Phenotype and secretory responses to oxidative stress in microglia

Abstract

The neonatal brain is particularly susceptible to oxidative stress. Our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (H2O2) levels rise significantly particularly in the neonatal brain and are sustained for up to 7 days. This rapidly accumulated H2O2 is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. To date, there is limited literature on the effects of increased H2O2 levels on microglial cells, which have been extensively implicated in the ensuing inflammatory injury. Microglial cultures were derived from the P1 mouse brain and exposed to either bolus concentrations of H2O2 (15 or 50 μM) or varying concentrations of continuous exposure for 4, 18 or 24 h. Continuous exposure of microglia to H2O2 was generated using the glucose oxidase-catalase system generating levels of H2O2 <10 μM. Reactive oxygen species and nitric oxide expression were measured. Conditioned medium was collected and analyzed for secreted cytokine levels. Treated cell extracts were processed for glutathione (oxidized and reduced) content and fixed cells were labeled for M1 and M2a phenotype markers. Overall, it is evident that microglial exposure to continuous H2O2 has pleiotropic and biphasic effects. Continuous exposure to very low levels of H2O2 is more damaging to cell survival than higher bolus doses at 18 h, and can produce considerably high levels of pro- and anti-inflammatory cytokines by 18 h. Significantly high levels of various chemokines/chemotactic molecules such as G-CSF, MIP-1b and MIP-2 are also produced in response to continuous low-dose H2O2 by 18 h. Interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. H2O2 exposure promotes an M2a microglial phenotype in the absence of IL-4/IL-13 signaling, suggesting a wound-healing role for microglia and a delayed activation mechanism for H2O2 after such an insult. Together, these specific effects can be used to clarify the microglial cell responses following injury in the immature brain.