Neuroprotective and Angiogenesis Effects of Levetiracetam Following Ischemic Stroke in Rats

Objective: The present study explored whether levetiracetam (LEV) could protect against experimental brain ischemia and enhance angiogenesis in rats, and investigated the potential mechanisms in vivo and in vitro.Methods: The middle cerebral artery was occluded for 60 min to induce middle cerebral artery occlusion (MCAO). The Morris water maze was used to measure cognitive ability. The rotation test was used to assess locomotor function. T2-weighted MRI was used to assess infarct volume. The neuronal cells in the cortex area were stained with cresyl purple. The anti-inflammatory effects of LEV on microglia were observed by immunohistochemistry. Enzyme-linked immunosorbent assays (ELISA) were used to measure the production of pro-inflammatory cytokines. Western blotting was used to detect the levels of heat shock protein 70 (HSP70), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF-1α) in extracts from the ischemic cortex. Flow cytometry was used to observe the effect of LEV on neuronal cell apoptosis.Results: LEV treatment significantly increased the density of the surviving neurons in the cerebral cortex and reduced the infarct size (17.8 ± 3.3% vs. 12.9 ± 1.4%, p < 0.01) after MCAO. Concurrently, the time required to reach the platform for LEV-treated rats was shorter than that in the saline group on day 11 after MCAO (p < 0.01). LEV treatment prolonged the rotarod retention time on day 14 after MCAO (84.5 ± 6.7 s vs. 59.1 ± 6.2 s on day 14 compared with the saline-treated groups, p < 0.01). It also suppressed the activation of microglia and inhibited TNF-α and Il-1β in the ischemic brain (135.6 ± 5.2 pg/ml vs. 255.3 ± 12.5 pg/ml, 18.5 ± 1.3 pg/ml vs. 38.9 ± 2.3 pg/ml on day 14 compared with the saline-treated groups, p < 0.01). LEV treatment resulted in a significant increase in HIF-1α, VEGF, and HSP70 levels in extracts from the ischemic cerebral cortex. At the same time, LEV reduced neuronal cell cytotoxicity and apoptosis induced by an ischemic stroke (p < 0.01).Conclusion: LEV treatment promoted angiogenesis and functional recovery after cerebral ischemia in rats. These effects seem to be mediated through anti-inflammatory and antiapoptotic activities, as well as inducing the expression of HSP70, VEGF, and HIF-1α.

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A review of experimental models of focal cerebral ischemia focusing on the middle cerebral artery occlusion model

F1000Research ◽

10.12688/f1000research.51752.2 ◽

2021 ◽

Vol 10 ◽

pp. 242

Author(s):

Melissa Trotman-Lucas ◽

Claire L. Gibson

Keyword(s):

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Middle Cerebral Artery Occlusion ◽

Cerebral Artery ◽

Artery Occlusion ◽

Lesion Volume ◽

Mechanistic Investigation ◽

Cerebral Artery Occlusion

Cerebral ischemic stroke is a leading cause of death and disability, but current pharmacological therapies are limited in their utility and effectiveness. In vitro and in vivo models of ischemic stroke have been developed which allow us to further elucidate the pathophysiological mechanisms of injury and investigate potential drug targets. In vitro models permit mechanistic investigation of the biochemical and molecular mechanisms of injury but are reductionist and do not mimic the complexity of clinical stroke. In vivo models of ischemic stroke directly replicate the reduction in blood flow and the resulting impact on nervous tissue. The most frequently used in vivo model of ischemic stroke is the intraluminal suture middle cerebral artery occlusion (iMCAO) model, which has been fundamental in revealing various aspects of stroke pathology. However, the iMCAO model produces lesion volumes with large standard deviations even though rigid surgical and data collection protocols are followed. There is a need to refine the MCAO model to reduce variability in the standard outcome measure of lesion volume. The typical approach to produce vessel occlusion is to induce an obstruction at the origin of the middle cerebral artery and reperfusion is reliant on the Circle of Willis (CoW). However, in rodents the CoW is anatomically highly variable which could account for variations in lesion volume. Thus, we developed a refined approach whereby reliance on the CoW for reperfusion was removed. This approach improved reperfusion to the ischemic hemisphere, reduced variability in lesion volume by 30%, and reduced group sizes required to determine an effective treatment response by almost 40%. This refinement involves a methodological adaptation of the original surgical approach which we have shared with the scientific community via publication of a visualised methods article and providing hands-on training to other experimental stroke researchers.

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A review of experimental models of focal cerebral ischemia focusing on the middle cerebral artery occlusion model

F1000Research ◽

10.12688/f1000research.51752.1 ◽

2021 ◽

Vol 10 ◽

pp. 242

Author(s):

Melissa Trotman-Lucas ◽

Claire L. Gibson

Keyword(s):

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Middle Cerebral Artery Occlusion ◽

Cerebral Artery ◽

Artery Occlusion ◽

Lesion Volume ◽

Mechanistic Investigation ◽

Cerebral Artery Occlusion

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Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies

Pharmaceutics ◽

10.3390/pharmaceutics12121207 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1207

Author(s):

Andrea Vítečková Wünschová ◽

Adam Novobilský ◽

Jana Hložková ◽

Peter Scheer ◽

Hana Petroková ◽

...

Keyword(s):

Middle Cerebral Artery ◽

Cerebral Artery ◽

Imaging Techniques ◽

Barium Sulphate ◽

Flow Conditions ◽

Preclinical Imaging ◽

Blood Clots ◽

3D Printed

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

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Abstract WMP76: Interleukin-33 Protects Against Transient Ischemic Stroke by Enhancing Regulatory T-Cell Expansion and Accumulation

Stroke ◽

10.1161/str.51.suppl_1.wmp76 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Xinjing Liu ◽

Ruiyao Hu ◽

Lulu Pei ◽

Yuming Xu ◽

Bo Song

Keyword(s):

Cell Death ◽

Ischemic Stroke ◽

T Cell ◽

Regulatory T Cell ◽

Cell Expansion ◽

Neuronal Apoptosis ◽

Infarct Volume ◽

Intraperitoneal Administration

Background: The interleukin (IL)-33 could promote proliferation of regulatory T lymphocytes (Tregs) which are negatively related with brain damage after ischemic stroke. How IL-33 works on Tregs after stroke is unclear. The purpose of this study was to investigate the role of IL-33 for Tregs-mediated neuroprotection and further expounded the mechanisms of protection in mice. Methods: In vitro study, primary mice neuronal cells were subjected to 3h oxygen-glucose deprivation (OGD). The vehicle or drug conditioned Tregs were applied to neurons at the time of induction of hypoxia respectively. Neuronal apoptosis, Tregs related cytokines were measured by MTT assay, Western blotting and enzyme-linked immune-sorbent assay (ELISA). In vivo study, Tregs were depleted by intraperitoneal administration of anti-CD25Ab. Intraperitoneal injection of IL-33 immediately post 60 min transient middle cerebral artery occlusion (tMCAO) modeling. The neurological function test at days 1, 3, 5, 7 and 14 after tMCAO. Infarct volume, Brain edema, cell death, percentage of Tregs and related cytokines were respectively measured by 2,3,5-triphenyltetrazolium chloride or MAP2 staining, dry-wet method, TUNEL staining, flow cytometry and immunofluorescence, Western blotting and ELISA. Results: The supernatant of IL-33-treated Tregs reduced neuronal apoptosis in the OGD model meanwhile elevated the production of Tregs related cytokines IL-10, IL-35 and TGF- β in vitro. Intraperitoneal administration of IL-33 significantly reduced infarct volume and stroke-induced cell death and improved sensorimotor functions. Notably, the protective effect of IL-33 was abolished in mice depleted of Tregs. IL-33 increased CD4+CD25+Foxp3+ Tregs in spleens, blood, and brain in vivo. Yet, ST2 blocking muted these IL-33 activities. Mechanistically, the protection of IL-33 was associated with reduced apoptosis protein and production of Tregs related cytokine. Conclusions: This study elucidated that IL-33 afforded neuroprotection against ischemic brain injury by enhancing ST2-dependent regulatory T-cell expansion and activation, which suggested a promising immune modulatory target for the treatment of stroke.

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Abstract WP430: Endothelial Cell Collection from Ipsilateral Middle Cerebral Artery in Acute Ischemic Stroke

Stroke ◽

10.1161/str.48.suppl_1.wp430 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Sunil A Sheth ◽

Abhishek Verma ◽

David S Liebeskind ◽

Jeffrey L Saver ◽

Gary Duckwiler ◽

...

Keyword(s):

Ischemic Stroke ◽

Endothelial Cell ◽

Middle Cerebral Artery ◽

Acute Ischemic Stroke ◽

Cerebral Artery ◽

Critical Role ◽

Early Time ◽

Stent Retriever ◽

Positive Controls

Introduction: The cerebrovascular endothelium plays a critical role in the pathogenesis of and response to acute ischemic stroke (AIS). To date, techniques to study its function have relied on animal and in vitro models. A robust method of endothelial cell (EC) capture in patients with AIS at early time points, from within the ischemic region, could greatly advance our understanding of cerebrovascular injury. Method: Patients undergoing thrombectomy for middle cerebral artery occlusion (MCA) within 8 hours of onset were offered enrollment if the pass of their stent-retriever device occurred directly into a distal access catheter in the proximal M1 segment, limiting exposure of the device to only the MCA. After retrieval, ECs adherent to the devices were retrieved and stained for EC (CD31) and leukocyte (CD45) markers. EC identity and yield were confirmed by flow cytometry with simultaneous immuno-fluorescence microscopy. Cultured human ECs were used as positive controls. The EC fraction was defined as CD31 + CD45 - with size and morphological features consistent with the positive controls. Results: ECs from stent-retriever devices (n=3) were collected and pooled. Approximately 8% of the collected cells represented ECs. EC collected from the stent-retrievers demonstrated highly similar shape, morphology and antibody staining patterns compared to the positive controls (Figure). Conclusions: Here we provide the first demonstration of a rapid post-thrombectomy method for reliable harvesting of cerebral ECs in humans. The ability to capture these cells in patients with AIS within hours of symptom onset opens many avenues of exploration for determining the role of ECs in AIS.

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Upregulation of TACE/ADAM17 after Ischemic Preconditioning is Involved in Brain Tolerance

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000033968.83623.d0 ◽

2002 ◽

Vol 22 (11) ◽

pp. 1297-1302 ◽

Cited By ~ 23

Author(s):

Antonio Cárdenas ◽

María A. Moro ◽

Juan C. Leza ◽

Esther O'Shea ◽

Antoni Dávalos ◽

...

Keyword(s):

Middle Cerebral Artery ◽

Middle Cerebral Artery Occlusion ◽

Brain Damage ◽

Cerebral Artery ◽

Ischemic Preconditioning ◽

Infarct Volume ◽

Artery Occlusion ◽

Tnf Α ◽

Cerebral Artery Occlusion

A short ischemic event (ischemic preconditioning [IPC]) can result in a subsequent resistance to severe ischemic injury (ischemic tolerance [IT]). Although tumor necrosis factor-α (TNF-α) contributes to the brain damage, its expression and neuroprotective role in models of IPC have also been described. However, the role of TNF-α convertase (TACE) in IPC and IT is not known. Using in vitro models, the authors previously demonstrated that TACE is upregulated after ischemic brain damage. In the present study, the authors used a rat model of transient middle cerebral artery occlusion as IPC to investigate TACE expression, its involvement in TNF-α release, and its role in IT. Western blot analysis showed that TACE expression is increased after IPC. Ischemic preconditioning caused TNF-α release, an effect that was blocked by the selective TACE inhibitor BB-1101 (10 mg · kg−1 · day−1; SHAM, 1,050 ± 180; IPC, 1,870 ± 290; IPC + BB, 1,320 ± 260 ng/mg; n = 4, P < 0.05). Finally, IPC produced a reduction in infarct volume, which was inhibited by treatment with BB-1101 and with anti–TNF-α (10 μg/5 doses; SHAM + permanent middle cerebral artery occlusion [pMCAO], 335 ± 20; IPC + pMCAO, 244 ± 14; IPC + BB + pMCAO, 300 ± 6; IPC + anti-TNF + pMCAO, 348 ± 22 mm3; n = 6–10, P < 0.05). Taken together, these data demonstrate that TACE is upregulated after IPC, plays a major role in TNF-α shedding in IPC, and has a neuroprotective role in IT.

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Middle Cerebral Artery Blood Flows by Combining TCD Velocities and MRA Diameters: In Vitro and In Vivo Validations

Ultrasound in Medicine & Biology ◽

10.1016/j.ultrasmedbio.2014.05.022 ◽

2014 ◽

Vol 40 (11) ◽

pp. 2692-2699 ◽

Cited By ~ 7

Author(s):

K.A. Yonan ◽

E.R. Greene ◽

J.M. Sharrar ◽

A. Caprihan ◽

C. Qualls ◽

...

Keyword(s):

Middle Cerebral Artery ◽

Cerebral Artery ◽

Blood Flows

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Autophagy is Involved in Neuroprotective Effect of Alpha7 Nicotinic Acetylcholine Receptor on Ischemic Stroke

Frontiers in Pharmacology ◽

10.3389/fphar.2021.676589 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhe-Qi Xu ◽

Jing-Jing Zhang ◽

Ni Kong ◽

Guang-Yu Zhang ◽

Ping Ke ◽

...

Keyword(s):

Ischemic Stroke ◽

Signaling Pathway ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Infarct Volume ◽

Neuroprotective Effect ◽

Autophagy Flux ◽

Nicotinic Acetylcholine

The α7 nicotinic acetylcholine receptor (α7nAChR) belongs to the superfamily of cys loop cationic ligand-gated channels, which consists of homogeneous α7 subunits. Although our lab found that activation of α7nAChR could alleviate ischemic stroke, the mechanism is still unknown. Herein, we explored whether autophagy is involved in the neuroprotective effect mediated by α7nAChR in ischemic stroke. Transient middle cerebral artery occlusion (tMCAO) and oxygen and glucose deprivation (OGD/R) exposure were applied to in vivo and in vitro models of ischemic stroke, respectively. Neurological deficit score and infarct volume were used to evaluate outcomes of tMCAO in the in vivo study. Autophagy-related proteins were detected by Western blot, and autophagy flux was detected by using tandem fluorescent mRFP-GFP-LC3 lentivirus. At 24 h after tMCAO, α7nAChR knockout mice showed worse neurological function and larger infarct volume than wild-type mice. PNU282987, an α7nAChR agonist, protected against OGD/R-induced neuronal injury, enhanced autophagy, and promoted autophagy flux. However, the beneficial effects of PNU282987 were eliminated by 3-methyladenine (3-MA), an autophagy inhibitor. Moreover, we found that PNU282987 treatment could activate the AMPK-mTOR-p70S6K signaling pathway in the in vitro study, while the effect was attenuated by compound C, an AMPK inhibitor. Our results demonstrated that the beneficial effect on neuronal survival via activation of α7nAChR was associated with enhanced autophagy, and the AMPK-mTOR-p70S6K signaling pathway was involved in α7nAChR activation–mediated neuroprotection.

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The Neuroprotective Effects of Insulin-Like Growth Factor 1 via the Hippo/YAP Signaling Pathway is Mediated by the PI3K/AKT Pathway Following Cerebral Ischemia-Reperfusion Injury

10.21203/rs.3.rs-187237/v1 ◽

2021 ◽

Author(s):

Pian Gong ◽

Yichun Zou ◽

Wei Zhang ◽

Qi Tian ◽

Shoumeng Han ◽

...

Keyword(s):

Ischemic Stroke ◽

Growth Factor ◽

Signaling Pathway ◽

Neuronal Death ◽

Infarct Volume ◽

Akt Signaling ◽

Akt Signaling Pathway ◽

Neuroprotective Effects

Abstract Insulin-like growth factor 1 (IGF-1) exhibits neuroprotective properties, such as vasodilatory and anti-inflammatory effects following ischemic stroke. However, the specific molecular mechanisms of action of IGF-1 following ischemic stroke remain elusive. We wanted to explore whether IGF-1 regulates Hippo/YAP signaling pathway, potentially via activation of the PI3K/AKT signaling pathway to exert its neuroprotective effects following ischemic stroke. In the in vitro study, we used oxygen–glucose deprivation to injure cultured PC12 and SH-5YSY cells, and cortical primary neurons. Cell viability was measured using CCK-8 assay. For the in vivo analyses, Sprague–Dawley rats were subjected to middle cerebral artery occlusion; neurological function was assessed using the neurological deficit score; infarct volume was measured using triphenyltetrazolium chloride staining, and neuronal death and apoptosis was evaluated by TUNEL staining, H&E staining and Nissl staining. Western blot was used to measure the levels of YAP/TAZ, PI3K and phosphorylated AKT (p-AKT) both in vitro and in vivo. We found that IGF-1 induced activation of YAP/TAZ, which resulted in improved cell viability in vitro, and decreased neurological deficits, neuronal death and apoptosis, and cerebral infarct volume in vivo. Notably, the neuroprotective effects of IGF-1 were reversed by an inhibitor of the PI3K/AKT signaling pathway, LY294002, which not only reduced expressions of PI3K and p-AKT, but also down-regulated expression of YAP/TAZ, leading to aggravation of neurological dysfunction. These findings indicate that neuroprotective effect of IGF-1 is partly realized by up-regulation of YAP/TAZ, which is mediated by activation of the PI3K/AKT signaling pathway following cerebral ischemic stroke.

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Role of long noncoding RNA MEG3/miR-378/GRB2 axis in neuronal autophagy and neurological functional impairment in ischemic stroke

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010946 ◽

2020 ◽

Vol 295 (41) ◽

pp. 14125-14139 ◽

Cited By ~ 2

Author(s):

Hong-Cheng Luo ◽

Ting-Zhuang Yi ◽

Fu-Gao Huang ◽

Ying Wei ◽

Xiao-Peng Luo ◽

...

Keyword(s):

Ischemic Stroke ◽

Middle Cerebral Artery ◽

Cerebral Artery ◽

Functional Impairment ◽

Molecular Mechanisms ◽

Glucose Deprivation ◽

Oxygen Glucose Deprivation ◽

Neuronal Autophagy

Autophagy has been shown to maintain neural system homeostasis during stroke. However, the molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. This study aims to investigate the regulatory mechanisms of the pathway consisting of MEG3 (maternally expressed gene 3), microRNA-378 (miR-378), and GRB2 (growth factor receptor-bound protein 2) in neuronal autophagy and neurological functional impairment in ischemic stroke. A mouse model of the middle cerebral artery occluded–induced ischemic stroke and an in vitro model of oxygen-glucose deprivation–induced neuronal injury were developed. To understand the role of the MEG3/miR-378/GRB2 axis in the neuronal regulation, the expression of proteins associated with autophagy in neurons was measured by Western blotting analysis, and neuron death was evaluated using a lactate dehydrogenase leakage rate test. First, it was found that the GRB2 gene, up-regulated in middle cerebral artery occluded–operated mice and oxygen-glucose deprivation–exposed neurons, was a target gene of miR-378. Next, miR-378 inhibited neuronal loss and neurological functional impairment in mice, as well as neuronal autophagy and neuronal death by silencing of GRB2. Confirmatory in vitro experiments showed that MEG3 could specifically bind to miR-378 and subsequently up-regulate the expression of GRB2, which in turn suppressed the activation of Akt/mTOR pathway. Taken together, these findings suggested that miR-378 might protect against neuronal autophagy and neurological functional impairment and proposed that a MEG3/miR-378/GRB2 regulatory axis contributed to better understanding of the pathophysiology of ischemic stroke.

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