Transcriptome Analysis Of Microglia After Subarachnoid Hemorrhage

Background and purposeSubarachnoid hemorrhage(SAH)is usually caused by the rupture of an intracranial aneurysm,which leads to blood accumulation in the subarachnoid space.It accounts for about 5%of all strokes.The onset age of SAH is mostly between 50-60 years old,and the annual incidence is about 6-10 per 100,000 people.The mortality rate is close to 30%,and 30-50%of survivors have delayed neurological dysfunction.For this reason,SAH imposes a heavy burden on society and economy.As previous studies related to cerebral vasospasm failed to improve the clinical outcomes of SAH patients.Recently,more and more scholars have focused their researches on the early brain injury(EBI)after SAH.It has been proven that EBI affects the prognosis of SAH patients.Among the pathological processes of EBI,the neuroinflammatory response,including the activation of immune cells and the release of proinflammatory cytokines,is considered to be the most important.Microglia are specilized resident innate immune cells of the central nervous system,that are mainly derived from the embryonic yolk sac.Microglia continuously monitor the brain milieu,and can be activated rapidly by injuries and pathogens.Activated microglia acquired M1(proinflammatory)and M2(anti-inflammatory)phenotypes.They can also have a role in antigen presentation,phagocytosis,expression and release of cytokines/chemokines,and other functions.Previous studies have suggested that microglia play an important role in neuroinflammation after SAH,and regulation of microglia-mediated neuroinflammation is a potential treatment strategy for SAH patients.So,further work is needed to explore the specific role of microglia after SAH,and the underlying mechanisms of how microglia mediate neuroinflammatory response.At present,the developments of transcriptomics,proteomics,and spatial transcriptomics are of great help in studying cell development,exploring molecular markers,and understanding the pathophysiological mechanisms of diseases.And transcriptomics analysis provides detailed information about the expression of each gene at mRNA level.Previous genomics studies on SAH included RNA-seq of intracranial aneurysm investigating its formation,or miRNA and IncRNA sequencing after SAH.However,transcriptional analysis of microglia after SAH has not been performed.Therefore,the main purpose of this study is to explore the role of microglia after SAH based on the experimental SAH model,and to further understand the neuroinflammatory response mediated by microglia after SAH by analyzing its transcriptomic changes.So as to provide certain ideas and directions for the treatment of SAH.Materials and methodsIn this research,adult male C57BL/6 mice were used to establish a SAH model by endovascular perforation through the internal carotid artery.Microglia depletion was performed by preoperative administration of PLX3397.Neurological scores and behavior tests were accessed after SAH with or without microglia depletion.Then,at 72 hours after SAH,mice were subjected to fluorescence activated cell sorting,subsequently the transcriptomic mRNA sequencing was performed on the sorted microglia.By detecting and analyzing the gene expression profile of microglia between the SAH group and the sham operation group,the differentially expressed genes(DEGs)were determined according to the fold change and adjusted p value.Then functional enrichment analysis were performed based on DEGs to understand the biological processes mediated by microglia after SAH.Then we screened out the relevant molecular receptors and transcription factors that regulated the microglia activation,and related verifications experiments were conducted.Result1.After SAH,the neurological function of the mice was significantly impaired.Microglia were increased significantly,and activated at 72 hours after operation.2.With PLX3397 administration,the number of microglia in the brain tissue after SAH decreased significantly,and the neurological function was partially improved.3.Compared with the sham group,the microglia purified from the injured hemispheres presented strong transcriptional changes at 72 hours after SAH.We identified 1576 DEGs,including 928 up-regulated and 648 down-regulated genes.4.After SAH,microglia cell division,inflammatory response,cytokine production,chemotaxis,and other biological processes are strongly activated,which promoted the infiltration of peripheral monocytes and neutrophils.5.IRF7 participates in the regulation of the immune inflammatory response,cytokine and chemokine-related pathways of microglia after SAH,and is an important transcription factor for microglia activation.6.TLR2 is involved in mediating the activation of microglia,immune-inflammatory response,cytokine and chemokine related pathways and other biological processes.It has protein-protein interaction relationship with IRF7.ConclusionsAfter experimental SAH,the number of microglia increased,and depletion of microglia could partially improve the neurological function of mice after SAH.After SAH,microglia activate immune inflammatory response,cytokine and chemokinerelated pathways,promoting the infiltration of peripheral inflammatory cells,and mediating neuroinflammatory response.The TLR2/IRF7 signaling pathway is considered to be able to regulate part of the pathophysiological process of neuroinflammatory response after SAH.