Middle Cerebral Artery Occlusion Mice Research Articles

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

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

Monocyte-Derived Macrophages Aggravate Cardiac Dysfunction After Ischemic Stroke in Mice.

Cardiac damage induced by ischemic stroke, such as arrhythmia, cardiac dysfunction, and even cardiac arrest, is referred to as cerebral-cardiac syndrome (CCS). Cardiac macrophages are reported to be closely associated with stroke-induced cardiac damage. However, the role of macrophage subsets in CCS is still unclear due to their heterogeneity. Sympathetic nerves play a significant role in regulating macrophages in cardiovascular disease. However, the role of macrophage subsets and sympathetic nerves in CCS is still unclear. In this study, a middle cerebral artery occlusion mouse model was used to simulate ischemic stroke. ECG and echocardiography were used to assess cardiac function. We used Cx3cr1GFPCcr2RFP mice and NLRP3-deficient mice in combination with Smart-seq2 RNA sequencing to confirm the role of macrophage subsets in CCS. We demonstrated that ischemic stroke-induced cardiac damage is characterized by severe cardiac dysfunction and robust infiltration of monocyte-derived macrophages into the heart. Subsequently, we identified that cardiac monocyte-derived macrophages displayed a proinflammatory profile. We also observed that cardiac dysfunction was rescued in ischemic stroke mice by blocking macrophage infiltration using a CCR2 antagonist and NLRP3-deficient mice. In addition, a cardiac sympathetic nerve retrograde tracer and a sympathectomy method were used to explore the relationship between sympathetic nerves and cardiac macrophages. We found that cardiac sympathetic nerves are significantly activated after ischemic stroke, which contributes to the infiltration of monocyte-derived macrophages and subsequent cardiac dysfunction. Our findings suggest a potential pathogenesis of CCS involving the cardiac sympathetic nerve-monocyte-derived macrophage axis.

Polydopamine-modified black phosphorus nanosheet drug delivery system for the treatment of ischemic stroke

Abstract Black phosphorus (BP), as a representative metal-free semiconductor, has been extensively explored. It has a higher drug loading capacity in comparison to conventional materials and also possesses excellent biocompatibility and biodegradability. Furthermore, BP nanosheets can enhance the permeability of the blood-brain barrier (BBB) upon near-infrared (NIR) irradiation, owing to their photothermal effect. However, the inherent instability of BP poses a significant limitation, highlighting the importance of surface modification to enhance its stability. Ischemic stroke (IS) is caused by the occlusion of blood vessels, and its treatment is challenging due to the hindrance caused by the BBB. Therefore, there is an urgent need to identify improved methods for bypassing the BBB for more efficient IS treatment. This research devised a novel drug delivery approach based on pterostilbene (Pte) supported by BP nanosheets, modified with polydopamine (PDA) to form BP-Pte@PDA. This system shows robust stability and traverses the BBB using effective photothermal mechanisms. This enables the release of Pte upon pH and NIR stimuli, offering potential therapeutic advantages for treating IS. In a middle cerebral artery occlusion (MCAO) mouse model, the BP-Pte@PDA delivery system significantly reduced infarct size, and brain water content, improved neurological deficits, reduced the TLR4 inflammatory factor expression, and inhibited cell apoptosis. In summary, the drug delivery system fabricated in this study thus demonstrated good stability, therapeutic efficacy, and biocompatibility, rendering it suitable for clinical application.

Aprepitant Alleviates Poststroke Pneumonia in a Mouse Model of Middle Cerebral Artery Occlusion.

Elevated substance P can be utilized to predict early mortality during the first week of cerebral infarction. Whether aprepitant, a substance P receptor blocker could be utilized to alleviate poststroke pneumonia which is investigated in this study. Intraluminal monofilament model of middle cerebral artery occlusion (MCAO) was constructed in C57BL/6J male mice, and the relative expression of substance P was detected in collected bronchoalveolar lavage fluid (BALF) and lung tissue homogenate at 24 hours, 48 hours, and 72 hours poststroke. On the other hand, different concentrations of aprepitant (0.5, 1, and 2 mg/kg) were atomized and inhaled into MCAO mice. Inflammation cytokines and bacterial load were detected in collected BALF and lung tissue homogenate at 72-hour poststroke, and lung injury was revealed by histological examination. Aprepitant administration decreased total proteins, total cells, neutrophils, and macrophages in BALF. The concentrations of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, interferon γ, monocyte chemoattractant protein-1, and IL-10 in lung tissue homogenates were also diminished by the administration of aprepitant. In conclusion, aprepitant could attenuate poststroke pneumonia in mice suggesting its potential therapeutic use in the clinic.

Mesenchymal stem cell-derived exosomes overexpressing SRC-3 protect mice from cerebral ischemia by inhibiting ferroptosis

BackgroundThe treatment for cerebral ischemia remains limited, and new therapeutic strategies are urgently needed. Exosome has shown great promise for the treatment of cerebral ischemia. Steroid receptor coactivator-3 (SRC-3) was reported to be involved in neurological performances. In this study, we aimed to investigate the protective effects of mesenchymal stem cell (MSC)-derived exosomes overexpressing SRC-3 on cerebral ischemia in mice. MethodsThe mice were treated with an intracerebroventricular injection of GFP-overexpressed exosomes (GFP-exo) and SRC-3-overexpressed exosomes (SRC3-exo) in a middle cerebral artery occlusion (MCAO) model of cerebral ischemia. ResultsThe results showed that SRC3-exo treatment significantly inhibited lipid peroxidation and ferroptosis of the neurons subjected to oxygen-glucose deprivation. It further suppressed the activation of microglia and astrocytes, and decreased the production of pro-inflammatory cytokines in the brains of MCAO mice. Furthermore, SRC3-exo treatment reduced the water content of brain tissue and infarct size, which alleviated the neurological damage and improved neurological performances in the MCAO mice. ConclusionsOur results suggest that MSC-derived exosomes expressing SRC3 can be a therapeutic strategy for cerebral ischemia by inhibiting ferroptosis.

Mitochondria-associated endoplasmic reticulum membranes tethering protein VAPB-PTPIP51 protects against ischemic stroke through inhibiting the activation of autophagy.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) serve as a crucial bridge connecting the endoplasmic reticulum (ER) and mitochondria within cells. Vesicle-associated membrane protein-associated protein B (VAPB) and protein tyrosine phosphatase interacting protein 51 (PTPIP51) are responsible for the formation and stability of MAMs, which have been implicated in the pathogenesis of various diseases. However, the role of MAMs in ischemic stroke (IS) remains unclear. We aimed to investigate the role of MAMs tethering protein VAPB-PTPIP51 in experimental cerebral ischemia. We simulated cerebral ischemia-reperfusion injury (CIRI) by using a mouse middle cerebral artery occlusion (MCAO) model. We observed a decrease in VAPB-PTPIP51 expression in the brain tissue. Our findings suggested compromised MAMs after MCAO, as a decreased mitochondria-ER contact (MERC) coverage and an increased distance were observed through the transmission electron microscope (TEM). Upon VAPB or PTPIP51 knockdown, the damage to MAMs was exacerbated, accompanied by excessive autophagy activation and increased reactive oxygen species (ROS) production, resulting in an enlarged infarct area and exacerbated neurological deficits. Notably, we observed that this damage was concomitant with the inhibition of the PI3K/AKT/mTOR pathway and was successfully mitigated by the treatment with the PI3K activator. Our findings suggest that the downregulation of VAPB-PTPIP51 expression after IS mediates structural damage to MAMs. This may exacerbate CIRI by inhibiting the PI3K pathway and activating autophagy, thus providing new therapeutic targets for IS.

Mechanism of microRNA regulating apoptosis after reperfusion in patients with mechanical thrombectomy.

Stroke is the second leading cause of death worldwide. Understanding of gene expression dynamics could bring new approaches in diagnostics and therapy of stroke. Small noncoding molecules termed 'microRNA' represent the most flexible network of gene expression regulators. To screen out miRNAs that are mainly regulated during reperfusion in mechanically embolized patients, and study their mechanisms of action in reperfusion injury after thrombectomy, in order to find new therapeutic targets for mechanically embolized patients. Serums from 30 patients with moderate to severe stroke after mechanical thrombectomy (MT) were collected to measure miRNA expressions. Clinical information of patients was analyze, and patients were divided into poor prognosis and good prognosis. Factors affecting prognosis was classified, and independent risk factors for poor prognosis were determined. Prognostic value of National Institutes of Health Stroke Scale (NIHSS) score on admission to patients with MT was assessed. ROC (receiver operating characteristic) curves were drawn, and Kaplan-Merier method determined whether different NIHSS scores at admission had any difference in the in-hospital survival rate of consistency index/random consistency index (CI/RI) patients treated with MT. An oxygen-glucose deprivation/reperfusion (OGD/R) cell model and an middle cerebral artery occlusion (MCAO)/reperfusion mouse model were established, in which miR-298 expression was tested. In OGD/R cells, proliferation, apoptosis, and autophagy were assessed after intervention with miR-298 and/or autophagy related gene 5 (ATG5). In MCAO mice, the infarct area was calculated, and neurological function was assessed. The relationship between miR-298 and ATG5 was explored and validated. Age, diabetes, hypertension, hemorrhage transformation, NIHSS score at admission, leukocyte, neutrophil count and neutrophil to lymphocyte ratio (NLR) level were associated with patient's prognosis. Diabetes, NIHSS score at admission, and hemorrhagic transformation were independent risk factors for predicting poor prognosis in patients treated with MT. NIHSS score on admission had a predictive value on patient's prognosis. miR-298 was upregulated in acute cerebral ischemia patients with MT (p<0.05), especially in those with poor prognosis. miR-298 was elevated in both cell and mouse models (p<0.05). Apoptosis and autophagy of cells were weakened after miR-298 knockdown, and infarction in the mouse brain tissues was reduced. ATG5 was a target of miR-298. Overexpressing ATG5 rescued miR-298-induced apoptosis and autophagy. In conclusion: regulation of miR-298 and ATG5 attenuates neuronal apoptosis and autophagy, providing a new strategy for brain injury after reperfusion in patients with MT.

Blocking cGAS-STING pathway promotes post-stroke functional recovery in an extended treatment window via facilitating remyelination.

Ischemic stroke is a major cause of worldwide death and disability, with recombinant tissue plasminogen activator being the sole effective treatment, albeit with a limited treatment window. The cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) pathway is emerging as the major DNA-sensing pathway to invoke immune responses in neuroinflammatory disorders. By performing a series of neurobehavioral assessments, electrophysiological analysis, high-throughput sequencing, and cell-based assays based on the transient middle cerebral artery occlusion (tMCAO) mouse stroke model, we examined the effects and underlying mechanisms of genetic and pharmacological inhibition of the cGAS-STING pathway on long-term post-stroke neurological functional outcomes. Blocking the cGAS-STING pathway, even 3days after tMCAO, significantly promoted functional recovery in terms of white matter structural and functional integrity as well as sensorimotor and cognitive functions. Mechanistically, the neuroprotective effects via inhibiting the cGAS-STING pathway were contributed not only by inflammation repression at the early stage of tMCAO but also by modifying the cell state of phagocytes to facilitate remyelination at the sub-acute phase. The activation of the cGAS-STING pathway significantly impeded post-stroke remyelination through restraining myelin debris uptake and degradation and hindering oligodendrocyte differentiation and maturation. Manipulating the cGAS-STING pathway has an extended treatment window in promoting long-term post-stroke functional recovery via facilitating remyelination in a mouse stroke model. Our results highlight the roles of the cGAS-STING pathway in aggregating stroke pathology and propose a new way for improving functional recovery after ischemic stroke. This work was primarily funded by the National Key R&D Program of China.

Glymphatic System Impairment Contributes to the Formation of Brain Edema After Ischemic Stroke.

Blood-brain barrier damage has traditionally been considered to determine the occurrence and development of poststroke brain edema, a devastating and life-threatening complication. However, no treatment strategy targeting blood-brain barrier damage has been proven clinically effective in ameliorating brain edema. In mice with stroke models induced by transient middle cerebral artery occlusion (MCAO), the changes in glymphatic system (GS) function impairment were detected by ex vivo fluorescence imaging, 2-photon in vivo imaging, and magnetic resonance imaging within 1 week after MCAO, and the effects of GS impairment and recovery on the formation and resolution of brain edema were evaluated. In addition, in patients with ischemic stroke within 1 week after onset, changes in GS function and brain edema were also observed by magnetic resonance imaging. We found that the extravasation of protein-rich fluids into the brain was not temporally correlated with edema formation after MCAO in mice, as brain edema reabsorption preceded blood-brain barrier closure. Strikingly, the time course of edema progression matched well with the GS dysfunction after MCAO. Pharmacological enhancement of the GS function significantly alleviated brain edema developed on day 2 after MCAO, accompanied by less deposition of Aβ (amyloid-β) and better cognitive function. Conversely, functional suppression of the GS delayed the absorption of brain edema on day 7 after MCAO. Moreover, patients with ischemic stroke revealed a consistent trend of GS dysfunction after reperfusion as MCAO mice, which was correlated with the severity of brain edema and functional outcomes. GS is a key contributor to the formation of brain edema after ischemic stroke, and targeting the GS may be a promising strategy for treating brain edema in ischemic stroke. URL: https://www.chictr.org.cn/showproj.html?proj=162857; Unique identifier: NFEC-2019-189.

Electroacupuncture Promotes Angiogenesis in Mice with Cerebral Ischemia by Inhibiting miR-7.

To observe the angiogenesis effect of electroacupuncture (EA) at Shuigou acupoint (GV 26) in the treatment of cerebral ischemia, and explore the value of miRNA-7 (miR-7) in it. First, 48 mice were randomly divided into sham operation, middle cerebral artery occlusion (MCAO) model, and EA treatment groups. Then 9 mice were divided into carrier control group, miR-7 knockout group and miR-7 overexpression group (n=3 each group). Finally, 20 mice were divided into model and carrier control group, model and miR-7 knockout group, EA treatment and carrier control group and EA treatment and miR-7 overexpression group, with 3-6 mice in each group. The MCAO model was established in the MCAO and EA groups. Neurological deficit score and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the severity of cerebral ischemia. Hematoxylin-eosin staining was used to describe basic pathological changes. Immunohistochemistry was used to quantify cerebral microvessel density. Real-time PCR and Western blot were used to detect the expression of miR-7 and its downstream target genes Krüppel-like factor 4/vascular endothelial growth factor (KLF4/VEGF) and angiopoietin-2 (ANG-2) in the ischemic cerebral cortex. After EA, neurological deficit scores and infarction volumes decreased, and the density of cerebral microvessels increased. In the MCAO group, miR-7 expression was higher than that in the sham group (P<0.01). After EA at GV 26, miR-7 expression decreased (P<0.01) and the expression of downstream target genes KLF4/VEGF and ANG-2 increased as compared with the MCAO group (P<0.01). After EA combined with overexpression of miR-7, the expression of downstream target genes KLF4/VEGF and ANG-2 decreased compared to the control EA group (P<0.01). After miR-7 knockdown, the expression of KLF4/VEGF and ANG-2 increased (P<0.05 or P<0.01). EA could promote angiogenesis in MCAO mice likely by inhibiting the expression of miR-7 and relieving inhibition of downstream target genes KLF4/VEGF and ANG-2.

Bone marrow mesenchymal stromal cell-derived exosomal NRF2 ameliorates cerebral ischemia-reperfusion injury by transcriptionally activating Lin28a.

Cerebral ischemia-reperfusion (I/R) injury (CIRI) have severe consequences on brain function, and the exciting evidence has revealed protective role of acyl-CoA synthetase long chain family member 4 (Lin28a) against cerebral ischemia-reperfusion injury. The present work aims to reveal its molecular mechanism in regulating CIRI, with the hope of providing a therapeutic method for cerebral I/R injury. We hypothesized that the exosomal nuclear factor erythroid 2-related factor 2 (NRF2) derived from bone marrow mesenchymal stromal cells (BMSCs) could transcriptionally activate Lin28a, and thereby alleviate cerebral ischemia-reperfusion injury. This hypothesis was validated in the present work. Middle cerebral artery occlusion (MCAO) model was established using C57BL/6 J mice, and the neurological deficit, infarct volume, and brain water content were assessed to evaluate neuron injury. Human glioblastoma cells (A172) were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) treatment to mimic a cerebral I/R injury cell model. Exosome isolation reagent was used to isolate exosomes from cell supernatant of bone marrow mesenchymal stromal cells (BMSCs) through sequential centrifugation and filtration steps. mRNA expression level of Lin28a was detected by quantitative real-time polymerase chain reaction. Protein expression was analyzed by western blotting assay. TUNEL cell apoptosis detection kit was used to analyze cell apoptosis in brain tissues. Enzyme-linked immunosorbent assays and commercial kits were used to detect levels of inflammatory markers and oxidative stress markers. Ferrous Iron Colorimetric Assay Kit and Fe2+ colorimetric assay kit were used to analyze Fe2+ level. The association of Lin28a and NRF2 was identified by Chromatin immunoprecipitation (ChIP) assay and dual-luciferase reporter assay. The treatment of MCAO substantially augmented infarct volume in mice, impaired neurological function, and elevated brain water content. Lin28a was lowly expressed in brain tissues of mice with CIRI, and its overexpression protected against cerebral I/R injury of MCAO mice. Moreover, Lin28a overexpression protected A172 cells against OGD/R treatment-induced injury. Additionally, NRF2 transcriptionally activated Lin28a in A172 cells. BMSC-derived exosomes increased Lin28a expression in a NRF2-dependent manner. BMSC-derived exosomal NRF2 improved OGD/R-induced A172 cell injury by inducing Lin28a production. BMSC-derived exosomal NRF2 improved CIRI by transcriptionally activating Lin28a.

Cepharanthine maintains integrity of the blood-brain barrier (BBB) in stroke via the VEGF/VEGFR2/ZO-1 signaling pathway.

Dysfunction of tight junctions such as zonula occludens protein-1 (ZO-1)-associated aggravation of blood-brain barrier (BBB) permeability plays an important role in the progression of stroke. Cepharanthine (CEP) is an extract from the plant Stephania cepharantha. However, the effects of CEP on stroke and BBB dysfunction have not been previously reported. In this study, we report that CEP improved dysfunction in neurological behavior in a middle cerebral artery occlusion (MCAO) mouse model. Importantly, CEP suppressed blood-brain barrier (BBB) hyperpermeability by increasing the expression of ZO-1. Notably, we found that CEP inhibited the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) in the cortex of MCAO mice. Additionally, the results of in vitro experiments demonstrate that treatment with CEP ameliorated cytotoxicity of human bEnd.3 brain microvascular endothelial cells against hypoxia/reperfusion (H/R). Also, CEP attenuated H/R-induced aggravation of endothelial permeability in bEND.3 cells by restoring the expression of ZO-1. Further study proved that the protective effects of CEP are mediated by inhibition of VEGF-A and VEGFR2. Based on the results, we conclude that CEP might possess a therapeutic prospect in stroke through protecting the integrity of the BBB mediated by the VEGF/VEGFR2/ZO-1 axis.

[PI3K/Akt/Erk signaling pathway mediates neuroprotection of CaMKⅡγ and CaMKⅡδ against ischemic reperfusion injury in mice].

To observe neuroprotective effects of Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)γ and CaMkII δ against acute neuronal ischemic reperfusion injury in mice and explore the underlying mechanism. Primary cultures of brain neurons isolated from fetal mice (gestational age of 18 days) were transfected with two specific siRNAs (si-CAMK2G and si-CAMK2D) or a control sequence (si-NT). After the transfection, the cells were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) conditions for 1 h followed by routine culture. The expressions of phosphatidylinositol-3-kinase/extracellular signal-regulated kinase (PI3K/Akt/Erk) signaling pathway components in the neurons were detected using immunoblotting. The expressions of the PI3K/Akt/Erk signaling pathway proteins were also detected in the brain tissues of mice receiving middle cerebral artery occlusion (MCAO) or sham operation. The neuronal cells transfected with siCAMK2G showed significantly lower survival rates than those with si-NT transfection at 12, 24, 48, and 72 h after OGD/R (P < 0.01), and si-CAMK2G transfection inhibited OGD/R-induced upregulation of CaMKⅡγ expression. Compared to si-NT, transfection with si-CAMK2G and si-CAMK2D both significantly inhibited the expressions of PI3K/Akt/Erk signaling pathway components (P < 0.01). In the mouse models of MCAO, the expressions of CaMKⅡδ and CaMKⅡγ were significantly increased in the brain, where activation of the PI3K/Akt/Erk signaling pathway was detected. The expression levels of CaMKⅡδ, CaMKⅡγ, Erk, phosphorylated Erk, Akt, and phosphorylated Akt were all significantly higher in MCAO mice than in the sham-operated mice at 24, 48, 72, and 96 h after reperfusion (P < 0.05). The neuroprotective effects of CaMKⅡδ and CaMKⅡγ against acute neuronal ischemic reperfusion injury are mediated probably by the PI3K/Akt/Erk pathway.

Induced Mesenchymal Stem Cells-Small Extracellular Vesicles Alleviate Post-stroke Cognitive Impairment by Rejuvenating Senescence of Neural Stem Cells.

Ischemic stroke is typified by hypoxia and a cascade of pathophysiological events, including metabolic dysfunction, ionic dysregulation, excitotoxicity, inflammatory infiltration, and oxidative stress. These ultimately result in neuronal apoptosis or necrosis with constrained neuroregenerative capabilities. In this study, neural stem cells (NSCs) under conditions of oxygen-glucose deprivation (OGD) in vitro and following middle cerebral artery occlusion (MCAO) in vivo were explored. Transcriptome sequencing revealed a decline in NSC differentiation and neurogenesis after OGD exposure, which was related to cellular senescence. This observation was corroborated by increased senescence markers in the MCAO mouse model, reduction in NSC numbers, and decline in neurogenesis. Importantly, iMSC-sEVs (induced mesenchymal stem cells-small extracellular vesicles) have the therapeutic potential to alleviate NSC senescence and rejuvenate their regenerative capacities both in vitro and in vivo. Moreover, iMSC-sEVs contribute to the recovery of cognitive function and synapse loss caused by MCAO.

Kaempferol regulates apoptosis and migration of neural stem cells to attenuate cerebral infarction by O-GlcNAcylation of β-catenin.

Ischemic stroke remains a major cause of disability and death. Kaempferol (Kae) is a neuroprotective flavonoid compound. Thus, this study aimed to explore the impact of Kae on cerebral infarction. We generated the middle cerebral artery occlusion (MCAO) mouse model to study the effects of Kae on infarction volume and neurological function. The oxygen and glucose deprivation (OGD)/reoxygenation (R) model of neural stem cells (NSCs) was established to study the effects of Kae on cell viability, migration, and apoptosis. Cell processes were assessed by cell counting kit-8, Transwell assay, flow cytometry, and TUNEL analysis. The molecular mechanism was assessed using the Western blot. The results indicated that Kae attenuated MCAO-induced cerebral infarction and neurological injury. Besides, Kae promoted cell viability and migration and inhibited apoptosis of OGD/R-treated NSCs. Moreover, OGD/R suppressed total O-GlcNAcylation level and O-GlcNAcylation of β-catenin, thereby suppressing the Wnt/β-catenin pathway, whereas Kae reversed the suppression. Inactivation of the Wnt/β-catenin pathway abrogated the biological functions of NSCs mediated by Kae. In conclusion, Kae suppressed cerebral infarction by facilitating NSC viability, migration, and inhibiting apoptosis. Mechanically, Kae promoted O-GlcNAcylation of β-catenin to activate the Wnt/β-catenin pathway. Kae may have a lessening effect on ischemic stroke.

A Neutrophil Hijacking Nanoplatform Reprograming NETosis for Targeted Microglia Polarizing Mediated Ischemic Stroke Treatment.

Precise and efficient regulation of microglia is vital for ischemic stroke therapy and prognosis. The infiltration of neutrophils into the brain provides opportunities for regulatory drugs across the blood-brain barrier, while hindered by neutrophil extracellular traps (NETs) and targeted delivery of intracerebral drugs to microglia. This study reports an efficient neutrophil hijacking nanoplatform (referred to as APTS) for targeted A151 (a telomerase repeat sequence) delivery to microglia without the generation of NETs. In the middle cerebral artery occlusion (MCAO)mouse model, the delivery efficiency to ischemic stroke tissues increases by fourfold. APTS dramatically reduces the formation of NETs by 2.2-fold via reprogramming NETosis to apoptosis in neutrophils via a reactive oxygen species scavenging-mediated citrullinated histone 3 inhibition pathway. Noteworthy, A151 within neutrophils is repackaged into apoptotic bodies following the death pattern reprogramming, which, when engulfed by microglia, polarizes microglia to an anti-inflammatory M2 phenotype. After four times treatment, the cerebral infarction area in the APTS group decreases by 5.1-fold. Thus, APTS provides a feasible, efficient, and practical drug delivery approach for reshaping the immune microenvironment and treating brain disorders in the central nervous system.

Circular RNA circEfnb2 promotes cell injury after cerebral infarction by sponging miR-202-5p and regulating TRAF3 expression

BackgroundExogenous neural cell transplantation may be therapeutic for stroke, cerebral ischemic injury. Among other mechanisms, increasing findings indicated circular RNAs (circRNAs) regulate the pathogenesis progression of cerebral ischemia. Mmu_circ_0015034 (circEfnb2) was upregulated in focal cortical infarction established by middle cerebral artery occlusion (MCAO) in mice. Our study was designed to probe the molecular mechanism of circEfnb2 in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal damage in cerebral ischemia. MethodsWe established an in vitro OGD/R cell model. CircEfnb2 and microRNA-202-5p (miR-202-5p) levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels were assessed using specific kits. Tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Flow cytometry analysis evaluated cell apoptosis. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved caspase 3, and Tumor necrosis factor receptor-associated factor 3 (TRAF3) were determined using Western blot assay. ResultsOverall, circEfnb2 was highly expressed whereas miR-202-5p was decreased in OGD/R-treated mouse hippocampal neuronal HT22 cells compared to normal controls (both p > 0.05). From an in vitro functional perspective, circEfnb2 knockdown attenuated an OGD/R-triggered neuronal injury compared to controls (p > 0.05). Mechanically, circEfnb2 acted as a sponge of miR-202-5p; downregulation of miR-202-5p annulled the inhibitory roles of circEfnb2 silencing in an OGD/R-caused neuronal injury model. Our analysis showed that miR-202-5p directly targeted TRAF3 as enhanced TRAF3 abolished the effects of miR-202-5p in the OGD/R-induced neuronal injury. In vivo, lentivirus with a short hairpin (sh)-circEfnb2 inhibited cerebral injury, when injected into cerebral cortex in MCAO mice (p > 0.05). ConclusionOur results suggest that circEfnb2 deficiency may decrease OGD/R-induced HT22 cell damage by modulating the miR-202-5p/TRAF3 axis. This explanation may provide a new direction for cerebral infarction potential therapeutic targets.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke. To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and increase of the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, nanocarriers targeted to VCAM achieved the highest level of brain delivery, nearly 2 orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (IL-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted nanocarriers. Thus, VCAM-targeted lipid nanocarriers represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating acute ischemic stroke.

4-Hydroxysesamin, a Modified Natural Compound, Attenuates Neuronal Apoptosis After Ischemic Stroke via Inhibiting MAPK Pathway.

The 4-hydroxysesamin (4-HS, a di-tetrahydrofuran lignin) is a modified sesamin that was prepared in the laboratory. This preclinical study was designed to preliminarily investigate the neuroprotective properties of 4-HS. In vitro, neuronal injury and inflammation were simulated by oxygen-glucose deprivation and lipopolysaccharide (LPS) exposure in mouse hippocampal neuronal HT22 cell line, and treated with 4-HS and/or metformin (MET, MAPK pathway activator for exploring mechanism). CCK-8, flow cytometry, and enzyme-linked immunosorbent assay were performed to evaluate cell viability, apoptosis, and inflammation. Apoptosis- and pathway-related proteins were detected by Western blotting. Middle cerebral artery occlusion (MCAO) was constructed as a stroke model and treated with 4-HS for in vivo confirmation. Histological staining was used for in vivo evaluation of 4-HS properties. The 4-HS showed similar anti-inflammatory activity to sesamin but did not affect the cell viability of HT22 cells. In vitro, 4-HS improved the cell viability, ameliorated neuronal apoptosis, along with the reversion of apoptotic proteins (Bax, cleaved-caspase 3/9, Bcl-2) expression and inflammatory cytokines (IL-6, TNF-α, IL-10) in LPS-treated HT22 cells. The 4-HS suppressed the phosphorylation of ERK, JNK, and p38 but the addition of MET reversed 4-HS-induced changes of phenotype and protein expression in LPS-treated cells. In vivo, 4-HS showed apparent improvement in cerebral infarction, brain tissue morphology, neuronal architecture, apoptosis, and inflammation of MCAO mice, and also showed inhibiting effects on the phosphorylation of ERK, JNK, and p38, confirming in vivo results. In this first pre-clinical study on 4-HS, we preliminarily demonstrated the neuroprotective properties of 4-HS both in cell and animal models, and proposed that the underlying mechanism might be associated with the MAPK pathway.

Dysregulated SYVN1 promotes CAV1 protein ubiquitination and accentuates ischemic stroke

BackgroundStroke is a major cause of death and severe disability, and there remains a substantial need for the development of therapeutic agents for neuroprotection in acute ischemic stroke (IS) to protect the brain against damage before and during recanalization. Caveolin-1 (CAV1), an integrated protein that is located at the caveolar membrane, has been reported to exert neuroprotective effects during IS. Nevertheless, the mechanism remains largely unknown. Here, we explored the upstream modifiers of CAV1 in IS. MethodsE3 ubiquitin ligases of CAV1 that are differentially expressed in IS were screened using multiple databases. The transcription factor responsible for the dysregulation of E3 ubiquitin-protein ligase synoviolin (SYVN1) in IS was predicted and verified. Genetic manipulations by lentiviral vectors were applied to investigate the effects of double-strand-break repair protein rad21 homolog (RAD21), SYVN1, and CAV1 in a middle cerebral artery occlusion (MCAO) mouse model and mouse HT22 hippocampal neurons induced by oxygen-glucose deprivation (OGD). ResultsSYVN1 was highly expressed in mice with MCAO, and knockdown of SYVN1 alleviated IS injury in mice, as evidenced by limited infarction volume, the lower water content in the brain, and repressed apoptosis and inflammatory response. RAD21 inhibited the transcription of SYVN1, thereby reducing the ubiquitination modification of CAV1. Overexpression of RAD21 elicited a neuroprotective role as well in mice with MCAO and HT22 induced with OGD, which was overturned by SYVN1. ConclusionTranscriptional repression of SYVN1 by RAD21 alleviates IS in mice by reducing ubiquitination modification of CAV1.