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

 Hyperhomocysteinemia (HHcy) has been considered as a risk factor for cardiovascular disease, Alzheimer's disease, nonalcoholic fatty liver, and many other pathological conditions. Vitamin B6, Vitamin B12, and folate have been used to treat HHcy in clinics. However, at present, clinical therapies of HHcy display unsatisfactory effects. Here, we would like to explore a new mechanism involved in homocysteine (Hcy) metabolic disorders and a novel target for HHcy treatment. The key enzymes involved in Hcy metabolism deserve more insightful investigation. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the intracellular Hcy metabolism. Until now, the effect of post-translational modification on the bioactivity of  still remains unclear. This study aimed at exploring the relationship between  S-sulfhydration and its bioactivity, and at identifying the contribution of an elevated Hcy level on  bioactivity.  By both  and  studies, we observed the following results: (i) The bioactivity of  was positively associated with its S-sulfhydration level; (ii)  was modified at Cys32, Cys130, Cys131, Cys193, and Cys306 by S-sulfhydration under physiological conditions; (iii) Hydrogen sulfide (HS) deficiency caused the decrease of  S-sulfhydration level and bioactivity in HHcy, which resulted in further aggravation of HHcy; and (iv) HS donors reversed the decreased bioactivity of  in HHcy, thus reducing the excessive Hcy level.  Our study suggested that HS could improve  bioactivity by S-sulfhydration, which might provide a candidate therapeutic strategy for HHcy. . 36, 1-14.

Insufficient S-Sulfhydration of Methylenetetrahydrofolate Reductase Contributes to the Progress of Hyperhomocysteinemia.

In women of childbearing age, extensive decidualization, shedding and remodeling of the endometrium during the menstrual cycle are fundamental for successful pregnancy. The role of prostaglandins (PGs) in menstruation has long been proposed in humans, and the rate-limiting enzyme cyclooxygenase was shown to play a key role in endometrial breakdown and shedding in a mouse menstrual-like model in our previous study. However, the specific types of PGs involved and their respective roles remain unclear. Therefore, our objective was to investigate the mechanism through which PGs regulate endometrial disintegration. In this study, the microscopy was observed by HE; the protein levels of prostaglandins E1 (PGE1), prostaglandins E2 (PGE2), prostaglandin F2α (PGF2α) and Prostaglandin I2 (PGI2) were detected by ELISA; the mRNA level of Pfgfr2, Vascular Endothelial Growth Factor(Vegf), Angiostatin and Hypoxia inducible factor-1α (Hif1α) were examined by real-time PCR; PTGFR Receptor (PTGFR), VEGF, Angiostatin and HIF-1α protein levels were investigated by western blotting; the locations of protein were observed by Immunohistochemistry; HIF-1α binding PTGFR promoter was detected by Chromatin Immunoprecipitation (ChIP) and real-time PCR. We found that the concentrations of PGE1, PGE2, and PGF2α all increased significantly during this process. Furthermore, Ptgfr mRNA increased soon after Progesterone (P4) withdrawal, and PTGFR protein levels increased significantly during abundant endometrial breakdown and shedding processes. PTGFR inhibitors AL8810 significantly suppressed endometrial breakdown and shedding, promoted Angiostatin expression, and reduced VEGF-A expressions and vascular permeability. And HIF-1α and PTGFR were mainly located in the luminal/gland epithelium, vascular endothelium, and pre-decidual zone. Interestingly, HIF-1α directly bound to Ptgfr promoter. Moreover, a HIF-1α inhibitor 2-methoxyestradiol (2ME) significantly reduced PTGFR expression and suppressed endometrial breakdown which was in accord with PTGFR inhibitor's effect. Similar changes occurred in human stromal cells relevant to menstruation in vitro. Our study provides evidence that PGF2α/PTGFR plays a vital role in endometrial breakdown via vascular changes that are regulated by HIF-1α during menstruation.

The Essential Role of PGF2α/PTGFR in Molding Endometrial Breakdown and Vascular Dynamics, Regulated by HIF-1α in a Mouse Menstrual-like Model.

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
Insufficient S-Sulfhydration of Methylenetetrahydrofolate Reductase Contributes to the Progress of Hyperhomocysteinemia. Antioxidants & redox signaling\| PubMed ID: 34409847	2022
The Essential Role of PGF2α/PTGFR in Molding Endometrial Breakdown and Vascular Dynamics, Regulated by HIF-1α in a Mouse Menstrual-like Model. Reproductive sciences (Thousand Oaks, Calif.)\| PubMed ID: 38637474	2024
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

Jiangxu Wu

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