Focal Middle Cerebral Artery Occlusion Research Articles

Maresin1 alleviates neuroinflammation by inhibiting caspase-3/ GSDME-mediated pyroptosis in mice cerebral ischemia-reperfusion model

ObjectiveTo explore the mechanism of Maresin1 in reducing cerebral ischemia-reperfusion injury. Materials and methodsMale C57BL/6 mice were randomly divided (n = 5 in each group), and focal middle cerebral artery occlusion (MCAO) model was used to simulate cerebral ischemia/reperfusion injury. TTC and the Longa score were used to detect the degree of neurological deficits. Western blot was used to detect the expression levels of GSDME, GSDME-N, caspase-3 and cleaved caspase-3 in cerebral ischemic penumbra tissue, and immunofluorescence was used to detect the expression levels of GSDME-N. The mRNA expression levels of GSDME and pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) were detected by RT-PCR. ResultsCompared with sham group, GSDME mRNA levels in MCAO group were significantly increased at 12 h and 24 h after reperfusion, and GSDME and GSDME-N significantly increased at 6–48 h after reperfusion. Compared with sham group, the percentage of infarct size, the Longa score, the mRNA expression levels of IL-1β, IL-6 and TNF-α, and GSDME, GSDME-N, caspase-3 and cleaved caspase-3 in MCAO group was significantly increased. Then, the percentage of infarct size and the Longa score significantly decreased after MaR1 administration, the mRNA expression levels of IL-1β and IL-6 downregulated, and GSDME, GSDME-N, caspase-3 and cleaved caspase-3 were also reduced. After administration of Z-DEVD-FMK(ZDF), the expression of caspase-3, cleaved caspase-3 and GSDME-N was decreased, which in MCAO+MaR1+ZDF group was not statistically significant compared with MCAO+ ZDF group. ConclusionMaresin1 alleviates cerebral ischemia/reperfusion injury by inhibiting pyroptosis mediated by caspase-3/GSDME pathway and alleviating neuroinflammation.

Matrix Metalloproteinase-9 Contributes to Epilepsy Development after Ischemic Stroke in Mice.

Epilepsy, a neurological disorder affecting over 50 million individuals globally, is characterized by an enduring predisposition and diverse consequences, both neurobiological and social. Acquired epilepsy, constituting 30% of cases, often results from brain-damaging injuries like ischemic stroke. With one third of epilepsy cases being resistant to existing drugs and without any preventive therapeutics for epileptogenesis, identifying anti-epileptogenic targets is crucial. Stroke being a leading cause of acquired epilepsy, particularly in the elderly, prompts the need for understanding post-stroke epileptogenesis. Despite the challenges in studying stroke-evoked epilepsy in rodents due to poor long-term survival rates, in this presented study the use of an animal care protocol allowed for comprehensive investigation. We highlight the role of matrix metalloproteinase-9 (MMP-9) in post-stroke epileptogenesis, emphasizing MMP-9 involvement in mouse models and its potential as a therapeutic target. Using a focal Middle Cerebral Artery occlusion model, this study demonstrates MMP-9 activation following ischemia, influencing susceptibility to seizures. MMP-9 knockout reduces epileptic features, while overexpression exacerbates them. The findings show that MMP-9 is a key player in post-stroke epileptogenesis, presenting opportunities for future therapies and expanding our understanding of acquired epilepsy.

Compatibility of ingredients of Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) and their protective effects on cerebral ischemia-reperfusion injury in rats.

Danshen (Radix Salviae Miltiorrhizae) and Honghua (Flos Carthami) (Danhong) are two drugs commonly prescribed together, which are often used in the treatment of cerebrovascular diseases in China. Due to the complexity of the ingredients of Danhong, the present study focused on performing the orthogonal compatibility method on the primary effective molecules of this drug: Tanshinol, salvianolic acid A, salvianolic acid B and hydroxysafflor yellow A. These four molecules were studied to determine their protective effects and to screen for the most compatible ingredients to improve cerebral ischemia-reperfusion injury (IR) in rats. Focal middle cerebral artery occlusion was performed to establish the cerebral IR model in rats. Male Sprague-Dawley rats were randomly divided into sham operation group, IR group and nine orthogonal administration groups with different ratios of Danhong effective ingredients and Danhong injection group. Neurological deficit score and cerebral infarction volume were measured postoperatively. Morphological pathological alterations were observed via H&E staining. Bcl-2 and Bax were quantified using ELISA. Immunohistochemistry was conducted to analyze the expression of caspase-3 in the hippocampus. The expression levels of cytochrome c, apoptotic peptidase activating factor 1 (apaf-1), caspase-9, caspase-3 and p53 mRNA in the hippocampus were assessed via reverse transcription-quantitative PCR. The results demonstrated that different compatibility groups significantly reduced the neurological function score and decreased the volume of cerebral infarct compared with the IR group. These groups were also indicated to improve the pathological damage to the brain tissue. In addition, certain compatibility groups significantly decreased the number of caspase-3 positive cells in the hippocampus and the expression levels of cytochrome c, apaf-1, caspase-9, caspase-3 and p53 mRNA in the brain tissue. Orthogonal group 4 (30 mg/kg tanshinol; 2.5 mg/kg salvianolic acid A; 16 mg/kg salvianolic acid B; 8 mg/kg hydroxysafflor yellow A) was indicated to be the most effective. The four effective ingredients of Danhong exhibited a protective effect on rats with cerebral IR injury, potentially through the inhibition of apoptosis via the downregulation of key targets upstream of the caspase-3 pathway. In addition, the present study provided novel insights for the continued study of the drug compatibility rules of TCM.

Inhibition of PDE1-B by Vinpocetine Regulates Microglial Exosomes and Polarization Through Enhancing Autophagic Flux for Neuroprotection Against Ischemic Stroke.

Exosomes contribute to cell–cell communications. Emerging evidence has shown that microglial exosomes may play crucial role in regulation of neuronal functions under ischemic conditions. However, the underlying mechanisms of microglia-derived exosome biosynthesis are largely unknown. Herein, we reported that the microglial PDE1-B expression was progressively elevated in the peri-infarct region after focal middle cerebral artery occlusion. By an oxygen-glucose-deprivation (OGD) ischemic model in cells, we found that inhibition of PDE1-B by vinpocetine in the microglial cells promoted M2 and inhibited M1 phenotype. In addition, knockdown or inhibition of PDE1-B significantly enhanced the autophagic flux in BV2 cells, and vinpocetine-mediated suppression of M1 phenotype was dependent on autophagy in ischemic conditions. Co-culture of BV2 cells and neurons revealed that vinpocetine-treated BV2 cells alleviated OGD-induced neuronal damage, and treatment of BV2 cells with 3-MA abolished the observed effects of vinpocetine. We further demonstrated that ischemia and vinpocetine treatment significantly altered microglial exosome biogenesis and release, which could be taken up by recipient neurons and regulated neuronal damage. Finally, we showed that the isolated exosome per se from conditioned BV2 cells is sufficient to regulate cortical neuronal survival in vivo. Taken together, these results revealed a novel microglia-neuron interaction mediated by microglia-derived exosomes under ischemic conditions. Our findings further suggest that PDE1-B regulates autophagic flux and exosome biogenesis in microglia which plays a crucial role in neuronal survival under cerebral ischemic conditions.

ShRNA‐mediated gene silencing of t‐PA prevents BBB disruption and elevation of MMP‐12 after ischemic stroke

Post‐stroke induction of endogenous tissue‐type plasminogen activator (t‐PA) is known to promote BBB disruption after ischemic stroke, which contributes to edema, inflammation, hemorrhagic transformations and brain damage. We presently evaluated if this effect of t‐PA leads to the elevation of MMP‐12 in the ischemic brain. Cohorts of adult, male rats were administered with plasmids that express either a t‐PA shRNA or a scrambled control shRNA (1 mg/Kg; i.v.; n=9–13/group) formulated as nanoparticles at 5 min of reperfusion following transient focal middle cerebral artery occlusion. We also have sham‐operated control and untreated ischemia‐induced cohorts. As compared to control shRNA cohort, t‐PA shRNA treated cohort prevented the degradation of tight junction proteins claudin‐5 and ZO‐1, curtailed BBB disruption (studied by Evans blue extravasation into brain), and showed significantly reduced MMP‐12 expression. These results indicate that post‐stroke induced endogenous t‐PA mediated BBB disruption contributes to elevation of MMP‐12 in the ischemic brain.Support or Funding InformationThis work was supported by the NIH Grant 1R01NS102573‐01A1 to KKV.

Left-right asymmetry influenced the infarct volume and neurological dysfunction following focal middle cerebral artery occlusion in rats.

ObjectiveTo investigate the differential effects of left versus right cerebral hemisphere on the infarct volume and behavioral function following focal cerebral ischemia in rats.Methods and MaterialsMiddle cerebral artery occlusion (MCAO) was induced in the right‐handed rats by filament insertion for 1.5 hr, and then reperfusion was established according to Zea‐Longa method. A total of 36 male Sprague Dawley rats were randomly divided into a left MCAO group or a right MCAO group. The modified neurological severity scores (mNSS), tapered beam‐walking test, and Morris water maze experiment were all carried out to evaluate the sensorimotor and cognitive outcomes at the 1d, 3d, and 7d after MCAO, respectively. Infarct volume of the brains was measured by triphenyltetrazolium chloride (TTC) staining.ResultsThe sensorimotor function was more worse in the left MCAO group than that in the right MCAO group at the 1d, 3d, and 7d after MCAO (p < 0.05). While the cognitive function was much better in the left MCAO group than that in the right MCAO group at the 1d and 3d after MCAO (p < 0.05). But no significant difference was achieved in cognitive function between the two groups at 7d after MCAO (p > 0.05). There was no significant difference in total infarct volume between the two groups at the 1d, 3d, and 7d after MCAO, respectively (p > 0.05).ConclusionThe infarct volume is not affected significantly by the left or right MCAO model in the early days. The lesions in the left hemisphere produce more severe sensorimotor impairments, while more severe cognitive impairments are produced by the right hemispherical lesions. These findings suggest that it is structural and functional asymmetry between the two hemispheres other than infarct volume that affects the outcomes of rat MCAO.

Therapeutic time window for conivaptan treatment against stroke-evoked brain edema and blood-brain barrier disruption in mice.

BackgroundIschemic stroke is often complicated by brain edema, disruption of blood-brain barrier (BBB), and uncontrolled release of arginine-vasopressin (AVP). Conivaptan, a V1a and V2 receptor antagonist, reduces brain edema and minimizes damage to the blood-brain barrier after stroke. Most stroke patients do not receive treatment immediately after the onset of brain ischemia. Delays in therapy initiation may worsen stroke outcomes. Therefore, we designed a translational study to explore the therapeutic time window for conivaptan administration.MethodsMice were treated with conivaptan beginning 3, 5, or 20 hours after 60-minute focal middle cerebral artery occlusion. Treatments were administered by continuous IV infusion for a total of 48 hours. Brain edema and blood-brain barrier (BBB) disruption were evaluated at endpoint.ResultsConivaptan therapy initiated at 3 hours following ischemia reduced edema in the ipsilateral hemisphere, which corresponded with improvements in neurological deficits. Stroke-triggered BBB disruption was also reduced in mice when conivaptan treatments were initiated at 3 hours of reperfusion. However, 5 and 20-hour delays of conivaptan administration failed to reduce edema or protect BBB.ConclusionTiming of conivaptan administration is important for successful reduction of brain edema and BBB disruption. Our experimental data open new possibilities to repurpose conivaptan, and make an important “bench-to-bedside translation” of the results into clinical practice.

Optimization of behavioural tests for the prediction of outcomes in mouse models of focal middle cerebral artery occlusion

Intraluminal middle cerebral artery occlusion (MCAO) is the most widely used model of stroke. We aimed to predict the outcome of MCAO using a combination of fine behavioural tests for the prediction of unsuccessful surgery in mice leading to no infarction, haemorrhage and unexpected death. MCAO was performed on adult mice under the guidance of laser-Doppler flowmetry (LDF) to warrant a decrease in regional cerebral blood flow (rCBF) in the MCA territory. Four outcomes of MCAO were defined according to histological analysis: infarction, no infarction, haemorrhage and unexpected death (death within 24h post-surgery). Fine behavioural tests including the rotarod, modified neurological severity score (mNSS), Clark general and Clark focal tests were performed separately at 6h, 12h and 24h post-stroke. A total of 94 mice were included in the analysis. The infarction rate associated with MCAO was 58.5% (55/94). After optimization of the timing and behavioural tests, we found that higher Clark focal (>17.5) or higher mNSS scores (>10) were markedly related to early death, whereas a lower mNSS score (<3.5) was indicative of a tendency to show no infarction at 6h post-stroke. After 24h post-stroke, there was a positive correlation between the infarct volume and Clark focal results. Behavioural tests could help to predict the outcomes in the MCAO mouse model.

Comparisons between Garcia, Modo, and Longa rodent stroke scales: Optimizing resource allocation in rat models of focal middle cerebral artery occlusion

The use of rodent stroke models allow for the understanding of stroke pathophysiology. There is currently no gold standard neurological assessment to measure deficits and recovery from stroke in rodent models. Agreement on a universal preclinical stroke scale allows for comparison of the outcomes among conducted studies. The present study aimed to compare three routinely used neurological assessments in rodent studies (i.e., Garcia, Modo, and Longa) to determine which is most effective for accurately and consistently quantifying neurological deficits in the context of focal middle cerebral artery occlusion (MCAo) in rats. Focal MCAo was induced in 22 male Wistar rats using a novel transfemoral approach. Rodents were assessed for neurological deficit pre-injury as well as 3 and 24h post-injury. Data was analyzed to determine Pearson correlation coefficients in addition to McNemar's χ2 values between each pair of neurological assessments. All three stroke scales, Garcia, Modo, and Longa, showed statistically significant changes between the baseline and the 3-hour neurological assessments. A trend towards neurological recovery was observed in all three stroke scales between the 3 and 24-hour endpoints. The three scales were highly correlated with each other, with Garcia and Modo having the strongest correlation. Of the three pairwise analyses, the comparison between the Garcia and Longa tests demonstrated the highest McNemar's χ2 value, indicating least marginal homogeneity between these two tests. The combination of high correlation between Garcia and Modo tests along with greatest marginal heterogeneity observed between the Garcia and Longa test lead us to recommend the use of Garcia and Longa neurological scales when researchers are hoping to capture the broadest range of neurological factors using only two stroke scales.

Combining Normobaric Oxygen with Ethanol or Hypothermia Prevents Brain Damage from Thromboembolic Stroke via PKC-Akt-NOX Modulation.

In a thromboembolic stroke model after reperfusion by recombinant tissue plasminogen activator (rt-PA), we aimed to determine whether therapeutic hypothermia (TH) and ethanol (EtOH) in combination with low concentration (60%) of normobaric oxygen (NBO) enhanced neuroprotection, as compared to using each of these agents alone. We further aimed to elucidate a potential role of the NADPH oxidase (NOX), phosphorylated protein kinase B (Akt), and protein kinase C-δ (PKC-δ) pathway in oxidative stress and neuroprotection. In Sprague-Dawley rats, a focal middle cerebral artery (MCA) occlusion was induced by an autologous embolus in the following experimental groups: rt-PA treatment alone, rt-PA + NBO treatment, rt-PA + TH at 33°C, rt-PA + EtOH, rt-PA + NBO + EtOH, rt-PA + NBO + TH, rt-PA + NOX inhibitor, rt-PA + EtOH + NOX inhibitor, or rt-PA + EtOH + Akt inhibitor. Control groups included sham-operated without stroke or stroke without treatment. Infarct volume and neurological deficit were assessed at 24h after rt-PA-induced reperfusion with or without treatments. ROS levels, NOX activity, and the protein expression of NOX subunits p22phox, p47phox, p67phox, gp91phox, as well as PKC-δ and phosphorylated Akt were measured at 3 and 24h after rt-PA-induced reperfusion. Following rt-PA in thromboembolic stroke rats, NBO combined with TH or EtOH more effectively decreased infarct volume and neurological deficit, as well as reactive oxygen species (ROS) production than with any of the used monotherapies. NOX activity and subunit expressions were downregulated and temporally associated with reduced PKC-δ and increased p-Akt expression. The present study demonstrated that combining NBO with either TH or EtOH conferred similar neuroprotection via modulation of NOX activation. The results suggest a role of Akt in NOX activation and implicate an upstream PKC-δ pathway in the Akt regulation of NOX. It is possible to substitute EtOH for TH, thus circumventing the difficulties in clinical application of TH through the comparatively easier usage of EtOH as a potential stroke management.

Effect of ephrin-B2 on the expressions of angiopoietin-1 and -2 after focal cerebral ischemia/reperfusion.

Ephrin-B2 has been shown to participate in angiogenesis, but the underlying mechanisms involved remain unclear. In this study, a rat model of focal cerebral ischemia was prepared by focal middle cerebral artery occlusion, followed by 24-hour reperfusion. Then, ephrin-B2 protein was administered intracerebroventricularly for 3 consecutive days via a micro-osmotic pump. Western blot assay and quantitative real-time reverse transcription PCR demonstrated the expression levels of angiopoietin-1 (Ang-1) mRNA and protein in the penumbra cortex of the ephrin-B2 treated group were decreased at day 4 after reperfusion, and increased at day 28, while the expression levels of angiopoietin-2 (Ang-2) were highly up-regulated at all time points tested. Double immunofluorescent staining indicated that Ang-1 and Ang-2 were both expressed in vascular endothelial cells positive for CD31. These findings indicate that ephrin-B2 influences the expressions of Ang-1 and Ang-2 during angiogenesis following transient focal cerebral ischemia.

A new method for focal middle cerebral artery occlusion in rats via transfemoral approach

A new method for focal middle cerebral artery occlusion in rats via transfemoral approach

Focal middle cerebral artery ischemia in rats via a transfemoral approach using a custom designed microwire

ObjectivesThe aim of this study was to develop a reliable and repeatable method of inducing focal middle cerebral artery occlusion (MCAo) in rats without ligation of the external carotid artery...

Expression of Ephrin-B2 after focal cerebral ischemia in rats

To explore the expression profile of Ephrin-B2 in the ischemic penumbra after transient focal cerebral ischemia in rats, and to clarify the mechanism of Ephrin-B2 triggering angiogenesis. Sprague-Dawley rats were randomly divided into a normal group, a sham operation group and ischemic-reperfusion 1, 3, 7, 14, and 28 d groups. Suture-occluded method was used to establish the focal middle cerebral artery occlusion model and the ischemic brain was reperfused 2 h after the occlusion. Western blot and quantitative real-time reverse-transcription polymerase chain reaction were used to detect the dynamic expression profile of Ephrin-B2 in the penumbra cortex. Double immunofluorescence was used to speculate the location and the co-expression of Ephrin-B2 in blood vessels, neurons and astrocytes. Microvessel density was quantified by the number of CD31+ cells. Rats were subjected to neurologic functional tests by modified neurological severity scores (mNSS) before sacrifice. Compared with the sham group, Ephrin-B2 protein and mRNA level of the penumbra cortex in the ischemic group increased 3 days (P<0.05) after the reperfusion, peaked at day 7 and 14 (P<0.01), and declined at day 28. Double immunofluorescence indicated that Ehprin-b2 was expressed in the neurons, blood vessels and astrocytes; mNSS peaked at day 7, and gradually declined at day 14. The microvessel density of penumbra cortex in the ischemic group increased 3 days (P<0.05) after the reperfusion, peaked at day 14 (P<0.01), and gradually declined at 48 h. Cerebral ischemia reperfusion induces the over-expression of Ephrin-B2, with a dynamic trend, suggesting that Ehprin-b2 may improve post-stroke functional recovery by enhancing angiogenesis and neurogenesis.

Neuroprotective effects of Asiaticoside.

In the central nervous system, Asiaticoside has been shown to attenuate in vitro neuronal damage caused by exposure to β-amyloid. In vivo studies demonstrated that Asiaticoside could attenuate neurobehavioral, neurochemical and histological changes in transient focal middle cerebral artery occlusion animals. In addition, Asiaticoside showed anxiolytic effects in acute and chronic stress animals. However, its potential neuroprotective properties in glutamate-induced excitotoxicity have not been fully studied. We investigated the neuroprotective effects of Asiaticoside in primary cultured mouse cortical neurons exposed to glutamate-induced excitotoxicity invoked by N-methyl-D-aspartate. Pretreatment with Asiaticoside decreased neuronal cell loss in a concentration-dependent manner and restored changes in expression of apoptotic-related proteins Bcl-2 and Bax. Asiaticoside pretreatment also attenuated the upregulation of NR2B expression, a subunit of N-methyl-D-aspartate receptors, but did not affect expression of NR2A subunits. Additionally, in cultured neurons, Asiaticoside significantly inhibited Ca2+ influx induced by N-methyl-D-aspartate. These experimental findings provide preliminary evidence that during excitotoxicity induced by N-methyl-D-aspartate exposure in cultured cortical neurons, the neuroprotective effects of Asiaticoside are mediated through inhibition of calcium influx. Aside from its anti-oxidant activity, down-regulation of NR2B-containing N-methyl-D-aspartate receptors may be one of the underlying mechanisms in Asiaticoside neuroprotection.

Taurine Protection against Endoplasmic Reticulum Stress in an Animal Stroke Model of Cerebral Artery Occlusion and Stroke Related conditions in Primary Neuronal Cell Culture

Taurine has been shown to provide protection against neurological diseases, such as Huntington's and stroke. We have shown that taurine can provide effective protection against Endoplasmic Reticulum (ER) stress induced by excitotoxicity or oxidative stress in PC12 cell line or primary neuronal cell cultures. In this study, we employed hypoxia/reoxygenation conditions for primary cortical neuronal cell cultures as well as the model of rat focal Middle Cerebral Artery Occlusion (MCAO). Using taurine greatly increased cell viability in primary neuronal cell culture and decreased the infarct area of sections at 6mm from 47.42±9.86% to 26.76 ± 6.91% in the MCAO model. Furthermore levels of the ER stress protein markers GRP78, Caspase12, CHOP and p‐IRE‐1 which were markedly increased in both the in vitro and in vivo models, significantly declined after taurine administration, suggesting that taurine may exert neuroprotection functions in both models. Moreover, taurine could downregulate the ratio of cleaved ATF6 and full length ATF6 in both models. In the animal model of stroke, taurine induced an upregulation of the Bcl‐2/Bax ratio and downregulation of Caspase‐3 protein activity indicating that it attenuates apoptosis in the core of the ischemic infarct. Our results show not only taurine elicits neuroprotection through the activation of the ATF6 and the IRE1 pathways, but also it can reduce apoptosis in these modelsGrant Funding Source: 09KW‐11, Department of Health, State of Florida

High-density Lipoprotein–based Therapy Reduces the Hemorrhagic Complications Associated With Tissue Plasminogen Activator Treatment in Experimental Stroke

We have previously reported that intravenous injection of high-density lipoproteins (HDLs) was neuroprotective in an embolic stroke model. We hypothesized that HDL vasculoprotective actions on the blood-brain barrier (BBB) may decrease hemorrhagic transformation-associated with tissue plasminogen activator (tPA) administration in acute stroke. We used tPA alone or in combination with HDLs in vivo in 2 models of focal middle cerebral artery occlusion (MCAO) (embolic and 4-hour monofilament MCAO) and in vitro in a model of BBB. Sprague-Dawley rats were submitted to MCAO, n=12 per group. The rats were then randomly injected with tPA (10 mg/kg) or saline with or without human plasma purified-HDL (10 mg/kg). The therapeutic effects of HDL and BBB integrity were assessed blindly 24 hours later. The integrity of the BBB was also tested using an in vitro model of human cerebral endothelial cells under oxygen-glucose deprivation. tPA-treated groups had significantly higher mortality and rate of hemorrhagic transformation at 24 hours in both MCAO models. Cotreatment with HDL significantly reduced stroke-induced mortality versus tPA alone (by 42% in filament MCAO, P=0.009; by 73% in embolic MCAO, P=0.05) and tPA-induced intracerebral parenchymal hematoma (by 92% in filament MCAO, by 100% in embolic MCAO; P<0.0001). This was consistent with an improved BBB integrity. In vitro, HDLs decreased oxygen-glucose deprivation-induced BBB permeability (P<0.05) and vascular endothelial cadherin disorganization. HDL injection decreased tPA-induced hemorrhagic transformation in rat models of MCAO. Both in vivo and in vitro results support the vasculoprotective action of HDLs on BBB under ischemic conditions.

Effects of aromatic resuscitation drugs on blood brain barrier in cerebral ischemia-reperfusion injury model rats

To research the effects of moschus, borneol, styrax and benzoinum on the structure and function of blood brain barrier in cerebral ischemia-reperfusion injury model rats. Focal middle cerebral artery occlusion (MCAO) was introduced as an in vivo ischemic model in rats. After 2 h MCAO, nylon suture was pulled up 1 cm to give blood reperfusion. After 22 h reperfusion, all animals were decapitated. The ultramicrostructure of blood brain barrier of ischemia hemisphere side in fronto-parietal cortex region by transmission electron microscope, and the content of VEGF and MMP-9 in ischemia side brain tissue were measured by ELISA. In model and solvent group rats, the capillary endothelium cells, astro-glial cells and nerve cells in ischemia hemisphere side in fronto-parietal region were emerged in different degree compared with sham-operated groups, which exhibited tight junction between endothelial cells being opened, basal lamina being dissolved, and permeability increasing, and cellularedema. In borneol (0.2 g x kg(-1)) group rats, the structure of three kinds of cells were nearly normal, which tight junction structure was clear, rough endoplasmic reticulum and polyribosome could be found in cytoplasm. In moschus (66.6 mg x kg(-1)) group rats, the structure of capillary endothelium cells and astrocytes were nearly normal as well as the basal lamina, but the electrons in neurons was maldistribution. In styrax (1.332 g x kg(-1)) group rats, astrocytes were nearly normal, while capillary endothelial cells and neurons exhibited oedema in different degrees. And the basal lamina was discontinuous, augmentation of cell spaces in endothelial cells increased the permeability, some endoplasmic reticulum broadened and ribosome ablated. In benzoinum (1.0 g x kg(-1)) group rats, oedema of capillary endothelial cells and astrocytes was significant, basal lamina broke. Meanwhile endoplasmic reticulum broadened as vacuole, the number of ribosome in rough endoplasmic reticulum decreased, crista mitochondriales in some neurons disappeared as vacuole which hint oedema happened. Results also showed that borneol decrease the level of VEGF in ischemia side brain tissue significantly, while has little influence on the level of MMP-9. Moschus showed the tendency to decrease the level of VEGF and MMP-9 in ischemia side brain tissue. Aromatic resuscitation drugs showed the protection effect on blood brain barrier in cerebral ischemia-reperfusion injury rats, which the protection effect of moschus and borneol were better than that of styrax and benzoinum. The mechanism of protection effect maybe related to decrease the level of VEGF and MMP-9.

Conditionally immortalised neural stem cells promote functional recovery and brain plasticity after transient focal cerebral ischaemia in mice

Cell therapy has enormous potential to restore neurological function after stroke. The present study investigated effects of conditionally immortalised neural stem cells (ciNSCs), the Maudsley hippocampal murine neural stem cell line clone 36 (MHP36), on sensorimotor and histological outcome in mice subjected to transient middle cerebral artery occlusion (MCAO). Adult male C57BL/6 mice underwent MCAO by intraluminal thread or sham surgery and MHP36 cells or vehicle were implanted into ipsilateral cortex and caudate 2 days later. Functional recovery was assessed for 28 days using cylinder and ladder rung tests and tissue analysed for plasticity, differentiation and infarct size. MHP36-implanted animals showed accelerated and augmented functional recovery and an increase in neurons (MAP-2), synaptic plasticity (synaptophysin) and axonal projections (GAP-43) but no difference in astrocytes (GFAP), oligodendrocytes (CNPase), microglia (IBA-1) or lesion volumes when compared to vehicle group. This is the first study showing a potential functional benefit of the ciNSCs, MHP36, after focal MCAO in mice, which is probably mediated by promoting neuronal differentiation, synaptic plasticity and axonal projections and opens up opportunities for future exploitation of genetically altered mice for dissection of mechanisms of stem cell based therapy.

Effect of procyanidin on neural cell apoptosis and expression of Caspase-3 following cerebral ischemic reperfusion injury in rats

Objective To study the effect of procyanidin on neural cell apoptosis and the expression of Caspase-3 of cerebral ische-mia reperfusion(I/R) injury in rats. Methods 40 rats were randomly divided into 4 groups, which were sham operated group, I/R group, low dose procyanidin treated group and high dose procyanidin treated group. The focal middle cerebral artery occlusion (MCAO) model was made by suture-occluded method. After MCAO for 90min following 24h of reperfusion, neural cell apoptosis and the expression of caspase-3 was investigated with TUNEL and immunohistochemistry. HE staining and Trc staining was also used. Result Compared with sham opera-ted group, neural cell apoptosis rate and the expression of caspase-3 were increased at the 24th hour of reperfusion in the ischemic territory(P < 0.05) . Compared with I/R group, low and high dose procyanidin treated group reduced expression of caspase-3 and neural cell apopto-sis rate in a dose-dependent manor (P <0.05). The change of ischemic impairment in procyanidin treated group was less than that of I/R group, and the change of high dose procyanidin treated group was less than that of low dose procyanidin treated group. Compared with that of I/R group, cerebral infarction volume of procyanidin treated group was decreased in a dose-dependent manor (P < 0.05). Conclusion Procyanidin may reduce cerebral ischemia-reperfusion injure by reducing expression of caspase-3 and neural cell apoptosis. Key words: Proanthocyanidins/PD ; Brain ischemia; Reperfusion injury/PC ; Caspases/BI/DE; Neurons/DE; Apoptosis