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Stroke has been the leading cause of adult morbidity and mortality over the past several years. After an ischemic stroke attack, many dormant or reversibly injured brain cells exist in the penumbra area. However, the pathological processes and unique cell information in the penumbra area of an acute ischemic stroke remain elusive. We applied unbiased single cell sequencing in combination with bulk RNA-seq analysis to investigate the heterogeneity of each cell type in the early stages of ischemic stroke and to detect early possible therapeutic targets to help cell survival. We used these analyses to study the mouse brain penumbra during this phase. Our results reveal the impact of ischemic stroke on specific genes and pathways of different cell types and the alterations of cell differentiation trajectories, suggesting potential pathological mechanisms and therapeutic targets. In addition to classical gene markers, single-cell genomics demonstrates unique information on subclusters of several cell types and metabolism changes in an ischemic stroke. These findings suggest that  Gadd45b  in microglia,  Cyr61  in astrocytes, and  Sgk3  in oligodendrocytes may play a subcluster-specific role in cell death or survival in the early stages of ischemic stroke. Moreover, RNA-scope multiplex  in situ  hybridization and immunofluorescence staining were applied to selected target gene markers to validate and confirm the existence of these cell subtypes and molecular changes during acute stage of ischemic stroke.

frontiers in cell and developmental biology

Single-Cell RNA Sequencing With Combined Use of Bulk RNA Sequencing to Reveal Cell Heterogeneity and Molecular Changes at Acute Stage of Ischemic Stroke in Mouse Cortex Penumbra Area