Translatome analysis in acute ischemic stroke: Astrocytes and microglia exhibit differences in poststroke alternative splicing of expressed transcripts.

Astrocytes and microglia undergo dynamic and complex morphological and functional changes following ischemic stroke, which are instrumental in both inflammatory responses and neural repair. While gene expression alterations poststroke have been extensively studied, investigations into posttranscriptional regulatory mechanisms, specifically alternative splicing (AS), remain limited. Utilizing previously reported Ribo-Tag-seq data, this study analyzed AS alterations in poststroke astrocytes and microglia from young adult male and female mice. Our findings reveal that in astrocytes, compared to the sham group, 109 differential alternative splicing (DAS) events were observed at 4 h poststroke, which increased to 320 at day 3. In microglia, these numbers were 316 and 266, respectively. Interestingly, the disparity between DAS genes and differentially expressed genes is substantial, with fewer than 10 genes shared at both poststroke time points in astrocytes and microglia. Gene ontology enrichment analysis revealed the involvement of these DAS genes in diverse functions, encompassing immune response (Adam8, Ccr1), metabolism (Acsl6, Pcyt2, Myo5a), and developmental cell growth (App), among others. Selective DAS events were further validated by semiquantitative RT-PCR. Overall, this study comprehensively describes the AS alterations in astrocytes and microglia during the hyperacute and acute phases of ischemic stroke and underscores the significance of certain hub DAS events in neuroinflammatory processes.

Microglial heterogeneity in the ischemic stroke mouse brain of both sexes.

BACKGROUND: Ischemic stroke elicits a complex and sustained immune response in the brain. Immunomodulatory treatments have long held promise for improving stroke outcomes, yet none have succeeded in the clinical setting. This lack of success is largely due to our incomplete understanding of how immune cells respond to stroke. The objective of the current study was to dissect the effect of permanent stroke on microglia, the resident immune cells within the brain parenchyma. METHODS: A permanent middle cerebral artery occlusion (pMCAO) model was used to induce ischemic stroke in young male and female mice. Microglia were sorted from fluorescence reporter mice after pMCAO or sham surgery and then subjected to single-cell RNA sequencing analysis. Various methods, including flow cytometry, RNA in situ hybridization, immunohistochemistry, whole-brain imaging, and bone marrow transplantation, were also employed to dissect the microglial response to stroke. Stroke outcomes were evaluated by infarct size and behavioral tests. RESULTS: First, we showed the morphologic and spatial changes in microglia after stroke. We then performed single-cell RNA sequencing analysis on microglia isolated from sham and stroke mice of both sexes. The data indicate no major sexual dimorphism in the microglial response to permanent stroke. Notably, we identified seven potential stroke-associated microglial clusters, including four major clusters characterized by a disease-associated microglia-like signature, a highly proliferative state, a macrophage-like profile, and an interferon (IFN) response signature, respectively. Importantly, we provided evidence that the macrophage-like cluster may represent the long-sought stroke-induced microglia subpopulation with increased CD45 expression. Lastly, given that the IFN-responsive subset constitutes the most prominent microglial population in the stroke brain, we used fludarabine to pharmacologically target STAT1 signaling and found that fludarabine treatment improved long-term stroke outcome. CONCLUSIONS: Our findings shed new light on microglia heterogeneity in stroke pathology and underscore the potential of targeting specific microglial populations for effective stroke therapies.

MMP-3 Knockout Induces Global Transcriptional Changes and Reduces Cerebral Infarction in Both Male and Female Models of Ischemic Stroke.

Ischemic stroke followed by reperfusion (IR) leads to extensive cerebrovascular injury characterized by neuroinflammation and brain cell death. Inhibition of matrix metalloproteinase-3 (MMP-3) emerges as a promising therapeutic approach to mitigate IR-induced stroke injury. We employed middle cerebral artery occlusion with subsequent reperfusion (MCAO/R) to model ischemic stroke in adult mice. Specifically, we investigated the impact of MMP-3 knockout (KO) on stroke pathophysiology using RNA sequencing (RNA-seq) of stroke brains harvested 48 h post-MCAO. MMP-3 KO significantly reduced brain infarct size following stroke. Notably, RNA-seq analysis showed that MMP-3 KO altered expression of 333 genes (252 downregulated) in male stroke brains and 3768 genes (889 downregulated) in female stroke brains. Functional pathway analysis revealed that inflammation, integrin cell surface signaling, endothelial- and epithelial-mesenchymal transition (EndMT/EMT), and apoptosis gene signatures were decreased in MMP-3 KO stroke brains. Intriguingly, MMP-3 KO downregulated gene signatures more profoundly in females than in males, as indicated by greater negative enrichment scores. Our study underscores MMP-3 inhibition as a promising therapeutic strategy, impacting multiple cellular pathways following stroke.

Mechanism of ferroptosis regulating ischemic stroke and pharmacologically inhibiting ferroptosis in treatment of ischemic stroke.

Ferroptosis is a newly discovered form of programmed cell death that is non-caspase-dependent and is characterized by the production of lethal levels of iron-dependent lipid reactive oxygen species (ROS). In recent years, ferroptosis has attracted great interest in the field of cerebral infarction because it differs morphologically, physiologically, and genetically from other forms of cell death such as necrosis, apoptosis, autophagy, and pyroptosis. In addition, ROS is considered to be an important prognostic factor for ischemic stroke, making it a promising target for stroke treatment. This paper summarizes the induction and defense mechanisms associated with ferroptosis, and explores potential treatment strategies for ischemic stroke in order to lay the groundwork for the development of new neuroprotective drugs.

The positive role of transforming growth factor-ß1 in ischemic stroke.

Ischemic stroke is one of the most disabling and fatal diseases around the world. The damaged brain tissues will undergo excessive autophagy, vascular endothelial cells injury, blood-brain barrier (BBB) impairment and neuroinflammation after ischemic stroke. However, there is no unified viewpoint on the underlying mechanism of brain damage. Transforming growth factor-ß1 (TGF-ß1), as a multi-functional cytokine, plays a crucial role in the intricate pathological processes and helps maintain the physiological homeostasis of brain tissues through various signaling pathways after ischemic stroke. In this review, we summarize the protective role of TGF-ß1 in autophagic flux, BBB, vascular remodeling, neuroinflammation and other aspects after ischemic stroke. Based on the review, we believe that TGF-ß1 could serve as a key target for treating ischemic stroke.

Soluble Triggering Receptors Expressed on Myeloid Cells (sTREM) in Acute Ischemic Stroke: A Potential Pathway of sTREM-1 and sTREM-2 Associated with Disease Severity.

In 2022, stroke emerged as the most significant cerebrovascular disorder globally, causing 6.55 million deaths. Microglia, crucial for CNS preservation, can exacerbate brain damage in ischemic stroke by triggering neuroinflammation. This process is mediated by receptors on microglia, triggering receptors expressed on myeloid cells (TREM-1 and TREM-2), which have contrasting roles in neuroinflammation. In this study, we recruited 38 patients within 4.5 h from the onset of ischemic stroke. The degree of severity was evaluated by means of the National Institutes of Health Stroke Scale (NIHSS) at admission (T0) and after one week of ischemic events (TW) and the Modified Rankin Scale (mRS) at three months. The plasma concentration of TREMs (sTREM) was analyzed by next-generation ELISA at T0 and TW. The sTREM-1 concentrations at T0 were associated with mRS, while the sTREM-2 concentrations at T0 were associated with both the NIHSS at T0 and the mRS. A strong correlation between sTREM-1 and sTREM-2 was observed, suggesting a dependent modulation of the levels. This study provides insights into the potential pathway of TREM-1 and TREM-2 as a future biomarker for stratifying high-risk patients with ischemic stroke.

ß-asarone induces viability and angiogenesis and suppresses apoptosis of human vascular endothelial cells after ischemic stroke by upregulating vascular endothelial growth factor A.

Ischemic stroke (IS) is a disease with a high mortality and disability rate worldwide, and its incidence is increasing per year. Angiogenesis after IS improves blood supply to ischemic areas, accelerating neurological recovery. ß-asarone has been reported to exhibit a significant protective effect against hypoxia injury. The ability of ß-asarone to improve IS injury by inducing angiogenesis has not been distinctly clarified. The experimental rats were induced with middle cerebral artery occlusion (MCAO), and oxygen-glucose deprivation (OGD) model cells were constructed using human microvascular endothelial cell line (HMEC-1) cells. Cerebral infarction and pathological damage were first determined via triphenyl tetrazolium chloride (TTC) and hematoxylin and eosin (H&E) staining. Then, cell viability, apoptosis, and angiogenesis were assessed by utilizing cell counting kit-8 (CCK-8), flow cytometry, spheroid-based angiogenesis, and tube formation assays in OGD HMEC-1 cells. Besides, angiogenesis and other related proteins were identified with western blot. The study confirms that ß-asarone, like nimodipine, can ameliorate cerebral infarction and pathological damage. ß-asarone can also upregulate vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) and induce phosphorylation of p38. Besides, the study proves that ß-asarone can protect against IS injury by increasing the expression of VEGFA. In vitro experiments affirmed that ß-asarone can induce viability and suppress apoptosis in OGD-mediated HMEC-1 cells and promote angiogenesis of OGD HMEC-1 cells by upregulating VEGFA. This establishes the potential for ß-asarone to be a latent drug for IS therapy.

Circulating levels of neurofilament light chain as a biomarker of infarct and white matter hyperintensity volumes after ischemic stroke.

Serum neurofilament light chain protein (sNfL) shows promise as a biomarker for infarct size in acute ischemic stroke and for monitoring cerebral small vessel disease (cSVD). However, distinguishing the cSVD contribution after stroke may not be possible due to post-stroke sNfL increase. Additionally, it remains unclear if etiologic subtype differences exist. We measured infarct and white matter hyperintensity (WMH) volumes using MRI at the index stroke in ischemic stroke patients (n = 316, mean age 53 years, 65% males) and at 7-year follow-up (n = 187). Serum NfL concentration was measured in the acute phase (n = 235), at 3-months (n = 288), and 7-years (n = 190) post stroke. In multivariable regression, acute and 3-month sNfL concentrations were associated with infarct volume and time since stroke, but not with stroke etiology or infarct location. Seven years post-stroke, sNfL was associated with WMHs and age, but not with stroke etiology. Nonlinear regression estimated that sNfL peaks around 1 month, and declines by 50% at 3 months, and 99% at 9 months. We conclude that sNfL can indicate infarct volume and time since brain injury in the acute and subacute phases after stroke. Due to the significant post-stroke sNfL increase, several months are needed for reliable assessment of cSVD activity.

Single-nucleus RNA sequencing reveals glial cell type-specific responses to ischemic stroke in male rodents.

Neuroglia critically shape the brain´s response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition of the early ischemic lesion. Here we present a single cell resolution transcriptomics dataset of the brain´s acute response to infarction. Oligodendrocyte lineage cells and astrocytes range among the most transcriptionally perturbed populations and exhibit infarction- and subtype-specific molecular signatures. Specifically, we find infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and reactive astrocytes, exhibiting transcriptional commonalities in response to ischemic injury. OPCs and reactive astrocytes are involved in a shared immuno-glial cross talk with stroke-specific myeloid cells. Within the perilesional zone, osteopontin positive myeloid cells accumulate in close proximity to CD44+ proliferating OPCs and reactive astrocytes. In vitro, osteopontin increases the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition of acutely infarcted brain tissue.

Bibliometric insights into the inflammation and mitochondrial stress in ischemic stroke.

BACKGROUND: Research in the areas of inflammation and mitochondrial stress in ischemic stroke is rapidly expanding, but a comprehensive overview that integrates bibliometric trends with an in-depth review of molecular mechanisms is lacking. OBJECTIVE: To map the evolving landscape of research using bibliometric analysis and to detail the molecular mechanisms that underpin these trends, emphasizing their implications in ischemic stroke. METHODS: We conducted a bibliometric analysis to identify key trends, top contributors, and focal research themes. In addition, we review recent research advances in mitochondrial stress and inflammation in ischemic stroke to gain a detailed understanding of the pathophysiological processes involved. CONCLUSION: Our integrative approach not only highlights the growing research interest and collaborations but also provides a detailed exploration of the molecular mechanisms that are central to the pathology of ischemic stroke. This synthesis offers valuable insights for researchers and paves the way for targeted therapeutic interventions.

Chronic consequences of ischemic stroke: Profiling brain injury and inflammation in a mouse model with reperfusion.

Stroke is a pervasive and debilitating global health concern, necessitating innovative therapeutic strategies, especially during recovery. While existing literature often focuses on acute interventions, our study addresses the uniqueness of brain tissue during wound healing, emphasizing the chronic phase following the commonly used middle cerebral artery (MCA) occlusion model. Using clinically relevant endpoints in male and female mice such as magnetic resonance imaging (MRI) and plasma neurofilament light (NFL) measurement, along with immunohistochemistry, we describe injury evolution. Our findings document significant alterations in edema, tissue remodeling, and gadolinium leakage through MRI. Plasma NFL concentration remained elevated at 30 days poststroke. Microglia responses are confined to the region adjacent to the injury, rather than continued widespread activation, and boron-dipyrromethene (BODIPY) staining demonstrated the persistent presence of foam cells within the infarct. Additional immunohistochemistry highlighted sustained B and T lymphocyte presence in the poststroke brain. These observations underscore potentially pivotal roles played by chronic inflammation brought on by the lipid-rich brain environment, and chronic blood-brain barrier dysfunction, in the development of secondary neurodegeneration. This study sheds light on the enduring consequences of ischemic stroke in the most used rodent stroke model and provides valuable insights for future research, clinical strategies, and therapeutic development.

The Therapeutic Effects of Blueberry-Treated Stem Cell-Derived Extracellular Vesicles in Ischemic Stroke.

An ischemic stroke, one of the leading causes of morbidity and mortality, is caused by ischemia and hemorrhage resulting in impeded blood supply to the brain. According to many studies, blueberries have been shown to have a therapeutic effect in a variety of diseases. Therefore, in this study, we investigated whether blueberry-treated mesenchymal stem cell (MSC)-derived extracellular vesicles (B-EVs) have therapeutic effects in in vitro and in vivo stroke models. We isolated the extracellular vesicles using cryo-TEM and characterized the particles and concentrations using NTA. MSC-derived extracellular vesicles (A-EVs) and B-EVs were round with a lipid bilayer structure and a diameter of ~150 nm. In addition, A-EVs and B-EVs were shown to affect angiogenesis, cell cycle, differentiation, DNA repair, inflammation, and neurogenesis following KEGG pathway and GO analyses. We investigated the protective effects of A-EVs and B-EVs against neuronal cell death in oxygen-glucose deprivation (OGD) cells and a middle cerebral artery occlusion (MCAo) animal model. The results showed that the cell viability was increased with EV treatment in HT22 cells. In the animal, the size of the cerebral infarction was decreased, and the behavioral assessment was improved with EV injections. The levels of NeuN and neurofilament heavy chain (NFH)-positive cells were also increased with EV treatment yet decreased in the MCAo group. In addition, the number of apoptotic cells was decreased with EV treatment compared with ischemic animals following TUNEL and Bax/Bcl-2 staining. These data suggested that EVs, especially B-EVs, had a therapeutic effect and could reduce apoptotic cell death after ischemic injury.

CEBPD aggravates apoptosis and oxidative stress of neuron after ischemic stroke by Nrf2/HO-1 pathway.

CCAAT enhancer binding protein delta (CEBPD) is a transcription factor and plays an important role in apoptosis and oxidative stress, which are the main pathogenesis of ischemic stroke. However, whether CEBPD regulates ischemic stroke through targeting apoptosis and oxidative stress is unclear. Therefore, to answer this question, rat middle cerebral artery occlusion (MCAO) reperfusion model and oxygen-glucose deprivation/reoxygenation (OGD/R) primary cortical neuron were established to mimic ischemic reperfusion injury. We found that CEBPD was upregulated and accompanied with increased neurological deficit scores and infarct size, and decreased neuron in MCAO rats. The siRNA targeted CEBPD inhibited CEBPD expression in rats, and meanwhile lentivirus system was used to blocked CEBPD expression in primary neuron. CEBPD degeneration decreased neurological deficit scores, infarct size and brain water content of MCAO rats. Knockdown of CEBPD enhanced cell viability and reduced apoptosis as well as oxidative stress in vivo and in vitro. CEBPD silencing promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the expression of heme oxygenase 1 (HO-1). Newly, CEBPD facilitated the transcription of cullin 3 (CUL3), which intensified ischemic stroke through Nrf2/HO-1 pathway that was proposed by our team in the past. In conclusion, targeting CEBPD-CUL3-Nrf2/HO-1 axis may be contributed to cerebral ischemia therapy.

The angiotensin II receptors type 1 and 2 modulate astrocytes and their crosstalk with microglia and neurons in an in vitro model of ischemic stroke.

BACKGROUND: Astrocytes are the most abundant cell type of the central nervous system and are fundamentally involved in homeostasis, neuroprotection, and synaptic plasticity. This regulatory function of astrocytes on their neighboring cells in the healthy brain is subject of current research. In the ischemic brain we assume disease specific differences in astrocytic acting. The renin-angiotensin-aldosterone system regulates arterial blood pressure through endothelial cells and perivascular musculature. Moreover, astrocytes express angiotensin II type 1 and 2 receptors. However, their role in astrocytic function has not yet been fully elucidated. We hypothesized that the angiotensin II receptors impact astrocyte function as revealed in an in vitro system mimicking cerebral ischemia. Astrocytes derived from neonatal wistar rats were exposed to telmisartan (angiotensin II type 1 receptor-blocker) or PD123319 (angiotensin II type 2 receptor-blocker) under normal conditions (control) or deprivation from oxygen and glucose. Conditioned medium (CM) of astrocytes was harvested to elucidate astrocyte-mediated indirect effects on microglia and cortical neurons. RESULT: The blockade of angiotensin II type 1 receptor by telmisartan increased the survival of astrocytes during ischemic conditions in vitro without affecting their proliferation rate or disturbing their expression of S100A10, a marker of activation. The inhibition of the angiotensin II type 2 receptor pathway by PD123319 resulted in both increased expression of S100A10 and proliferation rate. The CM of telmisartan-treated astrocytes reduced the expression of pro-inflammatory mediators with simultaneous increase of anti-inflammatory markers in microglia. Increased neuronal activity was observed after treatment of neurons with CM of telmisartan- as well as PD123319-stimulated astrocytes. CONCLUSION: Data show that angiotensin II receptors have functional relevance for astrocytes that differs in healthy and ischemic conditions and effects surrounding microglia and neuronal activity via secretory signals. Above that, this work emphasizes the strong interference of the different cells in the CNS and that targeting astrocytes might serve as a therapeutic strategy to influence the acting of glia-neuronal network in de- and regenerative context.

Dysfunction of astrocytic glycophagy exacerbates reperfusion injury in ischemic stroke.

Glycophagy has evolved from an alternative glycogen degradation pathway into a multifaceted pivot to regulate cellular metabolic hemostasis in peripheral tissues. However, the pattern of glycophagy in the brain and its potential therapeutic impact on ischemic stroke remain unknown. Here, we observed that the dysfunction of astrocytic glycophagy was caused by the downregulation of the GABA type A receptor-associated protein like 1 (GABARAPL1) during reperfusion in ischemic stroke patients and mice. PI3K-Akt pathway activation is involved in driving GABARAPL1 downregulation during cerebral reperfusion. Moreover, glycophagy dysfunction-induced glucosamine deficiency suppresses the nuclear translocation of specificity protein 1 and TATA binding protein, the transcription factors for GABARAPL1, by decreasing their O-GlcNAcylation levels, and accordingly feedback inhibits GABARAPL1 in astrocytes during reperfusion. Restoring astrocytic glycophagy by overexpressing GABARAPL1 decreases DNA damage and oxidative injury in astrocytes and improves the survival of surrounding neurons during reperfusion. In addition, a hypocaloric diet in the acute phase after cerebral reperfusion can enhance astrocytic glycophagic flux and accelerate neurological recovery. In summary, glycophagy in the brain links autophagy, metabolism, and epigenetics together, and glycophagy dysfunction exacerbates reperfusion injury after ischemic stroke.

Efficacy of bumetanide in animal models of ischemic stroke: a systematic review and meta-analysis.

This meta-analysis aimed to describe the efficacy of bumetanide in improving infarct volume, brain edema, and behavioral outcomes in animal models of cerebral ischemia. Embase, PubMed and Web of Science databases were searched from their inception to February 2024 (INPLASY:202430023). Data on the animal species, stroke model, drug dose, time of treatment, method of administration, study quality, and outcomes were extracted and pooled in a meta-analysis. The combined standardized mean difference (SMD) or mean difference (MD) estimates and 95% confidence intervals (CIs) were calculated using random- or fixed-effects models. Thirteen eligible studies involving >200 animals fulfilled the inclusion criteria and were included in this meta-analysis. Meta-analyses demonstrated that bumetanide treatment significantly reduced cerebral infarct volume (SMD: -0.42; 95% CI: -0.75, -0.09; p < 0.01; n = 186 animals) and consistently relieved brain edema (SMD: -1.39; 95% CI: -2.06, -0.72; p < 0.01; n = 64 animals). Subgroup analyses demonstrated that bumetanide treatment reduced infarct volume in transient but not permanent cerebral ischemia models. When administered after the stroke, it was more effective than treatment initiation before the stroke. Eight studies assessed the effect of bumetanide on behavioral function and the results showed that bumetanide treatment significantly improved neurobehavioral deficits (SMD: -2.35; 95% CI: -2.72, -1.97; p < 0.01; n = 250 animals). We conclude that bumetanide appears to be effective in reducing infarct volume and brain edema and improving behavioral recovery in animal models of cerebral ischemia. This mechanism needs to be confirmed through further investigation.

ARMC10 regulates mitochondrial dynamics and affects mitochondrial function via the Wnt/ß-catenin signalling pathway involved in ischaemic stroke.

Mitochondrial dynamics has emerged as an important target for neuronal protection after cerebral ischaemia/reperfusion. Therefore, the aim of this study was to investigate the mechanism by which ARMC10 regulation of mitochondrial dynamics affects mitochondrial function involved in ischaemic stroke (IS). Mitochondrial morphology was detected by laser scanning confocal microscopy (LSCM), and mitochondrial ultrastructural alterations were detected by electron microscopy. The expression of mitochondrial dynamics-related genes Drp1, Mfn1, Mfn2, Fis1, OPA1 and ARMC10 and downstream target genes c-Myc, CyclinD1 and AXIN2 was detected by RT-qPCR. Western blot was used to detect the protein expression of ß-catenin, GSK-3ß, p-GSK-3ß, Bcl-2 and Bax. DCFH-DA fluorescent probe was to detect the effect of ARMC10 on mitochondrial ROS level, Annexin V-FITC fluorescent probe was to detect the effect of ARMC10 on apoptosis, and ATP assay kit was to detect the effect of ARMC10 on ATP production. Mitochondrial dynamics was dysregulated in clinical IS samples and in the OGD/R cell model, and the relative expression of ARMC10 gene was significantly decreased in IS group (p < 0.05). Knockdown and overexpression of ARMC10 could affect mitochondrial dynamics, mitochondrial function and neuronal apoptosis. Agonist and inhibitor affected mitochondrial function and neuronal apoptosis by targeting Wnt/ß-Catenin signal pathway. In the OGD/R model, ARMC10 affected mitochondrial function and neuronal apoptosis through the mechanism that regulates Wnt/ß-catenin signalling pathway. ARMC10 regulates mitochondrial dynamics and protects mitochondrial function by activating Wnt/ß-catenin signalling pathway, to exert neuroprotective effects.

Hyperglycemia induces microglial pyroptosis by increasing oxygen extraction rate: Implication in neurological impairment during ischemic stroke.

Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia promotes microglial pyroptosis by increasing the oxygen extraction rate in an acute ischemic stroke model. C57BL/6 mice that underwent middle cerebral artery occlusion were used for assessment of blood glucose level and neurological function. The cerebral oxygen extraction ratio (CERO2), oxygen consumption rate (OCR) and partial pressure of brain tissue oxygen (PbtO2) were measured. To investigate the significance of the NOD­like receptor protein 3 (NLRP3) inflammasome, NLRP3­/­ mice were used, and the expression levels of NLRP3, caspase­1, full­length gasdermin D (GSDMD­FL), GSDMD­N domain (GSDMD­N), IL­1ß and IL­18 were evaluated. In addition, Z­YVAD­FMK, a caspase­1 inhibitor, was used to treat microglia to determine whether activation of the NLRP3 inflammasome was required for the enhancing effect of hyperglycemia on pyroptosis. It was revealed that hyperglycemia accelerated cerebral injury in the acute ischemic stroke model, as evidenced by decreased latency to fall and the percentage of foot fault. Hyperglycemia aggravated hypoxia by increasing the oxygen extraction rate, as evidenced by increased CERO2 and OCR, and decreased PbtO2 in response to high glucose treatment. Furthermore, hyperglycemia­induced microglial pyroptosis was confirmed by detection of increased levels of caspase­1, GSDMD­N, IL­1ß and IL­18 and a decreased level of GSDMD­FL. However, the knockout of NLRP3 attenuated these effects. Pharmacological inhibition of caspase­1 also reduced the expression levels of GSDMD­N, IL­1ß and IL­18 in microglial cells. These results suggested that hyperglycemia stimulated NLRP3 inflammasome activation by increasing the oxygen extraction rate, thus leading to the aggravation of pyroptosis following ischemic stroke.

Neuroprotective Effect and Mechanism of Tanreqing Injection on Ischemic Stroke: Insights from Network Pharmacology and in vivo Experiments.

OBJECTIVE: To explore the neuroprotective effects and mechanism of Tanreqing Injection (TRQ) on treating ischemic stroke based on network pharmacology and in vivo experimental validation. METHODS: The chemical compounds of TRQ were retrieved based on published data, with targets retrieved from PubChem, Therapeutic Target Database and DrugBank. Network visualization and analysis were performed using Cytoscape, with protein-protein interaction networks derived from the STRING database. Enrichment analysis was performed using Kyoto Encyclopedia of Genes Genomes pathway and Gene Ontology analysis. In in vivo experiments, the middle cerebral artery occlusion (MCAO) model was used. Infarct volume was determined by 2,3,5-triphenyltetrazolium hydrochloride staining and protein expressions were analyzed by Western blot. Molecular docking was performed to predict ligand-receptor interactions. RESULTS: We screened 81 chemical compounds in TRQ and retrieved their therapeutic targets. Of the targets, 116 were therapeutic targets for stroke. The enrichment analysis showed that the apelin signaling pathway was a key pathway for ischemic stroke. Furthermore, in in vivo experiment we found that administering with intraperitoneal injection of 2.5 mL/kg TRQ every 6 h could significantly reduce the infarct volume of MCAO rats (P<0.05). In addition, protein levels of the apelin receptor (APJ)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway were increased by TRQ (P<0.05). In addition, 41 chemical compounds in TRQ could bind to APJ. CONCLUSIONS: The neuroprotective effect of TRQ may be related to the APJ/PI3K/AKT signaling pathway. However, further studies are needed to confirm the findings.

Blood brain barrier-targeted lipid nanoparticles improved the neuroprotection of Ferrostatin-1 against cerebral ischemic damage in an experimental stroke model.

Cerebral ischemic stroke is a serious disease with high mortality and disability rates. However, few neuroprotective drugs have been used for ischemic stroke in the clinic. Two main reasons may be responsible for this failure: difficulty in penetrating the blood-brain barrier (BBB) and easily inactivated in the blood circulation. Ferroptosis, a lipid oxidation-related cell death, plays significant roles in cerebral ischemia-reperfusion injury. We utilized RVG29, a peptide derived from Rabies virus glycoprotein, to obtain BBB-targeted lipid nanoparticles (T-LNPs) in order to investigate whether T-LNPs improved the neuroprotective effects of Ferrostatin-1 (Fer1, an inhibitor of ferroptosis) against cerebral ischemic damage. T-LNPs significantly increased BBB penetration following oxygen/glucose deprivation exposure in an in vitro BBB model and enhanced the fluorescence distribution in brain tissues at 6 h post-administration in a cerebral ischemic murine model. Moreover, T-LNPs encapsulated Fer1 (T-LNPs-Fer1) significantly enhanced the inhibitory effects of Fer1 on ferroptosis by maintaining the homeostasis of NADPH oxidase 4 (NOX4) and glutathione peroxidase 4 (GPX4) signals in neuronal cells after cerebral ischemia. T-LNPs-Fer1 significantly suppressed oxidative stress [heme oxygenase-1 expression and malondialdehyde (the product of lipid ROS reaction)] in neurons and alleviated ischemia-induced neuronal cell death, compared to Fer1 alone without encapsulation. Furthermore, T-LNPs-Fer1 significantly reduced cerebral infarction and improved behavior functions compared to Fer1-treated cerebral ischemic mice after 45-min ischemia/24-h reperfusion. These findings showed that the T-LNPs helped Fer1 penetrate the BBB and improved the neuroprotection of Fer1 against cerebral ischemic damage in experimental stroke, providing a feasible translational strategy for the development of clinical drugs for the treatment of ischemic stroke.