Neuroprotection by hypoxic preconditioning involves oxidative stress-mediated expression of hypoxia-inducible factor and erythropoietin

Background and Purpose - Hypoxic preconditioning is an endogenous protection against subsequent lethal hypoxia, but the mechanism involved is not understood. Hypoxia is followed by reactive oxygen species (ROS) production and induces hypoxia-inducible factor (HIF) and its downstream factor erythropoietin (Epo), which is associated with neuroprotection. We hypothesized that these endogenous processes may contribute to hypoxic preconditioning. Methods - We used a mouse neuronal culture model, with 2 hours of hypoxia as preconditioning followed by 15 hours of hypoxic insult, and examined the expression of HIF-1 alpha, Epo, and their downstream proteins by Western blotting. Copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) mice were used to detect the effect of ROS. Cell survival and apoptosis were detected by mitogen-activated protein 2 quantification, apoptotic-related DNA fragmentation, and caspase-3 fragmentation. Antisense Epo was used to block endogenously produced Epo. Results - Hypoxic preconditioning was protective in wild-type (Wt) neurons but not in neurons obtained from SOD1 Tg mice. In Wt neurons, HIF-1 alpha and Epo expression showed a greater increase after hypoxia compared with Tg neurons and reached a higher level with preconditioned hypoxia, followed by pJak2, pStat5, and nuclear factor kappa B (NF-kappa B) expression. Antisense Epo decreased these downstream proteins and the neuroprotection of hypoxic preconditioning. Conclusions - Hypoxic preconditioning induces ROS, which may downregulate the threshold for production of HIF-1 alpha and Epo expression during subsequent lethal hypoxia, thus exerting neuroprotection through the Jak2 - Stat5 and NF-kappa B pathways.