Normobaric intermittent hypoxic training regulates microglia phenotype and enhances phagocytic activity

Abstract

The microglia are the resident immune cells in the central nerve system. In the various pathological conditions, prolonged activated microglia could deteriorate brain damage. The regulation of the microglia polarization should be considered in developing an intervention for ischemic stroke patients. Normobaric intermittent hypoxic training protects the brain from intensive ischemic stresses. This study examined the role of intermittent hypoxic training in the regulation of microglia polarization that occurs in the in vitro model of oxygen–glucose deprivation (OGD)–reoxygenation. EOC20 were assigned to the following groups; (1) Normoxia, (2) oxygen–glucose deprivation–reoxygenation, (3) intermittent hypoxic training, (4) oxygen–glucose deprivation–reoxygenation +  intermittent hypoxic training; 24 h after the intermittent hypoxic training, microglia were harvested to perform the following experiments; cell viability (Calcein AM and LDH activity assay), quantification of proteins (Western blot), cytokine (ELISA), and reactive oxygen species (ROS) (H₂DCFDA assays), phagocytic activity by using latex beads coated with FITC, and cell phenotype (immunocytochemistry and flow cytometric analysis, and immunoblot CD206 (M2)). One-way ANOVA with Tukey’s post hoc test was used for the statistical analysis. Oxygen–glucose deprivation/reoxygenation decreases cell viability to 50% of normoxia. Intermittent hypoxic training protects the microglia from oxygen–glucose deprivation/reoxygenation stress. Intermittent hypoxic training regulates the polarization of the microglial phenotype toward anti-inflammatory type M2 (vs. oxygen–glucose deprivation and reoxygenation). Intermittent hypoxic training increases phagocytic activity (about 12 folds) vs. normoxia. ROS in the oxygen–glucose deprivation/reoxygenation group is increased, but intermittent hypoxic training lowers the ROS generation by oxygen–glucose deprivation/reoxygenation. The protein content of the toll-like receptor (TLR2) was significantly elevated in the oxygen–glucose deprivation and reoxygenation group, and intermittent hypoxic training lowered to normoxia level. Anti-inflammatory cytokines, such as IL-10 and IL-4, were significantly increased in the intermittent hypoxic training groups. Due to the effect of intermittent hypoxic training on the microglia phenotype, intermittent hypoxic training could be considered as an effective intervention in the treatment or rehabilitation program for the ischemic stroke victims.

Impact statement

The effects of intermittent hypoxic training or conditioning on many pathological conditions have been widely investigated. One of the pathological conditions dealt with intermittent hypoxic training is ischemic stroke. Well-known mechanisms of intermittent hypoxia-induced protection are related to increased energy metabolism and the enhanced antioxidant effects. In the last decades, the role of microglia in the progress of ischemic stroke-related brain damage has been focused. The dual-edge function of microglia indicates that the microglia-mediated inflammatory response is definitely beneficial in the early stage of ischemic stroke, but long-term activation of microglia is rather detrimental during the recovery process. The effect of IHT on microglia polarization is not investigated. This study focused on whether IHT regulates the polarization of microglia without dampening its classic phagocytic function. This study will provide pivotal information regarding the effects of IHT on the long-term effects on the recovery process from ischemic stroke.