Generation of hydrogen peroxide during brief oxygen-glucose deprivation induces preconditioning neuronal protection in primary cultured neurons

Although reactive oxygen species (ROS) have been implicated in ischemic preconditioning (IPC)-induced neuronal protection, several key questions concerning ROS remain to be elucidated. The purpose of this study is to obtain direct evidence for the formation of specific ROS species generated by IPC, and to determine the specific species that is responsible for the observed neuronal protection. Primary cultured cortex neurons from rat embryos were preconditioned with 10 min of oxygen-glucose deprivation (OGD), which increased the intracellular levels of superoxide and hydrogen peroxide. This preconditioning markedly induced neuronal protection against 2-hr OGD stimuli. Preconditioning with exogenous ROS by the administration of xanthine/xanthine oxidase (X/XO), or hydrogen peroxide was also found to induce IPC-like neuronal protection. Administration of hydrogen peroxide scavengers, such as catalase, glutathione, or the thiol reductant N-(2-mercaptopriopionyl)-glycine, all reduced the increase in the intracellular hydrogen peroxide levels, which effectively eliminated IPC- or exogenous ROS-induced neuronal protection. In contrast, administration of the membrane-permeable superoxide dismutase mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride was able to block the increase of intracellular superoxide levels during IPC, but did not abolish either IPC- or exogenous X/XO preconditioning-induced neuronal protection. These findings strongly suggest that IPC enhances the generation of superoxide, which is then converted to hydrogen peroxide, and that hydrogen peroxide is likely the main trigger involved in the mechanism of IPC-induced neuronal protection.