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

Abstract

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.

Single-cell RNA-seq reveals the transcriptional landscape in ischemic stroke

“…In agreement, Guo et al. 52 recently profiled the single-cell transcriptional analysis of the mouse brain cortex penumbra area 24 h after MCAO, which identified 13 major cell types, each showing similar gene expression patterns to our study. However, the composition ratio of the cell types was different between the two studies (Guo et al.…”

“…Hence, cell-type specific regulatory genes, especially those involved in subcluster associated diseases, should be considered for biomarkers in stroke, and ischemic insult caused alteration of DEGs across multiple cell types may have a potential implication for the intervention. In agreement, Guo et al 52 recently profiled the single-cell transcriptional analysis of the mouse brain cortex penumbra area 24 h after MCAO, which identified 13 major cell types, each showing similar gene expression patterns to our study. However, the composition ratio of the cell types was different between the two studies (Guo et al versus our study), probably owing to several reasons, including in the subclusters classification of cell types, the different brain tissues sampled (cortex penumbra versus cerebral hemisphere), varying enzymatic methods for tissue dissociation and whether samples were mixed or not for library construction (yes versus no) and so on.…”

“…In agreement, Guo et al. 52 recently also identified 14 microglia subclusters in cortex penumbra, while, none of which fully expressed M1 or M2-type marker genes. In addition, the core marker genes of microglia including Gpr34, P2ry12, and Cx3cr1 were also downregulated after MCAO in both Guo et al and our study.…”

Interleukin 13 promotes long-term recovery after ischemic stroke by inhibiting the activation of STAT3

“…5 C). This phenotype has also been reported in stroke and many other diseases [ 50 , 52 , 53 ], indicating the inflammatory disease-associated phenotypes of MG 1/3 cluster. Strikingly, MG 3 cluster also demonstrated upregulated expression of Itgax, Lyz2 (Fig.…”

The Specific Role of Reactive Astrocytes in Stroke

“…Particularly, Hasel’s recent single-cell sequencing of LPS-induced RAs revealed that RAs could be divided into nine subpopulations with distinct gene expression and some subgroups appear in different CNS diseases ( Hasel et al, 2021 ). In the study of ischemic stroke, astrocytes in ischemic penumbra can be divided into seven subgroups and proposed related pathological mechanisms and therapeutic targets ( Guo et al, 2021 ). The spatiotemporal heterogeneity of RAs brings us many questions.…”