Hypomethylated leptin receptor reduces cerebral ischaemia-reperfusion injury by activating the JAK2/STAT3 signalling pathway.

OBJECTIVE: To investigate the cerebroprotective effects of leptin in vitro and in vivo via the Janus kinase-2 (JAK2)/transcription factor signal transducer and activators of transcription-3 (STAT3) pathway and leptin receptors (LEPR). METHODS: The study used the cellular oxygen-glucose deprivation (OGD) model in PC12 cells and the middle cerebral artery occlusion (MCAO) rat model of cerebral ischaemia-reperfusion injury (CIRI) to assess changes in gene expression and protein levels following leptin pretreatment. The methylated DNA immunoprecipitation (MeDIP) assay measured DNA methylation levels. RESULTS: The optimal leptin concentration for exerting neuroprotective effects against ischaemia-reperfusion injury in PC12 cells was 200 ng/ml in vitro, but excessive leptin diminished this effect. Leptin pretreatment in the MCAO rat model demonstrated a similar effect to previously reported leptin administration post-CIRI. In addition to regulating the expression of inflammation-related cytokines, Western blot analysis showed that leptin pretreatment upregulated BCL-2 and downregulated caspase 3 levels. The MeDIP analysis demonstrated that DNA methylation regulated LEPR gene expression in the MCAO rat model when leptin pretreatment was used. CONCLUSION: Exogenous leptin might bind to extra-activated LEPR by reducing the methylation level of the LEPR gene promoter region, which leads to an increase in phosphorylated JAK2/STAT3 and apoptotic signalling pathways.

The effect of salidroside in promoting endogenous neural regeneration after cerebral ischemia/reperfusion involves notch signaling pathway and neurotrophic factors.

BACKGROUND: Salidroside is the major bioactive and pharmacological active substance in Rhodiola rosea L. It has been reported to have neuroprotective effects on cerebral ischemia/reperfusion (I/R). However, whether salidroside can enhance neural regeneration after cerebral I/R is still unknown. This study investigated the effects of salidroside on the endogenous neural regeneration after cerebral I/R and the related mechanism. METHODS: Focal cerebral I/R was induced in rats by transient middle cerebral artery occlusion/reperfusion (MCAO/R). The rats were intraperitoneally treated salidroside once daily for 7 consecutive days. Neurobehavioral assessments were performed at 3 days and 7 days after the injury. TTC staining was performed to assess cerebral infarct volume. To evaluate the survival of neurons, immunohistochemical staining of Neuronal Nuclei (NeuN) in the ischemic hemisphere were conducted. Also, immunofluorescence double or triple staining of the biomarkers of proliferating neural progenitor cells in Subventricular Zone (SVZ) and striatum of the ischemia hemisphere were performed to investigate the neurogenesis. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of neurotrophic factors (NTFs) brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Expression of Notch1 and its target molecular Hes1 were also analyzed by western-blotting and RT-PCR. RESULTS: Salidroside treatment ameliorated I/R induced neurobehavioral impairment, and reduced infarct volume. Salidroside also restored NeuN positive cells loss after I/R injury. Cerebral I/R injury significantly increased the expression of 5-Bromo-2'-Deoxyuridine (BrdU) and doublecotin (DCX), elevated the number of BrdU/Nestin/DCX triple-labeled cells in SVZ, and BrdU/Nestin/glial fibrillary acidic protein (GFAP) triple-labeled cells in striatum. Salidroside treatment further promoted the proliferation of BrdU/DCX labeled neuroblasts and BrdU/Nestin/GFAP labeled reactive astrocytes. Furthermore, salidroside elevated the mRNA expression and protein concentration of BDNF and NGF in ischemia periphery area, as well. Mechanistically, salidroside elevated Notch1/Hes1 mRNA expression in SVZ. The protein levels of them were also increased after salidroside administration. CONCLUSIONS: Salidroside enhances the endogenous neural regeneration after cerebral I/R. The mechanism of the effect may involve the regulation of BDNF/NGF and Notch signaling pathway.

[Classification of childhood arterial ischemic stroke].

The etiology of childhood arterial ischemic stroke is complex, and identifying the underlying cause is crucial for optimizing treatment and preventing recurrence. Currently, the classification methods for childhood arterial ischemic stroke are largely based on data from international studies, but a unified consensus have not yet been reached. This paper reviews the existing classification methods and their subtype definitions, and points out some doubts and ambiguities. On this basisi, combined with the data collected by Beijing Children's Hospital on Chinese children with arterial ischemic stroke, a new classification method (COIST) was proposed according to the etiology and pathogenesis, namely: inflammation (I), abnormal vascular structure (S), thrombophilia (T), heart disease (C), other identifiable causes (O), and uncertain causes; and various subtypes are listed. It is hoped that this new classification method can attract the attention and discussion of domestic colleagues, with the aim of further refinement, in order to help clinicians better understand and quickly identify the etiologies of childhood ischemic stroke.

Bioinformatics screening and verification of ischemic stroke-related key genes and drug prediction.

Stroke is one of the major causes of disability and death worldwide. The lack of effective medical treatment for stroke heightens the need for new therapeutic targets. In this study, we obtained two microarray data sets from the Gene Expression Omnibus (GEO) database and identified differential genes (DEGs) between MCAO and control groups. Then, enrichment analysis of the DEGs was performed using DAVID and Metascape. The results show 27 DEGs shared between the two datasets. The functional enrichment analysis showed that these genes are mainly enriched in immune response, complement and coagulation cascades, apoptotic processes. The four hub genes (C1qc, Fcgr2b, C1qb, and Cd14) were screened out using the Cytoscape. Next, real-time PCR and Western blot analysis showed that expression of C1q and CD14 increased at 14 days after tMCAO. Furthermore, we took eight small molecule compounds with the lowest score using Cmap and studied their background characteristics. These results are built on a meta-analysis of data, which are generally accessible from the online space. Finally, we evaluated the protective effect of the rolipram through behavior tests after tMCAO, and results showed that the rolipram significantly attenuated neurobehavioral dysfunction at 14 days after brain ischemia. The present results provide novel insights into the biological process and potential therapeutic drugs involved in stroke.

Letter to the editor: "Beyond nimodipine: advanced neuroprotection strategies for aneurysmal subarachnoid hemorrhage vasospasm and delayed cerebral ischemia".

This letter commends the article by Luzzi et al. on alternative neuroprotection strategies for aneurysmal subarachnoid hemorrhage (SAH). It highlights the pharmacological advantages of nicardipine, cilostazol, and clazosentan over nimodipine in managing cerebral vasospasm and delayed cerebral ischemia. Emphasizing the need for personalized medicine, it advocates for integrating genetic screening and advanced monitoring techniques to tailor treatments to individual patient profiles. This approach could significantly improve clinical outcomes by optimizing drug efficacy and minimizing adverse effects.

Application and Development of Cell Membrane Functionalized Biomimetic Nanoparticles in the Treatment of Acute Ischemic Stroke.

Ischemic stroke is a serious neurological disease involving multiple complex physiological processes, including vascular obstruction, brain tissue ischemia, impaired energy metabolism, cell death, impaired ion pump function, and inflammatory response. In recent years, there has been significant interest in cell membrane-functionalized biomimetic nanoparticles as a novel therapeutic approach. This review comprehensively explores the mechanisms and importance of using these nanoparticles to treat acute ischemic stroke with a special emphasis on their potential for actively targeting therapies through cell membranes. We provide an overview of the pathophysiology of ischemic stroke and present advances in the study of biomimetic nanoparticles, emphasizing their potential for drug delivery and precision-targeted therapy. This paper focuses on bio-nanoparticles encapsulated in bionic cell membranes to target ischemic stroke treatment. It highlights the mechanism of action and research progress regarding different types of cell membrane-functionalized bi-onic nanoparticles such as erythrocytes, neutrophils, platelets, exosomes, macrophages, and neural stem cells in treating ischemic stroke while emphasizing their potential to improve brain tissue's ischemic state and attenuate neurological damage and dysfunction. Through an in-depth exploration of the potential benefits provided by cell membrane-functionalized biomimetic nanoparticles to improve brain tissue's ischemic state while reducing neurological injury and dysfunction, this study also provides comprehensive research on neural stem cells' potential along with that of cell membrane-functionalized biomimetic nanoparticles to ameliorate neurological injury and dysfunction. However, it is undeniable that there are still some challenges and limitations in terms of biocompatibility, safety, and practical applications for clinical translation.

Deadly cerebral ischemia due to carotid stenosis following a facial shot with a pellet gun: An autopsy case report.

INTRODUCTION: Facial gunshot wounds have devastating functional and aesthetic consequences for the patient. If associated with penetrating craniocerebral injuries, the prognosis is rather compromised even with appropriate medical and surgical treatment. Chop-off injuries with penetrating wounds constitute a challenging situation for the facial reconstructive surgeon in facial trauma. OBSERVATION: This case involved a 49-year-old man who sustained an accidental facial shot from a pellet gun. Radiological and clinical investigations revealed complex ballistic trauma to the maxillofacial region, with projectiles reaching the base of the skull. One of the projectiles migrated via the carotid canal towards a cerebral artery, leading to obstruction of the artery with cerebral infarction. An autopsy was performed which evaluated that the shooting distance was compatible with a long distance, causing the dispersion of lead grains with the absence of a wad inside the trauma site. CONCLUSION: In some cases of facial gunshot wounds, despite a complex and extensive lesion assessment, death may occur due to a neurological complication rather than sustaining hemodynamic shock, depending on the trajectory of the projectiles.

Function and mechanism of the human SOD2 gene in mice cerebral ischemia/ reperfusion injury.

PURPOSE: To investigate the neuroprotective effects of the SOD2 gene in cerebral ischemia reperfusion injury function and the underlying mechanisms in a mice model of middle cerebral artery ischemia reperfusion. METHODS: SOD2 transgenic mice were engineered using transcription activator-like effector nucleases, and the genotype was identified using PCR after every three generations. Transgenic and C57BL/6J wild type mice were simultaneously subjected to the middle cerebral artery occlusion model. RESULTS: SOD2 expression in the brain, heart, kidney, and skeletal muscle of transgenic mice was significantly higher than that in the wild type. Following ischemia reperfusion, the infarct volume of wild type mice decreased after treatment with fenofibrate compared to the CMC group. Infarction volume in SOD2 transgenic mice after CMC and fenofibrate treatment was significantly reduced. The recovery of cerebral blood flow in wild type mice treated with fenofibrate was significantly enhanced compared with that in the CMC group. CONCLUSIONS: The expression of SOD2 in transgenic mice was significantly higher than that in wild type mice, the neuroprotective role of fenofibrate depends on an increase in SOD2 expression.

Neuroprotective effects of the combined treatment of resveratrol and urapidil in experimental cerebral ischemia-reperfusion injury in rats.

PURPOSE: To evaluate the neuroprotective effect of resveratrol, urapidil, and a combined administration of these drugs against middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (IR) injury model in rats. METHODS: Thirty-five rats were divided into five groups of seven animals each. Animals in IR, IR resveratrol (IRr), IR urapidil (IRu), and IR + combination of resveratrol and urapidil (IRc) were exposed to MCAO induced cerebral ischemia reperfusion injury model. Rats in IRr and IRu groups received 30-mg/kg resveratrol and 5-mg/kg urapidil respectively. Animals in IRc received a combined treatment of both drugs. At the end of the study, brain tissues were used for oxidative stress (malondialdehyde, glutathione, and superoxide dismutase), pro-apoptotic caspase-3, anti-apoptotic Bcl-2, and pro-inflammatory tumor necrosis factor-α cytokine level measurements. RESULTS: The MCAO model successfully replicated IR injury with significant histopathological changes, elevated tissue oxidative stress, and upregulated apoptotic and inflammatory protein expression in IR group compared to control group (p < 0.001). All parameters were significantly alleviated in IRr group compared to IR group (all p < 0.05). In IRu group, all parameters except for caspase-3 and Bcl-2 were also significantly different than IR group (all p < 0.05). The IRc group showed the biggest difference compared to IR group in all parameters (all p < 0.001). The IRc had higher superoxide dismutase and Bcl-2 levels, and lower caspase-3 levels compared to both IRr and IRu groups (all p < 0.05). Also, the IRc group had lower MDA and TNF-α levels compared to IRu group (all p < 0.05). CONCLUSIONS: The results indicate that combined treatment of resveratrol and urapidil may be a novel strategy to downregulate neurodegeneration in cerebral IR injury.

Exosomal miR-486 derived from bone marrow mesenchymal stem cells promotes angiogenesis following cerebral ischemic injury by regulating the PTEN/Akt pathway.

Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have been shown to promote angiogenesis after ischemic stroke, in which microRNAs (miRs) are believed to play an important role in exosome-mediated therapeutic effects, though the mechanism is still not clear. In this study, a series of molecular biological and cellular assays, both in vitro and in vivo, were performed to elucidate the role of exosomal miR-486 in angiogenesis following cerebral ischemic and its molecular mechanisms. Our results revealed that BMSC-Exos significantly improved neurological function and increased microvessel density in ischemic stroke rats. In vitro assays showed that BMSC-Exos promoted the proliferation, migration, and tube formation ability of oxygen-glucose deprivation/reoxygenation (OGD/R) injured rat brain microvascular endothelial cells (RBMECs). Importantly, BMSC-Exos increased the expression of miR-486 and phosphorylated protein kinase B (p-Akt) and down-regulated the protein level of phosphatase and tensin homolog (PTEN) in vivo and in vitro. Mechanistic studies demonstrated that transfection with miR-486 mimic enhanced RBMECs angiogenesis and increased p-Akt expression, while inhibited PTEN expression. On the other hand, the miR-486 inhibitor induced an opposite effect, which could be blocked by PTEN siRNA. It was thus concluded that exosomal miR-486 from BMSCs may enhance the functional recovery by promoting angiogenesis following cerebral ischemic injury, which might be related to its regulation of the PTEN/Akt pathway.

Meldonium, as a potential neuroprotective agent, promotes neuronal survival by protecting mitochondria in cerebral ischemia-reperfusion injury.

BACKGROUND: Stroke is a globally dangerous disease capable of causing irreversible neuronal damage with limited therapeutic options. Meldonium, an inhibitor of carnitine-dependent metabolism, is considered an anti-ischemic drug. However, the mechanisms through which meldonium improves ischemic injury and its potential to protect neurons remain largely unknown. METHODS: A rat model with middle cerebral artery occlusion (MCAO) was used to investigate meldonium's neuroprotective efficacy in vivo. Infarct volume, neurological deficit score, histopathology, neuronal apoptosis, motor function, morphological alteration and antioxidant capacity were explored via 2,3,5-Triphenyltetrazolium chloride staining, Longa scoring method, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, rotarod test, transmission electron microscopy and Oxidative stress index related kit. A primary rat hippocampal neuron model subjected to oxygen-glucose deprivation reperfusion was used to study meldonium's protective ability in vitro. Neuronal viability, mitochondrial membrane potential, mitochondrial morphology, respiratory function, ATP production, and its potential mechanism were assayed by MTT cell proliferation and cytotoxicity assay kit, cell-permeant MitoTracker® probes, mitochondrial stress, real-time ATP rate and western blotting. RESULTS: Meldonium markedly reduced the infarct size, improved neurological function and motor ability, and inhibited neuronal apoptosis in vivo. Meldonium enhanced the morphology, antioxidant capacity, and ATP production of mitochondria and inhibited the opening of the mitochondrial permeability transition pore in the cerebral cortex and hippocampus during cerebral ischemia-reperfusion injury (CIRI) in rats. Additionally, meldonium improved the damaged fusion process and respiratory function of neuronal mitochondria in vitro. Further investigation revealed that meldonium activated the Akt/GSK-3ß signaling pathway to inhibit mitochondria-dependent neuronal apoptosis. CONCLUSION: Our study demonstrated that meldonium shows a neuroprotective function during CIRI by preserving the mitochondrial function, thus prevented neurons from apoptosis.

A neural cell automated analysis system based on pathological specimens in a gerbil brain ischemia model.

PURPOSE: Amid rising health awareness, natural products which has milder effects than medical drugs are becoming popular. However, only few systems can quantitatively assess their impact on living organisms. Therefore, we developed a deep-learning system to automate the counting of cells in a gerbil model, aiming to assess a natural product's effectiveness against ischemia. METHODS: The image acquired from paraffin blocks containing gerbil brains was analyzed by a deep-learning model (fine-tuned Detectron2). RESULTS: The counting system achieved a 79%-positive predictive value and 85%-sensitivity when visual judgment by an expert was used as ground truth. CONCLUSIONS: Our system evaluated hydrogen water's potential against ischemia and found it potentially useful, which is consistent with expert assessment. Due to natural product's milder effects, large data sets are needed for evaluation, making manual measurement labor-intensive. Hence, our system offers a promising new approach for evaluating natural products.

Bruton's tyrosine kinase inhibition ameliorated neuroinflammation during chronic white matter ischemia.

Chronic cerebral hypoperfusion (CCH), a disease afflicting numerous individuals worldwide, is a primary cause of cognitive deficits, the pathogenesis of which remains poorly understood. Bruton's tyrosine kinase inhibition (BTKi) is considered a promising strategy to regulate inflammatory responses within the brain, a crucial process that is assumed to drive ischemic demyelination progression. However, the potential role of BTKi in CCH has not been investigated so far. In the present study, we elucidated potential therapeutic roles of BTK in both in vitro hypoxia and in vivo ischemic demyelination model. We found that cerebral hypoperfusion induced white matter injury, cognitive impairments, microglial BTK activation, along with a series of microglia responses associated with inflammation, oxidative stress, mitochondrial dysfunction, and ferroptosis. Tolebrutinib treatment suppressed both the activation of microglia and microglial BTK expression. Meanwhile, microglia-related inflammation and ferroptosis processes were attenuated evidently, contributing to lower levels of disease severity. Taken together, BTKi ameliorated white matter injury and cognitive impairments induced by CCH, possibly via skewing microglia polarization towards anti-inflammatory and homeostatic phenotypes, as well as decreasing microglial oxidative stress damage and ferroptosis, which exhibits promising therapeutic potential in chronic cerebral hypoperfusion-induced demyelination.

E3 ubiquitin ligase MARCH1 reduces inflammation and pyroptosis in cerebral ischemia-reperfusion injury via PCSK9 downregulation.

Pyroptosis has been regarded as caspase-1-mediated monocyte death that induces inflammation, showing a critical and detrimental role in the development of cerebral ischemia-reperfusion injury (IRI). MARCH1 is an E3 ubiquitin ligase that exerts potential anti-inflammatory functions. Therefore, the study probed into the significance of MARCH1 in inflammation and pyroptosis elicited by cerebral IRI. Middle cerebral artery occlusion/reperfusion (MCAO/R)-treated mice and oxygen glucose deprivation/reoxygenation (OGD/R)-treated hippocampal neurons were established to simulate cerebral IRI in vivo and in vitro. MARCH1 and PCSK9 expression was tested in MCAO/R-operated mice, and their interaction was identified by means of the cycloheximide assay and co-immunoprecipitation. The functional roles of MARCH1 and PCSK9 in cerebral IRI were subsequently determined by examining the neurological function, brain tissue changes, neuronal viability, inflammation, and pyroptosis through ectopic expression and knockdown experiments. PCSK9 expression was increased in the brain tissues of MCAO/R mice, while PCSK9 knockdown reduced brain damage and neurological deficits. Additionally, inflammation and pyroptosis were inhibited in OGD/R-exposed hippocampal neurons upon PCSK9 knockdown, accompanied by LDLR upregulation and NLRP3 inflammasome inactivation. Mechanistic experiments revealed that MARCH1 mediated ubiquitination and degradation of PCSK9, lowering PCSK9 protein expression. Furthermore, it was demonstrated that MARCH1 suppressed inflammation and pyroptosis after cerebral IRI by downregulating PCSK9 both in vivo and in vitro. Taken together, the present study demonstrate the protective effect of MARCH1 against cerebral IRI through PCSK9 downregulation, which might contribute to the discovery of new therapies for improving cerebral IRI.

A predictive score for atrial fibrillation in poststroke patients.

BACKGROUND: Atrial fibrillation (AF) is a risk factor for cerebral ischemia. Identifying the presence of AF, especially in paroxysmal cases, may take time and lacks clear support in the literature regarding the optimal investigative approach; in resource-limited settings, identifying a higher-risk group for AF can assist in planning further investigation. OBJECTIVE: To develop a scoring tool to predict the risk of incident AF in the poststroke follow-up. METHODS: A retrospective longitudinal study with data collected from electronic medical records of patients hospitalized and followed up for cerebral ischemia from 2014 to 2021 at a tertiary stroke center. Demographic, clinical, laboratory, electrocardiogram, and echocardiogram data, as well as neuroimaging data, were collected. Stepwise logistic regression was employed to identify associated variables. A score with integer numbers was created based on beta coefficients. Calibration and validation were performed to evaluate accuracy. RESULTS: We included 872 patients in the final analysis. The score was created with left atrial diameter ≥ 42 mm (2 points), age ≥ 70 years (1 point), presence of septal aneurysm (2 points), and score ≥ 6 points at admission on the National Institutes of Health Stroke Scale (NIHSS; 1 point). The score ranges from 0 to 6. Patients with a score ≥ 2 points had a fivefold increased risk of having AF detected in the follow-up. The area under the curve (AUC) was of 0.77 (0.72-0.85). CONCLUSION: We were able structure an accurate risk score tool for incident AF, which could be validated in multicenter samples in future studies.

ANTECEDENTES: Fibrilação atrial (FA) é um fator de risco para isquemia cerebral. Identificar a presença de FA, especialmente em casos paroxísticos, pode demandar tempo, e não há fundamentos claros na literatura quanto ao melhor método de proceder à investigação; em locais de parcos recursos, identificar um grupo de mais alto risco de FA pode auxiliar no planejamento da investigação complementar. OBJETIVO: Desenvolver uma ferramenta de escore para prever o risco de FA no acompanhamento após acidente vascular cerebral (AVC). MéTODOS: Estudo longitudinal retrospectivo, com dados coletados dos prontuários eletrônicos de pacientes hospitalizados e acompanhados ambulatorialmente por isquemia cerebral, de 2014 a 2021, em um centro de AVC terciário. Foram coleados dados demográficos, clínicos, laboratoriais, de eletrocardiograma e ecocardiograma, além de dados de neuroimagem. Mediante uma regressão logística por stepwise, foram identificadas variáveis associadas. Um escore com números inteiros foi criado com base nos coeficientes beta. Calibração e validação foram realizadas para avaliar a precisão. RESULTADOS: Foram incluídos 872 pacientes na análise final. O escore foi criado com diâmetro de átrio esquerdo ≥ 42 mm (2 pontos), idade ≥ 70 anos (1 ponto), presença de aneurisma septal (2 pontos) e pontuação à admissão ≥ 6 na escala de AVC dos National Institutes of Health (National Institutes of Health Stroke Scale, NIHSS, em inglês; 1 ponto). O escore tem pontuação que varia de 0 a 6. Pacientes com escore ≥ 2 pontos tiveram cinco vezes mais risco de terem FA detectada no acompanhamento. A área sob a curva (area under curve, AUC, em inglês) foi de 0.77 (0.72­0.85). CONCLUSãO: Pudemos estruturar uma ferramenta precisa de escore de risco de FA, a qual poderá ser validada em amostras multicêntricas em estudos futuros.