isolation-flow-cytometric-assessment-neuroimmune-interactions-mini

Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model

Stroke is a leading cause of death and long-term disability worldwide, with ischemic stroke representing the majority of cases. Following an ischemic ...

Capital Medical University

Stroke is a major global cause of death and disability, with ischemic stroke being the most common type. The disruption of the blood-brain barrier (BBB) is a key factor in the pathophysiology of ischemic stroke, allowing immune cells to infiltrate and worsening neuroinflammation. This study uses single-cell RNA sequencing (scRNA-seq) to examine the transcriptional changes in neutrophils, endothelial cells, and T cells during ischemic stroke. Our findings indicate a significant increase in neutrophil and lymphocyte infiltration, along with a notable decrease in endothelial cell populations, demonstrating severe BBB disruption. Differential gene expression analysis shows that endothelial cells lose important characteristics post-stroke, while lymphocytes activate cytotoxic pathways that may lead to neuronal damage. Additionally, we reveal the contrasting roles of CXCR2 and CXCR4 in neutrophil movement and identify neutrophil-derived damage-associated molecular patterns (DAMPs) and matrix metalloproteinases (MMPs) as key drivers of endothelial cell apoptosis. Notably, the S100A8/A9 inhibitor paquinimod significantly protects neurons and reduces lymphocyte infiltration, suggesting that targeting S100A8/A9 could be a promising therapeutic strategy for reducing neurological deficits after ischemic stroke. Overall, these results enhance our understanding of the complex interactions between immune cells and the BBB in ischemic stroke, paving the way for innovative therapeutic approaches aimed at maintaining brain integrity and improving patient outcomes.

Single-cell RNA sequencing reveals S100A8/A9 neutrophils-induced endothelial cell death and lymphocyte infiltration after ischemic stroke.

Stroke is a worldwide leading cause of death and long-term disability, with ischemic strokes making up approximately 85% of all cases. There is a significant need for an ideal animal model that accurately replicates the disease's pathology to study the molecular mechanisms of brain injury. Various experimental models have been created to induce middle cerebral artery occlusion (MCAO), including intraluminal MCAO, photothrombotic models, endothelin-1 injections, and electrocoagulation. However, these often result in large infarct or lesion volumes accompanied by considerable variability. In this study, we present a ministroke model that specifically targets the mouse barrel cortex, making it suitable for investigating the mechanisms of minor strokes and stroke recurrence. In our model, the distal branch of the right middle cerebral artery (MCA), which supplies the sensorimotor cortex, is permanently ligated using 10-0 sutures. This is followed by a 7-min occlusion of the bilateral common carotid arteries (CCAs) and subsequent reperfusion. This approach produces a mild stroke characterized by small and consistent lesion volumes and very low mortality rates. A well-trained experimenter can achieve nearly zero mortality with this technique. Furthermore, this model of localized ischemia induces lesions in the functionally defined barrel cortex, allowing the use of the vibrissae-evoked forelimb placing test to assess functional outcomes. Key features • Introduces a novel ministroke model targeting the mouse barrel cortex, specifically designed for studying minor strokes. • Achieves consistently small infarct sizes and low mortality rates, allowing for long-term assessments of functional outcomes. • Requires specialized surgical skills for the permanent ligation of the right middle cerebral artery and temporary occlusion of common carotid arteries. • The adhesive removal test and the vibrissae-evoked forelimb placing test exhibit remarkable sensitivity in this model.

Article	Year
Single-cell RNA sequencing reveals S100A8/A9 neutrophils-induced endothelial cell death and lymphocyte infiltration after ischemic stroke. Biochemical and biophysical research communications\| PubMed ID: 39579533	2024
Developing a Ministroke Model in Mouse Barrel Cortex. Bio-protocol\| PubMed ID: 40395851	2025

Yuhan Sun

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