Background: Treating central nervous system (CNS) damage caused by stroke or traumatic injury currently has few therapeutic options. Neurons have traditionally been the main focus of investigations into understanding and treating CNS damage and repair. However, a growing body of literature indicates that knowledge about multicellular signaling interactions is needed.

Objective: To examine gene expression, molecular signaling and ligand-receptor interactions among different cell types during wound repair after focal ischemic stroke.

Methods: Ischemic stroke lesions were induced in young (2-4 months) and aged (>18 months) mice by injecting 1.5µl (27.4µg/µl) of the vasoconstrictor L-NIO (N5-(1-iminoethyl)-L-ornithine) into the striatum. Tissue sections were evaluated over a 0–14-day timeframe using immunohistochemistry (IHC) and single cell spatial transcriptomic (scST) using CosMx Spatial Molecular Imager. Bioinformatics and CellChat (v1.5) analyses were performed in R (v4.4) using Seurat package (v4).

Results: IHC and scST showed changes in many cells type during wound repair and neuroprotective astrocyte border formation around lesion cores between 3-14 days after stroke. UMAP analysis of 5- and 14-day samples identified cell clusters that were characterized as 11 unique cell types examined in this study. In both 5- and 14-day samples, astrocyte border and phagocytic microglia were present in stroke samples and essentially absent from controls. These differences persisted after stratifying by age. In both young and aged mice, ApoE and its receptors, Trem2+Tyrobp, were the most abundantly expressed ligand-receptor pair. ApoE was variably expressed by multiple cell types. Phagocytic microglia, macrophages, and microglia were the most prominent targets of ApoE signaling in both young and old mice. Additional ligand-receptor pairs and corresponding cell-cell interactions were also identified.

Conclusions: IHC and scST can identify cell-cell ligand-receptor interactions involved in wound repair after CNS ischemic stroke and can contribute to understanding molecular mechanisms potentially useful for developing targeted therapeutic strategies to improve outcomes.