Neuroprotective Effects And Mechanism Of Ischemic Postconditioning Against Cerebral Ischemia/reperfusion Injury

Objective: Both local brain inflammation and systemic Stroke induced immunodepression(SIID)play a critical role in stroke pathology,yet the effect of IPost C on brain inflammation has been poorly studied particularly the effect of IPost C on SIID.We have established an IPost C model in mice to provide for future studies on the underlying mechanisms of IPost C against stroke in mice.By using the novel technique of high-dimensional mass cytometry(Cy TOF),we quantified leukocyte phenotypes after IPost C,studied whether expression levels of cell surface makers reflect neuroinflammatory status,and whether changes in intracellular signaling pathways in the brain tissue and peripheral blood leukocytes are correlated with neuroinflammation after stroke.This study investigates how IPost C affects infiltration of leukocytes in the ischemic brain and lymphopenia associated with stroke-induced immunodepression.Oxidative stress plays an important role in cerebral ischemia-reperfusion injury.Dimethyl fumarate and monomethyl fumarate are antioxidant agents that activate the nuclear factor erythroid-2-related factor 2(Nrf2)/heme oxygenase-1 pathway and induce the expression of antioxidant proteins.The impact of dimethyl fumarate and monomethyl fumarate on the recovery from acute ischemia-induced brain injury is unknown.Methods: Stroke was induced by 45 min of middle cerebral artery occlusion in mice.IPost C was performed by a repeated series of brief occlusions of the middle cerebral artery(MCA)after reperfusion,in a focal ischemia model in mice.Infarct sizes,neurological scores were analyzed.Leukocytes were isolated from the ischemic brain,peripheral blood at 1,3 days after stroke,and stained with antibodies for 25 cell surface makers.The blood leukocytes were further stained with 9 antibodies defining intracellular signaling pathways.The samples were measured by the Cy TOF technique,and the acquired data were analyzed by manual gating strategy,unsupervised SAPDE(spanning-tree progression analysis of density-normalized events)algorithm,vi SEN and heatmap.Transient focal brain ischemia model was employed to examine the role of dimethyl fumarate and monomethyl fumarate in the treatment of acute ischemic stroke in wild type and Nrf2 mutant mice.Neurobehavioral evaluation,MRI,histology,neural cell death,glial activation and expression of genes related to the Nrf2 pathway were analyzed.Results: IPost C performed 2 min after reperfusion significantly reduced infarct sizes and attenuated neurological scores as measured up to 3 days post-stroke.In the group with IPost C,infarct sizes were reduced.The spared infarct areas were seen in the ischemic penumbra or ischemic margins,i.e.,the border zones between the cortical territories of the anterior cerebral artery(ACA)and those of the MCA,as well as in the ventromedial and dorsolateral striatum.Cy TOF analyses showed that The manual gating strategies identified 16 subtypes of leukocytes,including Mi DMs,Mo DMs,Neutrophil,?? T,CD4+T,CD8+T,Eff/mem CD4+T,Eff/mem CD8+T,NK,NK T cells,c DCs and so on.IPost C significantly blocked increases in the numbers of Mi DMs,Mo DMs,Neutrophil,?? T,CD4+T,CD8+T,Eff/mem CD4+T,Eff/mem CD8+T,NK,NK T as well as c DCs in the ischemic brain.Reduced-immune cell numbers in the peripheral blood were increased by IPost C,including monocyte,?? T,CD4+T,CD8+T,Eff/mem CD4+T,Eff/mem CD8+T,NK,NK T cells as well as c DCs.The SPADE analyses suggest that the protein levels of cell surface markers showed dynamic changes after stroke,including CD45,CD49 b,CD11c,Ly6 G,Ly6C,B220,CD3,CD4,CD8,CD44 and CD25.In addition,protein expressions of Ik B?,p AKT,p P38 and p EGFR were significantly upregulated in blood and brain neutrophils and monocytes/macrophage after ischemic stroke,and downregulated by IPost C.Dimethyl fumarate and monomethyl fumarate significantly reduced neurological deficits,infarct volume,brain edema,cell death and glial activation following transient focal brain ischemia.The protection of dimethyl fumarate and monomethyl fumarate was mostly evident during the subacute stage and was absent in Nrf2 deficient mice.Conclusions: IPost C reduced brain infarction and mitigated neurological deficits in mice,likely by blocking infiltration of both innate and adaptive immune cells in the ischemic brain.In addition,IPost C robustly attenuated peripheral lymphopenia and thus improved systemic immunodepression.We conclude that upregulation of cell surface markers or of cell signaling pathways are well correlated with neuroinflammation after ischemia stroke.Dimethyl fumarate and monomethyl fumarate may have therapeutic potential in acute cerebral ischemia-reperfusion injury and their protective role is likely mediated by Nrf2 pathway.