IHT‐induced neuronal protection is mediated by the microglia
Authors: Tantingco Genell , Ramirez Luis , Moore Colin , Mallet Robert , Ryou Myoung gwi 
Published: June 2021

Neuronal damage is the main cause of disability after various brain injuries, including ischemic stroke and traumatic brain injury. Recent years, it is being reported that microglia, a resident immune cell in the brain, shows dual edge roles. Intermittent hypoxic training (IHT) was introduced as a potential preventive or treatment program for the ischemic stroke and ethanol withdrawal syndrome. This study showed the effect of IHT on the function of microglia along with neuronal protection.MethodsEOC20, microglia, were randomly assigned to the following groups; 1) normoxic control, 2) oxygen‐glucose deprivation (OGD)‐reoxygenation, 3) IHT, and 4) IHT+ OGD‐reoxygenation. 3 days of IHT that consists of 5–8 daily and 5–10 min cycles of hypoxia with intervening 4 min of reoxygenation was applied to the microglia. OGD (0.1% O2, 90 min) and reoxygenation (24 hours) was used to simulate ischemic stroke and reperfusion. Additionally, the DMEM in which either normoxic or IHT‐treated microglia were grown was used to culture the neuronal cells. After word, the cell viability of neuronal cell exposed to the OGD and reoxygenation was measured. Calcein AM and the LDH assays have been used to compare the cell viability between groups. The microglia were characterized with staining CD45, 11b, CD68, and CD208 and by flow cytometry. Phagocytic activity of microglia was tested by using latex bead coated with FITC. ROS generation was also monitored by DCFDA assay. Both pro‐ and anti‐inflammatory cytokines were examined with commercial ELISA assay kits. Finally, the one‐way ANOVA followed by post hoc test was done to determine the statistical significance. The p‐value less than 0.05 was considered as a meaningful data.ResultsOGD and reoxygenation significantly decreased cell viability of microglia and interestingly, survived microglia from OGD and reoxygenation increased its phagocytic activity, which might benefit from the activated microglia. IHT was able to salvage the microglia from OGD and reoxygenation but also even more enhanced its phagocytic activity vs. OGD and reoxygenation group. This beneficial effect of IHT should be associated with increased antiinflammatory cytokine and shift of M1 to M2 stereotype. Last, it was observed that when neuronal cells were cultured in the DMEM in which microglia underwent 3 days of IHT, were able to be protected against OGD and reoxygenation. This might indicate humoral factors including cytokine released from microglia during IHT were an essential factor in maintaining cerebral circumstance, including neuron.ConclusionThis preliminary study suggests that microglia play a pivotal role in the neuronal damage in the ischemic stroke and can be a key target in developing ischemic stroke treatment. Furthermore, global intermittent hypoxic training might be potential and/or better intervention than an intervention that targets single cell type or organ in developing a treatment program for the ischemic stroke victims.Support or Funding InformationFaculty Development Grant, Tarleton State Univ Basic Research Program Grant, UNTHSCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.