Model Of Focal Cerebral Ischemia Research Articles

Research advances in the application of vagus nerve electrical stimulation in ischemic stroke.

Stroke seriously endangers human well-being and brings a severe burden to family and society. Different post-stroke dysfunctions result in an impaired ability to perform activities of daily living. Standard rehabilitative therapies may not meet the requirements for functional improvement after a stroke; thus, alternative approaches need to be proposed. Currently, vagus nerve stimulation (VNS) is clinically applied for the treatment of epilepsy, depression, cluster headache and migraine, while its treatment of various dysfunctions after an ischemic stroke is still in the clinical research stage. Recent studies have confirmed that VNS has neuroprotective effects in animal models of transient and permanent focal cerebral ischemia, and that its combination with rehabilitative training significantly improves upper limb motor dysfunction and dysphagia. In addition, vagus-related anatomical structures and neurotransmitters are closely implicated in memory-cognition enhancement processes, suggesting that VNS is promising as a potential treatment for cognitive dysfunction after an ischemic stroke. In this review, we outline the current status of the application of VNS (invasive and non-invasive) in diverse functional impairments after an ischemic stroke, followed by an in-depth discussion of the underlying mechanisms of its mediated neuroprotective effects. Finally, we summarize the current clinical implementation challenges and adverse events of VNS and put forward some suggestions for its future research direction. Research on VNS for ischemic stroke has reached a critical stage. Determining how to achieve the clinical transformation of this technology safely and effectively is important, and more animal and clinical studies are needed to clarify its therapeutic mechanism.

Establishment and Evaluation of Post-stroke Depression Rat Model

Objective To establish the rat model of post-stroke depression (PSD). Methods The focal cerebral ischemia model was set up by blocking the middle cerebral artery (MCA). Then the model rats were separately raised and put into chronic unpredictable mild stress (CUMS) to induce the PSD model and some of them were intervened by ? uoxertine. The rats were divided into control, stroke, depression, post-stroke depression and PSD treated with ? uoxertine groups and all of them were examined dynamically by Open-fi eld test (OFT), sucrose consumption test and forced swimming test. Results The animal models of PSD had significantly less weight gain than control group and stroke group(P0.01)at day 14 after CUMS. After given ? uoxertin the animals' body weight in PSD group increased signifi cantly than before. The scores of horizontal and vertical movement activities in OFT of PSD group were signifi cantly less than in control and stroke groups(P0.05 or P0.01). The fi xed time in forced-swimming test was signifi cantly longer in PSD group when compared with control and stroke groups(P0.05). Fluoxertin markedly increased open-fi eld activities(P0.05 or P0.01) and decreased the fi xed time in forced-swimming test(P0.01). Conclusion Anhedonia and underactivity, the core symptoms in the PSD patients, can be found completely and persistently in the PSD group rats. With good operability and repeatability, the rats PSD model is an ideal model for the PSD research. Fluoxetine can improve the behavior abnormality of the PSD rats.

Neuroprotective Effects of the Lithium Salt of a Novel JNK Inhibitor in an Animal Model of Cerebral Ischemia-Reperfusion.

The c-Jun N-terminal kinases (JNKs) regulate many physiological processes, including inflammatory responses, morphogenesis, cell proliferation, differentiation, survival, and cell death. Therefore, JNKs represent attractive targets for therapeutic intervention. In an effort to develop improved JNK inhibitors, we synthesized the lithium salt of 11H-indeno[1,2-b]quinoxaline-11-one oxime (IQ-1L) and evaluated its affinity for JNK and biological activity in vitro and in vivo. According to density functional theory (DFT) modeling, the Li+ ion stabilizes the six-membered ring with the 11H-indeno[1,2-b]quinoxaline-11-one (IQ-1) oximate better than Na+. Molecular docking showed that the Z isomer of the IQ-1 oximate should bind JNK1 and JNK3 better than (E)-IQ-1. Indeed, experimental analysis showed that IQ-1L exhibited higher JNK1-3 binding affinity in comparison with IQ-1S. IQ-1L also was a more effective inhibitor of lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) transcriptional activity in THP-1Blue monocytes and was a potent inhibitor of proinflammatory cytokine production by MonoMac-6 monocytic cells. In addition, IQ-1L inhibited LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. In a rat model of focal cerebral ischemia (FCI), intraperitoneal injections of 12 mg/kg IQ-1L led to significant neuroprotective effects, decreasing total neurological deficit scores by 28, 29, and 32% at 4, 24, and 48 h after FCI, respectively, and reducing infarct size by 52% at 48 h after FCI. The therapeutic efficacy of 12 mg/kg IQ-1L was comparable to that observed with 25 mg/kg of IQ-1S, indicating that complexation with Li+ improved efficacy of this compound. We conclude that IQ-1L is more effective than IQ-1S in treating cerebral ischemia injury and thus represents a promising anti-inflammatory compound.

Ruta graveolens water extract (RGWE) ameliorates ischemic damage and improves neurological deficits in a rat model of transient focal brain ischemia

Introduction and aimsThe limited therapeutic options for ischemic stroke treatment render necessary the identification of new strategies. In recent years, it has been shown that natural compounds may represent a valid therapeutic opportunity. Therefore, the present study aimed to evaluate the protective effect of Ruta graveolens water extract (RGWE) in an in vivo experimental model of brain ischemia. MethodsRGWE effects on ischemic damage and neurological function were evaluated in adult rats subjected to transient occlusion of the Middle Cerebral Artery (tMCAO), receiving two intraperitoneal injections of RGWE, 100 and 300 min after the induction of ischemia. In addition, astroglial and microglial activation was measured as GFAP and IBA-1 expression by immunofluorescence and confocal microscopy analysis. ResultsTreatment with RGWE containing 10 mg/kg of Rutin, the major component, ameliorates the ischemic damage and improves neurological performances. Interestingly, the pro-inflammatory states of astrocytes and microglia, respectively detected by using C3 and iNOS markers, were significantly reduced in ipsilateral cortical and striatal areas in ischemic RGWE-treated rats. ConclusionsRGWE shows a neuroprotective effect on brain infarct volume extent in a transient focal cerebral ischemia model and this effect was paralleled by the prevention of pro-inflammatory astroglial and microglial activation. Collectively, our findings support the idea that natural compounds may represent potential therapeutic opportunities against ischemic stroke.

A clinically relevant model of focal embolic cerebral ischemia by thrombus and thrombolysis in rhesus monkeys.

Over decades of research into the treatment of stroke, nearly all attempts to translate experimental treatments from discovery in cells and rodents to use in humans have failed. The prevailing belief is that it might be necessary to pretest pharmacological neuroprotection in higher-order brains, especially those of nonhuman primates (NHPs). Over the past few years, chemical thrombolysis and mechanical thrombectomy have been established as the standard of care for ischemic stroke in patients. The spotlight is now shifting towards emphasizing both focal ischemia and subsequent reperfusion in developing a clinically relevant stroke model in NHPs. This protocol describes an embolic model of middle cerebral artery occlusion in adult rhesus monkeys. An autologous clot is combined with a microcatheter or microwire through endovascular procedures, and reperfusion is achieved through local intra-artery thrombolysis with tissue plasminogen activator. These NHP models formed relatively stable infarct sizes, delivered predictable reperfusion and survival outcomes, and recapitulated key characteristics of patients with ischemic stroke as observed on MRI images and behavioral assays. Importantly, treated animals could survive 30 d after the surgery for post-stroke neurologic deficit analyses. Thus far, this model has been used in several translational studies. Here we describe in detail the teamwork necessary for developing stroke models of NHPs, including the preoperation preparations, endovascular surgery, postoperation management and histopathological analysis. The model can be established by the following procedures over a 45-d period, including preparation steps (14 d), endovascular operation (1 d) and evaluation steps (30 d).

Proteomics on the role of muscone in the “consciousness-restoring resuscitation” effect of musk on ischemic stroke

Ethnopharmacological relevanceMusk is a representative drug of aroma-relieving traditional Chinese medicine, and it is a commonly used traditional Chinese medicine for the treatment of ischemic stroke. Muscone is the core medicinal component of musk. Aim of the studyWe sought to identify the target of muscone in the treatment of ischemic stroke using network pharmacology, an animal model of ischemic stroke, and differential proteomics. Materials and methodsThe drug targets of muscone in the treatment of ischemic stroke were predicted and analyzed using information derived from sources such as the Traditional Chinese Medicine Systems Pharmacology database and Swiss Target Prediction tool. The animal model of focal cerebral ischemia was established by suture-based occlusion of the middle cerebral artery of rats. The rats were divided into six groups: sham-operated control, model, musk, muscone1, muscone2, and muscone3. Neurological deficit scores were calculated after intragastric administration of musk or muscone. The microcirculation blood flow of the pia mater was detected using a laser speckle blood flow meter. The cerebral infarction rate was detected by 2,3,5-triphenyltetrazolium chloride staining. The necrosis rate of the cerebral cortex and the hippocampal neurons was detected by hematoxylin and eosin staining. Blood-brain barrier damage was detected by the Evans blue method. Quantitative proteomics analysis in the sham-operated control, model, and muscone groups was performed using tandem-mass-tags. Considering fold changes exceeding 1.2 as differential protein expression, the quantitative values were compared among groups by analysis of variance. Furthermore, a protein-protein interaction network was constructed, and differentially expressed proteins were analyzed by gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. ResultsNetwork pharmacology identified 339 targets for the intersection of 17 components of musk and cerebral ischemia-reperfusion injury. The GO and KEGG enrichment items mainly identified regulation of neuronal synaptic structure and transfer function, synaptic neurotransmitters, and receptor activity. Zoopery showed that the model group had a higher behavioral score, cerebral infarction rate, cortical and hippocampal neuron death rate, Evans blue exudation in the brain, and bilateral pia mater microcirculation blood flow differences than the sham-operated control group (P <0.01). Compared with the model group, the behavioral score, infarction rate, hippocampal neuronal mortality, and Evans blue content decreased significantly in the musk, muscone2, and muscone3 groups (P <0.05). Proteomic analysis showed that 160 genes were differentially expressed among the sham-operated control, model, and muscone groups. GO items with high enrichment included neuronal synapses, postsynaptic signal transduction, etc. KEGG items with high enrichment included cholinergic synapses, calcium signaling pathway, dopaminergic synapses, etc. Protein interaction analysis revealed that the top three protein pairs were Ndufa10/Ndufa6, Kcna2/Kcnab2, and Gsk3b/Traf6. ConclusionsMuscone can reduce neuronal necrosis, protect the blood-brain barrier, and improve the neurological damage caused by cerebral ischemia via molecular mechanisms mainly involving the regulation of neuronal synaptic connections. Muscone is an important active component responsible for the “consciousness-restoring resuscitation” effect of musk on ischemic stroke.

Molecular Hydrogen Neuroprotection in Post-Ischemic Neurodegeneration in the Form of Alzheimer's Disease Proteinopathy: Underlying Mechanisms and Potential for Clinical Implementation-Fantasy or Reality?

Currently, there is a lot of public interest in naturally occurring substances with medicinal properties that are minimally toxic, readily available and have an impact on health. Over the past decade, molecular hydrogen has gained the attention of both preclinical and clinical researchers. The death of pyramidal neurons in especially the CA1 area of the hippocampus, increased permeability of the blood-brain barrier, neuroinflammation, amyloid accumulation, tau protein dysfunction, brain atrophy, cognitive deficits and dementia are considered an integral part of the phenomena occurring during brain neurodegeneration after ischemia. This review focuses on assessing the current state of knowledge about the neuroprotective effects of molecular hydrogen following ischemic brain injury. Recent studies in animal models of focal or global cerebral ischemia and cerebral ischemia in humans suggest that hydrogen has pleiotropic neuroprotective properties. One potential mechanism explaining some of the general health benefits of using hydrogen is that it may prevent aging-related changes in cellular proteins such as amyloid and tau protein. We also present evidence that, following ischemia, hydrogen improves cognitive and neurological deficits and prevents or delays the onset of neurodegenerative changes in the brain. The available evidence suggests that molecular hydrogen has neuroprotective properties and may be a new therapeutic agent in the treatment of neurodegenerative diseases such as neurodegeneration following cerebral ischemia with progressive dementia. We also present the experimental and clinical evidence for the efficacy and safety of hydrogen use after cerebral ischemia. The therapeutic benefits of gas therapy open up new promising directions in breaking the translational barrier in the treatment of ischemic stroke.

Retraction Note to: Alleviation of glutamate-mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia: a possible mechanism of GABA agonism.

Retraction Note to: Alleviation of glutamate-mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia: a possible mechanism of GABA agonism.

Liraglutide Ameliorates Cerebral Ischemia in Mice via Antipyroptotic Pathways.

It was recently shown that pyroptosis, an inflammatory form of programmed cell death, is critically involved in the pathogenesis of ischemic stroke. Liraglutide (Lg) is a novel long-acting analog of glucagon-like peptide-1 that has potential protective effects against stroke. However, the relationship between Lg and pyroptosis in the brain is not well defined. In this study, we found that injection of Lg significantly improved the recovery of motor function, increased cerebral blood flow and ameliorated cerebral damage in a mouse model of focal cerebral cortical ischemia. Our results revealed that Lg treatment significantly reduced the levels of NLRP3, Caspase1, IL-1β and the pore-forming protein gasdermin D in microglial cells in vitro, suggesting that the neuroprotective effect of Lg may be achieved through the inhibition of pyroptosis. Furthermore, by using a specific inhibitor of NOD-like receptor protein 3 (NLRP3), we confirmed that the antipyroptotic mechanism of Lg may be mediated by NLRP3 in vivo. Our present study unveils a novel neuroprotective mechanism through which Lg alleviates ischemia by exerting NLRP3-dependent antipyroptotic effects.

Therapeutic Potential of a Combination of Electroacupuncture and Human iPSC-Derived Small Extracellular Vesicles for Ischemic Stroke.

This paper aimed to explore the roles of the combination of electroacupuncture (EA) and induced pluripotent stem cell-derived small extracellular vesicles (iPSC-EVs) on mice with ischemic stroke and the underlying mechanisms. A focal cerebral ischemia model was established in C57BL/6 mice through middle cerebral artery occlusion (MCAO). After 3 days, neurological impairment and motor function were examined by performing behavioral tests. The infarct volume and neuronal apoptosis were examined using TTC staining and TUNEL assays. Flow cytometry was performed to assess the proliferation of T lymphocytes. The changes in the interleukin (IL)-33/ST2 axis were evaluated by immunofluorescence and Western blotting. The combination of EA and iPSC-EVs treatment ameliorated neurological impairments and reduced the infarct volume and neuronal apoptosis in MCAO mice. EA plus iPSC-EVs suppressed T helper (Th1) and Th17 responses and promoted the regulatory T cell (Treg) response. In addition, EA plus iPSC-EVs exerted neuroprotective effects by regulating the IL-33/ST2 axis and inhibiting the microglia and astrocyte activation. Taken together, the study shows that EA and iPSC-EVs exerted a synergistic neuroprotective effect in MCAO mice, and this treatment may represent a novel potent therapy for ischemic stroke and damage to other tissues.

Vagus nerve stimulation alleviated cerebral ischemia and reperfusion injury in rats by inhibiting pyroptosis via \u03b17 nicotinic acetylcholine receptor

Cumulative evidence suggests that pyroptosis, a new sort of programmed cell death, is closely related to cerebral ischemia/reperfusion (I/R) injury. Our previous studies have testified that vagus nerve stimulation (VNS) was involved in many different neuroprotective and neuroplasticity pathways via α7 nicotinic acetylcholine receptor (α7nAchR), a vital node of the cholinergic anti-inflammatory pathway during cerebral I/R injury. We aimed to determine the neuroprotective effects of VNS through α7nAchR-mediated inhibition of pyroptosis. Focal cerebral ischemic stroke rat models were obtained by middle cerebral artery occlusion for 120 min. Expression of the NLRP3 inflammasome was evaluated using western blotting and immunofluorescence (IF) staining. The neurological deficit score, infarct volume, TUNEL staining findings, transmission electron microscopy findings, and expression of inflammatory cytokines were assessed 3 days after I/R injury. Our findings suggested that the protein expression levels of NLRP3, GSDMD-N, cleaved caspase-1, and ASC gradually increased until they peaked on day 3 after I/R injury. VNS inhibited the expression of pyroptosis-related molecules and decreased the number of pyroptotic cells and membrane pores. Administration of α7nAchR-antagonist and agonist helped in further assessment of the role of α7nAchR in pyroptosis. α7nAchR-agonist mimicked VNS’s neuroprotective effects on the improvement of neurological deficits, the reduction of infarct volumes, and the inhibition of neuronal pyroptosis after cerebral I/R injury. Conversely, the neuroprotection provided by VNS could be reversed by the administration of α7nAchR-antagonist. In conclusion, VNS-induced neuroprotection via inhibition of neuronal pyroptosis was α7nAchR-dependent, highlighting the pivotal role of α7nAChR in suppressing cellular pyroptosis and neuroinflammation. These findings may allow a better understanding of treatment principles for cerebral I/R injury.

Abstract WP263: Effective Combination Therapy For Experimental Stroke: Synergistic Neuroprotection By Lau-0901 And At-npd1

Objective: Cerebral ischemia causes irreversible brain damage, which involves oxidative stress, the release of endogenous substances and inflammation. We tested the hypothesis that blocking pro-inflammatory platelet-activating factor receptor (PAF-R) plus administering a selected docosanoid after middle cerebral artery occlusion (MCAo) would lead to neurological recovery. Following small bioactive molecules were investigated: LAU-0901 (LAU), PAF-R antagonist that blocks pro-inflammatory signaling, and aspirin-triggered neuroprotectin D1 (AT-NPD1), which activates cell-survival pathways, and their combination that exerts potent anti-inflammatory activity in the brain. This study investigated the dose-response and therapeutic window in a model of focal cerebral ischemia. Methods: Male SD rats were subjected to 2h of MCAo, behavior was evaluated on days 1-7, and an ex vivo MRI was conducted on day 7. In Dose-response, rats were treated with LAU (45 and 60mg/kg, IP, at 3h), AT-NPD1 (111, 222, and 333μg/kg, IV, at 3.15 h after onset of stroke), LAU+AT-NPD1 or vehicle (n=5-6 per group). In Therapeutic window, LAU (60mg/kg, IP), AT-NPD1 (222μg/kg, IV), LAU+AT-NPD1 were administered at 3, 4, 5, and 6 h after onset of MCAo. Results: Treatments with LAU (45 and 60 mg/kg) alone improved behavior on days 1-7 by 32-38%; AT-NPD1 (all doses) by 20-30% compared to vehicle groups. The neuroprotective effect was enhanced using the LAU+AT-NPD1, which resulted in improved behavior up to 35-40% on days 1-7. Total lesion volumes, which were computed using T2WI, were significantly reduced with LAU-0901 (45 and 60mg/kg) by 76-85% and AT-NPD1 (all doses) alone by 62-77% compared to the vehicle group. Ischemic core, penumbra, and total lesion volumes were dramatically reduced with LAU+AT-NPD1 by 92-94% compared to the vehicle. LAU and AT-NPD1 alone, improved behavior on day 7 by 35 and 28%. In contrast, LAU+AT-NPD1 improved behavior by 46% when administered at 3h, by 52% at 4h, 34% at 5h, and 19% at 6h. Conclusion: LAU-0901 and AT-NPD1 alone provide high-grade neuroprotection. Combination therapy with LAU-0901 and AT-NPD1 is more effective than the single therapy, affording synergistic neuroprotection with improved behavior when administered up to 6 h after MCAo in rats.

Abstract WP235: Docosanoids Protect Inflammation-induced Brain Damage And Lead To Neurological Recovery In Experimental Stroke

Objective: Cerebral ischemia initiates lipid peroxidation, uncompensated oxidative stress, and neuronal damage. We investigated the neuroprotective bioactivity of docosanoid (DOC) mediators: Neuroprotectin D1 (NPD1), Resolvin D1 (RvD1), and their combination (NPD1+RvD1) against experimental ischemic stroke. These lipid mediators are biosynthesized “on-demand” in response to stroke onset to resolve neuroinflammation and restore homeostasis. This study investigated the dose-response and therapeutic window in a model of focal cerebral ischemia. Methods: Male SD rats received 2h of middle cerebral artery occlusion (MCAo). Neurological score was evaluated on days 1, 2, 3, and 7, followed by an ex vivo MRI on day 7. In dose-response study, rats were treated with NPD1 (111, 222, and 333μg/kg), RvD1 (111, 222, and 333μg/kg), NPD1 + RvD1 or vehicle (n=4-5 per group). All treatments were administered IV at 3h after the onset of MCAo. In the therapeutic window study, vehicle, NPD1 (222 μg/kg), RvD1 (222 μg/kg), NPD1+RvD1 were administered IV at 3, 4, 5, and 6 h after onset of MCAo. Results: No adverse behavioral side effects were observed after drugs administration. NPD1 (111, 222, and 333μg/kg) treatment alone greatly improved neurological scores (by 32-35%) and RvD1 (111, 222, and 333μg/kg) alone (by 31-35%) vs vehicle group on day 7. The neuroprotective effect was enhanced using the NPD1+ RvD1, which improved total neurological scores on days 1-7 by 25-40% vs vehicle. Ischemic core, penumbra, and total lesion volumes (computed from T2WI) were reduced with NPD1+RvD1 by 69, 70, and 67% vs vehicle. Therapeutic window: treatments with NPD1 and RvD1 alone when administered at 3h improved behavior on day 7 by 35%. Combinatory NPD1+RvD1 treatment improved behavior when administered at 3h (by 56%), 4h (by 23%), 5h (by 26%), and 6 h (by 28%), vs vehicle. Conclusion: We show that NPD1, RvD1 alone provides high-grade neuroprotection against inflammation and elicit neurological recovery in the MCAo model. Combination therapy with NPD1 and RvD1 is more effective than the single therapy when administered up to 6h after focal cerebral ischemia in rats. Thus, we hypothesize that these treatments may potentially be used in treating focal ischemic stroke in the clinical setting.

Gastrodin injection alleviates lung injury caused by focal cerebral ischemia in rats through NGF/TrkA pathway-mediated activation of the anti-inflammatory pathway

To investigate the therapeutic mechanism of gastrodin injection for alleviating lung injury caused by focal cerebral ischemia in rats and the role of the NGF-TrkA pathway in mediating this effect. Forty SD rats were equally randomized into normal group, sham-operated group, model group and gastrodin group, and in the latter two groups, rat models of focal cerebral ischemia were established by embolization of the right middle cerebral artery. After successful modeling, the rats were treated with intraperitoneal injection of gastrodin injection at the daily dose of 10 mg/kg for 14 days. After the treatment, the wet/dry weight ratio of the lung tissue was determined, the pathological changes in the lung tissue were observed using HE staining, and the levels of IL-10 and TNF-α in the arterial blood were detected with ELISA. The expressions of NF-κB p65 and TNF-α in the lung tissue were detected with Western blotting, and the expressions of NGF and TrkA were detected using immunohistochemical staining and Western blotting. Compared with the normal control and sham-operated groups, the rats in the model group showed obvious inflammatory lung injury, significantly increased wet/ dry weight ratio of the lungs (P < 0.01), increased TNF-α level in arterial blood (P < 0.01), and significantly up-regulated protein expressions of NF-κB p65 (P < 0.01), TNF-α (P < 0.01), NGF (P < 0.05) and TrkA(P < 0.05) in the lung tissue. Treatment with gastrodin injection obviously alleviated lung inflammation, decreased the wet/dry weight ratio of the lungs (P < 0.05), and significantly lowered TNF-α level (P < 0.01) and increased IL-10 level in the arterial blood in the rat models (P < 0.01); gastrodin injection also significantly decreased the protein expressions of NF-κB p65 and TNF-α (P < 0.05) and up-regulated the expressions of NGF and TrkA in the lung tissue of the rats (P < 0.05). The NGF/TrkA pathway may participate in cerebral ischemia-induced inflammatory lung injury, which can be obviously alleviated by gastrodin through the activation of the anti-inflammatory pathway mediated by the NGF/TrkA pathway.

Effects of Panax notoginseng Saponin on Neurons Based on Oxygen-Glucose Deprivation/Reoxygenation after Focal Cerebral Ischemia

We aimed to evaluate the effects of Panax notoginseng saponin on neuronal apoptosis and neuroprotection based on oxygen-glucose deprivation/reoxygenation in rats with focal cerebral ischemia. 100 rats were randomly divided into sham operation, middle cerebral artery occlusion model, low-dose Panax notoginseng saponin (10 mg/kg), medium-dose Panax notoginseng saponin (25 mg/kg) and high-dose Panax notoginseng saponin (50 mg/kg) groups. The model of focal cerebral ischemia was established by middle cerebral artery occlusion. For sham group, the right common, external and internal carotid arteries were only dissociated, without ligation or insertion. After modeling, Panax notoginseng saponin groups were intraperitoneally injected with Panax notoginseng saponin. Hippocampal neurons isolated from normal rats were randomly divided into control, oxygen-glucose deprivation/reoxygenation model, 1 μmol/l, 5 μmol/l and 20 μmol/l oxygen-glucose deprivation/reoxygenation+Panax notoginseng saponin groups. Neurological function was scored by Longa method and neuron morphology was observed. Neuronuclear antigen and neuroepithelial stem protein in brain tissues were detected by immunohistochemistry. The survival rate of neurons, lactate dehydrogenase leakage, neuronal apoptosis and expression levels of protein kinase B, phosphorylatedprotein kinase B and apoptosis-related proteins B-cell lymphoma 2, Bcl-2-associated X protein and caspase-3 were examined by cell counting kit-8 assay, lactate dehydrogenase kit, Hoechst 33 342 staining and Western blotting, respectively. Compared with model group, Panax notoginseng saponin groups had significantly lower neurological function scores (p<0.05). Panax notoginseng saponin significantly relieved neuronal injury and increased neuronuclear and neuroepithelial stem protein-positive cells (p<0.05). It significantly raised the survival rate of neurons, reduced lactate dehydrogenase leakage and inhibited neuronal apoptosis dose-dependently. Panax notoginseng saponin up-regulated the protein expressions of phosphorylated-protein kinase B/protein kinase B and B-cell lymphoma 2 and down-regulated those of Bcl-2-associated X protein and caspase-3 dose-dependently. It can protect against and repair neuronal injury caused by oxygen-glucose deprivation/reoxygenation, probably by activating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway to suppress neuronal apoptosis.

Fucoidan (a Sulfated Polysaccharide) and Cerebroprotein in Combination Alleviates the Neuroinflammation Mediated Neural Damage and Functional Deficits in Focal Cerebral Ischemia Model of Rat

Fucoidan (a Sulfated Polysaccharide) and Cerebroprotein in Combination Alleviates the Neuroinflammation Mediated Neural Damage and Functional Deficits in Focal Cerebral Ischemia Model of Rat

Melatonin and calpeptin synergistically protect against post-reperfusion injury in a rat middle cerebral artery occlusion stroke model

Cerebral ischemia induces oxidative injury and increases the intracellular calcium ion concentration to activate several calcium-dependent proteases such as calpains. Calpain activation leads to various necrotic and apoptotic processes. Calpeptin is a potent, cell-permeable calpain inhibitor. As a strong antioxidant and free radical scavenger, melatonin shows beneficial effect in rodent models of focal cerebral ischemia when given prior to ischemia or reperfusion. This study was focused on the neuroprotective effects of melatonin and/or calpeptin given after onset of reperfusion. For this purpose, right-sided middle cerebral artery occlusion (MCAO) for 90 minutes followed by 24 or 72 hours of reperfusion was performed in male Sprague Dawley rats, then, melatonin 50 or 150 µg/kg, calpeptin 10, 15 or 50 µg/kg or a combination of melatonin 50 µg/kg plus calpeptin 15 or 50 µg/kg were injected via an intracerebroventricular route at 15 minutes after onset of reperfusion. Melatonin or calpeptin tended to reduce the relative infarct volume and significantly decreased the neurological deficit at 24 hours. The combination achieved a greater protection than each of them alone. Melatonin, calpeptin or the combination all decreased Fluoro-Jade B (FJB)+ degenerative neurons and cleaved/total caspase-3 ratio at 24 hours. These treatments did not significantly impact the density of surviving neurons and ED-1+ macrophage/activated microglia. At the 72-hour-reperfusion, melatonin or the combination decreased the relative infarct volume and neurological deficit. Nevertheless, only the combination reduced FJB+ degenerating neurons at 72 hours. In conclusion, a combination of melatonin and calpeptin exerted synergistic protection against post-reperfusion injury in a rat MCAO stroke model.

JLX001 ameliorates cerebral ischemia injury by modulating microglial polarization and compromising NLRP3 inflammasome activation via the NF-κB signaling pathway

Ischemic stroke is a devastating disease with high morbidity and mortality rates, and the proinflammatory microglia-mediated inflammatory response directly affects stroke outcome. Previous studies have reported that JLX001, a novel compound with a structure similar to that of cyclovirobuxine D (CVB-D), exerts antiapoptotic, anti-inflammatory and antioxidative effects on ischemia-induced brain injury. However, the role of JLX001 in microglial polarization and nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome regulation after ischemic stroke has not been fully investigated. In this study, we used the middle cerebral artery occlusion (MCAO) method to establish a focal cerebral ischemia model and found that JLX001 attenuated the brain infarct size and improved cerebral damage. Moreover, the expression levels of proinflammatory cytokines (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) were significantly reduced while those of the anti-inflammatory cytokine IL-10 were increased in the JLX001-treated group. Immunofluorescence staining and flow cytometry revealed an increased number of anti-inflammatory phenotypic microglia and a reduced number of proinflammatory phenotypic microglia in JLX001-treated MCAO mice. Western blotting analysis showed that JLX001 inhibited the expression of NLRP3 and proteins related to the NLRP3 inflammasome axis in vivo. Furthermore, JLX001 reduced the number of NLRP3/Iba1 cells in ischemic penumbra tissues. Finally, mechanistic analysis revealed that JLX001 significantly inhibited the expression of proteins related to the NF-κB signaling pathway. Additionally, pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, ameliorated cerebral ischemia–reperfusion injury by suppressing microglial polarization towards the proinflammatory phenotype and NLRP3 activation in vivo, further suggesting that these protective effects of JLX001 were mediated by inhibition of the NF-κB signaling pathway. These results suggest that JLX001 is a promising therapeutic approach for ischemic stroke.

A Novel Phenylpyrrolidine Derivative: Synthesis and Effect on Cognitive Functions in Rats with Experimental Ishemic Stroke

We performed an in silico, in vitro, and in vivo assessment of a potassium 2-[2-(2-oxo-4-phenylpyrrolidin-1-yl) acetamido]ethanesulfonate (compound 1) as a potential prodrug for cognitive function improvement in ischemic brain injury. Using in silico methods, we predicted the pharmacological efficacy and possible safety in rat models. In addition, in silico data showed neuroprotective features of compound 1, which were further supported by in vitro experiments in a glutamate excitotoxicity-induced model in newborn rat cortical neuron cultures. Next, we checked whether compound 1 is capable of crossing the blood–brain barrier in intact and ischemic animals. Compound 1 improved animal behavior both in intact and ischemic rats and, even though the concentration in intact brains was low, we still observed a significant anxiety reduction and activity escalation. We used molecular docking and molecular dynamics to support our hypothesis that compound 1 could affect the AMPA receptor function. In a rat model of acute focal cerebral ischemia, we studied the effects of compound 1 on the behavior and neurological deficit. An in vivo experiment demonstrated that compound 1 significantly reduced the neurological deficit and improved neurological symptom regression, exploratory behavior, and anxiety. Thus, here, for the first time, we show that compound 1 can be considered as an agent for restoring cognitive functions.

Exploring cell membrane water exchange in aquaporin-4-deficient ischemic mouse brain using diffusion-weighted MRI

BackgroundAquaporin-4 is a membrane channel protein that is highly expressed in brain astrocytes and facilitates the transport of water molecules. It has been suggested that suppression of aquaporin-4 function may be an effective treatment for reducing cellular edema after cerebral infarction. It is therefore important to develop clinically applicable measurement systems to evaluate and better understand the effects of aquaporin-4 suppression on the living body.MethodsAnimal models of focal cerebral ischemia were created by surgically occluding the middle cerebral artery of wild-type and aquaporin-4 knockout mice, after which multi-b-value multi-diffusion-time diffusion-weighted imaging measurements were performed. Data were analyzed with both the apparent diffusion coefficient (ADC) model and a compartmental water-exchange model.ResultsADCs were estimated for five different b value ranges. The ADC of aquaporin-4 knockout mice in the contralateral region was significantly higher than that of wild-type mice for each range. In contrast, aquaporin-4 knockout mice had significantly lower ADC than wild-type mice in ischemic tissue for each b-value range. Genotype-dependent differences in the ADC were particularly significant for the lowest ranges in normal tissue and for the highest ranges in ischemic tissue. The ADCs measured at different diffusion times were significantly different for both genotypes. Fitting of the water-exchange model to the ischemic region data found that the water-exchange time in aquaporin-4 knockout mice was approximately 2.5 times longer than that in wild-type mice.ConclusionsMulti-b-value multi-diffusion-time diffusion-weighted imaging may be useful for in vivo research and clinical diagnosis of aquaporin-4-related diseases.