Experimental Ischemic Stroke Research Articles

STING orchestrates microglia polarization via interaction with LC3 in autophagy after ischemia.

Autophagy has both protective and pathogenetic effects on injury caused by cerebral ischemia/reperfusion (I/R). Our previous research has indicated that stimulator of interferon genes (STING) could orchestrate microglia polarization following middle cerebral artery occlusion. However, it remains largely unexplored whether STING balances microglial polarization by regulating autophagy in brain I/R injury. Here, STING was observed to show an up-regulation in the microglia from mice subjected to experimental ischemic stroke. Strikingly, the deletion of STING led to the significant skewness of microglia activated by ischemia from a pro- to anti-inflammatory state and substantially alleviated ischemia-induced infarction and neuronal injury. In addition, STING-null mice can restore long-term neurobehavioral function. Then, the crosstalk between neuroinflammation and microglia autophagy was analyzed. The differential activity of autophagy in wild-type and STING-knockout (KO) mice or primary microglia was largely reversed when STING was restored in microglia. Irritating autophagy by rapamycin skewed the anti‑inflammatory state induced by STING-KO to a pro‑inflammatory state in microglia. Furthermore, microtubule-associated protein light-chain-3 (LC3) was identified as the key factor in the STING regulation of autophagy by glutathione-S-transferase (GST) pull-down analysis. Mechanically, STING can directly interact with LC3 through the STING transmembrane domain (1-139aa). Herein, current data determine the pivotal role of autophagy, specifically via LC3 protein, in the regulation of microglial phenotypic transformation by STING. These findings may provide a possible treatment target for delaying the progression of ischemic stroke.

Comparative study of the neuroprotective potential of semaglutide injectable preparations in experimental ischemic stroke

Introduction. Stroke remains one of the major causes of death in type diabetes mellitus (DM). Injectable form of glucagon-like peptide-1 receptor agonist semaglutide, Ozempic® decreases the stroke risk. In Russia there appeared a biosimilar Semavic® but its effects on the brain are not yet studied.Aim. To compare neuroprotective properties of Semavic® and Ozempic® while used before ischemic stroke in rats without DM.Materials and methods. The study was conducted in male Wistar rats that were divided into the following groups: “Control” (n = 10) – 0.9% NaCl, “MET” (n = 9) – metformin 200 mg/kg once daily per os, “Ozempic” (n = 10) – Ozempic® 0.012 mg/kg s.c. once daily, “Semavic” (n = 9) – Semavic® 0.012 mg/kg s.c. once daily. After 7 days ischemic stroke was modelled, after 48 hour of reperfusion neurological deficit and brain damage volume were evaluated. Glycemia was measured on the 3rd, 7th days as well as during and after ischemia.Results. None of the study drugs caused hypoglycemia including in poststroke period. Neurological deficit in “MET” group did not differ from that in the “Control” (11.00 [6.50; 12.50] and 10.0 [6.25; 12.00] scores). Both semaglutide drugs caused comparable improvement in neurological status (14.00 [12.00; 18.00] and 14.00 [11.00; 18.00] scores in “Ozempic” and “Semavic” groups). Brain necrosis volume in “Control” group was 16.60 [13.40; 28.58] %. All the study drugs had infarct-limiting effect but brain damage volume in “Ozempic” (6.00 [4.32; 8.44] %) and “Semavic” (7.69 [2.99; 11.33] %) was smaller than in “MET” group (13.07 [8.67; 29.94] %). There were no differences between semaglutide drugs.Conclusions. Biosimilar Semavic® and original Ozempic® demonstrate comparable neuroprotective effect while used in animals without DM prior to ischemic stroke modelling. This protective effect is not due to the drugs’ influence on glycemic profile.

Activation of spleen tyrosine kinase (SYK) contributes to neuronal pyroptosis and cognitive impairment in diabetic mice via the NLRP3/Caspase-1/GSDMD signaling pathway.

Diabetes mellitus (DM) patients are at increased risk of cognitive impairment. The precise mechanisms underlying the association between DM and cognitive impairment remain unclear. Spleen tyrosine kinase (SYK), a crucial regulator of signal transduction, has been implicated in microglial pyroptosis in experimental ischemic stroke models. The present study investigated the potential role of SYK in DM-associated cognitive impairment. Diabetes was induced by streptozotocin (STZ) in C57BL/6 mice, and cognitive function and cerebral injury were assessed 12weeks later using the Morris water maze (MWM), TUNEL assay and Western blotting. In vitro, the inhibition of SYK was investigated in a mouse hippocampal neuronal cell line cultured with high glucose. Compared with control mice, DM mice presented impaired spatial learning and memory. Additionally, SYK activation was linked to neuronal pyroptosis, as evidenced by increases in the number of TUNEL-positive cells and protein levels of NLRP3, ASC, procaspase-1, caspase-1, GSDMD, the GSDMD N-terminal fragment, pro-IL-1β, and IL-1β in the hippocampus of DM mice. Compared with no treatment, SYK knockdown markedly attenuated cognitive impairment and histologic and ultrastructural pathological changes in the hippocampus of DM mice. The increased expression of pyroptosis-associated proteins and the increased number of TUNEL-positive cells were also significantly reduced. In vitro, high glucose significantly activated SYK to trigger the canonical pyroptotic pathway in cultured HT22 cells. The inhibition of SYK with a small interfering RNA or specific inhibitor significantly ameliorated the neuronal pyroptosis mediated by high glucose. Our findings demonstrate that SYK activation plays a pivotal role in promoting the cognitive impairment associated with DM. This effect is mediated by triggering neuronal pyroptosis through the canonical NLRP3/Caspase-1/GSDMD pathway. These results suggest that SYK may serve as a potential target for preventing or mitigating cognitive impairment in patients with DM.

Systematic review and Meta-analysis of brain plasticity associated with electroacupuncture in experimental ischemic stroke.

To systematically evaluate the role of electroacupuncture in maintaining brain plasticity in ischemic stroke mediated brain damage. We searched for all relevant trials published through Oct 7, 2022 from seven databases. Metho-dological quality was assessed using the CAMARADES Risk of Bias Tool. A Meta-analysis of comparative effects was performed using Review Manager v.5.3 software. A total of 101 studies involving 2148 animals were included. For most studies, primary outcomes results of the Meta-analysis indicate that EA significantly improved ischemic stroke rat's postsynaptic density thickness [Standardized Mean Difference (SMD) = 1.41, 95% confidence interval (CI) (0.59, 2.23), P = 0.0008], numerical density of synapses [SMD = 1.55, 95% CI (0.48, 2.63), P = 0.005] compared with non-EA-treated. Similarly, EA could improve parts of biomarkers of synapses, neurogenesis, angiogenesis and neurotrophin activity than the control group (P < 0.05). The existing evidence suggests EA regulating ischemic stroke may be through brain plasticity. More rigorous and high quality studies should be conducted in the future.

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.

Macrophage Scavenger Receptor 1 attenuates ischemic white matter injury via HRH1-mediated microglial phagocytosis

The removal of axonal and myelin debris by macrophages is crucial for safeguarding nerves and facilitating functional recuperation in cerebral ischemic stroke. However, the physiological function of macrophage scavenger receptor 1 (MSR1) in ischemic white matter injury remains poorly de-fined. In this study, we observed an elevation in Msr1 expression levels in mice with experimental cerebral ischemic stroke. Msr 1-deficient (Msr1-/-) mice exhibited exacerbated behavioral deficits and aggravated white matter injury after ischemic stroke. Furthermore, the overexpression of Msr1 led to an increase in the phosphorylation of Akt via Hrh1, which in turn expedited the clearance of myelin debris through the PI3K/AKT pathway. In conclusion, our findings underscore the essential role of MSR1 in microglial phagocytosis and its ability to mitigate ischemic white matter injury in cerebral ischemic stroke.

Identification and analysis of key immunity-related genes in experimental ischemic stroke

The regulation of the immune system and the occurrence of inflammation are vital factors in the pathophysiology of ischemic stroke. This study aims to screen target molecules which play key roles in alleviating the brain injury following ischemic stroke via regulating neuroinflammation. Several bioinformatics methods were used to identify immune-related genes in ischemic stroke. A total of 218 genes were identified as differentially expressed genes within the GSE97537 dataset. By performing GO, KEGG, and GSEA analyses, DEGs were mainly enriched in pathways related to immunity and inflammation. By utilizing the MCODE plugin in conjunction with Cytoscape software, a total of six crucial genes were identified, including C1qb, C1qc, Fcer1g, Fcgr3a, Tyrobp, and CD14. Based on the above crucial genes, 13 miRNAs were predicted. Furthermore, 71 potential drugs with therapeutic properties that target the crucial genes were screened, including lovastatin, ASPIRIN, and PREDNISOLONE. Moreover, the results of RT-qPCR showed that compared with Sham group, the expressions of C1qb, C1qc, Fcer1g, Fcgr3a, Tyrobp, and CD14 in MCAO group were significantly increased, which was consistent with the expression trend of validation dataset and training dataset. In conclusion, immune-related genes may play a key role in ischemic stroke. In addition, six crucial genes were identified as potential biomarkers and 71 promising drugs were screened to treat ischemic stroke patients.

Dissecting glial scar formation by spatial point pattern and topological data analysis

Glial scar formation represents a fundamental response to central nervous system (CNS) injuries. It is mainly characterized by a well-defined spatial rearrangement of reactive astrocytes and microglia. The mechanisms underlying glial scar formation have been extensively studied, yet quantitative descriptors of the spatial arrangement of reactive glial cells remain limited. Here, we present a novel approach using point pattern analysis (PPA) and topological data analysis (TDA) to quantify spatial patterns of reactive glial cells after experimental ischemic stroke in mice. We provide open and reproducible tools using R and Julia to quantify spatial intensity, cell covariance and conditional distribution, cell-to-cell interactions, and short/long-scale arrangement, which collectively disentangle the arrangement patterns of the glial scar. This approach unravels a substantial divergence in the distribution of GFAP+ and IBA1+ cells after injury that conventional analysis methods cannot fully characterize. PPA and TDA are valuable tools for studying the complex spatial arrangement of reactive glia and other nervous cells following CNS injuries and have potential applications for evaluating glial-targeted restorative therapies.

Benserazide is neuroprotective and improves functional recovery after experimental ischemic stroke by altering the immune response

Stroke is a leading cause of permanent disability worldwide. Despite intensive research over the last decades, key anti-inflammatory strategies that have proven beneficial in pre-clinical animal models have often failed in translation. The importance of neutrophils as pro- and anti-inflammatory peripheral immune cells has often been overlooked in ischemic stroke. However, neutrophils rapidly infiltrate into the brain parenchyma after stroke and secrete an array of pro-inflammatory factors including reactive oxygen species, proteases, cytokines, and chemokines exacerbating damage. In this study, we demonstrate the neuroprotective and anti-inflammatory effect of benserazide, a clinically used DOPA decarboxylase inhibitor, using both in vitro models of inflammation and in vivo mouse models of focal cerebral ischemia. Benserazide significantly attenuated PMA-induced NETosis in isolated human neutrophils. Furthermore, benserazide was able to protect both SH-SY5Y and iPSC-derived human cortical neurons when challenged with activated neutrophils demonstrating the clinical relevance of this study. Additional in vitro data suggest the ability of benserazide to polarize macrophages towards M2-phenotypes following LPS stimulation. Neuroprotective effects of benserazide are further demonstrated by in vivo studies where peripheral administration of benserazide significantly attenuated neutrophil infiltration into the brain, altered microglia/macrophage phenotypes, and improved the behavioral outcome post-stroke. Overall, our data suggest that benserazide could serve as a drug candidate for the treatment of ischemic stroke. The importance of our results for future clinical trials is further underlined as benserazide has been approved by the European Medicines Agency as a safe and effective treatment in Parkinson’s disease when combined with levodopa.

Hippocampal infarction and generalized seizures predict early mortality after endovascular middle cerebral artery occlusion in mice

Endovascular middle cerebral artery occlusion (MCAO) is a widely used experimental ischemic stroke model. However, the model carries high early mortality. Our aim was to investigate the factors that influence early mortality within 48 h of reperfusion after transient MCAO. Using C57BL/6 mice, we induced 1-hour endovascular filament MCAO. To introduce heterogeneity of infarct volumes, a subset of animals had additional tandem common carotid artery occlusion (MCAO+CCAO). Continuous video monitoring was used to gain insight into the cause of death. Mortality within 48 h was 25% in the pooled cohort. All animals with early mortality suffered from infarcts in the hippocampus, sometimes accompanied by infarcts in the thalamus and midbrain, which occurred exclusively in the MCAO+CCAO group. All animals with early mortality developed convulsive seizures captured on video monitoring. None of the animals that did not develop convulsive seizures died. Among the three regions, hippocampal infarction appeared necessary for convulsive seizures and early mortality. Our data highlight seizures as the primary cause of mortality within the first 48 h after endovascular filament MCAO, linked to hippocampal infarction. Since hippocampal blood supply is mainly from the posterior cerebral artery (PCA), avoiding concurrent PCA ischemia can decrease mortality in proximal MCAO models.

Proximal and distant expression of growth differentiation factor 15 (GDF15) correlate with neurological deficit following experimental ischemic stroke.

Growth differentiation factor 15 (GDF15) has emerged as a promising biomarker in cerebro-cardiovascular disease, particularly in acute and chronic inflammatory stress situations. However, understanding the origins, targets and functions of GDF15 in clinical situations, such as ischemic stroke, remains a complex challenge. This study aims to assess the sources of GDF15 production following an experimental ischemic stroke. Adult male Wistar rats underwent cerebral embolization through microspheres injection into the left or right internal carotid artery. Two hours post-surgery, GDF15 expression was analyzed in the brain, blood, lungs, liver and heart using quantitative RT-PCR and Western blotting. Stroke model induced large cerebral infarcts accompanied by severe neurological deficits. GDF15 gene expression exhibited a substantial increase in the ipsilateral cortex and cerebellum, with a lesser extent in the contralateral cortex. Regarding GDF15 protein expression, proGDF15 levels were elevated in the 3 aforementioned organs mentioned and the heart. However, the mature form of GDF15 was exclusively present and increased in the heart. Finally, the expression of GDF15 expression was correlated with the neurological deficit score. Our findings suggest that both the GDF15 gene and pro-protein are expressed in the ischemic brain after a stroke, while only its mature form is expressed remotely in in the heart. The impact of increased GDF15 in the heart following a stroke remains to be established. This is particularly relevant in understanding its relationships with poor neurological outcomes, determining whether it may contribute to stroke-induced cardiac dysfunction.

Selective deletion of interleukin-1 alpha in microglia does not modify acute outcome but regulates neurorepair processes after experimental ischemic stroke.

Inflammation is a key contributor to stroke pathogenesis and exacerbates brain damage leading to poor outcome. Interleukin-1 (IL-1) is an important regulator of post-stroke inflammation, and blocking its actions is beneficial in pre-clinical stroke models and safe in the clinical setting. However, the distinct roles of the two major IL-1 receptor type 1 agonists, IL-1α and IL-1β, and the specific role of IL-1α in ischemic stroke remain largely unknown. Here we show that IL-1α and IL-1β have different spatio-temporal expression profiles in the brain after experimental stroke, with early microglial IL-1α expression (4 h) and delayed IL-1β expression in infiltrated neutrophils and a small microglial subset (24-72 h). We examined for the first time the specific role of microglial-derived IL-1α in experimental permanent and transient ischemic stroke through microglial-specific tamoxifen-inducible Cre-loxP-mediated recombination. Microglial IL-1α deletion did not influence acute brain damage, cerebral blood flow, IL-1β expression, neutrophil infiltration, microglial nor endothelial activation after ischemic stroke. However, microglial IL-1α knock out (KO) mice showed reduced peri-infarct vessel density and reactive astrogliosis at 14 days post-stroke, alongside long-term impaired functional recovery. Our study identifies for the first time a critical role for microglial IL-1α on neurorepair and functional recovery after stroke, highlighting the importance of targeting specific IL-1 mechanisms in brain injury to develop more effective therapies.

Long-term grape powder feed attenuates experimental ischemic stroke by altering gut microbiota and metabolome

Long-term grape powder feed attenuates experimental ischemic stroke by altering gut microbiota and metabolome

Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions

Ischemic stroke induces neovascularization of the injured tissue as an attempt to promote structural repair and neurological recovery. Angiogenesis is regulated by pericytes that potently react to ischemic stroke stressors, ranging from death to dysfunction. Platelet-derived growth factor (PDGF) receptor (PDGFR)β controls pericyte survival, migration, and interaction with brain endothelial cells. PDGF-D a specific ligand of PDGFRβ is expressed in the brain, yet its regulation and role in ischemic stroke pathobiology remains unexplored. Using experimental ischemic stroke mouse model, we found that PDGF-D is transiently induced in brain endothelial cells at the injury site in the subacute phase. To investigate the biological significance of PDGF-D post-ischemic stroke regulation, its subacute expression was either downregulated using siRNA or upregulated using an active recombinant form. Attenuation of PDGF-D subacute induction exacerbates neuronal loss, impairs microvascular density, alters vascular permeability, and increases microvascular stalling. Increasing PDGF-D subacute bioavailability rescues neuronal survival and improves neurological recovery. PDGF-D subacute enhanced bioavailability promotes stable neovascularization of the injured tissue and improves brain perfusion. Notably, PDGF-D enhanced bioavailability improves pericyte association with brain endothelial cells. Cell-based assays using human brain pericyte and brain endothelial cells exposed to ischemia-like conditions were applied to investigate the underlying mechanisms. PDGF-D stimulation attenuates pericyte loss and fibrotic transition, while increasing the secretion of pro-angiogenic and vascular protective factors. Moreover, PDGF-D stimulates pericyte migration required for optimal endothelial coverage and promotes angiogenesis. Our study unravels new insights into PDGF-D contribution to neurovascular protection after ischemic stroke by rescuing the functions of pericytes.

Pharmacological inhibition of receptor-interacting protein kinase 2 (RIPK2) elicits neuroprotective effects following experimental ischemic stroke

Ischemic stroke induces a debilitating neurological insult, where inflammatory processes contribute greatly to the expansion and growth of the injury. Receptor-interacting protein kinase 2 (RIPK2) is most well-known for its role as the obligate kinase for NOD1/2 pattern recognition receptor signaling and is implicated in the pathology of various inflammatory conditions. Compared to a sham-operated control, ischemic stroke resulted in a dramatic increase in the active, phosphorylated form of RIPK2, indicating that RIPK2 may be implicated in the response to stroke injury. Here, we assessed the effects of pharmacological inhibition of RIPK2 to improve post-stroke outcomes in mice subjected to experimental ischemic stroke. We found that treatment at the onset of reperfusion with a RIPK2 inhibitor, which inhibits the phosphorylation and activation of RIPK2, resulted in marked improvements in post-stroke behavioral outcomes compared to the vehicle-administered group assessed 24 h after stroke. RIPK2 inhibitor-treated mice exhibited dramatic reductions in infarct volume, concurrent with reduced damage to the blood-brain barrier, as evidenced by reduced levels of active matrix metalloproteinase-9 (MMP-9) and leakage of blood-borne albumin in the ipsilateral cortex. To explore the protective mechanism of RIPK2 inhibition, we next pretreated mice with RIPK2 inhibitor or vehicle and examined transcriptomic alterations occurring in the ischemic brain 6 h after stroke. We observed a dramatic reduction in neuroinflammatory markers in the ipsilateral cortex of the inhibitor-treated group while also attaining a comprehensive view of the vast transcriptomic alterations occurring in the brain with inhibitor treatment through bulk RNA-sequencing of the injured cortex. Overall, we provide significant novel evidence that RIPK2 may represent a viable target for post-stroke pharmacotherapy and potentially other neuroinflammatory conditions.

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

Elabela ameliorates neuronal pyroptosis and mitochondrial fission via APJ/ZBP1 signaling in ischemic stroke

Pyroptosis signifies a significant form of programmed neuronal demise subsequent to ischemic stroke. In our prior investigations, we demonstrated that the Elabela (ELA)–Apelin receptor (APJ) axis alleviated neuronal death by improving collateral circulation and mitigating ferroptosis in a murine model of middle cerebral artery occlusion (MCAO). However, the connection between ELA and neuronal pyroptosis remains further elucidation. Here, we observed an upregulation of ELA and APJ expression in both murine brain specimens and cultured HT-22 hippocampal neurons exposed to experimental ischemic stroke. ELA administration markedly diminished the infarct size in comparison to controls. ELA treatment ameliorated neurological deficits and anxiety-like symptoms in mice with stroke, concurrently inhibiting pyroptosis and mitochondria fission in neurons. Conversely, ELA knockdown yielded the opposite effects. Utilizing RNA-sequencing analysis, we identified a candidate for pyroptosis priming, Z-DNA-binding protein 1 (ZBP1), which was suppressed in ELA-treated HT-22 neurons during oxygen-glucose deprivation/reperfusion (OGD/R). Subsequent co-immunoprecipitation analyses demonstrated the binding between APJ and ZBP1. Specifically, APJ suppressed ZBP1 to inhibit NLRP3 inflammasome activation and dynamin-related protein 1-mediated mitochondrial fission in neurons. In summary, our findings suggest that ELA functions as a stroke-induced signal limiting neuronal pyroptosis and mitochondrial fission via APJ/ZBP1 signaling, thereby underscoring ELA as a potential therapeutic target for ischemic stroke treatment.

Overexpression of low-density lipoprotein receptor prevents neurotoxic polarization of astrocytes via inhibiting NLRP3 inflammasome activation in experimental ischemic stroke.

JOURNAL/nrgr/04.03/01300535-202502000-00027/figure1/v/2024-05-28T214302Z/r/image-tiff Neurotoxic astrocytes are a promising therapeutic target for the attenuation of cerebral ischemia/reperfusion injury. Low-density lipoprotein receptor, a classic cholesterol regulatory receptor, has been found to inhibit NLR family pyrin domain containing protein 3 (NLRP3) inflammasome activation in neurons following ischemic stroke and to suppress the activation of microglia and astrocytes in individuals with Alzheimer's disease. However, little is known about the effects of low-density lipoprotein receptor on astrocytic activation in ischemic stroke. To address this issue in the present study, we examined the mechanisms by which low-density lipoprotein receptor regulates astrocytic polarization in ischemic stroke models. First, we examined low-density lipoprotein receptor expression in astrocytes via immunofluorescence staining and western blotting analysis. We observed significant downregulation of low-density lipoprotein receptor following middle cerebral artery occlusion reperfusion and oxygen-glucose deprivation/reoxygenation. Second, we induced the astrocyte-specific overexpression of low-density lipoprotein receptor using astrocyte-specific adeno-associated virus. Low-density lipoprotein receptor overexpression in astrocytes improved neurological outcomes in middle cerebral artery occlusion mice and reversed neurotoxic astrocytes to create a neuroprotective phenotype. Finally, we found that the overexpression of low-density lipoprotein receptor inhibited NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation injured astrocytes and that the addition of nigericin, an NLRP3 agonist, restored the neurotoxic astrocyte phenotype. These findings suggest that low-density lipoprotein receptor could inhibit the NLRP3-meidiated neurotoxic polarization of astrocytes and that increasing low-density lipoprotein receptor in astrocytes might represent a novel strategy for treating cerebral ischemic stroke.

Experimental Ischemic Stroke Induces Secondary Bihemispheric White Matter Degeneration and Long-Term Cognitive Impairment.

Clinical studies have identified widespread white matter degeneration in ischemic stroke patients. However, contemporary research in stroke has predominately focused on the infarct and periinfarct penumbra regions. The involvement of white matter degeneration after ischemic stroke and its contribution to post-stroke cognitive impairment and dementia (PSCID) has remained less explored in experimental models. In this study, we examined the progression of locomotor and cognitive function up to 4 months after inducing ischemic stroke by middle cerebral artery occlusion in young adult rats. Despite evident ongoing locomotor recovery, long-term cognitive and affective impairments persisted after ischemic stroke, as indicated by Morris water maze, elevated plus maze, and open field performance. At 4 months after stroke, multimodal MRI was conducted to assess white matter degeneration. T2-weighted MRI (T2WI) unveiled bilateral cerebroventricular enlargement after ischemic stroke. Fluid Attenuated Inversion Recovery MRI (FLAIR) revealed white matter hyperintensities in the corpus callosum and fornix across bilateral hemispheres. A positive association between the volume of white matter hyperintensities and total cerebroventricular volume was noted in stroke rats. Further evidence of bilateral white matter degeneration was indicated by the reduction of fractional anisotropy and quantitative anisotropy at bilateral corpus callosum in diffusion-weighted MRI (DWI) analysis. Additionally, microglia and astrocyte activation were identified in the bilateral corpus callosum after stroke. Our study suggests that experimental ischemic stroke induced by MCAO in young rat replicate long-term cognitive impairment and bihemispheric white matter degeneration observed in ischemic stroke patients. This model provides an invaluable tool for unraveling the mechanisms underlying post-stroke secondary white matter degeneration and its contribution to PSCID.

Involvement of Calpain in Neurovascular Unit Damage through Up-regulating PARP-NF-κB Signaling during Experimental Ischemic Stroke.

Calpain and PARP-NF-κB signaling are reported to participate in the ischemic brain injury. In this study, it was investigated whether calpain was contributed to the neurovascular unit (NVU) damage through up-regulating PARP-NF-κB signaling during experimental ischemic stroke. Male Sprague-Dawley rats were suffered from 90min of middle cerebral artery occlusion, followed by reperfusion. The NVU damage was evaluated by the permeability of blood-brain barrier (BBB), the degradation of proteins in extracellular matrix and tight junctions, and ultrastructural changes. The inflammatory response was determined by the expression of inflammatory genes driven by PARP-NF-κB signaling and the activities of myeloperoxidase (MPO). Treatment with MDL 28,170, a calpain inhibitor, improved neurological functions, reduced TUNEL staining index, lessened brain swelling, and decreased infarct volume in ischemic rats. Moreover, it reduced the BBB permeability, enhanced the levels of laminin, collagen IV and occludin, and attenuated the ultrastructural damage of NVU in penumbra and core after induction of ischemia. Meanwhile, it enhanced the levels of cytosolic IκBα, lessened the levels of nuclear PARP and NF-κB p65, reduced the levels of ICAM-1, TNF-α, IL-1β, MMP-9, and MMP-2,and suppressed the activities of MPO in penumbra and core. These data showed that calpain inhibition suppressed PARP-NF-κB signaling-mediated inflammatory response, reduced NVU damage, and protected brain against ischemic stroke, suggesting the involvement of calpain in the NVU damage through up-regulating PARP-NF-κB signaling during brain ischemia.