Model Of Global Cerebral Ischemia Research Articles

Pre- and Post-Treatment with Novel Antiepileptic Drug Oxcarbazepine Exerts Neuroprotective Effect in the Hippocampus in a Gerbil Model of Transient Global Cerebral Ischemia.

Oxcarbazepine, an antiepileptic drug, has been reported to modulate voltage-dependent sodium channels, and it is commonly used in epilepsy treatment. In this study, we investigated the neuroprotective effect of oxcarbazepine in the hippocampus after transient ischemia in gerbils. Gerbils randomly received oxcarbazepine 100 or 200 mg/kg before and after transient ischemia. We examined its neuroprotective effect in the cornu ammonis 1 subfield of the gerbil hippocampus at 5 days after transient ischemia by using cresyl violet staining, neuronal nuclei immunohistochemistry and Fluoro-Jade B histofluorescence staining for neuroprotection, and by using glial fibrillary protein and ionized calcium-binding adapter molecule 1 immunohistochemistry for reaction of astrocytes and microglia, respectively. Pre- and post-treatment with 200 mg/kg of oxcarbazepine, but not 100 mg/kg of oxcarbazepine, protected pyramidal neurons of the cornu ammonis 1 subfield from transient ischemic damage. In addition, pre- and post-treatment with oxcarbazepine (200 mg/kg) significantly ameliorated astrocytes and microglia activation in the ischemic cornu ammonis 1 subfield. In brief, our current results indicate that post-treatment as well as pre-treatment with 200 mg/kg of oxcarbazepine can protect neurons from ischemic insults via attenuation of the glia reaction.

Changes in electrolyte concentrations alter the impedance during ischemia-reperfusion injury in rat brain

Objective: Ischemic stroke is a major cause of death and disability worldwide. Nowadays, electrical impedance spectroscopy is an emerging tool to differentiate between normal and stroke conditions. Approach: In this study, changes in the bio-impedance spectroscopy using a two-electrode method with varying frequencies from 100 to 35 kHz have been assessed in a model of global cerebral ischemia in anesthetized rats during normal, occlusion and reperfusion conditions. Global cerebral ischemia was induced by bilateral common carotid artery occlusion for 40 min following 40 min of reperfusion. The concentration of sodium, potassium, calcium and chloride ions in the whole rat brain was determined by electrolyte analyzer. For the interpretation of in vivo results, changes in electrical impedance with varying concentrations of sodium, potassium and calcium ions in artificial cerebrospinal fluid (aCSF) were also observed using the bio-impedance spectroscopy method. Main results: The in vivo bio-impedance analysis suggests that the impedance is consistently increased during occlusion as compared to the normal condition. The in vitro study revealed that the impedance escalates with an increase in the concentration of potassium and calcium ions and reduces with an increase in the concentration of sodium ions in aCSF. A further electrolyte analysis suggested that the level of sodium and chloride ions is significantly decreased and the level of calcium and potassium is significantly increased during occlusion as compared to the normal condition. Significance: These findings suggest that the increase in impedance during occlusion may be due to changes in the ionic concentration of the rat brain. The above in vivo and in vitro studies successfully demonstrated and interrelated the change in impedance with corresponding changes in ionic concentration.

Sodium Hydrosulfide Post-conditioning Protects Hippocampal CA1 Neurons from Neuronal Cell Injury in the Rat Model of Transient Global Cerebral Ischemia Through Activation of Extracellular-regulated Kinases Signaling.

The effect of hydrogen sulfide (H2S) on global cerebral ischemia remains partially understood. This study aimed to investigate the neuroprotective effect of sodium hydrosulfide (NaHS, a donor of H2S) post-conditioning and its underlying mechanism in a transient global cerebral ischemia (tGCI) model. The tGCI rat model was established by the four-vessel occlusion method. Wistar rats were randomly assigned into 6 groups: sham, tGCI, tGCI +NaHS, tGCI+vehicle, tGCI+U0126 and tGCI+U0126+NaHS groups. Neurons survival was assessed by Nissl staining and NeuN immunostaining. Levels of extracellular extracellular-regulated kinases (ERK)1/2 and p-ERK1/2 were determined by western blot and immunohistochemistry (IHC). Intraperitoneal injection of NaHS (24 µmol/kg) at 24 h post-tGCI attenuated tGCI-induced decrease of the survival and NeuN-positive neurons in the hippocampal CA1 subregion. Compared to the sham group, tGCI significantly up-regulated p-ERK1/2 protein at 26 and 48 h post-tGCI. NaHS post-conditioning further enhanced the phosphorylation of ERK1/2 at 26, 48 and 168 h post-tGCI. Nevertheless, U0126 (an inhibitor of MEK1/2) pre-treatment reduced the p-ERK1/2 level in both the tGCI+ U0126 group and the tGCI+ U0126+ NaHS group. IHC staining revealed that p-ERK1/2-positive cell could be observed in several hippocampal subregions of the rats receiving NaHS post-conditioning. Immunofluorescence staining showed that some neurons were double-stained with p-ERK1/2 and NeuN. Furthermore, U0126 pre-treatment significantly attenuated the protective effect of NaHS post-conditioning on the neurons survival and NeuNpositive neurons in CA1 subregion. These results suggested that NaHS post-conditioning can protect hippocampal CA1 neurons from tGCI-induced injury, at least partially, through activation of ERK1/2 signaling.

Effects of total saponins of panax notoginseng on depressive behavior and hippocampal nerve regeneration in rats with following global cerebral ischemia depression

Objective To explore the effect of the total saponins of panax notoginseng (TSPN) on depression-like behavior following global cerebral ischemia depression in rats and its mechanism. Methods Using four-vessel occlusion method to build the global cerebral ischemia model, then the cerebral ischemia rats were given solitary breeding with chronic unpredictable mild stress(CUMS) to prepare depression model.Seventy rats were divided into sham group (n=10), model group (n=20), PSD group (n=20) and TSPN group (n=20). The rats in the TSPN group were administered TSPN intraperitoneally 30 min post-brain ischemia.The dose of TSPN (75mg/kg) was suspended in 0.9% saline 10g/L, once per day for 30 days after reperfusion.While rats in the vehicle group and PSD group was treated with equal volume of 0.9% saline, one injection per day until the rats were sacrificed at 30 days after brain ischemia.The BrdU, doublecortin (DCX) expression in the hippocampus was assessed by immunohistochemistry. Results In comparison with the model group, the sucrose preference percentage in the PSD group was significantly lower ((46.2±9.2)%, (61.2±7.6)%; t=3.18, P<0.05), then the PSD rats were administered TSPN intraperitoneally, the sucrose preference percentage increased significantly ((62.4±3.4)%, (46.2±9.2)%; t=3.43, P<0.05). During the forced swimming test, the immobility time of PSD group was significantly increased compared with the model group ((119.4±9.7)s, (88.0±15.6)s ; t=4.30, P<0.01), while after PSD rats administering TSPN intraperitoneally, the immobility time was shorten remarkably ((97.4±6.7)s, (119.4±9.7)s; t=3.01, P<0.05). Compared with the Model group(BrdU+ : (12.6±2.2)/mm2, DCX+ : (38.6±4.2)/mm2), the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in PSD group decreased (BrdU+ : (8.8±1.5)/mm2, DCX+ : (27.2±2.8)/mm2; t=3.25, 4.29, both P<0.01). And compared with PSD group, the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in TSPN group increased significantly (BrdU+ : (14.8±2.8)/mm2, DCX+ : (37.0±3.3)/mm2; t=4.68, 3.69, both P<0.05). Conclusion TSPN can improve the depression-like behavior of rats following global cerebral ischemia, which may be related with promoting hippocampal nerve regeneration. Key words: Total saponins of panax notoginseng; Hippocampus; Nerve regeneration; Following global cerebral ischemia depression; Rats

Transient Bilateral Common Carotid Artery Occlusion (tBCCAO) of Rats as a Model of Global Cerebral Ischemia

Background: Transient bilateral common carotid artery occlusion (tBCCAO) has been performed in rats as a model of global ischemia. However, the technique varied between laboratories and produced difficulties in the comparison of results. Variations such as rat strain, age, ischemic and reperfusion duration could affect the results. This review aims to provide a general overview of the variation of animal strains, duration of tBCCAO, reported cerebral ischemic area produced by tBCCAO, use of TTC staining for measurement of volume of brain ischemia and functional neurological tests.&#x0D; Method: The data of this review were obtained from abstracts in PubMed database and Google Scholars and were not limited by publication time. Keywords used to search the abstracts were (BCCAO OR “bilateral common carotid artery occlusion” OR “stroke” OR “cerebral ischemia” OR “brain ischemia”) AND (rat OR rats). The research method of each study was identified from the collected abstracts. The abstracts were chosen for further study on the basis that they met the inclusion criteria which were English language articles; original research article; animal model used were adolescent, adult, and elderly rats; ischemic finding in rats’ cerebrum by BCCAO technique was presented; ischemic size were assessed and the result was described; studies that had control group; and studies that induced transient global ischemic to the rats’ cerebrum. Data that were extracted to the datasheet were references; animal model strain; ischemic duration; reperfusion duration; ischemic area; 2,3,5 Triphenyltetrazolium Chloride (TTC) staining; Cavalieri method; and rats’ neurological functional tests.&#x0D; Results and Conclusions: There were differences in the ischemic area between Wistar and Sprague-Dawley rats after transient BCCAO. There were differences in the TTC staining solution concentration that was used to identify ischemic area of the brain following transient BCCAO. There was a very limited number of studies using Cavalieri method for the quantification of ischemic volume of rats’ brain after transient BCCAO. Neurological functional tests in animal models post transient BCCAO did not include sensory and memory functions tests.&#x0D; Bangladesh Journal of Medical Science Vol.18(3) 2019 p.491-500

Critical Role of Nrf2 in Experimental Ischemic Stroke.

Ischemic stroke is one of the leading causes of death and long-term disability worldwide; however, effective clinical approaches are still limited. The transcriptional factor Nrf2 is a master regulator in cellular and organismal defense against endogenous and exogenous stressors by coordinating basal and stress-inducible activation of multiple cytoprotective genes. The Nrf2 network not only tightly controls redox homeostasis but also regulates multiple intermediary metabolic processes. Therefore, targeting Nrf2 has emerged as an attractive therapeutic strategy for the prevention and treatment of CNS diseases including stroke. Here, the current understanding of the Nrf2 regulatory network is critically examined to present evidence for the contribution of Nrf2 pathway in rodent ischemic stroke models. This review outlines the literature for Nrf2 studies in preclinical stroke and focuses on the in vivo evidence for the role of Nrf2 in primary and secondary brain injuries. The dynamic change and functional importance of Nrf2 signaling, as well as Nrf2 targeted intervention, are revealed in permanent, transient, and global cerebral ischemia models. In addition, key considerations, pitfalls, and future potentials for Nrf2 studies in preclinical stroke investigation are discussed.

MiRNA-210 induces the apoptosis of neuronal cells of rats with cerebral ischemia through activating HIF-1α-VEGF pathway.

This study aims to explore the expression of micro-ribonucleic acid (miRNA)-210 in the cerebral cortex of rat model with global cerebral ischemia, and determine its function on the regulation of the apoptosis of neuronal cells. Rat models of global cerebral ischemia were established in vitro. Rats were euthanized at 24 h after reperfusion and the cerebral cortex was collected. The expression of miRNA-210 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Neuronal cells were transfected by liposomes in vitro and cells were divided into neuronal cell group (group i), neuronal cell + miRNA-210 mimic group (group ii) and neuronal cell + miRNA-210 inhibitor group (group iii). The cell apoptosis rate and gene and protein expressions of HIF-1α, VEGF and Caspase-3 were measured. The level of miRNA-210 in rats with global cerebral ischemia was remarkably higher than that in rats from sham operation group (p<0.05). The apoptosis rate of neuronal cells was increased evidently when miRNA-210 was overexpressed, and the expressions of HIF-1α, VEGF and Caspase-3 were elevated markedly at both mRNA and protein levels. Our data indicate that miRNA-210 expression is upregulated in the rats with global cerebral ischemia, and the rise of miRNA-210 level increases the apoptosis of neuronal cells through the activation of HIF-1α-VEGF signaling pathway.

Neuroprotective effects of the peel of &lt;i&gt;Citrus kawachiensis&lt;/i&gt; (Kawachi Bankan) and auraptene in the hippocampus of hyperglycemia mice and global cerebral ischemia/reperfusion injury mice.

The peel of Citrus kawachiensis (Kawachi Bankan), a citrus species grown in Ehime, Japan, is abundant in auraptene. Auraptene, a coumarin compound, have been shown to exert anti-inflammatory effects in peripheral tissues, but it was still unclear of the effect in the brain. Hyperglycemia and brain ischemia induce inflammation and oxidative stress and cause massive damage in the brain; therefore, we examined the anti-inflammatory and other effects of the dried peel powder of C. kawachiensis and auraptene in a hyperglycemia and global cerebral ischemia models. The C. kawachiensis treatment inhibited astroglial activation in the hippocampus and the hyperphosphorylation of tau protein in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in the type 2 diabetic db/db mice. The C. kawachiensis treatment inhibited microglial and astroglial activation, and neuronal cell death in the hippocampus of transient global cerebral ischemia mice. It was suggested that the dried peel powder of C. kawachiensis exerts anti-inflammatory and neuroprotective effects in the brain. We attempted to demonstrate the effect of auraptene in the brains in streptozotocin-induced hyperglycemic mice and transient global cerebral ischemia mice. Auraptene administration showed the similar effects as the peel of C. kawachiensis in the hippocampus of these mice models. These results suggested that auraptene have potential effects as a neuroprotective agent in the peel of C. kawachiensis.

Loss of interneurons and disruption of perineuronal nets in the cerebral cortex following hypoxia-ischaemia in near-term fetal sheep

Hypoxia-ischaemia (HI) in term infants is a common cause of brain injury and neurodevelopmental impairment. Development of gamma-aminobutyric acid (GABA)ergic circuitry in the cerebral cortex is a critical event in perinatal brain development. Perineuronal nets (PNNs) are specialised extracellular matrix structures that surround GABAergic interneurons, and are important for their function. Herein, we hypothesised that HI would reduce survival of cortical interneurons and disrupt PNNs in a near-term fetal sheep model of global cerebral ischaemia. Fetal sheep (0.85 gestation) received sham occlusion (n = 5) or 30 min of reversible cerebral ischaemia (HI group; n = 5), and were recovered for 7 days. Expression of interneurons (glutamate decarboxylase [GAD]+; parvalbumin [PV]+) and PNNs (Wisteria floribunda agglutinin, WFA) was assessed in the parasagittal cortex by immunohistochemistry. HI was associated with marked loss of both GAD+ and PV+ cortical interneurons (all layers of the parasagittal cortex and layer 6) and PNNs (layer 6). The expression and integrity of PNNs was also reduced on surviving GAD+ interneurons. There was a trend towards a linear correlation of the proportion of GAD+ neurons that were WFA+ with seizure burden (r2 = 0.76, p = 0.0534). Overall, these data indicate that HI may cause deficits in the cortical GABAergic system involving loss of interneurons and disruption of PNNs, which may contribute to the range of adverse neurological outcomes following perinatal brain injury.

Carvacrol Attenuates Hippocampal Neuronal Death after Global Cerebral Ischemia via Inhibition of Transient Receptor Potential Melastatin 7.

Over the last two decades, evidence supporting the concept of zinc-induced neuronal death has been introduced, and several intervention strategies have been investigated. Vesicular zinc is released into the synaptic cleft, where it then translocates to the cytoplasm, which leads to the production of reactive oxygen species and neurodegeneration. Carvacrol inhibits transient receptor potential melastatin 7 (TRPM7), which regulates the homeostasis of extracellular metal ions, such as calcium and zinc. In the present study, we test whether carvacrol displays any neuroprotective effects after global cerebral ischemia (GCI), via a blockade of zinc influx. To test our hypothesis, we used eight-week-old male Sprague–Dawley rats, and a GCI model was induced by bilateral common carotid artery occlusion (CCAO), accompanied by blood withdrawal from the femoral artery. Ischemic duration was defined as a seven-minute electroencephalographic (EEG) isoelectric period. Carvacrol (50 mg/kg) was injected into the intraperitoneal space once per day for three days after the onset of GCI. The present study found that administration of carvacrol significantly decreased the number of degenerating neurons, microglial activation, oxidative damage, and zinc translocation after GCI, via downregulation of TRPM7 channels. These findings suggest that carvacrol, a TRPM7 inhibitor, may have therapeutic potential after GCI by reducing intracellular zinc translocation.

Palmitic acid methyl ester is a novel neuroprotective agent against cardiac arrest

We previously discovered that palmitic acid methyl ester (PAME) is a potent vasodilator first identified and released from the superior cervical ganglion and remain understudied. Thus, we investigated PAME's role in modulating cerebral blood flow (CBF) and neuroprotection after 6 min of cardiac arrest (model of global cerebral ischemia). Our results suggest that PAME can enhance CBF under normal physiological conditions, while administration of PAME (0.02 mg/kg) immediately after cardiopulmonary resuscitation can also enhance CBF in vivo. Additionally, functional learning and spatial memory assessments (via T-maze) 3 days after asphyxial cardiac arrest (ACA) suggest that PAME-treated rats have improved learning and memory recovery versus ACA alone. Furthermore, improved neuronal survival in the CA1 region of the hippocampus were observed in PAME-treated, ACA-induced rats. Altogether, our findings suggest that PAME can enhance CBF, alleviate neuronal cell death, and promote functional outcomes in the presence of ACA.

Low Doses of G-CSF Prevent Cerebral Infarction and Maintain Muscle Strength in an Experimental Model of Global Ischemic Stroke.

Stroke is a major cause of severe and long-term disability in adult individuals. Treatment of this disease is limited by the narrow therapeutic window in which intervention is crucial. An alternative therapy for stroke could be cellular growth factors, which participate in several pathways that mediate neuronal cell death. We evaluated the neuroprotective ability of different doses of granulocyte colonystimulating factor (G-CSF; 5, 50 and 100 μg/kg/day) in the mouse model of global cerebral ischemia induced by bilateral occlusion of the common carotid arteries for 80 minutes. The control group received vehicle (5% glucose solution) and the treated group was administered with G-CSF at two postsurgery time-points: immediately after and 24 hours after. Subsequently, muscle strength, leukocyte count, infarcted cortical area, and apoptosis/TUNEL were evaluated. The global ischemia promoted an impairment of the strength (16%) and a cerebral infarction (0.437±0.08 cm2) which were accompanied by apoptosis evaluated by TUNEL in control mice. In mice treated with G-CSF the strength function was maintained, the infarcted area (~70%) and apoptosis were decreased in a similar magnitude in all treated groups. Accordingly, the cytokine activities were confirmed by blood leukocyte count that was increased approximately 2-fold than that observed in the control group. The results indicate a neuroprotective effect of G-CSF, even in small doses, in mice subjected to global cerebral ischemia, thereby reducing the neurofunctional impairment caused by stroke, when considering the maintenance of muscle strength in the treated animals.

Effects of Lipoxin A4 Pretreatment on Cognitive Function of Aged Rats after Global Cerebral Ischemia Reperfusion.

The aim of the present study was to investigate the effect of lipoxin A4 (LXA4) pretreatment on cognitive function of aged rats after global cerebral ischemia reperfusion, and to explore its possible mechanism. Thirty-six aged male Sprague-Dawley rats were randomly divided into three groups (n=2 each): sham-operation group (S group), global cerebral ischemia reperfusion group (I/R group) and LXA4-pretreatment group (L group). The rat model of global cerebral ischemia reperfüsion was established by occlusion of the bilateral common carotid artery with hypotension. The cognitive function of rats was determined by a step-down type passive avoidance test and Morris Water Maze test on the third day after reperfUsion. Rats were sacrificed after Water Maze test and the pathological changes of hippocampal CAI region were observed and the related inflammatory mediators were determined. As compared with S group, the escape latency in I/R group was prolonged from the first day to the fifth day, while that in L group was prolonged from the first day to the third day. The retention time in I/R group and L group in the first quadrant was shortened. The reaction time, frequency of reaction mistake and frequency of escape mistake in I/R group increased, and the latent period shortened. The frequency of escape mistake in L group increased, and the damage in the hippocampal CAI region of I/R group and L group was obvious. The levels of S-100β, TNP-α, IL-lβ, IL-10 and NF-κB in I/R group and L group increased. As compared with I/R group, the escape latency in L group was shortened from the first day to the fifth day, and the retention time in the first quadrant prolonged. The reaction time, frequency of reaction mistake and frequency of escape mistake in L group decreased, and the latent period prolonged. The damage in the hippocampal CAI region of L group was alleviated as well. The levels of S-100β, TNP-α, IL-lβ and NF-κB in L group decreased, and those of IL-10 increased. It can be concluded that LXA4 pretreatment can improve the cognitive function in aged rats after global cerebral ischemia reperfusion probably by inhibiting the inflammatory reaction.

Mild hypothermia inhibits the Notch 3 and Notch 4 activation and seizure after stroke in the rat model

Ischemic brain injury is an important cause for seizure. Mild hypothermia of the brain or the whole body is an effective way to remit the post-stroke seizure. Our previous study revealed an implication of Notch 1 and 2 in the post-stroke seizure. This study further investigated the involvement of Notch 3 and 4 in post-stroke seizure and the effect of mild hypothermia on these two factors. A global cerebral ischemia (GCI) model was conducted in Sprague Dawley rats. Seizure activity was evaluated by the frequency of seizure attacks, seizure severity scores, and seizure discharges. Seizures were frequently occurred in the first and the second 24 h after GCI, however active whole-body cooling (mild hypothermia) and DAPT (Notch inhibitor) injection into the hippocampus, alone or in combination, alleviated seizure activity after GCI. Immunohistochemistry and Western blot assays revealed the up-regulation of Notch intracellular domain (NICD) 3 and 4 in the cerebral cortex and hippocampus following GCI, but mild hypothermia and DAPT inhibited the up-regulation of NICD 3 and 4. NF-κB, PPARα, PPARγ, cyclin D1, Sox2 and Pax6 are associated with the pathogenesis of diverse type of seizures. GCI induced NF-κB, cyclin D1, and Pax6 activity, but suppressed PPARγ. These effects of GCI were abolished by both mild hypothermia and DAPT treatment. This indicated the implication of Notch signaling in the effects of GCI. Collectively, mild hypothermia inhibits Notch 3 and Notch 4 activation and seizure after stroke in the rat model. This study adds to the further understanding of the pathogenesis of post-stroke seizures and the protective mechanism of mild hypothermia.

Changes in the hippocampal and peripheral phospholipid profiles are associated with neurodegeneration hallmarks in a long-term global cerebral ischemia model: Attenuation by Linalool

Phospholipid alterations in the brain are associated with progressive neurodegeneration and cognitive impairment after acute and chronic injuries. Various types of treatments have been evaluated for their abilities to block the progression of the impairment, but effective treatments targeting long-term post-stroke alterations are not available. In this study, we analyzed changes in the central and peripheral phospholipid profiles in ischemic rats and determined whether a protective monoterpene, Linalool, could modify them. We used an in vitro model of glutamate (125 μM) excitotoxicity and an in vivo global ischemia model in Wistar rats. Linalool (0.1 μM) protected neurons and astrocytes by reducing LDH release and restoring ATP levels. Linalool was administered orally at a dose of 25 mg/kg every 24 h for a month, behavioral tests were performed, and a lipidomic analysis was conducted using mass spectrometry. Animals treated with Linalool displayed faster neurological recovery than untreated ischemic animals, accompanied by better motor and cognitive performances. These results were confirmed by the significant reduction in astrogliosis, microgliosis and COX-2 marker, involving a decrease of 24:0 free fatty acid in the hippocampus. The altered profiles of phospholipids composed of mono and polyunsaturated fatty acids (PC 36:1; 42:1 (24:0/18:1)/LPC 22:6)/LPE 22:6) in the ischemic hippocampus and the upregulation of PI 36:2 and other LCFA (long chain fatty acids) in the serum of ischemic rats were prevented by the monoterpene. Based on these data, alterations in the central and peripheral phospholipid profiles after long-term was attenuated by oral Linalool, promoting a phospholipid homeostasis, related to the recovery of brain function.

Tau pathology and neurochemical changes associated with memory dysfunction in an optimised murine model of global cerebral ischaemia - A potential model for vascular dementia?

Cerebral ischemia is known to be a major cause of death and the later development of Alzheimer's disease and vascular dementia. However, ischemia induced cellular damage that initiates these diseases remain poorly understood. This is primarily due to lack of clinically relevant models that are highly reproducible. Here, we have optimised a murine model of global cerebral ischaemia with multiple markers to determine brain pathology, neurochemistry and correlated memory deficits in these animals. Cerebral ischaemia in mice was induced by bilateral common carotid artery occlusion. Following reperfusion, the mice were either fixed with 4% paraformaldehyde or decapitated under anaesthesia. Brains were processed for Western blotting or immunohistochemistry for glial (GLT1) and vesicular (VGLUT1, VGLUT2) glutamate transporters and paired helical filament (PHF1) tau. The PHF1 tau is the main component of neurofibrillary tangle, which is the pathological hallmark of Alzheimer's disease and vascular dementia. The novel object recognition behavioural assay was used to investigate the functional cognitive consequences in these mice. The results show consistent and selective neuronal and glial cell changes in the hippocampus and the cortex together with significant reductions in GLT1 (***P < 0.001), VGLUT1 (**P < 0.01) and VGLUT2 (***P < 0.001) expressions in the hippocampus in occluded mice as compared to sham-operated animals. These changes are associated with increased PHF1 (***P < 0.0001) protein and a significant impairment of performance (*p < 0.0006, N = 6/group) in the novel object recognition test. This model represents a useful tool for investigating cellular, biochemical and molecular mechanisms of global cerebral ischaemia and may be an ideal preclinical model for vascular dementia.

Recovery of Neurovascular Unit Integrity by CDK5-KD Astrocyte Transplantation in a Global Cerebral Ischemia Model.

Astrocytes play metabolic and structural support roles and contribute to the integrity of the blood-brain barrier (BBB), linking communication between neurons and the endothelium. Cyclin-dependent kinase 5 (CDK5) likely exerts a dual effect on the endothelium and astrocytes due to its involvement in migration and angiogenesis; the overactivation of CDK5 is associated with dysfunction in glutamate recapture and hypoxia. Recently, we proposed that CDK5-targeted astrocytes facilitate the recovery of neurological and motor function in transplanted ischemic rats. In the current study, we treated cerebral ischemic rats and endothelial cells exposed to glutamate toxicity with CDK5 knock-down (CDK5-KD) astrocytes to determine the role of CDK5 in neurovascular integrity. We found that the effects of CDK5-KD were sustained for 4months, preventing neuronal and astrocyte loss, facilitating the recovery of the BBB via the production of BDNF by endogenous astrocytes (GFP-) surrounding vessels in the motor cortex and the corpus callosum of global ischemic rats, and improving neurological performance. These findings were supported by the in vitro findings of increased transendothelial resistance, p120-ctn+ adhesion and reduced intercellular gaps induced by a CDK5 inhibitor (roscovitine) in bEnd.3 cells in a glutamate-toxicity model. Additionally, CDK5-KD astrocytes in co-culture protected the endothelial cell viability, increased BDNF release from astrocytes, increased BDNF immunoreactivity in neighboring astrocytes and endothelial cells and enhanced cell adhesion in a glutamate-toxicity model. Altogether, these findings suggest that a CDK5 reduction in astrocytes protects the endothelium, which promotes BDNF release, endothelial adhesion, and the recovery of neurovascular unit integrity and brain function in ischemic rats.

Salubrinal and robenacoxib treatment after global cerebral ischemia. Exploring the interactions between ER stress and inflammation

BackgroundBlood reperfusion of the ischemic tissue after stroke promotes increases in the inflammatory response as well as accumulation of unfolded/misfolded proteins in the cell, leading to endoplasmic reticulum (ER) stress. Both Inflammation and ER stress are critical processes in the delayed death of the cells damaged after ischemia. The aim of this study is to check the putative synergic neuroprotective effect by combining anti-inflammatory and anti-ER stress agents after ischemia. MethodsThe study was performed on a two-vessel occlusion global cerebral ischemia model. Animals were treated with salubrinal one hour after ischemia and with robenacoxib at 8 h and 32 h after ischemia. Parameters related to the integrity of the blood–brain barrier (BBB), such as matrix metalloproteinase 9 and different cell adhesion molecules (CAMs), were analyzed by qPCR at 24 h and 48 h after ischemia. Microglia and cell components of the neurovascular unit, including neurons, endothelial cells and astrocytes, were analyzed by immunofluorescence after 48 h and seven days of reperfusion. ResultsPharmacologic control of ER stress by salubrinal treatment after ischemia, revealed a neuroprotective effect over neurons that reduces the transcription of molecules involved in the impairment of the BBB. Robenacoxib treatment stepped neuronal demise forward, revealing a detrimental effect of this anti-inflammatory agent. Combined treatment with robenacoxib and salubrinal after ischemia prevented neuronal loss and changes in components of the neurovascular unit and microglia observed when animals were treated only with robenacoxib. ConclusionCombined treatment with anti-ER stress and anti-inflammatory agents is able to provide enhanced neuroprotective effects reducing glial activation, which opens new avenues in therapies against stroke.

MTOR is involved in stroke-induced seizures and the anti-seizure effect of mild hypothermia.

Stroke is considered an underlying etiology of the development of seizures. Stroke leads to glucose and oxygen deficiency in neurons, resulting in brain dysfunction and injury. Mild hypothermia is a therapeutic strategy to inhibit stroke-induced seizures, which may be associated with the regulation of energy metabolism of the brain. Mammalian target of rapamycin (mTOR) signaling and solute carrier family 2, facilitated glucose transporter member (GLUT)-1 are critical for energy metabolism. Furthermore, mTOR overactivation and GLUT-1 deficiency are associated with genetically acquired seizures. It has been hypothesized that mTOR and GLUT-1 may additionally be involved in seizures elicited by stroke. The present study established global cerebral ischemia (GCI) models of rats. Convulsive seizure behaviors frequently occurred during the first and the second days following GCI, which were accompanied with seizure discharge reflected in the EEG monitor. Expression of phosphor (p)-mTOR and GLUT-1 were upregulated in the cerebral cortex and hippocampus, as evidenced by immunohistochemistry and western blot analyses. Mild hypothermia and/or rapamycin (mTOR inhibitor) treatments reduced the number of epileptic attacks, seizure severity scores and seizure discharges, thereby alleviating seizures induced by GCI. Mild hypothermia and/or rapamycin treatments reduced phosphorylation levels of mTOR and the downstream effecter p70S6 in neurons, and the amount of GLUT-1 in the cytomembrane of neurons. The present study revealed that mTOR is involved in stroke-induced seizures and the anti-seizure effect of mild hypothermia. The role of GLUT-1 in stroke-elicited seizures appears to be different from the role in seizures induced by other reasons. Further studies are necessary in order to elucidate the exact function of GLUT-1 in stroke-elicited seizures.

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.