Mouse Middle Cerebral Artery Occlusion Model Research Articles

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.

Targeted mRNA Nanoparticles Ameliorate Blood-Brain Barrier Disruption Postischemic Stroke by Modulating Microglia Polarization.

The ischemic stroke is a major global health concern, with high mortality and disability rates. Unfortunately, there is a dearth of effective clinical interventions for managing poststroke neuroinflammation and blood-brain barrier (BBB) disruption that are crucial for the brain injury evolving and neurological deficits. By leveraging the pathological progression of an ischemic stroke, we developed an M2 microglia-targeting lipid nanoparticle (termed MLNP) approach that can selectively deliver mRNA encoding phenotype-switching interleukin-10 (mIL-10) to the ischemic brain, creating a beneficial feedback loop that drives microglial polarization toward the protective M2 phenotypes and augments the homing of mIL-10-loaded MLNPs (mIL-10@MLNPs) to ischemic regions. In a transient middle cerebral artery occlusion (MCAO) mouse model of an ischemic stroke, our findings demonstrate that intravenously injected mIL-10@MLNPs induce IL-10 production and enhance the M2 polarization of microglia. The resulting positive loop reinforces the resolution of neuroinflammation, restores the impaired BBB, and prevents neuronal apoptosis after stroke. Using a permanent distal MCAO mouse model of an ischemic stroke, the neuroprotective effects of mIL-10@MLNPs have been further validated by the attenuation of the sensorimotor and cognitive neurological deficits. Furthermore, the developed mRNA-based targeted therapy has great potential to extend the therapeutic time window at least up to 72 h poststroke. This study depicts a simple and versatile LNP platform for selective delivery of mRNA therapeutics to cerebral lesions, showcasing a promising approach for addressing an ischemic stroke and associated brain conditions.

CircRBM33 induces endothelial dysfunction by targeting the miR-6838-5p/PDCD4 axis affecting blood-brain barrier in mice with cerebral ischemia-reperfusion injury.

circRNAs (circRNAs) are involved in the process of cerebral ischemia-reperfusion injury (CI/RI). Our study aims to explore circRBM33 in the endothelial function of the blood-brain barrier (BBB). The mouse middle cerebral artery occlusion model (MCAO) was established and restored to perfusion, and OGD/R-induced endothelial cells were used to simulate CI/RI. circRBM33, miR-6838-5p and PDCD4, as well as Occludin, ZO-1 and Claudin-5 TJs were evaluated by quantitative PCR and Western blot. The ring structure of circRBM33 was verified by RNAse R and actinomycin D experiments. MTT and LDH Cytotoxicity assay determined viability and toxicity, and flow cytometry determined apoptosis rate. Inflammatory cytokines and the number of microglia in brain tissue were measured by ELISA and IHC. The interaction between genes was verified by RIP and dual luciferase reporter assay. circRBM33 was a circrRNA present in the cytoplasm and up-regulated in the brain tissue of MCAO mice and OGD/R-induced endothelial cells. Silenced circRBM33 promoted Occludin, ZO-1, and Claudin-5 expression and cell proliferation, and inhibited cytotoxicity, inflammatory response, and apoptosis. Functionally, circRBM33-absorbed miR-6838-5p was involved in regulating PDCD4, leading to endothelial cell dysfunction, and thus affecting the function of the BBB. circRBM33 by mediating miR-6838-5p/PDCD4 axis induces endothelial dysfunction, thereby affecting the BBB in mice with CI/RI.

Lipocalin-2-mediated astrocyte pyroptosis promotes neuroinflammatory injury via NLRP3 inflammasome activation in cerebral ischemia/reperfusion injury

BackgroundNeuroinflammation is a vital pathophysiological process during ischemic stroke. Activated astrocytes play a major role in inflammation. Lipocalin-2 (LCN2), secreted by activated astrocytes, promotes neuroinflammation. Pyroptosis is a pro-inflammatory form of programmed cell death that has emerged as a new area of research in stroke. Nevertheless, the potential role of LCN2 in astrocyte pyroptosis remains unclear.MethodsAn ischemic stroke model was established by middle cerebral artery occlusion (MCAO) in vivo. In this study, in vitro, oxygen–glucose deprivation and reoxygenation (O/R) were applied to cultured astrocytes. 24p3R (the LCN2 receptor) was inhibited by astrocyte-specific adeno-associated virus (AAV-GFAP-24p3Ri). MCC950 and Nigericin sodium salt (Nig) were used to inhibit or promote the activation of NLRP3 inflammasome pharmacologically, respectively. Histological and biochemical analyses were performed to assess astrocyte and neuron death. Additionally, the neurological deficits of mice were evaluated.ResultsLCN2 expression was significantly induced in astrocytes 24 h after stroke onset in the mouse MCAO model. Lcn2 knockout (Lcn2−/−) mice exhibited reduced infarct volume and improved neurological and cognitive functions after MCAO. LCN2 and its receptor 24p3R were colocalized in astrocytes. Mechanistically, suppression of 24p3R by AAV-GFAP-24p3Ri alleviated pyroptosis-related pore formation and the secretion of pro-inflammatory cytokines via LCN2, which was then reversed by Nig-induced NLRP3 inflammasome activation. Astrocyte pyroptosis was exacerbated in Lcn2−/− mice by intracerebroventricular administration of recombinant LCN2 (rLCN2), while this aggravation was restricted by blocking 24p3R or inhibiting NLRP3 inflammasome activation with MCC950.ConclusionLCN2/24p3R mediates astrocyte pyroptosis via NLRP3 inflammasome activation following cerebral ischemia/reperfusion injury.

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Advanced Glycation End-Products (AGEs) Promote Endothelial Cell Pyroptosis Under Cerebral Ischemia and Hypoxia via HIF-1α-RAGE-NLRP3.

This work mainly aimed to explore the role and mechanism of advanced glycation end-products (AGEs) in inducing cerebrovascular endothelial cell pyroptosis under oxygen glucose deprivation (OGD) condition. The mouse cerebral microvascular endothelial cells (BMECs and bEnd.3) were used as the objects to construct the OGD model in vitro. Then, cells were pretreated with AGE-modified human serum albumin (AGE-HSA). Thereafter, CCK-8 assay was conducted to detect cell viability, and flow cytometry (FCM) was performed to measure cell pyroptosis level. Meanwhile, the expression of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA). The expression of HIF-α, NLRP3, and RAGE was detected by fluorescence staining. The opening status of cell membrane pore was observed under the electron microscope, and the expression levels of FL-GSDMD, NT-GSDMD, and caspase-1 were measured through Western Blot (WB) assay. Moreover, bEnd.3 cells were treated with siRAN-silenced NLRP3 and HIF-α inhibitor, so as to observe the effect of AGEs on cell pyroptosis level. In the mouse model, the middle cerebral artery occlusion (MCAO) model was constructed by the suture-occluded method. After intraperitoneal injection of AGEs, the pathological changes in mouse brain tissues were detected; the expression levels of NLRP3, ZO-1, and CD31 were determined by histochemical staining, and the levels of inflammatory factors and pyroptosis-related proteins were also detected. Under OGD condition, AGEs induced the pyroptosis of bEnd.3 cells, and the cell pyroptosis rate increased, higher than that of the OGD group. Meanwhile, the levels of inflammatory factors were up-regulated; the expression of HIF-α, NLRP3, and RAGE in cells increased; and the levels of NT-GSDMD and caspase-1 were markedly higher than those of the control and OGD groups. siRNA-NLRP3 or HIF-α inhibitor treatment suppressed pyroptosis and reduced the inflammatory factor levels. In mouse experiments, AGE injection aggravated brain injury in the MCAO mouse model, decreased the expression of ZO-1 and CD31, and elevated the levels of NLRP3 and inflammatory factors. Under cerebral ischemia condition, AGEs can induce endothelial cell pyroptosis via HIF-α-RAGE-NLRP3, thereby further aggravating brain injury.

Transmembrane p24 trafficking protein 10 (TMED10) inhibits mitochondrial damage and protects neurons in ischemic stroke via the c-Jun N-terminal kinase (JNK) signaling pathway.

Stroke, a type of acute cerebrovascular disease, is a global disease with high mortality. Neuronal ischemia and hypoxia are closely related to occurrence and development of cognitive impairment. Transmembrane p24 trafficking protein 10 (TMED10) as a transmembrane protein involves in vesicle protein transport in the secretory pathways. However, the function and mechanism of TMED10 on ischemic stroke and cognitive impairments remain unclear. In current study, TMED10 was highly expressed in cerebral ischemic penumbra of middle cerebral artery occlusion (MCAO) mouse model. Downregulation of TMED10 suppressed cell survival and facilitated apoptosis in primary cortical neurons, which were grown under oxygen glucose deprivation/reoxygenation (OGD/R) condition. Upregulation of TMED10 protected neurons form apoptosis induced by OGD/R. Further research indicated that the decrease of TMED10 resulted in neuronal mitochondrial injury through increasing reactive oxygen species (ROS) production. Meanwhile, TMED10 reduction induced neuronal apoptosis and mitochondrial damage through activating the c-Jun N-terminal kinase (JNK) pathway. Moreover, the knockdown of TMED10 increased cerebral infarction area, aggravated neuronal injury and promoted neuronal apoptosis through activating the JNK pathway in the cerebral ischemic penumbra of MCAO mouse model. Additionally, Morris water maze test verified that the severity of cognitive impairment increased with the decline of TMED10. Collectively, this study reveals that TMED10 inhibits mitochondrial damage, and protects neurons from apoptosis in MCAO-induced ischemic stroke and cognitive impairment via blocking the JNK pathway.

Comparison of ketamine/xylazine and isoflurane anesthesia on the establishment of mouse middle cerebral artery occlusion model.

The middle cerebral artery occlusion model (MCAO) is one of the most common stroke models in neuroscience research. The establishment of the mouse MCAO model in terms of animal survival depends on anesthesia, which is an important part of the entire surgical process. The 7-day survival rate of the MCAO model under isoflurane (ISO) anesthesia (35%) was lower than ketamine/xylazine (KX) anesthesia (70%), which demonstrated that the success rate of the MCAO model under KX anesthesia would be significantly higher than that under ISO anesthesia. As confirmed by TTC staining and MRI, the cerebral infarction area of mice successfully modeled under ISO anesthesia was significantly smaller than that of KX anesthesia. The diameter of cerebral blood vessels under ISO anesthesia was significantly larger than that under KX, and the blood perfusion volume was also significantly increased in the same area. ISO has proven to delay the coagulation time and affect the activation of coagulation factors. ISO anesthesia may cause bleeding, vasodilation, respiratory depression, and other phenomena that affect the success rate and death of diseased animal models. In conclusion, compared with ISO anesthesia, KX anesthesia is a safer and more suitable method for the establishment of a mouse MCAO model. The data will inform safer and more detailed anesthesia recommendations forthe establishment of animal models of vascular-related major injury diseases.

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.

Selenium Alleviates Cerebral Ischemia/Reperfusion Injury by Regulating Oxidative Stress, Mitochondrial Fusion and Ferroptosis

To clarify the potential role of selenium (Se) on cerebral ischemia/reperfusion (I/R) injury, we utilized mouse middle cerebral artery occlusion (MCAO) followed by reperfusion as an animal model and oxygen–glucose deprivation and reoxygenation (OGD/R) to treat N2a cells as a cell model, respectively. MCAO model was established in mice and then divided into different groups with or without Se treatment. TTC staining was used to observe whether the cerebral I/R modeling was successful, and the apoptosis level was determined by TUNEL staining. The expression of GPx-4 and p22phox was assessed by western blot. In vitro experiments, the OGD/R induced oxidative stress in N2a cells was assessed by levels of GSH/GSSG, malondialdehyde, superoxide dismutase and iron content, respectively. QRT-PCR was used to detect the mRNA levels of Cox-2, Fth1, Mfn1 and mtDNA in N2a cells. JC-1 staining and flow cytometry was performed to detect the mitochondrial membrane potential. Se treatment alleviated cerebral I/R injury and improved the survival rate of mice. Additionally, Se treatment apparently attenuated oxidative stress and inhibited iron accumulation in MCAO model mice and OGD/R model of N2a cells. In terms of its mechanism, Se could up-regulate Mfn1 expression to alleviate oxidative stress and ferroptosis by promoting mitochondrial fusion in vivo and vitro. These findings suggest that Se may have great potential in alleviating cerebral I/R injury.

Serum small extracellular vesicles promote M1 activation of microglia after cerebral ischemia/reperfusion injury

Microglial M1 activation is detrimental to stroke outcomes. Recent studies have shown that circulating small extracellular vesicles (sEVs) can deliver miRNAs to target cells and regulate recipient cell functions. Herein, we tested the hypothesis that miRNA delivery by serum sEVs after cerebral ischemia/reperfusion (I/R) injury promote microglial M1 activation, demonstrating that serum sEVs from middle cerebral artery occlusion (MCAO) mice promoted proliferation and M1 activation of BV2 microglia. To explore the underlying mechanism of serum sEVs-mediated microglial activation in the early phase of cerebral I/R injury, we examined the effects of ischemic brain injury on the serum sEVs miRNAs profile in a mouse MCAO model using small RNAseq. Of the 1257 detected miRNA replications, the levels of 72 were significantly modulated. Bioinformatics analysis revealed that a panel of miRNAs was closely associated with inflammation, and in vitro experiments demonstrated that serum sEVs from MCAO mice could effectively transfer inflammatory miRNAs to BV2 microglia. Collectively, our data suggested that miRNAs delivered by serum sEVs after cerebral I/R injury promoted microglial M1 activation. The identification of microglial activation regulators in future studies will give rise to more effective treatments for stroke.

Single-cell RNA-seq reveals the transcriptional landscape in ischemic stroke

Ischemic stroke (IS) is a detrimental neurological disease with limited treatments options. It has been challenging to define the roles of brain cell subsets in IS onset and progression due to cellular heterogeneity in the CNS. Here, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively map the cell populations in the mouse model of MCAO (middle cerebral artery occlusion). We identified 17 principal brain clusters with cell-type specific gene expression patterns as well as specific cell subpopulations and their functions in various pathways. The CNS inflammation triggered upregulation of key cell type-specific genes unpublished before. Notably, microglia displayed a cell differentiation diversity after stroke among its five distinct subtypes. Importantly, we found the potential trajectory branches of the monocytes/macrophage’s subsets. Finally, we also identified distinct subclusters among brain vasculature cells, ependymal cells and other glia cells. Overall, scRNA-seq revealed the precise transcriptional changes during neuroinflammation at the single-cell level, opening up a new field for exploration of the disease mechanisms and drug discovery in stroke based on the cell-subtype specific molecules.

Orexin-A inhibits cerebral ischaemic inflammatory injury mediated by the nuclear factor-κB signalling pathway and alleviates stroke-induced immunodepression in mice

Cerebral ischaemia is accompanied by infectious complications due to immunosuppression, known as stroke-induced immunodepression (SIID). Orexin-A (OXA), a neuropeptide produced in the hypothalamus, has been reported to have neuroprotective properties after stroke and is known to modulate inflammatory processes in peripheral tissues. The aim of this study was to determine the effects of orexin-A (OXA) on cerebral ischaemic inflammatory injury and SIID following experimental stroke. Cerebral ischaemia was induced in C57/BL6 mice by middle cerebral artery occlusion (MCAO). A mouse model of pneumonia and poststroke pneumococcal pneumonia was established by intratracheal inoculation with S. pneumoniae in a normal mouse or MCAO mouse model on the third day. We found that OXA postconditioning inhibited cerebral ischaemic inflammatory injury. The mechanism involved downregulation of the NF-κB signalling pathway. In addition, OXA may serve as a potential treatment target for attenuating stroke-induced immunodepression in mice.

ΜicroRNA-122 protects against ischemic stroke by targeting Maf1.

The protection of brain tissue against damage and the reduction of infarct size is crucial for improving patient prognosis following ischemic stroke. Therefore, the present study aimed to investigate the regulatory effect of microRNA (miR)-122 and its target gene repressor of RNA polymerase III transcription MAF1 homolog (Maf1) on the infarct area in ischemic stroke. Reverse transcription-quantitative PCR (RT-qPCR) was performed to determine miR-122 expression levels in an ischemic stroke [middle cerebral artery occlusion (MCAO)] mouse model. Nissl staining was conducted to measure the infarct area of the MCAO mouse model. Moreover, RT-qPCR was performed to investigate the relationship between the expression of Maf1 and miR-122 in the MCAO mouse model. Dual-luciferase reporter assay in vitro and miR-122 mimic or inhibitor treatment in vivo were conducted to verify that miR-122 targeted and inhibited Maf1 expression. The results suggested that miR-122 was upregulated in the brain tissue of MCAO model mice. miR-122 overexpression effectively reduced the size of the infarct area in comparison with a control and miR-122 knockdown in brain tissue resulted in the opposite effect. Moreover, Maf1 was confirmed to be a direct target of miR-122. The results of a dual-luciferase reporter assay indicated that miR-122 bound to the 3'-untranslated region of Maf1. Maf1 expression decreased after stroke model induction in comparison with that in sham animals, and Maf1 expression was negatively associated with the expression of miR-122. In addition, miR-122 knockdown increased Maf1 expression levels, whereas miR-122 overexpression decreased Maf1 expression levels in comparison with a control. In conclusion, the results suggested that miR-122 improved the outcome of acute ischemic stroke by reducing the expression of Maf1.

Involvement of the Microglial Aryl Hydrocarbon Receptor in Neuroinflammation and Vasogenic Edema after Ischemic Stroke.

Microglia are activated after ischemic stroke and induce neuroinflammation. The expression of the aryl hydrocarbon receptor (AhR) has recently been reported to elicit cytokine expression. We previously reported that microglial activation mediates ischemic edema progression. Thus, the purpose of this study was to examine the role of AhR in inflammation and edema after ischemia using a mouse middle cerebral artery occlusion (MCAO) model. MCAO upregulated AhR expression in microglia during ischemia. MCAO increased the expression of tumor necrosis factor α (TNFα) and then induced edema progression, and worsened the modified neurological severity scores, with these being suppressed by administration of an AhR antagonist, CH223191. In THP-1 macrophages, the NADPH oxidase (NOX) subunit p47phox was significantly increased by AhR ligands, especially under inflammatory conditions. Suppression of NOX activity by apocynin or elimination of superoxide by superoxide dismutase decreased TNFα expression, which was induced by the AhR ligand. AhR ligands also elicited p47phox expression in mouse primary microglia. Thus, p47phox may be important in oxidative stress and subsequent inflammation. In MCAO model mice, P47phox expression was upregulated in microglia by ischemia. Lipid peroxidation induced by MCAO was suppressed by CH223191. Taken together, these findings suggest that AhR in the microglia is involved in neuroinflammation and subsequent edema, after MCAO via p47phox expression upregulation and oxidative stress.

CGAS knockdown promotes microglial M2 polarization to alleviate neuroinflammation by inhibiting cGAS-STING signaling pathway in cerebral ischemic stroke

Inflammation plays a pivotal role in promoting the pathophysiology of ischemic stroke (IS). Microglia is the major immunocompetent cells involved in different neuropathologies. The activation of cyclic GMP-AMP synthase (cGAS) and its downstream signaling protein-stimulator of interferon genes (STING) is increasingly recognized as a crucial determinant of neuropathophysiology. However, the mechanisms underlying cGAS-STING signaling regulating inflammatory response during IS remains to be elucidated. In this study, HT22 cells was used to establish an oxygen-glucose deprivation (OGD) cell model in vitro, and then this cell culture supernatant containing OGD-induced DAMPs (OIDs) was employed to stimulate BV2 microglia. Furthermore, a middle cerebral artery occlusion (MCAO) mouse model was established. Cells and MCAO mice were treated with si-cGAS or si-NC lentivirus. The expression levels of STING, cGAS and p-IRF3 in BV2 cells or MCAO mouse brain; the microglial M1/M2 polarization of BV2 microglia or isolated microglial cells from MCAO mouse brain; the contents of iNOS, TNF-α, TGF-β and IL-10 in the culture medium of BV2 cells or in murine brain homogenates, were all detected. In addition, the severity of cerebral infarction with or without the knockdown of cGAS in a MCAO mouse model was also determined by TTC staining. Results showed that OGD-induced DAMPs strongly activated cGAS-STING pathway and triggered microglia polarization in BV2 cells, reflecting as the accumulation of a plethora of pro-inflammatory factors in activated microglia. However, these effects could be inhibited by cGAS knockdown. In the MCAO mouse model, the inhibition of cGAS-STING pathway resulted from cGAS knockdown could effectively diminish cell apoptosis in mouse brain stimulated by MIDs (MCAO-induced DAMPs), reduced the area ratio of cerebral infarction and ultimately improved the injured nerve function during IS. Taken together, our elucidation of underlying mechanisms involved in the microglial inflammatory response, triggered by cGAS-STING signaling, highlights this pathway as a potential therapeutic target in IS.

Tenacissoside H promotes neurological recovery of cerebral ischaemia/reperfusion injury in mice by modulating inflammation and oxidative stress via TrkB pathway.

Cerebral ischaemia/reperfusion (I/R)-induced acute brain injury remains a troublesome condition in clinical practice. The present study aimed to investigate the protective effect of tenacissoside H (TH) on I/R-induced cerebral injury in mice. Here, a mouse model of middle cerebral artery occlusion (MCAO) was established by an improved Longa-Zea method. TH was given by intraperitoneal injection once a day within 1week before establishing the mouse MCAO model. The neurological functions of mice were evaluated and the apoptosis of neurons was also detected by the TUNEL method and Nissl's staining. ELISA and western blot were used to detect the expression of inflammatory factors, oxidation factors and proteins in the cerebral ischaemic cortex. The results revealed that TH dose-dependently reduced neurological impairment, neuron apoptosis and brain oedema induced by MCAO. Furthermore, TH attenuated the expression of pro-inflammatory cytokines (including interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α), iNOS and nuclear factor (NF)-κB while increased production of anti-inflammatory cytokines (IL-4, IL-10 and BDNF) and proteins of tropomyosin-related kinase receptor B (TrkB) and PPARγ. Nevertheless, after the addition of TrkB inhibitor, the effects of TH above were mostly restrained. In conclusion, TH can protect mice against I/R-induced neurological impairments via modulating inflammation and oxidative stress through TrkB signalling.

Long Noncoding RNA SNHG14 Promotes Ischemic Brain Injury via Regulating miR-199b/AQP4 Axis.

Ischemic stroke is the leading cause of disability worldwide. Long noncoding RNAs (lncRNAs) play important roles in the pathogenesis of cerebral ischemia. This study aimed to investigate the role and mechanism of lncRNA small nucleolar RNA host gene 14 (SNHG14) in ischemic brain injury. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in mice. The expression of SNHG14 in MCAO mouse model was detected by quantitative real-time PCR (qRT-PCR). The levels of SNHG14, microRNA-199b (miR-199b) and aquaporin 4 (AQP4) in oxygen-glucose deprivation (OGD)-stimulated BV2 cells were determined by qRT-PCR or western blot assay. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The levels of pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The levels of oxidative stress markers were examined using commercial kits. The relationships among SNHG14, miR-199b and AQP4 were confirmed by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. SNHG14 was up-regulated in MCAO mouse model. Depletion of SNHG14 lessened cerebral ischemia in MCAO mouse model. SNHG14 silencing inhibited inflammation and oxidative stress in OGD-exposed BV2 cells. MiR-199b level was decreased, while AQP4 level was increased in OGD-treated BV2 cells. Knockdown of miR-199b reversed the effect of SNHG14 knockdown on ischemic damage in OGD-stimulated BV2 cells. Moreover, AQP4 overexpression abolished the effect of miR-199b on ischemic injury in OGD-treated BV2 cells. Furthermore, SNHG14 indirectly regulate AQP4 expression by sponging miR-199b. Knockdown of SNHG14 attenuated ischemic brain injury by inhibiting inflammation and oxidative stress through the miR-199b/AQP4 axis.

Expression and Distribution Pattern of Pnn in Ischemic Cerebral Cortex and Cultured Neural Cells Exposed to Oxygen-Glucose Deprivation.

Pinin (Pnn), a multifunctional protein, participates in embryonic development as well as in cellular apoptosis, proliferation, and migration through regulating mRNA alternative splicing and gene transcription. Previous studies have shown that Pnn plays important roles in neural system development and the expression level of Pnn in astrocytes is altered by ischemic stress and associated with cellular apoptosis. In the present study, we further utilized primary cultured rat neurons and astrocytes with oxygen-glucose deprivation (OGD) and a mouse model with middle cerebral artery occlusion (MCAO)-induced ischemic stroke to examine the effect of ischemic stress on Pnn expression and distribution in different types of neural cells. Under normoxia, Pnn is mainly localized in the nuclear speckle of primary cultured neurons. The expression level of Pnn was increased after the OGD treatment and then decreased in the reoxygenation period. Moreover, the cytoplasmic expression of Pnn was observed in neurons with OGD and reoxygenation (OGD/R). Unlike that in neurons, the Pnn expression in astrocytes was decreased after OGD treatment and then gradually increased during the reoxygenation period. Of interest, the nuclear–cytoplasmic translocation of Pnn was not observed in astrocytes with OGD/R. In the MCAO mouse model, the neuronal expression of Pnn in the peri-ischemic region was reduced by three days post induction of ischemic stroke. However, the Pnn expression in astrocytes was not altered. Moreover, the nuclear speckle distribution of Pnn in neurons was also diminished following ischemic stroke. In conclusion, the Pnn expression and distribution after OGD and during reoxygenation showed distinct manners in neurons and astrocytes, implying that Pnn may play different roles in different types of neural cells in the stress response to ischemic injury.

Remote ischemic conditioning enhances oxygen supply to ischemic brain tissue in a mouse model of stroke: Role of elevated 2,3-biphosphoglycerate in erythrocytes

Oxygen supply for ischemic brain tissue during stroke is critical to neuroprotection. Remote ischemic conditioning (RIC) treatment is effective for stroke. However, it is not known whether RIC can improve brain tissue oxygen supply. In current study, we employed a mouse model of stroke created by middle cerebral artery occlusion (MCAO) to investigate the effect of RIC on oxygen supply to the ischemic brain tissue using a hypoxyprobe system. Erythrocyte oxygen-carrying capacity and tissue oxygen exchange were assessed by measuring oxygenated hemoglobin and oxygen dissociation curve. We found that RIC significantly mitigated hypoxic signals and decreased neural cell death, thereby preserving neurological functions. The tissue oxygen exchange was markedly enhanced, along with the elevated hemoglobin P50 and right-shifted oxygen dissociation curve. Intriguingly, RIC markedly elevated 2,3-biphosphoglycerate (2,3-BPG) levels in erythrocyte, and the erythrocyte 2,3-BPG levels were highly negatively correlated with the hypoxia in the ischemic brain tissue. Further, adoptive transfusion of 2,3-BPG-rich erythrocytes prepared from RIC-treated mice significantly enhanced the oxygen supply to the ischemic tissue in MCAO mouse model. Collectively, RIC protects against ischemic stroke through improving oxygen supply to the ischemic brain tissue where the enhanced tissue oxygen delivery and exchange by RIC-induced 2,3-BPG-rich erythrocytes may play a role.