Time Course Of Microglial Polarization State After Intracerebral Hemorrhage In Mice

Objective:Microglia are extremely important immune cells in the central nervous system.Under normal circumstances,microglia are branched and immune surveillance is performed on the central nervous system.After intracerebral hemorrhage,hematoma components activate microglial cells.At first,microglia are activated and transformed into M1 phenotype through classical pathways,secreting a large number of pro-inflammatory factors to exert cellular inflammatory effects;on the other hand,microglial cells shift to M2 phenotype by alternative pathways,and secrete some neuroprotective factors to promote neural repair.The transition of microglia from a resting state to an activated state is a dynamic process.Polarized state refers to the ability of microglia to be affected by the surrounding environment,present different phenotypes,and retain varying functions to maintain tissue homeostasis.Compared with peripheral macrophages,microglia exhibit similar and unique characteristics in terms of phenotypic polarization,and thus have innate immune functions.The state of polarization of microglia in different time periods after cerebral hemorrhage is still clear.If the harmful effects of M1 polarization of microglia are suppressed,the neuroprotective effect of M2 polarization is of great significance for the intervention of neurological diseases.This study explores the transformation of microglial M1 and M2 types at different time points after cerebral hemorrhage in mice,and provides a potential basis for possible future interventions in the transformation of pro-inflammatory M1 to anti-inflammatory repair M2.Methods:Forty-eight ICR mice were randomly divided into a surgical control group and a cerebral hemorrhage model group,and the two groups were divided into three subgroups at 1d,3d,and 7d according to different postoperative time points.Cerebral hemorrhage models were prepared by type Ⅳ collagenase in each model group,and 0.9% sodium chloride injection was given at the same time in the surgical control group.Nerve function scores were performed at different time points after operation.Western blot was used to determine the expression of TNF-α,IL-6,BDNF,and IGF1.Immunofluorescence staining was used to label microglia M1 type(Iba1 + CD80)and M2 type(Iba1 + CD206),to evaluate the activation status of microglia in the tissue surrounding the hematoma after bleeding.Correlation analysis of TNF-α,IL-6,BDNF,IGF1 expression and microglia number.Results:Compared with the surgery control group at each time point,the intracerebral hemorrhage group at each time point had different degrees of neurological deficits,which was most obvious at 1 day(P <0.01).At the same time,the expression levels of TNF-α and IL-6 protein increased,which was most obvious at 3 days(P <0.01).Compared with the surgical control group in the same period,the expression of BDNF and IGF1 protein increased in the cerebral hemorrhage group,and the expression of BDNF and IGF1 protein increased significantly on the 3rd and 7th day of cerebral hemorrhage(P <0.01).Immunofluorescence staining showed that M1 was predominant in 1-3 days after intracerebral hemorrhage,and the peak of M1 microglia reached the peak on the 3rd day;M2 microglia was predominant on the 7th day;that is,M1 was predominantly early after hemorrhage(1-3 days);M2 increased at the recovery period after bleeding(3-7 days).After intracerebral hemorrhage,the number of M1 microglia was highly positively correlated with the expression of TNF-α and IL-6,and the number of M2 microglia was highly positively correlated with the expression of BDNF and IGF1.Conclusion:M1 microglia dominate the acute phase of intracerebral hemorrhage(1-3d),and M2 microglia dominate the subacute phase of intracerebral hemorrhage(7d),suggesting that M1 microglia may be associated with post-intracerebral hemorrhage early edema,and M2 microglia may be involved in neural repair after intracerebral hemorrhage.Intervention on M1 microglia to M2 phenotype in the early stage of intracerebral hemorrhage may have important significance through promoting hematoma clearance,reducing edema formation after early intracerebral hemorrhage,and improving neurological deficits.