Spatial Transcriptomics and Proteomics Profiling After Ischemic Stroke Reperfusion: Insights Into Vascular Alterations

Abstract

Background: More than half of patients with ischemic stroke experience futile reperfusion, increasing the risk of death and disabilities despite a successful recanalization. The reason behind this is debated, and we aim to investigate cerebrovascular changes toward a broader understanding of these conditions. We hypothesize that ischemic stroke reperfusion modifies the expression profile in the microvasculature in a spatial manner toward peri-infarct brain edema and circulatory failure.

Methods: We investigated the early (24-hour) changes in spatial gene expression in the brain parenchymal endothelial cells and mural cells following ischemia stroke reperfusion in 13- to 14-week-old C57BL/6JRj male mice (n=5). Ischemia was induced by occlusion of the middle cerebral artery for 60 minutes, and Nissl staining was used to validate infarct size. Spatial transcriptomics complemented by bulk proteomics was conducted in the peri-infarct cortex region and validated with immunohistochemical semiquantification of proteins of interest. To avoid individual biological variations, changes in the peri-infarct cortex region were expressed relatively to the matching contralateral hemisphere region.

Results: Ischemic stroke reperfusion impaired the blood-brain barrier integrity through junctional Cldn5 (claudin-5) downregulation, changes of the actin cytoskeleton adhesion, and high expression of the proinflammatory Il-6 (interleukin-6). Molecules important for extracellular Ca²⁺ influx and intracellular Ca²⁺ release, Cacna1e (R-type Ca²⁺ channels), Orai2, Ryr3, Itpr1, and Itpka (inositol-trisphosphate 3-kinase A), were markedly reduced. Furthermore, reduced Grm5 (glutamate receptor 5) associated with upregulated Nfatc3 and Stat3 implicates suppression of the contractile phenotype, suggesting reduced poststroke vascular resistance due to loss of mural cell tone. The complete spatial transcriptomics map over the ipsilateral and contralateral hemispheres is available online as a Web tool.

Conclusions: Emphasizing the spatial molecular pattern behind blood-brain barrier disruption and loss of the vascular tone in the acute phase following ischemic stroke reperfusion suggests the gene expression contribution for a therapeutic target in ischemia-reperfusion abnormalities.

Keywords: blood-brain barrier; endothelial cells; ischemic stroke; pericytes; proteomics; transcriptomics; vascular smooth muscle.