Intermittent Normobaric Hypoxic Training Protects the Brain by Regulating Microglia Phenotype

Abstract

IntroductionEthanol Withdrawal (EW) syndrome is an etiologically important factor linked to the ischemia and reperfusion injuries, including ischemic stroke. In the US, ischemic stroke is the 4th leading cause of death and the first cause of adult disability. Previously we showed that intermittent normobaric hypoxic training (IHT) protects the brain from EW syndrome by reducing ROS and increasing protective proteins. Pro‐inflammatory microglia has been studied in various CNS diseases. However, the function of microglia in the IHT‐induced brain protective mechanism is not yet thoroughly explored. In here, we focused on how IHT influence on regulating the phenotype of microglia, a pivotal resident phagocytic cell in the brain, in the in vitro IHT model.MethodsMouse microglia cells, EOC20 cell, underwent three days of IHT program which composed of 5–7 cycles of hypoxia/normoxia. O2 concentration was gradually decreased from 2.5% to 2 % each daily training cycle. Two sessions of EW treatment was done as following; EtOH (100 mM) for 16 hrs followed by 6 hrs of EtOH cessation. Cells were assigned into four groups; 1) non‐treated control, 2) IHT, 3) EW, 4) IHT+EW. Cell viability was evaluated by Calcein AM assay. Immunocytochemistry and flow cytometry were applied to determine the phenotype of microglia; CD 68 (M1), CD 206 (M2). The expression of mRNA of pro‐ and anti‐inflammatory cytokines were examined with quantitative real‐time rt‐PCR. Additionally, protein contents, which play a role in cerebral protection, were quantified by immunoblot assays. Data were expressed as mean ± SEM. Two factor ANOVA analysis and Tukey's multiple comparison test was used. P value < 0.05 was considered as a significant value.ResultsEW reduced cell viability and IHT was able to salvage the microglia from EW stress. IHT‐induced protection was mediated by increased HIF‐1α and activated Erk, an anti‐apoptotic protein kinase. The expression of CD 68 was not reduced with IHT vs. EW, but CD 206 was significantly increased in the IHT group vs. non‐IHT groups. An increased pro‐inflammatory phenotype of microglia was observed in the flow cytometry. IHT significantly decreased the expression of TNF‐α and IL‐6, pro‐inflammatory cytokines.ConclusionIHC drives the microglia toward anti‐inflammatory phenotype, which could be an important factor in the IHC‐induced cerebral protective mechanism.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.