Single-nuclei RNA sequencing unveils astrocyte and oligodendrocyte lineage cells in post-stroke human brain

BACKGROUND: Ischemic stroke (IS) induces profound, region-specific shifts in cellular composition and gene expression, particularly within glial populations such as astrocytes, oligodendrocytes, and oligodendrocyte precursor cells (OPCs). These glial responses are central to post-stroke neuroprotection, axonal stability, and myelin repair. METHODS: We performed single-nucleus RNA sequencing (snRNA-seq) on surgically obtained human cortical tissues: two IS cases (superficial frontotemporal peri-infarct cortex resected during decompressive craniectomy within ∼24 h of onset) and three comparator samples (cortical tissue from craniocerebral trauma surgery). Key glial transcriptional alterations were validated in a mouse photothrombotic middle cerebral artery occlusion (pMCAO) model using Western blot (WB), quantitative PCR (qPCR), and immunofluorescence. RESULTS: We analyzed 23,638 nuclei, resolving eight major cell types. IS samples showed relative increases in astrocytes and OPCs and decreases in mature oligodendrocytes, consistent with reactive gliosis and oligodendrocyte loss. Differential expression identified NMNAT2, HIF3A, NRP1, SEZ6L, and GPM6A as key upregulated genes enriched in astrocyte and oligodendrocyte lineage cells. Pseudotime analysis indicated a shift of OPCs toward earlier lineage states and trajectories consistent with remyelination after IS. Synaptic pathway enrichment with unchanged PSD95 and SYN levels suggests transcriptional glial-neuronal regulation rather than large-scale synaptic protein alteration. Validation by WB, qPCR, and IF confirmed the translational relevance of these findings across species. CONCLUSIONS: This study provides a peri-infarct human cortical snRNA-seq atlas after IS, revealing coordinated glial transcriptional responses that promote cellular adaptation and repair. The upregulation of NMNAT2, HIF3A, NRP1, SEZ6L, and GPM6A highlights potential therapeutic targets for enhancing neuroprotection and remyelination after stroke.