Mechanism Of Glial Polarization After Intracerebral Hemorrhage,a Research Based On Single-Cell RNA Sequencing

OBJECTIVES:Here we investigated the rule of microglia（MG）and astrocyte（ASC）phenotype transition and the possible mechanism of glia cell interaction after intracer-ebral hemorrhage（ICH）through the establishment of time-serial ICH mice models and single cell RNA-sequencing（sc RNA-seq）.MATERIALS AND METHODS:Stereotactic injection of 0.4u L collagen IV was used to produce ICH mouse model.Shams and peri-hematoma brain tissues（n=8）at12h,1d,3d,7d and 14d post-ICH were cut in fresh and lysed into single cell solution for sc RNA-seq based on BD Rhapsody platform.Fastq data were processed with fastp and UMI-tools with default parameters to create single-cell expression matrix.Seurat was used to filter out low quality cells and features,dimension reduction and cell clus-tering.Single R was utilized for cell type identification.Cell trajectory pseudotime anal-ysis was done using Monocle3.For sub-cluster analysis,Gene Ontology and KEGG enrichment were performed and gene set enrichment analysis（GSEA）and gene set var-iation analysis（GSVA）were done to explore cell cluster functions,differences and the tendency of cell polarization.Cell Chat was used to explore the cell-cell interaction in microglia and astrocytes.RESULTS:33392 cells with transcriptomic data from 6 time-serial peri-hema-toma samples were retrieved from sc RNA-seq.17468 microglia and 2574 astrocytes were identified respectively.Based on the expression of C1q,MG were clustered into C1q ^high MG and C1q ^low MG.Pseudotime analysis uncovered the upregulation of Arg1and S100a6 and the downregulation of C1qa,C1qb,C1qc and Apoe in the development of C1q^low MG.C1q^high MG could be seen in all brain samples.In 12h to 1d post-ICH,the activation of AP-1 complex can be seen;upregulation of Mki67 and Top2a was noticed in 3d post-ICH and C1qa/C1qb/C1qc/Apoe were upregulated in 7d and 14d post-ICH.MAPK pathway was enriched in C1q^high MG,while pathways associated with neurodegeneration were enriched in C1q^low MG.GSVA indicated that the pathway of glycolysis,pentose-phosphate pathway and phenylalaline metabolism are enriched in microglia activation.C1q^high MG was enriched in glycosaminoglycan degradation while C1q^low MG was not.2574 astrocytes were classified into 5 types,including:（1）immature ASC（A0）;（2）Mesenchymal ASC（Am）;（3）C3 and Serping1 elevated ASC（A1a）,and C1q elevated ASC（A1b）;（4）S100a10 and Ptx3 elevated ASC A2a and A2b;（5）intermittent ASC（Ai）.Pseudotime trajectory analysis indicated that A1a de-velop via A0→Am→A1a axis,the upregulation of Vim,Gfap,C3 and Serping1 and downregulation of AP-1 components Fos,Fosb,Jun and Junb can be seen.A2a develop from A0→A2 axis,Vim、Gfap、S100a6、S100a10、Ptx3 were upregulated and Gja1,Bcan and Tspan were downregulated.Enrichment analysis indicated the enrichment of proteasome associated pathway in A1 astrocytes in compare with the pathway of neu-rodegeneration in A2 astrocytes.Cell Chat uncoverd that MG interact with ASC via VISTA associated pathway and C1q^low MG to A0 and A1 are interacted through Tgfb pathway,Spp1 pathway,Psap-Gpr37l1 pathway,Osm-Osmr,Nampt and Mif pathways.CONCLUSIONS:This study revealed that pro-inflammatory and anti-inflamma-tory glia cells are derived from different cell proportion.C1q and Apoe is a promising marker for the pro-or anti-inflammatory phenotype of MG.Astrocyte mesenchymal polarization can only be seen in A1a polarization.MG interacts with ASC via VISTA associated signaling pathway and C1q^lowMG alters ASC polarization via Tgfb,Spp1,Psap-Gpr37l1,Osm-Osmr pathways,etc.