Ischemic Stroke In Mice Research Articles

Abstract TMP110: Impact of Microplastics and Nanoplastics on Ischemic Stroke in Mice

Introduction: Over the past few decades, plastics have been widely used in many products. Recently, the potential ingestion of plastic debris called microplastics and nanoplastics (MNPs) and their toxicity to organs and tissues have been reported. They can enter the bloodstream not only by ingestion, but also by inhalation and dermal contact. MNPs are emerging as a potential risk factor for cerebrovascular events such as myocardial infarction and stroke in recent clinical study. However, the relationship between MNPs exposure and ischemic stroke is not well elucidated. In this study, we investigated the effect of MNPs on ischemic stroke using the transient middle cerebral artery occlusion (tMCAO) model in mice. Methods: We used 12-week-old male C57BL/6J mice. Mice (n = 48) were divided into four groups based on the materials administered. After daily pretreatment with either pure water as vehicle, polystyrene (PS, 150 mg/kg/day), polyethylene (PE, 150 mg/kg/day), and polyvinyl chloride (PVC, 150 mg/kg/day) for 35 days, mice were subjected to tMCAO for 60 minutes. Treatment with MNPs was continued until postoperative day 2. As a safety study, we measured the body weight and blood pressure during the pretreatment. We compared Garcia score, neurological deficit score (NDS), rotarod performance (% baseline), and infarction volume (% ipsilateral) between the groups 3 days after tMCAO. Results: No significant side effects such as weight loss or blood pressure changes were observed during pretreatment. Garcia scores were significantly lower in the PE (7.5 ± 4.0) and PVC (9.1 ± 2.1) groups compared to the vehicle group (11.9 ± 3.7) on day 3 (Fig. A, P <0.05, respectively). Similarly, NDS was significantly higher in the PE (2.5 ± 1.3) and PVC (2.3 ± 0.5) groups compared to the vehicle group (1.4 ± 1.0) on day 3 (Fig. B, P <0.05). There was a significant difference in rotarod performance between PE (55.6%) and vehicle (100.2%) groups on day 3 (Fig. C, P <0.05). Infarct volumes were significantly larger in the PE (30.2%) and PVC (27.3%) groups compared to the vehicle group (14.2%) on day 3 (Fig. D, P <0.05). These results were not confirmed in the PS group. Conclusions: This preliminary study revealed the potential negative effects of microplastics and nanoplastics on ischemic stroke in mice. Future research should evaluate the long-term outcomes and the mechanisms by which microplastics and nanoplastics exacerbate the ischemic stroke.

Abstract 159: Cerebral endothelial cells generate ceramides that worsen outcomes of acute ischemic stroke in mice.

Abstract 159: Cerebral endothelial cells generate ceramides that worsen outcomes of acute ischemic stroke in mice.

Molecular Mediators of Neutrophil Primary Granule Release Following Acute Ischemic Stroke and their Associated Epigenetic Modulation by HDAC2.

High concentrations of neutrophil degranulation products in the plasma and thrombi are poor prognostic indicators in patients with acute ischemic stroke (AIS). This study aimed to identify candidate effectors capable of mediating neutrophil degranulation post-AIS, and to reveal their underlying epigenetic mechanisms. Microarrays and ChIP-seq were applied to analyze the neutrophils of patients with AIS. Cerebral ischemia was induced in C57/BL6 mice by middle cerebral artery occlusion (MCAO). Lipopolysaccharide was used to induce inflammation in HL-60 Cells. Protein and mRNA levels were assessed using flow cytometry, ELISA, western blotting, and RT-PCR. Degranulation was identified as a significant pathway in the neutrophils of patients with AIS, while Rho GTPase and the SNARE complex also showed importance. HDAC2 differentially binds to genes involved in neutrophil degranulation in patients with AIS. SYT9, SH3BP1, and STXBP1 were identified in two sequencing experiments, for which HDAC2 bound to their promoter, intron, and upstream regions, respectively. Consistently, candidate degranulation effectors and products showed substantially increased expression and co-localization in the neutrophils of thrombi obtained from patients with middle cerebral artery stenosis with poor prognosis, a mouse model of MCAO, and an HL-60 cell-based model of inflammation. Knockdown of SYT9, SH3BP1, and STXBP1 impaired primary granule release in vitro, whereas HDAC2 activity was decreased following LPS induction and ischemic stroke in mice. Furthermore, HDAC2 inhibition upregulated SYT9, SH3BP1, and STXBP1. Our findings suggest that these three molecules may be indispensable in the process of neutrophil degranulation following AIS, and are targeted by HDAC2, paving the way for the development of new drugs.

CaMKIIα hub ligands are unable to reverse known phenotypes in Angelman syndrome mice.

Angelman Syndrome (AS) is a neurodevelopmental disorder caused by the loss of function of ubiquitin-protein ligase E3A (UBE3A), resulting in marked changes in synaptic plasticity. In AS mice, a dysregulation of Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) was previously described. This has been convincingly validated through genetic rescue of prominent phenotypes in mouse cross-breeding experiments. Selective ligands that specifically stabilize the CaMKIIα central association (hub) domain and affect different conformational states in vitro are now available. Two of these ligands, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and (E)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (Ph-HTBA), confer neuroprotection after ischemic stroke in mice where CaMKIIα is known to be dysregulated. Here, we sought to investigate whether pharmacological modulation with these prototypical CaMKIIα hub ligands presents a viable approach to alleviate AS symptoms. We performed an in vivo functional evaluation of AS mice treated for a total of 14 days with either HOCPCA or Ph-HTBA (7days pre-treatment and 7days of behavioural assessment). Both compounds were well-tolerated but unable to revert robust phenotypes of motor performance, anxiety, repetitive behaviour or seizures in AS mice. Biochemical experiments subsequently assessed CaMKIIα autophosphorylation in AS mouse brain tissue. Taken together our results indicate that pharmacological modulation of CaMKIIα via the selective hub ligands used here is not a viable treatment strategy in AS.

Electroacupuncture Pretreatment Reduces Ischemic Brain Injury by Inhibiting the Lactate Production and Its Derived Protein Lactylation Formation.

Given that electroacupuncture (EA) pretreatment inhibits lactate production and lactate-derived lysine lactation (Kla) aggravates ischemic brain injury, we aimed to investigate whether the formation of Kla protein is involved in EA pretreatment to alleviate ischemic brain injury. EA was performed on the Baihui acupoint (GV20) of male C57BL/6J mice before receiving the permanent middle cerebral artery occlusion (pMCAO) surgery. Western blot and immunofluorescent staining were used to observe neuronal survival, astrocyte activation, and protein Kla levels, and the lactate levels in ischemic brains were assayed with a commercial kit. TTCstaining and neurological function scores are performed to evaluate the brain damage in mice. We found that the increased lactate content and protein Kla levels were significantly decreased in ischemic brain tissue of mice after receiving EA pretreatment, and accompanied by the reduction of astrocyte activation and neuronal injury and death. Meantime, we found that EA pretreatment was effective in reversing the worsening of ischemic brain injury caused by lactate supplementation. However, EA pretreatment did not further reduce the lactate content and protein Kla levels and ameliorate brain injury in ischemic stroke mice after inhibition of glycolysis. Our study reveals that EA pretreatment reduced ischemic brain damage by inhibiting lactate production and its derived protein Kla formation in mice with ischemic stroke.

Electroacupuncture promotes neural function recovery by alleviating mitochondria damage in cerebral ischemia mice.

This study aimed to observe the effect of electroacupuncture (EA) at Zusanli point (ST36) on motor function of cerebral ischemia mice, and to observe the effect of EA on mitochondrial morphology of peri-infarct cortex neurons in cerebral ischemia mice. Middle cerebral artery occlusion (MCAO) was used to develop an ischemic stroke mice model. EA treatment was performed for three consecutive days for 15 min per day after MCAO modeling. We investigated the therapeutic effects of EA on MCAO mice by performing neurobehavioral assessment (modified Neurological Severity Score, Rotarod test, Open-field test and Gait analysis) and TTC staining. The morphology and function of neuronal mitochondria were evaluated by transmission electron microscopy, qRT-PCR, chemiluminescence, and western blot. Nissl staining, TUNEL staining and immunofluorescence staining were used to observe neuronal morphology and apoptosis. Furthermore, ELISA was employed to measure the expression levels of inflammatory factors in mouse serum. EA alleviated motor dysfunction and infarct volume in mice with cerebral ischemia. It improved the neuronal mitochondria damage in MCAO mice, and decreased the protein and mRNA expression level of mitochondrial fission related proteins (FIS1 and Drp1). In addition, EA can reduce neuronal damage and apoptosis of nerve cells, and decrease the level of inflammatory factors (IL-1β, TNF-α, IL-6 and IL-8) in cerebral ischemia mice. EA therapy can improve motor dysfunction and alleviate the damage of neuron mitochondria in cerebral ischemic mice.

Hif-1α ablation reduces the efficiency of NeuroD1 gene-based therapy and aggravates the brain damage following ischemic stroke

ABSTRACT Introduction Hypoxia-inducible factor 1α [HIF1α] regulates gene expression, allowing the organism to respond to low oxygen levels. Meanwhile, astrocytes participate in inflammatory processes and are associated with neurotoxic chemicals that can increase stroke volume, contributing considerably to the devastating effects of a stroke. Objective To evaluate whether Hif-1α ablation from the central nervous system is implicated in motor dysfunction and ischemic brain damage following stroke. Furthermore, to explore if Hif-1α ablation affects the therapeutic impact of NeuroD1 gene-based therapy. Methods Endothelin-1 [ET-1] was injected to induce ischemic stroke in mice. Both wild-type and Hypoxia-inducible factor 1α conditional knockout [Hif-1α CKO] mice were used. The effect of Hif-1α ablation was assessed by the neuron numbers, astrocyte activity, vascular endothelial growth factor [VEGF] expression, and behavioral tests. Moreover, western blot, ELISA, and RNA sequencing were used. Then, we used pAAV2/9-GfaABC1D-NeuroD1-P2A-EGFP-WPRE injection to examine the impact of NeuroD1 in Hif-1α CKO mice following ischemic stroke. Results We found that following stroke, motor dysfunction significantly increased in Hif-1α CKO mice. Furthermore, elevation of apoptosis and activation in both microglia and astrocytes were observed, consequently up-regulating neuroinflammation. Meanwhile, Hif-1α ablation significantly decreased the efficiency of NeuroD1 gene-based therapy. Conclusion Our findings demonstrate that Hif-1α ablation from the nervous system is implicated in ischemic stroke pathogenesis mainly by increasing neuron cell death and inducing astrocytes as well as decreasing the efficiency of NeuroD1. These data support the idea that manipulating HIF-1α is a viable therapeutic for ischemic stroke.

Mef2c Exacerbates Neuron Necroptosis via Modulating Alternative Splicing of Cflar in Ischemic Stroke With Hyperlipidemia.

Hyperlipidemia is a common comorbidity of stroke patients, elucidating the mechanism that underlies the exacerbated ischemic brain injury after stroke with hyperlipidemia is emerging as a significant clinical problem due to the growing proportion of hyperlipidemic stroke patients. Mice were fed a high-fat diet for 12 weeks to induce hyperlipidemia. Transient middle cerebral artery occlusion was induced as a mouse model of ischemic stroke. Emx1Cre mice were crossed with Mef2cfl/fl mice to specifically deplete Mef2c in neurons. We reported that hyperlipidemia significantly aggravated neuronal necroptosis and exacerbated long-term neurological deficits following ischemic stroke in mice. Mechanistically, Cflar, an upstream necroptotic regulator, was alternatively spliced into pro-necroptotic isoform (CflarR) in ischemic neurons of hyperlipidemic mice. Neuronal Mef2c was a transcription factor modulating Cflar splicing and upregulated by hyperlipidemia following stroke. Neuronal specific Mef2c depletion reduced cerebral level of CflarR and cFLIPR (translated by CflarR), while mitigated neuron necroptosis and neurological deficits following stroke in hyperlipidemic mice. Our study highlights the pathogenic role of CflarR splicing mediated by neuronal Mef2c, which aggravates neuron necroptosis following stroke with comorbid hyperlipidemia and proposes CflarR splicing as a potential therapeutic target for hyperlipidemic stroke patients.

New TIPARP inhibitor rescues mitochondrial function and brain injury in ischemic stroke

Ischemic stroke is a high-mortality disease that urgently requires new therapeutic strategies. Insufficient cerebral blood supply can induce poly (ADP-ribose) polymerase (PARP) activation and mitochondrial dysfunction, leading to tissue damage and motor dysfunction. We demonstrate that the expression of TCDD inducible PARP (TIPARP) is elevated in ischemic stroke patients and mice. Knockdown of Tiparp reduces brain infarction and promotes recovery of motor function in ischemic stroke mice. A rationally designed TIPARP inhibitor, XG-04-B1, promotes repair of brain injury and recovery of motor function in ischemic stroke mice. Mechanistically, XG-04-B1 increases neuronal plasticity and inhibits astrocyte activation in ischemic stroke mice. In addition, eukaryotic translation initiation factor 3 subunit B (EIF3B) is a direct target of TIPARP. TIPARP interacts with EIF3B through nucleoplasmic redistribution, leading to mitochondrial dysfunction. Knockdown of Tiparp and inhibition of TIPARP via XG-04-B1 restore mitochondrial homeostasis in ischemic stroke mice. Taken together, TIPARP activation contributes to mitochondrial dysfunction and subsequent brain injury, and is therefore a promising therapeutic target for stroke.

Molecular Imaging Reveals Antineuroinflammatory Effects of HDAC6 Inhibition in Stroke Models.

Ischemic stroke is a devastating disease that causes neuronal death, neuroinflammation, and other cerebral damage. However, effective therapeutic strategies for ischemic stroke are still lacking. Histone deacetylase 6 (HDAC6) has been implicated in the pathogenesis of ischemic stroke, and the pharmacological inhibition of HDAC6 has shown promising neuroprotective effects. In this study, we utilized positron emission tomography (PET) imaging with the HDAC6-specific radioligand [18F]PB118 to investigate the dynamic changes of HDAC6 expression in the brain after ischemic injury. The results revealed a significant decline in [18F]PB118 uptake in the ipsilateral hemisphere on the first day after ischemia, followed by a gradual increase on days 4 and 7. To evaluate the therapeutic potential of HDAC6 inhibitors, we developed a novel brain-permeable and potent HDAC6 inhibitor, PB131, and assessed its neuroprotective effects in an ischemic stroke mouse model. PET imaging studies demonstrated that PB131 treatment alleviated the decline in [18F]PB118 uptake and reduced the infarct size in middle cerebral artery occlusion mice. Furthermore, PET imaging with the TSPO-specific radioligand [18F]FEPPA revealed that PB131 significantly suppressed neuroinflammation in the ischemic brain. These findings provide insights into the dynamic changes of HDAC6 in ischemic stroke and the potential of HDAC6 inhibitors as novel therapeutic agents for this condition.

Anisodine hydrobromide injection promotes neural remodeling and recovery after ischemic stroke in mice.

Anisodine hydrobromide injection has shown promising therapeutic effects in treating patients with cerebral infarction, improving recovery of neurological function during the post-cerebral infarction period. However, the effects of anisodine hydrobromide on brain recovery and neuroplasticity are unclear. This study explores the therapeutic effects and underlying mechanisms of anisodine hydrobromide in mice experiencing the chronic phase of an ischemia stroke. The electrocautery method established a distal middle cerebral artery occlusion (MCAO) model in healthy male C57BL/6 mice. Neurological deficits were evaluated using Golgi and immunofluorescence staining to measure the effects of anisodine hydrobromide on neural proliferation, migration and remodeling. DAPT (dipeptidic γ-secretase-specific inhibitor) was employed to explore the involvement of the Notch signaling pathway post-anisodine hydrobromide treatment. Compared to the control and MCAO groups, mice treated with anisodine hydrobromide showed improved post-stroke neurological function, increased neurite intersections, and dendritic spine density in the peri-infarct cortex. Anisodine hydrobromide also promoted neural cell regeneration which is dendritic and axonal structures and synaptic vesicle protein restructuring. Gap43, NGF, Notch1, and Hes1 protein level increased significantly in the ANI group provided inhibitor DAPT was absent. Anisodine hydrobromide can promote neurological function, neurotrophic factors, and neuroplasticity. Notch signaling pathways also impact the effects of anisodine hydrobromide on neural plasticity in ischemia stroke.

Membrane Associated RNA-Containing Vesicles Regulate Cortical Astrocytic Microdomain Calcium Transients in Awake Ischemic Stroke Mice.

Astrocytic processes minutely regulate neuronal activity via tripartite synaptic structures. The precision-tuning of the function of astrocytic processes is garnering increasing attention because of its significance in promoting brain repair following ischemic stroke. Microdomain calcium (Ca2+) transients in astrocytic processes are pivotal for the functional regulation of these processes. However, the understanding of the alterations and regulatory mechanism of microdomain Ca2+ transients during stroke remains limited. In the present study, a fast high-resolution, miniaturized two-photon microscopy is used to show that the levels of astrocytic microdomain Ca2+ transients are significantly reduced in the peri-infarct area of awake ischemic stroke mice. This finding correlated with the behavioral deficits shown by these mice under freely-moving conditions. Mitochondrial Ca2+ activity is an important factor driving the microdomain Ca2+ transients. DEAD Box 1 (DDX1) bound to circSCMH1 (a circular RNA involved in vascular post-stroke repair) facilitates the formation of membrane-associated RNA-containing vesicles (MARVs) and enhances the activity of astrocytic microdomain Ca2+ transients, thereby promoting behavioral recovery. These results show that targeting astrocytic microdomain Ca2+ transients is a potential therapeutic approach in stroke intervention.

Enriched environment enhances angiogenesis in ischemic stroke through SDF-1/CXCR4/AKT/mTOR pathway

Environmental-gene interactions significantly influence various bodily functions. Enriched environment (EE), a non-pharmacological treatment method, enhances angiogenesis in ischemic stroke (IS). However, underlying the role of EE in angiogenesis in aged mice post-IS remain unclear. This study aimed to determine the potential mechanism by which EE mediates angiogenesis in 12-month-old IS mice and oxygen-glucose deprivation/reperfusion (OGD/R)-induced bEnd.3 cells. In vivo, EE treatment alleviated the neurological deficits, enhanced angiogenesis, upregulated SDF-1, VEGFA, and the AKT/mTOR pathway. In addition, exogenous SDF-1 treatment had a protective effect similar to that of EE treatment in aged mice with IS. However, SDF-1 neutralizing antibody, AMD3100 (CXCR4 inhibitor), ARQ092 (AKT inhibitor), and rapamycin (mTOR inhibitor) treatment blocked the neuroprotective effect of EE treatment and inhibited angiogenesis in IS mice. In vitro, exogenous SDF-1 promoted migration of OGD/R-induced bEnd.3 cells and activated the AKT/mTOR pathway. AMD3100, ARQ092, and rapamycin inhibited SDF-1-induced cell migration. Collectively, these findings demonstrate that EE enhances angiogenesis and improves the IS outcomes through SDF-1/CXCR4/AKT/mTOR pathway.

Impaired Peripheral Vascular Function Following Ischemic Stroke in Mice: Potential Insights into Blood Pressure Variations in the Post-Stroke Patient.

High systolic blood pressure and increased blood pressure variability after the onset of ischemic stroke are associated with poor clinical outcomes. One of the key determinants of blood pressure is arteriolar size, determined by vascular smooth muscle tone and vasodilatory and vasoconstrictor substances that are released by the endothelium. The aim of this study is to outline alterations in vasomotor function in isolated peripheral arteries following ischemic stroke. The reactivity of thoracic aortic segments from male C57BL/6 mice to dilators and constrictors was quantified using wire myography. Acetylcholine-induced endothelium-dependent vasodilation was impaired after ischemic stroke (LogIC50 Sham = -7.499, LogIC50 Stroke = -7.350, p = 0.0132, n = 19, 31 respectively). The vasodilatory responses to SNP were identical in the isolated aortas in the sham and stroke groups. Phenylephrine-induced vasoconstriction was impaired in the aortas isolated from the stroke animals in comparison to their sham treatment counterparts (Sham LogEC50= -6.652 vs. Stroke LogEC50 = -6.475, p < 0.001). Our study demonstrates that 24 h post-ischemic stroke, peripheral vascular responses are impaired in remote arteries. The aortas from the stroke animals exhibited reduced vasoconstrictor and endothelium-dependent vasodilator responses, while the endothelium-independent vasodilatory responses were preserved. Since both the vasodilatory and vasoconstrictor responses of peripheral arteries are impaired following ischemic stroke, our findings might explain increased blood pressure variability following ischemic stroke.

Guanxinning Tablet Alleviates Post-Ischemic Stroke Injury Via Regulating Complement and Coagulation Cascades Pathway and Inflammatory Network Mobilization.

Currently, ischemic stroke (IS) continues to significantly contribute to functional deterioration and reduced life quality. Regrettably, the choice of neuro-rehabilitation interventions to enhance post-IS outcomes is limited. Guanxinning tablet (GXNT), a multi-component medicine composed of Danshen and Chuanxiong, has demonstrated neuroprotective potential against ischemic brain injury and diabetic encephalopathy. However, the therapeutic impact of GXNT on post-IS functional outcomes and pathological injury, as well as the underlying molecular mechanisms and anti-IS active substances, remain unclear. To answer the above questions, neurological and behavioral assessment, cerebral lesions, and blood-brain barrier (BBB) integrity were combined to comprehensively investigate GXNT's pharmacodynamic effects against post-IS injury. The possible molecular mechanisms were revealed through transcriptome sequencing coupled with experimental verification. Furthermore, the brain tissue distribution of main components in GXNT, behavioral changes of IS zebrafish, and molecular docking were integrated to identify the anti-IS active compounds. Treatment with GXNT significantly mitigated the functional deficits, cerebral cortex lesions, and BBB disruption following IS. Transcriptome sequencing and bioinformatics analysis suggested that complement and coagulation cascades as well as inflammation might play crucial roles in the GXNT's therapeutic effects. Molecular biology experiments indicated that GXNT administration effectively normalized the abnormal expression of mRNA and protein levels of key targets related to complement and coagulation cascades (eg C3 and F7) and inflammation (eg MMP3 and MMP9) in the impaired cortical samples of IS mice. The locomotor promotion in IS zebrafish as well as favorable affinity with key proteins (C3, F7, and MMP9) highlighted anti-IS activities of brain-permeating constituents (senkyunolide I and protocatechuic acid) of GXNT. Taken together, these intriguing findings indicate that GXNT intervention exerts a beneficial effect against post-IS injury via regulating the complement and coagulation cascades pathway and mobilizing inflammatory network. Senkyunolide I and protocatechuic acid show promise as anti-IS active compounds.

Apolipoprotein E deficiency exacerbates blood-brain barrier disruption and hyperglycemia-associated hemorrhagic transformation after ischemic stroke

BackgroundThe polymorphism of the apolipoprotein E (ApoE) gene has been implicated in both the susceptibility to neurodegenerative disease and the prognosis of traumatic brain injury (TBI). However, the influence of ApoE on the risk of hemorrhagic transformation (HT) after acute ischemic stroke remains inconclusive. The present study aimed to investigate the potential impact of ApoE deficiency on the risk of hyperglycemia-associated HT and to elucidate the underlying mechanisms. MethodsWild-type (WT) and ApoE knockout (ApoE−/−) mice were injected with 50 % glucose to induce hyperglycemia and subsequently subjected to 90 min of intraluminal middle cerebral artery occlusion (MCAO). The mortality, neurological function, HT incidence and HT grading-score were evaluated at 24 hours after reperfusion. To evaluate the integrity of blood-brain barrier (BBB), the immunoglobulin G (IgG) leakage and the protein expressions of tight junctions (TJs) were detected using immunofluorescent staining and western blotting. Finally, the levels of matrix metalloproteinases (MMP)-2/9, microglial activation and proinflammatory mediators were investigated using immunofluorescent staining and western blotting. ResultsApoE−/− mice exhibited increased mortality and exacerbated neurological impairment, concomitant with more severe hyperglycemia-associated HT 24 hours post-reperfusion. Meanwhile, ApoE deficiency exacerbated the disruption of BBB, characterized by increased leakage of IgG, aggravated degradation of TJs and microvascular basement membranes. Furthermore, ApoE deficiency further aggravated the upregulation of MMP-2/9 and microglia-triggered neuroinflammation. ConclusionsOur findings demonstrate that the absence of ApoE exacerbates neurological impairment and hyperglycemia-associated HT in ischemic stroke mice, which is closely associated with MMP-2/9 signaling and neuroinflammation-mediated disruption of BBB.

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.

The humanized platelet glycoprotein VI Fab inhibitor EMA601 protects from arterial thrombosis and ischaemic stroke in mice.

Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, KD: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk.

Neuroprotective Effects of AER-271 in a tMCAO Mouse Model: Modulation of Autophagy, Apoptosis, and Inflammation.

Following ischemic stroke, aquaporin 4 (AQP4) expression modifications have been associated with increased inflammation. However, the underlying mechanisms are not fully understood. This study aims to elucidate the mechanistic basis of post-cerebral ischemia-reperfusion (I/R) inflammation by employing the AQP4-specific inhibitor, AER-271. The middle cerebral artery occlusion (MCAO) model was used to induce ischemic stroke in mice. C57BL/6 mice were randomly allocated into four groups: sham operation, I/R, AER-271, and 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020) treatment, with observations recorded at 1day, 3days, and 7days post-tMCAO. Each group consisted of 15 mice. Procedures included histological examination through HE staining, neurological scoring, Western blot analysis, and immunofluorescence staining. AER-271 treatment yielded significant improvements in post-stroke weight recovery and neurological scores, accompanied by a reduction in cerebral infarction volume. Moreover, AER-271 exhibited a noticeable influence on autophagic and apoptotic pathways, affecting the activation of both pro-inflammatory and anti-inflammatory cytokines. Alterations in the levels of inflammatory biomarkers MCP-1, NLRP3, and caspase 1 were also detected. Finally, a comparative assessment of the effects of AER-271 and TGN-020 in mitigating apoptosis and microglial polarization in ischemic mice revealed neuroprotective effects with no significant difference in efficacy. This study provides essential insights into the neuroprotective mechanisms of AER-271 in cerebral ischemia-reperfusion injury, offering potential clinical applications in the treatment of ischemic cerebrovascular disorders.

Microglial heterogeneity in the ischemic stroke mouse brain of both sexes

BackgroundIschemic 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.MethodsA 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.ResultsFirst, 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.ConclusionsOur findings shed new light on microglia heterogeneity in stroke pathology and underscore the potential of targeting specific microglial populations for effective stroke therapies.