Characterizing stroke-related cellular changes in the surviving neurons of mouse ischemic stroke

Rapid restoration of cerebral blood flow through endovascular therapy is crucial for minimizing neuronal injury in ischemic stroke. This study characterized cellular and molecular alterations during the acute and subacute phases of distal middle cerebral artery occlusion (dMCAO) in mice using single-nucleus (snRNA-seq) and single-cell (scRNA-seq) RNA sequencing. C57BL/6 mice were assigned to control, sham, dMCAO 3-day, and dMCAO 14-day groups. snRNA-seq identified diverse cell populations, including neurons (glutamatergic and GABAergic), fibroblast-like cells, astrocytes, oligodendrocytes, microglia, endothelial cells, and pericytes. Microglia shifted from homeostatic ( Siglech , P2ry12 ) to acute-phase ( Lgals1 , Top2a , Mki67 ) and disease-associated states, consistent with previous evidence confirming that our dataset captured stroke-related dynamics. snRNA-seq enabled efficient recovery and analysis of neurons, revealing stroke-induced cell state changes.; notably, glutamatergic neurons declined on day 3, while endothelial cells increased. Gene ontology analysis indicated neuronal death, autophagy, and cAMP biosynthesis pathways. Elevated Syngap1 , Ikbkb , and Rock1 expression across glutamatergic subclusters suggested roles in cell death–related mechanisms and vulnerability to ischemic injury. Dissociation of SynGAP1 from PSD-95 after ischemia may enhance ERK1/2 phosphorylation, whereas ischemic preconditioning suppresses this dissociation and prevents ERK1/2 overactivation. Immunohistochemistry confirmed Syngap1 and cAMP response element-binding (CREB) pathway activation at 3 and 14 days post-ischemia, aligning with sequencing results. Suppressing CREB with pAAV-A-CREB reduced neuronal survival, underscoring its role in autophagy and neuroprotection. These findings provide mechanistic insight into stroke-induced molecular alterations and identify autophagy and cAMP pathways within the penumbra as promising therapeutic targets. • snRNA-seq revealed stroke-induced cell state changes with efficient recovery of neurons. • snRNA-seq identified distinct cell types, including neurons, fibroblast-like cells, astrocytes, oligodendrocytes, microglia, endothelial cells, and pericytes. • Microglia transitioned from homeostatic to acute-phase and disease-associated states. • SynGAP1 dissociation linked ERK overactivation to ischemic neuronal injury. • Gene ontology analysis revealed neuronal death, autophagy, and cAMP biosynthesis pathways across all three glutamatergic subclusters.