Role of RNA polymerase III transcription and regulation in ischaemic stroke.

Ischaemic stroke is a leading cause of death and life-long disability due to neuronal cell death resulting from interruption of glucose and oxygen supplies. RNA polymerase III (Pol III)-dependent transcription plays a central role in protein synthesis that is necessary for normal cerebral neuronal functions, and the survival and recovery under pathological conditions. Notably, Pol III transcription is highly sensitive to ischaemic stress that is known to rapidly shut down Pol III transcriptional activity. However, its precise role in ischaemic stroke, especially during the acute and recovery phases, remains poorly understood. The microenvironment within the ischaemic brain undergoes dynamic changes in different phases after stroke. Emerging evidence highlights the distinct roles of Pol III transcription in neuroprotection during the acute phase and repair during the recovery phase of stroke. Additionally, investigations into the mTOR-MAF1 signalling pathway, a conserved regulator of Pol-III transcription, reveal its therapeutic potential in enhancing acute phase neuroprotection and recovery phase repair.

Hypoxanthine is a metabolic biomarker for inducing GSDME-dependent pyroptosis of endothelial cells during ischemic stroke.

Rationale: Stroke induces metabolic changes in the body, and metabolites have become potential biomarkers for stroke. However, the specific metabolites involved in stroke and the mechanisms underlying brain injury during stroke remain unclear. Methods: Surface-enhanced Raman spectroscopy (SERS) and liquid chromatography-mass spectrometry (LC‒MS) analysis of clinical serum samples from 69 controls and 51 ischemic stroke patients who underwent reperfusion within 24 hours were performed to identify differentially abundant metabolites. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) and then intravenously injected with hypoxanthine. The infarct area was evaluated via tetrazolium chloride (TTC) staining, and behavior tests were conducted. Blood-brain barrier (BBB) leakage was assessed by Evans blue and IgG staining. Human blood vessel organoids were used to investigate the mechanism of hypoxanthine-induced pyroptosis of endothelial cells. Results: SERS and LC‒MS revealed the metabolic profiles of serum from stroke patients and controls with high sensitivity, speed and accuracy. Hypoxanthine levels were significantly elevated in the acute stage of ischemic stroke in both patients and mice (p < 0.001 after Bonferroni correction). In addition, increasing hypoxanthine increased the infarct area and aggravated BBB leakage and neurobehavioral deficits in mice after ischemic stroke. Further mechanistic studies using endothelial cells, human blood vessel organoids, and stroke mice demonstrated that hypoxanthine-mediated gasdermin E (GSDME)-dependent pyroptosis of endothelial cells occurs through intracellular Ca2+ overload. Conclusion: Our study identified hypoxanthine as an important metabolite that induces vascular injury and BBB disruption in stroke through triggering GSDME-dependent pyroptosis of endothelial cells.

MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathway.

OBJECTIVE: To explore whether miR-199a-5p regulated BBB integrity through PI3K/Akt pathway after ischemia stroke. METHODS: Adult male Sprague-Dawley rats with permanent middle cerebral artery occlusion(MCAO) were used in experiment. The Ludmila Belayev 12-point scoring was used to measure the neurological function of MCAO rats. The Evans Blue Stain, immunofluorescence staining, western-blotting and RT-PCR were performed to evaluate the effects of miR-199a-5p mimic on BBB integrity in rats following MCAO. RESULTS: The result suggested that miR-199a-5p mimic treatment possessed the potential to boost proprioception and motor activity of MCAO rats. MiR-199a-5p decreased the expression of PIK3R2 after MCAO, activated Akt signaling pathway, and increased the expression of Claudin-5 and VEGF in the ischemic penumbra. Furthermore, miR-199a-5p alleviated inflammation after cerebral ischemia. BBB leakage and neurocyte apoptosis were cut down in MCAO rats treated with miR-199a-5p mimic. CONCLUSIONS: MiR-199a-5p mimic decreased the expression of PIK3R2 and activated Akt signaling pathway after ischemia stroke, reduced the expression of inflammatory cytokines, and attenuated BBB disruption after ischemic stroke.

Neural and Glial Regulation of Angiogenesis in CNS in Ischemic Stroke.

CNS diseases associated with compromised blood supply and/or vascular integrity are one of the leading causes of mortality and disability in adults worldwide and are also among 10 most common causes of death in children. Angiogenesis is an essential element of regeneration processes upon nervous tissue damage and can play a crucial role in neuroprotection. Here we review the features of cerebral vascular regeneration after ischemic stroke, including the complex interactions between endothelial cells and other brain cell types (neural stem cells, astrocytes, microglia, and oligodendrocytes). The mechanisms of reciprocal influence of angiogenesis and neurogenesis, the role of astrocytes in the formation of the blood-brain barrier, and roles of microglia and oligodendrocytes in vascular regeneration are discussed. Understanding the mechanisms of angiogenesis regulation in CNS is of critical importance for the development of new treatments of neurovascular pathologies.

Ischemic Microenvironment-Targeted Bioinspired Lipoprotein Sequentially Penetrates Cerebral Ischemic Lesions to Rescue Ischemic Stroke.

Reperfusion injury represents a significant impediment to recovery after recanalization in an ischemic stroke and can be alleviated by neuroprotectants. However, inadequate drug delivery to ischemic lesions impairs the therapeutic effects of neuroprotectants. To address this issue, an ischemic microenvironment-targeted bioinspired lipoprotein system encapsulating lipoic acid (LA@PHDL) is herein designed to sequentially penetrate ischemic lesions and be readily taken up by neurons and microglia. In transient middle cerebral artery occlusion (tMCAO) mouse models, LA@PHDL accumulates rapidly and preferentially in the ischemic brain, with a 2.29-fold higher than the nontargeted nanoplatform in the early stage. Furthermore, LA@PHDL effectively restores neurological function, reduces infarct volume to 17.70%, prevents brain cell necrosis and apoptosis, and attenuates inflammation in tMCAO mouse models. This design presents new opportunities for delivering neuroprotectants to cerebral ischemic lesions to improve the outcome of an ischemic stroke.

Differences in Acute Expression of Matrix Metalloproteinases-9, 3, and 2 Related to the Duration of Brain Ischemia and Tissue Plasminogen Activator Treatment in Experimental Stroke.

Matrix metalloproteinases (MMPs) such as MMP-9, 3, and 2 degrade the cellular matrix and are believed to play a crucial role in ischemic stroke. We examined how the duration of ischemia (up to 4 h) and treatment with recombinant tissue plasminogen activator altered the comparative expression of these MMPs in experimental ischemic stroke with reperfusion. Both prolonged ischemia and r-tPA treatment markedly increased MMP-9 expression in the ischemic hemisphere (all p < 0.0001). The duration of ischemia and r-tPA treatment also significantly increased MMP-2 expression (p < 0.01-0.001) in the ischemic hemisphere (p < 0.01) but to a lesser degree than MMP-9. In contrast, MMP-3 expression significantly decreased in the ischemic hemisphere (p < 0.001) with increasing duration of ischemia and r-tPA treatment (p < 0.05-0001). MMP-9 expression was prominent in the vascular compartment and leukocytes. MMP-2 expression was evident in the vascular compartment and MMP-3 in NeuN+ neurons. Prolonging the duration of ischemia (up to 4 h) before reperfusion increased brain hemorrhage, infarction, swelling, and neurologic disability in both saline-treated (control) and r-tPA-treated mice. MMP-9 and MMP-2 expression were significantly positively correlated with, and MMP-3 was significantly negatively correlated with, infarct volume, swelling, and brain hemorrhage. We conclude that in experimental ischemic stroke with reperfusion, the duration of ischemia and r-tPA treatment significantly altered MMP-9, 3, and 2 expression, ischemic brain injury, and neurological disability. Each MMP showed unique patterns of expression that are strongly correlated with the severity of brain infarction, swelling, and hemorrhage. In summary, in experimental ischemic stroke in male mice with reperfusion, the duration of ischemia, and r-tPA treatment significantly altered the immunofluorescent expression of MMP-9, 3, and 2, ischemic brain injury, and neurological disability. In this model, each MMP showed unique patterns of expression that were strongly correlated with the severity of brain infarction, swelling, and hemorrhage.

Hypoxia-Responsive Biomimetic Nanobubbles for Oxygen Delivery Promote Synergistic Ischemic Stroke Protection.

Effective, precise, and controllable oxygen delivery is crucial for regulating the oxygenation balance of brain tissue at the early stages of acute ischemic stroke (AIS) because the absence of oxygen may result in a series of highly interconnected vascular-neural pathological events, including oxidative stress, inflammation, and neuroapoptosis. In this study, platelet membrane-reassembled oxygen nanobubbles (PONBs) were constructed for oxygen delivery to protect AIS. Benefiting from the preserved natural targeting ability of platelet membranes, oxygen can be controlled release into the hypoxia lesion at the preperfusion stage due to vascular injury targeting and oxygen sustained diffusion capability after PONBs administration. Furthermore, synergizing with bioactive components carried by platelet membranes, PONBs can inhibit post-AIS vascular occlusion and maintain blood-brain barrier integrity, thereby facilitating enhanced oxygen delivery of PONBs, establishing a positive feedback loop between oxygen delivery and AIS protection. Additionally, the accumulation of PONBs enhances the ultrasound imaging contrast, enabling precise localization and dynamic monitoring of AIS lesions. Thus, PONBs represent a promising strategy for the diagnosis and treatment of AIS.

Effects of white matter hyperintensity burden on functional outcome after mild versus moderate-to-severe ischemic stroke.

It is uncertain whether the prognostic power of white matter hyperintensity (WMH) on post-stroke outcomes is modulated as a function of initial neurological severity, a critical determinant of outcome after stroke. This multi-center MRI study tested if higher WMH quintiles were associated with 3-month poor functional outcome (modified Rankin Scale ≥ 3) for mild versus moderate-to-severe ischemic stroke. Mild and moderate-to-severe stroke were defined as admission National Institute of Health Stroke Scale scores of 1-4 and ≥ 5, respectively. Mean age of the enrolled patients (n = 8918) was 67.2 ± 12.6 years and 60.1% male. The association between WMH quintiles and poor functional outcome was modified by stroke severity (p-for-interaction = 0.008). In mild stroke (n = 4994), WMH quintiles associated with the 3-month outcome in a dose-dependent manner for the 2nd to 5th quintile versus the 1st quintile, with adjusted-odds-ratios (aOR [95% confidence interval]) being 1.29 [0.96-1.73], 1.37 [1.02-1.82], 1.60 [1.19-2.13], and 1.89 [1.41-2.53], respectively. In moderate-to-severe stroke (n = 3924), however, there seemed to be a threshold effect: only the highest versus the lowest WMH quintile was significantly associated with poor functional outcome (aOR 1.69 [1.29-2.21]). WMH burden aggravates 3-month functional outcome after mild stroke, but has a lesser modulatory effect for moderate-to-severe stroke, likely due to saturation effects.

Synergism of salvianolic acid B and ginsenoside Rg1 magnifies the therapeutic potency against ischemic stroke.

Even though considerable progress has been made to reduce insult, ischemic stroke is still a significant cause of mortality and morbidity in the world, and new therapeutic strategies are urgently needed. In the present study, the magnesium salt of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) combination as a multicomponent strategy against stroke was evaluated. The synergistic effect of Sa1B and Rg1 was evaluated by Bliss independence analysis on the middle cerebral artery occlusion model. The infarct volume, neuroethology, cerebral structure, and neurocyte number were evaluated by 3,5-triphenyltetrazolium chloride staining, Longa score, Garcia score, hematoxylin-eosin staining, and Nissl staining, respectively. Metabolomics was used to search for potential biomarkers and explore the mechanism of Sa1B/Rg1. First, the superior effects of SalB/Rg1 than SalB or Rg1 at the same dose were evaluated. Compared with SalB ( P  < 0.001) or Rg1 ( P  < 0.01), SalB/Rg1 significantly decreased infarct volume through 3,5-triphenyltetrazolium chloride staining and protected the structural integrity of cortex and striatum. The superior effect of SalB/Rg1 on neurological behavior was also detected compared with SalB or Rg1 significantly. Accompanying behavioral improvement, a considerable increase of SalB/Rg1 on neurons detected by Nissl staining was found on the cortex compared with SalB ( P  < 0.05) or Rg1 ( P  < 0.01). Second, the synergistic effect between SalB and Rg1 was strictly verified by Bliss independence analysis ( P  < 0.01) based on infarct volume. Finally, alleviation of cerebral metabolic disorders may be the possible mechanism of SalB/Rg1. Our study provided a multicomponent strategy against ischemic stroke, with not only dose reduction but also improved efficacy relative to single agents.

Physical exercise-induced circAnks1b upregulation promotes protective endoplasmic reticulum stress and suppresses apoptosis via miR-130b-5p/Pak2 signaling in an ischemic stroke model.

AIMS: Physical exercise (PE) can accelerate post-stroke recovery. This study investigated contributions of circRNAs to PE-induced improvements in post-stroke neurological function. METHODS: Rats subjected to transient middle cerebral artery occlusion were left sedentary or provided running-wheel access for 4 weeks during recovery. CircRNAs from peri-infarct cortex were identified by high-throughput sequencing, and interactions with miRNAs by immunoprecipitation, fluorescence in suit hybridization, and dual-luciferase reporter assays. In vivo circRNA knockdown was achieved using shRNA-AAVs and in vitro overexpression by plasmid transfection. Transmission electron microscopy, western blotting, and TUNEL assays were conducted to explore circRNA contributions to endoplasmic reticulum (ER) stress and neuronal apoptosis. CircRNA levels were measured in plasma from stroke patients by qRT-PCR and associations with neurological scores assessed by Pearson's correlation analysis. RESULTS: PE upregulated circAnks1b, reduced infarct volume, and mitigated neurological dysfunction, while circAnks1b knockdown exacerbated neurological dysfunction and increased infarct size despite PE. CircAnks1b sponged miR-130b-5p, thereby disinhibiting Pak2 expression. Conversely, Pak2 downregulation disrupted PE-mediated protective ER stress, leading to reduced IRE1/XBP1 and heightened apoptosis. Plasma circAnks1b was higher in stroke patients receiving PE than sedentary patients and correlated negatively with neurological scores. CONCLUSIONS: CircAnks1b upregulation may be an effective therapeutic strategy for post-stroke recovery.

The Impact of miR-34a on Endothelial Cell Viability and Apoptosis in Ischemic Stroke: Unraveling the MTHFR-Homocysteine Pathway.

INTRODUCTION: Ischemic stroke (IS) is a global health concern, often tied to dyslipidemia and vascular endothelial dysfunction. MicroRNA-34a (miR-34a) was reported to be up-regulated in the blood samples of patients with IS, but the specific role of miR-34a and methylenetetrahydrofolate reductase (MTHFR) in IS remains to be elucidated. METHODS: We studied 143 subjects: 71 IS patients, and 72 healthy controls. Human umbilical vein endothelial cells (HUVECs) were cultured and transfected with a miR-34a mimic, inhibitor, or negative control. The miR-34a expression in serum and HUVECs was quantified via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Viability and apoptosis of HUVECs were assessed using CCK-8 assay and flow cytometry. The expression levels of bcl-2, bax, cyt-c, cleaved caspase 3, MTHFR, and homocysteine were measured by Western blot or enzyme-linked immunosorbent assay (ELISA). The relationship between miR-34a and MTHFR was verified by luciferase reporter assay. The levels of MTHFR and homocysteine in serum were examined by ELISA. RESULTS: MiR-34a expression was increased in IS patients and inhibited viability of HUVECs while promoting their apoptosis. Overexpression of miR-34a up-regulated pro-apoptotic proteins (bax, cyt-c and cleaved caspase 3) and down-regulated anti-apoptotic protein bcl-2 in HUVECs. MTHFR was identified as the downstream target of miR-34a and its expression was reduced by miR-34a overexpression, while homocysteine levels increased. Consistently, MTHFR levels were lower and homocysteine levels were higher in IS patients compared with controls. DISCUSSION: Our results suggest that up-regulated miR-34a plays a role in the pathogenesis of IS, potentially through inhibiting MTHFR expression and increasing homocysteine in endothelial cells. Therefore, miR-34a might be a therapeutic target for IS.

Lipocalin-2 aggravates blood-brain barrier dysfunction after intravenous thrombolysis by promoting endothelial cell ferroptosis via regulating the HMGB1/Nrf2/HO-1 pathway.

BACKGROUND: Disruption of the blood-brain barrier (BBB) is a major contributor to hemorrhagic transformation (HT) in patients with acute ischemic stroke (AIS) following intravenous thrombolysis (IVT). However, the clinical therapies aimed at BBB protection after IVT remain limited. METHODS: One hundred patients with AIS who underwent IVT were enrolled (42 with HT and 58 without HT 24 h after IVT). Based on the cytokine chip, the serum levels of several AIS-related proteins, including LCN2, ferritin, matrix metalloproteinase-3, vascular endothelial-derived growth factor, and X-linked inhibitor of apoptosis, were detected upon admission, and their associations with HT were analyzed. After finding that LCN2 was related to HT in patients with IVT, we clarified whether the modulation of LCN2 influenced BBB dysfunction and HT after thrombolysis and investigated the potential mechanism. RESULTS: In patients with AIS following IVT, logistic regression analysis showed that baseline serum LCN2 (p = 0.023) and ferritin (p = 0.046) levels were independently associated with HT. A positive correlation between serum LCN2 and ferritin levels was identified in patients with HT. In experimental studies, recombinant LCN2 (rLCN2) significantly aggravated BBB dysfunction and HT in the thromboembolic stroke rats after thrombolysis, whereas LCN2 inhibition by ZINC006440089 exerted opposite effects. Further mechanistic studies showed that, LCN2 promoted endothelial cell ferroptosis, accompanied by the induction of high mobility group box 1 (HMGB1) and the inhibition of nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins. Ferroptosis inhibitor ferrostatin-1 (fer-1) significantly restricted the LCN2-mediated BBB disruption. Transfection of LCN2 and HMGB1 siRNA inhibited the endothelial cell ferroptosis, and this effects was reversed by Nrf2 siRNA. CONCLUSION: LCN2 aggravated BBB disruption after thrombolysis by promoting endothelial cell ferroptosis via regulating the HMGB1/Nrf2/HO-1 pathway, this may provide a promising therapeutic target for the prevention of HT after IVT.

Ferroptosis in Ischemic Stroke and Related Traditional Chinese Medicines.

Stroke is a severe neurological disorder resulting from the rupture or blockage of blood vessels, leading to significant mortality and disability worldwide. Among the different types of stroke, ischemic stroke (IS) is the most prevalent, accounting for 70-80% of cases. Cell death following IS occurs through various mechanisms, including apoptosis, necrosis, and ferroptosis. Ferroptosis, a recently identified form of regulated cell death characterized by iron overload and lipid peroxidation, was first described by Dixon in 2012. Currently, the only approved pharmacological treatment for IS is recombinant tissue plasminogen activator (rt-PA), which is limited by a narrow therapeutic window and often results in suboptimal outcomes. Recent research has identified several traditional Chinese medicines (TCMs) that can inhibit ferroptosis, thereby mitigating the damage caused by IS. This review provides an overview of stroke, the role of ferroptosis in IS, and the potential of certain TCMs to inhibit ferroptosis and contribute to stroke treatment.

In vitro modelling of the neurovascular unit for ischemic stroke research: Emphasis on human cell applications and 3D model design.

Ischemic stroke has garnered global medical attention as one of the most serious cerebrovascular diseases. The mechanisms involved in both the development and recovery phases of ischemic stroke are complex, involving intricate interactions among different types of cells, each with its own unique functions. To better understand the possible pathogenesis, neurovascular unit (NVU), a concept comprising neurons, endothelial cells, mural cells, glial cells, and extracellular matrix components, has been used in analysing various brain diseases, particularly in ischemic stroke, aiming to depict the interactions between cerebral vasculature and neural cells. While in vivo models often face limitations in terms of reproducibility and the ability to precisely mimic human pathophysiology, it is now important to establish in vitro NVU models for ischemic stroke research. In order to accurately portray the pathological processes occurring within the brain, a diverse array of NVU 2D and 3D in vitro models, each possessing unique characteristics and advantages, have been meticulously developed. This review presents a comprehensive overview of recent advancements in in vitro models specifically tailored for investigating ischemic stroke. Through a systematic categorization of these developments, we elucidate the intricate links between NVU components and the pathogenesis of ischemic stroke. Furthermore, we explore the distinct advantages offered by innovative NVU models, notably 3D models, which closely emulate in vivo conditions. Additionally, an examination of current therapeutic modalities for ischemic stroke developed utilizing in vitro NVU models is provided. Serving as a valuable reference, this review aids in the design and implementation of effective in vitro models for ischemic stroke research.

Role of Microglia in Stroke.

Ischemic stroke is a complex brain pathology caused by an interruption of blood supply to the brain. It results in neurological deficits which that reflect the localization and the size of the compromised brain area and are the manifestation of complex pathogenic events triggered by energy depletion. Inflammation plays a prominent role, worsening the injury in the early phase and influencing poststroke recovery in the late phase. Activated microglia are one of the most important cellular components of poststroke inflammation, appearing from the first few hours and persisting for days and weeks after stroke injury. In this chapter, we will discuss the nature of the inflammatory response in brain ischemia, the contribution of microglia to injury and regeneration after stroke, and finally, how ischemic stroke directly affects microglia functions and survival.

[Research progress on the pyroptosis mediated by the NLRP3 inflammasome in the pathology of ischemic stroke].

Ischemic stroke is one of the diseases which pose a significant threat to human health. Recent studies have suggested that programmed cell death plays an important role in brain tissues affected by ischemia and hypoxia. Pyroptosis, which is characterized by both apoptosis and necrosis, is mediated by activation of inflammasomes, such as NOD-like receptor family, pyrin domain containing 3 (NLRP3). Pyroptosis, which depends on caspase-1 activation and the release of pro-inflammatory cytokines, such as Interleukin 1ß and Interleukin 18, plays a vital role in regulating cell survival and death following ischemic injury. Previous studies have shown that pyroptosis is closely related to the activation of the NLRP3 inflammasome in ischemic stroke tissues. This distinctive form of cell death mainly occurs in microglia, neurons, astrocytes and endothelial cells. This paper reviews the role of pyroptosis mediated by inflammasomes in ischemic stroke, discussing the targets and substances that affect the activation of the NLRP3 inflammasome, which can provide a new theoretical and experimental basis for the treatment of ischemic stroke.

Simvastatin exerts neuroprotective effects post-stroke by ameliorating endoplasmic reticulum stress and regulating autophagy/apoptosis balance through pAMPK/LC3B/ LAMP2 axis.

Statins have evident neuroprotective role in acute ischemic stroke(AIS). The pleiotropic effect by which statin exerts neuroprotective effects, needs to be explored for considering it as one of the future adjunctive therapies in AIS. Endoplasmic reticulum(ER) assists cellular survival by reducing protein aggregates during ischemic conditions. ER-stress mediated apoptosis and autophagy are predominant reasons for neuronal death in AIS. Statin exerts both anti-apoptotic and anti-autophagic effect in neurons under ischemic stress. Although the influence of statin on autophagic neuroprotection has been reported with contradictory results. Thus, in our study we have attempted to understand its influence on autophagic protection while inhibiting upregulation of autophagic death(autosis). Previously we reported, statin can alleviate apoptosis via modulating cardiolipin mediated mitochondrial dysfunction. However, the clearance of damaged mitochondria is essential for prolonged cell survival. In our study, we tried to decipher the mechanism by which statin leads to neuronal survival by the mitophagy mediated cellular clearance. Simvastatin was administered to Sprague Dawley(SD) rats both as prophylaxis and treatment. The safety and efficacy of the statin was validated by assessment of infarct size and functional outcome. A reduction in oxidative and ER-stress were observed in both the prophylactic and treatment groups. The influence of statin on autophagy/apoptosis balance was evaluated by molecular assessment of mitophagy and cellular apoptosis. Statin reduces the post-stroke ER-stress and predominantly upregulated autophagolysosome mediated mitophagy than apoptotic cell death by modulating pAMPK/LC3B/LAMP2 axis. Based on the above findings statin could be explored as an adjunctive therapy for AIS in future.

Brain border-derived CXCL2+ neutrophils drive NET formation and impair vascular reperfusion following ischemic stroke.

BACKGROUND: The brain border compartments harbor a diverse population of immune cells and serve as invasion sites for leukocyte influx into the brain following CNS injury. However, how brain-border myeloid cells affect stroke pathology remains poorly characterized. METHODS AND RESULTS: Here, we showed that ischemic stroke-induced expansion of CXCL2+ neutrophils, which exhibit highly proinflammatory features. We tracked CXCL2+ neutrophils in vivo by utilizing a photoconvertible Kik-GR mouse (fluorescent proteins Kikume Green Red, Kik-GR) and found that brain-infiltrating CXCL2+ neutrophils following ischemic stroke were mainly derived from the brain border rather than the periphery. We demonstrated that CXCL2 neutralization inhibited the formation and releasing of neutrophil extracellular traps (NETs) from in vitro cultured primary neutrophils. Furthermore, CXCL2-neutralizing antibody treatment reduced brain infarcts and improved vascular reperfusion at day 3 postischemic stroke. CONCLUSIONS: Collectively, brain border-derived CXCL2+ neutrophil expansion may impair vascular reperfusion by releasing NETs following ischemic stroke.

S100A9 deletion in microglia/macrophages ameliorates brain injury through the STAT6/PPARγ pathway in ischemic stroke.

BACKGROUND: Microglia and infiltrated macrophages (M/M) are integral components of the innate immune system that play a critical role in facilitating brain repair after ischemic stroke (IS) by clearing cell debris. Novel therapeutic strategies for IS therapy involve modulating M/M phenotype shifting. This study aims to elucidate the pivotal role of S100A9 in M/M and its downstream STAT6/PPARγ signaling pathway in neuroinflammation and phagocytosis after IS. METHODS: In the clinical study, we initially detected the expression pattern of S100A9 in monocytes from patients with acute IS and investigated its association with the long-term prognosis. In the in vivo study, we generated the S100A9 conditional knockout (CKO) mice and compared the stroke outcomes with the control group. We further tested the S100A9-specific inhibitor paqunimod (PQD), for its pharmaceutical effects on stroke outcomes. Transcriptomics and in vitro studies were adopted to explore the mechanism of S100A9 in modulating the M/M phenotype, which involves the regulation of the STAT6/PPARγ signaling pathway. RESULTS: S100A9 was predominantly expressed in classical monocytes and was correlated with unfavorable outcomes in patients of IS. S100A9 CKO mitigated infarction volume and white matter injury, enhanced cerebral blood flow and functional recovery, and prompted anti-inflammation phenotype and efferocytosis after tMCAO. The STAT6/PPARγ pathway, an essential signaling cascade involved in immune response and inflammation, might be the downstream target mediated by S100A9 deletion, as evidenced by the STAT6 phosphorylation inhibitor AS1517499 abolishing the beneficial effect of S100A9 inhibition in tMCAO mice and cell lines. Moreover, S100A9 inhibition by PQD treatment protected against neuronal death in vitro and brain injuries in vivo. CONCLUSION: This study provides evidence for the first time that S100A9 in classical monocytes could potentially be a biomarker for predicting IS prognosis and reveals a novel therapeutic strategy for IS. By demonstrating that S100A9-mediated M/M polarization and phagocytosis can be reversed by S100A9 inhibition in a STAT6/PPARγ pathway-dependent manner, this study opens up new avenues for drug development in the field.

Hsp90aa1/JUN/Ccl2 regulatory axis mediates migration and differentiation of NSPCs, promoting the onset and progression of early post-ischemic stroke epilepsy.

Early-onset epilepsy following ischemic stroke is a severe neurological condition, the pathogenesis of which remains incompletely understood. Recent studies suggest that Neural stem/progenitor cells (NSPCs) play a crucial role in the disease process, yet the precise molecular mechanisms regulating NSPCs have not been thoroughly investigated. This study utilized single-cell transcriptome sequencing and bioinformatics analysis to identify disease-related genes, which were subsequently validated in both in vitro and in vivo experiments. The findings revealed that Hsp90aa1 (heat shock protein 90 kDa alpha, class A member 1), Jun proto-oncogene (JUN), and CC Motif Ligation 2 (Ccl2) constitute an important regulatory axis influencing the migration and differentiation of NSPCs, potentially impacting the onset and progression of early-onset epilepsy post-ischemic stroke. Additionally, the expression of Hsp90aa1 was found to influence the likelihood of seizure occurrence and the severity of brain ischemia.