effect on the signal decay rate in the sham-operated group. The results of this study suggest that the enhanced signal decay observed in MCAO rats is caused, not by the Fenton reaction but through a reaction that involves SOD. Nitroxyl radicals are known to act as antioxidants that reduce oxidative damage. Therefore, the presence of carbamoyl-PROXYL should reduce the degree of transient MCAO damage, assuming that the enhanced signal decay of carbamoyl-PROXYL, which was used to confirm in vivo ROS generation, is related pathologically to the transient MCAO injuries. Brain damage was barely discernible until 3 h of reperfusion, and was clearly suppressed with the probe of intermediate permeability. The antioxidant MCI-186 (edaravon) completely suppressed the enhanced in vivo signal decay following transient MCAO. These results clearly demonstrate that ROS are generated at the interface of the cerebrovascular cell membrane when reperfusion follows MCAO in rats, and that the ROS generated during the initial stages of transient MCAO cause brain injury (See Figure 1 and Figure 2).