Experimental Study Of Zinc Modulation Of Brain Ischemia-induced Inflammatory Response Affecting Neurological Damage

ObjectiveThe inflammatory response after cerebral ischemia is the main cause of disease progression,and different phenotypes of T cells affect inflammation in the brain through multiple pathways,such as modulating immune responses and interacting with immune cells in the brain.Zinc plays an important role in neurological repair in the brain during different periods of cerebral ischemia,but studies have mainly focused on the central nervous system,while little is known about the role of zinc in systemic immune regulation after cerebral ischemia.Therefore,the aim of this study was to investigate the effects of zinc on cerebral and splenic CD4~+T cells during the acute phase of cerebral ischemia and to determine that zinc ions affect the proliferation and activation of peripheral splenic CD4~+T cells during the acute phase of cerebral ischemia by modulating the microglial inflammatory response,which further aggravates post-ischemic brain injury.Methods1.In this study,male SD(Spregue-Dawley)rats were selected to make an animal model of MCAO(Middle cerebral artery occlusion)to study the effect of zinc ions on T-cell infiltration in rat spleen and brain tissue during the acute phase of cerebral ischemia.The experimental groups were: sham-operated group(Sham),model group(MCAO),and three time points of ischemia two-hour reperfusion for 1,3 and 7 days were selected for each treatment.The number or proportion of CD4~+T cells,CD8+T cells,Th1(CD4~+IFN-γ+)cells,Th2(CD4~+IL-4~+)cells in the spleen and brain tissues of rats were detected by flow cytometry,and the changes of zinc ions in the brain were detected by applying immunofluorescence staining.After that,on the basis of rat MCAO model,TPEN(15 mg/kg)or equal volume of DMSO was injected,and the neurological function score and brain infarct volume were examined for assessing the protective effect of chelated zinc on brain injury in ischemic rats in the acute phase;after that,we chose the time point of two hours of ischemia and three days of reperfusion to perform flow cytometry on CD4~+T,Th1 and Th2 in the spleen and brain of rats.We then performed flow cytometry on CD4~+T,Th1 and Th2 in the spleen and brain.We also observed the microglia typing and the changes of COX2,iNOS,IFN-γ,IL-1β and TNF-α in the brain tissue grinds by ELISA.2.Culture rat microglia(RM)to study the effect of zinc ions on the secretion of inflammatory factors by microglia,and the proliferation and activation of CD4~+T cells promoted by their secreted inflammatory factors.Experimental groupings: Con,OGD,OGD+Zinc.The concentration and time of zinc administration were firstly determined by CCK-8 assay.ELISA was performed to detect the content of IL-1β,TNF-α and IFN-γ in the culture medium.After that,rat spleen CD4~+T cells were sorted out and CD4~+T cells were cultured with microglia medium.CD4~+T cell proliferation was detected after 5 d of CFSE labeling,and the ability of CD4~+T cells to secrete IFN-γ was detected by ELISA.Results1.During the acute phase of cerebral ischemia,zinc ions accumulate in large numbers on the ischemic side of the brain.The proportion of CD4~+ T cells and their subtype Th1 cells was increased in the spleen and brain compared to Sham.Compared with the model group,rats had reduced brain infarct volume and neurological scores after zinc chelation,and the number of CD4~+ T cells and the proportion of Th1 cells in spleen and brain tissues were decreased.These results suggest that zinc chelation promotes neurological repair in the acute phase of cerebral ischemia in rats,while the proportion of pro-inflammatory cells decreased after zinc chelation,suggesting that zinc ions are involved in the changes of CD4~+T cell number and function in the periphery and brain.Subsequently,in an assay of microglia in the brain,it was found that the proportion of microglia of M2 type increased and the secretion of pro-inflammatory cytokines decreased in rats after zinc chelation.This result suggests that zinc ions are involved in the activation process of microglia.2.Zinc promotes the proliferation and activation of CD4~+T cells by inducing microglia activation and secretion of pro-inflammatory factors.We first determined the optimal concentration and time of zinc administration by CCK-8,which were 50 μM and 48 h.After that,we confirmed that zinc could activate microglia to release pro-inflammatory inflammatory factors IL-1β,TNF-α,and IFN-γ.To further verify that zinc ions stimulate the activation and proliferation of CD4~+T cells by first activating microglia in the brain and thus stimulating CD4~+T cells.We first labeled CD4~+T cells with CFSE,and after 5 d of culture,flow cytometry revealed that CD4~+T cells cultured with microglia medium containing zinc ions had proliferation and increased IFN-γ secretion,whereas CD4~+T cells directly added with zinc ions did not have proliferation.This result suggests that the activation of CD4~+T in peripheral immune organs by zinc ions after ischemia is not produced directly,but that zinc activates microglia to release pro-inflammatory cytokines before causing changes in CD4~+T number and function.ConclusionsIn conclusion,the results of the present study showed that zinc chelation during the acute phase after cerebral ischemia in rats reduced post-ischemic brain injury,and the proportion of CD4~+T and its fractional Th1 cells was downregulated after zinc chelation.At the same time,this study illustrated that zinc ions first activated microglia in the brain,and then stimulated CD4~+T differentiation and proliferation.This study reveals the mechanism of zinc affecting peripheral immune cells in the brain during the acute phase of cerebral ischemia,and provides new ideas to improve patient prognosis.