Exploration Of Microenvironment After Cerebral Ischemia-reperfusion Based On Single-cell Transcriptome Sequencing Technology

Background:Stroke has the characteristics of high morbidity,high disability and high mortality.It is the second leading cause behind acute myocardial infarction.The incidence of ischemic stroke is higher,accounting for 87%of all cerebrovascular diseases.Cerebral ischemia-reperfusion injury(CIRI),ischemic stroke returns to normal blood circulation after a period of time,when hypoperfusion ischemia and hypoxic brain tissue regains blood supply,the ischemic injury further aggravates,More serious brain injury phenomena include nerve cell death and fatal brain edema.As a result,most patients are unable to fully recover normal neurological functions,receive timely and comprehensive rehabilitation treatment in time,and still have a certain degree of functional impairment,including limb paralysis,swallowing disorders,or aphasia.Therefore,finding potential therapeutic targets for ischemic stroke has become an urgent scientific problem.The central nervous system is very complicated,in part because there are many types of cells and they have specific gene expression patterns,and single-cell transcriptomics can accurately identify the classification of different cells in the central nervous system.By accurately classifying different cells in the central nervous system and clarifying their biological functions,a deeper study of central nervous system diseases is possible.Single-cell sequencing technology can quickly and accurately determine the gene expression patterns of thousands of cells,analyze the genetic information heterogeneity of cells of the same phenotype,and reveal the composition,distribution,function,and development status of cell subpopulations in the microenvironment in a panoramic manner.Discover new subgroups and their functions.It has been applied in many fields such as neurobiology,organ growth,cancer biology,and embryology.However,there is no research to explore cerebral ischemia-reperfusion injury at the single cell level.Therefore,this study intends to draw a map of the brain tissue microenvironment after cerebral ischemia-reperfusion injury by using single-cell sequencing technology.Methods:This study intends to establish a rat cerebral ischemia-reperfusion model(MCAO)to perform enzymatic hydrolysis and cell suspension of edema cerebral cortex tissues.Single-cell transcriptome sequencing was used,combined with cluster analysis and t-SNE visualization and other bioinformatics methods to distinguish cell subgroups,while using GO function enrichment and KEGG pathway enrichment to reveal the biological function of each cell subgroup.Results:The results showed that rat brain tissue can be roughly divided into 20 cell subgroups by single cell sequencing analysis,including 3 types of microglial cell subgroups,3 types of oligodendrocyte subgroups,5 types of endothelial cell subgroups,and Ependymal cells,neuronal cells,astrocytes,etc.;Comparing the cell subgroup gene expression in the experimental group(MCAO)and the control group(Sham),it was found that 9 subgroup cells had significant differences between the two samples.Including cell subpopulation 7(log2FC=-1.159,p<0.001),cell subpopulation 3(log2FC=0.535,p<0.001),etc.Comparing different cell subpopulations to find differential genes,and interpreting them by enrichment analysis For its biological function,this study confirms that the most significant biological processes of cell subgroup 1 enrichment include T cell activation,regulation of lymphocyte activation and angiogenesis.Cell subgroup 2 is mainly enriched in intracellular protein transmembrane transport,autophagy,regulation of apoptosis signal pathways and other biological processes.Conclusion:In summary,we used single-cell sequencing technology to draw a preliminary map of the brain microenvironment of cerebral ischemia-reperfusion injury.We not only performed cluster analysis of brain tissue cells,but also found subcellular cells that changed after cerebral ischemia-reperfusion injury.Bioinformatics analysis of the top 40 genes of cell subpopulations revealed important functional interactions between cells group and provided potential therapeutic targets for ischemic stroke.