Mouse Model Of Cerebral Ischemia Research Articles

The foam cell-derived exosomal miRNA Novel-3 drives neuroinflammation and ferroptosis during ischemic stroke.

Large artery atherosclerosis (LAA) is a prevalent cause of acute ischemic stroke (AIS). Understanding the mechanisms linking atherosclerosis to stroke is essential for developing appropriate intervention strategies. Here, we found that the exosomal miRNA Novel-3 is selectively upregulated in the plasma of patients with LAA-AIS. Notably, Novel-3 was predominantly expressed in macrophage-derived foam cells, and its expression correlated with atherosclerotic plaque vulnerability in patients undergoing carotid endarterectomy. Exploring the function of Novel-3 in a mouse model of cerebral ischemia, we found that Novel-3 exacerbated ischemic injury and targeted microglia and macrophages expressing ionized calcium-binding adapter molecule 1 in peri-infarct regions. Mechanistically, Novel-3 increased ferroptosis and neuroinflammation by interacting with striatin (STRN) and downregulating the phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin signaling pathway. Blocking Novel-3 activity or overexpressing STRN provided neuroprotection under ischemic conditions. Our findings suggest that exosomal Novel-3, which is primarily derived from macrophage-derived foam cells, targets microglia and macrophages in the brain to induce neuroinflammation and could serve as a potential therapeutic target for patients with stroke who have atherosclerosis.

Neuroprotective effects of Daphnetin on hippocampal neurons and blood-brain barrier integrity in a mouse model of cerebral ischemia

The purpose of this research was to assess the impact of different doses of Daphnetin (DAP, a natural compound derived from coumarin) on hippocampus neuronal injury, neurobehavioral function, blood-brain barrier (BBB) integrity, expression of claudin-5, brain-derived neurotrophic factor (BDNF), superoxide dismutase (SOD), and inflammatory markers in a mouse model of cerebral ischemia. Cerebral ischemia was induced in mice through 30 minutes of bilateral common carotid occlusion (BCCAO), followed by 48 hours of reperfusion. The viability of hippocampal neurons was assessed using Cresyl violet staining and BBB function was determined by measuring Evans blue (E.B) dye leakage. Spatial memory was tested using the Radial Arm Water Maze (RAWM) task. Claudin-5 and BDNF were measured by immunofluorescence, while SOD, interleukin-1 beta (IL-1β), and nuclear factor-κB (NF-κB) expression were determined through western blotting. Administering DAP significantly increased neuron survival in the hippocampus CA1, CA3, and dentate gyrus (DG) regions and improved spatial memory dose-dependently (P<0.0001). Treatment with DAP (40 mg/kg IP) significantly reduced E.B leakage and brain water content (P<0.001). Furthermore, it increased the claudin-5, BDNF, and SOD levels and diminished NF-κB and IL-1β expression (P<0.0001). The research found that DAP protected neurons in the CA1, CA3, and DG areas of the hippocampus, enhanced behavioral functions, and preserved BBB integrity in a cerebral ischemia model. This positive impact is achieved by increasing the expression of claudin-5, BDNF, and SOD and diminishing neuroinflammation. Further research is required to clarify the mechanisms and possible clinical uses.

Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state.

JOURNAL/nrgr/04.03/01300535-202410000-00024/figure1/v/2024-02-06T055622Z/r/image-tiff Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

Integrated transcriptomic and proteomic profiling reveals the key molecular signatures of brain endothelial reperfusion injury.

Reperfusion therapy after ischemic stroke often causes brain microvascular injury. However, the underlying mechanisms are unclear. Transcriptomic and proteomic analyses were performed on human cerebral microvascular endothelial cells following oxygen-glucose deprivation (OGD) or OGD plus recovery (OGD/R) to identify molecules and signaling pathways dysregulated by reperfusion. Major findings were further validated in a mouse model of cerebral ischemia and reperfusion. Transcriptomic analysis identified 390 differentially expressed genes (DEGs) between the OGD/R and OGD group. Pathway analysis indicated that these genes were mostly associated with inflammation, including the TNF signaling pathway, TGF-β signaling pathway, cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, and NF-κB signaling pathway. Proteomic analysis identified 201 differentially expressed proteins (DEPs), which were primarily associated with extracellular matrix destruction and remodeling, impairment of endothelial transport function, and inflammatory responses. Six genes (DUSP1, JUNB, NFKBIA, NR4A1, SERPINE1, and THBS1) were upregulated by OGD/R at both the mRNA and protein levels. In mice with cerebral ischemia and reperfusion, brain TNF signaling pathway was activated by reperfusion, and inhibiting TNF-α with adalimumab significantly attenuated reperfusion-induced brain endothelial inflammation. In addition, the protein level of THBS1 was substantially upregulated upon reperfusion in brain endothelial cells and the peri-endothelial area in mice receiving cerebral ischemia. Our study reveals the key molecular signatures of brain endothelial reperfusion injury and provides potential therapeutic targets for the treatment of brain microvascular injury after reperfusion therapy in ischemic stroke.

The effects of protein-rich extract from Rhizoma Gastrodiae against cerebral ischemia/reperfusion injury via regulating MAPK and PI3K/AKT signaling pathway

BackgroundRhizoma Gastrodiae is a highly valuable traditional Chinese medicine and functional health food that has been used in China to treat neurological disorders for thousands of years. Rhizoma Gastrodiae contains various of biological activities, such as antioxidative, neuroprotective, learning improvement, anxiolytic, and antidepressant effects. However, no studies have been conducted to explore the effects of the protein components in Rhizoma Gastrodiae (GEPS) and its potential protective effects against ischemic stroke.Our main goal was to investigate the effects of GEPS on ischemia/reperfusion (I/R) injury and its possible mechanisms. MethodsA middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia mouse model and an oxygen-glucose deprivation (OGD/R) injury model in HT22 cells were established. A neurobehavioral test was performed 24 h after MCAO, and brain infarction was measured. A Morris water maze experiment was conducted on Day 14 after reperfusion in mice. Hematoxylin and eosin (HE) and TUNEL staining were performed to assess apoptotic neuronal death. Immunohistochemical analysis was used to detect BDNF and GAP43 expression. The content of SOD, MDA, GSH-PX and ROS were detected. The protein expression was analyzed using Western blotting. Cell viability was determined by MTT assay. Cell apoptosis was examined by flow cytometry. ResultsGEPS reduced apoptosis, decreased cerebral infarction, improved neurological defects, and ameliorated oxidative stress in the ischemic penumbra. In addition, GEPS increased the expression of BDNF and GA43 in the penumbra. Mechanistically, GEPS counteracted MCAO-induced PI3K/AKT inhibition and activation of MAPK signaling pathways. ConclusionGEPS has a clear neuroprotective effect on I/R injury, and its mechanism may be linked to the PI3K/AKT and MAPK signaling pathways.

The Aldose Reductase Inhibitor Epalrestat Maintains Blood–Brain Barrier Integrity by Enhancing Endothelial Cell Function during Cerebral Ischemia

Excessive activation of aldose reductase (AR) in the brain is a risk factor for aggravating cerebral ischemia injury. Epalrestat is the only AR inhibitor with proven safety and efficacy, which is used in the clinical treatment of diabetic neuropathy. However, the molecular mechanisms underlying the neuroprotection of epalrestat remain unknown in the ischemic brain. Recent studies have found that blood–brain barrier (BBB) damage was mainly caused by increased apoptosis and autophagy of brain microvascular endothelial cells (BMVECs) and decreased expression of tight junction proteins. Thus, we hypothesized that the protective effect of epalrestat is mainly related to regulating the survival of BMVECs and tight junction protein levels after cerebral ischemia. To test this hypothesis, a mouse model of cerebral ischemia was established by permanent middle cerebral artery ligation (pMCAL), and the mice were treated with epalrestat or saline as a control. Epalrestat reduced the ischemic volume, enhanced BBB function, and improved the neurobehavior after cerebral ischemia. In vitro studies revealed that epalrestat increased the expression of tight junction proteins, and reduced the levels of cleaved-caspase3 and LC3 proteins in mouse BMVECs (bEnd.3 cells) exposed to oxygen–glucose deprivation (OGD). In addition, bicalutamide (an AKT inhibitor) and rapamycin (an mTOR inhibitor) increased the epalrestat-induced reduction in apoptosis and autophagy related protein levels in bEnd.3 cells with OGD treatment. Our findings suggest that epalrestat improves BBB function, which may be accomplished by reducing AR activation, promoting tight junction proteins expression, and upregulating AKT/mTOR signaling pathway to inhibit apoptosis and autophagy in BMVECs.

Mechanism of neocryptotanshinone in protecting against cerebral ischemic injury: By suppressing M1 polarization of microglial cells and promoting cerebral angiogenesis

AimThis study explored the protective function and mechanism of neocryptotanshinone (NEO) on cerebral ischemia. MethodsLipopolysaccharide/γ-interferon(LPS/IFN-γ)was employed to mimic the polarization of mouse microglial cells BV2. After NEO treatment, the M1 polarization level of BV2 cells was identified using flow cytometry (FCM), fluorescent cell staining and enzyme linked immunosorbent assay(ELISA). Moreover, the mouse endothelial cells bEnd.3 were applied to be the study objects, which were intervened with NEO under the hypoxic condition. Thereafter, based on in-vitro tubule formation assay and fluorescence staining, the in-vitro tubule formation ability of bEnd.3 cells was detected. By adopting middle cerebral artery occlusion(MCAO) method, we constructed the mouse model of cerebral ischemia. After NEO intervention, the pathological changes of brain tissues were identified, while CD34 expression was measured by immunohistochemical (IHC) staining, nerve injury was detected by Nissl staining, and the changes in neurological behaviors of mice were also detected. ResultsOur results showed that NEO suppressed M1 polarization of BV2 cells, which exerted its effect through suppressing NF-κB and STAT3 signals, thereby decreasing the levels of iNOS, CD11b and inflammatory factors. NEO stimulated tubule formation in bEnd.3 cells based on the hypoxic situation, which exerted its effect through activating the Vascularendothelial growth factor-Vascular Endothelial Growth Factor Receptor 2-Notch homolog 1(VFGF-VEGFR2-Notch1) signal. Furthermore, NEO suppressed cerebral ischemia in mice and lowered the ischemic penumbra. NEO also improved the neurological behaviors of mice, increased the CD34 levels and decreased the expression of inflammatory factors. ConclusionNEO has well protective effect against cerebral ischemia, and its mechanisms are related to suppressing M1 polarization of microglial cells and promoting cerebral angiogenesis, which are the mechanisms of NEO in treating ischemic encephalopathy.

The protective effect of Buzhong Yiqi decoction on ischemic stroke mice and the mechanism of gut microbiota.

Buzhong Yiqi decoction (BZYQD) has been developed for preventing or reducing the recurrence of ischemic stroke for a long time in China. However, the mechanism of action of the BZYQD is not completely understood. Our research aims to determine whether the mechanism of action of BZYQD is by regulating gut microbiota using 16SR RNA and fecal microbiota transplantation. In a cerebral ischemia mouse model, the results showed that prophylactic administration of BZYQD could reduce brain infarct volume and improve neurological function and behavior. The prophylactic administration of BZYQD could regulate intestinal microbiota and increase the abundance of butyrate-producing Prevotellaceae_NK3B31_group and probiotic Akkermansia in mice 72 h after surgery. Transplanting BZYQD-administered bacterial flora into antibiotic-depleted mice could reproduce the therapeutic effects of BZYQD. Overall, our study provided molecular insights into the mechanism and impact of BZYQD in the prevention of cerebral ischemic damage and highlighted the potential of regulation of intestinal microbiota as a therapeutic approach for ischemic stroke.

Protein phosphatase 2A regulation of GABA B receptors normalizes ischemia-induced aberrant receptor trafficking and provides neuroprotection.

One major factor regulating the strength of GABAB receptor signaling and thereby neuronal excitability is the dynamic control of their cell surface expression. GABAB receptors are constitutively internalized and recycled back to the plasma membrane to maintain a stable number of receptors at cell surface for appropriate signaling. Protein phosphatase 2A (PP2A) dependent dephosphorylation of serine 783 (S783) in the GABAB2 subunit is a key event for downregulating GABAB receptor cell surface expression particularly under conditions associated with excitotoxicity. Here, we investigated the role of PP2A in regulating GABAB receptor cell surface expression under physiological and excitotoxic conditions. For this purpose, we developed an interfering peptide (PP2A-Pep) that inhibits the interaction of GABAB receptors with PP2A. Using cultured cortical neurons, we found that PP2A downregulates GABAB receptor cell surface expression by inhibiting recycling of the receptors and thereby promoting degradation of the receptors. Inhibition of the GABAB receptor/PP2A interaction by PP2A-Pep in cultured cortical neurons restored GABAB receptor cell surface expression after excitotoxic stress and inhibited progressing neuronal death even when added 48 h after the insult. To explore the therapeutic potential of PP2A-Pep, we further analyzed effect of PP2A-Pep in the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia. Incubation of brain slices prepared from MCAO-treated mice with PP2A-Pep restored normal GABAB receptor expression and GABAB receptor-mediated inhibition, reduced ischemic-induced overexcitability of neurons, and prevented neuronal death in the ischemic penumbra. This data illustrates the crucial role of regulating GABAB receptor phosphorylation by PP2A for controlling neuronal inhibition and excitability. The results further suggest that interfering with the GABAB receptor/PP2A interaction is a promising strategy for the development of specific therapeutic interventions to treat neurological diseases associated with a disturbed excitation/inhibition balance and downregulation of GABAB receptors.

Characterization of the Sphingolipidome of the Peri-Infarct Tissue during Hemorrhagic Transformation in a Mouse Model of Cerebral Ischemia.

Cardiovascular diseases like stroke cause changes to sphingolipid mediators like sphingosine 1-phosphate (S1P) or its ceramide analogs, which bear the potential to either alleviate or exacerbate the neurological damage. Therefore, the precise identification of alterations within the sphingolipidome during ischemic stroke (IS) and hemorrhagic transformation (HT) harbors a putative therapeutic potential to orchestrate local and systemic immunomodulatory processes. Due to the scarcity of research in this field, we aimed to characterize the sphingolipidome in IS and HT. C57BL/6 mice underwent middle cerebral artery occlusion (MCAO) and specimens of the peri-infarct tissue were taken for sphingolipid profiling. Ischemic stroke resulted in reduced S1P whilst ceramides were elevated six hours post ischemia onset. However, these differences were nearly revoked at 24 hours post ischemia onset. Moreover, the topmost S1P and ceramide levels were linked to the presence of HT after MCAO. In this study we show the characterization of the sphingolipidomic landscape of the peri-infarct tissue after ischemic stroke and HT. Especially, highest values of S1P, C 18 lactosylceramide, C 18 glucosylceramide, and C 24:1 ceramide were nearly entirely expressed by mice with HT. Our results warrant further investigations into the immunomodulatory consequences of altered sphingolipid species for the development of HT after IS.

Bone marrow-derived mesenchymal stem cells overexpressed with miR-182-5p protects against brain injury in a mouse model of cerebral ischemia

BackgroundToll-like receptor 4 (TLR4) plays a critical role in ischemic brain injury by mediating the inflammatory response. The microRNA miR-185-5p suppresses inflammatory signaling by targeting TLR4. This study investigates whether overexpressing miR-182-5p in bone marrow-derived mesenchymal stem cells (BM-MSCs) could potentiate the neuroprotective effects of BM-MSCs in a mouse model of ischemic brain injury. MethodsWe isolated BM-MSCs from mice, transfected the cells with miR-182-5p mimic, determined their MSC lineage through flow cytometry analysis of surface markers, examined miR-182-5p and TLR4 expression levels, and injected them into mice undergone middle cerebral artery occlusion (MCAO). MSC transplanted mice were subjected to behavior assays to determine cognitive and motor functions and biochemical analysis to determine neuroinflammation and TLR4/NF-κB in the ischemic hemisphere. ResultsWe found that BM-MSCs overexpressing miR-182-5p showed reduced TLR4 expression without affecting their MSC lineage. Mice transplanted with miR-182-5p overexpressing BM-MSCs after MCAO showed significantly improved cognitive and motor functions and reduced neuroinflammation, including suppressed microglial M1 polarization, reduced inflammatory cytokines, and inhibited TLR4/ NF-κB signaling. ConclusionOur findings suggest that overexpressing miR-182-5p in BM-MSCs can enhance the neuroprotective effects of BM-MSCs against ischemic brain injury by suppressing TLR4-mediated inflammatory response.

Astragaloside IV Improve Neurological Function of Cerebral Ischemia

This study intends to assess astragaloside IV’s effect on neurological function in mice cerebral ischemia model. The mouse model of cerebral ischemia was established by photochemistry and then assigned into sham operation group (photochemical building do not accept cold light irradiation) and control group (10 ug/ml by intraperitoneal injection of saline solution), drug group (10 ug/ml by intraperitoneal injection of Astragaloside IV) followed by analysis of neurological severity, cerebral infarction area, loss of neurons, glial cell activation and the activities of LC3, Beclin1, Caspase-3, P62 and mTOR by Western Blot. The neurons in cerebral infarction were missing and marginal area and penumbra appeared. The tissue in cerebral infarction became white, and the modeling was successful. The drug group showed significantly reduced scores and decreased infarct area of brain tissue compared with control group on day 14, 21 and 28 (P &lt; 0.05). TUNEL staining showed increased number of TUNEL cells at the ischemic edge in the drug group (0.35±0.07)% (P &lt; 0.05), while the IBAL staining of (27.12±3.01)% and GFAP staining of (0.08±0.02)% in the drug group showed significant inhibition of astrocytes (P &lt; 0.05). The activity of LC3, Beclin1, Caspase-3 and P62 in drug group was inhibited, while the activity of mTOR was promoted. In conclusion, Astragaloside IV improves the balance ability and the neural function of cerebral ischemia repair in mice model.

Erinacine A attenuates glutamate transporter 1 downregulation and protects against ischemic brain injury

Maintaining glutamate homeostasis through astrocyte-enriched glutamate transporter 1 (GLT-1) is critical for neuronal survival, but it is often disrupted after brain injury. Hericium erinaceus (HE), an edible mushroom, was reported to be anti-inflammatory and neuroprotective against brain ischemia, but its effect on glutamate homeostasis was unknown. Here we investigated the neuroprotective effect of erinacine A (EA), an active component of HE, with special focus on the GLT-1 function in the in vitro and in vivo cerebral ischemia mouse models. By using oxygen-glucose deprivation (OGD) to challenge mouse glia-neuron (GN) mixed culture as the in vitro model, we found that EA treatment significantly improved neuronal/astroglial survival and attenuated OGD-induced proinflammatory NFκB and AKT signaling activations. Notably, EA attenuated OGD-induced GLT-1 downregulation, and a selective GLT-1 inhibitor WAY-213613 reversed these EA-mediated neuroprotection. EA also ameliorated glutamate excitotoxicity effectively. In a transient hypoxia-ischemia (tHI) brain injury mouse model, we examined an EA treatment strategy by performing a pre-tHI daily oral gavage of EA (oEA) for 7 days followed by a post-tHI intranasal injection of EA (nEA) for 3 days, and found that this treatment significantly protected sensorimotor cortex and improved the post-tHI forepaw grip strength. Western blotting results further revealed that EA treatment also preserved astrocyte-enriched glutamate and aspartate transporter (GLAST) as well as a GLT-1 function-associated potassium channel Kir4.1 in the cerebral cortex and striatum after tHI. These results suggest that EA is effective for preserving GLT-1 and glutamate clearance machinery to protect against excitotoxicity after ischemic brain injury.

Salvianolic acid C improves cerebral ischemia reperfusion injury through suppressing microglial cell M1 polarization and promoting cerebral angiogenesis

This study aimed to investigate the mechanism of salvianolic acid C (SAC), the active ingredient in Salvia miltiorrhiza, in improving cerebral ischemia injury.The mouse microglial cells BV2 and mouse endothelial cells bEnd.3 were used as the objects of study. LPS/IFN-γ was applied to simulate the BV2 polarization, and bEnd.3 cells were treated under hypoxic condition. The BV2 cell polarization level was measured through flow cytometry (FCM), the TLR4 and MyD88 expression levels were detected by fluorescence staining, whereas the expression of inflammatory factors TNF-α, IL-6 and IL-1β was analyzed through ELISA. Tubule formation assay was also conducted to observe the tubule formation ability of bEnd.3 cells in vitro, and the level of VEGFR2 was detected by fluorescence staining. Cells were treated with the PKM2 inhibitor IN3, aiming to observe the influence of SAC on glycolysis of BV2 cells. In addition, the mouse model of cerebral ischemia was constructed through the middle cerebral artery occlusion (MCAO) method, and the pathological changes in brain tissues were detected after SAC intervention. Meanwhile, the levels of IBA-1, CD31 and ZO-1 were determined through histochemical staining. Nissl staining to detect nerve cell damage.In BV2 cell experiment, SAC suppressed the M1 polarization of BV2 cells, reduced the inflammatory factor levels, and inhibited the activation of TLR4 signal through suppressing glycolysis. When PKM2 was suppressed, the effects of SAC were antagonized. In the bEnd.3 model, SAC promoted tubule formation in bEnd.3 cells under hypoxic condition, and increased the expression of VEGFR2 and Notch1. In the mouse model, SAC improved the neurological function in MCAO mice, and inhibited the activation of microglial cells and the expression of inflammatory factors. At the same time, SAC up-regulated the expression of ZO-1 and CD31, and maintained the blood-brain barrier (BBB) function.As a major component of Salvia miltiorrhiza, SAC can suppress microglial cell polarization and promote tubule formation in endothelial cells to exert the neurological repair function in cerebral ischemia. SAC is a multi-functional neuroprotective small molecule.

Effects of wheel-running on anxiety and depression-relevant behaviours in the MCAO mouse model of stroke: moderation of brain-derived neurotrophic factor and serotonin receptor gene expression

Stroke continues to be a major cause of mortality globally. Post-stroke treatment is complicated by the heterogenous nature of pathology and the emergence of secondary psychological symptoms are an additional challenge to the recovery process. Poststroke depression (PSD) is a common co-morbidity and is a major impediment to recovery. While selective serotonin reuptake inhibitors (SSRIs) have proven to be clinically efficacious in treating PSD, the pathogenic processes that underlie the manifestation of depressive mood post-stroke remains unclear. Furthermore, the use of SSRIs is associated with risks of intracerebral haemorrhage, so alternative treatment options need to be continuously explored. Exercise has been demonstrated to be beneficial for improving mood in humans and preclinical models of neurological conditions. Little is known of the mood-related benefits of physical exercise post-stroke. Using the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischaemia, we investigated whether behavioural deficits emerge post-MCAO and could be rescued by voluntary wheel-running. We report that MCAO induced hypo-locomotion and anhedonia-related behaviours, with some improvements conferred by wheel-running. Serotonin transporter gene expression was increased in the MCAO hippocampus and frontal cortex, but this increase remained despite wheel-running. Wheel-running associated up-regulation of BDNF gene expression was unaffected in MCAO mice, reflecting conservation of key neuroplasticity molecular pathways. Taken together, our results highlight the need for further research into serotonergic modulation of the affective symptoms of stroke.

A3 adenosine receptor agonist IB-MECA reverses chronic cerebral ischemia-induced inhibitory avoidance memory deficit

BackgroundChronic cerebral ischemia (CCI) is a major cause of subcortical ischemic vascular dementia. Here, we examined the neuroprotective action of the A3 adenosine receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA) on white matter lesions following CCI. MethodsA CCI mouse model was established using unilateral common carotid artery occlusion. IB-MECA and 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS1523), an antagonist of the A3 adenosine receptor, were administered by intraperitoneal injection. The inhibitory avoidance test was used to examine changes in memory performance. Microtubule-associated protein 2 (MAP-2) and neurofilament were assessed by immunohistochemistry, while expressions of phosphorylated extracellular signal-regulated kinase (ERK), ERK, interferon-beta (IFN-β), and glial fibrillary acidic protein (GFAP) were assessed by western blot assay. ResultsThe memory retention score was reduced in vehicle-treated mice compared with IB-MECA-treated (p < 0.05) mice. Compared to sham-operated mice, p-ERK, GFAP and IFN-β were increased, while MAP-2 and neurofilament were reduced in vehicle-treated mice (p < 0.01 for each). IB-MECA reduced ERK phosphorylation (p < 0.01) and GFAP expression (p < 0.05), but upregulated MAP-2 and IFN-β (p < 0.01 for both), compared with vehicle. MRS1523 suppressed the effects of IB-MECA on the memory deficit, ERK phosphorylation, and on MAP-2, neurofilament, GFAP and IFN-β levels. ConclusionOur results suggest that A3 adenosine receptor stimulation ameliorates CCI-induced memory deficits, modulates the ERK signaling pathway, preserves MAP-2 and neurofilament expression, regulates GFAP expression, and upregulates the anti-inflammatory cytokine IFN-β. Thus, the A3 adenosine receptor may be a therapeutic target for treatment of cognitive disorders and cerebral inflammatory diseases.

Abstract 164: Bet Protein Degradation With The Proteolysis-targeting Chimera, Dbet1, Ameliorates Brain Damage And Neurological Deficits After Transient Cerebral Ischemia

Introduction: Accumulating evidence indicates that b romodomain and e xtra- t erminal domain (BET) proteins play a pivotal role in the transcriptional regulation of the inflammatory response, and consequently, BET inhibition has emerged as a promising novel therapeutic strategy to reduce inflammation linked to many diseases, including ischemic brain injury. dBET1 is the first BET-targeted proteolysis-targeting chimera (PROTAC) that induces a rapid and efficient deletion of BET proteins in vivo . Hypothesis: In this study, we hypothesized that dBET1 protects against brain damage and neurological deficits in a transient cerebral ischemia mouse model through the regulation of the inflammatory process. Methods: Adult C57BL/6 WT mice were subjected to transient cerebral ischemia surgery (tMCAO) and were administered intraperitoneally with dBET1 (30 mg/kg) or vehicle at 4 and 24 hours after the stroke onset. Infarct volume, neurological deficits, blood-brain barrier integrity, and several inflammatory mediators were examined 48h after tMCAO. Results: The expression level of the BET protein, BRD4, in the cerebral cortex was remarkably lower in the dBET1-treated animals compared to the controls. dBET1 significantly reduced infarct volume (56.2±7.9%, P≤0.01, n=13-14) and improved the neurological performance in the open field test, vertical grid test, and neurological deficit scoring (P≤0.05, n=19-20). In dBET1-treated mice, in both ischemic striatum and cerebral cortex, the inflammation-associated reactive gliosis in astrocytes and microglia was significantly attenuated (P≤0.05, n=6), along with a reduction in stroke-mediated blood-brain barrier damage and neutrophil infiltration. Mechanistically, dBET1 produced a dramatic decrease in the expression levels of pro-inflammatory mediators, while it enhanced levels of anti-inflammatory mediators (P≤0.05, n=6). Conclusion: Overall, this study provides direct in vivo evidence that BET protein degradation with dBET1 suppresses neuroinflammation and leads to anatomical and functional protection against ischemic brain damage. Blocking BET proteins is a promising new strategy to reduce neuroinflammation and eventually improve functional outcomes in stroke.

Prunus cerasoides Extract and Its Component Compounds Upregulate Neuronal Neuroglobin Levels, Mediate Antioxidant Effects, and Ameliorate Functional Losses in the Mouse Model of Cerebral Ischemia.

Prunus cerasoides (PC) has been reported to have antimicrobial and anti-inflammatory properties, but its potential as a neuroprotective agent in a mouse model of cerebral ischemia has not been explored. Considering neuroglobin (Ngb), an endogenous neuroprotective factor, as a novel approach to neuroprotection, in this study, Ngb promoter activity, Ngb expression changes, and antioxidant protection by PC extract (PCE) and PC component compounds (PCCs) were analyzed in oxygen–glucose deprivation (OGD)-treated neurons. In vivo analysis involved transient middle cerebral artery occlusion (tMCAO) in mice with pre- and post-treatment exposure to PCE. Following ischemic stroke induction, neurological behavior scores were obtained, and cellular function-related signals were evaluated in the ischemic infarct areas. In addition to PCE, certain component compounds from PCE also significantly increased Ngb levels and attenuated the intracellular ROS production and cytotoxicity seen with OGD in primary neurons. Administration of PCE reduced the infarct volume and improved neurological deficit scores in ischemic stroke mice compared with the vehicle treatment. Increased Ngb levels in infarct penumbra with PCE treatment were also accompanied by decreased markers of apoptosis (activated p38 and cleaved caspase-3). Our findings point to the benefits of Ngb-mediated neuroprotection via PCE and its antioxidant activity in an ischemic stroke model.

Artesunate attenuates inflammatory injury and inhibits the NF-κB pathway in a mouse model of cerebral ischemia.

ObjectiveInflammation is an important factor in the pathological process of cerebral ischemia. Artesunate exhibits a broad range of anti-inflammatory properties in many diseases. We investigated the potential protective effect of artesunate against cerebral ischemia and the related mechanisms.MethodsMice were divided into distal middle cerebral artery occlusion (dMCAO), sham, low dose, and high dose groups and subjected to dMCAO, except for the sham group. The low and high dose groups were administered artesunate (15 and 30 mg/kg), and the neuroprotective effects were analyzed by evaluating infarct volumes and neurological deficits. Microglial activation and neutrophil infiltration were evaluated by immunofluorescence, immunohistochemical staining, and western blotting. Inflammatory mediators were measured by enzyme-linked immunosorbent assays. Nuclear factor (NF)-κB nuclear translocation was detected by immunofluorescence and western blotting.ResultsCompared with the dMCAO group, artesunate significantly improved neurological deficit scores and infarct volumes and ameliorated inflammation by reducing neutrophil infiltration, suppressing microglial activation, and downregulating tumor necrosis factor-α and interleukin-1β expression. Furthermore, artesunate inhibited nuclear translocation of NF-κB and inhibitor protein α proteolysis.ConclusionsArtesunate protected against inflammatory injury by reducing neutrophil infiltration and microglial activation, suppressing inflammatory cytokines, and inhibiting the NF-κB pathway. Therefore, artesunate is a potential ischemic stroke treatment.