| Background and objective:Stroke is a common disease that seriously endangers human health and life.With its high morbidity,mortality,disability and recurrence rate,stroke,cardiovascular diseases and malignant tumor are the three major causes of human death.In recent years,the epidemiological data of our country showed that ischemic stroke accounts for about 85%of stroke and has become the first fatal disease in our country.However,even the most surviving stroke patients also have neurological deficits,which seriously affect the physical and mental health of patients and their families and it gives a huge economic burden to the patient family and society.Therefore,we urgently need to find new targets for the prevention and treatment of stroke.Repulsive guidance molecule a(RGMa)is a member of the repulsive guidance molecule family.RGMa play multiple roles in the central nervous system.Our research team has been focused on the role of RGMa in ischemic stroke for a long time.In the middle cerebral artery occlusion/reperfusion(MCAO/R)model of rats,RGMa began to rise 12 hours after ischemic injury and 48 hours increased to the peak,then it began to decline,but it was still higher than the normal level for 14 days.Inhibition of RGMa expression by virus or RGMa specific antibody could significantly improve neurological deficits after MCAO/R.But after stroke,the whole system of the body often reacts to stress.So,the central nervous system is also not a single cell or tissue involved in the combat.The purpose of this study is to explore the overall expression and distribution of RGMa in brain tissue after MCAO/R injury and to reveal the stress response of RGMa in CNS after MCAO/R injury.Methods:1.MCAO/R model was performed in adult male SD rats at 240-280 g.RGMa was co-stained by immunofluorescence staining with neural stem cell marker Nestin,oligodendrocyte precursor cell marker NG2,mature oligodendrocyte marker CC1,microglia marker Ibal,neuron marker NeuN,vascular endothelial cell marker CD31,vascular smooth muscle cell marker?SMA,Fibronectin and Collagen I-the markers of extracellular matrix after MCAO/R injury at day 7 and day 14.2.MCAO/R model was constructed in adult male SD rats at 240-280 g.The marker of M1 and M2 microglia-interleukin-1?(IL-1?),inducible nitric oxide synthase(iNOS),arginase-1(Arg-1)and CD206 were detected by quantitative real-time polymerase chain reaction(qRT-PCR)at day 7 and day 14 after MCAO/R injury.3.Microglia cells were cultured in vitro.After 24 hours of induction with lipopolysaccharide(LPS)100 ug/L and interleukin-4(IL-4)20 ug/L,the expression of M1 and M2 microglia marker molecules(IL-1?,iNOS,Arg-1,CD206)was detected by qRT-PCR and the expression of RGMa in M1 and M2 microglia was detected by Western blot.Results:1.RGMa was expressed in neural stem cells,oligodendrocyte precursor cells,oligodendrocyte,microglia,neurons,vascular endothelial cells and vascular smooth muscle cells at day 7 and day 14 after MCAO/R injury,but it did not exist in extracellular matrix.2.The expression of marker molecules of M1 and M2 microglia increased at day 7 and day 14 after MCAO/R injury.RGMa was expressed in activated and polarized microglia.3.The expression of RGMa in LPS-induced polarized M1 microglia and IL-4-induced polarized M2 microglia increased significantly in vitro.Conclusion:After MCAO/R injury,RGMa is widely expressed in central nervous system(CNS).With the prolongation of ischemia time,microglia were activated and polarized and the expression of RGMa increases significantly in both M1 and M2 microglia.RGMa may play an important role in the activation and polarization of microglia. |
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