Microglia Phenotype Changes At The Acute Stage After Ischemic Stroke: A Study From Single-cell Dimension

Background and objective:Acute large-vessel occlusion ischemic stroke is a severe central nervous system disease with poor outcomes.Due to the fast deterioration and limited time window for acute treatment,recognition of pathological mechanisms during the early hours is the key to finding effective treatment.As the resident immune cells,microglia play important roles in the acute phase of ischemic stroke,while the classification of M1/M2 polarization at very early stages after ischemic stroke remained controversial,which impeded the further exploration of acute neuroprotective strategies.This research aims to explore the phenotype change of microglia at the super early phase after ischemic stroke and blood reperfusion from single-cell dimension.Method:We simulate the acute pathological processes of ischemic stroke from normal condition to acute cerebral ischemia and then to the reperfusion period using the middle cerebral artery occlusion model in mice.Blood flow and pathological changes of the ischemic region and the state of microglia re-distribution were evaluated using laser doppler perfusion imaging,triphenyl tetrazolium chloride staining,hematoxylin-eosin staining,Nissl staining,and immunofluorescence staining.The temporal change of genes profile,cell subtypes,and microglial subpopulation were analyzed using single-cell RNA sequencing.Bioinformatics analysis methods including differentially expressed genes,functional pathway analysis,transcription factor regulatory network Analysis,and cell communication analysis were performed to further explain the heterogeneity of microglia during the acute phase of ischemic stroke.Double-immunofluorescence staining was performed to verify the subpopulation classification.Results:This study found that for long-time vascular occlusion stroke,despite the restored perfusion in the ischemic hemisphere after recanalization,long-term reperfusion was not satisfactory,and the blood flow distribution was not identical to that before the experiment.A total of 128,477 single-cells were enrolled in this analysis after qualified control,labeled by astrocyte,neuron,microglia,oligodendrocyte,vessel endothelial cell,pericyte,B cell,T cell,neutrophil,boarder-associated macrophage,and peripheral-origin macrophage.In this system,we identified 37,614 microglial cells divided into eight distinct subpopulations.Three subpopulations mainly constructed by cells from control samples were found including one homeostatic subpopulation characterized by high expression of Hpgd,Tagap,and Cited2,and two preliminary activated phenotypes characterized by P2ry13 and Wsb1 respectively,which suggested microglia did not entirely show homogeneous under the normal state.After ischemic stroke,two clusters of microglia both exhibited M1-like polarization manifested by upregulation of inflammatory genes,whereas they were not identical and the intrinsic differentiation was observed in pseudotime analysis,presented as diminished neurotrophic property,and amplified inflammation responses.Meanwhile,we identified three special clusters with a low inflammation level,classified by high expression of Arhgap45,Rgs10,and Pkm separately.However,no evident M2-like characteristics were observed in these cells and the classic microglia function was also attenuated.These subpopulations exhibited higher activation of neuropeptide functional pathways.Furthermore,we performed cell-cell communication analysis and identified major couplings contributing to the interaction between microglia and other cell populations.Conclusion:In this study,based on the blood flow and pathological changes after ischemic stroke,the single-cell expression landscape of early neural cells was established.Eight microglia subsets were identified revealing the temporal heterogeneity of microglia in the acute stage of ischemic stroke,which provided potential intervention targets for microglia protection in the early stage of stroke.