| BackgroundStroke contributes to the most of disability and death in the world. Ischemic stroke constitutes 87% of all strokes. The past studies about stroke mainly focused on the neurons which are target cells in ischemia. Further researches on central nervous system investigate glial cells, which account for 90% of the cells in the central nervous system. The glial cells not only support and supply nutrients to neurons, but also interact with neurons, participating in many physiological and pathological processes.As the resident immune cell in the brain, microglia plays a vital role in regulating the inflammation. Inflammation is crucial to the functional recovery of cerebral ischemia injury. Studies have found that microglia can be activated into different phenotypes and exert different functions due to various injuries. Microglia can be activated into the M1 type after cerebral ischemia to initiate an immune cascade reaction and produce the inflammatory cytokines and reactive oxygen species. M2 is a state of alternative activation, in which microglia shows an anti-inflammatory phenotype that is involved in damage repair. How to regulate the activation of microglia becomes the focus of the research on the neuroprotective mechanism.There is CB2 receptor on the microglia in the central nervous system. Activation of CB2 receptor on the microglia exerts a neuroprotective effect. CB2 receptor not only regulates microglial proliferation, differentiation and migration, but also reduces the neurotoxicity of microglia. The electroacupuncture preconditioning can activate the endogenous cannabinoid system, promote the expression of the endogenous cannabinoid receptors and play a protective role in the cerebral ischemia. But it is not clear whether electroacupuncture pretreatment regulates the activation of microglia through CB2 receptor in cerebral ischemia.In this experiment, we assessed the role of CB2 receptor in the activation of microglia after the electroacupucture preconditioning on cerebral ischemia from the multiple perspectives of molecular, cell and tissue by Western Blot, immunofluorescence staining and ELISA.Experiment 1. The time-course change in the number and morphology of activated microglia in the cerebral ischemic penumbra during ischemia and reperfusion.Objective: To observe the change in the number and morphology of microglia in the cerebral ischemic penumbra during ischemia and reperfusion. Methods: Male Sprague-Dawley rats subjected to transient focal cerebral ischemia were sacrificed at 6 hours, 24 hours, 3 days and 7 days after reperfusion. The number and morphology of microglia was demonstrated by immunofluorescent confocal image and rebuilt in three dimensions. Results: Microglia was activated after ischemia and reperfusion. The expression of microglial marker Iba1 in I/R group increased(P<0.05, vs. sham). Resting microglia with many thin processes extending away from their soma turned into a rounded amoeboid-like appearance at the 7 days after reperfusion. Conclusion: There was a spatiotemporal relationship between the number and morphology of microglia and cerebral injury in the ipsilateral hemisphere after ischemia and reperfusion.Experiment 2. The time-course shift of the microglia phenotype in the cerebral ischemic penumbra during ischemia and reperfusion.Objective: To detect the switch of the microglia phenotype in the cerebral ischemic penumbra during ischemia and reperfusion. Methods: Male Sprague-Dawley rats subjected to transient focal cerebral ischemia were sacrificed at 6 hours, 24 hours, 3 days, and 7 days after reperfusion. The shift of microglia phenotype from M1 to M2 in the cerebral ischemic penumbra was measured by western blot, confocal images and ELISA. Results: Quantitative analysis showed the peak expression of M1 phenotype markers i NOS, IL-1β, TNF-α and CD68 occurred at 6 hours or 24 hours after reperfusion, while Arginase, BDNF, IL-10 and CD206, which are markers of M2 microglia, peaked at the 3 days or 7 days after reperfusion(P<0.05, vs. sham). Conclusion: During the evolution of cerebral injury, microglia switched from M1 to M2 state, which was measured by the expression of different specific markers.Experiment 3. The effect of electroacupuncture pretreatment on the activation of microglia after ischemia and reperfusion.Objective: To detect the effect of electroacupuncture pretreatment on the activation of microglia after ischemia and reperfusion.Methods: Adult male Sprague-Dawley rats were divided into 3 groups: sham, MCAO and EA+MCAO. They were sacrificed at 3 days after reperfusion respectively. The expression of M1/M2 phenotypes of microglia in the cerebral ischemic penumbra was analyzed by western blot.Results: Electroacupuncture pretreatment increased the expression of Arginase and BDNF and decreased the expression of i NOS and IL-1β(P<0.05, vs. MCAO). Conclusion: Electroacupuncture pretreatment made the activated microglia shift from M1 to M2 phenotype.Experiment 4. Electroacupuncture preconditioning regulates the activation of microglia after ischemia and reperfusion through CB2 receptor. Objective: To detect whether electroacupuncture preconditioning regulates the activation of microglia after ischemia and reperfusion through CB2 receptor. Methods: 1. The effect of CB2 R agonist AM1241 on the activation of microglia after reperfusion. The adult male Sprague-Dawley rats were divided into 4 groups: sham, MCAO, AM1241+MCAO and Vehicle+MCAO. The rats were sacrificed at 3 days after reperfusion respectively. The expression of M1/M2 phenotypes of microglia in the cerebral ischemic penumbra was analyzed by western blot. 2. The effect of CB2 R antagonists AM630 on the activation of microglia after reperfusion. The adult male Sprague-Dawley rats were divided into 5 groups: sham, MCAO, EA+MCAO, AM630+EA+MCAO and Vehicle+EA+MCAO. The rats were sacrificed at 3 days after reperfusion respectively. The expression of M1/M2 phenotypes of microglia in the cerebral ischemic penumbra was analyzed by western blot. Results: Compared with MCAO, AM1241 increased the expression of Arginase and reduced the expression of IL-1β. There was no significant difference between MCAO and Vehicle+MCAO(P<0.05, vs. MCAO). Compared with EA+MCAO, AM630 increased the expression of i NOS and reduced the expression of BDNF(P<0.05, vs. EA+MCAO). Conclusion: Electroacupuncture preconditioning regulated the activation of microglia through CB2 receptor.Summary After ischemia and reperfusion, the microglia was activated. Resting microglia with many thin processes extending away from their soma turned into a rounded amoeboid-like appearance. There was a spatiotemporal relationship between microglia phenotype and cerebral injury in the ipsilateral hemisphere. During the evolution of cerebral injury, microglia switched from M1 to M2 state, which was measured by the expression of different specific markers. Electroacupuncture pretreatment made the activated microglia shift from M1 to M2 phenotype through CB2 receptor after cerebral ischemia. |
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