| Traumatic brain injury(TBI)is one of the major diseases threatening human health,and there is no effective treatment at present.The pathological process of TBI can be divided into primary injury and secondary injury.Primary injury occurs at the time of trauma and can not be intervened,while secondary injury occurs minutes to weeks after injury,and it is mediated by a variety of cytokines and inflammatory factors.More and more studies have shown that microglia-mediated inflammation plays an important role in secondary brain injury.Activated microglia are divided into two phenotypes:M1 and M2 phenotype,which have opposing functions in inflammatory response.Inhibiting M1 and promoting M2 has demonstrated neuroprotective effects in animal models of diseases.However,whether the polarization of microglia to M2 benefits TBI is unknown.In this study,we performed in vivo and in vitro experiments to examine the effects of microglia polarization to M2 on neuroinflammation and outcome of TBI,which will provide evidence for new treatments of TBI.This study is divided into three parts,the first part:isolation,culture and identification of primary microglia in mice.The second part:the effects of PPAR-y mediated microlgia polarization to M2 on neuroinflammation after LPS stimulation in vitro.The third part:the effects of microlgia polarization to M2 on the outcome of TBI in mice.Part I Isolation,culture and identification of primary mouse microgliaObjective:To isolate and culture primary microglia from neonatal mice,to evaluate the purity of isolated microglia,and then to lay a foundation for further study on the effect of microglia polarization on inflammatory response in vitro.Methods:The neonatal C57BL/6 mice(1-2 d)were used,the cerebral cortex was separated under a microscope.The brain tissue was cut,digested,centrifuged and suspended.The cells were inoculated into polylysine-coated culture bottles.Primary mixed glial cells were cultured until the 9th day,when the cells showed obvious stratified growth.After two days of complete stratification,the cells were shaken and separated.Then the cells were detected by Iba-1 immunofluorescence assay.Results:Microglia were successfully isolated and cultured.The purity of microglia was 92.58%by Iba-1 immunofluorescence assay,which met the requirements of in vitro experiments.Conclusions:The purity of primary culture microglia is relatively high,which can be used to further study the effect of microglia polarization on neuroinflammation in vitro.Part Ⅱ Effects of microglia polarization to M2 phenotype onneuroinflammation in vitroObjective:To study the effects of microglia polarization to M2 on microglia-mediated neuroinflammatory response after LPS stimulation in vitro.Methods:The microglia was divided into the following experimental groups:control group,microglia+LPS group,microglia+LPS+rosiglitazone(PPAR-gamma agonist)group,microglia+LPS+GW9662(PPAR-gamma antagonist)group.The expression levels of inflammatory factors TNF-a,IL-1β,IL-6 and IL-10 were detected by ELISA,mRNA expression levels of microglia polarization markers Ml(CD16/32,CD86),M2(CD206,Ym-1)were detected by qRT-PCR.Results:Compared with control group,M1 markers increased and M2 markers decreased after LPS stimulation,and the expressions of inflammatory factors TNF-α,IL-1β,IL-6 and IL-10 also increase significantly after LPS stimulation.M1 markers decreased and M2 markers increased after rosiglitazone treatment,and the expressions of TNF-α,IL-1β,IL-6 and IL-10 in rosiglitazone treatment group were lower than those in LPS stimulation group.However,M1 markers increased and M2 markers decreased after GW9662 treatment,and the expressions of TNF-α,IL-1β,IL-6 and IL-10 in GW9662 treatment group were higher than those in LPS stimulation group.Conclusions:After LPS stimulation,M1 polarization increases,and the expression of inflammatory cytokines in microglia was up-regulated.Rosiglitazone promoted polarization of microglia to M2 phenotype and the expression of inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-10 were down-regulated.In conclusion,polarization of microglia to M2 phenotype attenuated inflammatory responses after LPS stimulation.Part Ⅲ Effects of microglia polarization to M2 phenotype on the outcome of traumatic brain injury in miceObjective:To study the effects of microglia polarization to M2 on neuroinflammation,axonal injury and neurological outcome after traumatic brain injury in mice.Methods:C57BL/6 mice were used to establish the model of TBI using lateral fluid percussion injury device(LFPI),and the mice were divided into the following experimental groups:sham group,TBI group,TBI+ rosightazone(PPAR-gamma agonist)group,TBI+GW9662(PPAR-gamma antagonist)group.Neurological function of mice was evaluated by neurological severity score(NSS),the expression levels of inflammatory factors TNF-α,IL-1β,IL-6 and IL-10 were detected by ELISA.Axonal injury was assessed by silver staining and β-APP Immunohistochemical staining,mRNA expression of microglial polarization markers CD 16/32,CD86,CD206,Ym-1 were detected by qRT-PCR,and microglial polarization phenotype was detected by immunofluorescence.Results:Microglia polarized to M1 phenotype increases after TBI,and the expression of inflammatory factors TNF-α,IL-1β,IL-6 and IL-10 also increased significantly after TBI.Rosiglitazone promote microglia polarization to M2 phenotype and reduce the expression of inflammatory factors TNF-α,IL-1β,IL-6 and IL-10 in cerebral cortex,and also attenuated axonal injury and reduced NSS score.While GW9662 promote microglia polarization to Ml phenotype and increased the expression of inflammatory factors,aggravated axonal injury and NSS score incresed after TBI.Conclusions:TBI promote microglia polarization to M1 phenotype accompanied by up-regulation of inflammatory factors.Rosiglitazone promoted microglia polarization to M2 phenotype and attenuate inflammatory,alleviate axonal injury and improve the neurological function outcome after traumatic brain injury. |
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