Animal Models Of Cerebral Ischemia Research Articles

Abstract TP72: Transcranial Direct Current Stimulation (tDCS) for Neuroprotection in Acute Cerebral Ischemia: Meta-analysis of Preclinical Studies and Implications for Human Clinical Trials

Introduction: Transcranial direct current stimulation (tDCS) is a noninvasive neuromodulation technique safe and promising in early human studies of nonacute disorders (depression, OCD, stroke recovery), and also reported to confer direct neuroprotection and vasodilation in animal models of acute cerebral ischemia. Systematic review of preclinical acute cerebral ischemia studies would aid planning for potential initial human clinical trials. Method: A systematic Medline search identified controlled, preclinical studies of tDCS in acute ischemic stroke. Studies were categorized according to stimulation type (hemispheric cathodal; hemispheric anodal; targeted cerebellar fastigial nucleus). Stratified meta-analysis assessed effect of tDCS on final infarct volume. Results: The systematic search identified 17 controlled comparisons of tDCS in preclinical acute cerebral ischemia models, including a total of 256 animals, all with middle cerebral artery occlusion. Hemispheric cathodal stimulation was used in 3 experiments (32 animals), hemispheric anodal stimulation in 1 experiment (8 animals) and fastigial nucleus stimulation in 13 experiments (91 animals). Overall, tDCS reduced final infarct volume by 27.5 cc (95% CI 25.33-29.74, P<0.00001). Heterogeneity of effect by stimulation type was noted (I 2 =90.5%), with the greatest infarct reduction with cathodal - 34.9 cc (95% CI 30.60-39.18, P< 0.00001), followed by targeted fastigial nucleus - 26.8 cc (95% CI 25.33-29.53, P< 0.00001), and anodal - 17.8 cc (95% CI 11.42-24.18, P< 0.00001). In the cathodal studies, stimulation was started 30-45 minutes after ischemia onset. Conclusions: Transcranial direct current stimulation reduces final infarct volume across animal preclinical studies. The greatest neuroprotective effect is observed with hemispheric cathodal stimulation, with treatment initiation in a time frame achievable in human prehospital clinical trials. Human clinical trials of hemispheric cathodal tDCS are warranted.

Neuronal loss and gliosis in the rat striatum subjected to 15 and 30 minutes of middle cerebral artery occlusion.

Selective neuronal death or loss in certain brain regions has been well characterized in animal models of transient global cerebral ischemia. However, selective neuronal death in transient focal cerebral ischemia needs more investigation. Therefore, in this study, we studied selective neuronal death in the striatum (caudate putamen) of rats subjected to 15 or 30min middle cerebral artery occlusion (MCAO). Neuronal death occurred in the dorsolateral field, not in the medial field in 30min, not 15min, MCAO-operated rats 5days after MCAO using neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In this group, immunoreactivity of glial fibrillary acidic protein in astrocytes was hardly shown in the dorsolateral field, although the immunoreactivity increased in the medial field. In addition, immunoreactivity of ionized calcium binding adapter molecule 1 in microglia was dramatically increased in the dorsolateral, not in the medial, field only in 30min MCAO-operated rats. Briefly, these results show that at least 30min of MCAO can evoke selective neuronal death, astrocytic dysfunction and microglial activation in the dorsolateral field of the rat striatum and suggest that a rat model of 30min MCAO can be used to investigate mechanisms of neuronal death and gliosis following brief transient focal cerebral ischemic events for acute transient ischemic attack.

Preclinical Evidence and Mechanism of Xingnaojing Injection for Cerebral Ischemia: A Systematic Review and Meta-Analysis of Animal Studies.

Objectives Cerebral ischemia can cause severe harm to people's health with the characteristics of high incidence, high disability, and high mortality. Xingnaojing injection (XNJI) is widely used in the treatment of cerebral ischemia. The aim of this review is to evaluate the efficacy and mechanism of XNJI in animal models of cerebral ischemia. Methods Total seven electronic databases in English or Chinese (CNKI, Wanfang, VMIS, PubMed, MEDLINE, Embase, and the Cochrane Library) about most experiments and studies which came out before June 2018 of XNJI for cerebral ischemia have been searched. Data extraction, quality assessment, and meta-analysis are conducted according to the Cochrane standards and RevMan 5.3 software. Results We have identified 23 eligible studies and made a meta-analysis based on these studies. Meta-analysis shows that XNJI contributes significantly to reduction in neurological deficit score (P = 0.0002, MD = −1.25, 95% CI: −1.92, −0.58) compared with the control group of cerebral ischemia. Subgroup analytic results demonstrate that XNJI has been more effective in animal model of cerebral ischemia-reperfusion injury (P = 0.009, MD = −1.35, 95%CI: −2.36, −0.34) than that of permanent cerebral ischemia (P = 0.0002, MD = −1.08, 95%CI: −1.66, −0.51). Compared with control group, XNJI could remarkably reduce cerebral infarction area (P < 0.00001, MD = −14.98, 95%CI: −21.36, −8.59), brain edema (P < 0.00001, MD = −4.64, 95%CI: −5.38, −3.90), and neuronal cell apoptosis (P < 0.0001, MD = −12.21, 95%CI: 18.05, −6.37). Meanwhile, the meta-analysis shows that XNJI has a significant anti-inflammatory effect, and the levels of TNF-α, IL-6, and IL-1β are significantly reduced by XNJI (P = 0.001, MD = −4.13, 95%CI:−6.68, −1.58; P < 0.00001, MD = −119.23, 95%CI: −138.04, −100.43; P = 0.21, MD = −228.69, 95% CI: −586.20, 128.83). Additionally, XNJI could raise the body's antioxidant function and the level of SOD and GSH-Px (P = 0.002, MD = 53.02, 95% CI: −20.52, 85.78; P = 0.01, MD = 8.65, 95% CI: 1.77, 15.48) and decrease the level of MDA (P < 0.00001, MD = −4.16, 95% CI: −5.50, −2.82). Conclusion XNJI might be effective in cerebral ischemia by regulating oxidative stress and inflammatory reaction.

Multi-target effects of 3-n-Butylphalide, a component of Apium graveolens L., in treatment of neurodegenerative diseases

L-3-n-Butylphalide (l-NBP) was isolated from seeds of Apium graveolens L. by the Institute of Materia Medica. CAMS in the 1980s. It was showed to be effective in anti-epilepsy. Soon it was synthesized as a racemic form, dl-3-n-butylphalide (dl-NBP) and demonstrated that dl-NBP had the similar pharmacological effects compared to l-NBP. In 1990s dl-NBP was found to be potent in protection of brain injury from cerebral ischemia in animal models. It was approved by SFDA for clinical trial and was approved as a new drug for acute ischemic stroke in 2005. Today, dl-NBP is a frequently used drug for treatment of ischemic stroke in China.

Effects of Vitamin D3 on the NADPH Oxidase and Matrix Metalloproteinase 9 in an Animal Model of Global Cerebral Ischemia.

Decreased blood flow in the brain leads to a rapid increase in reactive oxygen species (ROS). NADPH oxidase (NOX) is an enzyme family that has the physiological function to produce ROS. NOX2 and NOX4 overexpression is associated with aggravated ischemic injury, while NOX2/4-deficient mice had reduced stroke size. Dysregulation of matrix metalloproteinases (MMPs) contributes to tissue damage. The active form of vitamin D3 expresses neuroprotective, immunomodulatory, and anti-inflammatory effects in the CNS. The present study examines the effects of the vitamin D3 pretreatment on the oxidative stress parameters and the expression of NOX subunits, MMP9, microglial marker Iba1, and vitamin D receptor (VDR), in the cortex and hippocampus of Mongolian gerbils subjected to ten minutes of global cerebral ischemia, followed by 24 hours of reperfusion. The ischemia/reperfusion procedure has induced oxidative stress, changes in the expression of NOX2 subunits and MMP9 in the brain, and increased MMP9 activity in the serum of experimental animals. Pretreatment with vitamin D3 was especially effective on NOX2 subunits, MMP9, and the level of malondialdehyde and superoxide anion. These results outline the significance of the NOX and MMP9 investigation in brain ischemia and the importance of adequate vitamin D supplementation in ameliorating the injury caused by I/R.

Glycoprotein NMB: an Emerging Role in Neurodegenerative Disease.

Neurodegeneration is characterized by severe neuronal loss leading to the cognitive and physical impairments that define various neurodegenerative diseases. Neuroinflammation is one hallmark of neurodegenerative diseases and can ultimately contribute to disease progression. Increased inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1 β), and tumor necrosis factor-α (TNF-α) are associated with Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Unfortunately, current therapeutic options lack ability to stop or effectively slow progression of these diseases and are primarily aimed at alleviating symptoms. Thus, it is crucial to discover novel treatment candidates for neurodegenerative diseases. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type-I transmembrane glycoprotein first identified in a melanoma cell line. GPNMB augments bone mineral deposition by stimulating osteoblast differentiation. Aside from its anabolic function in the bone, emerging evidence suggests that GPNMB has anti-inflammatory and reparative functions. GPNMB has also been demonstrated to be neuroprotective in an animal model of ALS, cerebral ischemia, and other disease models. Given these discoveries, GPNMB should be investigated as a potential therapeutic option for multiple neurodegenerative diseases.

L-3-n-Butylphthalide Activates Akt/mTOR Signaling, Inhibits Neuronal Apoptosis and Autophagy and Improves Cognitive Impairment in Mice with Repeated Cerebral Ischemia-Reperfusion Injury.

L-3-n-Butylphthalide (L-NBP) exerts neuroprotective effects in animal models of cerebral ischemia, but its potential benefits in repeated cerebral ischemia-reperfusion (RCIR) injury remain unknown. We investigated the effect of L-NBP on cognitive impairment induced by RCIR in mice. Male C57Bl/6 mice received sham surgery or bilateral common carotid artery occlusion (3 times, 20min each) and were orally administered preoperative L-NBP (30mg/kg/day, 7 days), postoperative L-NBP (30 or 60mg/kg/day, 28 days) or postoperative vehicle (28 days). Learning and memory were assessed by the Morris water maze task and step-down passive avoidance test. Nissl staining was used to identify pathologic changes in the hippocampal CA1 region. The expressions of proteins associated with signaling, apoptosis and autophagy were assessed by quantitative PCR and western blot. RCIR induced deficits in learning and memory that were alleviated by preoperative or postoperative L-NBP administration. Pathologic lesions in the hippocampal CA1 region induced by RCIR were less severe in mice treated with L-NBP. Preoperative or postoperative L-NBP administration in mice receiving RCIR promoted hippocampal expression of phospho-Akt and phospho-mTOR (suggesting activation of Akt/mTOR signaling), increased the Bcl-2/Bax ratio (indicating suppression of apoptosis) and reduced the LC3-II/LC3-I ratio (implying inhibition of autophagy). Preoperative or postoperative L-NBP administration also depressed hippocampal levels of beclin-1 mRNA (indicating suppression of autophagy). These findings suggest that the effect of L-NBP to alleviate learning and memory deficits in mice following RCIR may involve activation of Akt/mTOR signaling and regulation of the expressions of proteins related to apoptosis and autophagy.

Tentative exploration of acupoints’ biology effects to the cerebral ischemia model

Objective The molecular biology mechanism of cerebral ischemia animal models be analyzed from acupuncture experimental literatures by using data mining methods as a tentative exploration. Methods Retrieve the literatures of acupuncture animal experiments during 1978-2015, and screen the literatures about acupuncture effect on cerebral ischemia animal model of gene protein expression. Acupoint and gene/protein targets were extracted and normalized, and then the frequency statistics, association rule, calculation of complex networks were analyzed and visually displayed. Results A total of 350 papers were included. Seventy-nine acupoints were summarized, and the most frequent acupoint was baihui; A total of 180 genes/proteins were found, and the most frequent genes/proteins was BCL2. The most common group acuppoints zusanli and quchi for the association rule results. The results of complex networks analysis showed the HSP70, BCL2, CASP3, Bax were more closely associated with cerebral ischemia injury or rehabilitation mechanism. Conclusions The mechanism of acupuncture treatment for cerebral ischemia was that acupuncture adjusting ischemic brain damage and improve the nervous system disorders from many channels and multiple targets. Key words: Brain ischemia; Acupuncture points; Gene expression; Complex network; Asocciation rules

Dl-3-n-butylphthalide promotes neuroplasticity and motor recovery in stroke rats

Backgrounds and aimsRacemic l-3-n-butylphthalide (dl-NBP), is able to achieve a functional recovery in animal models of cerebral ischemia, vascular dementia, and Alzheimer’s disease. In this study, we investigated the effect of dl-NBP on axonal growth, neurogenesis and behavioral performances in rats with cerebral ischemia. MethodsFocal cerebral ischemia in rats was produced by intracerebral injection of endothelin-1. Starting from postoperative day 7, the experimental rats were administered 70mg/kg dl-NBP by oral gavage for two weeks. Biotinylated dextran amine (BDA) was injected into the contralateral sensorimotor cortex on day 14 after ischemia to trace the sprouting of corticospinal tract (CST) fibers into the denervated cervical spinal cord. The expressions of Nogo-A, Nogo-R, Rho-A, and ROCK in the perilesional cortex, the expressions of BDA, PSD-95, and vGlut1 in the denervated spinal cord, 5-bromo-20-deoxyuridine (BrdU)/DCX-positive cells in the subventricular zone (SVZ) of the injured hemisphere were detected by immunofluorescence. The rats’ behavioral abilities were measured on postoperative days 30–32 in the beam-walking, cylinder and sticky label tests. Resultsdl-NBP treatment significantly increased the number and length of crossing CST fibers, enhanced significantly the expression levels of synapse-associated proteins including PSD95 and VGlut-1 in the denervated cervical spinal cord, elevated the number of BrdU+/DCX+ cells in SVZ, and reduced markedly those of Rho-A+, ROCK+, Nogo-A+ and Nogo-R+ cells in perilesional cortex. In addition, dl-NBP improved the behavioral performance of the ischemic rats. Conclusiondl-NBP enhanced the behavioral recovery after cerebral ischemia in rats, possibly by increasing axonal growth and neurogenesis.

Preconditioning with melatonin improves therapeutic outcomes of bone marrow-derived mesenchymal stem cells in targeting liver fibrosis induced by CCl4.

Preconditioning of mesenchymal stem cells (MSCs) with melatonin (MT) has shown promising results in animal models of myocardial infarction, renal ischemia and cerebral ischemia. Here, we use this strategy in the liver fibrosis induced by CCl4. There were five groups: normal, CCl4, CCl4 + vehicle, CCl4 + BMMSCs and CCl4 + MT-bone marrow (BM)-derived MSCs (MT-BMMSCs). CCl4 was injected twice weekly for 8weeks and treatment either with cells or vehicle was performed at the beginning of week 5 with a single dose. BMMSCs were preconditioned with MT for 24h before injection. MT-BMMSCs had a high ability of homing into the injured liver (P ≤ 0.05 vs. BMMSCs). The CCl4 + MT-BMMSCs group showed higher percentage of glycogen storage but lower percentage of collagen and lipid accumulation (P ≤ 0.05 vs. CCl4 + BMMSCs). The CCl4 + MT-BMMSCs group showed lower expressions of transforming growth factor-β1 (TGF-β1) and Bax and lower content of sera alanine aminotransferase (ALT) but higher expressions of matrix metalloproteinases (MMPs) and Bcl2 compared with the BMMSCs group (P ≤ 0.05). The results showed the better therapeutic outcomes of MT preconditioning by probably improving cell homing and also better maintenance of the balance between matrix degrading and accumulating factors.

Transient cerebral ischemia induces albumin expression in microglia only in the CA1 region of the gerbil hippocampus.

Albumin, the most abundant plasma protein, is known to exhibit a neuroprotective effect in animal models of focal and global cerebral ischemia. In the present study, the expression and immunoreactivity of albumin was examined in the hippocampus following 5 min of transient cerebral ischemia in gerbils. Albumin immunoreactivity was observed in microglia of the CA1 hippocampal region 2 days post-ischemic insult, and it was significantly increased at 4 days following ischemia-reperfusion. In addition, at 4 days post-ischemic insult, albumin-immunoreactive microglia were abundant in the stratum pyramidale of the CA1 region. The present results demonstrated that albumin was newly expressed post-injury in microglia in the CA1 region, suggesting ischemia-induced neuronal loss. Albumin expression may therefore be associated with ischemia-induced delayed neuronal death in the CA1 region following transient cerebral ischemia.

Hyperglycemia exacerbates downregulation of dynamin-like protein 1 in ischemic cerebral injury

Ischemic stroke is one of the leading causes of adult disability and death. Hyperglycemia is associated with an increased risk of stroke and poor outcomes after brain injury. Dynamin-like protein I (DLP-1) regulates mitochondrial fission and promotes mitochondrial dynamics. Neurodegenerative diseases are associated with mitochondrial dysfunction, and the downregulation of DLP-1 has been previously identified in a stroke animal model. Here, we investigated the changes in DLP-1 protein expression in an animal model of focal cerebral ischemia with induced hyperglycemia. Streptozotocin (40 mg/kg) was intraperitoneally injected into male rats to induce hyperglycemia, and middle cerebral artery occlusion (MCAO) was surgically induced 4 weeks after streptozotocin treatment. Brain tissue was isolated 24 hours after MCAO, and cerebral cortex samples were used for this study. Proteomics revealed a decrease in DLP-1 expression in MCAO animals when compared with controls, and this downregulation was more prominent in MCAO animals with hyperglycemia. Reverse-transcription polymerase chain reaction and Western blot analyses confirmed that DLP-1 was significantly downregulated in MCAO-injured animals with hyperglycemia compared to those without hyperglycemia. The decrease in DLP-1 indicates mitochondrial morphological changes and dysfunction. Together, these results suggest that the severe decrease of DLP-1 seen after brain injury under hyperglycemic conditions may exacerbate the damage to the brain.

Raf Kinase Inhibitor Protein Attenuates Ischemic-Induced Microglia Cell Apoptosis and Activation Through NF-κB Pathway

Background: Acute ischemic stroke is one of the most important factors leading to disability and death with the characterization of accumulated neuron death and injured supportive neurovascular structures. Raf-1 kinase inhibitory protein (RKIP) is a key molecule in cell response to survival or death stimuli. However, the role of RKIP in stroke is worthy to be further studied. Methods: We used lentivirus mediated RKIP knockdown and overexpression to investigate the effect of RKIP on animal models of focal cerebral ischemia. Cell Counting Kit-8 assay, lactate dehydrogenase release analysis, and Annexin V-APC apoptosis assay were used to detect the effect RKIP on microglial cell apoptosis and survival. Transwell migration assay was carried out to evaluate the migration of microglia cells. The releases of inflammatory cytokines were determined by ELISA. The activation of NF-kappaB signaling pathway was determined by western blot. Results: Overexpression of RKIP reduced focal cerebral ischemia injury. RKIP knockdown and overexpression regulated survival, activation, and motility via the NF-κB pathway. NF-κB inhibitor BAY 11-7082 blocked the changes caused by RKIP down-regulation after oxygen-glucose deprivation (OGD). RKIP overexpression inhibited the upregulation of phosphorylation of NF-κB induced by OGD and cerebral ischemia. Conclusions: The present study showed that RKIP protects against ischemic stroke through inhibition of microglial excessive activation, inhibits its motility, and promotes neuronal survival partly though IKKβ-IκBα-NF-κB signaling axis and indicate that RKIP is a new target for the treatment of ischemic stroke.

Intravenously Infused F3.Olig2 Improves Memory Deficits via Restoring Myelination in the Aged Hippocampus Following Experimental Ischemic Stroke.

Oligodendrocytes play a crucial role in creating the myelin sheath that is an important component in neural transmission. In an animal model of transient cerebral ischemia, application of oligodendrocyte progenitor cells (OPCs) has not yet been reported. In this study, the effects of F3.Olig2 transplantation on memory and cognitive dysfunction were investigated in the aged gerbil in which ischemic stroke was induced. To investigate the possible mechanisms underlying repair, changes in the expression of myelin basic protein (MBP), oligodendrocyte-specific protein (OSP), and brain-derived neurotrophic factor (BDNF) were examined. Experimental ischemic stroke was induced by occlusion of bilateral common carotid arteries in aged gerbils. Gerbils (n=31 per group) were randomly divided into three groups: (1) vehicle sham group, (2) vehicle ischemia group, and (3) F3.Olig2 ischemia group. After 1, 3, and 7 days of ischemiareperfusion (I-R), saline or F3.Olig2 cells (1106 cells in 100 l) were injected into the gerbils intravenously. The gerbils were sacrificed 10 days after I-R for identification of grafted F3.Olig2 cells, and 15 and 30 days after I-R for tissue analysis after conducting passive avoidance and novel object recognition test. Injected F3.Olig2 cells and MBP, OSP, and BDNF were detected by specific antibodies using immunohistochemistry and/or Western blots. Memory and cognition were significantly increased in the F3.Olig2 ischemia group compared with the vehicle ischemia group. In the F3.Olig2 ischemia group, the neurons were not protected from ischemic damage; however, MBP, OSP, and BDNF expressions were significantly increased. Our results show that injection of F3.Olig2 cells significantly improved impaired memory and cognition, which might be related to increased MBP expression via increasing OSP and BDNF expression in the aged gerbil hippocampus following transient cerebral ischemia.

Anti-excitotoxic effects of cannabidiol are partly mediated by enhancement of NCX2 and NCX3 expression in animal model of cerebral ischemia

Excitotoxicity and imbalance of sodium and calcium homeostasis trigger pathophysiologic processes in cerebral ischemia which can accelerate neuronal death. Neuroprotective role of cannabidiol (CBD), one of the main non-psychoactive phytocannabinoids of the cannabis plant, has attracted attention of many researchers in the neurodegenerative diseases studies. The present investigation was designed to determine whether cannabidiol can alleviate the severity of ischemic damages and if it is able to exert its anti-excitotoxic effects through sodium and calcium regulation. By using stereotaxic surgery, a guide cannula was implanted into the lateral ventricle. Cannabidiol (50, 100, and 200ng/rat; i.c.v.) was administrated for 5 consecutive days. After pretreatment, the rats were subjected to 60min of right middle cerebral artery occlusion (MCAO). After 24h, neurological deficits score, infarct volume, brain edema, and blood–brain barrier (BBB) permeability in total of hemisphere, cortex, piriform cortex-amygdala, and striatum were assessed. The expression of Na+/Ca2+ exchangers (NCXs) protein as an endogenous target in these regions was also studied. The present results indicate that administration of cannabidiol (100 and 200ng/rat) in the MCAO-induced cerebral ischemia caused a remarkable reduction in neurological deficit, infarction, brain edema, and BBB permeability in comparison with the vehicle group. Up-regulation of NCX2 and NCX3 in cannabidiol-received groups was also observed. These findings support the view that the reduction of ischemic injuries elicited by cannabidiol can be at least partly due to the enhancement of NCX protein expression and its cerebro-protective role in those cerebral territories supplied by MCA.

Tissue-type Plasminogen Activator (tPA) Modulates the Postsynaptic Response of Cerebral Cortical Neurons to the Presynaptic Release of Glutamate.

Tissue-type plasminogen activator (tPA) is a serine proteinase released by the presynaptic terminal of cerebral cortical neurons following membrane depolarization (Echeverry et al., 2010). Recent studies indicate that the release of tPA triggers the synaptic vesicle cycle and promotes the exocytosis (Wu et al., 2015) and endocytic retrieval (Yepes et al., 2016) of glutamate-containing synaptic vesicles. Here we used electron microscopy, proteomics, quantitative phosphoproteomics, biochemical analyses with extracts of the postsynaptic density (PSD), and an animal model of cerebral ischemia with mice overexpressing neuronal tPA to study whether the presynaptic release of tPA also has an effect on the postsynaptic terminal. We found that tPA has a bidirectional effect on the composition of the PSD of cerebral cortical neurons that is independent of the generation of plasmin and the presynaptic release of glutamate, but depends on the baseline level of neuronal activity and the extracellular concentrations of calcium (Ca2+). Accordingly, in neurons that are either inactive or incubated with low Ca2+ concentrations tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-mediated phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in neurons with previously increased baseline levels of pCaMKIIα in the PSD due to neuronal depolarization in vivo or incubation with high concentrations of either Ca2+ or glutamate in vitro, tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. We found that these effects of tPA are mediated by synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. Our data indicate that by regulating the pCaMKIIα/PP1 balance in the PSD tPA acts as a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate.

Identification of Reversible Disruption of the Human Blood-Brain Barrier Following Acute Ischemia.

Animal models of acute cerebral ischemia have demonstrated that diffuse blood-brain barrier (BBB) disruption can be reversible after early reperfusion. However, irreversible, focal BBB disruption in humans is associated with hemorrhagic transformation in patients receiving intravenous thrombolytic therapy. The goal of this study was to use a magnetic resonance imaging biomarker of BBB permeability to differentiate these 2 forms of BBB disruption. Acute stroke patients imaged with magnetic resonance imaging before, 2 hours after, and 24 hours after treatment with intravenous tissue-type plasminogen activator were included. The average BBB permeability of the acute ischemic region before and 2 hours after treatment was calculated using a T2* perfusion-weighted source images. Change in average permeability was compared with percent reperfusion using linear regression. Focal regions of maximal BBB permeability from the pretreatment magnetic resonance imaging were compared with the occurrence of parenchymal hematoma (PH) formation on the 24-hour magnetic resonance imaging scan using logistic regression. Signals indicating reversible BBB permeability were detected in 18/36 patients. Change in average BBB permeability correlated inversely with percent reperfusion (P=0.006), indicating that early reperfusion is associated with decreased BBB permeability, whereas sustained ischemia is associated with increased BBB disruption. Focal regions of maximal BBB permeability were significantly associated with subsequent formation of PH (P=0.013). This study demonstrates that diffuse, mild BBB disruption in the acutely ischemic human brain is reversible with reperfusion. This study also confirms prior findings that focal severe BBB disruption confers an increased risk of hemorrhagic transformation in patients treated with intravenous tissue-type plasminogen activator.

L‐3‐n‐butylphthalide Rescues Hippocampal Synaptic Failure and Attenuates Neuropathology in AgedAPP/PS1 Mouse Model of Alzheimer's Disease

Our previous studies showed that L-3-n-butylphthalide (L-NBP), an extract from seeds of Apium graveolens Linn (Chinese celery), improved cognitive ability in animal models of cerebral ischemia, vascular dementia, and Alzheimer's disease (AD). It is well known that cognitive deficit of AD is caused by synaptic dysfunction. In this study, we investigated the effect of L-NBP on hippocampal synaptic function in APP/PS1 AD transgenic mice and related mechanisms. Eighteen-month-old APP/PS1 transgenic (Tg) mice were administrated 15 mg/kg L-NBP by oral gavage for 3 months. Synaptic morphology and the thickness of postsynaptic density (PSD) in hippocampal neurons were investigated by electron microscope. The dendritic spines, Aβ plaques, and glial activation were detected by staining. The expressions of synapse-related proteins were observed by Western blotting. L-NBP treatment significantly increased the number of synapses and apical dendritic thorns and the thickness of PSD, increased the expression levels of synapse-associated proteins including PSD95, synaptophysin (SYN), β-catenin, and GSK-3β, and attenuated Aβ plaques and neuroinflammatory responses in aged APP/PS1 Tg mice. L-NBP may restore synaptic and spine function in aged APP Tg mice through inhibiting Aβ plaques deposition and neuroinflammatory response. Wnt/β-catenin signaling pathway may be involved in L-NBP-related restoration of synaptic function.

Zinc contributes to acute cerebral ischemia-induced blood–brain barrier disruption

Zinc ions are stored in synaptic vesicles and cerebral ischemia triggers their release from the terminals of neurons. Zinc accumulation in neurons has been shown to play an important role in neuronal death following ischemia. However, almost nothing is known about whether zinc is involved in ischemia-induced blood–brain barrier (BBB) disruption. Herein, we investigated the contribution of zinc to ischemia-induced acute BBB disruption and the possible molecular mechanisms using both cellular and animal models of cerebral ischemia. Zinc greatly increased BBB permeability and exacerbated the loss of tight junction proteins (Occludin and Claudin-5) in the endothelial monolayer under oxygen glucose deprivation conditions. In cerebral ischemic rats, a dramatically elevated level of zinc accumulation in microvessels themselves was observed in isolated microvessels and in situ, showing the direct interaction of zinc on ischemic microvessels. Treatment with a specific zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), even at 60-min post-ischemia onset, could greatly attenuate BBB permeability in the ischemic rats as measured by Evan's Blue extravasation, edema volume and magnetic resonance imaging. Furthermore, zinc accumulation in microvessels activated the superoxide/matrix metalloproteinase-9/-2 pathway, which leads to the loss of tight junction proteins (Occludin and Claudin-5) and death of endothelial cells in microvessels themselves. Our findings reveal a novel mechanism of cerebral ischemia-induced BBB damage, and implicate zinc as an effective and viable new target for reducing acute BBB damage following ischemic stroke.

Endothelin Receptors, Mitochondria and Neurogenesis in Cerebral Ischemia

Background: Neurogenesis is most active during pre-natal development, however, it persists throughout the human lifespan. The putative role of mitochondria in neurogenesis and angiogenesis is gaining importance. Since, ETB receptor mediated neurogenesis and angiogenesis has been identified, the role of these receptors with relevance to mitochondrial functions is of interest. Methods: In addition to work from our laboratory, we undertook an extensive search of bibliographic databases for peer-reviewed research literature. Specific technical terms such as endothelin, mitochondria and neurogenesis were used to seek out and critically evaluate literature that was relevant. Results: The ET family consists of three isopeptides (ET-1, ET-2 and ET-3) that produce biological actions by acting on two types of receptors (ETA and ETB). In the central nervous system (CNS) ETA receptors are potent constrictors of the cerebral vasculature and appear to contribute in the causation of cerebral ischemia. ETA receptor antagonists have been found to be effective in animal model of cerebral ischemia; however, clinical studies have shown no efficacy. Mitochondrial functions are critically important for several neural development processes such as neurogenesis, axonal and dendritic growth, and synaptic formation. ET appears to impair mitochondrial functions through activation of ETA receptors. On the other hand, blocking ETB receptors has been shown to trigger apoptotic processes by activating intrinsic mitochondrial pathway. Mitochondria are important for their role in molecular regulation of neurogenesis and angiogenesis. Stimulation of ETB receptors in the adult ischemic brain has been found to promote angiogenesis and neurogenesis mediated through vascular endothelial growth factor and nerve growth factor. It will be interesting to investigate the effect of ETB receptor stimulation on mitochondrial functions in the CNS following cerebral ischemia. Conclusion: The findings of this review implicate brain ETB receptors in angiogenesis and neurogenesis following cerebral ischemia, it is possible that the positive effect of stimulating ETB receptors in cerebral ischemia may be mediated through mitochondrial functions.