metallothionein and Infarction--Middle-Cerebral-Artery

This study investigated the protective effects and mechanisms of sevoflurane preconditioning (SPC) on neurons in ischemic mice. After SPC, mice were subjected to middle cerebral artery occlusion (MCAO). Cerebral infarction area, cell apoptosis, and metallothionein-1 (MT-1) and metallothionein-2 (MT-2) expressions in MCAO mice were analyzed. Mouse primary neurons were isolated and cultured to determine the location of metallothioneins (MTs) using immunofluorescence. Neurons transfected with MT-siRNA, exogenous MTs, or sh-MTF-1 were subjected to SPC and/or oxygen-glucose deprivation (OGD), and MT-1/MT-2 expression and neurotoxin release were assayed. Meanwhile, neurons were treated with the nitric oxide donor SNAP, degraded SNAP, or the peroxide initiator paraquat, and alterations in MT-1/MT-2 expression and neurotoxicity release were observed. SPC attenuated neuronal injury and apoptosis in MCAO mice. SPC could protect neurons against OGD injury and resulted in upregulated MT-1/MT-2 expression. MT-siRNA transfection led to the downregulated expression of MT-1/MT-2 and increased neurotoxicity, and the expression patterns of these neurons were different from those of neurons transfected with exogenous MTs. The knockdown of MTs could hinder the protective effect of SPC against OGD. Pretreatment with SNAP or paraquat could increase MTF-1 expression in the nucleus of neurons, protecting against OGD injury. The inhibition of nitric oxide and peroxide inhibited the protective role of SPC in OGD by downregulating MTF-1 expression. sh-MTF-1 transfection downregulated MT-1/MT-2 expression and enhanced neurotoxicity in neurons. SPC confers neuroprotection in focal cerebral ischemia mouse models by upregulating the expression of MT-1 and MT-2 by activating NO and peroxide and increasing MTF-1 expression in the nucleus.

Topics: Animals; Apoptosis; Cells, Cultured; Glucose; Infarction, Middle Cerebral Artery; Metallothionein; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Nitric Oxide; Oxygen; Sevoflurane

Intracellular zinc overload causes neuronal injury during the course of neurological disorders, whereas mild levels of zinc are beneficial to neurons. Previous reports indicated that non-steroidal anti-inflammatory drugs, including indomethacin and aspirin, can reduce the risk of ischemic stroke. This study found that chronic pretreatment of rats with indomethacin, a non-selective cyclooxygenase inhibitor, provided tolerance to ischemic injuries in an animal model of stroke by eliciting moderate zinc elevation in neurons. Consecutive intraperitoneal injection of indomethacin (3mg/kg/day for 28 days) led to modest increases in intraneuronal zinc as well as synaptic zinc content, with no significant stimulation of neuronal death. Furthermore, indomethacin induced the expressions of intracellular zinc homeostatic and neuroprotective proteins, rendering the brain resistant against ischemic damages and improving neurological outcomes. However, administration of a zinc-chelator, N,N,N',N'-tetra(2-picolyl)ethylenediamine (TPEN; 15 mg/kg/day), immediately after indomethacin administration eliminated the beneficial actions of the drug. Therefore, indomethacin preconditioning can modulate intracellular zinc availability, contributing to ischemic tolerance in the brain after stroke.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Brain; Carrier Proteins; Cell Death; Chelating Agents; Disease Models, Animal; Drug Administration Schedule; Ethylenediamines; HSP70 Heat-Shock Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Indomethacin; Infarction, Middle Cerebral Artery; Male; Metallothionein; Neurons; Rats; Rats, Sprague-Dawley; Time Factors; Zinc

Free radicals are known to cause secondary neuronal damage in cerebral ischemia/reperfusion (I/R). We investigated here the neuroprotective effect of resveratrol, a potent antioxidant present in grape seed, against cerebral I/R-induced mitochondrial dysfunctions in hippocampus. Transient rat middle cerebral artery occlusion (MCAO) model of brain ischemia was used to induce brain infarction. Resveratrol (10(-7) g/kg) was given twice intravenously: 15 min pre-occlusion and at the time of reperfusion (2 h post-occlusion). Resveratrol significantly restored ATP content and the activity of mitochondrial respiratory complexes in resveratrol treated group which were severely altered in MCAO group. Western blot analysis showed a marked decrease in cytochrome c release as a result of resveratrol treatment. Electrophoretic migration of hippocampal genomic DNA showed a marked decrease in DNA fragmentation after resveratrol treatment. Notably, expression of Hsp70 and metallothionein (MT) was significantly higher in MCAO group but their expression was more significant in resveratrol treated group. The status of mitochondrial glutathione (GSH), glucose 6-phosphate dehydrogenase (G6-PD) and serum lactate dehydrogenase (LDH) was restored by resveratrol treatment with a significant decrease in mitochondrial lipid peroxidation (LPO), protein carbonyl and intracellular H(2)O(2) content. Resveratrol significantly improved neurological deficits assessed by different scoring methods. Also, the brain infarct volume and brain edema were significantly reduced. Histological analysis of CA1 hippocampal region revealed that resveratrol treatment diminished intercellular and pericellular edema and glial cell infiltration. The findings of this study highlight the ability of resveratrol in anatomical and functional preservation of ischemic neurovascular units and its relevance in the treatment of ischemic stroke.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Brain Ischemia; Cell Death; Cytochromes c; DNA Fragmentation; Glucosephosphate Dehydrogenase; Glutathione; Hippocampus; HSP70 Heat-Shock Proteins; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; Lactate Dehydrogenases; Lipid Peroxidation; Male; Metallothionein; Mitochondria; Neuroprotective Agents; Protein Carbonylation; Rats; Rats, Wistar; Resveratrol; Stilbenes

Metallothioneins (MTs) are small cysteine-rich proteins found widely throughout the mammalian body, including the CNS. MT-1 and -2 protect against reactive oxygen species and free radicals. We investigated the role of MT-1 and -2 using MT-1,-2 knockout (KO) mice. MT-1,-2 KO mice exhibited greater neuronal damage after permanent middle cerebral artery occlusion (MCAO) than wild-type mice. MT-2 mRNA was significantly increased at 6, 12, and 24 h after MCAO in the wild-type mouse brain [as detected by real-time reverse-transcription polymerase chain reaction (RT-PCR)], while MT-1 and MT-3 were decreased at 12 and 24 h. In an immunohistochemical study, MT expression displayed colocalization with glial fibrillary acidic protein (GFAP)-positive cells (astrocytes) in the penumbra area in wild-type mice. Since erythropoietin (EPO) has been reported to induce MT-1 and -2 gene expression in vitro, we examined its effect after permanent MCAO, and explored the possible underlying mechanism by examining MT-1 and -2 induction in vivo. In wild-type mice, EPO significantly reduced both infarct area and volume at 24 h after the ischemic insult. However, in MT-1,-2 KO mice EPO-treatment did not alter infarct volume (vs. vehicle-treatment). In wild-type mice at 6 h after EPO administration, real-time RT-PCR revealed increased MT-1 and -2 mRNA expression in the cerebral cortex (without MCAO). Further, MT-1 and -2 immunoreactivity was increased in the cortex of EPO-treated mice. These findings indicate that MTs are induced, and may be neuroprotective against neuronal damage, after MCAO. Furthermore, EPO is neuroprotective in vivo during permanent MCAO, and this may be at least partly mediated by MTs.

Topics: Animals; Astrocytes; Brain; Brain Infarction; Brain Ischemia; Cytoprotection; Erythropoietin; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Infarction, Middle Cerebral Artery; Male; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Neuroprotective Agents; RNA, Messenger; Up-Regulation