Rat Middle Cerebral Artery Occlusion Model Research Articles

Intracarotid cold saline infusion contributes to neuroprotection in MCAO‑induced ischemic stroke in rats via serum and glucocorticoid‑regulated kinase 1.

Intracarotid cold saline infusion (ICSI) brings about neuroprotective effects in ischemic stroke. However, the involvement of serum and glucocorticoid‑regulated kinase1 (SGK1) in the underlying mechanism of ICSI is not fully understood; therefore, we used the rat middle cerebral artery occlusion (MCAO) model to investigate the neuroprotective effects of ICSI on ischemic stroke in rats, as well as the involvement of SGK1 in these effects. ICSI decreased infarct size and brain swelling, as determined by 2,3,5‑triphenyltetrazolium chloride staining and the dry‑wet weight method, respectively. The results of terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and Nissl staining showed that ICSI also suppressed apoptosis and increased the relative integral optical density (IOD) values of Nissl bodies in the rat MCAO model. Regarding the mechanism, the results of immunohistochemistry and western blotting revealed that ICSI upregulated SGK1 expression and downregulated beclin‑1 and LC‑3 expression in the rat MCAO model. In addition, SGK1 knockdown increased ICSI‑mediated infarct size and brain swelling, promoted apoptosis, and reduced the IOD values of Nissl bodies in the rat MCAO model. In addition, we found that SGK1 knockdown upregulated beclin‑1 and LC‑3 expression mediated by ICSI. Overall, ICSI had a neuroprotective effect on ischemic stroke after reperfusion by upregulating SGK1 and inhibiting autophagy.

Therapeutic effect of transplantation of human bone marrow‑derived mesenchymal stem cells on neuron regeneration in a rat model of middle cerebral artery occlusion.

Human bone marrow-derived mesenchymal stromal cells (hBMSCs) have been revealed to be beneficial for the regeneration of tissues and cells in several diseases. The present study aimed to elucidate the mechanisms underlying the effect of hBMSC transplantation on neuron regeneration in a rat model of middle cerebral artery occlusion (MCAO). The hBMSCs were isolated, cultured and identified. A rat model of MCAO was induced via the modified Longa method. Neurological severity scores (NSS) were adopted for the evaluation of neuronal function in the model rats after cell transplantation. Next, the expression levels of nestin, β-III-tubulin (β-III-Tub), glial fibrillary acidic protein (GFAP), HNA and neuronal nuclear antigen (NeuN) were examined, as well as the positive expression rates of human neutrophil alloantigen (HNA), nestin, NeuN, β-III-Tub and GFAP. The NSS, as well as the mRNA and protein expression of nestin, decreased at the 1st, 2nd, 4 and 8th weeks, while the mRNA and protein expression of NeuN, β-III-Tub and GFAP increased with time. In addition, after treatment, the MCAO rats showed decreased NSS and mRNA and protein expression of nestin, but elevated mRNA and protein expression of NeuN, β-III-Tub and GFAP at the 2nd, 4 and 8th weeks, and decreased positive expression of HNA and nestin with enhanced expression of NeuN, β-III-Tub and GFAP. Therefore, the present findings demonstrated that hBMSC transplantation triggered the formation of nerve cells and enhanced neuronal function in a rat model of MCAO.

Polydatin Attenuates Neuronal Loss via Reducing Neuroinflammation and Oxidative Stress in Rat MCAO Models.

Ischemic stroke is characterized by permanent or transient obstruction of blood flow, which initiates a cascading pathological process, starting from acute ATP loss and ionic imbalance to subsequent membrane depolarization, glutamate excitotoxicity, and calcium overload. These initial events are followed by neuroinflammation and oxidative stress, eventually causing neuronal neurosis and apoptosis. Complicated interplays exist between these steps happening across various stages, which not only represent the complicated nature of ischemic pathology but also warrant a detailed delineation of the underlying molecular mechanisms to develop better therapeutic options. In the present study, we examined the neuroprotective effects of polydatin against ischemic brain injury using a rat model of permanent middle cerebral artery occlusion (MCAO). Our results demonstrated that polydatin treatment reduced the infarction volume and mitigated the neurobehavioral deficits, sequentially rescued neuronal apoptosis. Ischemic stroke induced an elevation of neuroinflammation and reactive oxygen species, which could be attenuated by polydatin via the reduced activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. In addition, polydatin upregulated the endogenous antioxidant nuclear factor erythroid 2-related factor 2, heme oxygenase-1, the thioredoxin pathway, and eventually reversed ischemic-stroke-induced elevation of ROS and inflammation in ischemic cortical tissue. The diverse and broad actions of polydatin suggested that it could be a multiple targeting neuroprotective agent in ameliorating the detrimental effects of MCAO, such as neuroinflammation, oxidative stress, and neuronal apoptosis. As repetitive clinical trials of neuroprotectants targeting a single step of stroke pathological process have failed previously, our results suggested that a neuroprotective strategy of acting at different stages may be more advantageous to intervene in the vicious cycles in MCAO.

The differential neuroprotection of HSP70-hom gene single nucleotide polymorphisms: In vitro (neuronal hypoxic injury model) and in vivo (rat MCAO model) studies

To investigate the effect of HSP70-hom+2437 single nucleotide polymorphisms (SNPs) on hypoxia and ischemia condition, we constructed the neuronal hypoxic injury model and the rat middle cerebral artery occlusion (MCAO) model to compare the inhibition rate of neurons and detect the infarct volume as well as the expression of related apoptotic proteins in order to explore the possible mechanisms. The neuroblastoma cells SHSY5Y were divided into the OE (transfected with the C allele) group, OEmu (transfected with the T allele) group and negative control (NC, transfected with the empty lentiviral vector CON195) group. Varying degrees of hypoxia were induced by deferoxamine (DFO). The inhibition rate of hypoxic neurons and the expression of related apoptotic proteins were detected in the three genotype groups. While in the rat MCAO model, we built five groups including the sham group, the blank control group (injected with physiological saline), the negative control group (injected with lentivirus and physiological saline), the C allele group and the T allele group (injected with lentivirus overexpressing C and T allele). The MCAO model operation was then underwent in all five groups, the infarct volume by TTC staining and the expression of related apoptotic proteins were detected after 24 h. The results in neuronal hypoxic injury model showed a significant difference in the inhibition rate between the three groups (P < 0.05), and the average inhibition rates for the OEmu, OE and NC groups were 13.2%, 19.2% and 23.3%, respectively. The inhibition rates also differed between lower and higher DFO concentrations (P < 0.05). Compared with the NC group, Bax decreased significantly in the OE and OEmu groups, whereas PI3K and HSPA1L (HSP70-hom) increased. However, the expression of Bax in the OEmu group decreased significantly more than in the OE group, whereas PI3K and HSPA1L levels showed no difference between the two groups. Corresponding with the results above, overexpressing HSP70-hom could reduce the infarct volume of ischemic injury by TTC staining in rat MCAO model and the T allele group also had less infarct volume than C allele group. Compared with the sham group, blank control group and negative control group, Bax decreased significantly in the C and T allele groups, while HSPA1L and p- AKT increased. Furthermore, the expression of Bax in the T allele group decreased significantly more than that in the C allele group, while there were no significant differences in HSPA1L and p-AKT levels between the two groups. Therefore, the overexpression of HSP70-hom+2437 could play a protective role in hypoxic neurons and ischemic brain tissue by upregulating the expression of HSPA1L and PI3K/p-AKT and downregulating the expression of BAX. The neuroprotective effect of the T allele was stronger than that of the C allele, which may be related to the strengthened downregulation of BAX.

A NMR-Based Metabonomics Approach to Determine Protective Effect of a Combination of Multiple Components Derived from Naodesheng on Ischemic Stroke Rats.

Naodesheng (NDS) is a widely used traditional Chinese medicine (TCM) prescription for the treatment of ischemic stroke. A combination of 10 components is derived from NDS. They are: Notoginsenoside R1, ginsenoside Rg1, ginsenoside b1, ginsenoside Rd, hydroxysafflor yellow A, senkyunolide I, puerarin, daidzein, vitexin, and ferulic acid. This study aimed to investigate the protective effect of the ten-component combination derived from NDS (TCNDS) on ischemic stroke rats with a middle cerebral artery occlusion (MCAO) model by integrating an NMR-based metabonomics approach with biochemical assessment. Our results showed that TCNDS could improve neurobehavioral function, decrease the cerebral infarct area, and ameliorate pathological features in MCAO model rats. In addition, TCNDS was found to decrease plasma lactate dehydrogenase (LDH) and malondialdehyde (MDA) production and increase plasma superoxide dismutase (SOD) production. Furthermore, 1H-NMR metabonomic analysis indicated that TCNDS could regulate the disturbed metabolites in the plasma, urine, and brain tissue of MCAO rats, and the possible mechanisms were involved oxidative stress, energy metabolism, lipid metabolism, amino acid metabolism, and inflammation. Correlation analysis were then performed to further confirm the metabolites involved in oxidative stress. Correlation analysis showed that six plasma metabolites had high correlations with plasma LDH, MDA, and SOD. This study provides evidence that an NMR-based metabonomics approach integrated with biochemical assessment can help to better understand the underlying mechanisms as well as the holistic effect of multiple compounds from TCM.

Neuroprotection by 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside extracts from Polygonum multiflorum against cerebral ischemia/reperfusion injury through the 5-hydroxytryptamine/5-hydroxytryptamine receptor pathway.

Objective: To investigate the therapeutic effect of 2,3,5,4ʹ-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) on the expression of 5-hydroxytryptamine (5-HT)/5-HT receptor 2A (5-HT2A), 5-HT transporter (5-HTT), and uncoupling protein 4 (UCP4) after cerebral ischemia/reperfusion (I/R) injury.Methods: Sprague–Dawley rats were randomly divided into control, model and 125 (low-dose), 250 (middle-dose), and 500 (high-dose) mg/mL TSG groups. Rat cerebral I/R injury model was established by middle cerebral artery occlusion (MCAO). After successful establishment of rat MCAO model, rats in control and model groups were decapitated immediately. Rats in TSG group were orally administered 125, 250, and 500 mg/mL TSG in corresponding groups at a dose of 1 mL/100 g per day for 7 continuous days, and then the rats were decapitated. The infarct size was determined using triphenyl tetrazolium chloride staining and the expression of UCP4 and 5-HT2A in the hippocampus and thalamic nucleus was detected using immunohistochemistry and western blot assay. The expression of 5-HTT in brain tissue was detected using western blot assay. Serum 5-HT levels were detected using ELISA.Results: After treatment, the infarct size due to cerebral I/R injury decreased with increased concentrations of TSG. Synchronous reduction of 5-HT in the blood and 5-HTT in the brain was observed, and 5-HT2A was expressed in normal brain tissue but its level was increased in rats after cerebral I/R injury. A high level of UCP4 was found in normal brain tissue, which rose by 6 hrs after cerebral I/R injury but reduced to minimal levels 24 hrs after injury. With increasing TSG concentration, the levels of 5-HT, 5HTT, and UCP4 were increased, while the level of 5-HT2A was decreased.Conclusion: TSG is effective in treating cerebral I/R injury in rats, and its mechanism may be implemented through the 5-HT/5-HTR pathway, by increasing 5-HT release, enhancing the activity of 5-HTT, increasing expression of UCP4, and inhibiting 5-HT2A activity.

Reduced tongue force and functional swallowing changes in a rat model of post stroke dysphagia

PurposeDysphagia is a common problem after stroke that is often associated with tongue weakness. However, the physiological mechanisms of post-stroke tongue muscle weakness and optimal treatments have not been established. To advance understanding of physiological mechanisms of post stroke dysphagia, we sought to validate the unilateral transient middle cerebral artery occlusion (MCAO) rat model of ischemic stroke as a translational model of post stroke dysphagia. Our goal was to establish clinically relevant measures and chronicity of functional deficits; criteria that increase the likelihood that findings will translate to the clinic. We hypothesized that MCAO would cause tongue weakness and functional swallowing changes. MethodsMaximum voluntary tongue forces and videofluoroscopic swallowing studies were collected in 8-week old male Sprague-Dawley rats prior to receiving either a left MCAO (N = 10) or sham (N = 10) surgery. Tongue forces and VFSS were reassessed at 1 and 8 weeks post-surgery. ResultsMaximum voluntary tongue force, bolus area, and bolus speed were significantly reduced in the MCAO group at the 1 and 8-week timepoints. ConclusionClinically relevant changes to swallowing and tongue force support the use of the MCAO rat model as a translational model of post stroke dysphagia. This model will allow for future studies to improve our understanding of the physiology contributing to these functional changes as well as the impact of therapeutic interventions on physiological targets and function.

Post-stroke pain caused by peripheral sensory hypersensitization after transient focal cerebral ischemia in rats

The mechanisms underlying central post-stroke pain are not well understood and there is no satisfactory treatment. Here, in a rat model of stroke, we measured nociceptive threshold using current stimulation of primary afferent neurons in both hind paws. Male Wistar rats underwent middle cerebral artery occlusion (MCAO) for 50 min. Nociceptive thresholds for Aβ, Aδ and C fiber stimulation (at 2000, 250, and 5 Hz, respectively, using a Neurometer), and neurological deficits, were measured for 23 days after MCAO. Sensory thresholds in both hind paws were significantly lower in MCAO model rats than in control rats for 23 days after MCAO, with the greatest difference seen in Aδ fibers and the smallest in C fibers. Brain infarct area was measured histologically, and the correlation between neurological deficit and infarct size was examined. Neurological deficits were worse in animals with larger infarcts. Furthermore, correlations were observed between infarct size, neurological deficit, and sensory threshold of Aδ fibers 1 day after MCAO. These findings indicate that rats develop hyperalgesia after MCAO and that sensory abnormalities in Aδ fibers after cerebral ischemia may play an important role in post-stroke pain.

MiR‑335 promotes stress granule formation to inhibit apoptosis by targeting ROCK2 in acute ischemic stroke.

Under harmful environmental conditions, stress granules (SGs), macromolecular aggregates that are associated with cell survival and death, are produced in the eukaryotic cytoplasm. However, whether and how microRNAs (miRNAs/miRs) modulate SG formation induced by acute ischemic stroke has not been investigated. In the present study, a rat model of middle cerebral artery occlusion (MCAO) was utilized and miRNA array profiling and reverse transcription-quantitative polymerase chain reaction were performed. The results revealed that miR-335 was downregulated during acute ischemic stroke, which was concomitant with reduced SG formation, enhanced apoptosis levels and increased Rho associated protein kinase 2 (ROCK2) expression. In the MCAO rat and serum-free cell models, miR-335 treatment upregulated SG formation, alleviated the ischemia-induced infarction, and decreased ROCK2 protein expression and apoptosis levels. By contrast, when compared with miR-335 treatment, the inhibition of miR-335 resulted in reduced SG formation and higher ROCK2 expression and apoptosis levels. Target prediction analysis and luciferase 3′-untranslated region reporter assay identified ROCK2 as the direct target of miR-335. Furthermore, ROCK2 silencing enhanced SG formation and attenuated the level of apoptosis in the serum-free cell model. In addition, ROCK2 silencing markedly inhibited the effect of miR-335 on SG formation and apoptosis levels. Unexpectedly, the phosphorylation of T-cell intracellular antigen-1 was significantly inhibited by miR-335 in the MCAO rat model, which provides a reasonable explanation for the promotional effect of miR-335 on SG formation by specifically targeting ROCK2. In conclusion, these results demonstrate that miR-335 promotes SG formation and inhibits apoptosis by reducing ROCK2 expression in acute ischemic stroke, which provides a possible therapeutic target for brain injury.

Excessive Expression of Microglia/Macrophage and Proinflammatory Mediators in Olfactory Bulb and Olfactory Dysfunction After Stroke.

Olfactory dysfunction can be caused by stroke but the pathogenesis is still unclear. Previous studies have proved that olfactory dysfunction could be caused by microglia activation in the olfactory bulb and that middle cerebral artery occlusion (MCAO) may induce ipsilateral olfactory bulb microglia activation. This study aimed to explore the possible pathogenesis of ischemic stroke-induced olfactory dysfunction. We used a rat model of MCAO to simulate ischemic stroke. Olfactory function tests were performed using buried food test. The mRNA expression of olfactory marker protein (OMP), microglia/macrophage activation, and proinflammatory mediators were measured using reverse transcription-quantitative polymerase chain reaction. Following MCAO, rats had poorer olfactory performance. In the olfactory bulb of the rats, the mRNA expression of OMP decreased and the mRNA expression of microglia/macrophage activation and proinflammatory mediators increased. Ischemic stroke causes microglia/macrophage activation and promotes neuroinflammation in the olfactory bulb, causing olfactory dysfunction.

Intranasal Calcitonin Gene-Related Peptide Protects Against Focal Cerebral Ischemic Injury in Rats Through the Wnt/β-Catenin Pathway.

BackgroundIntranasal calcitonin gene-related peptide (CGRP) delivery offers a noninvasive method of bypassing the blood-brain barrier for the delivery of CGRP to the brain. Here, we first reported the therapeutic benefits of intranasal CGRP delivery in rats following middle cerebral artery occlusion (MCAO).Material/MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) assay, enzyme-linked immunosorbent assay (ELISA), rat MCAO model, TTC (2, 3, 5-triphenyltetrazolium chloride) staining, hematoxylin and eosin (H & E) staining, Morris water maze test, TUNEL assay, immunofluorescence, and western blot assay were used to investigate the role of CGRP in rats. Cell Counting Kit-8 assay, colony formation assay, cell cycle assay, apoptosis assay, western blot assay, and TOP/FOP assay were used to investigate the role of CGRP in normal human astrocytes (NHA) cells.ResultsThe CGRP-MCAO-NDDS (nasal drug delivery system) group showed a significant reduction in the infarct volume and improvement in neurologic deficit tests of motor, sensory, reflex and vestibulo-motor functions compared to those rats in the CGRP-MCAO-IV group. CGRP markedly inhibited apoptosis and increased the expression of vascular endothelial growth factor (VEGF) and bFGF and decreased the expression of GAP43 in the cortex of MCAO rats. CGRP promoted cell proliferation and cell cycle process and inhibited cell apoptosis through the Wnt/β-catenin pathway in NHA cells.ConclusionsThis noninvasive, simple, and cost-effective method is a potential treatment strategy for focal cerebral ischemic injury.

Targeted Temperature Management at 33°C or 36°C Produces Equivalent Neuroprotective Effects in the Middle Cerebral Artery Occlusion Rat Model of Ischemic Stroke.

Targeted temperature management (TTM, 32°C to 36°C) is one of the most successful achievements in modern resuscitation medicine. It has become standard treatment for survivors of sudden cardiac arrest to minimize secondary brain damage. TTM at 36°C is just as effective as TTM at 33°C and is actually preferred because it reduces adverse TTM-associated effects. TTM also likely has direct neuroprotective effects in ischemic brains in danger of stroke. It remains unclear, however, whether higher temperature TTM is equally effective in protecting the brain from the effects of stroke. Here, we asked whether TTM at 36°C is as effective as TTM at 33°C in improving outcomes in a middle cerebral artery occlusion (MCAO) model of ischemic stroke. After dividing rats randomly into MCAO, MCAO+33°C TTM, MCAO+36°C TTM, and sham groups, we subjected all of them except for the sham group to MCAO for 3 h (for the behavioral tests) or 4 h (for all other biochemical analyses). We found TTM protocols at both 33°C and 36°C to produce comparable reductions of infarct volumes in the MCAO territory and equally attenuate the extracellular release of high mobility group box 1 in postischemic brains. Both the TTM conditions prevent the mRNA induction of a major pro-inflammatory cytokine, tissue necrosis factor-α, in the ischemic penumbra region. Finally, both the TTM protocols produce similar improvements in neurological outcomes in rats, as measured by a battery of behavior tests 21 h after the start of reperfusion. These data acquired in a rat MCAO model suggest TTM at 36°C has excellent therapeutic potential for improving clinical outcomes for patients with acute ischemic stroke.

Reliability of behavioral tests in the middle cerebral artery occlusion model of rat.

Stroke is one of the leading causes of death and disability worldwide, and its incidence is increasing. To overcome impairment from stroke, translational research for developing new therapeutic technologies has been conducted and middle cerebral artery occlusion (MCAo) in rat is the representative model. Since recovery from neurological impairment in contralateral limbs caused by brain damage is the major goal of treatment, behavioral tests that assess the relevant function are used. To determine therapeutic effect, obtaining reliable results of behavioral assessment is a prerequisite. However, studies on the reliability of behavioral tests in the MCAo rat model and necessity of prior training have not yet been reported. In this study, the authors investigate relative and absolute inter-rater reliabilities of modified neurological severity score (mNSS), cylinder test, and grid-walking test before training and repeated training every week until the reliability of results reached a satisfactory level. The training included repeated learning of the scoring system and decreasing disagreements among the raters. For MCAo modeling, adult male Sprague-Dawley rats were subjected to 90 min of transient MCAo. Six raters conducted behavioral tests via observation of video-recording on sham-operated and MCAo model rats at 3 or 7 days after the intervention. An independent experimenter randomly numbered each video clip to blind the experiment. The results of reliabilities were unacceptable before training and improved to a satisfactory level after 6 weeks of training in all of the tests. In conclusion, mNSS, cylinder test, and grid-walking test on the MCAo rat model are reliable evaluation methods after conducting appropriate training.

A Mesenchymal stem cell line (B10) increases angiogenesis in a rat MCAO model

A human mesenchymal stem cell line (B10) transplantation has been shown to improve ischemia-induced neurological deficits in animal stroke models. To understand the underlying mechanism, we have investigated the effects of B10 transplantation on cerebral angiogenesis in a rat middle cerebral artery occlusion (MCAO) model. B10 cells were transplanted intravenously 24 h after MCAO. Immunofluorescence staining results showed that compared to PBS-groups, vWF positive vessel and endoglin positive new vessels were increased in B10-transplanted MCAO groups in the lesion areas. The mRNA of angiogenesis factors including placental growth factor and hypoxia inducible factor (HIF)-1α were increased 3 days after MCAO in the core and IBZ areas of B10-transplanted group. Angiopoetin1 mRNA was increased only in the IBZ. Western blotting results showed that HIF-1α and vascular endothelial growth factor (VEGF) proteins were increased in B10-transplanted group. Both HIF-1α and VEGF were expressed in macrophage/microglia in the core area. In the IBZ, however, HIF-1α was expressed both in astrocytes and macrophage/microglia, while VEGF was expressed only in macrophage/microglia. Moreover, TGFβ protein levels were found to be increased in B10-transplanted group in the core and IBZ regions. Cell culture experiments using a human microglia cell line (HMO6) and B10 showed that IL-1β induced VEGF mRNA expression in both cell types. IL-1β was found to be highly expressed in B10 cells, and its co-culture with HMO6 further increased that in B10. Co-culture increased VEGF mRNA in both B10 and HMO6. In the rat brains, IL-1β was expressed in macrophage/microglia and transplanted-B10 cells in the core. IL-1β positive cell number was increased slightly, but significantly in B10-transplanted rats. To explore further, IL-1β expression was silenced in B10 cells by transfecting mRNA specific siRNA, and then transplanted in MCAO rats. Immunostaining result showed that endoglin positive area was decreased in IL-1β-silenced B10 transplanted groups compared to nonsilenced-B10 transplanted groups. Interestingly, vessel-like structure appeared as early as 3 days after MCAO in IL-1β-silenced B10-transplanted group. Thus our results demonstrated that B10 cells increased angiogenesis in MCAO rat model, through the regulation of HIF-1α and VEGF expression, where IL-1β might play a role.

Oncostatin M-induced upregulation of SDF-1 improves Bone marrow stromal cell migration in a rat middle cerebral artery occlusion stroke model

Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit potential regenerative effects on the injured brain. However, these effects are constrained by their limited ability to migrate to the injured site. Oncostatin M (OSM) has been shown to affect the proliferation and migration of mesenchymal stem cells. Therefore, in the present study, we explored whether OSM improves BMSC migration and secretion of growth factors and cytokines in a rat middle cerebral artery occlusion (MCAO) stroke model. The effect of OSM on the proliferation and apoptosis of rat BMSCs was first assessed in vitro, and the gene and secretion levels of factors related to cell nutrition and migration, such as SDF-1 and VEGF, were detected. To further explore underlying pathways triggered by OSM, BMSCs were treated with OSM in the presence or absence of inhibitors of the STAT3 and ERK pathways. Effects of OSM on SDF-1 expression in astrocytes and BMSC migration were also evaluated. In the rat MCAO model, OSM secretion levels were detected in the brain for up to 72 h after model establishment. Ventricle injection of OSM alone or OSM combined with caudal vein graft of BMSCs was then performed in MCAO stroke rats. After 72 h, production of SDF-1 and grafted BMSCs was detected in the lesion areas of the brain, and the nerve function score was evaluated. We found that the production of OSM continually increased in the brains of MCAO rats from 12 h to 72 h. OSM significantly upregulated SDF-1 in BMSCs via the STAT3 and ERK pathways and significantly promoted the expression of VEGF and MMP-2. OSM also promoted the secretion of SDF-1 in astrocytes through the STAT3 and ERK pathways to in turn enhance BMSC migration. Combination treatment with OSM and BMSCs in MCAO rats increased the migration efficiency of BMSCs in the brain, which significantly improved neurofunctional recovery while reducing the expression of inflammatory mediators and promoting the secretion of nutrition factors. Overall, these results show that OSM is highly expressed in the brains of MCAO stroke rats and can upregulate SDF-1 to promote BMSC migration. Thus, combination treatment with OSM and BMSCs improves the graft efficiency of BMSCs and neurofunctional recovery.

Salvia miltiorrhiza enhances the survival of mesenchymal stem cells under ischemic conditions.

ObjectivesTo validate the enhanced therapeutic effect of Salvia miltiorrhiza Bunge (SM) for brain ischemic stroke through the anti‐apoptotic and survival ability of mesenchymal stem cells (MSCs).MethodsThe viability and the expression level of cell apoptotic and survival‐related proteins in MSCs by treatment of SM were assessed in vitro. In addition, the infarcted brain region and the behavioural changes after treatment of MSCs with SM were confirmed in rat middle cerebral artery occlusion (MCAo) models.Key findingsWe demonstrated that SM attenuates apoptosis and improves the cell viability of MSCs. In the rat MCAo model, the recovery of the infarcted region and positive changes of behaviour are observed after treatment of MSCs with SM.ConclusionsThe therapy using SM enhances the therapeutic effect for brain ischemic stroke by promoting the survival of MSCs. This synergetic effect thereby proposes a new experimental approach of traditional Chinese medicine and stem cell‐based therapies for patients suffering from a variety of diseases.

18F]DPA-714 PET imaging shows immunomodulatory effect of intravenous administration of bone marrow stromal cells after transient focal ischemia

BackgroundThe potential application of bone marrow stromal cell (BMSC) therapy in stroke has been anticipated due to its immunomodulatory effects. Recently, positron emission tomography (PET) with [18F]DPA-714, a translocator protein (TSPO) ligand, has become available for use as a neural inflammatory indicator. We aimed to evaluate the effects of BMSC administration after transient middle cerebral artery occlusion (MCAO) using [18F]DPA-714 PET.The BMSCs or vehicle were administered intravenously to rat MCAO models at 3 h after the insult. Neurological deficits, body weight, infarct volume, and histology were analyzed. [18F]DPA-714 PET was performed 3 and 10 days after MCAO.ResultsRats had severe neurological deficits and body weight loss after MCAO. Cell administration ameliorated these effects as well as the infarct volume. Although weight loss occurred in the spleen and thymus, cell administration suppressed it. In both vehicle and BMSC groups, [18F]DPA-714 PET showed a high standardized uptake value (SUV) around the ischemic area 3 days after MCAO. Although SUV was increased further 10 days after MCAO in both groups, the increase was inhibited in the BMSC group, significantly. Histological analysis showed that an inflammatory reaction occurred in the lymphoid organs and brain after MCAO, which was suppressed in the BMSC group.ConclusionsThe present results suggest that BMSC therapy could be effective in ischemic stroke due to modulation of systemic inflammatory responses. The [18F]DPA-714 PET/CT system can accurately demonstrate brain inflammation and evaluate the BMSC therapeutic effect in an imaging context. It has great potential for clinical application.

Vitexin reverses the autophagy dysfunction to attenuate MCAO-induced cerebral ischemic stroke via mTOR/Ulk1 pathway

Stroke, as a kind of acute cerebrovascular diseases, has greatly influenced the patients’ quality of life and left a huge public health burden. Vitexin is a flavone C-glycoside (apigenin-8-C-β-D-glucopyranoside) present in several medicinal and other plants. This study aims to explore the role of vitexin in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic stroke. The results showed that the MCAO-induced brain infarction was obviously decreased by vitexin. And the abnormal protein levels of Caspase-3, Bcl-2-associated X protein (Bax), antigen identified by monoclonal antibody (Ki-67) and B cell lymphoma 2 (Bcl-2) in MCAO model rats were reversed by vitexin. Further research indicated that vitexin alleviated MCAO-induced oxidative injury by reducing the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA) and nitric Oxide (NO). In addition, vitexin attenuated the secretion of pro-inflammatory cytokine (interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α)) and increased anti-inflammatory cytokine (IL-10) production to ameliorate MCAO-induced inflammation. What’s more, vitexin repressed the MCAO-induced autophagy through mechanistic target of rapamycin (mTOR)/Ulk1 pathway. Specifically, the MCAO-induced decreased expression of mTOR, peroxisome proliferator-activated receptor γ (PPARγ) and p62 were inhibited by vitexin. At the same time, MCAO-induced increased expression of Ulk1, Beclin1 and rate of LC3Ⅱ/LC3Ⅰ also were repressed by vitexin. But the inhibition of vitexin on the MCAO-induced oxidative injury, apoptosis and inflammation were reversed by rapamycin. These results implied that vitexin suppressed the autophagy dysfunction to attenuate MCAO-induced cerebral ischemic stroke via mTOR/Ulk1 pathway.

Lig4-4 selectively inhibits TREK-1 and plays potent neuroprotective roles in vitro and in rat MCAO model

Two-pore domain potassium channel TREK-1 was known to play an important role in neuroprotection, especially in acute cerebral ischemia. In the present study we found that 3-(nitromethyl) isobenzofuran-1(3H)-one (lig4-4) could robustly inhibit TREK-1 currents with an IC50 of 2.06 μM. However, the IC50 of lig4-4 for other ion channel subtypes such as Kv2.1, Kv1.5, Kv3.1, hERG and neuronal Na+ and Ca2+ channels were more than 30 μM, suggesting a specific inhibition of TREK-1 channel. MTT assay showed that lig4-4 significantly enhanced cell viability of cultured neurons under the condition of oxygen and glucose deprivation followed by reoxygenation (OGD/R). Annexin V/Propidium Iodide apoptosis assay also demonstrated that lig4-4 obviously reduced cell apoptosis in OGD/R-injured neurons. Western blotting results indicated that OGD/R-induced up-regulation of cleaved-caspase-3 expression and down-regulation of Bcl-2 could be notably reversed by lig4-4. The in vivo study showed that after oral administration of lig4-4 at 50 mg/kg, the infarct volume in middle cerebral artery occlusion (MCAO) rat model reduced from a vehicle control of 38.8% to 28%. Both in vitro and in vivo studies suggested that inhibition of TREK-1 with lig4-4 might produce a neuroprotective effect against cerebral ischemia. In conclusion, we demonstrated that lig4-4 selectively inhibited TREK-1 and protected brain from cerebral ischemic injury. The mechanisms might relate to block TREK-1 and inhibit neuronal apoptosis by modulating the expressions of Bcl-2 and cleaved-caspase-3.

By Activating Akt/eNOS Bilobalide B Inhibits Autophagy and Promotes Angiogenesis Following Focal Cerebral Ischemia Reperfusion

Background/Aims: Ischemic stroke is a leading cause of long-term disability. To date, there is no effective treatment for stroke. Previous studies have shown that Ginkgo biloba extract has protective effects against neurodegenerative disorders. In this present study, we sought to test the potential protective role of an active component of Ginkgo biloba extract, bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). Methods: A rat model of MCAO was used to test the potential protective effects of Bilobalide B on stroke protection. TTC staining was performed to evaluate infarct size of the brains. Neurological deficit score was measured to reveal the effects of the treatments on animal behavior and cognition. Immunohistochemical staining and transmission electronic microscope analysis were performed to measure the cellular responses to drug treatment. Western blotting and ELISA were performed. The expression of Cleaved- Casepase 3, Beclin-1, p62 and LC3I/II were quantified, and the Phosphorylation of eNOS and Akt were evaluated. The ratio of Bcl-2/ Bax was determined to reveal the molecular pathways that are involved in the drug treatment. Results: We found that intraperitoneal delivery of various Bilobalide doses during ischemia can protect against brain injury, as evidenced by reduced infarct size and improved neurological scores after surgery. Histochemical analysis revealed that treatment with bilobalide can significantly reduce apoptosis, autophagy, and promote angiogeneis following ischemia/reperfusion injury to the brain. The performence of increased phosphorylation of eNOS and Akt suggested that bilobalide can activate Akt prosurvival and eNOS pathways to promote cell survival and angiogenesis, respectively. Conclusions: Our results suggested that bilobalide benefits stroke symptoms by reducing cell death pathways and promoting angiogenesis. As such, bilobalide may be a potential agent for improving self-repair after ischemic stroke.