Exogenous low dose hydrogen peroxide increases hypoxia-inducible factor-1alpha protein expression and induces preconditioning protection against ischemia in primary cortical neurons

HIF-1 is believed to play a critical role in hypoxia/ischemia (H/I) preconditioning protection in neonatal brain. Recently, it has been shown that hydrogen peroxide (H(2)O(2)) may contribute to H/I preconditioning in rat primary neurons. We hypothesize that H(2)O(2) produced during H/I preconditioning may increase HIF-1alpha protein expression and contribute to H/I preconditioning protection in the immature brain. To test this hypothesis, we used 6-8 days in vitro (DIV) primary cortical neurons from embryonic day 16 CD1 mouse brains and preconditioned them with 10 min of oxygen and glucose deprivation (OGD) or exogenous H(2)O(2) at doses from 5 to 50 microM. Both OGD and low dose H(2)O(2) (15 microM) preconditioning provided neuronal protection 24 h later against a 2 h OGD insult. Cell survival was 34.9+/-1.8% and 35.8+/-3.8% with OGD and H(2)O(2) preconditioning respectively vs. 20.0+/-0.4% without preconditioning (P<0.01). After OGD preconditioning, HIF-1alpha protein increased at 4 h and peaked at 8h, then declined at 18 h and increased again to reach another peak at 32 h. HIF-1alpha protein following H(2)O(2) preconditioning increased at 8h and peaked at 32 h. For both preconditioning paradigms, HIF-1alpha expression level declined to baseline at 72 h. Our results suggest that low levels of H(2)O(2) may up-regulate HIF-1alpha protein and thereby mediate H/I preconditioning protection.