Cerebral Ischemia Research Articles

Effects of Anesthesia with Pentobarbital/Ketamine on Mitochondrial Permeability Transition Pore Opening and Ischemic Brain Damage.

The alteration of mitochondrial functions, especially the opening of the mitochondrial permeability transition pore (mPTP), has been proposed as a key mechanism in the development of lesions in cerebral ischemia, wherefore it is considered as an important target for drugs against ischemic injury. In this study, we aimed to investigate the effects of mitochondrial complex I inhibitors as possible regulators of mPTP using an in vitro brain ischemia model of the pentobarbital/ketamine (PBK)-anesthetized rats. We found that PBK anesthesia itself delayed Ca2+-induced mPTP opening and partially recovered the respiratory functions of mitochondria, isolated from rat brain cortex and cerebellum. In addition, PBK reduced cell death in rat brain slices of cerebral cortex and cerebellum. PBK inhibited the adenosine diphosphate (ADP)-stimulated respiration of isolated cortical and cerebellar mitochondria respiring with complex I-dependent substrates pyruvate and malate. Moreover, pentobarbital alone directly increased the resistance of isolated cortex mitochondria to Ca2+-induced activation of mPTP and inhibited complex I-dependent respiration and mitochondrial complex I activity. In contrast, ketamine had no direct effect on functions of isolated normal cortex and cerebellum mitochondria. Altogether, this suggests that modulation of mitochondrial complex I activity by pentobarbital during PBK anesthesia may increase the resistance of mitochondria to mPTP opening, which is considered the key event in brain cell necrosis during ischemia.

Botch improves cognitive impairment after cerebral ischemia associated with microglia-induced A1-type astrocyte activation

Vascular cognitive impairment (VCI) is a clinical syndrome that arises from cerebrovascular issues and associated risk factors, resulting in difficulties in at least one area of cognitive function. VCI has emerged as the second most prevalent type of dementia following Alzheimer's disease, yet there is no effective clinical treatment. Botch, an endogenous Notch1 antagonist, demonstrates neuroprotective effects by inhibiting neuroinflammatory responses mediated through the Notch pathway. While its role in stroke-induced neuroinflammation is well-established, its involvement in VCI remains largely unexplored. This study investigates the role and potential mechanisms of Botch in a rat model of cognitive impairment caused by bilateral common carotid artery occlusion (BCCAO). Firstly, we observed that Botch levels were down-regulated in BCCAO rats, which correlated with increased release of inflammatory cytokines and neuronal damage. Microglia in BCCAO rats released interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and complement component 1q (C1q), leading to the activation of neurotoxic C3+ A1 reactive astrocytes. Then, the down-regulation of Botch exacerbated microglia-mediated inflammation, activated C3+ A1 astrocytes, worsened neuronal damage, and led to a decline in cognitive function. Conversely, the re-expression of Botch alleviated C3+ astrocyte activation, inhibited neuronal damage, and improved mental function. In conclusion, Botch plays a crucial role in inhibiting neuroinflammation induced by type A1 reactive astrocytes. It achieves this by blocking the activation of microglia triggered by the Notch pathway. Ultimately, it inhibits neuronal damage to play a neuroprotective role. These findings suggest that Botch may represent a novel potential target for treating VCI.

Tetrahydroxy Stilbene Glucoside Promotes Mitophagy and Ameliorates Neuronal Injury after Cerebral Ischemia Reperfusion via Promoting USP10-Mediated YBX1 Stability.

Tetrahydroxy stilbene glucoside (TSG) from Polygonum multiflorum exerts neuroprotective effects after ischemic stroke. We explored whether TSG improved ischemic stroke injury via PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy. Oxygen glucose deprivation/reoxygenation (OGD/R) in vitro model and middle cerebral artery occlusion (MCAO) rat model were established. Cerebral injury was assessed by neurological score, hematoxylin and eosin staining, 2,3,5-triphenyltetrazolium chloride staining, and brain water content. Apoptosis, cell viability, and mitochondrial membrane potential were assessed by flow cytometry, cell counting kit-8, and JC-1 staining, respectively. Colocalization of LC3-labeled autophagosomes with lysosome-associated membrane glycoprotein 2-labeled lysosomes or translocase of outer mitochondrial membrane 20-labeled mitochondria was observed with fluorescence microscopy. The ubiquitination level was determined using ubiquitination assay. The interaction between molecules was validated by coimmunoprecipitation and glutathione S-transferase pull-down. We found that TSG promoted mitophagy and improved cerebral ischemia/reperfusion damage in MCAO rats. In OGD/R-subjected neurons, TSG promoted mitophagy, repressed neuronal apoptosis, upregulated Y-box binding protein-1 (YBX1), and activated PINK1/Parkin signaling. TSG upregulated ubiquitin-specific peptidase 10 (USP10) to elevate YBX1 protein. Furthermore, USP10 inhibited ubiquitination-dependent YBX1 degradation. USP10 overexpression activated PINK1/Parkin signaling and promoted mitophagy, which were reversed by YBX1 knockdown. Moreover, TSG upregulated USP10 to promote mitophagy and inhibited neuronal apoptosis. Collectively, TSG facilitated PINK1/Parkin pathway-mediated mitophagy by upregulating USP10/YBX1 axis to ameliorate ischemic stroke.

PEP-1-PIN1 Promotes Hippocampal Neuronal Cell Survival by Inhibiting Cellular ROS and MAPK Phosphorylation.

Background: The peptidyl-prolyl isomerase (PIN1) plays a vital role in cellular processes, including intracellular signaling and apoptosis. While oxidative stress is considered one of the primary mechanisms of pathogenesis in brain ischemic injury, the precise function of PIN1 in this disease remains to be elucidated. Objective: We constructed a cell-permeable PEP-1-PIN1 fusion protein and investigated PIN1's function in HT-22 hippocampal cells as well as in a brain ischemic injury gerbil model. Methods: Transduction of PEP-1-PIN1 into HT-22 cells and signaling pathways were determined by Western blot analysis. Intracellular reactive oxygen species (ROS) production and DNA damage was confirmed by DCF-DA and TUNEL staining. Cell viability was determined by MTT assay. Protective effects of PEP-1-PIN1 against ischemic injury were examined using immunohistochemistry. Results: PEP-1-PIN1, when transduced into HT-22 hippocampal cells, inhibited cell death in H2O2-treated cells and markedly reduced DNA fragmentation and ROS production. This fusion protein also reduced phosphorylation of mitogen-activated protein kinase (MAPK) and modulated expression levels of apoptosis-signaling proteins in HT-22 cells. Furthermore, PEP-1-PIN1 was distributed in gerbil hippocampus neuronal cells after passing through the blood-brain barrier (BBB) and significantly protected against neuronal cell death and also decreased activation of microglia and astrocytes in an ischemic injury gerbil model. Conclusions: These results indicate that PEP-1-PIN1 can inhibit ischemic brain injury by reducing cellular ROS levels and regulating MAPK and apoptosis-signaling pathways, suggesting that PIN1 plays a protective role in H2O2-treated HT-22 cells and ischemic injury gerbil model.

Ischemia-induced endogenous Nrf2/HO-1 axis activation modulates microglial polarization and restrains ischemic brain injury.

Cerebral ischemic stroke accounts for more than 80% of all stroke cases. During cerebral ischemia, reactive oxygen species produced in the ischemic brain induce oxidative stress and inflammatory responses. Nrf2 is a transcription factor responsible for regulating cellular redox balance through the induction of protective antioxidant and phase II detoxification responses. Although the induction of endogenous Nrf2/HO-1 axis activation has been observed in the ischemic brain, whether ischemia-induced endogenous Nrf2/HO-1 axis activation plays a role in modulating microglia (MG) phenotypes and restraining ischemic brain injury is not characterized and requires further exploration. To investigate that, we generated mice with Nrf2 knockdown specifically in MG to rigorously assess the role of endogenous Nrf2 activation in ischemic brain injury after stroke. Our results showed that MG-specific Nrf2 knockdown exacerbated ischemic brain injury after stroke. We found that Nrf2 knockdown altered MG phenotypes after stroke, in which increased frequency of inflammatory MG and decreased frequency of anti-inflammatory MG were detected in the ischemic brain. Moreover, we identified attenuated Nrf2/HO-1 axis activation led to increased CD68/IL-1β and suppressed CD206 expression in MG, resulting in aggravated inflammatory MG in MG-specific Nrf2 knockdown mice after stroke. Intriguingly, using type II diabetic preclinical models, we revealed that diabetic mice exhibited attenuated Nrf2/HO-1 axis activation in MG and exacerbated ischemic brain injury after stroke that phenocopy mice with MG-specific Nrf2 knockdown. Finally, the induction of exogenous Nrf2/HO-1 axis activation in MG through pharmacological approaches ameliorated ischemic brain injury in diabetic mice. In conclusion, our findings provide cellular and molecular insights demonstrating ischemia-induced endogenous Nrf2/HO-1 axis activation modulates MG phenotypes and restrains ischemic brain injury. These results further strengthen the therapeutic potential of targeting Nrf2/HO-1 axis in MG for the treatment of ischemic stroke and diabetic stroke.

Feasibility and radial artery occlusion rate of sheathless distal transradial access using balloon guide catheters.

Distal transradial access (dTRA), performed through an anatomical snuffbox, minimizes post-procedural burdens of endovascular treatments. However, despite the benefits of balloon-guide catheters (BGCs), their use in dTRA is limited by their small radial artery diameter. Herein, we evaluated the feasibility and radial artery occlusion (RAO) rate of 8Fr BGCs used in sheathless dTRA. This retrospective study reviewed patients treated with sheathless dTRA using an 8Fr Optimo at a single center between July 2023 and May 2024. dTRA procedures were performed under general anesthesia in patients not requiring urgent treatment. The RAO was assessed using ultrasonography 24h after the procedure. The demographic and procedural characteristics were compared between the radial artery patency and occlusion groups. Of 170 patients, 50 underwent dTRA, and 43 (86%) completed the procedure. RAO occurred in 12/43 (28%) patients with dTRA. Univariate and receiver operating characteristic curve analyses demonstrated that the median radial artery diameter was significantly smaller in the RAO group (P < 0.001), with an optimal cut-off value of 2.4mm to predict RAO. Complications included minor cerebral ischemia in two patients, but no severe ischemia was observed. Sheathless dTRA using an 8Fr Optimo BGC is feasible, but the risk of RAO should be noted, particularly in patients with small radial artery diameters. This study suggests a radial artery diameter cutoff value of 2.4mm to predict RAO, aiding access decisions for large-bore BGC. Further multicenter prospective studies are warranted to confirm these findings and assess long-term outcomes.

Nose-to-brain delivery of stem cells in stroke: the role of extracellular vesicles.

Stem cell transplantation offers a promising therapy that can be administered days, weeks, or months after a stroke. We recognize 2 major mitigating factors that remain unresolved in cell therapy for stroke, notably: (1) well-defined donor stem cells and (2) mechanism of action. To this end, we advance the use of ProtheraCytes, a population of non-adherent CD34+ cells derived from human peripheral blood and umbilical cord blood, which have been processed under good manufacturing practice, with testing completed in a phase 2 clinical trial in post-acute myocardial infarction (NCT02669810). We also reveal a novel mechanism whereby ProtheraCytes secrete growth factors and extracellular vesicles (EVs) that are associated with angiogenesis and vasculogenesis. Our recent data revealed that intranasal transplantation of ProtheraCytes at 3 days after experimentally induced stroke in adult rats reduced stroke-induced behavioral deficits and histological damage up to 28 days post-stroke. Moreover, we detected upregulation of human CD63+ EVs in the ischemic brains of stroke animals that were transplanted with ProtheraCytes, which correlated with increased levels of DCX-labeled neurogenesis and VEGFR1-associated angiogenesis and vasculogenesis, as well as reduced Iba1-marked inflammation. Altogether, these findings overcome key laboratory-to-clinic translational hurdles, namely the identification of well-characterized, clinical grade ProtheraCytes and the elucidation of a potential CD63+ EV-mediated regenerative mechanism of action. We envision that additional translational studies will guide the development of clinical trials for intranasal ProtheraCytes allografts in stroke patients, with CD63 serving as a critical biomarker.

Reactive Oxygen Species-Responsive Chitosan-Bilirubin Nanoparticles Loaded with Statin for Treatment of Cerebral Ischemia.

Cerebral ischemia impairs blood circulation, leading to elevated reactive oxygen species (ROS) production. A ROS-responsive delivery of drugs can enhance the therapeutic efficacy and minimize the side effects. There is insufficient evidence on the impact of ROS-responsive nanoparticles on ischemic stroke. We developed ROS-responsive chitosan-bilirubin (ChiBil) nanoparticles to target acute ischemic lesions and investigated the effect of atorvastatin-loaded ROS-responsive ChiBil. We randomly assigned rats with transient middle cerebral artery occlusion (MCAO) to 4 groups: saline, Statin, ChiBil, and ChiBil-Statin. These groups were treated daily via the tail vein for 7 d. Behavioral assessment, magnetic resonance (MR) imaging, evaluation of neuroinflammation, blood-brain barrier (BBB) integrity, apoptosis, and neurogenesis after stroke were conducted. Invitro, results showed nanoparticle uptake and reduced intracellular ROS, lipid peroxidation, and inflammatory cytokines (IL-6 and TNF-α). Invivo, results showed improved motor deficits and decreased infarct volumes on MR images in the ChiBil-Statin group compared with the Control group on day 7 (P < 0.05). Furthermore, the expression of inflammatory cytokines such as IL-1β and IL-6 was reduced in the ChiBil-Statin group compared with the Control group (P < 0.05). Improvements in BBB integrity, apoptosis, and neurogenesis were observed in the ChiBil-Statin group. The findings demonstrated that intravenous ROS-responsive multifunctional ChiBil-Statin could effectively deliver drugs to the ischemic brain, exerting marked synergistic pleiotropic neuroprotective effects. Therefore, ChiBil-Statin holds promise as a targeted therapy for ischemic vascular diseases characterized by increased ROS production, leading to new avenues for future research and potential clinical applications.

IL-17A-producing γδ T cells: A novel target in stroke immunotherapy.

The activation of the immune system is crucial for the fate of the ischemic brain tissue and neurological outcome in experimental stroke. Rapidly after stroke γδ (γδ17), T cells release IL-17A in the ischemic brain and thereby amplify the early detrimental immune response. Notably, IL-17A levels in γδ17 T cells are modulated by the intestinal microbiota which is, in turn, shaped by the diet. Importantly, besides their proinflammatory effects, meningeal γδ17 T cells have been recently implicated in regulating neuronal signaling, behavior, and cognition under homeostatic and pathological conditions at the brain-meningeal interface. Against this background, we propose that a dietary intervention represents a promising treatment option to improve poststroke outcomes by the modulation of the microbiota composition and IL-17A levels in γδ T cells.

Berberine alleviates cerebellar damage in global cerebral ischemia: A comprehensive study with stereological analysis, evaluation of the antioxidant response, and locomotor function in rat models

AbstractBackgroundGlobal cerebral ischemia (GCI) leads to significant oxidative damage in the cerebellum, mainly affecting Purkinje cells. This study aimed to investigate the neuroprotective effects of berberine chloride (BBR), a compound known for its antioxidant properties against GCI.Materials and methodsFourty‐two adult male Wistar rats were divided into three groups: Sham, GCI, and GCI + BBR. Rats received BBR (50 mg/kg) seven days before and six hours after inducing GCI via bilateral common carotid artery occlusion for 20 min. They were assessed for locomotor activity by the open‐field test, the cerebellar biochemical factors malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), cerebellum volume and Purkinje neuron count by stereological analysis.ResultsThe BBR treatment reduced the concentration of MDA and increased the activity of antioxidant enzymes SOD, CAT and GPx in the GCI + BBR group compared to the GCI group. Stereological analysis revealed higher Purkinje cell count, cerebellum, white matter, and gray matter volume in the GCI + BBR group compared to the GCI group. Furthermore, GCI + BBR showed enhanced locomotor function compared to the GCI group.ConclusionBBR showed therapeutic benefits and improved locomotor function. It demonstrated antioxidative effects by lowering MDA levels, boosting enzymatic activities, and significantly mitigating Purkinje cell death and cerebellar volume loss.

RiboTag RNA Sequencing Identifies Local Translation of HSP70 In Astrocyte Endfeet After Cerebral Ischemia.

Brain ischemia causes disruption in cerebral blood flow and blood-brain barrier (BBB) integrity which are normally maintained by the astrocyte endfeet. Emerging evidence points to dysregulation of the astrocyte translatome during ischemia, but its effects on the endfoot translatome are unknown. In this study, we aimed to investigate the early effects of ischemia on the astrocyte endfoot translatome in a rodent model of cerebral ischemia-reperfusion. To do so, we immunoprecipitated astrocyte-specific tagged ribosomes (RiboTag IP) from mechanically isolated brain microvessels. In mice subjected to middle cerebral artery occlusion and reperfusion and contralateral controls, we sequenced ribosome-bound RNAs from perivascular astrocyte endfeet and identified 205 genes that were differentially expressed in the translatome after ischemia. Pathways associated with the differential expressions included proteostasis, inflammation, cell cycle, and metabolism. Transcription factors whose targets were enriched amongst upregulated translating genes included HSF1, the master regulator of the heat shock response. The most highly upregulated genes in the translatome were HSF1-dependent Hspa1a and Hspa1b , which encode the inducible HSP70. We found that HSP70 is upregulated in astrocyte endfeet after ischemia, coinciding with an increase in ubiquitination across the proteome. These findings suggest a robust proteostasis response to proteotoxic stress in the endfoot translatome after ischemia. Modulating proteostasis in endfeet may be a strategy to preserve endfeet function and BBB integrity after ischemic stroke.

A stroke organoids-multiomics platform to study injury mechanism and drug response

Stroke is one of the top causes of death and disability worldwide, and its pathogenesis and mechanism have not been fully elucidated. Several agents have shown protective effects against stroke in animal models; however, few studies have shown obvious effects in clinical practice. This might be due to differences in brain structure and physiological function between humans and rodents. In this study, we established a hypoxic stroke model in human-induced pluripotent stem cell (hiPSC)-derived brain organoids to simulate the hypoxic stroke caused by ischemia. Then, by combining proteomics, single-cell transcriptome analysis, and histopathological analysis, a significant increase of three types of astrocytes was identified and they showed different responses to hypoxic environments; also the main type of astrocyte that cause brain tissue hyperplasia in ischemia brains was identified. In addition, the cortical excitatory neurons had signs of apoptosis and aging after hypoxia both in vivo and in vitro. Most importantly, we identified a possible role of a traditional Chinese medicine formula called DengZhanShengMai capsule in ischemic and hypoxic stroke treatment through regulation of lipid metabolism related biological functions. These results indicate that the combination of brain organoids and multiomics method is helpful for developing a new strategy to direct study stroke, and could provide a promising platform for drug screening of stroke in the future.

The ABCs of Subarachnoid Hemorrhage Blood Volume Measurement: A Simplified Quantitative Method Predicts Outcomes and Delayed Cerebral Ischemia.

We developed a simplified ABC/2-derived method to estimate total subarachnoid hemorrhage volume (SAHV) on noncontrast computed tomography in patients with aneurysmal SAH and compared the clinical and radiographic outcomes. In this retrospective observational cohort study, we analyzed 277 patients with SAH admitted to our Comprehensive Stroke Center between 2012 and 2022. We derived a mathematical model (model 1) by measuring SAH basal cisternal blood volume using an ABC/2-derived ellipsoid formula (A=width/thickness, B=length, C=vertical extension) on head noncontrast computed tomography in 5 major SAH cisternal compartments. We compared model 1 against a manual segmentation method (model 2) on noncontrast computed tomography. Data were analyzed using logistic regression analysis, t test, receiver operator characteristic curves, and area under the curve analysis. There was no significant difference in cisternal SAHV analysis between the 2 models (P=0.14). Mean SAHV by the simplified method was 7.0 mL (95% CI, 5.89-8.09) for good outcome and 16.6 mL (95% CI, 13.49-19.77) for poor outcome. Patients with delayed cerebral ischemia had higher SAHV, with a cutoff value of 10 mL. Our simplified ABC/2-derived method to estimate SAHV is comparable to manual segmentation and can be performed in low-resource settings. Higher total SAHV was associated with worse outcomes and higher risk of delayed cerebral ischemia. A potential dose-response relationship was observed, with SAHV >10 mL predicting worse outcomes and higher risk of DCI.

Improved Door-to-Needle Time After Implementation of Tenecteplase as the Preferred Thrombolytic for Acute Ischemic Stroke at a Large Community Teaching Hospital Emergency Department

Background: Acute ischemic stroke is a leading cause of death and long-term disability. To improve patient outcomes, timely restoration of blood flow to the ischemic brain tissue is vital. One reperfusion strategy includes the administration of thrombolytics. Historically, alteplase has been the thrombolytic of choice for acute ischemic stroke; however, given recent safety and efficacy data, tenecteplase has gained popularity due to its optimal pharmacokinetic profile. Objectives: This study compares outcomes between adult patients with acute ischemic stroke who received tenecteplase as the preferred thrombolytic versus alteplase. Methods: This was a single center, retrospective cohort study that included adult patients who received intravenous thrombolytic therapy, either tenecteplase 0.25 mg/kg or alteplase 0.9 mg/kg, for acute ischemic stroke from May 2021 to December 2023. The primary outcome was door-to-needle time. Secondary safety outcomes included the incidence of symptomatic intracerebral hemorrhage (ICH), any ICH on 24-hour follow-up imaging, major extracranial bleeding, and angioedema. Secondary efficacy outcomes included discharge with a favorable neurological outcome, discharge disposition, ICU length of stay, and overall length of stay. Secondary stroke metric times evaluated include door-to-computed tomography (CT) time, CT-to-needle time, neurologist notification-to-needle time, and thrombolytic decision-to-needle time. Results: Fifty patients were included in the alteplase group and 50 patients were included in the tenecteplase group. The primary outcome, door-to-needle time, was significantly shorter in the tenecteplase group (36 vs 30 minutes, P = .006). There were no statistically significant differences found in the secondary safety and efficacy outcomes. Patients who received tenecteplase experienced significantly faster CT-to-needle times (17 vs 11 minutes, P = .006), neurologist notification-to-needle times (32 vs 25 minutes, P = .001), and thrombolytic decision-to-needle times (9 vs 5 minutes, P &lt; .001). Conclusions: In this retrospective, observational study, there was a statistically significant decrease in door-to-needle time with tenecteplase compared to alteplase. No significant differences in secondary safety and efficacy outcomes were observed.

Oxygen Extraction Fraction Mapping on Admission Magnetic Resonance Imaging May Predict Recovery of Hyperacute Ischemic Brain Lesions After Successful Thrombectomy: A Retrospective Observational Study.

In acute stroke, diffusion-weighted imaging (DWI) is used to assess the ischemic core. Dynamic-susceptibility contrast perfusion magnetic resonance imaging allows an estimation of the oxygen extraction fraction (OEF), but the outcome of DWI lesions with increased OEF postrecanalization is unclear. This study investigated the impact of OEF on the fate of DWI lesions in patients achieving recanalization after thrombectomy. This was a retrospective analysis of the HIBISCUS-STROKE cohort (Cohort of Patients to Identify Biological and Imaging Markers of Cardiovascular Outcomes in Stroke; NCT: 03149705), a single-center observational study that prospectively enrolled patients who underwent magnetic resonance imaging triage for thrombectomy and a day-6 T2-fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging. Automated postprocessing of admission dynamic-susceptibility contrast perfusion magnetic resonance imaging generated OEF maps. At visual analysis, the OEF status within DWI lesions was assessed in comparison to the contralateral side and correlated with volume changes (difference of ischemic lesion between admission DWI and registered day-6 T2-FLAIR). At voxel-based analysis, recovered DWI regions (lesions present on the admission DWI but absent on the registered day-6 T2-FLAIR) and nonrecovered regions were segmented to extract semiquantitative OEF values. Of the participants enrolled from 2016 to 2022, 134 of 321 (41.7%) were included (median age, 71.0 years; 58.2% male; median baseline National Institutes of Health Scale score, 15.0). At visual analysis, 46 of 134 (34.3%) patients had increased OEF within DWI lesions. These patients were more likely to show a reduction in ischemic lesion volumes compared with those without increased OEF (median change, -4.0 versus 4.8 mL; P<0.0001). Multivariable analysis indicated that increased OEF within DWI lesions was associated with a reduction in ischemic lesion volumes from admission DWI to day-6 T2-FLAIR (odds ratio, 0.68 [95% CI, 0.49-0.87]; P=0.008). At voxel-based analysis, recovered DWI regions had increased OEF, while nonrecovered regions had decreased OEF (median, 126.9% versus -27.0%; P<0.0001). Increased OEF within hyperacute DWI lesions was associated with ischemic lesion recovery between admission DWI and day-6 T2-FLAIR in patients achieving recanalization after thrombectomy. URL: https://www.clinicaltrials.gov; Unique identifier: NCT03149705.

Different expression patterns of inflammation-related genes and serum microRNAs in young-onset ischemic stroke

Brain ischemia results in the activation of a cascade of inflammatory responses, contributing to the pathogenesis of stroke. This study aimed to assess the patterns of possible changes in the expression of specific inflammation-associated protein-encoding genes and miRNAs in the peripheral blood between the acute and chronic phase of young-onset cryptogenic (Cryp) and large-artery atherosclerotic (LAA) stroke. Blood and serum were collected from patients with cryptogenic and large-artery atherosclerotic stroke at the stroke onset and 1-year follow-up. The relative expression of inflammation-related genes was analysed at the mRNA and miRNA levels using real-time quantitative PCR. Moreover, the relationship between the relative gene expression levels and clinical data was assessed using several different statistical approaches. Seventy-three patients were included in this study, with a median age of 47 (IQR, 9) years. Approximately 72% were men. In patients with cryptogenic stroke, at the mRNA level, ICAM1, CXCL8, TNF, NFKBIA, PYCARD, IL1B, and IL18 were observed to be upregulated at the stroke onset compared to the 1-year follow-up. In patients with LAA stroke, only the expression of NFKBIA was significantly higher during acute stroke. Further, the miRNA serum levels of miR-21, miR-122, and miR-155 were higher at the onset of stroke in patients with cryptogenic stroke but not in those with LAA stroke. The differences between the relative gene expression levels during acute stroke and at the 1-year follow-up were more pronounced in patients with cryptogenic stroke with no cardiovascular risk factors. The expression changes of inflammatory genes in whole blood and miRNAs in the serum differ in patients with cryptogenic and LAA stroke.

Edaravone Dexborneol provides neuroprotective effect by inhibiting neurotoxic activation of astrocytes through inhibiting NF-κB signaling in cortical ischemia

Edaravone Dexborneol (EDB), comprised of edaravone and (+)- bornel, has been demonstrated to have synergistic effects of antioxidant and anti-inflammatory, which makes it to be applied for stroke as a protectant. However, the underlying mechanism of neuroprotection of EDB has not been fully elucidated. Increasing evidence has shown that neurotoxic A1 astrocytes were closely related to neuronal death after cerebral ischemia. However, whether EDB could provide neuroprotection by modulating the activation of astrocytes has not yet been elucidated. The present study aimed to explore whether EDB afforded neuroprotection by modulating A1 polarization of astrocytes and the down-stream signaling after cerebral ischemia. We first validated the neuroprotective effects of EDB in mice suffering focal cerebral ischemia via evaluating behavioral test, infarct volumes and neuronal survival. As for the down-stream signaling, our data further showed that EDB alleviated neuronal death by suppressing activation of neurotoxic A1 astrocytes via inhibition of NF-κB signaling pathway in vitro. Additionally, administration of EDB reduced the number of A1 reactive astrocytes in mice of focal cerebral ischemia. The above findings demonstrated that EDB provided neuroprotective effect by inhibiting neurotoxic activation of A1 astrocytes in animal model of cerebral ischemia, which indicated that EDB-mediated phenotypic regulation of astrocytes is a potential research direction to promote neurological recovery in central nervous system (CNS) diseases.

Changes in early endosomes in rat hippocampal CA1 neurons after transient global cerebral ischaemia.

Transient global ischaemia in rodents causes selective loss of hippocampal CA1neurons, but the potential involvement of endocytic pathways has not been fully explored. The aim of this study was to investigate the changes in early endosomes in the CA1 subfield after ischaemia and reperfusion. A four-vessel occlusion (4-VO) model was established in Wistar rats to induce 13 minutes of global cerebral ischaemia. Neuronal death was detected by Fluoro-Jade B (FJ-B) staining at various intervals after reperfusion, and intracellular membrane changes in ischaemic neurons were revealed using DiOC6(3), a lipophilic fluorescent probe. Ras-related protein Rab5 (Rab5) immunostaining was performed to detect changes in early endosomes in ischaemic neurons. Western blot analysis was used to confirm the morphological observations on Rab5 in the CA1 hippocampal subfield. FJ-B staining confirmed progressive neuronal death in the CA1 subfield in ischaemic rats after reperfusion. DiOC6(3) staining revealed abnormally increased membranous components in ischaemic CA1 neurons. Specifically, early endosomes, as labelled by Rab5 immunostaining, significantly increased in number and size in CA1 neurons at 1.5 and 2 days post-reperfusion, followed by rupture at day 3 and a decrease in staining intensity at day 7 post-reperfusion. Western blot analysis confirmed a significant upregulation of Rab5 protein levels at day 2, which returned to near control levels by day 7. Our study revealed significant changes in the dynamics of early endosomes in CA1 neurons after ischaemia-reperfusion injury. The initial increase in the area fraction of early endosomes in CA1 neurons may reflect an upregulation of endocytic activity, whereas the fragmentation and reduction of early endosomes at the later stage may indicate a failure of adaptive mechanisms of ischaemic neurons against ischaemia-induced death. Understanding the temporal dynamics of early endosomes provides critical insights into the cellular mechanisms that govern fate of CA1 hippocampal neuronsl after ischaemia/reperfusion.

Ferroptosis-associated alterations in diabetes following ischemic stroke: Insights from RNA sequencing

ObjectiveFerroptosis is an iron-dependent form of programmed cell death associated with lipid peroxidation. Though diabetes worsens cerebral injury and clinical outcomes in stroke, it is poorly understood whether ferroptosis contributes to diabetes-exacerbated stroke. This study aimed to identify ferroptosis-associated differentially expressed genes in ischemic stroke under diabetic condition and then explore their roles using comprehensive bioinformatics analyses. MethodsType 1 diabetes (T1D) model was established in male mice at 8–10 weeks of age by one intraperitoneal injection of streptozotocin (110 mg/kg). Ischemic stroke was induced by a transient 45-minute middle cerebral artery occlusion and evaluated three days thereafter. Ischemic brain cortex was dissected 24 h after the reperfusion and subjected to bulk tissue RNA sequencing followed by bioinformatics analysis and verification of key findings via quantitative real-time PCR. ResultsEnlarged infarct size was seen in diabetic, as compared with non-diabetic mice, in conjunction with worsened neurological behaviors. Both body and spleen weights were reduced in diabetic as compared with non-diabetic mice. There was a trend for reduced survival rate in diabetic mice following the stroke. In RNA sequencing analysis, we identified 1299 differentially expressed genes in ischemic brain between diabetic and non-diabetic mice, with upregulation and downregulation for 732 and 567 genes, respectively. Among these genes, 27 genes were associated with ferroptosis. Further analysis reveals that solute carrier family 25 member 28(SLC25A28) and sterol carrier protein 2(SCP2) were the top genes associated with ferroptosis in diabetic mice following ischemic stroke. In several bioinformatics analyses, we found SLC25A28, one of the top ferroptosis-related genes, is involved in several metabolic and regulatory pathways as well as the regulatory complexity of microRNAs and circular RNAs, which demonstrates the potential role of SLC25A28 in diabetes-exacerbated stroke. Drug network analysis suggests SLC25A28 as a potential therapeutic target for ameliorating ischemic injury in diabetes. ConclusionsOur bulk RNA sequencing and bioinformatics analyses show that altered ferroptosis signaling pathway was associated with the exacerbation of experimental stroke injury under diabetic condition. Especially, additional investigation into the mechanisms of SLC25A28 and SCP2 in diabetes-exacerbated stroke will be explored in the future study.

Drp1 acetylation mediated by CDK5-AMPK-GCN5L1 axis promotes cerebral ischemic injury via facilitating mitochondrial fission

The aberrant acetylation of mitochondrial proteins is involved in the pathogenesis of multiple diseases including neurodegenerative diseases and cerebral ischemic injury. Previous studies have shown that depletion of mitochondrial NAD+, which is necessary for mitochondrial deacetylase activity, leads to decreased activity of mitochondrial deacetylase and thus causes hyperacetylation of mitochondrial proteins in ischemic brain tissues, which results in altered mitochondrial dynamics. However, it remains largely unknown about how mitochondrial dynamics-related protein Drp1 is acetylated in ischemic neuronal cells and brain tissues. Here, we showed that Drp1 and GCN5L1 expression was up-regulated in OGD-treated neuronal cells and ischemic brain tissues induced by dMCAO, accompanied by the increased mitochondrial fission, mtROS accumulation, and cell apoptosis. Further, we confirmed that ischemia/hypoxia promoted Drp1 interaction with GCN5L1 in neuronal cells and brain tissues. GCN5L1 knockdown attenuated, while its overexpression enhanced Drp1 acetylation and mitochondrial fission, indicating that GCN5L1 plays a crucial role in ischemia/hypoxia-induced mitochondrial fission by acetylating Drp1. Mechanistically, ischemia/hypoxia induced Drp1 phosphorylation by CDK5 upregulation-mediated activation of AMPK in neuronal cells, which in turn facilitated the interaction of GCN5L1 with Drp1, thus enhancing Drp1 acetylation and mitochondrial fission. Accordingly, inhibition of AMPK alleviated ischemia/hypoxia- induced Drp1 acetylation and mitochondrial fission and protected brain tissues from ischemic damage. These findings provide a novel insight into the functional roles of GCN5L1 in regulating Drp1 acetylation and identify a previously unrecognized CDK5-AMPK-GCN5L1 pathway that mediates the acetylation of Drp1 in ischemic brain tissues.