Stroke triggers dynamic m6A reprogramming of cerebral circular RNAs.

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N6-methyladenosine (m6A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m6A methylation of circRNAs. Changes in m6A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m6A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m6A modification patterns. The majority of circRNAs that showed post-stroke differential m6A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m6A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m6A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology.

Targeting MAD2B as a strategy for ischemic stroke therapy.

INTRODUCTION: Post-stroke cognitive impairment is one of the major causes of disability due to cerebral ischemia. MAD2B is an inhibitor of Cdh1/APC, and loss of Cdh1/APC function in mature neurons increases ROCK2 activity, leading to changes in synaptic plasticity and memory loss in mouse neurons. Whether MAD2B regulates learning memory capacity through ROCK2 in cerebral ischemia is not known. OBJECTIVES: We investigated the role and mechanism of MAD2B in cerebral ischemia-induced cognitive dysfunction. METHODS: The expression of MAD2B and its downstream related molecules was detected by immunoblotting and intervened with neuroprotectants after middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R). We constructed MAD2B-cKO-specific knockout mice, knocked down and overexpressed MAD2B in mouse hippocampus by lentiviral injection in brain stereotaxis, modeled cerebral ischemia by using MCAO, and explored the role of MAD2B in post-stroke cognitive impairment (PSCI) by animal behaviors such as Y-maze and Novel object recognition test. Then the expression of MAD2B/ROCK2, downstream molecules and apoptosis-related molecules was detected. Finally, ROCK2 expression was intervened using its inhibitor and shRNA-ROCK2 lentivirus. RESULTS: The expression of MAD2B and its downstream molecules increased after MCAO and OGD/R. Nonetheless, this expression underwent a decline post-therapy with neuroprotective agents. Deletion of MAD2B in the hippocampus ameliorated memory and learning deficits and improved motor coordination in MCAO mice. Conversely, the overexpression of MAD2B in the hippocampus exacerbated learning and memory deficits. Deletion of MAD2B resulted in the downregulation of ROCK2/LIMK1/cofilin. It effectively reduced ischemia-induced upregulation of BAX and cleaved caspase-3, which could be reversed by MAD2B overexpression. Inhibition or knockdown of ROCK2 expression in primary cultured neurons led to the downregulation of LIMK1/cofilin expression and reduced the expression of apoptosis-associated molecules induced by ischemia. CONCLUSIONS: Our findings suggest that MAD2B affects neuronal apoptosis via Rock2, which affects neurological function and cerebral infarction.

An Unusual Presentation of Moyamoya Disease in a Pediatric Patient.

Moyamoya disease (MMD) is a chronic cerebro-vasculopathy that is extremely rare in the pediatric population. The main characteristic feature is the progressive stenosis in the internal carotid artery with or without the involvement of its main branches in the circle of Willis leading to ischemic stroke. Patients have clinical manifestations related to cerebral ischemia in the carotid branch territories, such as sensory impairment, hemiparesis, and aphasia/dysarthria. Herein, we report a case of MMD in a six-year-old Emirati female who presented with unusual manifestations of MMD in the form of headache, vomiting, and double vision and was diagnosed with MMD based on a brain MRI with angiography. To our knowledge, this is the first reported case of MMD in the United Arab Emirates.

Beyond nimodipine: advanced neuroprotection strategies for aneurysmal subarachnoid hemorrhage vasospasm and delayed cerebral ischemia.

The clinical management of aneurysmal subarachnoid hemorrhage (SAH)-associated vasospasm remains a challenge in neurosurgical practice, with its prevention and treatment having a major impact on neurological outcome. While considered a mainstay, nimodipine is burdened by some non-negligible limitations that make it still a suboptimal candidate of pharmacotherapy for SAH. This narrative review aims to provide an update on the pharmacodynamics, pharmacokinetics, overall evidence, and strength of recommendation of nimodipine alternative drugs for aneurysmal SAH-associated vasospasm and delayed cerebral ischemia. A PRISMA literature search was performed in the PubMed/Medline, Web of Science, ClinicalTrials.gov, and PubChem databases using a combination of the MeSH terms "medical therapy," "management," "cerebral vasospasm," "subarachnoid hemorrhage," and "delayed cerebral ischemia." Collected articles were reviewed for typology and relevance prior to final inclusion. A total of 346 articles were initially collected. The identification, screening, eligibility, and inclusion process resulted in the selection of 59 studies. Nicardipine and cilostazol, which have longer half-lives than nimodipine, had robust evidence of efficacy and safety. Eicosapentaenoic acid, dapsone and clazosentan showed a good balance between effectiveness and favorable pharmacokinetics. Combinations between different drug classes have been studied to a very limited extent. Nicardipine, cilostazol, Rho-kinase inhibitors, and clazosentan proved their better pharmacokinetic profiles compared with nimodipine without prejudice with effective and safe neuroprotective role. However, the number of trials conducted is significantly lower than for nimodipine. Aneurysmal SAH-associated vasospasm remains an area of ongoing preclinical and clinical research where the search for new drugs or associations is critical.

Effect of pretreatment with Devil's Claw on locomotor activity, infarct volume, and neuronal density in focal cerebral ischemia in rats.

Objective: Stroke is a highly prevalent and devastating condition affecting millions worldwide. The Devil's Claw (DCW) plant is a native African plant whose anti-inflammatory, antioxidant, and neuroprotective properties have been investigated. We postulated that DCW could protect the brain injury caused by cerebral ischemia. Materials and Methods: The rats were randomly divided into four groups. The sham and control (Ctrl) groups received pretreatment with a distilled water vehicle. Doses of 200 and 400 mg/kg were selected for pretreatment with DCW. The filament or intravascular occlusion method was used for middle cerebral artery occlusion (MCAO). The Triphenyl tetrazolium chloride (TTC) staining method was used to investigate the infarct zone and penumbra volume. The neuroprotective effect of DCW was measured by hematoxylin staining. Movement performance was evaluated from neurological deficit score, rotarod performance, and open field tests. Results: TTC staining showed that the DCW/400 group could maintain the penumbra's structure and reduce the infarct volume compared to the Ctrl group (p<0.001). Histological studies confirmed the neuroprotective properties of DCW at doses of 200 and 400 mg/kg compared to the Ctrl group (p<0.01 and p<0.0001, respectively). The results of behavioral tests showed an improvement in behavioral performance in pretreatment 400 mg/kg doses compare to Ctrl group (p<0.0001). Conclusion: The study showed that pretreatment with DCW with its neuron protection potential reduces the infarct area and restores motor function after MCAO.

Research progress of propofol in alleviating cerebral ischemia/reperfusion injury.

Ischemic stroke is a leading cause of adult disability and death worldwide. The primary treatment for cerebral ischemia patients is to restore blood supply to the ischemic region as quickly as possible. However, in most cases, more severe tissue damage occurs, which is known as cerebral ischemia/reperfusion (I/R) injury. The pathological mechanisms of brain I/R injury include mitochondrial dysfunction, oxidative stress, excitotoxicity, calcium overload, neuroinflammation, programmed cell death and others. Propofol (2,6-diisopropylphenol), a short-acting intravenous anesthetic, possesses not only sedative and hypnotic effects but also immunomodulatory and neuroprotective effects. Numerous studies have reported the protective properties of propofol during brain I/R injury. In this review, we summarize the potential protective mechanisms of propofol to provide insights for its better clinical application in alleviating cerebral I/R injury.

Diagnostic value of transcranial doppler to predict delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage : To predict delayed cerebral ischemia.

BACKGROUND: Transcranial Doppler (TCD) is a technique to assess blood flow velocity in the cerebral arteries. TCD is frequently used to monitor aneurysmal subarachnoid hemorrhage (aSAH) patients. This study compares TCD-criteria for vasospasm and its association with Delayed Cerebral Ischemia (DCI). An overall score based on flow velocities of various intracranial arteries was developed and evaluated. METHODS: A retrospective diagnostic accuracy study was conducted between 1998 and 2017 with 621 patients included. Mean flow velocity (MFV) of the cerebral artery was measured between 2-5 days and between 6-9 days after ictus. Cutoff values from the literature, new cutoff values, and a new composite score (Combined Severity Score) were used to predict DCI. Sensitivity, specificity, and area under the curve (AUC) were determined, and logistic regression analysis was performed. RESULTS: The Combined Severity Score showed an AUC 0.64 (95%CI 0.56-.71) at days 2-5, with sensitivity 0.53 and specificity 0.74. The Combined Severity Score had an adjusted Odds Ratio of 3.41 (95CI 1.86-6.32) for DCI. MCA-measurements yielded the highest AUC to detect DCI at day 2-5: AUC 0.65 (95%CI 0.58-0.73). Optimal cutoff MFV of 83 cm/s for MCA resulted in sensitivity 0.73 and specificity 0.50 at days 2-5. CONCLUSION: TCD-monitoring of aSAH patients may be a valuable strategy for DCI risk stratification. Lower cutoff values can be used in the early phase after the ictus (day 2-5) than are commonly used now. The Combined Severity Score incorporating all major cerebral arteries may provide a meaningful contribution to interpreting TCD measurements.

Effect of berberine on the hippocampal structure, biochemical factors, memory, and blood-brain barrier in rat model of transient global cerebral ischemia.

Global cerebral ischemia (GCI) results in damage to the neurons and leads to cognitive impairments. Berberine (BBR) is known for its neuroprotective qualities. This study aimed to investigate the effects of BBR on memory, Blood-brain barrier (BBB) permeability, biochemical factors, and neuronal structure. Sixty-three adult male Wistar rats were divided randomly into Sham (21), GCI (21), and GCI + BBR (21) groups. The GCI + BBR group received 50 mg/kg of BBR for 7 days before and 6 h after 20 min of GCI induction. After 24 h, assessments included hippocampal neuronal structure, catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GPX) levels, memory performance, and BBB permeability. The GCI + BBR group reduced volume loss in the CA1 and its sublayers (oriens, pyramidal, and radiatum) compared to the GCI group (p < 0.0001, p < 0.001, p < 0.01 and p < 0.001, respectively). Additionally, the GCI + BBR group showed higher pyramidal neuron density (p < 0.0001) and number (p < 0.0001) compared to the GCI group. BBR also decreased MDA levels (p < 0.0001) and increased CAT activity (p < 0.0001) in the GCI + BBR group compared to the GCI group, with GPX and SOD activity approaching Sham levels (p < 0.0001, both). BBR demonstrated significant improvements in short and long-term memory compared to the GCI group (p < 0.01, p < 0.0001, respectively). Furthermore, BBB permeability in the GCI + BBR group was significantly reduced compared to the GCI group (p < 0.0001). These findings demonstrated BBR's potential to protect the neurons in the CA1 and BBB structures, enhance antioxidant activity, and alleviate GCI-induced memory impairments.

RNA sequencing reveals the potential mechanism of exercise preconditioning for cerebral ischemia reperfusion injury in rats.

INTRODUCTION: Cerebral ischemia reperfusion injury (CIRI) often leads to deleterious complications after stroke patients receive reperfusion therapy. Exercise preconditioning (EP) has been reported to facilitate brain function recovery. We aim to explore the specific mechanism of EP in CIRI. METHODS: Sprague-Dawley rats were randomized into Sham, middle cerebral artery occlusion (MCAO), and EP groups (n = 11). The rats in the EP group received adaptive training for 3 days (10 m/min, 20 min/day, with a 0° incline) and formal training for 3 weeks (6 days/week, 25 m/min, 30 min/day, with a 0° incline). Then, rats underwent MCAO surgery to establish CIRI models. After 48 h, neurological deficits and cerebral infarction of the rats were measured. Neuronal death and apoptosis in the cerebral cortices were detected. Furthermore, RNA sequencing was conducted to investigate the specific mechanism of EP on CIRI, and qPCR and Western blotting were further applied to confirm RNA sequencing results. RESULTS: EP improved neurological deficit scores and reduced cerebral infarction in MCAO rats. Additionally, pre-ischemic exercise also alleviated neuronal death and apoptosis of the cerebral cortices in MCAO rats. Importantly, 17 differentially expressed genes (DEGs) were identified through RNA sequencing, and these DEGs were mainly enriched in the HIF-1 pathway, cellular senescence, proteoglycans in cancer, and so on. qPCR and Western blotting further confirmed that EP could suppress TIMP1, SOCS3, ANGPTL4, CDO1, and SERPINE1 expressions in MCAO rats. CONCLUSION: EP can improve CIRI in vivo, the mechanism may relate to TIMP1 expression and HIF-1 pathway, which provided novel targets for CIRI treatment.

Promotion of mature angiogenesis in ischemic stroke by Taohong Siwu decoction through glycolysis activation.

Backgrounds: Mature angiogenesis plays a critical role in improving cerebral ischemia-reperfusion injury (CIRI). Glycolysis serves as the primary energy source for brain microvascular endothelial cells (BMECs), whereas other vascular cells rely on aerobic respiration. Therefore, intercellular variations in energy metabolism could influence mature angiogenesis. Taohong Siwu Decoction (THSWD) has demonstrated efficacy in treating ischemic stroke (IS), yet its potential to promote mature angiogenesis through glycolysis activation remains unclear. Methods: In this study, we established a middle cerebral artery occlusion/reperfusion (MCAO/R) model in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. We assessed neuroprotective effects using neurobehavioral scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin-eosin (HE) staining, and Nissl staining in MCAO/R rats. Additionally, we evaluated mature angiogenesis and glycolysis levels through immunofluorescence, immunohistochemistry, and glycolysis assays. Finally, we investigated THSWD's mechanism in linking glycolysis to mature angiogenesis in OGD/R-induced BMECs. Results: In vivo experiments demonstrated that THSWD effectively mitigated cerebral damage and restored neurological function in MCAO/R rats. THSWD significantly enhanced CD31, Ang1, PDGFB, and PDGFR-ß expression levels, likely associated with improved glucose, pyruvate, and ATP levels, along with reduced lactate and lactate/pyruvate ratios. In vitro findings suggested that THSWD may boost the expression of mature angiogenesis factors (VEGFA, Ang1, and PDGFB) by activating glycolysis, increasing glucose uptake and augmenting lactate, pyruvate, and ATP content, thus accelerating mature angiogenesis. Conclusion: THSWD could alleviate CIRI by activating the glycolysis pathway to promote mature angiogenesis. Targeting the glycolysis-mediated mature angiogenesis alongside THSWD therapy holds promise for IS treatment.

Transcranial Doppler in the Detection of Cerebral Vasospasm After Subarachnoid Hemorrhage.

Background Transcranial Doppler (TCD) is a simple, noninvasive, nonionizing, portable technique but not widely practiced to detect cerebral vasospasm after subarachnoid hemorrhage (SAH). Objective The aim of this study was to assess the performance of TCD in the detection of cerebral vasospasm in patients with SAH considering CT angiography (CTA) as a gold standard. Methods and material This cross-sectional study included 50 patients with acute SAH admitted to the National Institute of Neurosciences & Hospital (NINS & H), Dhaka, Bangladesh, from February to June 2021. The neurological status, severity of SAH, and initial CT findings were recorded. All patients were screened for cerebral vasospasm with TCD on the 4th, 7th, 10th, and 14th days after the event. Screening of cerebral vasospasm by CTA was done on the 14th day of the event or earlier if TCD suggested vasospasm. Results The mean age of the participants was 51.4 ±13.4 years (mean ± SD), and females were predominant (N=29, 58%). CTA detected cerebral vasospasm in 18 (36%) participants, but TCD could detect it in only 13 (26%) cases. Among the participants who had no vasospasm by CTA, all but one were also found to have no vasospasm by TCD. The agreement between TCD and CTA in detecting cerebral vasospasm was significant (p<0.001, κ=0.726). TCD shows good specificity (96.9%) and positive predictive value (92.8%), but sensitivity (72.2%) and negative predictive value (81.6%) were comparatively lower. Overall, the diagnostic accuracy of TCD in detecting cerebral vasospasm was 88%. Conclusions Although compared to CTA, TCD is a highly specific but less sensitive tool in detecting vasospasm, TCD remains a reliable screening tool for detecting vasospasm following SAH.

Glycyrrhetinic Acid Triggers a Protective Autophagy by Inhibiting the JAK2/STAT3 Pathway in Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia/reperfusion injury (CIRI) is a common feature of ischemic stroke leading to a poor prognosis. Effective treatments targeting I/R injury are still insufficient. The study aimed to investigate the mechanisms, by which glycyrrhizic acid (18ß-GA) in ameliorates CIRI. Our results showed that 18ß-GA significantly decreased the infarct volume, neurological deficit scores, and pathological changes in the brain tissue of rats after middle cerebral artery occlusion. Western blotting showed that 18ß-GA inhibited the expression levels of phosphorylated JAK2 and phosphorylated STAT3. Meanwhile, 18ß-GA increased LC3-II protein levels in a reperfusion duration-dependent manner, which was accompanied by an increase in the Bcl-2/Bax ratio. Inhibition of 18ß-GA-induced autophagy by 3-methyladenine (3-MA) enhanced apoptotic cell death. In addition, 18ß-GA inhibited the JAK2/STAT3 pathway, which was largely activated in response to oxygen-glucose deprivation/reoxygenation. However, the JAK2/STAT3 activator colivelin TFA abolished the inhibitory effect of 18ß-GA, suppressed autophagy, and significantly decreased the Bcl-2/Bax ratio. Taken together, these findings suggested that 18ß-GA pretreatment ameliorated CIRI partly by triggering a protective autophagy via the JAK2/STAT3 pathway. Therefore might be a potential drug candidate for treating ischemic stroke.

The Effects and Mechanisms of AM1241 in Alleviating Cerebral Ischemia-Reperfusion Injury.

OBJECTIVE: Research has shown that cerebral ischemia-reperfusion injury (CIRI) involves a series of physiological and pathological mechanisms, including inflammation, oxidative stress, and cell apoptosis. The cannabinoid receptor 2 agonist AM1241 has been found to have anti-inflammatory and anti-oxidative stress effects. However, it is unclear whether AM1241 has a protective effect against brain ischemia-reperfusion injury, and its underlying mechanisms are not yet known. METHODS: In this study, we investigated the anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects of AM1241 and its mechanisms in BV2 cells stimulated with H2O2 and in a C57BL/6 mouse model of CIRI in vitro and in vivo, respectively. RESULTS: In vitro, AM1241 significantly inhibited the release of pro-inflammatory cytokines TNF-α and IL-6, reactive oxygen species (ROS), and the increase in Toll-like receptor 4/myeloid differentiation protein 2 (MD2/TLR4) complex induced by H2O2. Under H2O2 stimulation, MD2 overexpression resulted in increased levels of MD2/TLR4 complex, TNF-α, IL-6, NOX2, BAX, and Cleaved-Caspase3 (C-Caspase3), as well as the activation of the MAPK pathway and NF-κB, which were reversed by AM1241. In addition, molecular docking experiments showed that AM1241 directly interacted with MD2. Surface Plasmon Resonance (SPR) experiments further confirmed the binding of AM1241 to MD2. In vivo, AM1241 significantly attenuated neurofunctional impairment, brain edema, increased infarct volume, oxidative stress levels, and neuronal apoptosis in CIRI mice overexpressing MD2. CONCLUSION: Our study demonstrates for the first time that AM1241 alleviates mouse CIRI by inhibiting the MD2/TLR4 complex, exerting anti-inflammatory, anti-oxidative stress and anti-apoptotic effects.

Mesenchymal Stem Cell-Loaded Hydrogel Improves Surgical Treatment for Chronic Cerebral Ischemia.

Chronic cerebral ischemia (CCI) results in a prolonged insufficient blood supply to the brain tissue, leading to impaired neuronal function and subsequent impairment of cognitive and motor abilities. Our previous research showed that in mice with bilateral carotid artery stenosis, the collateral neovascularization post Encephalo-myo-synangiosis (EMS) treatment could be facilitated by bone marrow mesenchymal stem cells (MSCs) transplantation. Considering the advantages of biomaterials, we synthesized and modified a gelatin hydrogel for MSCs encapsulation. We then applied this hydrogel on the brain surface during EMS operation in rats with CCI, and evaluated its impact on cognitive performance and collateral circulation. Consequently, MSCs encapsulated in hydrogel significantly augment the therapeutic effects of EMS, potentially by promoting neovascularization, facilitating neuronal differentiation, and suppressing neuroinflammation. Furthermore, taking advantage of multi-RNA-sequencing and in silico analysis, we revealed that MSCs loaded in hydrogel regulate PDCD4 and CASP2 through the overexpression of miR-183-5p and miR-96-5p, thereby downregulating the expression of apoptosis-related proteins and inhibiting early apoptosis. In conclusion, a gelatin hydrogel to enhance the functionality of MSCs has been developed, and its combination with EMS treatment can improve the therapeutic effect in rats with CCI, suggesting its potential clinical benefit.

The cornel Iridoid glycoside attenuated brain edema of the cerebral ischemia/reperfusion rats by modulating the polarized aquaporin 4.

Brain edema after ischemic stroke could worsen cerebral injury in patients who received intravenous thrombolysis. Cornus officinalis Sieb. et Zucc., a long-established traditional Chinese medicine, is beneficial to the treatment of neurodegenerative diseases including ischemic stroke. In particular, its major component, cornel iridoid glycoside (CIG), was evidenced to exhibit neuroprotective effects against cerebral ischemic/reperfusion injury (CIR/I). Aimed to explore the effects of the CIG on brain edema of the CIR/I rats, the CIG was analyzed with the main constituents by using HPLC. The molecular docking analysis was performed between the CIG constituents and AQP4-M23. TGN-020, an AQP4 inhibitor, was used as a comparison. In the in vivo experiments, the rats were pre-treated with the CIG and were injured by performing middle cerebral artery occlusion/reperfusion (MCAO/R). After 24 h, the rats were examined for neurological function, pathological changes, brain edema, and polarized Aqp4 expressions in the brain. The HPLC analysis indicated that the CIG was composed of morroniside and loganin. The molecular docking analysis showed that both morroniside and loganin displayed lower binding energies to AQP4-M23 than TGN-020. The CIG pre-treated rats exhibited fewer neurological function deficits, minimized brain swelling, and reduced lesion volumes compared to the MCAO/R rats. In the peri-infarct and infarct regions, the CIG pre-treatment restored the polarized Aqp4 expression which was lost in the MCAO/R rats. The results suggested that the CIG could attenuate brain edema of the cerebral ischemia/reperfusion rats by modulating the polarized Aqp4 through the interaction of AQP4-M23 with morroniside and loganin.

Dystrophin 71 deficiency causes impaired aquaporin-4 polarization contributing to glymphatic dysfunction and brain edema in cerebral ischemia.

OBJECTIVE: The glymphatic system serves as a perivascular pathway that aids in clearing liquid and solute waste from the brain, thereby enhancing neurological function. Disorders in glymphatic drainage contribute to the development of vasogenic edema following cerebral ischemia, although the molecular mechanisms involved remain poorly understood. This study aims to determine whether a deficiency in dystrophin 71 (DP71) leads to aquaporin-4 (AQP4) depolarization, contributing to glymphatic dysfunction in cerebral ischemia and resulting in brain edema. METHODS: A mice model of middle cerebral artery occlusion and reperfusion was used. A fluorescence tracer was injected into the cortex and evaluated glymphatic clearance. To investigate the role of DP71 in maintaining AQP4 polarization, an adeno-associated virus with the astrocyte promoter was used to overexpress Dp71. The expression and distribution of DP71 and AQP4 were analyzed using immunoblotting, immunofluorescence, and co-immunoprecipitation techniques. The behavior ability of mice was evaluated by open field test. Open-access transcriptome sequencing data were used to analyze the functional changes of astrocytes after cerebral ischemia. MG132 was used to inhibit the ubiquitin-proteasome system. The ubiquitination of DP71 was detected by immunoblotting and co-immunoprecipitation. RESULTS: During the vasogenic edema stage following cerebral ischemia, a decline in the efflux of interstitial fluid tracer was observed. DP71 and AQP4 were co-localized and interacted with each other in the perivascular astrocyte endfeet. After cerebral ischemia, there was a notable reduction in DP71 protein expression, accompanied by AQP4 depolarization and proliferation of reactive astrocytes. Increased DP71 expression restored glymphatic drainage and reduced brain edema. AQP4 depolarization, reactive astrocyte proliferation, and the behavior of mice were improved. After cerebral ischemia, DP71 was degraded by ubiquitination, and MG132 inhibited the decrease of DP71 protein level. CONCLUSION: AQP4 depolarization after cerebral ischemia leads to glymphatic clearance disorder and aggravates cerebral edema. DP71 plays a pivotal role in regulating AQP4 polarization and consequently influences glymphatic function. Changes in DP71 expression are associated with the ubiquitin-proteasome system. This study offers a novel perspective on the pathogenesis of brain edema following cerebral ischemia.

[Soy isoflavones alleviates calcium overload in rats with cerebral ischemia-reperfusion by inhibiting the Wnt/Ca2+ signaling pathway].

OBJECTIVE: To explore the mechanism by which soybean isoflavone (SI) reduces calcium overload induced by cerebral ischemia-reperfusion (I/R). METHODS: Forty-eight SD rats were randomized into 4 groups to receive sham operation, cerebral middle artery occlusion for 2 h followed by 24 h of reperfusion (I/R model group), or injection of adeno-associated virus carrying Frizzled-2 siRNA or empty viral vector into the lateral cerebral ventricle after modeling.Western blotting was used to examine Frizzled-2 knockdown efficiency and changes in protein expressions in the Wnt/Ca2+ signaling pathway.Calcium levels and pathological changes in the ischemic penumbra (IP) were measured using calcium chromogenic assay and HE staining, respectively.Another 72 SD randomly allocated for sham operation, I/R modeling, or soy isoflavones pretreatment before modeling were examined for regional cerebral blood flow using a Doppler flowmeter, and the cerebral infarct volume was assessed using TTC staining.Pathologies in the IP area were evaluated using HE and Nissl staining, and ROS level, Ca2+ level, cell apoptosis, and intracellular calcium concentration were analyzed using immunofluorescence assay or flow cytometry; the protein expressions of Wnt5a, Frizzled-2, and P-CaMK Ⅱ in the IP were detected with Western blotting and immunohistochemistry. RESULTS: In rats with cerebral I/R, Frizzled-2 knockdown significantly lowered calcium concentration (P < 0.001) and the expression levels of Wnt5a, Frizzled-2, and P-CaMK Ⅱ in the IP area.In soy isoflavones-pretreated rats, calcium concentration, ROS and MDA levels, cell apoptosis rate, cerebral infarct volume, and expression levels of Wnt/Ca2+ signaling pathway-related proteins were all significantly lower while SOD level was higher than those in rats in I/R model group. CONCLUSION: Soy isoflavones can mitigate calcium overload in rats with cerebral I/R by inhibiting the Wnt/Ca2+ signaling pathway.

[Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes].

OBJECTIVE: To investigate the protective effect of exogenous leptin against focal cerebral ischemia-reperfusion (I/R) injury in mice and explore the underlying mechanism. METHODS: A total of 100 C57BL/6 mice were randomly divided into 5 groups, including a sham-operated group, cerebral I/R model group, and 3 leptin treatment groups with intraperitoneal injections of 0.5, 1.0 or 2.0 leptin immediately after occlusion of the internal carotid artery. At 24 h after reperfusion, neurological function scores of the mice were assessed, and TTC staining was used to determine the area of cerebral infarction. The pathological changes in the cortical brain tissue of the mice were observed using HE staining, and degenerative damage of the cortical neurons were assessed with Fluoro-Jade C staining. The expression of glial fibrillary acidic protein in cortical brain tissues was detected using immunohistochemistry and Western blotting. In another 45 C57BL/6 mice with sham operation, I/R modeling, or leptin (1 mg/kg) treatment, glutamic acid in the cortical brain tissue was detected using glutamate assay, and cortical glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) protein expressions were detected using immunohistochemistry. RESULTS: Compared with the I/R model mice, the leptin-treated mice had significantly lower neurological deficit scores, smaller cerebral infarct area, milder pathologies in the cortical brain tissue, and lessened cortical neuronal damage with normal morphology and less excessive proliferation of the astrocytes. Leptin treatment significantly up-regulated the expressions of GLT-1 and GLAST and lowered the content of glutamic acid in the brain tissue of the I/R mice. CONCLUSION: Exogenous leptin has obvious neuroprotective effect against cerebral I/R injury in mice, mediated probably by controlling excessive astrocyte proliferation and up-regulating cortical GLT-1 and GLAST expressions to reduce glutamate-mediated excitotoxic injury of the astrocytes.

Early Hydrogen-Oxygen Gas Mixture Inhalation in Patients with Aneurysmal Subarachnoid Hemorrhage (HOMA): study protocol for a randomized controlled trial.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (aSAH) is a life-threatening neurosurgical emergency with a high mortality rate. Delayed cerebral ischemia (DCI) and cerebral vasospasm (CVS) are delayed products of early brain injury (EBI), which may constitute the principal determinant of an unfavorable patient prognosis. Consequently, the mitigation of DCI and CVS assumes paramount significance in the pursuit of enhanced patient outcomes. However, except for oral nimodipine, there is no effective therapy available in the current guideline. Hence, the exigency arises to proffer novel treatment paradigms. The diversity of hydrogen therapeutic targets has been largely reported in basic research, unveiling its latent capacity to ameliorate EBI in aSAH patients. METHODS: Early Hydrogen-Oxygen Gas Mixture Inhalation in Patients with Aneurysmal Subarachnoid Hemorrhage (HOMA), a single-center, prospective, open-labeled, randomized controlled clinical trial, endeavors to evaluate the efficacy and safety of hydrogen-oxygen gas mixture inhalation therapy in aSAH patients. A cohort of 206 patients will be randomized to either hydrogen-oxygen gas mixture inhalation group (8 h per day, 3 L/min, hydrogen concentration of 67%, oxygen concentration of 33%) or oxygen inhalation group (8 h per day, 3 L/min, oxygen concentration of 33%) within 72 h after aSAH and treated for 7 days in the ICU ward. The primary outcomes are the incidence of DCI and CVS during hospitalization. DISCUSSION: The HOMA aims to evaluate the effectiveness of hydrogen-oxygen gas mixture inhalation therapy in preventing DCI or CVS and improving outcomes in aSAH patients. Notably, this is the first large-scale trial of hydrogen therapy in aSAH patients. Given that the Chinese population represents a significant portion of the global population and the increasing incidence of stroke due to aging, optimizing patient care is vital. Given the current challenges in aSAH patient outcomes, initiating more prospective clinical trials is essential. Recent research has shown hydrogen's therapeutic potential, aligning with EBI in aSAH, driving our exploration of hydrogen therapy's mechanisms in post-aneurysm rupture damage. ETHICS AND DISSEMINATION: The protocol for the HOMA study was approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University (KY 2022-020-02). All results of the present study will be published in peer-reviewed journals and presented at relevant conferences. TRIAL REGISTRATION: ClinicalTrials.gov NCT05282836. Registered on March 16, 2022.