Single-nucleus RNA sequencing reveals glial cell type-specific responses to ischemic stroke in male rodents

Abstract

Neuroglia critically shape the brain´s response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition of the early ischemic lesion. Here we present a single cell resolution transcriptomics dataset of the brain´s acute response to infarction. Oligodendrocyte lineage cells and astrocytes range among the most transcriptionally perturbed populations and exhibit infarction- and subtype-specific molecular signatures. Specifically, we find infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and reactive astrocytes, exhibiting transcriptional commonalities in response to ischemic injury. OPCs and reactive astrocytes are involved in a shared immuno-glial cross talk with stroke-specific myeloid cells. Within the perilesional zone, osteopontin positive myeloid cells accumulate in close proximity to CD44⁺ proliferating OPCs and reactive astrocytes. In vitro, osteopontin increases the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition of acutely infarcted brain tissue.

Fig. 1. snRNAseq reveals differential cell cluster abundance and cluster-specific transcriptional perturbations 48 h after ischemic stroke.

a Illustration of study design, depicting brain regions sampled for snRNAseq, from n = 4 Sham control rats and n = 7 middle cerebral artery occlusion (MCAO) group rats. MRI images of brain tissue from Sham-operated, moderate (mMCAO) and severe (sMCAO) MCAO group rats are presented, ischemic lesions are highlighted in orange. Rat schematic created with BioRender.com. MCAO ipsi MCAO group, hemisphere ipsilateral to ischemic lesion, MCAO contra MCAO group, hemisphere contralateral to ischemic lesion. b Uniform Manifold Approximation and Projection (UMAP) plot depicting 68616 nuclei annotated to 29 major cell clusters in the integrated dataset. Cell cluster AC astrocyte cluster, CHOL_IN cholinergic interneurons, EP_M_C ependymal and mural cell cluster, GABA_Amb Ambiguous GABAergic neuronal cluster, GABA_IN_Adarb2 + , GABA_IN_Adarb2- GABAergic interneurons, Adarb2 positive/negative, respectively, GABA_MSN GABAergic medium spiny neurons, GLU_Satb2 + , GLU_Satb2- Glutamatergic neurons, Satb2 positive/negative, respectively, OLIGO_1 immature oligodendrocyte lineage cluster, OLIGO_2 myelinating and mature oligodendrocyte lineage cluster. c–e Dotplots depicting curated marker genes for all major cell clusters. The dendrogram in (c) represents overarching taxons of identified major cell clusters. The dotplot in (d) depicts curated cluster markers of glutamatergic neurons. Colored bars next to the gene names denote established associations to cortical layers. Representative corresponding RNA in situ hybridization (ISH) results are depicted next to the colored bars. All RNA ISH studies were taken from Allen Brain Atlas database, and are referenced in detail in Supplementary Tab. 1. L = layer, CLA = claustrum, ec = external capsule, LSr = lateral septal nucleus, PIR = piriform cortex. e Dotplot depicting marker gene expression in cholinergic and GABAergic neurons. f Stacked bar plot depicting the relative abundance of each cell cluster within each sample. g Nuclei distribution coloured by treatment group. h Gene module score derived from the stroke-associated myeloid cell (SAMC) gene set. i Strip plots depicting distribution of DEGs derived from MCAO ipsi vs Sham and MCAO ipsi vs MCAO contra comparisons, for all major cell clusters. DEGs were calculated using the MAST statistical framework as specified within the Methods section. Source data are provided within Supplementary data.

Fig. 2. Emergence of transcriptionally distinct OPCs and mature oligodendrocytes within infarcted brain tissue.

a–d Subclustering of oligodendrocyte lineage clusters. a Uniform Manifold Approximation and Projection (UMAP) plot depicting 10240 nuclei annotated to 10 subclusters. b Dotplot depicting curated sub-cluster markers. c Stacked bar plot depicting the relative abundance of each subcluster within each group. d Nuclei distribution coloured by treatment group. Subcluster OPC oligodendrocyte precursor cell, COP committed oligodendrocyte precursor, NFOLIGO newly formed oligodendrocyte, MFOLIGO myelin-forming oligodendrocyte, MOLIGO mature oligodendrocyte, MC_OLIGO myeloid cell oligodendrocyte mixed cluster. e Projection of Monocle3 generated pseudotime trajectory onto subcluster UMAP plot, with subcluster OPC_0 as root. f Feature Plots depicting S-phase and G2/M-phase gene module scores. g, h Volcano plots depicting DEGs derived from the comparison of clusters OPC_1 to OPC_0 (g) and MOLIGO_1 to MOLIGO_2 (h), DEGs were calculated using the MAST statistical framework as specified within the Methods section. i Heatmap depicting the average scaled gene expression of curated DEGs, split by subcluster and treatment group. Functional annotations are given on the left side of the gene names. MCAO ipsi MCAO group, hemisphere ipsilateral to ischemic lesion, MCAO contra MCAO group, hemisphere contralateral to ischemic lesion, PIP3 Phosphatidylinositol (3,4,5)-trisphosphate, TK Tyrosine kinase, ECM Extracellular matrix, DAO Diseases associated oligodendrocyte, GAG Glycosaminoglycan. j Clustered heatmap depicting aggregate gene expressions of Monocle3-derived co-regulated gene modules. Modules associated with OPC_1 and MOLIGO_1 are highlighted in orange and magenta, respectively. k The average aggregate expression of the OPC_1 and MOLIGO_1 associated modules is plotted along the pseudo-time trajectory. The Top 25 module-defining genes, as sorted by descending Moran´s I, are depicted in boxes on the right side of the respective gene module feature plots. l Heatmap depicting the top 100 most variable decoupleR-derived transcription factor activities within the oligodendrocyte lineage sub-clustering analysis, split by sub-cluster and treatment group. Color code on top of the heatmap is defined in (i). Source data are provided within Supplementary data.

Fig. 3. Proliferating, VIM-positive OPCs accumulate in the perilesional zone 48 h after MCAO in rats.

a Overview of a representative coronal rat brain section 48 h post filament induced permanent middle cerebral artery occlusion (MCAO), stained for NG2, VIM and Ki67. Grey matter ROIs (GM) are highlighted in violet, white matter ROIs (WM) in lime green, lower right inset depicts a corresponding T2 weighted MRI image from the same animal. Bar = 2 mm b Representative images from GM and WM ROIs of Sham, MCAO contra and MCAO ipsi sections, split by antigen. Ki67 = magenta, NG2 = Cyan, VIM = yellow, DAPI (nuclei) = blue, bars = 50 µm. White arrowheads point to triple-positive cells. MCAO ipsi MCAO group, hemisphere ipsilateral to ischemic lesion, MCAO contra MCAO group, hemisphere contralateral to ischemic lesion. c–f Cell counts within GM (c) and WM (d), respectively, are presented as box plots for NG2⁺/VIM⁺/Ki67⁺ triple positive cells. Box plots depict medians, 25th to 75th percentiles as hinges, minimal and maximal values as whiskers, and individual counts, for each animal as dots. Cell counts for NG2⁺/VIM⁺/Ki67⁺, NG2⁺/VIM^-/Ki67⁺, NG2⁺/VIM⁺/Ki67^-, NG2⁺/VIM^-/Ki67^- are also jointly shown as colored stacked bar plots, for GM (e) and WM (f) ROIs. Data derived from n = 4 animals per group, p values derived from Kruskal–Wallis-H-Tests, followed by Dunn’s post-hoc comparisons. Statistical comparison of all subsets within all ROIs is reported in Supplementary Data 7. Source data are provided as a Source Data file.

Fig. 4. Transcriptional heterogeneity of reactive astrocytes within infarcted brain tissue.

a–d Subclustering analysis of astrocytes. a Uniform Manifold Approximation and Projection (UMAP) plot depicting 1233 nuclei annotated to 5 astrocyte (AC) subclusters. b Dotplot depicting curated homeostatic and reactive astrocyte marker genes. c Stacked bar plots depicting the absolute and relative abundance of each subcluster within each group. MCAO ipsi MCAO group, hemisphere ipsilateral to ischemic lesion, MCAO contra MCAO group, hemisphere contralateral to ischemic lesion. d Nuclei distribution coloured by group. e–g Volcano plots depicting DEGs derived from the comparison of the reactive astrocyte subclusters AC_3 (e), AC_4 (f) and AC_5 (g) to the homeostatic astrocyte subclusters (AC_1 and AC_2, pooled). DEGs were calculated using the MAST statistical framework as specified within the Methods section. h–j Heatmaps depicting the average scaled gene expression of curated upregulated DEGs, derived from the comparison of AC_3 and AC_4 (h) and AC_5 (i) to homeostatic astrocytes, as well as DEGs downregulated in reactive astrocytes (j), split by subcluster and group. BBB Blood brain barrier, ECM Extracellular matrix, Slc Solute carrier, AS Amino acid (k) Clustered heatmap depicting the top 50 most variable transcription factor (TF) activities, within the astrocyte subclustering analysis, split by subcluster and treatment group. Source data are provided within Supplementary data.

Fig. 5. Reactive astrocytes and proliferating OPCs are CD44 positive and abundant in the perilesional zone 48 h after permanent MCAO in rats.

a Overview of a representative coronal rat brain section 48 h post middle cerebral artery occlusion MCAO, stained for GFAP, CD44 and VIM. Grey matter ROIs (GM) are highlighted in violet, white matter ROIs (WM) in lime green, lower right inset depicts a corresponding T2 weighted MRI image from the same animal. Bar = 2 mm. b Representative images taken from GM and WM ROIs of Sham, MCAO contra and MCAO ipsi sections, split by antigen. VIM = magenta, GFAP = Cyan, CD44 = yellow, all overlaid with DAPI (nuclei) = blue. Bars = 50 µm. MCAO ipsi MCAO group, hemisphere ipsilateral to ischemic lesion, MCAO contra MCAO group, hemisphere contralateral to ischemic lesion. c–f Cell counts within GM (c) and WM (d) are presented as box plots for GFAP⁺/CD44⁺/VIM⁺ triple positive cells. Cell counts for GFAP⁺/CD44⁺/VIM⁺, GFAP⁺/CD44^-/VIM⁺, GFAP⁺/CD44⁺/VIM^-, GFAP⁺/CD44^-/VIM^- are jointly shown as colored stacked bar plots, for GM (e) and WM (f) ROIs. g Representative coronal overview, 48 h post MCAO, stained for NG2, CD44, Ki67. GM ROIs in violet, WM ROIs in lime green, lower right inset shows corresponding MRI image from the same animal. Bar = 2 mm. h Representative images from GM and WM ROIs taken from Sham, MCAO contra and MCAO ipsi groups, split by antigen. Ki67 = magenta, NG2 = Cyan, CD44 = yellow. Bars = 50 µm. White arrowheads point to NG2⁺/CD44⁺/Ki67⁺ triple positive cells. i–l Cell counts within GM (i) and WM (j), respectively are presented as box plots for NG2⁺/CD44⁺/Ki67⁺. Cell counts for NG2⁺/CD44⁺/Ki67⁺, NG2⁺/CD44^-/Ki67⁺, NG2⁺/CD44⁺/Ki67^-, NG2⁺/CD44^-/Ki67^- are also jointly shown as colored stacked bar plots, for GM (k) and WM (l) ROIs. Data derived from n = 4 Sham control and n = 5 MCAO group animals, p values derived from Kruskal–Wallis-H-Tests, followed by Dunn’s post hoc comparisons. All box plots depict medians, 25th to 75th percentiles as hinges, minimal and maximal values as whiskers, and individual counts, for each animal as dots. Statistical comparison of all subsets within all ROIs is reported in Supplementary Data 7. Source data are provided as a Source Data file.

Fig. 6. Osteopontin-positive myeloid cells accumulate in the perilesional zone in close proximity to CD44-positive cells 48 h after permanent MCAO in rats.

a Overview of a representative coronal brain section 48 h post MCAO, stained for Iba1, CD44 and osteopontin (OPN). Grey matter ROIs (GM) are highlighted in violet, white matter ROIs (WM) in lime green, lower right inset depicts a corresponding T2 weighted MRI image from the same animal. Bar = 2 mm. b Representative images from GM and WM ROIs of Sham, MCAO contra and MCAO ipsi sections, split by antigen. OPN = magenta, Iba1 = cyan, CD44 = yellow. Bars = 50 µm. MCAO ipsi MCAO group, hemisphere ipsilateral to ischemic lesion, MCAO contra MCAO group, hemisphere contralateral to ischemic lesion. c–f Cell counts within GM (c) and WM (d) are presented as box plots for Iba1⁺/OPN⁺ double positive cells, cell counts for Iba1⁺/CD44⁺/OPN⁺, Iba1⁺/CD44^-/OPN⁺, Iba1⁺/CD44⁺/OPN^-, Iba1⁺/CD44^-/OPN^- are jointly shown as colored stacked bar plots, for GM (e) and WM (f) ROIs. Data derived from n = 4 Sham control and n = 5 MCAO group animals, p values derived from Kruskal–Wallis-H-Tests, followed by Dunn’s post hoc comparisons. All box plots depict medians, 25th to 75th percentiles as hinges, minimal and maximal values as whiskers, and individual counts, for each animal as dots. Statistical comparison of all subsets within all ROIs is reported in Supplementary Data 7. Source data are provided as a Source Data file.

Fig. 7. Osteopontin induces OPC migration but not proliferation in vitro.

a–c In vitro cell migration assay. Cells were seeded in 2 well culture inserts, creating defined 500 µm gaps. NG2 positive cells which migrated into the 500 µm gap were quantified after 48 h of treatment. a Representative images of OPC cell cultures 48 h after incubation without (upper panel: untreated control = UC), or with 1 µg/ml osteopontin (OPN) (lower panel), stained for DAPI (nuclei) = blue, Ki67 = magenta and NG2 = cyan, split by channel. Scale bars denote 500 µm gaps. b Box plot depicting the number of NG2 positive cells, which migrated into 500 µm gaps, for each condition, p-values derived from two-sided unpaired Student´s t test (t = 2.400, df=20, n = 11 replicates per group (independent wells), from 3 independent experiments). In n = 7 replicates (independent wells) per group from 2 independent experiments Ki67 was visualized (c). c Box plot depicting the percentages of Ki67⁺ cells within the 500 µm gap, for each condition, p values derived from unpaired two-sided Student´s t test (t = 0.3612, df = 12). d–e Bromodeoxyuridine (BrdU) incorporation assay. d BrdU incorporation was visualized 24 h after incubation without (UC) (upper panel) or with 1 µg/ml OPN (lower panel). Representative x20 magnification images are shown, stained for DAPI (Nuclei) = blue, BrdU = magenta and NG2 = cyan, split by channel. Scale bars = 100 µm. e Box plot depicting the percentages of BrdU⁺ cells, for each group, p values derived from unpaired two-sided Student´s t test (t = 0.5119, df = 12, n = 7 replicates per group, from 2 independent experiment). All box plots depict medians, 25th to 75th percentiles as hinges, minimal and maximal values as whiskers, and individual counts, for each replicate as dots. Source data are provided as a Source Data file.