Influence Of TREM2 On Microglia And Possible Mechanism Involved In Ischemic Stroke

Background: Effective treatments for ischemic stroke are insufficient.Inflammatory response plays an important role in the pathological process of ischemic stroke.Microglia,as an innate immune cell of central nervous system,is widely involved in this process.TREM2(Triggering receptor expressed on myeloid cells 2)is mainly expressed in microglia and has broad effects on the function of microglia.However,there are few studies focus attention on effects of TREM2 on microglia in ischemic stroke.Purpose: To evaluate the effects of TREM2 on the function of microglia in ischemic stroke,and the possible mechanisms underlying were further explored.Methods:As to experiments in vivo,the middle cerebral artery occlusion(MCAO)model of mice was established by intraluminal suture method;Western blot was used to detect the changes of TREM2 protein in brain tissue after MCAO,and the time of TREM2 reaching to peak was selected as the observation time point for follow-up experiments;immunofluorescence staining was used to observe the distribution of TREM2 in brain tissue.Immunofluorescence staining was performed to assess the phagocytic function,proliferation level and autophagy level of microglia in wild-type mice and TREM2-knockout mice,on the 7th day after MCAO;3D reconstruction and stereological analysis of microglia was performed using imaris software.Microglia were obtained by Percoll gradient centrifugation followed with FACS from brain tissue.Transcriptomics and bioinformatics analyses were performed to explore the possible mechanisms underlying TREM2 affecting microglia function.As to experiments in vitro,primary microglia were cultured using wild-type and TREM2-knockout neonatal mice within 3 days.Microglia were treated with OGD to mimic cerebral infarction in vitro.Mito-Tracker staining and ATP level detection were conducted to assess the mitochondrial quality.Energy was supplemented via cyclocreatine administration to carry out rescue experiment.Then,the phagocytic function was explored by phagocytizing Zymosan A Bio Particles test,Ed U and Ki67 immunofluorescence staining performed to assess proliferation level,annexin-V/PI staining and flow cytometry conducted to evaluate the apoptosis of microglia.Results: 1.MCAO model was successfully established by intraluminal suture method.TREM2 protein level increased gradually and reached the peak on the 7th day after MCAO model,and TREM2 was specifically expressed in microglia,in brain tissue.2.Compared with wild-type mice,phagocytic function,proliferation and autophagy levels,cell surface area,branch length and the number of microglia around the infarction,were all decreased after TREM2 knockout,in MCAO model mice.3.Compared with wild-type mice,1790 genes expression were up-regulated and 1279 were down-regulated in TREM2-knockout mice,on the 7th day after MCAO.Some metabolic pathways were significant in KEGG pathway enrichment analysis,and the up-regulated genes were related to glycolysis,while the down-regulated genes were related to oxidative phosphorylation.4.Compared with the wild-type primary microglia,the number of mitochondria,ATP level,the number of Zymosan A Bio Particles being phagocytized and the level of proliferation were all decreased,and the proportion of apoptotic cells increased,in TREM2-knockout primary microglia,after OGD treatment.The phagocytosis and proliferation level were restored in varying degrees,and the proportion of apoptosis was decreased,after cyclocreatine supplement in TREM2-knockout primary microglia.Conclusion: TREM2 could affect the functions of microglia via the energy metabolism pathway in ischemic stroke.