Genell Tantingco, Tarleton State University

Intermittent Hypoxic Conditioning: Potential Treatment for Ischemic Stroke

Background: Intermittent, normobaric hypoxia (IH) conditioning increases cerebral resistance to subsequent ischemia-reperfusion, resulting in near prevention of cerebral infarct after ischemic stroke. The cerebroprotective mechanisms of IH are unknown. Cerebral ischemia-reperfusion triggers activation of microglia might contribute to injuring cells by over-activating proinflammatory cytokines and chemokine.  The regulation of the phenotype of microglia will provide a pivotal clue in developing an effective treatment for the ischemic stroke.  Furthermore, it will help to develop an intervention the victims of ischemic stroke.  Currently, FDA-approved treatment for the ischemic stroke is only rtPA.

Purpose: This study examined a mechanism how IH-derived phenotype switches of microglia occur in the in vitro OGD-reoxygenation model.

Methods: EOC 20 microglia cells (n=30) were conditioned by a three days IH program consisting of 5-8 daily, 5-10 min cycles of hypoxia with intervening 4 min reoxygenation, previously shown to produce robust cardioprotection [Exp Biol Med 2004; 229:806-812; Basic Res Cardiol 2006; 101:436-446].  24 h after the last IH, Microglia was harvested to perform a series of experiment; content changes of a series of proteins and cytokine (immunoblot and ELISA), ROS generation (DCFDA assays), phagocytic activity (FITC conjugated particle phagocytosis assay), and cell phenotype (immunocytochemistry, CD68 (M1) and CD206 (M2)).

Results: Immunocytochemistry and western blot indicates that IH shifts the microglial phenotype relatively toward anti-inflammatory type (vs. control).   Phagocytic activity of EOC 20 was significantly enhanced vs. control group.  EGF alpha and IL-10 content were elevated in the IH group.  Microscopic examination and flow cytometry quantification showed IH enhanced phagocytic activity (vs. non-IHT groups), Last, ROS generation in the OGD groups was significantly increased, but IH was able to dampen the ROS generation.

Conclusion: IH shifts the microglia to the anti-inflammatory phenotype.  A possible mechanism of IH-induced phenotype change of microglia could be related to the regulating ROS and anti-inflammatory cytokine.

Presentation Author(s):
Genell Tantingco*

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