Min Of Bilateral Carotid Artery Occlusion Research Articles

Inhibition of microRNA-200c preserves astrocyte sirtuin-1 and mitofusin-2, and protects against hippocampal neurodegeneration following global cerebral ischemia in mice.

Memory impairment remains a leading disability in survivors of global cerebral ischemia, occurring secondary to delayed neurodegeneration of hippocampal cornu ammonis-1 (CA1) neurons. MicroRNA-200c (miR-200c) is induced following ischemic stress and we have previously demonstrated that pre-treatment with anti-miR-200c is protective against embolic stroke in mice. In the present study we assessed the role of miR-200c on CA1 neurodegeneration, sirtuin-1 (SIRT1), and mitochondrial dynamic protein expression in a mouse model of transient global cerebral ischemia and in vitro in primary mouse astrocyte cultures after simulated ischemia. Mice were subjected to 10 min bilateral common carotid artery occlusion plus hypotension with 5% isoflurane. After 2 h recovery mice were treated with intravenous injection of either anti-miR-200c or mismatch control. Memory function was assessed by Barnes maze at post-injury days 3 and 7. Mice were sacrificed at post-injury day 7 for assessment of brain cell-type specific expression of miR-200c, SIRT1, and the mitochondrial fusion proteins mitofusin-2 (MFN2) and OPA1 via complexed fluorescent in situ hybridization and fluorescent immunohistochemistry. Global cerebral ischemia induced significant loss of CA1 neurons, impaired memory performance and decreased expression of CA1 SIRT1, MFN2, and OPA1. Post-injury treatment with anti-miR-200c significantly improved survival, prevented CA1 neuronal loss, improved post-injury performance in Barnes maze, and was associated with increased post-injury expression of CA1 SIRT1 and MFN2 in astrocytes. In vitro, primary mouse astrocyte cultures pre-treated with miR-200c inhibitor prior to oxygen/glucose deprivation preserved expression of SIRT1 and MFN2, and decreased reactive oxygen species generation, whereas pre-treatment with miR-200c mimic had opposite effects that could be reversed by co-treatment with SIRT1 activator. These results suggest that miR-200c regulates astrocyte mitochondrial homeostasis via targeting SIRT1, and that CA1 astrocyte mitochondria and SIRT1 represent potential post-injury therapeutic targets to preserve cognitive function in survivors of global cerebral ischemia.

Abatement of neurobehavioral and neurochemical dysfunctions in cerebral ischemia/reperfusion injury by Tetrapleura tetraptera fruit extract

Ethnopharmacological relevanceTetrapleura tetraptera Taub. (family Fabaceae), is generally found in the lowland forest of tropical Africa. Its leaves and fruits are traditionally used in West Africa for the management of brain disorders. Aim of the studyThis study evaluated the effect of Tetrapleura tetraptera methanol fruit extract (TT) on bilateral common carotid artery occlusion-induced cerebral ischemia/reperfusion (I/R) injury in male Wistar rats. Materials and methodsRats pretreated with TT for 7 days before a 30 min bilateral common carotid artery occlusion and reperfusion for 24 h were assessed for neurobehavioural deficits. Cortical, striatal and hippocampal oxidative stress, pro-inflammatory events, electrolyte imbalance and neurochemical dysfunctions, as well as hippocampal histopathological alterations, were also evaluated. HPLC-DAD analysis was performed to identify likely compounds contributing to the bioactivity of the extract. ResultsTT reduced I/R-induced behavioral deficits and ameliorated I/R-induced oxidative stress by restoring reduced glutathione level, increasing catalase and superoxide dismutase activities, and also reducing both lipid peroxidation and xanthine oxidase activity in the brain. TT attenuated I/R-increased myeloperoxidase and lactate dehydrogenase activities as well as disturbances in Na+ and K+ levels. Alterations elicited by I/R in the activities of Na+/K+ ATPase, complex I, glutamine synthetase, acetylcholinesterase, and dopamine metabolism were abated by TT pretreatment. TT prevented I/R-induced histological changes in the hippocampus. HPLC-DAD analysis revealed the presence of aridanin, a marker compound for Tetrapleura tetraptera, and other phytochemicals. ConclusionsThese findings indicate that Tetrapleura tetraptera fruit has a protective potential against stroke through modulation of redox and electrolyte imbalances, and attenuation of neurotransmitter dysregulation and other neurochemical dysfunctions. Tetrapleura tetraptera fruit could be a promising source for the discovery of bioactives for stroke therapy.

Regional-specific effects of cerebral ischemia/reperfusion and dehydroepiandrosterone on synaptic NMDAR/PSD-95 complex in male Wistar rats

Excessive glutamate efflux and N-methyl-D-aspartate receptor (NMDAR) over-activation represent well-known hallmarks of cerebral ischemia/reperfusion (I/R) injury, still, expression of proteins involved in this aspect of I/R pathophysiology show inconsistent data. Neurosteroid dehydroepiandrosterone (DHEA) has been proposed as potent NMDAR modulator, but its influence on I/R-induced changes up to date remains questionable. Therefore, I/R-governed alteration of vesicular glutamate transporter 1 (vGluT1), synaptic NMDAR subunit composition, postsynaptic density protein 95 (PSD-95) and neuronal morphology alone or following DHEA treatment were examined. For that purpose, adult male Wistar rats were treated with a single dose of vehicle or DHEA (20 mg/kg i.p.) 4 h following sham operation or 15 min bilateral common carotid artery occlusion. Western blot was used for analyses of synaptic protein expressions in hippocampus and prefrontal cortex, while neuronal morphology was assessed using Nissl staining. Regional-specific postischemic changes were detected on protein level i.e. signs of neuronal damage in CA1 area was accompanied with hippocampal vGluT1, NR1, NR2B enhancement and PSD-95 decrement, while histological changes observed in layer III were associated with decreased NR1 subunit in prefrontal cortex. Under physiological conditions DHEA had no effect on protein and histological appearance, while in ischemic milieu it restored hippocampal PSD-95 and NR1 in prefrontal cortex to the control level. Along with intact neurons, ones characterized by morphology observed in I/R group were also present. Future studies involving NMDAR-related intracellular signaling and immunohistochemical analysis will reveal precise effects of I/R and DHEA treatment in selected brain regions.

Laser Speckle Imaging of Rat Pial Microvasculature during Hypoperfusion-Reperfusion Damage.

The present study was aimed to in vivo assess the blood flow oscillatory patterns in rat pial microvessels during 30 min bilateral common carotid artery occlusion (BCCAO) and 60 min reperfusion by laser speckle imaging (LSI). Pial microcirculation was visualized by fluorescence microscopy. The blood flow oscillations of single microvessels were recorded by LSI; spectral analysis was performed by Wavelet transform. Under baseline conditions, arterioles and venules were characterized by blood flow oscillations in the frequency ranges 0.005–0.0095 Hz, 0.0095–0.021 Hz, 0.021–0.052 Hz, 0.052–0.150 Hz and 0.150–0.500 Hz. Arterioles showed oscillations with the highest spectral density when compared with venules. Moreover, the frequency components in the ranges 0.052–0.150 Hz and 0.150–0.500 were predominant in the arteriolar total power spectrum; while, the frequency component in the range 0.150–0.500 Hz showed the highest spectral density in venules. After 30 min BCCAO, the arteriolar spectral density decreased compared to baseline; moreover, the arteriolar frequency component in the range 0.052–0.150 Hz significantly decreased in percent spectral density, while the frequency component in the range 0.150–0.500 Hz significantly increased in percent spectral density. However, an increase in arteriolar spectral density was detected at 60 min reperfusion compared to BCCAO values; consequently, an increase in percent spectral density of the frequency component in the range 0.052–0.150 Hz was observed, while the percent spectral density of the frequency component in the range 0.150–0.500 Hz significantly decreased. The remaining frequency components did not significantly change during hypoperfusion and reperfusion. The changes in blood flow during hypoperfusion/reperfusion caused tissue damage in the cortex and striatum of all animals. In conclusion, our data demonstrate that the frequency component in the range 0.052–0.150 Hz, related to myogenic activity, was significantly impaired by hypoperfusion and reperfusion, affecting cerebral blood flow distribution and causing tissue damage.

MODULATION OF KEY BIOCHEMICAL MARKERS RELEVANT TO STROKE BY ANTIARIS AFRICANA LEAF EXTRACT FOLLOWING CEREBRAL ISCHEMIA/REPERFUSION INJURY

Background:Oxidative stress plays a significant role in stroke pathogenesis. Hence, plants rich in antioxidant phytochemicals have been suggested as effective remedies for prevention and treatment of stroke and other neurological diseases. Antiaris africana Engl. (Moraceae) is traditionally used for the management of brain-related problems but there is paucity of data on its anti-stroke potential.Materials and Methods:Ischemia/reperfusion injury was induced by a 30 min bilateral common carotid artery occlusion/ 2 h reperfusion (BCCAO/R) in the brain of male Wistar rats. A sham-operated group which was not subjected to BCCAO/R and a group subjected to BCCAO/R without treatment with MEA served as controls. The ameliorative effect of 14 days of pretreatment with 50 mg/kg or 100 mg/kg A. africana methanol leaf extract (MEA) on BCCAO/R-mediated alterations to key markers of oxidative stress (malondialdehyde, reduced glutathione, xanthine oxidase, superoxide dismutase, catalase and glutathione peroxidase) and neurochemical disturbances and excitotoxicity (myeloperoxidase, glutamine synthetase, Na+/K+ ATPase, acetylcholinesterase and tyrosine hydroxylase), was evaluated and compared with the effect produced by treatment with 20 mg/kg quercetin as a reference standard.Results:Results show that pretreatment with MEA significantly mitigated or reversed BCCAO/R-induced changes in the level or activity of the evaluated biochemical markers of oxidative stress, neurochemical dysfunction and excitotoxicity compared with the BCCAO/R untreated control group (p < 0.05). The effect produced by 100 mg/kg MEA was similar to that of the reference standard, quercetin.Conclusion:These results revealed the neuroprotective potential of A. africana in stroke and other ischemia-related pathologies.

Malvidin's Effects on Rat Pial Microvascular Permeability Changes Due to Hypoperfusion and Reperfusion Injury.

The present study was aimed to evaluate the malvidin’s protective effects on damage induced by 30 min bilateral common carotid artery occlusion (BCCAO) and 60 min reperfusion (RE) in rat pial microcirculation. Rat pial microcirculation was observed using fluorescence microscopy through a closed cranial window. Western blotting analysis was performed to investigate the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS) and matrix metalloproteinase 9 (MMP-9) expression. Moreover, MMP-9 activity was evaluated by zymography. Finally, neuronal damage and radical oxygen species (ROS) formation were assessed. In all animals, pial arterioles were classified in five orders of branching according to Strahler’s method. In hypoperfused rats, 30 min BCCAO and 60 min RE caused a decrease in arteriolar diameter, an increase in microvascular leakage and leukocyte adhesion, accompanied by decreased capillary perfusion and red blood cell velocity (VRBC). Moreover, marked neuronal damage and evident ROS generation were detected. Conversely, malvidin administration induced arteriolar dilation in dose-related manner, reducing microvascular leakage as well as leukocyte adhesion. Capillary perfusion and VRBC were protected. Nitric oxide (NO) synthase inhibition significantly attenuated malvidin’s effects on arteriolar diameter. Western blotting analysis revealed an increase in eNOS and p-eNOS expression, while zymography indicated a decrease in MMP-9 activity after malvidin’s administration. Furthermore, malvidin was able to prevent neuronal damage and to decrease ROS generation. In conclusion, malvidin protects rat pial microcirculation against BCCAO/RE injury, preventing blood-brain impairment and neuronal loss. Malvidin’s effects appear to be mediated by eNOS activation and scavenger activity.

Delayed hyperoxic ventilation attenuates oxygen-induced free radical accumulation during early reperfusion after global brain ischemia.

To compare the effect of immediate and delayed administration of oxygen on the accumulation of free radicals in ischemia-reperfusion animal models. Thirty-two adult male Mongolian gerbils with microdialysis probes implanted in the right hippocampal CA1 were divided randomly into four groups (eight each). One group was sham-operated (Sham group) whereas the other three groups were subjected to 10 min bilateral carotid artery occlusion (BCAO). BCAO-treated animals were then subjected to the following: (a) immediate 30% O2 (near normoxia, NO group), (b) immediate 100% O2 (hyperoxia, HO group), and (c) 30% O2 for 60 min, followed by 100% O2 for 60 min (delayed hyperoxia, DHO group). Hippocampal accumulation of hydroxyl radicals (•OH) during reperfusion was estimated by measuring 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA in microdialysis perfusate. Hippocampi were removed 2 h after perfusion to measure malondialdehyde, pyruvate dehydrogenase activity, indices of lipid peroxidation, and cellular respiration. At 24 h after BCAO, the histology of hippocampi was analyzed to rate the injury. Immediately after the onset of reperfusion, all groups showed markedly elevated DHBA, which returned to baseline over 1-2 h. Compared with the NO group, the HO group showed significantly higher peak DHBA and slower recovery. In contrast, the DHO group was not significantly different from the NO group in terms of the DHBA level. DHO animals also showed significantly lower hippocampal malondialdehyde accumulation and higher pyruvate dehydrogenase activity at 2 h after reperfusion versus the HO group. Histology analysis also showed animals in the DHO group with ameliorated injury compared with the HO group. Hydroxyl radical accumulation was more sensitive to O2 during early reperfusion. Delayed hyperoxia may re-establish oxidative metabolism while minimizing oxidative stress after CA.

Dietary essential oil components in the prevention of hypoperfusion/reperfusion-induced tissue damage in the rat cerebral cortex

To extend our previous observations on the beneficial effect of dietary Pistacia lentiscus L. essential oil during cerebral bilateral common carotid artery occlusioninduced injury, we evaluated the activity of one of its major components, beta-caryophyllene (BCP), already known to possess peculiar biological activities, in Wistar rat cerebral cortex. Cerebral hypoperfusion was produced by a 30 min bilateral common carotid artery occlusion followed by 60 min reperfusion (BCCAO/R). Animals were starved for 12 hours before surgery and, 6 hours prior to hypoperfusion, BCP (40 mg/kg/0, 45 ml of sunflower oil as vehicle) was administered via gavage. Biological samples of brain tissue, plasma and cerebrospinal fluid (CSF) were examined by HPLC, western blot, gel zymography and immunohistochemistry and analyzed for fatty acids, expression of the enzyme ciclooxygenase-2 (COX-2), CB receptors for endocannabinoids (eCBs), and peroxisome proliferator-activated receptor (PPAR)-alpha and enzymatic activity of matrix-metalloprotease-9 (MMP9). Data obtained indicate that BCP appears to influence the outcome of BCCAO/R cerebral injury by modulating changes in levels of polyunsaturated fatty acids, biosynthesis of eCBs and eCB congeners, expression of CB1 and CB2 receptors, COX-2 protein levels and enzymatic activity of MMP9. Brain tissue response to the hypoperfusion/reperfusion-induced cerebral insult is modulated by dietary administration of BCP, suggesting the possible use of this molecule as nutritional treatment in neuroprevention. Work funded by Fondazione Banco di Sardegna.

Prostaglandin I2 IP Receptor Agonist, Beraprost, Prevents Transient Global Cerebral Ischemia Induced Hippocampal CA1 Injury in Aging Mice.

Beraprost sodium is a new stable, orally active Prostaglandin I2 analogue. The aim of this study was to determine the effect of beraprost on cognitive dysfunction and locomotor impairment induced by bilateral common carotid artery occlusion in mice. We investigated the ameliorating effect of beraprost through PGI2 IP receptor by studying neurologic deficit assessment and T-maze testing in young and old male C57Bl/6 wild-type (WT) and IP receptor knockout (IP KO) mice following a 12 min bilateral common carotid artery occlusion (BCCAo) and 7 days of reperfusion. Beraprost reversed BCCAo induced cognitive impairment and neurological deficit in a dose dependent manner. Immunohistochemical studies showed attenuation of neuronal cell death, astrogliosis, microglial invasion, and myeloperoxidase (MPO) activity in both young and old WT mice after post treatment with beraprost. Moreover, after BCCAo, phosphorylated cAMP response element binding protein positive cell numbers were increased with beraprost treatment over vehicle treated controls. These results show that beraprost treatment attenuated cognitive dysfunction and neurological deficits induced by BCCAo, and suggest that this effect may be mediated by the neuroprotective effects of treatment.

Genetic Deletion of Prostacyclin IP Receptor Exacerbates Transient Global Cerebral Ischemia in Aging Mice

Transient global cerebral ischemia causes delayed neuronal death in the hippocampal CA1 region. It also induces an up regulation of cyclooxygenase 2 (COX-2), which generates several metabolites of arachidonic acid, known as prostanoids, including Prostaglandin I2 (PGI2). The present study investigated whether the PGI2 IP receptor plays an important role in brain injury after global cerebral ischemia in aged mice. Adult young (2–3 months) and aged (12–15 months) male C57Bl/6 wild-type (WT) or IP receptor knockout (IP KO) mice underwent a 12 min bilateral common carotid artery occlusion (BCCAO) or a sham surgery. Behavior tests (neurologic deficit and T-maze) were performed 3 and 7 days after BCCAO. After seven days of reperfusion, the numbers of cells positive for markers of neurons, astrocytes, microglia, myeloperoxidase (MPO) and phosphorylated CREB (p-CREB) were evaluated immunohistochemically. Interestingly, in young and aged IP KO ischemic mice, there was a significant increase (p < 0.01) in cognitive deficit, hippocampal CA1 pyramidal neuron death, microglia and MPO activation, while p-CREB was reduced as compared to their corresponding WT controls. These data suggest that following ischemia, IP receptor deletion contributes to memory and cognitive deficits regulated by the CREB pathway and that treatment with IP receptor agonists could be a useful target to prevent harmful consequences.

Increased phosphorylation of mTOR is involved in remote ischemic preconditioning of hippocampus in mice

Different signaling pathways are involved in tissue protection against ischemia reperfusion (IR) injury, among them mammalian target of rapamycin (mTOR) and related pathways have been examined in many recent studies. Present study evaluated the role of mTOR in remote ischemic preconditioning (RIPC) of hippocampus. Renal ischemia was induced (3 cycles of 5min occlusion and 5min reperfusion of unilateral renal artery) 24h before global brain ischemia (20min bilateral common carotid artery occlusion). Saline or rapamycin (mTOR inhibitor; 5mg/kg, i.p.) was injected 30min before RIPC. mTOR and phosphorylated mTOR (p-mTOR) expression, superoxide dismutase (SOD) activity and retention trial of passive avoidance test were determined 24h after global ischemia. Apoptosis and neuronal cell density were assessed 72h after hippocampal ischemia. RIPC decreased apoptosis (p<0.05 vs. IR), improved memory (p<0.05 vs. IR), and augmented p-mTOR expression and SOD activity after hippocampal ischemia (p<0.05 vs. IR). Rapamycin abolished all protective effects of RIPC (p<0.05 vs. RIPC+IR) suggesting a role for mTOR in RIPC induced hippocampal protection.

Deleterious Effects of High Dose Connexin 43 Mimetic Peptide Infusion After Cerebral Ischaemia in Near-Term Fetal Sheep

Hypoxic-ischaemic brain injury at birth is associated with 1–3/1000 cases of moderate to severe encephalopathy. Previously, we have shown that connexin 43 hemichannel blockade, with a specific mimetic peptide, reduced the occurrence of seizures, improved recovery of EEG power and sleep state cycling, and improved cell survival following global cerebral ischaemia. In the present study, we examined the dose response for intracerebroventricular mimetic peptide infusion (50 μmol/kg/h for 1 h, followed by 50 μmol/kg/24 h (low dose) or 50 μmol/kg/h for 25 h (high dose) or vehicle only (control group), starting 90 min after the end of ischaemia), following global cerebral ischaemia, induced by 30 min bilateral carotid artery occlusion, in near-term fetal sheep (128 ± 1 days gestation). Both peptide infusion groups were associated with a transient significant increase in EEG power between 2–12 h after ischaemia. The ischaemia-low dose group showed a significant recovery of EEG power from day five compared to the ischaemia-vehicle and -high dose groups. In contrast, the high dose infusion was associated with greater secondary increase in impedance (brain cell swelling), as well as a trend towards a greater increase in lactate concentration and mortality. These data suggest that higher doses of connexin mimetic peptide are not beneficial and may be associated with adverse outcomes, most likely attributable to uncoupling of connexin 43 gap junctions leading to dysfunction of the astrocytic syncytium.

Effect of acute administration of dietary Pistacia lentiscus L. essential oil on the ischemia-reperfusion-induced changes in rat frontal cortex and plasma

In this study Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, was tested for its protective effects in cerebral ischemia/reperfusion-induced injury in Wistar rat frontal cortex and plasma. Cerebral ischemia was produced by a 20 min bilateral common carotid artery occlusion followed by 30 min reperfusion. Pistacia lentiscus L. essential oil (E.O.) (200 mg/0, 45 ml of sunflower oil as vehicle) was administered via gavage 6 hours prior to ischemia. Rats were randomly assigned to four groups, ischemic/reperfused (I/R) and sham-operated rats treated with the vehicle or with E.O.. Different brain areas were analysed for fatty acid changes and expression of the enzyme cyclooxygenase-2 (COX-2). Ischemia/reperfusion triggered in frontal cortex a decrease of docosahexaenoic acid (DHA), the membrane highly polyunsaturated fatty acid (HPUFA) most susceptible to oxidation. Pre-treatment with E.O. prevented this change and led further to decreased levels of COX-2, as assessed by Western Blot. In plasma of ischemic/reperfused rats, E.O. administration increased both the DHA-to-eicosapentaenoic acid (EPA) ratio and levels of the endocannabinoid congeners palmytoylethanolamide (PEA) and oleoylethanolamide (OEA). The results obtained suggest that ischemia/reperfusion triggers a cerebral insult sufficient to cause a a region specific lipid peroxidation as evidenced by the detectable, significant decrease in the tissue level of DHA, the most abundant essential fatty acid of neuronal membrane phospholipids. Acute dietary pre-treatment with E.O. triggers modifications both in the frontal cortex, where COX-2 expression decreases and the decrease of DHA is apparently prevented, and in plasma, where PEA and OEA levels increase. We suggest that the activity of PEA and OEA, as endogenous ligands of the peroxisome proliferator-activated receptor (PPAR)-alpha, by inducing the peroxisomal beta oxidation, may explain the observed increase in the DHA/EPA ratio. The latter, in fact, might account for an increased metabolism of n-3 aimed at restoring DHA within damaged brain tissue. The possibility that changes in fatty acid metabolism and plasmatic availability of PEA and OEA are correlated events represents an issue worth future investigations.

Protein-Energy Malnutrition Alters Hippocampal Plasticity-Associated Protein Expression following Global Ischemia in the Gerbil

Previously it has been demonstrated that protein-energy malnutrition (PEM) impairs habituation in the open field test following global ischemia. The present study examined the hypothesis that PEM exerts some of its deleterious effects on functional outcome by altering the post-ischemic expression of the plasticity-associated genes brain-derived neurotrophic factor (BDNF), its receptor tropomyosin-related kinase B (trkB), and growth-associated protein-43 (GAP-43). Male, Mongolian gerbils (11-12 wk) were randomized to either control diet (12.5% protein) or PEM (2% protein) for 4 wk, and then underwent 5 min bilateral common carotid artery occlusion or sham surgery. Tympanic temperature was maintained at 36.5 ± 0.5°C during surgery. Brains collected at 1, 3 and 7 d post-surgery were processed by in-situ hybridization or immunofluorescence. BDNF and trkB mRNA expression was increased in hippocampal CA1 neurons after ischemia at all time points and was not significantly influenced by diet. However, increased trkB protein expression after ischemia was exacerbated by PEM at 7 d in the CA1 region. Post-ischemic GAP-43 protein increased at 3 and 7 d in the CA1 region, and PEM intensified this response and extended it to the CA3 and hilar regions. PEM exerted these effects without exacerbating CA1 neuron loss caused by global ischemia. The findings suggest that PEM increases the stress response and/or hyper-excitability in the hippocampus after global ischemia. Nutritional care appears to have robust effects on plasticity mechanisms important to recovery after brain ischemia.

Do alterations in temperature regulation contribute to protein‐energy malnutrition‐induced increase in reactive gliosis after global ischemia?

Protein‐energy malnutrition (PEM) impairs functional outcome after global ischemia, and this is associated with an increase in reactive gliosis. Since temperature is a key determinant of brain damage following an ischemic insult, the objective of this study was to determine if PEM modifies post‐ischemic temperature regulation. Bio‐electrical sensor transmitters were implanted into the peritoneal cavity of male, Sprague‐Dawley rats (31d) for continuous monitoring of core temperature. Rats were randomized for 7d to modified AIN‐93G control diet (CON; 18% protein) or PEM (2% protein). Animals were exposed to global ischemia (I) induced by 10 min bilateral carotid artery occlusion and hypotension (38 ± 0.2 mm Hg) or sham surgery (S) with tympanic temperature maintained at 37.4 ± 0.2°C. Physiological values were as follows: pH 7.29–7.41, pO2 119–144 mm Hg, pCO2 34–51 mm Hg, blood glucose 3.8–6.7 mmol/L. Preliminary results indicate mean (± SEM) core and daily temperature fluctuation from 5d of postsurgical monitoring as follows: CON‐S: 37.4 ± 0.04°C, 2.0 ± 0.1°C (n=3); PEM‐S: 37.4 ± 0.1°C, 2.6 ± 0.1°C (n=3); CON‐I: 37.7 ± 0.1°C, 1.9 ± 0.2 °C (n=3); PEM‐I: 37.5 ± 0.2°C, 2.3 ± 0.3°C (n=2). These data suggest that although PEM does not cause marked hypothermia or hyperthermia during the postsurgical period, it reduces thermoregulatory function in both sham and ischemic animals. Funded by the Heart and Stroke Foundation of SK.

Pretreatment with the ciclosporin derivative NIM811 reduces delayed neuronal death in the hippocampus after transient forebrain ischaemia

There have been several previous studies showing that ciclosporin, a ligand for cyclophilin D (CypD), reduces mitochondrial permeability transition (mPT) and ameliorates delayed neuronal death. NIM811 is a non-immunosuppressive ciclosporin derivative that also inhibits mPT, but has significantly less cytotoxicity than ciclosporin. Actually, in animal experiments, several investigators have reported that NIM811 ameliorates central nervous system disorders, such as traumatic brain injury, transient focal cerebral ischaemia and spinal cord injury. Therefore, we evaluated whether the ciclosporin derivative, NIM811 reduces mPT and ameliorates delayed neuronal death in the hippocampal CA1 sectors in mice when subjected to transient forebrain ischaemia. Male C57BL/6 mice were treated with 50 mg/kg ciclosporin, 10, 50 or 100 mg/kg NIM811 or phosphate-buffered saline. At 30 min post-injection, all mice were subjected to 20 min bilateral common carotid artery occlusion (BCCAO). To estimate delayed neuronal death, the sections were prepared for HE staining and terminal deoxynucleotidyl transferase-mediated dUTP end-labelling (TUNEL) staining at 72 h after 20 min BCCAO. Furthermore, using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) staining technique, we evaluated whether NIM811 (1, 10, 100 or 1000 microm) inhibited mPT in the neurons exposed to 100 microm glutamate. Both delayed neuronal injury and apoptosis in the hippocampal CA1 sectors were significantly ameliorated at 72 h after transient forebrain ischaemia in the mice treated with 100 mg/kg NIM811 or 50 mg/kg ciclosporin. The treatments with 100 microm and 1,000 microm NIM811 significantly inhibited the reduction of mitochondrial membrane potential in the neurons exposed to 100 microm glutamate. These findings strongly suggest that NIM811 inhibits mPT and ameliorates delayed neuronal death in mice subjected to transient forebrain ischaemia.

Caloric restriction provided after global ischemia does not reduce hippocampal cornu ammonis injury or improve functional recovery

Since caloric restriction (CR) can modify multiple pathways central to the ischemic cascade and enhance neuroplasticity mechanisms, we hypothesized that CR should exert protective effects following brain ischemia. Previous studies have suggested benefit when CR was administered prior to ischemia. We investigated whether prolonged CR beginning after global ischemia would result in lasting protection as assessed by performance in the open field, as a measure of functional outcome, and hippocampal CA1 neuronal counts. Adult, male Mongolian gerbils were subjected to 5 min bilateral carotid artery occlusion (ISCH) or sham surgery (SHAM) with tympanic temperature maintained at 36.5±0.2 °C during the intra-ischemic period. After screening out gerbils with incomplete ischemia, each of the two surgical groups were randomly assigned to control diet (CON) or 30% CR for the duration of the study (60 d). Gerbils were tested in the open field on d3, 7, 10, 30 and 60. ISCH-CON animals showed a significantly higher level of activity in the open field (impaired habituation) compared to SHAM-CON gerbils on all test days ( P<0.001). Open field activity was significantly lower in the ISCH-CR group than in ISCH-CON gerbils only on d7 ( P=0.024). Open field activity of the SHAM-CR gerbils showed a trend to increase relative to that of SHAM-CON gerbils during the last 30 d of the study ( P=0.055 on d60), raising the question of suitability of the open field test for long-term studies of CR and ischemia. Brain sections obtained at d60 were stained with hematoxylin and eosin. Hippocampal CA1 neuron counts were significantly reduced by ischemia ( P<0.001), and there was no sparing effect of CR. Our findings suggest that prolonged 30% CR administered beginning after global ischemia cannot diminish brain injury or enhance long-term recovery.

TIMP-3 and MMP-3 contribute to delayed inflammation and hippocampal neuronal death following global ischemia

Hippocampal neuronal death following transient global ischemia in the mouse takes days to occur, providing a potential timeframe for therapeutic intervention. Since matrix metalloproteinase-3 (MMP-3) enhances inflammation and tissue inhibitor of metalloproteinases-3 (TIMP-3) promotes apoptosis in ischemia, we hypothesized that they are involved in neuronal death secondary to transient global ischemia. Timp-3 knockout (T3KO) and wild type (T3WT) mice underwent 30 min bilateral carotid artery occlusion (BCAO), which causes hippocampal neuronal death 7 days after reperfusion. Mice lacking the Timp-3 gene have significantly less astrocytosis, microglial reactivity, MMP-3 activity and neuronal cell death. In addition, T3KO mice had decreased tumor necrosis factor (TNF) receptor-1 (TNFR1) expression and increased TNF-α converting enzyme (TACE) activity. Mmp-3 KO mice with a similar BCAO showed significantly fewer microglial cells, reduced TNF-α expression, and less neuronal death than the Mmp-3 WT. To see if TIMP-3 and MMP-3 cell death pathways were independent, we blocked MMPs with the broad-spectrum MMP inhibitor, BB-94, on days 3 through 6 of reperfusion in T3WT and T3KO mice. BB-94 rescued hippocampal neurons at 7 days in both T3WT and T3KO mice, but significantly fewer neurons died in T3KO mice treated with BB-94. Our results indicate a novel additive role for TIMP-3 and MMP-3 in delayed neuronal death, and show that delayed treatment with MMP inhibitors can be used to reduce hippocampal death.

The time‐dependency of post‐conditioning for neuroprotection against global cerebral ischemia and reperfusion injury in C57BL/6 mice

To establish (a) suitable treatment protocol(s) for neuroprotection in mice, the post‐conditioning treatments were performed by 3 cycles of reperfusion and occlusion (5s/5s, 10s/10s, 15s/15s, 30s/30s and 45s/45s, respectively) on bilateral common carotid artery immediately after 10 min bilateral common carotid artery occlusion (BCCAO) in male C57BL/6 mice. The escape latency was the shortest in Morris water maze test and the recovery of cerebral blood flow after reperfusion was complete in 15s/15s post‐conditioning group while the mice in 30s/30s group performed best in beam walking test. Calcium overload as well as neuron death rate were depressed most significantly in hippocampus and striatum in 15s/15s group and 30s/30s group respectively. Additionally, we tested the neuroprotective efficiency of 15s/15s postconditioning applied at 5min, 15min, 30min or 1h after the reperfusion and found that the protective effect was decreasing when increasing the delay time of postconditioning. Delay for more than 30min eliminated the protective effect of post‐conditioning. We concluded that appropriate post‐conditioning (15s/15s and 30s/30s, 3 cycles) provides better neuroprotection against global cerebral ischemia and reperfusion injury in mice.

Hypoxia induces caspase‐9 and caspase‐3 activation without neuronal death in gerbil brains

To investigate the in vivo apoptotic machinery in oxygen deprived brain, we examined the expression of caspase-9 and caspase-3 in the hippocampus of Mongolian gerbils subjected to either transient hypoxia (4% O2 for 6 min) or forebrain ischemia (10 min bilateral carotid artery occlusion) followed by 8 h to 7 days of reoxygenation or blood recirculation. Apoptotic death was characterized by isolating hippocampal genomic DNA and analysing DNA fragmentation as well as histological studies including TUNEL assay and toluidine blue staining of brain sections. The results showed that both hypoxic and ischemic gerbil brains exhibited an increase in caspase-9 and caspase-3 gene expression. However, no cell damage was detectable following hypoxia, while marked DNA fragmentation and extensive cell death was observed following ischemia. Moreover, although hypoxia did not lead to cell death, both hypoxia and ischemia were associated with cleavage of procaspase-9 and procaspase-3 and increases in their activities as well as cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a major caspase-3 substrate. These results indicate that, in vivo, even late apoptotic events such as caspase activation and PARP-1 cleavage in hypoxic brains do not necessarily induce an irreversible commitment to apoptotic neuronal death.