Cerebral Artery Occlusion In Mice Research Articles

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.

Brain‐Targeted 9‐Phenanthrol‐Loaded Lipid Nanoparticle Prevents Brain Edema after Cerebral Ischemia‐Reperfusion Injury by Inhibiting the Trpm4 Channel in Mice

AbstractBrain edema robustly increases mortality and hinders functional recovery after acute ischemic stroke. However, there are currently no effective therapies available for treating or preventing it. The unchecked opening of the transient receptor potential M4 (TRPM4) channel results in an excessive influx of Na+ and water, which contributes significantly to the formation of brain edema after ischemic stroke. 9‐phenanthrol (9‐Phe), a potent TRPM4 inhibitor, has limited clinical applicability due to its potential cytotoxicity and poor solubility. A brain‐targeting T7 (HAIYPRH)‐modified lipid nanoparticle (LNP) encapsulated 9‐Phe (9‐Phe@T7‐LNP) is designed and synthesized to improve the physicochemical properties and pharmacokinetic properties of 9‐Phe for treating brain edema in vivo. These results demonstrated that 9‐Phe@T7‐LNP can penetrate the intact blood‐brain barrier (BBB) in normal mice and target the brain parenchyma. Moreover, 9‐Phe@T7‐LNP effectively reduced infarct volume and brain edema, prevented neuronal loss and BBB disruption, improved survival, and facilitated neurological function recovery after transient middle cerebral artery occlusion in mice. Additionally, 9‐Phe@T7‐LNP scavenged oxygen‐free radicals and prevented neuronal apoptosis in cultured neurons subjected to oxygen and glucose deprivation/reperfusion. In summary, these findings showed that 9‐Phe@T7‐LNP holds strong potential as a promising targeted therapy for brain edema after stroke, providing superior pharmacological neuroprotection against brain edema.

A pilot study of a novel brain penetrant Mn(III) porphyrin-based SOD mimic on middle cerebral artery occlusion in mice

Introduction: Oxidative stress exacerbates acute ischemic stroke (AIS). Manganese Porphyrin (MnP)-based superoxide dismutase (SOD) mimics have shown therapeutic benefits for AIS. However, earlier MnP versions faced limited blood-brain barrier (BBB) penetration and hypotension. A novel SOD mimic MnP-05 is designed to overcome the limitations of previous generation structural analogs. Our study aimed to assess whether MnP-05 improves transient middle cerebral artery occlusion (tMCAO) outcomes in mice. Methods: Male and female C57BL/6J mice aged 12-14 weeks were used and data collection was aggregated. Initially, a safety study was conducted using uninjured mice to establish safe dosage levels independent of hypotension. Subsequently, pharmacokinetic (PK) analysis assessed BBB penetration of MnP-05. Finally, effcacy studies of MnP-05 were assessed following a 60-minute tMCAO. Intravenous administration of 1 mg/kg of MnP-05 occurred 10 minutes after recanalization, followed by intraperitoneal implantation of an osmotic pump delivering 5 mg/kg/day of MnP-05 for 72 hours after tMCAO. The concentration of MnP-05 was evaluated in ischemic core and contralateral normal brain by LC-MS/MS. The neurological symptoms were assessed by Garcia score and neurological deficit score (NDS) and compared at 72 hours between the control and MnP-05 groups in the effcacy study. Results: The safety study revealed that doses of 10mg/kg or less were considered safe, not causing hypotension (Fig.1). In the PK study, MnP-05 demonstrated preferential distribution to ischemic lesions compared to contralateral side (p = 0.03, Fig.2). In the effcacy study, MnP-05 group demonstrated improved neurological symptoms at 72 hours in Garcia score (p = 0.02), NDS (p = 0.006), and probability of survival (p = 0.03)(Fig.3). Conclusions: We established the safety dose of MnP-05 in tMCAO mice. It penetrates in ischemic lesions through BBB. Our findings suggest that MnP-05 is a promising therapeutic agent for AIS. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury: a promising target for intervention.

JOURNAL/nrgr/04.03/01300535-202419110-00028/figure1/v/2024-03-08T184507Z/r/image-tiff Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury. Various calcium channels are involved in cerebral ischemia/reperfusion injury. Cav3.2 channel is a main subtype of T-type calcium channels. T-type calcium channel blockers, such as pimozide and mibefradil, have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury. However, the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear. Here, in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons. The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons. We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury. Cav3.2 knockout markedly reduced infarct volume and brain water content, and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury. Additionally, Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress, inflammatory response, and neuronal apoptosis. In the hippocampus of Cav3.2-knockout mice, calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury. These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling. Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

Ketamine improves neuronal recovery following spreading depolarization in peri-infarct tissues.

Spreading depolarization (SD) has emerged as an important contributor to the enlargement of acute brain injuries. We previously showed that the N-methyl-D-aspartate receptor antagonist ketamine was able to prevent deleterious consequences of SD in brain slices, under conditions of metabolic compromise. The current study aimed to extend these observations into an in vivo stroke model, to test whether gradients of metabolic capacity lead to differential accumulation of calcium (Ca2+) following SD. In addition, we tested whether ketamine protects vulnerable tissuewhile allowing SD to propagate through surrounding undamaged tissue. Focal lesions were generated using a distal middle cerebral artery occlusion in mice, and clusters of SD were generated at 20 min intervals with remote microinjection of potassium chloride. SDs invading peri-infarct regions had significantly different consequences, depending on the distance from the infarct core. Proximal to the lesion, Ca2+ transients were extended, as compared with responses in better-perfused tissue more remote from the lesion. Extracellular potential shifts were also longer and hyperemia responses were reduced in proximal regions following SDs. Consistent with in vitro studies, ketamine, at concentrations that did not abolish the propagation of SD, reduced the accumulation of intracellular Ca2+ in proximal regions following an SD wave. These findings suggest that deleterious consequences of SD can be targeted in vivo, without requiring outright block of SD initiation and propagation.

URB597 exerts neuroprotective effects against transient brain ischemia injury in mice by regulating autophagic flux and necroptosis

Ischemic stroke is a leading cause of death and disability, and medical treatments for ischemic stroke are very limited. URB597 is a potent and selective inhibitor of fatty acid amide hydrolase (FAAH). However, the effect of URB597 on ischemic stroke and the underlying molecular mechanisms remain little known. In this study, focal cerebral ischemia was induced by transient middle cerebral artery occlusion in mice. Our results showed that URB597 dose-dependently improved neurological function and reduced brain infarct volume and brain edema 24 h after brain ischemia. The most effective dose was 1 mg/kg and the therapeutic time window was within 3 h after ischemic stroke. To further investigate the underlying mechanism, necroptosis and autophagy flux were detected by Western blot and/or immunofluorescence staining with or without chloroquine, an autophagic flux inhibitor. Our results showed that URB597 promoted autophagic flux and reduced neuronal necroptosis after brain ischemia and these effects could be abolished by chloroquine. In addition, we found that peroxisome proliferator-activated receptor α (PPARα) antagonist GW6471 partly abolished the effect of URB597 against brain ischemia and URB597 upregulated the expressions of PPARα. In conclusion, URB597 exerts a neuroprotective effect in a dose- and time-dependent manner, and this effect may be related to its restoration of autophagic flux and inhibition of neuronal necroptosis. PPARα is involved in the neuroprotective effect of URB597. This study provides novel evidence that URB597 may be a promising agent for the clinical treatment of ischemic stroke.

A novel specific aptamer targets cerebrovascular endothelial cells after ischemic stroke

Cell specific-targeted therapy (CSTT) for acute ischemic stroke remains underdeveloped. Cerebrovascular endothelial cells (CECs) are key components of the blood–brain barrier and are the first brain cells affected by ischemic stroke. After stroke, CEC injury causes insufficient energy supply to neurons and leads to cytotoxic and vasogenic brain edema. Aptamers are short single-stranded RNA or DNA molecules that can bind to specific ligands for cell specific delivery. The expression of vascular cell adhesion molecule-1 (VCAM-1) is increased on CECs after stroke. Herein, we report that an RNA-based VCAM-1-aptamer can specifically target CECs in stroke brains following transient middle cerebral artery occlusion in mice. Our data demonstrate the potential of an RNA-based aptamer as an effective delivery platform to target CECs after stroke. We believe this method will allow for the development of CSTT for treatment of patients with stroke.

Immp2l Mutation Induces Mitochondrial Membrane Depolarization and Complex III Activity Suppression after Middle Cerebral Artery Occlusion in Mice.

We previously reported that mutations in inner mitochondrial membrane peptidase 2-like (Immp2l) increase infarct volume, enhance superoxide production, and suppress mitochondrial respiration after transient cerebral focal ischemia and reperfusion injury. The present study investigated the impact of heterozygous Immp2l mutation on mitochondria function after ischemia and reperfusion injury in mice. Mice were subjected to middle cerebral artery occlusion for 1 h followed by 0, 1, 5, and 24 h of reperfusion. The effects of Immp2l+/- on mitochondrial membrane potential, mitochondrial respiratory complex III activity, caspase-3, and apoptosis-inducing factor (AIF) translocation were examined. Immp2l+/- increased ischemic brain damage and the number of TUNEL-positive cells compared with wild-type mice. Immp2l+/- led to mitochondrial damage, mitochondrial membrane potential depolarization, mitochondrial respiratory complex III activity suppression, caspase-3 activation, and AIF nuclear translocation. The adverse impact of Immp2l+/- on the brain after ischemia and reperfusion might be related to mitochondrial damage that involves depolarization of the mitochondrial membrane potential, inhibition of the mitochondrial respiratory complex III, and activation of mitochondria-mediated cell death pathways. These results suggest that patients with stroke carrying Immp2l+/- might have worse and more severe infarcts, followed by a worse prognosis than those without Immp2l mutations.

Characterization of the Involvement of Tumour Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6) in Ischemic Brain Injury Caused by Middle Cerebral Artery Occlusion in Mouse.

The identification of novel targets to modulate the immune response triggered by cerebral ischemia is crucial to promote the development of effective stroke therapeutics. Since tumour necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), a hyaluronate (HA)-binding protein, is involved in the regulation of immune and stromal cell functions in acute neurodegeneration, we aimed to characterize its involvement in ischemic stroke. Transient middle cerebral artery occlusion (1 h MCAo, followed by 6 to 48 of reperfusion) in mice resulted in a significant elevation in cerebral TSG-6 protein levels, mainly localized in neurons and myeloid cells of the lesioned hemisphere. These myeloid cells were clearly infiltrating from the blood, strongly suggesting that brain ischemia also affects TSG-6 in the periphery. Accordingly, TSG-6 mRNA expression was elevated in peripheral blood mononuclear cells (PBMCs) from patients 48 h after ischemic stroke onset, and TSG-6 protein expression was higher in the plasma of mice subjected to 1 h MCAo followed by 48 h of reperfusion. Surprisingly, plasma TSG-6 levels were reduced in the acute phase (i.e., within 24 h of reperfusion) when compared to sham-operated mice, supporting the hypothesis of a detrimental role of TSG-6 in the early reperfusion stage. Accordingly, systemic acute administration of recombinant mouse TSG-6 increased brain levels of the M2 marker Ym1, providing a significant reduction in the brain infarct volume and general neurological deficits in mice subjected to transient MCAo. These findings suggest a pivotal role of TSG-6 in ischemic stroke pathobiology and underscore the clinical relevance of further investigating the mechanisms underlying its immunoregulatory role.

NAD + precursor nutritional supplements sensitize the brain to future ischemic events

Nicotinamide adenine dinucleotide (NAD+) is a redox cofactor critical for oxidative phosphorylation. Nicotinamide (NAM) and nicotinamide riboside (NR) are NAD+ precursors widely used as nutritional supplements to augment oxidative phosphorylation. Indeed, NAD+ precursors have been reported to improve outcomes in ischemic stroke when administered as a rescue therapy after stroke onset. However, we have also reported that enhanced reliance on oxidative phosphorylation before ischemia onset might worsen outcomes. To address the paradox, we examined how NAD+ precursors modulate the outcome of middle cerebral artery occlusion in mice, when administered either 20 minutes after reperfusion or daily for three days before ischemia onset. A single post-ischemic dose of NAM or NR indeed improved tissue and neurologic outcomes examined at 72 hours. In contrast, pre-ischemic treatment for three days enlarged the infarcts and worsened neurological deficits. As a possible explanation for the diametric outcomes, a single dose of NAM or NR augmented tissue AMPK, PGC1α, SIRT1, and ATP in both naïve and ischemic brains, while the multiple-dose paradigm failed to do so. Our data suggest that NAD+ precursor supplements may sensitize the brain to subsequent ischemic events, despite their neuroprotective effect when administered after ischemia onset.

Photothrombotic Middle Cerebral Artery Occlusion in Mice: A Novel Model of Ischemic Stroke.

Stroke is one of the main causes of death and disability worldwide. Over the past decades, several animal models of focal cerebral ischemia have been developed allowing to investigate pathophysiological mechanisms underlying stroke progression. Despite intense preclinical research efforts, the need for noninvasive mouse models of vascular occlusion targeting the middle cerebral artery yet avoiding mechanical intervention is still pressing. Here, by applying the photothrombotic stroke model to the distal branch of the middle cerebral artery, we developed a novel strategy to induce a targeted occlusion of a large blood vessel in mice. This approach induces unilateral damage encompassing most of the dorsal cortex from the motor up to the visual regions 1 week after stroke. Pronounced limb dystonia one day after the damage is partially recovered after one week. Furthermore, we observe the insurgence of blood vessel leakage and edema formation in the peri-infarct area. Finally, this model elicits a notable inflammatory response revealed as a strong increase in astrocyte density and morphologic complexity in the perilesional region of the cortex compared with both other regions of the ipsilesional and contralesional hemispheres, and in sham-operated mice. To conclude, the stroke model we developed induces in mice the light-mediated occlusion of one of the main targets of human ischemic stroke, the middle cerebral artery, free from the limitations of commonly used preclinical models.

Regulation and Release of Vasoactive Endoglin by Brain Endothelium in Response to Hypoxia/Reoxygenation in Stroke.

In large vessel occlusion stroke, recanalization to restore cerebral perfusion is essential but not necessarily sufficient for a favorable outcome. Paradoxically, in some patients, reperfusion carries the risk of increased tissue damage and cerebral hemorrhage. Experimental and clinical data suggest that endothelial cells, representing the interface for detrimental platelet and leukocyte responses, likely play a crucial role in the phenomenon referred to as ischemia/reperfusion (I/R)-injury, but the mechanisms are unknown. We aimed to determine the role of endoglin in cerebral I/R-injury; endoglin is a membrane-bound protein abundantly expressed by endothelial cells that has previously been shown to be involved in the maintenance of vascular homeostasis. We investigated the expression of membranous endoglin (using Western blotting and RT-PCR) and the generation of soluble endoglin (using an enzyme-linked immunosorbent assay of cell culture supernatants) after hypoxia and subsequent reoxygenation in human non-immortalized brain endothelial cells. To validate these in vitro data, we additionally examined endoglin expression in an intraluminal monofilament model of permanent and transient middle cerebral artery occlusion in mice. Subsequently, the effects of recombinant human soluble endoglin were assessed by label-free impedance-based measurement of endothelial monolayer integrity (using the xCELLigence DP system) and immunocytochemistry. Endoglin expression is highly inducible by hypoxia in human brain endothelial monolayers in vitro, and subsequent reoxygenation induced its shedding. These findings were corroborated in mice during MCAO; an upregulation of endoglin was displayed in the infarcted hemispheres under occlusion, whereas endoglin expression was significantly diminished after transient MCAO, which is indicative of shedding. Of note is the finding that soluble endoglin induced an inflammatory phenotype in endothelial monolayers. The treatment of HBMEC with endoglin resulted in a decrease in transendothelial resistance and the downregulation of VE-cadherin. Our data establish a novel mechanism in which hypoxia triggers the initial endothelial upregulation of endoglin and subsequent reoxygenation triggers its release as a vasoactive mediator that, when rinsed into adjacent vascular beds after recanalization, can contribute to cerebral reperfusion injury.

Vespakinin-M, a natural peptide from Vespa magnifica, promotes functional recovery in stroke mice

Acute ischemic stroke triggers complex systemic pathological responses for which the exploration of drug resources remains a challenge. Wasp venom extracted from Vespa magnifica (Smith, 1852) is most commonly used to treat rheumatoid arthritis as well as neurological disorders. Vespakinin-M (VK), a natural peptide from wasp venom, has remained largely unexplored for stroke. Herein, we first confirmed the structure, stability, toxicity and distribution of VK as well as its penetration into the blood–brain barrier. VK (150 and 300 µg/kg, i.p.) was administered to improve stroke constructed by middle cerebral artery occlusion in mice. Our results indicate that VK promote functional recovery in mice after ischemia stroke, including an improvement of neurological impairment, reduction of infarct volume, maintenance of blood-brain barrier integrity, and an obstruction of the inflammatory response and oxidative stress. In addition, VK treatment led to reduced neuroinflammation and apoptosis associated with the activation of PI3K–AKT and inhibition of IκBα–NF-κB signaling pathways. Simultaneously, we confirmed that VK can combine with bradykinin receptor 2 (B2R) as detected by molecular docking, the B2R antagonist HOE140 could counteract the neuro-protective effects of VK on stroke in mice. Overall, targeting the VK–B2R interaction can be considered as a practical strategy for stroke therapy.

Transcranial focused ultrasound stimulation reduces vasogenic edema after middle cerebral artery occlusion in mice.

Blood-brain barrier (BBB) disruption underlies the vasogenic edema and neuronal cell death induced by acute ischemic stroke. Reducing this disruption has therapeutic potential. Transcranial focused ultrasound stimulation has shown neuromodulatory and neuroprotective effects in various brain diseases including ischemic stroke. Ultrasound stimulation can reduce inflammation and promote angiogenesis and neural circuit remodeling. However, its effect on the BBB in the acute phase of ischemic stroke is unknown. In this study of mice subjected to middle cerebral artery occlusion for 90 minutes, low-intensity low-frequency (0.5 MHz) transcranial focused ultrasound stimulation was applied 2, 4, and 8 hours after occlusion. Ultrasound stimulation reduced edema volume, improved neurobehavioral outcomes, improved BBB integrity (enhanced tight junction protein ZO-1 expression and reduced IgG leakage), and reduced secretion of the inflammatory factors tumor necrosis factor-α and activation of matrix metalloproteinase-9 in the ischemic brain. Our results show that low-intensity ultrasound stimulation attenuated BBB disruption and edema formation, which suggests it may have therapeutic use in ischemic brain disease as a protector of BBB integrity.

Oligodendrocyte precursor cell transplantation promotes angiogenesis and remyelination via Wnt/β-catenin pathway in a mouse model of middle cerebral artery occlusion

White matter injury is a critical pathological characteristic during ischemic stroke. Oligodendrocyte precursor cells participate in white matter repairing and remodeling during ischemic brain injury. Since oligodendrocyte precursor cells could promote Wnt-dependent angiogenesis and migrate along vasculature for the myelination during the development in the central nervous system, we explore whether exogenous oligodendrocyte precursor cell transplantation promotes angiogenesis and remyelination after middle cerebral artery occlusion in mice. Here, oligodendrocyte precursor cell transplantation improved motor and cognitive function, and alleviated brain atrophy. Furthermore, oligodendrocyte precursor cell transplantation promoted functional angiogenesis, and increased myelin basic protein expression after ischemic stroke. The further study suggested that white matter repairing after oligodendrocyte precursor cell transplantation depended on angiogenesis induced by Wnt/β-catenin signal pathway. Our results demonstrated a novel pathway that Wnt7a from oligodendrocyte precursor cells acting on endothelial β-catenin promoted angiogenesis and improved neurobehavioral outcomes, which facilitated white matter repair and remodeling during ischemic stroke.

Abstract 12536: Nanoliposomal Treatment Reduces Stroke Injury Following Middle Cerebral Artery Occlusion in Mice

Introduction: Neuroprotective strategies for stroke are lacking leading to significant disability and death. Liposomes (phospholipid particles) are commonly used as molecular carriers of therapeutic agent cargos. We previously showed that cargo-less nanoliposomes (NL, <100 nm size particles) composed of phosphatidylcholine, cholesterol and monosialoganglioside (70:25:5 molar ratios) induced an antioxidant protective response in endothelial cells exposed to oxidative stress-mediated amyloid injury. Hypothesis: NL will preserve neuroblastoma cell viability in the setting of hypoxic injury and will reduce functional and histologic injury in mice following middle cerebral artery occlusion (MCAO). Methods: SH-SY5Y neuroblastoma cells were exposed to 20-hour physoxic (5% oxygen) condition or hypoxic (1% oxygen) condition without or with NL (100 or 300 μg/mL). Viability was measured using calcein-AM fluorescence and SH-SY5Y gene expression of antioxidant proteins heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1) and superoxide dismutase 1 (SOD1) were measured by quantitative polymerase chain reaction. C57BL/6J mice were treated with saline (N=8) or NL (10000 ug/mL, N=7) while undergoing 60-minute MCAO followed by reperfusion. Day 2 post-injury neurologic impairment scoring was compared as well as post-mortem brain infarction size. Results: Neuroblastoma cells showed reduced viability following hypoxia that was reversed by treatment with NL. NL increased gene expression of HO-1, NQO1 and SOD1 compared to untreated controls. NL-treated mice showed reduced neurologic function impairment and reduced brain infarct size compared to controls (18.8±2% versus 27.3±2.3%, p=0.017, see Figure). Conclusions: Cargo-less NL reduced functional and structural stroke injury in mice subjected to MCAO likely mediated in part through induction of an antioxidant stress response. NL is a candidate novel agent to mitigate stroke injury.

Nanoliposomes Reduce Stroke Injury Following Middle Cerebral Artery Occlusion in Mice.

Neuroprotective strategies for stroke remain inadequate. Nanoliposomes comprised of phosphatidylcholine, cholesterol, and monosialogangliosides (nanoliposomes) induced an antioxidant protective response in endothelial cells exposed to amyloid insults. We tested the hypotheses that nanoliposomes will preserve human neuroblastoma (SH-SY5Y) and human brain microvascular endothelial cells viability following oxygen-glucose deprivation (OGD)-reoxygenation and will reduce injury in mice following middle cerebral artery occlusion. SH-SY5Y and human brain microvascular endothelial cells were exposed to oxygen-glucose deprivation-reoxygenation (3 hours 0.5%-1% oxygen and glucose-free media followed by 20-hour ambient air/regular media) without or with nanoliposomes (300 µg/mL). Viability was measured (calcein-acetoxymethyl fluorescence) and protein expression of antioxidant proteins HO-1 (heme oxygenase-1), NQO1 (NAD[P]H quinone dehydrogenase 1), and SOD1 (superoxide dismutase 1) were measured by Western blot. C57BL/6J mice were treated with saline (n=8) or nanoliposomes (10 mg/mL lipid, 200 µL, n=7) while undergoing 60-minute middle cerebral artery occlusion followed by reperfusion. Day 2 postinjury neurological impairment score and infarction size were compared. SH-SY5Y and human brain microvascular endothelial cells showed reduced viability post-oxygen-glucose deprivation-reoxygenation that was reversed by nanoliposomes. Nanoliposomes increased protein expressions of HO-1, NQO1 in both cell types and SOD1 in human brain microvascular endothelial cells. Nanoliposomes-treated mice showed reduced neurological impairment and brain infarct size (18.8±2% versus 27.3±2.3%, P=0.017) versus controls. Nanoliposomes reduced stroke injury in mice subjected to middle cerebral artery occlusion likely through induction of an antioxidant protective response. Nanoliposome is a candidate novel agent for stroke.

Integrated Bioinformatics Analysis of Potential mRNA and miRNA Regulatory Networks in Mice With Ischemic Stroke Treated by Electroacupuncture.

Background: The complicated molecular mechanisms underlying the therapeutic effect of electroacupuncture (EA) on ischemic stroke are still unclear. Recently, more evidence has revealed the essential role of the microRNA (miRNA)–mRNA networks in ischemic stroke. However, a systematic analysis of novel key genes, miRNAs, and miRNA–mRNA networks regulated by EA in ischemic stroke is still absent.Methods: We established a middle cerebral artery occlusion (MCAO) mouse model and performed EA therapy on ischemic stroke mice. Behavior tests and measurement of infarction area were applied to measure the effect of EA treatment. Then, we performed RNA sequencing to analyze differentially expressed genes (DEGs) and functional enrichment between the EA and control groups. In addition, a protein–protein interaction (PPI) network was built, and hub genes were screened by Cytoscape. Upstream miRNAs were predicted by miRTarBase. Then hub genes and predicted miRNAs were verified as key biomarkers by RT-qPCR. Finally, miRNA–mRNA networks were constructed to explore the potential mechanisms of EA in ischemic stroke.Results: Our analysis revealed that EA treatment could significantly alleviate neurological deficits in the affected limbs and reduce infarct area of the MCAO model mice. A total of 174 significant DEGs, including 53 upregulated genes and 121 downregulated genes, were identified between the EA and control groups. Functional enrichment analysis showed that these DEGs were associated with the FOXO signaling pathway, NF-kappa B signaling pathway, T-cell receptor signaling pathway, and other vital pathways. The top 10 genes with the highest degree scores were identified as hub genes based on the degree method, but only seven genes were verified as key genes according to RT-qPCR. Twelve upstream miRNAs were predicted to target the seven key genes. However, only four miRNAs were significantly upregulated and indicated favorable effects of EA treatment. Finally, comprehensive analysis of the results identified the miR-425-5p-Cdk1, mmu-miR-1186b-Prc1, mmu-miR-434-3p-Prc1, and mmu-miR-453-Prc1 miRNA–mRNA networks as key networks that are regulated by EA and linked to ischemic stroke. These networks might mainly take place in neuronal cells regulated by EA in ischemic stroke.Conclusion: In summary, our study identified key DEGs, miRNAs, and miRNA–mRNA regulatory networks that may help to facilitate the understanding of the molecular mechanism underlying the effect of EA treatment on ischemic stroke.

Targeting platelet glycoprotein VI attenuates progressive ischemic brain damage before recanalization during middle cerebral artery occlusion in mice

In acute ischemic stroke due to large vessel occlusion (LVO) infarcts rapidly grow into the penumbra, which represents dysfunctional, but still viable brain tissue amenable to rescue by vessel recanalization. However, infarct progression and/or delayed patient presentation are serious and frequent limitations of this so far only acute therapy. Thus, a major goal of translational research is to “freeze” the penumbra already during LVO (before opening the vessel) and thereby extend individual time windows for non-futile recanalization. We used the filament occlusion model of the middle cerebral artery (MCAO) in mice and assessed progressive infarction under occlusion at 2, 3, and 4 h after onset. We show that blocking the activatory platelet receptor glycoprotein (GP)VI substantially delayed progressive neocortical infarction compared to isotype control antibody treated mice. Moreover, the local vascular recruitment of infiltrating neutrophils and T-cells was mitigated. In conclusion, our experimental data support ongoing clinical trials blocking platelet GPVI in acute ischemic stroke.

Contribution of ginsenosides Rg1, Rb1 to the neuroprotective effect of Panax notoginseng in mouse organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation

We previously demonstrated that Panax notoginseng (pNG) root extract treatments exertedneuroprotective effects on brain injuries using middle cerebral artery occlusion in mice. The present study aims to investigate the neuroprotective effects of PNG extract and its ginsenosides Rg1 and Rb1 on ischemic neuronal damage caused by oxygen and glucose deprivation (OGD) in mouse organotypic hippocampal slice cultures (OHSCs). Before the experiments, hippocampal slices collected from 7-day-old Swiss mice were cultured for 7 days. OGD was triggered in OHSCs for 30, 60, or 90 min with the aim of finding the optimal period of OGD for drug testing. PNG extract (10, 30 μg/ml), ginsenosides Rg1 and Rb1 (5, 25 μM), or MK-801 25 μM, a reference drug, was added to the culture medium 24 h before OGD and these treatments were continued for 24 h after the optimum 60-min period of OGD. After 24 h of OGD exposure, the measurement of propidium iodide uptake was analysed in OHSCs to evaluate neuronal cell damage. The results showed that OGD time-dependently increased PI uptake of the OHSCs. PNG 30 μg/ml treatment reduced the OGD-induced neuronal cell damage in OHSCs. Ginsenosides Rg1 25 μM, Rb1 (5, 25 μM), as well as MK-801 (25 μM) significantly inhibited PI uptake 24 h after OGD exposure. However, ginsenoside Rg1 5 μM did not show any significant effects on the OGD-induced neuronal cell damage. These findings indicated that ginsenosides Rg1 and Rb1 contributed to the neuroprotective effects of PNG against ischemic damage in OHSCs and the neuroprotective effect of ginsenoside Rb1 was stronger than that of ginsenoside Rg1.