Early Transcriptional Changes in Neutrophil-Mediated Processes Following Recanalization After Ischemic Stroke

Ischemic stroke is a leading cause of death and long-term disability worldwide. Recanalization therapies, including thrombolysis and mechanical thrombectomy, restore blood flow, yet many patients experience poor outcomes, a phenomenon known as futile recanalization. Given the short therapeutic window for ischemic stroke, identifying early biomarkers to guide targeted interventions and improve outcomes is critical.

Using a murine middle cerebral occlusion model that mimics a large vessel occlusion with recanalization, a comprehensive microarray analysis from blood samples collected immediately and 3 hours after recanalization (N=44) was performed. Differentially expressed genes, enrichment pathways, immune cell proportions, enriched cell markers, predicted micro-RNAs, and transcription factors were identified using RStudio. Findings in mice were validated with rat middle cerebral artery occlusion (GSE21136) and patients with stroke (GSE16561) data sets to confirm transcriptional changes in peripheral blood postrecanalization.

Il1r2, Cd55, Mmp8, Cd14, and Cd69 were early biomarkers poststroke and postrecanalization. Cross-validation revealed Vcan as a differentially expressed gene conserved across species, making it a novel ischemic marker detected as early as 3 hours postrecanalization (4 hours after middle cerebral artery occlusion) in mice, 24 hours after recanalization in rats (middle cerebral artery occlusion-thrombectomy), and within 24 hours from onset in humans receiving recombinant tissue plasminogen activator-thrombolysis. CIBERSORTx and ImmuCellAI-mouse deconvolution showed neutrophil elevation postrecanalization. Leukocyte and neutrophil activation pathways were enriched early after stroke in mice and humans, with stronger upregulation in the female sex. Several regulatory micro-RNAs were identified, and Nuclear Factor Erythroid 4 (NFE4) and Metal Regulatory Transcription Factor 1 (MTF1) emerged as key transcription factors. A coregulatory network underlying neutrophil activity was constructed, highlighting its central role in early responses to ischemia and recanalization, which was enriched in the female sex.

We identified novel early genomic markers for ischemia and recanalization, including the conserved marker Vcan, and highlighted age- and sex-specific immune responses. Mapping a neutrophil-centered coregulatory network provides mechanistic insight into futile recanalization and supports the development of targeted therapies to improve clinical outcomes.