cytochrome-c-t and Brain-Ischemia

We review research investigating mitochondrial damage during heart and brain ischaemia, focusing on the mechanisms and consequences of ischaemia-induced and/or reperfusion-induced: (a) inhibition of mitochondrial respiratory complex I; (b) release of cytochrome c from mitochondria; (c) changes to mitochondrial phospholipids; and (d) nitric oxide inhibition of mitochondria. Heart ischaemia causes inhibition of cytochrome oxidase and complex I, release of cytochrome c, and induction of permeability transition and hydrolysis and oxidation of mitochondrial phospholipids, but some of the mechanisms are unclear. Brain ischaemia causes inhibition of complexes I and IV, but other effects are less clear.

Topics: Animals; Brain Ischemia; Cytochromes c; Electron Transport Complex IV; Humans; Hypoxia; Mitochondria; Mitochondria, Heart; Multienzyme Complexes; Myocardial Ischemia; Nitric Oxide; Oxidation-Reduction; Oxidative Phosphorylation; Phospholipids; Reactive Oxygen Species

Due to a lack of efficient treatments, searching for novel therapies against acute ischemic stroke represents one of the main fields in neuropharmacology. In this review we summarize and discuss the role of mitochondrial participation in ischemia-induced neuronal death. Mitochondria are regarded as the main link between cellular stress signals and the execution of programmed death of nerve cells. Since it was described that the release of mitochondrial proteins such as cytochrome c, apoptosis inducing factor and endonuclease G are key elements in cell death pathways, they have been the focus of cell death studies. Changes in the permeability of the mitochondrial outer membrane result in a non-reversible step in cell death processes. Cytochrome c released from mitochondria binds in the cytoplasm to Apaf-1 to initiate the formation of an apoptosome, which then binds pro-caspase-9. Active caspase-9 cleaves "executioner" caspases, which in turn proceed to cleave key substrates in the cell. Thus, the identification of new targets might enable establishment of novel strategies for therapeutic research, in this case based on the molecular mechanisms of mitochondrial pathways, to improve the development of compounds for treatment of ischemia.

Topics: Anti-Bacterial Agents; Apoptotic Protease-Activating Factor 1; Brain Ischemia; Calcium; Cell Death; Cytochromes c; Energy Metabolism; Homeostasis; Minocycline; Mitochondria; Mitochondrial Membranes; Neurons; Permeability; Reactive Oxygen Species

There is growing evidence that preservation of mitochondrial respiratory function during cerebral ischemia-reperfusion predicts the ultimate extent of tissue injury. Because neurons are selectively vulnerable to ischemic injury, many studies have focused on neuronal mitochondrial dysfunction in ischemia. However, positron emission tomography (PET) studies in animals and humans suggest that non-neuronal cells such as astrocytes may also experience mitochondrial metabolic compromise that contributes to ischemic necrosis. Astrocytes carry out a number of functions that are critical to normal nervous system function, including uptake of neurotransmitters, regulation of pH and ion concentrations, and metabolic support of neurons. Mitochondria are important for many of these actions. We have used a cell culture model of stroke, oxygen-glucose deprivation (OGD), to study the response of astrocyte mitochondria to ischemia, and to evaluate how changes in astrocyte mitochondrial function might affect neuronal survival and recovery after ischemia.

Topics: Animals; Apoptosis; Astrocytes; Brain; Brain Ischemia; Cell Hypoxia; Cell Membrane Permeability; Cytochromes c; Disease Models, Animal; Energy Metabolism; Humans; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Neuroprotective Agents; Oxidative Stress; Reactive Oxygen Species; Signal Transduction

Ischemic stroke is divided into acute, subacute and convalescent phases according to the time of onset. Clinically, Mailuoning oral liquid (MLN O) is a traditional Chinese patent medicine for treating ischemic stroke. Previous studies have shown that MLN O could prevent acute cerebral ischemia-reperfusion. However, its underlying mechanism remains unclear.. To investigate the relationship between neuroprotection and apoptosis for clarifying MLN O mechanism in the recovery phase of ischemic stroke.. We imitated stroke using middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro models. The infarct volume, neurological deficit scores, HE staining, Nissl staining, TUNEL staining, immunohistochemistry, and Western blot were correspondingly performed to find pathological changes and detect neuronal apoptosis in rat cerebral cortex. The contents of LDH, Cyt-c, c-AMP and BDNF in rat plasma and cerebral cortex were detected by ELISA. Cell viability was measured by CCK8 assay. Cell morphology, Hoechst 33342 staining and Annexin-V-Alexa Fluor 647/PI staining were performed to assess neuronal apoptosis. The expression levels of proteins were evaluated by western blotting.. MLN O obviously reduced brain infarct volume and neurological deficit scores in MCAO rats. MLN O inhibited inflammatory cell infiltration and neuronal apoptosis, but promoted gliosis, neuronal survival, and neuroprotection in the cortical region of MCAO rats. Additionally, MLN O decreased the amount of LDH and cytochrome c, while increasing the expression of c-AMP in the plasma and ischemic cerebral cortex of MCAO rats, and promoting the expression of BDNF in the cortical tissue of MCAO rats. Besides, MLN O improved cell viability, restored cell morphology, while attenuating cell damage, inhibiting neuronal apoptosis following OGD/R in PC-12 cells. Moreover, MLN O inhibited apoptosis by suppressing the expression of pro-apoptotic-associated proteins, including Bax, cytochrome c, Cleaved caspase 3 and HIF-1α, whereas accelerating the expression of Bcl-2 in vivo and in vitro. Furthermore, MLN O inhibited the activity of AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR), but activated the signaling pathway of cAMP-response element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) in MCAO rats and OGD/R-stimulated PC-12 cells.. These results demonstrated that MLN O inhibited AMPK/mTOR to affect apoptosis associated with mitochondria, leading to improve CREB/BDNF-mediated neuroprotection in the recovery period of ischemic stroke in vivo and in vitro.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain Ischemia; Brain-Derived Neurotrophic Factor; Cytochromes c; Infarction, Middle Cerebral Artery; Ischemic Stroke; Neuroprotection; Rats; Reperfusion Injury; TOR Serine-Threonine Kinases

Calenduloside E (CE) isolated from Aralia elata (Miq.) Seem. is a natural triterpenoid saponin that can reportedly ameliorate myocardial ischemia/reperfusion injury. However, its potential roles and mechanism in cerebral ischemia/reperfusion injury are barely understood. In this study, we established an oxygen-glucose deprivation/reoxygenation (OGD/R) model in HT22 cells. We found that CE significantly attenuated the OGD/R-induced inhibition of cell viability and apoptotic cell death in HT22 cells. Moreover, CE treatment significantly ameliorated OGD/R-induced mitochondrial fission by inhibiting mitochondrial dynamin-related protein 1 (Drp1) recruitment and increasing Drp1 phosphorylation at Ser637. CE treatment significantly ameliorated OGD/R-induced mitochondrial dysfunction by increasing the mitochondrial membrane potential and reducing the mitochondrial ROS and cellular calcium accumulation. Moreover, CE treatment significantly inhibited the OGD/R-induced release of mitochondrial Cytochrome C and increase in Bax, Cleaved-caspase3 and Cleaved-caspase9 protein levels, whereas CE treatment significantly reversed the OGD/R-induced decrease in Bcl-2 and full length of caspase3 and caspase9 protein levels. In vivo, we found that CE treatment significantly ameliorated ischemic/hypoxic-induced brain infarct volume, neurological deficits, and neuronal apoptosis in mice after middle cerebral artery occlusion and reperfusion. CE treatment also significantly ameliorated the mitochondrial transmembrane potential, decreased Cytochrome C release, and reversed the increase in Bax, Cleaved-caspase3 and Cleaved-caspase9 protein levels and the decrease in Bcl-2 and full length of caspase3 and caspase9 protein levels induced by cerebral ischemia/reperfusion (I/R). All these results indicated that CE treatment exerted a neuroprotective effect by ameliorating mitochondrial dysfunction during cerebral I/R injury.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Cytochromes c; Glucose; Mice; Mitochondria; Oleanolic Acid; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; Saponins

Paeoniflorin (PF), a bioactive monoterpene glucoside, has shown a variety of pharmacological effects such as anti-inflammation and autophagy modulation etc. In this study, we investigated whether and how PF exerted a protective effect against ischemic brain injury in vivo and in vitro. Primary rat cortical neurons underwent oxygen/glucose deprivation/reperfusion (OGD/R) for 90 min. We showed that after OGD/R, a short fragment of histone deacetylase 4 (HDAC4) produced by caspase3-mediated degradation was markedly accumulated in the nucleus and the activity of caspase3 was increased. Treatment with PF (100 nM, 1 μM) significantly improved the viability of cortical neurons after OGD/R. Furthermore, PF treatment could maintain HDAC4 intrinsic subcellular localization and reduce the caspase3 activity without changing the HDAC4 at the transcriptional level. PF treatment significantly reduced OGD/R-caused inhibition of transcriptional factor MEF2 expression and increased the expression of downstream proteins such as GDNF, BDNF, and Bcl-xl, thus exerting a great anti-apoptosis effect as revealed by TUNEL staining. The beneficial effects of PF were almost canceled in HDAC4 (D289E)-transfected PC12 cells after OGD/R. In addition, PF treatment reduced the caspase9 activity, rescued the release of cytochrome c from mitochondria, and maintained the integrity of mitochondria membrane. We conducted in vivo experiments in 90-min-middle cerebral artery occlusion (MCAO) rat model. The rats were administered PF (20, 40 mg/kg, ip, 3 times at the reperfusion, 24 h and 48 h after the surgery). We showed that PF administration dose-dependently reduced infarction area, improved neurological symptoms, and maintained HDAC4 localization in rats after MCAO. These results demonstrate that PF is effective in protecting against ischemic brain injury and inhibit apoptosis through inhibiting the cytochrome c/caspase3/HDAC4 pathway.

Topics: Animals; Anti-Inflammatory Agents; Brain Ischemia; Caspase 3; Cytochromes c; Disease Models, Animal; Glucosides; Histone Deacetylases; Male; Monoterpenes; Morris Water Maze Test; Open Field Test; Rats; Rats, Sprague-Dawley; Rotarod Performance Test; Signal Transduction

Stroke is a major public health problem with an imperative need for a more effective and tolerated therapy. Neuroprotective therapy may be an effective therapeutic intervention for stroke. The morbidity and mortality of stroke-induced secondary brain injury is mainly caused by neuronal apoptosis, which can be executed in a caspase-dependent or apoptosis inducing factor (AIF)-dependent manner. As apoptosis is an energy-dependent process with a relative time delay, abnormal energy metabolism could be a significant and fundamental pathophysiological basis of stroke. To our knowledge, convincible evidences that AMPK inhibition exerts neuroprotection in cerebral ischemia injury via anti-apoptosis remain to be investigated. Accordingly, the aims of this study were to investigate the protective effects of AMPK inhibitor BML-275 on cerebral ischemic/reperfusion (I/R) injury and to elucidate the underlying mechanisms. Cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in male C57BL/6 mice. The therapeutic effects of BML-275 were evaluated by infarct sizes, neurological scores and the proportion of apoptotic neurons after 24 h of reperfusion. The cell apoptosis markers cyt c and AIF were also evaluated. The results showed that intraperitoneally administration of BML-275 alleviate the cerebral infarction, neurological deficit and neuronal apoptosis induced by MCAO. BML-275 simultaneously induces anti-apoptosis and decreases the expression of cyt c and AIF. This study supports the hypothesis that anti-apoptosis is one of potential neuroprotective strategies for the treatment of stroke.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Apoptosis Inducing Factor; Brain Ischemia; Cytochromes c; Dose-Response Relationship, Drug; Male; Mice; Mice, Inbred C57BL; Molecular Structure; Neurons; Neuroprotective Agents; Pyrazoles; Pyrimidines; Structure-Activity Relationship

Exercise training has a neuroprotective effect against ischaemic injury, but the underlying mechanism is not completely clear. This study explored the potential mechanisms underlying the protective effects of treadmill training and caveolin-1 regulation against mitochondrial dysfunction in cerebral ischaemic injury.. After middle cerebral artery occlusion (MCAO) surgery, rats were subjected to treadmill training and received daidzein injections and combined therapy. A series of analyses, including neurological function scoring; body weight measurement; Nissl, haematoxylin and eosin staining; cerebral infarction volume assessment; mitochondrial morphology examination; caveolin-1, cytoplasmic and mitochondrial cytochrome C (CytC), and translocase of outer membrane 20 (TOM20) expression analysis; apoptosis index analysis; and transmission electron microscopy were conducted.. Treadmill training increased caveolin-1 expression, reduced neurobehavioral scores and cerebral infarction volumes, improved tissue morphology, reduced neuronal loss, inhibited mitochondrial outer membrane permeabilization (MOMP) through the caveolin-1 pathway, prevented excessive Cyt-C release from mitochondria, and reduced the degrees of apoptosis and mitochondrial damage. In addition, treadmill training increased the expression of TOM20 through the caveolin-1 pathway and maintained import signal function, thereby protecting mitochondrial integrity.. Treadmill exercise protected mitochondrial integrity and inhibited the endogenous mitochondrial apoptosis pathway. The damage of cerebral ischaemia was alleviated in rats through enhancement of caveolin-1 by treadmill exercise.

Topics: Animals; Apoptosis; Body Weight; Brain Ischemia; Caveolin 1; Cytochromes c; Exercise Test; Isoflavones; Male; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Neuroprotective Agents; Physical Conditioning, Animal; Rats; Rats, Sprague-Dawley; Signal Transduction

Mitochondrial dynamics and function are important for cell survival regulation under stress. In this study, we report that cerebral ischemia/reperfusion (I/R) injury significantly reduced mitochondrial function through reduced PTEN-induced kinase 1 (PINK1) expression, ATP (Adenosine triphosphate) levels, and increased oxidative stress compared to sham rats. PINK1 overexpression mice significantly improved mitochondrial function by increased mitochondrial complex I, II, and III activities and ATP levels with concomitant decline in reactive oxygen species levels. PINK1 overexpression mice after I/R injury significantly reduced apoptosis through downregulation of cytochrome c, p53 expressions compared to cerebral I/R injury rats. Furthermore, we showed from parkin siRNA studies that PINK1 regulated phosphorylation parkin is critical to the protection against cerebral I/R injury. Altogether, we show that PINK1 mediated parkin regulation is key to the protection against cerebral I/R injury through regulation of mitochondrial function and apoptosis.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Brain Ischemia; Cell Survival; Cytochromes c; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria; Oxidative Stress; Protein Kinases; Rats; Reactive Oxygen Species; Reperfusion Injury

Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC) in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Cytochromes c; Disease Models, Animal; Mice, Inbred C57BL; MicroRNAs; Stroke; Tight Junctions; Treatment Outcome

Brain ischemia represents a leading cause of death and disability in industrialized countries. To date, therapeutic intervention is largely unsatisfactory and novel strategies are required for getting better protection of neurons injured by cerebral blood flow restriction. Recent evidence suggests that brain insulin leads to protection of neuronal population undergoing apoptotic cell death via modulation of oxidative stress and mitochondrial cytochrome c (CytC), an effect to be better clarified. In this work, we investigate on the effect of insulin given intracerebroventricular (ICV) before inducing a transient global ischemia by bilateral occlusion of the common carotid arteries (BCCO) in Mongolian gerbils (MG). The transient (3 min) global ischemia in MG is observed to produce neurodegenerative effect mainly into CA3 hippocampal region, 72 h after cerebral blood restriction. Intracerebroventricular microinfusion of insulin significantly prevents the apoptosis of CA3 hippocampal neurons. Histological observation, after hematoxylin and eosin staining, puts in evidence the neuroprotective role of insulin, but Raman microimaging provides a clearer insight in the CytC mechanism underlying the apoptotic process. Above all, CytC has been revealed to be an outstanding, innate Raman marker for monitoring the cells status, thanks to its resonant scattering at 530 nm of incident wavelength and to its crucial role in the early stages of cells apoptosis. These data support the hypothesis of an insulin-dependent neuroprotection and antiapoptotic mechanism occurring in the brain of MG undergoing transient brain ischemia. The observed effects occurred without any peripheral change on serum glucose levels, suggesting an alternative mechanism of insulin-induced neuroprotection.

Topics: Animals; Apoptosis; Brain Ischemia; CA3 Region, Hippocampal; Cytochromes c; Disease Models, Animal; Gerbillinae; Infusions, Intraventricular; Insulin; Male; Mitochondria; Neurons; Neuroprotection; Neuroprotective Agents; Spectrum Analysis, Raman

Topics: Animals; bcl-2-Associated X Protein; Brain Ischemia; Calcium; Cell Death; Cytochromes c; Down-Regulation; Energy Metabolism; Hand Strength; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Swelling; Motor Activity; Oxygen Consumption; Pramipexole; Rats, Wistar; Reactive Oxygen Species; Recovery of Function; Signal Transduction; Stroke

At abnormally elevated levels of intracellular Ca

Topics: Animals; Brain; Brain Ischemia; Calcium; Cytochromes c; Dogs; Female; Heart Arrest; Mitochondria

Cannabinoid type 1 (CB1) receptor agonist arachidonyl-2-chloroethylamide (ACEA) induces neuroprotection against brain ischemia, and the mechanism, however, is still elusive. In this study, we used bilateral common carotid artery occlusion (BCCAO) in mice and oxygen-glucose deprivation (OGD) in primary cultured neurons to mimic brain ischemic injury, and hypothesized that cannabinoid CB1 receptor agonist ACEA protects ischemic neurons via inhibiting the opening of mitochondrial permeability transition pore (MPTP). In vivo, we found that BCCAO treatment reduced the neurological functions, increased the number of apoptotic neuronal cells and deteriorated the mitochondrial morphology in the ischemic brain tissue. And in vitro, we observed that OGD injury reduced cell viability, mitochondrial function and anti-oxidant SOD2 expression, increased lactate dehydrogenase (LDH), mitochondrial cytochrome C (Cyto C) and apoptosis-inducing factor (AIF) releases, elevated the cell apoptosis and mitochondrial superoxide level. And the CB1 receptor agonist ACEA significantly abolished the BCCAO and OGD-induced neuronal injury above. However, the MPTP opener atractyloside (Atr) markedly reversed the ACEA-induced neuroprotective effects, inhibited the mitochondrial Cyto C and AIF releases and relieved the mitochondrial swelling, but the MPTP inhibitor cyclosporin A (CsA) did not cause significant effects on the ACEA-induced neuroprotection above. These findings indicated that inhibition of MPTP opening may be involved in the cannabinoid CB1 receptor agonist ACEA-induced neuroprotection.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Arachidonic Acids; Atractyloside; Brain Ischemia; Cell Survival; Cyclosporine; Cytochromes c; L-Lactate Dehydrogenase; Male; Mice; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Neuroprotective Agents; Primary Cell Culture; Superoxide Dismutase; Superoxides

 To explore the potential effect of miR-125b on p53-mediated regulation of Bax/Cytochrome C/Caspase-3 apoptotic signaling pathway in rats with cerebral ischemia-reperfusion (CIR) injury..  Sprague-Dawley (SD) rats were used to conduct CIR injury and injected with miR-125b mimic/inhibitor or p53 inhibitor (Pifithrin-α, PFT-α). Dual-luciferase reporter gene assay was used to analyze the targeting relationship between miR-125b and p53. Longa scoring and Triphenyl tetrazolinm chloride (TTC) staining were used to test the neurologic function and determine infarct size, respectively. Hematoxylin-eosin (HE) and Nissl's stainings were conducted to observe the morphology of cortical neurons. Neuronal nuclei (NeuN) expression was detected by immunohistochemical staining. QRT-PCR was performed to detect the expressions of miR-125b and p53. TUNEL staining and Western blotting was used to determine neuronal apoptosis and expressions of Bax/Cytochrome C/Caspase-3 signaling pathway-related proteins, respectively..  Our results showed that miR-125b could directly target p53. As observed, overexpression of miR-125b could obviously reduce the neurological score, infarct size, and brain water content after CIR in rats, which also improved the morphology of cortical neurons, increased the number of neurons, reduced neuronal apoptosis, and inhibited the expressions of Bax/Cytochrome C/Caspase-3 pathway. Moreover,the similar results were observed in rats with CIR after injected with PFT-α. But no significant differences in each index were found in CIR group and CIR + anti-miR-125b + PFT-α group.. MiR-125b exerts protective effects on CIR injury through inhibition of Bax/Cytochrome C/Caspase-3signaling pathway via targeting p53, which is likely to be a promising treatment for CIR.. 3'-UTR: 3-untranslated region; CIR: cerebral ischemia-reperfusion; CIS: cerebral ischemic stroke; PFT-α: Pifithrin-α; PVDF: polyvinylidene fluoride; SD: Sprague-Dawley; TBST: tris buffered saline with tween. TTC staining: Triphenyl tetrazolinm chloride staining; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Brain Infarction; Brain Ischemia; Caspase 3; Caspases; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Male; MicroRNAs; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Toluene; Tumor Suppressor Protein p53

Organelle damage and increases in mitochondrial permeabilization are key events in the development of cerebral ischemic tissue injury because they cause both modifications in ATP turnover and cellular apoptosis/necrosis. Early restoration of blood flow and improvement of mitochondrial function might reverse the situation and help in recovery following an onset of stroke. Mitochondria and related bioenergetic processes can be effectively used as pharmacological targets. Progesterone (P4), one of the promising neurosteroids, has been found to be neuroprotective in various models of neurological diseases, through a number of mechanisms. This influenced us to investigate the possible role of P4 in the mitochondria-mediated neuroprotective mechanism in an ischemic stroke model of rat. In this study, we have shown the positive effect of P4 administration on behavioral deficits and mitochondrial health in an ischemic stroke injury model of transient middle cerebral artery occlusion (tMCAO). After induction of tMCAO, the rats received an initial intraperitoneal injection of P4 (8 mg/kg body weight) or vehicle at 1 h post-occlusion followed by subcutaneous injections at 6, 12 and 18 h. Behavioral assessment for functional deficits included grip strength, motor coordination and gait analysis. Findings revealed a significant improvement with P4 treatment in tMCAO animals. Staining of isolated brain slices from P4-treated rats with 2,3,5-triphenyltetrazolium chloride (TTC) showed a reduction in the infarct area in comparison to the vehicle group, indicating the presence of an increased number of viable mitochondria. P4 treatment was also able to attenuate mitochondrial reactive oxygen species (ROS) production, as well as block the mitochondrial permeability transition pore (mPTP), in the tMCAO injury model. In addition, it was also able to ameliorate the altered mitochondrial membrane potential and respiration ratio in the ischemic animals, thereby suggesting that P4 has a positive effect on mitochondrial bioenergetics. In conclusion, these results demonstrate that P4 treatment is beneficial in preserving the mitochondrial functions that are altered in cerebral ischemic injury and thus can help in defining better therapies.

Topics: Animals; Behavior, Animal; Biomarkers; Brain Ischemia; Cytochromes c; Dopamine; Energy Metabolism; Infarction, Middle Cerebral Artery; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Swelling; Neuroprotection; Oxidation-Reduction; Oxidative Stress; Progesterone; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Serotonin

Oxidative stress has been implicated in the pathogenesis of neurodegenerative disorders, such as vascular cognitive impairment (VCI). The present study was performed to investigate the potential neuroprotective effect of the antioxidant astaxanthin (ATX) in a mouse model of VCI. VCI was induced in male ICR mice by repeated occlusion of the bilateral common carotid artery, leading to repeated cerebral ischemia/reperfusion (IR) injury. After surgery, the mice received ATX or an equal volume of vehicle by daily intragastric administration for 28days. The results showed that ATX treatment ameliorated learning and memory deficits after repeated cerebral IR. ATX administration rescued the number of surviving pyramidal neurons in the CA1 and CA3 regions. The concentration of malondialdehyde was decreased, and the levels of reduced glutathione and superoxide dismutase in the hippocampus were increased. Electron microphotography revealed that damage to the ultrastructure of neurons was also reduced by ATX administration. In addition, the expression levels of Cytochrome C (Cyt C), cleaved Caspase-3 and Bax were lower and the expression of Bcl-2 was higher compared to control IR mice. Our findings demonstrate that ATX is able to suppresse learning and memory impairment caused by repeated cerebral IR and that this effect is associated with attenuation of oxidative stress.

Topics: Animals; Antioxidants; Apoptosis; Brain Ischemia; Cytochromes c; Dementia, Vascular; Disease Models, Animal; Glutathione; Hippocampus; Learning; Male; Malondialdehyde; Maze Learning; Memory Disorders; Mice; Mice, Inbred ICR; Neurons; Neuroprotective Agents; Oxidative Stress; Reperfusion; Reperfusion Injury; Superoxide Dismutase; Xanthophylls

Ischemic postconditioning has been demonstrated to alleviate brain ischemia/reperfusion-induced neuronal apoptosis; however, the protective mechanisms underlying the improved and more convenient method of remote ischemic postconditioning (RIPostC) are only recently beginning to be elucidated. Mitochondria are important in the regulation of cell apoptosis, and the B‑cell lymphoma 2 (Bcl‑2) homology 3 interacting‑domain death agonist (BID) promotes the insertion/oligomerization of Bcl‑2‑associated X protein into the mitochondrial outer membrane, leading to the release of proapoptotic proteins from the mitochondria. The present study hypothesized that RIPostC targets the BID‑mediated mitochondrial apoptotic pathway to exert neuroprotective effects, and the optimal time window for RIPostC application was investigated. RIPostC was conducted as follows: Three 10‑min cycles of bilateral femoral artery occlusion with intervals of 10 min reperfusion after 0, 10 or 30 min of brain reperfusion. The results revealed that reperfusion induced significant activation of BID, via proteolytic cleavage and translocation to the mitochondria, as determined using western blot analysis and immunofluorescence staining. Mitochondrial release of cytochrome c was additionally detected during BID activation, all of which were inhibited by the application of RIPostC. When RIPostC was applied during reperfusion, it demonstrated a significant protective effect. Furthermore, the infarct volume, neurological function and the degree of neuronal apoptosis were improved with application of RIPostC. These results suggested that the protective mechanisms of RIPostC may be associated with inhibition of the BID‑mediated mitochondrial apoptotic pathway, which may act as a potential molecular target for therapeutic intervention in the future.

Topics: Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Brain Ischemia; Cytochromes c; Disease Models, Animal; Gene Expression; Ischemic Postconditioning; Male; Mitochondria; Neurons; Protein Transport; Rats; Signal Transduction

Ginkgolide K (GK) belongs to the ginkgolide family of natural compounds found in Ginkgo biloba leaves, which have been used for centuries to treat cerebrovascular and cardiovascular diseases. We evaluated the protective effects of GK against neuronal apoptosis by assessing its ability to sustain mitochondrial integrity and function. Co-immunoprecipitation showed that Drp1 binding to GSK-3β was increased after an oxygen-glucose deprivation/reperfusion (OGD/R) insult in cultured neuroblastoma cells. This induced Drp1 and GSK-3β translocation to mitochondria and mitochondrial dysfunction, which was attenuated by GK. GK also reduced mitochondrial fission by increasing Drp1 phosphorylation at Ser637 and inhibiting mitochondrial Drp1 recruitment. In addition, GK exposure induced GSK-3β phosphorylation at Ser9 and enhanced the interaction between adenine nucleotide translocator (ANT) and p-GSK-3β. This interaction suppressed the interaction between ANT and cyclophilin D (CypD), which inhibited mitochondrial permeability transition pore (mPTP) opening. Similarly, suppression of mitochondrial fission by Mdivi-1 also inhibited GSK-3β-induced mPTP opening. Treating mice with GK prevented GSK-3β and Drp1 translocation to mitochondria and attenuated mitochondrial dysfunction after middle cerebral artery occlusion. We therefore propose that by inhibiting mitochondrial fission and attenuating mPTP opening, GK exerts neuroprotective effects that mitigate or prevent neuronal damage secondary to ischemic stroke.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Line; Cytochromes c; Dynamins; Ginkgolides; Glucose; Glycogen Synthase Kinase 3 beta; Ion Channel Gating; Lactones; Male; Mice; Mitochondrial Dynamics; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Neurons; Neuroprotective Agents; Oxygen; Protein Transport; Reactive Oxygen Species; Reperfusion Injury; Stroke

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT

Topics: Animals; Brain Injuries; Brain Ischemia; Cytochromes c; Male; Melatonin; Mice; Mitochondria; Receptor, Melatonin, MT1; Signal Transduction

To explore the effects and molecular mechanisms of the combination between total Astragalus extract (TAE) and total Panax notoginseng saponins (TPNS) against cerebral ischemia-reperfusion injury.. C57BL/6 mice were randomly divided into sham-operated group, model group, TAE (110 mg/kg) group, TPNS (115 mg/kg) group, TAE-TPNS combination group and Edaravone (4 mg/kg) group, treated for 4 days, then, cerebral ischemia-reperfusion injury was established by bilateral common carotid artery (CCA) ligation for 20 min followed by reperfusion for 1 and 24 h.. The combination of TAE 110 mg/kg and TPNS 115 mg/kg could strengthen protective effects on cerebral ischemia injury, the mechanism underlying might be related to improving jointly the early energy metabolism, and relieving the delayed apoptosis via inhibiting the mitochondrial apoptosis pathway of JNK signal transduction.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Apoptosis; Astragalus Plant; Brain; Brain Ischemia; CA1 Region, Hippocampal; Caspase 3; Caspase 9; Cell Survival; Cytochromes c; Energy Metabolism; JNK Mitogen-Activated Protein Kinases; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Panax notoginseng; Plant Extracts; Reperfusion Injury; Saponins; Sodium-Potassium-Exchanging ATPase

Preconditioning by brief ischemic episode induces tolerance to a subsequent lethal ischemic insult, and it has been suggested that reactive oxygen species are involved in this phenomenon. Thioredoxin 2 (Trx2), a small protein with redox-regulating function, shows cytoprotective roles against oxidative stress. Here, we had focused on the role of Trx2 in ischemic preconditioning (IPC)-mediated neuroprotection against oxidative stress followed by a subsequent lethal transient cerebral ischemia. Animals used in this study were randomly assigned to six groups; sham-operated group, ischemia-operated group, IPC plus (+) sham-operated group, IPC + ischemia-operated group, IPC + auranofin (a TrxR2 inhibitor) + sham-operated group and IPC + auranofin + ischemia-operated group. IPC was subjected to a 2 minutes of sublethal transient ischemia 1 day prior to a 5 minutes of lethal transient ischemia. A significant loss of neurons was found in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-group 5 days after ischemia-reperfusion; in the IPC + ischemia-operated-group, pyramidal neurons in the SP were well protected. In the IPC + ischemia-operated-group, Trx2 and TrxR2 immunoreactivities in the SP and its protein level in the CA1 were not significantly changed compared with those in the sham-operated-group after ischemia-reperfusion. In addition, superoxide dismutase 2 (SOD2) expression, superoxide anion radical ( O2-) production, denatured cytochrome c expression and TUNEL-positive cells in the IPC + ischemia-operated-group were similar to those in the sham-operated-group. Conversely, the treatment of auranofin to the IPC + ischemia-operated-group significantly increased cell damage/death and abolished the IPC-induced effect on Trx2 and TrxR2 expressions. Furthermore, the inhibition of Trx2R nearly cancelled the beneficial effects of IPC on SOD2 expression, O2- production, denatured cytochrome c expression and TUNEL-positive cells. In brief, this study shows that IPC conferred neuroprotection against ischemic injury by maintaining Trx2 and suggests that the maintenance or enhancement of Trx2 expression by IPC may be a legitimate strategy for therapeutic intervention of cerebral ischemia.

Topics: Animals; Auranofin; Brain Ischemia; CA1 Region, Hippocampal; Cell Death; Cytochromes c; Enzyme Inhibitors; Gerbillinae; Ischemic Preconditioning; Male; Neurons; Neuroprotection; Oxidative Stress; Random Allocation; Superoxide Dismutase; Superoxides; Thioredoxin Reductase 2; Thioredoxins

Therapeutic hypothermia is effective to attenuate brain ischemia/reperfusion (I/R) injury after cardiac arrest, and multiple mechanisms have been proposed. Dynamin-related protein 1 (Drp1), a large GTPases of dynamin superfamily, predominantly controls mitochondrial fission and is related to IR-induced Cyt C release and apoptosis. However, the effect of therapeutic hypothermia on Drp1 and mitochondrial fission after cardiac arrest remains still unclear. In this study, non-cardiac arrest and post-cardiac arrest rats received 6-h normothermia (37-38°C) or therapeutic hypothermia (32-34°C), and the hippocampus was harvested at 6h and 72h after cardiac arrest. Results showed the expression of Drp1 and Cyt C increased after cardiac arrest, but therapeutic hypothermia partially reversed this increase at 6h after cardiac arrest. Transmission electron microscopy (TEM) also showed a change in morphology following therapeutic hypothermia after cardiac arrest. Moreover, therapeutic hypothermia could decrease the histopathological damage, inhibit the apoptosis of CA1 neurons and improve the survival and neurological outcomes at 72h after cardiac arrest. Taken together, our study demonstrates that therapeutic hypothermia is neuroprotective against global cerebral I/R injury, which is, at least partially, ascribed to the inhibition Drp1 and Cyt C expression and the protection of mitochondrial structure.

Topics: Animals; Apoptosis; Brain Ischemia; Cytochromes c; Dynamins; Heart Arrest; Hippocampus; Hypothermia, Induced; Male; Mitochondria; Neurons; Rats, Sprague-Dawley; Reperfusion Injury

Although a substantial number of pre-clinical and experimental studies have investigated effects of 17β-estradiol, its precise molecular mechanism of action in the early state of chronic cerebral hypoperfusion remains controversial. The present study attempted to verify whether post-ischemic estradiol treatment (33.3 μg/kg for seven consecutive days) affects previously reported number of hippocampal apoptotic cells and amount of DNA fragmentation characteristic for apoptosis as well as the expression of key elements within synaptosomal Akt and Erk signal transduction pathways (NF-κB, Bax, Bcl-2, cytochrome C, caspase 3, and PARP). Additionally, alterations of aforementioned molecules linked to protection in various neurodegenerative disorders were monitored in the cytosolic, mitochondrial, and nuclear fractions associating investigated kinases and NF-κB with gene expression of their downstream effectors-Bcl-2, Bax, and caspase 3. The results revealed that an initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by chronic cerebral hypoperfusion was significantly reduced by 17β-estradiol. In synaptic regions, an altered profile with respect to the protein expression of Bcl-2 and phosphorylated Akt was detected, although the level of other examined proteins was not modified. In other investigated sub-cellular fractions, 17β-estradiol elicited phosphorylation and translocation of Akt and Erk along with modulation of the expression of their subsequent effectors. Our findings support the concept that repeated post-ischemic 17β-estradiol treatment attenuates neurodegeneration induced by chronic cerebral hypoperfusion in hippocampus through the activation of investigated kinases and regulation of their downstream molecules in sub-cellular manner indicating a time window and regime of its administration as a valid therapeutic intervention.

Topics: Animals; Apoptosis; Brain Ischemia; Caspase 3; Cytochromes c; Estradiol; Hippocampus; Male; Mitochondria; Neurons; Neuroprotective Agents; Proto-Oncogene Proteins c-akt; Rats, Wistar; Signal Transduction

Excessive mitochondrial fission activation has been implicated in cerebral ischemia-reperfusion (IR) injury. Hypothermia is effective in preventing cerebral ischemic damage. However, effects of hypothermia on ischemia-induced mitochondrial fission activation is not well known. Therefore, the aim of this study was to investigate whether hypothermia protect the brain by inhibiting mitochondrial fission-related proteins activation following cerebral IR injury. Adult male C57BL/6 mice were subjected to transient forebrain ischemia induced by 15min of bilateral common carotid artery occlusion (BCCAO). Mice were divided into three groups (n=48 each): Hypothermia (HT) group, with mild hypothermia (32-34°C) for 4h; Normothermia (NT) group, similarly as HT group except for cooling; Sham group, with vessels exposed but without occlusion or cooling. Hematoxylin and eosin (HE), Nissl staining, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and behavioral testing (n=6 each) demonstrated that hypothermia significantly decreased ischemia-induced neuronal injury. The expressions of Dynamin related protein 1 (Drp1) and Cytochrome C (Cyto C) (n=6 each) in mice hippocampus were measured at 3, 6, 24, and 72h of reperfusion. IR injury significantly increased expressions of total Drp1, phosphorylated Drp1 (P-Drp1 S616) and Cyto C under normothermia. However, mild hypothermia inhibited Drp1 activation and Cyto C cytosolic release, preserved neural cells integrity and reduced neuronal necrosis and apoptosis. These findings indicated that mild hypothermia-induced neuroprotective effects against ischemia-reperfusion injury is associated with suppressing mitochondrial fission-related proteins activation and apoptosis execution.

Topics: Animals; Apoptosis; Brain Ischemia; Cytochromes c; Dynamins; Hippocampus; Hypothermia, Induced; Male; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Dynamics; Pyramidal Cells; Recognition, Psychology; Reperfusion Injury

When given prior to brain ischemia, mitochondrial division inhibitor-1 (mdivi-1) attenuates the brain damage caused by ischemia. Here, we investigated the potential effects of post-ischemia mdivi-1 treatment (1mg/kg, i.p., administered immediately after 2h of ischemia and prior to reperfusion) using a MCAO rat model. Mdivi-1 treatment decreased infarct volume and improved neurological function. In addition, cytochrome C release was attenuated, and neuronal apoptosis was decreased. The mitochondrial fission protein dynamin-related protein 1 (Drp1) was decreased in the mitochondrial fraction but increased in the cytosolic fraction. Mdivi-1 treatment augmented the increases in the mRNA expression of peroxisome proliferator-activated receptor coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcriptional factor A. In conclusion, when given after ischemia and prior to reperfusion, mdivi-1 can protect against brain damage by inhibiting the mitochondria-mediated apoptosis induced by mitochondrial fission. Post-ischemia mdivi-1 treatment might promote I/R-induced mitochondrial biogenesis.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cytochromes c; Disease Models, Animal; Dynamins; Male; Mitochondria; Mitochondrial Dynamics; Neurons; Neuroprotective Agents; Organelle Biogenesis; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury

Providing adequate protection against cerebral ischemia remains an unrealized goal. The present study was aimed at testing whether chronic intermittent hypobaric hypoxia (CIHH) would have protective effects against cerebral ischemia and investigating the potential role of mitochondrial membrane ATP-sensitive potassium channel (mitoK. Ischemia was induced in rats by occlusion of bilateral common carotid arteries for 8min on day 2 after bilateral vertebral arteries were permanently electrocauterized and CIHH was simulated in a hypoxic chamber. Learning and memory impairments were analyzed using the Morris water maze. The delay neuronal death (DND) in the hippocampus CA1 was observed by thionine staining. The expression of the two subunits of mitoK. CIHH pretreatment ameliorated the learning and memory impairments produced by ischemia, concomitant with reduced DND in the hippocampus CA1 area. Expression levels of SUR1 and Kir6.2 both increased for at least one week after CIHH pretreatment. Levels of the two subunits were higher in the CIHH pretreatment combined with ischemia group than the ischemia only group at 2 d and 7 d after ischemia. Furthermore, the concentration of Cyt c was decreased in mitochondria and increased in the cytoplasm after ischemia which was prevented by CIHH. The decrease of Δψm and the destruction of mitochondrial ultrastructure were both rescued by CIHH pretreatment. The above protective effects of CIHH were blocked by 5-HD intraperitoneal injection 30min before ischemia.. CIHH pretreatment can reduce cerebral ischemic injury, which is mediated by upregulating the expression and activity of mitoK

Topics: Animals; Brain Ischemia; CA1 Region, Hippocampal; Cytochromes c; Hypoxia; Male; Maze Learning; Membrane Potential, Mitochondrial; Memory Disorders; Mitochondria; Potassium Channels; Potassium Channels, Inwardly Rectifying; Pressure; Pyramidal Cells; Rats, Wistar; Spatial Memory; Sulfonylurea Receptors

The neuroprotective effect of polydatin (PD) against hemorrhagic shock-induced mitochondrial injury has been described previously, and mitochondrial dysfunction and apoptosis were reportedly involved in ischemic stroke. In the present study the neuroprotective effect of PD in preventing apoptosis was evaluated following induction of focal cerebral ischemia by middle cerebral artery occlusion (MCAO) in rats. PD (30 mg/kg) was administered by caudal vein injection 10 min prior to ischemia/reperfusion (I/R) injury. 24 h following I/R injury, ameliorated modified neurological severity scores (mNSS) and reduced infarct volume were observed in the PD treated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Annexin V/propidium iodide assays demonstrated the anti-apoptotic effect of PD in the ischemic cortex. In addition, PD improved I/R injury‑induced mitochondrial dysfunction, reflected by morphological observations and measurements of mitochondrial membrane potential and intracellular ATP measurement. Western blot analysis revealed an increase in B‑cell lymphoma 2 apoptosis regulator (Bcl-2) expression, and a decrease in Bcl‑2‑associated protein X apoptosis regulator expression in the PD group in comparison with the vehicle treated group. PD treatment also prevented the release of cytochrome c from mitochondria into the cytoplasm, and blunted the activities of caspase‑9 and caspase‑3. Furthermore, PD treatment decreased the levels of reactive oxygen species in neurons isolated from the ischemic cortex. The findings of this study, therefore, suggest that PD has a dual effect, ameliorating both oxidative stress and mitochondria‑dependent apoptosis, making it a promising new therapy for the treatment of ischemic stroke.

Topics: Adenosine Triphosphate; Animals; Apoptosis; bcl-2-Associated X Protein; Biomarkers; Brain Ischemia; Cerebral Cortex; Cytochromes c; Disease Models, Animal; Glucosides; Male; Membrane Potential, Mitochondrial; Mitochondria; Neurons; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Stilbenes

In this study we sought to determine whether NO donor NOC-18 can protect brain mitochondria against ischemia-induced dysfunction, particularly opening of mitochondrial permeability transition pore (MPTP), and cell death. We found that inhibition of respiration with NAD-dependent substrates, but not with succinate, was observed after 30 min ischemia indicating that complex I of the mitochondrial respiratory chain is the primary site affected by ischemia. There was no loss of mitochondrial cytochrome c during 30-120 min of brain ischemia. Prolonged, 90 min ischemia substantially decreased calcium retention capacity of brain mitochondria suggesting sensitization of mitochondria to Ca(2+)-induced MPTP opening, and this was prevented by NOC-18 infusion prior to ischemia. NOC-18 did not prevent ischemia-induced inhibition of mitochondrial respiration, however, it partially protected against ischemia-induced necrosis. Protective effects of NOC-18 were abolished in the presence of selective inhibitors of protein kinase G (PKG) and protein kinase C (PKC). These results indicate that pre-treatment with NOC-18 protected brain mitochondria against ischemia-induced MPTP opening by decreasing mitochondrial sensitivity to calcium and partly protected brain cells against necrotic death in PKG- and PKC-depending manner.

Topics: Animals; Brain Ischemia; Calcium; Cell Death; Cyclic GMP-Dependent Protein Kinases; Cytochromes c; Male; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Necrosis; Neuroprotective Agents; Nitric Oxide Donors; Nitroso Compounds; Protein Kinase C; Rats, Wistar

P38 mitogen-activated protein kinase (MAPK) is a pro-apoptotic and pro-inflammatory protein that is activated in response to cellular stress. While p38 is known to be activated in response to cerebral ischemia, the precise role of p38 and its isoforms in ischemia-induced neuronal apoptosis remains unclear. In the current study, we examined the differential activation and functional roles of p38α and p38β MAPK isoforms in short-term ovariectomized female rats treated with either the neuroprotective ovarian hormone 17beta-estradiol (E2) or placebo in a model of global cerebral ischemia (GCI). GCI induced biphasic activation of total p38 in the hippocampal CA1, with peaks at 30 min and 1 day after 10-min ischemia-reperfusion. Further study demonstrated that activated p38α, but not p38β, translocated to the nucleus 30 min and 3 h post reperfusion, and that this event coincided with increased phosphorylation of activating transcription factor 2 (ATF2), a p38 target protein. Intriguingly, activated p38α was also enhanced in mitochondrial fractions of CA1 neurons 1 day after GCI, and there was loss of mitochondrial membrane potential, as well as enhanced cytochrome c release and caspase-3 cleavage at 2 days post GCI. Importantly, E2 prevented the biphasic activation of p38, as well as both nuclear and mitochondrial translocation of p38α after GCI, and these findings correlated with attenuation of mitochondrial dysfunction and delayed neuronal cell death in the hippocampal CA1. Furthermore, administration of a p38 inhibitor was able to mimic the neuroprotective effects of E2 in the hippocampal CA1 region by preventing nuclear and mitochondrial translocation of p38α, loss of mitochondrial membrane potential, and neuronal apoptosis. As a whole, this study suggests that changes in subcellular localization of the activated p38α isoform are required for neuronal apoptosis following GCI, and that E2 exerts robust neuroprotection, in part, through dual inhibition of activation and subcellular trafficking of p38α.

Topics: Activating Transcription Factor 2; Animals; Apoptosis; Brain Ischemia; CA1 Region, Hippocampal; Caspase 3; Cell Nucleus; Cytochromes c; Estradiol; Female; Gene Expression Regulation; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase 11; Mitogen-Activated Protein Kinase 14; Neurons; Neuroprotective Agents; Ovariectomy; Phosphorylation; Protein Kinase Inhibitors; Protein Transport; Rats; Rats, Sprague-Dawley; Signal Transduction

Ischemic stroke is a major macrovascular complication of diabetes mellitus. Sitagliptin, a dipeptidyl peptidase-IV inhibitor, was recently shown to improve cognitive functions in diabetic rats; hence the present study was conducted to evaluate its protective effect against transient ischemia-reperfusion (I/R) in diabetic animals.. Diabetes was induced by streptozotocin (40 mg/kg). Six weeks later, cerebral I/R was induced by bicommon carotid occlusion for 15 min followed by 1h reperfusion. Sitagliptin (250 mg/kg; p.o.) was administered daily during the last 2 weeks before I/R.. The drug alleviated hippocampal injury inflicted by diabetes and/or I/R injury where it suppressed nuclear factor kappa (NF-κ)B, and consequently the downstream inflammatory cytokines tumor necrosis factor-α and interleukin-6. In parallel, the anti-inflammatory cytokine interleukin-10 was elevated. Antioxidant potential of sitagliptin was depicted, where it reduced neutrophil infiltration, lipid peroxides and nitric oxide associated with replenished reduced glutathione. Decline of excitatory amino acid glutamate content is a main finding which is probably mediated by the NF-κB signaling pathway as well as improved oxidant status. Sitagliptin exerted an anti-apoptotic effect as reflected by the reduction of the mitochondrial matrix component cytochrome -C and the key downstream executioner caspase-3. Histopathological examination corroborated the biochemical data.. These findings suggest that sitagliptin is endowed with neuroprotective properties which are probably mediated by its antioxidant, anti-inflammatory, and anti-apoptotic mechanisms and hence may provide a novel agent for the management of ischemic stroke in diabetics.

Topics: Animals; Brain Ischemia; Caspase 3; Cytochromes c; Diabetes Complications; Diabetes Mellitus, Experimental; Hippocampus; Hypoglycemic Agents; Interleukin-10; Male; Neuroprotective Agents; Neutrophil Infiltration; Pyrazines; Rats; Rats, Wistar; Reperfusion Injury; Sitagliptin Phosphate; Triazoles

Sulfiredoxin 1 (Srxn1), an endogenous antioxidant protein, plays an important neuroprotective role in cerebral ischemia. However, the exact mechanisms of action of Srxn1 in cerebral ischemia have not yet been fully elucidated. Therefore, in the present study, rat primary cortical astrocytes transfected with a lentiviral vector encoding short hairpin RNA (shRNA) were exposed to oxygen-glucose deprivation (OGD) for 4 h or to 100 µM hydrogen peroxide (H2O2) for 6 h, in order to construct an in vitro model of cerebral ischemia-induced damage. We found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in cell viability, as shown by MTS assay, an increase in cell damage, as shown by lactate dehydrogenase assay and an increase in cellular apoptosis, as shown by Hoechst 33342 staining and flow cytometry. Furthermore, we found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in mitochondrial transmembrane potential (Δψm) as indicated by JC-1 staining, an increase in the cytoplasmic expression of cytochrome c (Cyt.C), caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP) and Bax protein at the protein level, but a decrease in the expression of the anti-apoptotic Bcl-2 protein; these effects were tightly associated with the mitochondrial apoptotic pathway. However, we found that there was no obvious change in the intracellular calcium ([Ca2+]i) levels and caspase-12 expression following the knockdown of Srxn1. Taken together, the results from the present study demonstrate that Srxn1 protects primary rat cortical astrocytes from OGD- or H2O2-induced apoptosis and that involves the activation of the mitochondrial apoptotic pathway, which suggests that Srxn1 may be a potential target in the treatment of cerebral ischemia.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Astrocytes; bcl-2-Associated X Protein; Brain Ischemia; Caspase 12; Caspase 3; Caspase 9; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytochromes c; Glucose; Hydrogen Peroxide; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Neuroprotective Agents; Oxidative Stress; Oxidoreductases Acting on Sulfur Group Donors; Poly(ADP-ribose) Polymerases; Primary Cell Culture; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering

Brain ischemia/reperfusion injury results in death of vulnerable neurons and extensive brain damage. It is well known that mitochondrial release of cytochrome c (cyto c) is a hallmark of neuronal death, however the molecular events underlying this release are largely unknown. We tested the hypothesis that cyto c release is regulated by breakdown of the cristae architecture maintenance protein, optic atrophy 1 (OPA1), located in the inner mitochondrial membrane. We simulated ischemia/reperfusion in isolated primary rat neurons and interrogated OPA1 release from the mitochondria, OPA1 oligomeric breakdown, and concomitant dysfunction of mitochondrial dynamic state. We found that ischemia/reperfusion induces cyto c release and cell death that corresponds to multiple changes in OPA1, including: (i) translocation of the mitochondrial fusion protein OPA1 from the mitochondria to the cytosol, (ii) increase in the short isoform of OPA1, suggestive of proteolytic processing, (iii) breakdown of OPA1 oligomers in the mitochondria, and (iv) increased mitochondrial fission. Thus, we present novel evidence of a connection between release of cyto c from mitochondria and disruption of the mitochondrial fusion.

Topics: Animals; Brain Ischemia; Cell Death; Cell Hypoxia; Cytochromes c; Mitochondrial Dynamics; Neurons; Optic Atrophy, Autosomal Dominant; Rats; Rats, Sprague-Dawley

The aims of this study are to clarify the role of mTOR in mediating cerebral ischemic brain damage and the effects of rapamycin on ischemic outcomes. Ten minutes of forebrain ischemia was induced in rats, and their brains were sampled after 3 h, 16 h, and 7 days reperfusion for histology, immunohistochemistry and biochemical analysis. Our data demonstrated that cerebral ischemia resulted in both apoptotic and necrotic neuronal death; cerebral ischemia and reperfusion led to significant increases of mRNA and protein levels of p-mTOR and its downstream p-P70S6K and p-S6; elevation of LC3-II, and release of cytochrome c into the cytoplasm in both the cortex and hippocampus. Inhibition of mTOR by rapamycin markedly reduced ischemia-induced damage; suppressed p-Akt, p-mTOR, p-P70S6K and p-S6 protein levels; decreased LC3-II and Beclin-1; and prevented cytochrome c release in the two structures. All together, these data provide evidence that cerebral ischemia activates mTOR and autophagy pathways. Inhibition of mTOR deactivates the mTOR pathway, suppresses autophagy, prevents cytochrome c release and reduces ischemic brain damage.

Topics: Animals; Brain Ischemia; Cytochromes c; In Situ Nick-End Labeling; Prosencephalon; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases

Mitochondria and oxidative stress play important roles in neuronal cell death associated with cerebral ischemia. Elevated level of reactive oxygen species (ROS) and mitochondrial dysfunction are thought to be responsible for cerebral ischemia injury along with neural cells death through several apoptotic mechanisms. In this study, exposure of rat pheochromocytoma (PC12) cells to hydrogen peroxide (H2O2) at the concentration of 0.3 mM for 24 h caused significant loss of cell viability, lactate dehydrogenase (LDH) release from cells, ascent of ROS level and mitochondrial membrane potential (MMP) decrease. Moreover, the activities of caspase-9, caspase-8 and caspase-3 all were increased in H2O2-induced PC12 cells. However, pretreatment with ginkgolide K (GK) solutions of different concentrations (10, 50, 100 µM) for 24 h prior to exposuring to H2O2 significantly increased cells viability, suppressed LDH release, attenuated ROS level, prevented cytochrome c release from mitochondria and boosted MMP expression. In addition, ginkgolide K notably inhibited the caspase-3 and caspase-9 but not caspase-8 activities in exogenous H2O2-treated PC12 cells. These results demonstrated that ginkgolide K protected PC12 cells from H2O2-induced apoptosis by restoring MMP expression, ameliorating oxidative stress and subsequently leading to inhibit the activity of caspase-3 protein. Therefore, the present study supported that ginkgolide K may be a promising neuroprotective compound for cerebral ischemia treatment.

Topics: Animals; Antioxidants; Apoptosis; Brain Ischemia; Caspases; Cell Survival; Cytochromes c; Ginkgo biloba; Ginkgolides; Hydrogen Peroxide; L-Lactate Dehydrogenase; Lactones; Membrane Potential, Mitochondrial; Mitochondria; Neuroprotective Agents; Oxidative Stress; PC12 Cells; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species

The ischemic stroke cascade is composed of several pathophysiological events, providing multiple targets for pharmacological intervention. JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel hybrid molecule, in which a benzodiazepine portion is covalently linked to a dihydropyridine ring, forming a new chemical entity with potential multisite neuroprotective activity. In the present study, JM-20 prevented PC-12 cell death induced either by glutamate, hydrogen peroxide or KCN-mediated chemical hypoxia. This molecule also protected cerebellar granule neurons from glutamate or glutamate plus pentylenetetrazole-induced damage at very low micromolar concentrations. In rat liver mitochondria, JM-20, at low micromolar concentrations, prevented the Ca2+-induced mitochondrial permeability transition, as assessed by mitochondrial swelling, membrane potential dissipation and organelle release of the pro-apoptotic protein cytochrome c. JM-20 also inhibited the mitochondrial hydrolytic activity of F1F0-ATP synthase and Ca2+ influx. Therefore, JM-20 may be a multi-target neuroprotective agent, promoting reductions in neuronal excitotoxic injury and the protection of the mitochondria from Ca2+-induced impairment as well as the preservation of cellular energy balance.

Topics: Animals; Benzodiazepines; Brain Ischemia; Calcium; Cell Death; Cerebellum; Cytochromes c; Dihydropyridines; Glutamic Acid; Hydrogen Peroxide; Hydrolysis; Liver; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proton-Translocating ATPases; Mitochondrial Swelling; Neurons; Neuroprotective Agents; Niacin; Oxidative Stress; PC12 Cells; Pentylenetetrazole; Phosphates; Potassium Cyanide; Rats; Stroke

We have documented that tissue kallikrein (TK) prevents neurons from hypoxia/reoxygenation injury through the B2R-ERK1/2 pathway and the antihypoxic function of TK through Homer1b/c-ERK1/2 signaling pathways. The present study investigates the molecular mechanisms of exogenous TK activation of the B2R-ERK1/2 pathway through the β-arrestin-2 assembled B2R-Raf-MEK1/2 signaling module in vivo. The cresyl violet staining results indicated that exogenous TK protected the rat hippocampal CA1 neurons against cerebral ischemia/reperfusion (I/R) injury. The immunoprecipitation (IP) and immunoblotting (IB) results revealed that exogenous TK upregulated the β-arrestin-2 assembled B2R-Raf-MEK1/2 signaling module and upregulated the phosphorylation of Raf (p-Raf), MEK1/2 (p-MEK1/2), and ERK1/2 (p-ERK1/2). Meanwhile, exogenous TK upregulated the expression of nuclear factor-κB (NF-κB), depressed the release of cytochrome c (Cyt c) and bax from mitochondria to the cytosol, and depressed the activation of caspase-3. Take together, our results suggest that exogenous TK attenuated the cerebral I/R induced rat hippocampal CA1 neurons injury through activating the β-arrestin-2 assembled B2R-Raf-MEK1/2 signaling module and that the activated B2R-Raf-MEK1/2 signaling module could upregulate the expression of NF-κB, decrease the release of cytochrome c and bax from mitochondria to the cytosol, and depress the activation of caspase-3.

Topics: Animals; Arrestins; bcl-2-Associated X Protein; beta-Arrestin 2; beta-Arrestins; Brain Ischemia; CA1 Region, Hippocampal; Caspase 3; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Male; MAP Kinase Signaling System; Nerve Degeneration; Neurons; Oligodeoxyribonucleotides, Antisense; raf Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue Kallikreins; Up-Regulation

Hydrogen, a popular antioxidant gas, can selectively reduce cytotoxic oxygen radicals and has been found to protect against ischemia-reperfusion (I/R) injury of multiple organs. Acute neuronal death during I/R has been attributed to loss of mitochondrial permeability transition coupled with mitochondrial dysfunction. This study was designed to investigate the potential therapeutic effect of hydrogen-rich saline on neuronal mitochondrial injury from global cerebral I/R in rats.. We used a four-vessel occlusion model of global cerebral ischemia and reperfusion, with Sprague-Dawley rats. The rats were divided randomly into six groups (n = 90): sham (group S), I/R (group I/R), normal saline (group NS), atractyloside (group A), hydrogen-rich saline (group H), and hydrogen-rich saline + atractyloside (group HA). In groups H and HA, intraperitoneal hydrogen-rich saline (5 mL/kg) was injected immediately after reperfusion, whereas the equal volume of NS was injected in the other four groups. In groups A and HA, atractyloside (15 μL) was intracerebroventricularly injected 10 min before reperfusion, whereas groups NS and H received equal NS. The mitochondrial permeability transition pore opening and mitochondrial membrane potential were measured by spectrophotometry. Cytochrome c protein expression in the mitochondria and cytoplasm was detected by western blot. The hippocampus mitochondria ultrastructure was examined with transmission electron microscope. The histologic damage in hippocampus was assessed by hematoxylin and eosin staining.. Hydrogen-rich saline treatment significantly improved the amount of surviving cells (P < 0.05). Furthermore, hydrogen-rich saline not only reduced tissue damage, the degree of mitochondrial swelling, and the loss of mitochondrial membrane potential but also preserved the mitochondrial cytochrome c content (P < 0.05).. Our study showed that hydrogen-rich saline was able to attenuate neuronal I/R injury, probably by protecting mitochondrial function in rats.

Topics: Animals; Antioxidants; Apoptosis; Brain Ischemia; Calcium; Cell Survival; Cytochromes c; Hydrogen; Infusions, Intraventricular; Male; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Neurons; Neuroprotective Agents; Random Allocation; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride

The aim of this study was to investigate the influence of atorvastatin on the opening of the mitochondrial permeability transition pore (MPTP) and the expression of cytochrome C (Cyt C) in Sprague-Dawley rats with cerebral ischemia-reperfusion (I/R). The rat model of cerebral artery ischemia was established by the suture-occluded method with ischemia for 2 h and reperfusion for 72 h. Thirty-four male rats were randomly divided into four groups: the normal group and the sham-operation group without any treatment, the I/R group with only intragastric administration of normal saline, and the intervention group, which received intragastric administration of 10 mg/kg atorvastatin at different times. All rats were sacrificed at 72 h. Compared with the I/R group, the morphology of nerve cells in the intervention group was reduced, the number of TUNEL-positive cells decreased, the expression of cortical cytoplasm Cyt C decreased, and the mitochondrial absorbance value increased. All of these differences were statistically significant. Atorvastatin could inhibit neuronal apoptosis and alleviate the cerebral I/R injury. The mechanism may be related to the blocking of the MPTP opening and the subsequent reduction of Cyt C release.

Topics: Animals; Anticholesteremic Agents; Apoptosis; Atorvastatin; Brain Ischemia; Cytochromes c; Disease Models, Animal; Heptanoic Acids; Infarction, Middle Cerebral Artery; Male; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Pyrroles; Rats, Sprague-Dawley; Reperfusion Injury

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A 1, Atg7 knockdown or in atg5(-/-) MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Brain Ischemia; Cytochromes c; Cytoprotection; Glucose; Hypoxia; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Mitochondria; Mitophagy; Neurons; Quinazolinones; Rats; Reperfusion Injury; Ubiquitin-Protein Ligases

In this study, we investigated the neuroprotective effect of oxysophoridine on ischemia and ischemia-like insults. Protection by oxysophoridine was studied at the in vivo level using a model of middle cerebral artery occlusion in mice and at the in vitro level using primary rat hippocampal neuronal cultures exposed to oxygen-glucose deprivation, a model of ischemia-like injury. The behavioral test was performed by using the neurological scores. The infarction volume of brain was assessed in the brain slices stained with 2,3,5-triphenyl tetrazolium chloride. The neuron apoptosis was evaluated by Hoechst 33342 staining. The morphological change in the neurons was examined using a Transmission Electron Microscope (TEM or EM). To evaluate neuron apoptosis, caspase-3, -9, and - 8 activities were measured using assay kits with an ELISA reader. The Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2, and Bax proteins. The quantitative real-time PCR assay was used to evaluate the release of cytochrome c and the expression of caspase-3 mRNA. Oxysophoridine-treated groups (62.5, 125, 250 mg/kg) markedly reduced neurological deficit scores and infarct volumes. Treatment with oxysophoridine (5, 20, 80 µmol/L) significantly attenuated neuronal damage, with evidence of decreased cell apoptosis and decreased cell morphologic impairment. Furthermore, treatment with oxysophoridine could effectively downregulate the expression of cytochrome c and caspase-3 in both mRNA and protein levels, and Bax in the protein level, and induce an increase of Bcl-2 in the protein level. The caspase-3, -9, and -8 activities were also inhibited. These findings suggested that oxysophoridine may be a potential neuroprotective agent for cerebral ischemia injury.

Topics: Alkaloids; Animals; Apoptosis; bcl-2-Associated X Protein; Brain; Brain Ischemia; Caspase 3; Cytochromes c; Hippocampus; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred ICR; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Specific Pathogen-Free Organisms

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Topics: Animals; Brain Ischemia; CA1 Region, Hippocampal; Cell Survival; Cytochromes c; Gene Expression; Insulin; Mitochondria; Neurons; Neuroprotective Agents; Phosphorylation; Protein Processing, Post-Translational; Rats; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Swine; Tyrosine

Previous studies have indicated that bone morphogenetic protein-7 (BMP-7) is neuroprotective against cerebral ischemia/reperfusion (IR) injury. The present study was undertaken to determine the molecular mechanisms involved in this effect. Adult male Wistar rats were subjected to 2 h of transient middle cerebral artery occlusion (MCAO), followed by 24 h of reperfusion. BMP-7 (10-4 g/kg) or vehicle was infused into rats at the onset of reperfusion via the tail vein. Neurological deficits, infarct volume, histopathological changes, oxidative stress-related biochemical parameters, neuronal apoptosis, and apoptosis-related proteins were assessed. BMP-7 significantly improved neurological and histological deficits, reduced the infarct volume, and decreased apoptotic cells after cerebral ischemia. BMP-7 also markedly enhanced the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and reduced the level of malondialdehyde (MDA) in IR rats. In addition, Western blot analysis indicated that BMP-7 prevented cytochrome c release, inhibited activation of caspase-3, caspase-9 and caspase-8. Our data suggested that BMP-7 has protective effects against cerebral IR injury in rats, and the neuroprotective effects may be attributed to attenuating oxidative stress and inhibiting neuronal apoptosis.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 7; Brain Ischemia; Caspase 3; Caspase 8; Caspase 9; Cytochromes c; Glutathione Peroxidase; Male; Malondialdehyde; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

Mitochondrial division inhibitor (mdivi-1) is a derivative of quinazolinone that acts as a selective inhibitor of a mitochondrial fission protein Drp1. A previous study demonstrated that as a selective inhibitor of Drp1, mdivi-1 has a protective effect in an experimental model of heart ischemia/reperfusion injury. In this study, we investigated the protective effects of mdivi-1 on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that mdivi-1 (1.2mg/kg) significantly reduced cerebral damage induced by ischemia/reperfusion. This neuroprotective effect was dose-dependent. Mdivi-1 treatment blocked apoptotic cell death in cerebral ischemia/reperfusion injury, and significantly decreased the expression of Drp1 and Cytochrome C. These results suggest that mdivi-1 exerts neuroprotective effects against nerve injury after cerebral ischemia/reperfusion, and the underlying mechanism may be through the prevention of Cytochrome C release and suppression of the mitochondrial apoptosis pathway.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cytochromes c; Dynamins; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger

High levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy), contribute to autophagy and ischemia/reperfusion injury (I/R). Previous studies have shown that I/R injury and HHcy cause increased cerebrovascular permeability; however, the associated mechanism remains obscure. Interestingly, during HHcy, cytochome-c becomes homocysteinylated (Hcy-cyto-c). Cytochrome-c (cyto-c) transports electrons and facilitates bioenergetics in the system. However, its role in autophagy during ischemia/reperfusion injury is unclear. Tetrahydrocurcumin (THC) is a major herbal antioxidant and anti-inflammatory agent. Therefore, the objective of this study was to determine whether THC ameliorates autophagy during ischemia/reperfusion injury by reducing homocysteinylation of cyto-c in hyperhomocysteinemia pathological condition. To test this hypothesis, we employed 8-10-week-old male cystathionine-beta-synthase heterozygote knockout (CBS⁺/⁻) mice (genetically hyperhomocystemic mice). Experimental group was: CBS⁺/⁻, CBS⁺/⁻ + THC (25 mg/kg in 0.1% DMSO dose); CBS ⁺/⁻/I/R, and CBS⁺/⁻/I/R + THC (25 mg/kg in 0.1% DMSO dose). Ischemia was performed for 30 min and reperfusion for 72 h. THC was injected intra-peritoneally (I.P.) once daily for a period of 3 days after 30 min of ischemia. The infarct area was measured using 2,3,5-triphenyltetrazolium chloride staining. Permeability was determined by brain edema and Evans Blue extravasation. The brain tissues were analyzed for oxidative stress, matrix metalloproteinase-9 (MMP-9), damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3) by Western blot. The mRNA levels of S-adenosyl-L-homocysteine hydrolases (SAHH) and methylenetetrahydrofolate reductase (MTHFR) genes were measured by quantitative real-time polymerase chain reaction. Co-immunoprecipitation was used to determine the homocysteinylation of cyto-c. We found that brain edema and Evans Blue leakage were reduced in I/R + THC-treated groups as compared to sham-operated groups along with reduced brain infarct size. THC also decreased oxidative damage and ameliorated the homocysteinylation of cyto-c in-part by MMP-9 activation which leads to autophagy in I/R groups as compared to sham-operated groups. This study suggests a potential therapeutic role of dietary THC in cerebral ischemia.

Topics: Animals; Antioxidants; Autophagy; Brain Ischemia; Curcumin; Cytochromes c; Hyperhomocysteinemia; Male; Mice; Mice, Knockout; Reperfusion Injury

This aim of this study was to explore the effects and molecular mechanisms of Astragalus extract against cerebral ischemia injury through the energy metabolism and apoptosis pathways of c‑Jun N-terminal kinase (JNK) signal transduction. After the bilateral common carotid artery of C57BL/6 mice was occluded for 20 min followed by 1-h reperfusion, the ATP content, total adenine nucleotides (TAN), energy charge (EC), and sodium potassium ATPase (Na(+)-K(+)‑ATPase) activity were decreased markedly in brain tissues. Astragalus extract markedly increased the ATP and ADP levels, EC value, and Na(+)-K(+)-ATPase activity. Twenty-four and 48 h after reperfusion, the neurocyte survival rate decreased and apoptosis rate increased, while the expression of phosphorylated JNK1/2, cytochrome c (Cyt C), and cysteine aspartic acid-specific protease (caspase)-9 and -3 were significantly enhanced in brain tissues. Astragalus extract significantly increased neurocyte survival and decreased the apoptosis rate as well as down-regulated the expression of p-JNK1/2, Cyt C, caspase-9, and caspase-3. These results suggest that Astragalus extract has neuroprotective effects against nerve injury after cerebral ischemia-reperfusion, and the underlying mechanism may be associated with improved cellular energy metabolism, inhibition of JNK signal transduction pathway activation, and then suppression of the mitochondrial apoptosis pathway.

Topics: Animals; Apoptosis; Astragalus Plant; Brain Ischemia; CA1 Region, Hippocampal; Caspase 3; Caspase 9; Cytochromes c; Energy Metabolism; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Plant Extracts; Reperfusion Injury

Recently, the treatment of stroke has focused on antioxidant therapies, where oxidative stress is implicated. The preventive and therapeutic potential of plant compounds on ischemic stroke has been intensively studied because many of them contain antioxidant properties. Genistein, one of the active ingredients in soybean, possesses many bioactivities. In this study, we investigated the potential neuroprotective effects of genistein and its possible mechanism of action in a cerebral ischemia mouse model. Mice were pretreated with genistein (2.5, 5, and 10mg/kg) or vehicle orally once daily for 14 consecutive days before transient middle cerebral artery occlusion was performed. Genistein at doses of 2.5-10mg/kg significantly reduced the infarct volume, improved the neurological deficit and prevented cell apoptosis after ischemia. In addition, genistein pretreatment was shown to inhibit the ischemia-induced reactive oxygen species (ROS) production, enhance the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), and decrease levels of malondialdehyde (MDA) in stroke mice. Moreover, genistein reversed the mitochondria dysfunction after ischemia, as evidenced by decreasing mitochondria ROS levels, preventing cytochrome C release to the cytoplasm and inhibiting caspase-3 activation. Western blotting showed ischemia activated the ROS-dependent nuclear factor-κB (NF-κB) signaling pathway, and genistein suppressed phosphorylation and activation of the NF-κB p65 subunit, as well as the phosphorylation and degradation of the inhibitor protein of κBα (IκBα). Our findings suggested that genistein has a neuroprotective effect in transient focal ischemia, which may involve regulation of mitochondria-dependent apoptosis pathways and suppression of ROS-induced NF-κB activation.

Topics: Animals; Apoptosis; Blotting, Western; Brain Ischemia; Caspase 3; Cytochromes c; Disease Models, Animal; Enzyme Activation; Genistein; Glutathione Peroxidase; In Situ Nick-End Labeling; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Mitochondria; Neuroprotective Agents; NF-kappa B; Reactive Oxygen Species; Superoxide Dismutase

High levels of reactive oxygen species inflict oxidative damage on various cellular components that eventually culminate in a variety of diseases. This study investigated the cytoprotective effects of a nucleic acid-based health product (Squina [Hong Kong, China] DNA) against oxidative stress, particularly in neuronal cells. Adult female Sprague-Dawley rats were treated with Squina DNA, and changes in mitochondrial antioxidant status and functional capacity were assessed by the activities of antioxidant enzymes and ATP generation capacity in brain, heart ventricular, kidney, skeletal muscle, and liver tissues of control and Squina DNA-treated rats. The effects of Squina DNA pretreatment on brain/neuronal cell injury were investigated in a rat model of cerebral ischemia/reperfusion (I/R) injury and a neuroblastoma SH-SY5Y cell model of β-amyloid (Aβ) protein fragment 25-35-induced toxicity. Long-term Squina DNA treatment caused dose-dependent increases in mitochondrial antioxidant status and functional capacity in rat brain, heart ventricular, kidney, skeletal muscle, and liver tissues. Squina DNA pretreatment significantly prevented I/R injury in brain tissue. The cerebroprotection was associated with a reversal of I/R-induced impairment in mitochondrial antioxidant status and disruption in membrane integrity. Squina DNA ethanol extract also significantly inhibited the Aβ-induced apoptosis in SH-SY5Y neuronal cells, as evidenced by less caspase 3 and caspase 9 activation as well as mitochondrial cytochrome c release in Aβ-challenged cells. Squina DNA may enhance the resistance of tissues and cells to oxidative stress, particularly in pathological conditions such as stroke and aging-related neurodegenerative diseases.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; Biological Products; Brain; Brain Ischemia; Caspases; Cell Line, Tumor; Cytochromes c; DNA; Dose-Response Relationship, Drug; Female; Heart Ventricles; Kidney; Liver; Mitochondria; Mitochondrial Membranes; Muscle, Skeletal; Neurodegenerative Diseases; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Salmon; Stroke

The overproduction of reactive oxygen species and the resulting damage are central to the pathology of many diseases. The study of the temporal and spatial accumulation of reactive oxygen species has been limited because of the lack of specific probes and techniques capable of continuous measurement. We demonstrate the use of a miniaturized electrochemical cytochrome c (Cyt c) biosensor for real-time measurements and quantitative assessment of superoxide production and inactivation by natural and engineered antioxidants in acutely prepared brain slices from mice. Under control conditions, superoxide radicals produced from the hippocampal region of the brain in 400-μm-thick sections were well within the range of detection of the electrode. Exposure of the slices to ischemic conditions increased the superoxide production twofold and measurements from the slices were stable over a 3- to 4-h period. The stilbene derivative and anion channel inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilbene markedly reduced the extracellular superoxide signal under control conditions, suggesting that a transmembrane flux of superoxide into the extracellular space may occur as part of normal redox signaling. The specificity of the electrode for superoxide released by cells in the hippocampus was verified by the exogenous addition of superoxide dismutase (SOD), which decreased the superoxide signal in a dose-dependent manner. Similar results were seen with the addition of the SOD mimetic cerium oxide nanoparticles (nanoceria), in that the superoxide anion radical scavenging activity of nanoceria with an average diameter of 15 nm was equivalent to 527 U of SOD for each 1 μg/ml of nanoceria added. This study demonstrates the potential of electrochemical biosensors for studying real-time dynamics of reactive oxygen species in a biological model and the utility of these measurements in defining the relative contribution of superoxide to oxidative injury.

Topics: Animals; Antioxidants; Biosensing Techniques; Brain Ischemia; Calibration; Cattle; Cell Hypoxia; Cerium; Cytochromes c; Electrochemical Techniques; Electrodes; Female; Hippocampus; Horses; Hypoxanthine; Immobilized Proteins; In Vitro Techniques; Male; Mice; Nanoparticles; Superoxide Dismutase; Superoxides; Voltage-Dependent Anion Channels; Xanthine Oxidase

Paeoniflorin (PF), the principal component of Paeoniae Radix prescribed in traditional Chinese medicine, has been reported to exhibit many pharmacological effects including protection against ischemic injury. However, the mechanisms underlying the protective effects of PF on cerebral ischemia are still under investigation. The present study showed that PF treatment for 14 days could significantly inhibit transient middle cerebral artery occlusion (MCAO)-induced over-activation of astrocytes and microglia, and prevented up-regulations of pro-inflamamtory mediators (TNFα, IL-1β, iNOS, COX(2) and 5-LOX) in plasma and brain. Further study demonstrated that chronic treatment with PF suppressed the activations of JNK and p38 MAPK, but enhanced ERK activation. And PF could reverse ischemia-induced activation of NF-κB signaling pathway. Moreover, our in vitro study revealed that PF treatment protected against TNFα-induced cell apoptosis and neuronal loss. Taken together, the present study demonstrates that PF produces a delayed protection in the ischemia-injured rats via inhibiting MAPKs/NF-κB mediated peripheral and cerebral inflammatory response. Our study reveals that PF might be a potential neuroprotective agent for stroke.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Astrocytes; bcl-2-Associated X Protein; Benzoates; Brain; Brain Ischemia; Bridged-Ring Compounds; Cerebral Infarction; Cyclooxygenase 2; Cytochromes c; Disease Models, Animal; Gene Expression Regulation; Glucosides; Hippocampus; Inflammation; Interleukin-1beta; Lipoxygenase; Male; Microglia; Mitogen-Activated Protein Kinases; Monoterpenes; Neurons; NF-kappa B; Nitric Oxide Synthase Type II; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Tumor Necrosis Factor-alpha

Taurine, an abundant amino acid in the nervous system, is reported to reduce ischemic brain injury in a dose-dependent manner. This study was designed to investigate whether taurine protected brain against experimental stroke through affecting mitochondria-mediated cell death pathway. Rats were subjected to 2-h ischemia by intraluminal filament, and then reperfused for 22 h. It was confirmed again that taurine (50 mg/kg) administered intravenously 1 h after ischemia markedly improved neurological function and decreased infarct volume at 22 h after reperfusion. In vehicle-treated rats, the levels of intracellular ATP and the levels of cytosolic and mitochondrial Bcl-xL in the penumbra and core were markedly reduced, while the levels of cytosolic Bax in the core and mitochondrial Bax in the penumbra and core were enhanced significantly. There was a decrease in cytochrome C in mitochondria and an increase in cytochrome C in the cytosol of the penumbra and core. These changes were reversed by taurine. Furthermore, taurine inhibited the activation of calpain and caspase-3, reduced the degradation of αII-spectrin, and attenuated the necrotic and apoptotic cell death in the penumbra and core. These data demonstrated that preserving the mitochondrial function and blocking the mitochondria-mediated cell death pathway may be one mechanism of taurine's action against brain ischemia.

Topics: Adenosine Triphosphate; Animals; bcl-2-Associated X Protein; bcl-X Protein; Brain Ischemia; Calpain; Caspase 3; Cell Death; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Mitochondria; Nervous System Diseases; Rats; Rats, Sprague-Dawley; Stroke; Taurine

It is demonstrated that the c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Our previous studies have suggested that K252a can obviously inhibit JNK activation induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. Here, we further discussed the potential mechanism of ischemic brain injury induced by the activation of JNK after 15?min of transient global cerebral ischemia. As a result, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and 14-3-3 protein (a cytoplasmic anchor of Bax) induced by the activation of JNK, K252a decreased the release of Bax from Bcl-2/Bax and 14-3-3/Bax dimers, further attenuating the translocation of Bax from cytosol to mitochondria and the release of cytochrome c induced by ischemia/reperfusion, which related to mitochondria-mediated apoptosis. Importantly, pre-infusion of K2525a 20?min before ischemia showed neuroprotective effect against neuronal cells apoptosis. These findings imply that K252a induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 subregion via inhibiting the mitochondrial apoptosis pathway induced by JNK activation.

Topics: 14-3-3 Proteins; Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; CA1 Region, Hippocampal; Carbazoles; Cytochromes c; Enzyme Inhibitors; Indole Alkaloids; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase Kinases; Mitochondria; Mitogen-Activated Protein Kinase Kinase Kinase 11; Neurons; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Signal Transduction

The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.. We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.. We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.

Topics: Animals; bcl-2-Associated X Protein; bcl-Associated Death Protein; Brain Ischemia; Cytochromes c; Enzyme Activation; Glucose; Male; Mitochondria; Models, Biological; Oxygen; Phospholipid Transfer Proteins; Phosphorylation; Protein Kinase C-delta; Protein Phosphatase 2; Protein Transport; Proteomics; Rats; Rats, Sprague-Dawley; Signal Transduction; Substrate Specificity

Neuronal apoptosis following ischemia can be mediated by a caspase-dependent pathway, which involves the mitochondrial release of cytochrome c that initiates a cascade of caspase activation. In addition, there is a caspase-independent pathway, which is mediated by the release of apoptosis-inducing factor (AIF). Using caspase inhibitor gene therapy, we investigated the roles of caspases on the mitochondrial release of cyt c and the release of AIF. Specifically, we used herpes simplex virus-1 amplicon vectors to ectopically express a viral caspase inhibitor (crmA or p35) in mixed cortical cultures exposed to oxygen/glucose deprivation. Overexpression of either crmA or p35 (but not the caspase-3 inhibitor DEVD) inhibited the release of AIF; this suggests that there can be cross-talk between the caspase-dependent and the ostensibly caspase-independent pathway. In addition, both crmA overexpression and DEVD inhibited cyt c release, suggesting a positive feedback loop involving activated caspases stimulating cyt c release.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Brain Ischemia; Caspase 3; Caspase Inhibitors; Caspases; Cell Hypoxia; Cells, Cultured; Cerebral Cortex; Cytochromes c; Disease Models, Animal; Gene Transfer Techniques; Genetic Vectors; Glucose; Herpesvirus 1, Human; Mitochondria; Neurons; Rats; Rats, Sprague-Dawley; Signal Transduction

Acetaminophen, a popular analgesic and antipyretic, has been found to be effective against neuronal cell death in in vivo and in vitro models of neurological disorders. Acute neuronal death has been attributed to loss of mitochondrial permeability transition coupled with mitochondrial dysfunction. The potential impact of acetaminophen on acute injury from cerebral ischemia-reperfusion has not been studied. We investigated the effects of acetaminophen on cerebral ischemia-reperfusion-induced injury using a transient global forebrain ischemia model. Male Sprague-Dawley rats received 15mg/kg of acetaminophen intravenously during ischemia induced by hypovolemic hypotension and bilateral common carotid arterial occlusion, which was followed by reperfusion. Acetaminophen reduced tissue damage, degree of mitochondrial swelling, and loss of mitochondrial membrane potential. Acetaminophen maintained mitochondrial cytochrome c content and reduced activation of caspase-9 and incidence of apoptosis. Our data show that acetaminophen reduces apoptosis via a mitochondrial-mediated mechanism in an in vivo model of cerebral ischemia-reperfusion. These findings suggest a novel role for acetaminophen as a potential stroke therapeutic.

Topics: Acetaminophen; Animals; Apoptosis; Brain; Brain Ischemia; Carotid Artery Diseases; Caspase 9; Cytochromes c; Disease Models, Animal; Hypotension; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Swelling; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors

To determine the effects of diazoxide on oxygen free radicals and cell apoptosis in brain tissue after deep hypothermia cerebral ischemia reperfusion injury in young rats.. Fifty-four 3-week-old Sprague-Dawley rats were randomly and equitably divided into sham-operated group, model group and diazoxide group respectively (n = 18). The model of hypothermia cerebral ischemia reperfusion injury was made. After 24 hours of operation, the brains of rats were removed and preserved. The content of superoxide dismutase (SOD) and malonaldehyde (MDA) in brain tissue were detected. Cytosolic C release of cytochrome was confirmed by Western Blot. The protein expression of Caspase-3 was determined by immunohistochemistry.. In the model group, the content of SOD was (198 +/- 41) U/mg, lower than the sham-operated group's (321 +/- 36) U/mg (P < 0.01). The content of MDA was (212 +/- 21) nmol/mg, was higher than the sham-operated group's (100 +/- 23) nmol/mg (P < 0.01), and the expressions of cytochrome C (0.72 +/- 0.09) and Caspase-3 (83 +/- 10) were all significantly higher than those in the sham-operated group (0.17 +/- 0.02 and 115 +/- 9) (P < 0.01). Compared with the model group, the content of SOD in the diazoxide group [(264 +/- 34) U/mg] was markedly increased (P < 0.05). In addition, diazoxide provided significant reductions in the content of MDA [(174 +/- 19) nmol/mg] and the expressions of cytochrome C (0.41 +/- 0.05) and Caspase-3 (99 +/- 11) (P < 0.05).. The neuroprotective effects of diazoxide against brain injury induced by deep hypothermia cerebral ischemia reperfusion through inhibiting oxygen free radicals and cell apoptosis. Diazoxide may become a new neuroprotective drug after infant complicated congenital cardiac operation.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Caspase 3; Circulatory Arrest, Deep Hypothermia Induced; Cytochromes c; Diazoxide; Disease Models, Animal; Female; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion; Superoxide Dismutase

The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca(2+) and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol Ca(2+) × mg protein(-1). ADP or ATP also prevented Ca(2+)-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K(i)) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca(2+)-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Brain; Brain Ischemia; Calcium; Cytochromes c; Female; Mitochondria; Mitochondrial Swelling; Oxidative Phosphorylation; Rats; Rats, Wistar

Mitochondrial dysfunctions generating from cerebral ischemia-reperfusion exert a potential threat on neuronal cell survival and hence, accelerate the aging process and age dependent neuropathology. Thirty min moderate cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) followed by 30 min reperfusion caused an increased diene production, depleted glutathione (GSH) content, reduced superoxide dismutase (SOD) and catalase activities and pyramidal neuronal loss in young (2 months old) and aged (20 months old) rat brain compared to sham operated controls. Cytidine 5' diphosphocholine (CDP-Choline) is a known neuroprotective drug. CDP-Choline after metabolism in the liver suffers hydrolysis and splits into cytidine and choline before entering systemic circulation and hardly circumvents blood brain barrier (BBB) as such. Previous reports show CDP-Choline liposomes significantly increased in vivo uptake compared to "free drug" administration in cerebral ischemia. To enhance the therapeutic concentration build up in brain we sought to formulate mannosylated liposomal CDP-Choline (MLCDP) utilizing the mannose receptors. We tested the therapeutic supremacy of MLCDP over liposomal CDP-Choline (LCDP) in global moderate cerebral ischemia reperfusion induced neuronal damage. CDP-Choline in MLCDP delivery system was found potent to exert substantial protection against global moderate cerebral ischemia reperfusion induced mitochondrial damage in aged rat brain. Membrane lipid peroxidation, GSSG/GSH ratio and reactive oxygen species (ROS) generation in cerebral tissue were found to be higher in aged, compared to young rat. Further decline of those parameters was observed in aged rat brain by the induction of global moderate cerebral ischemia and reperfusion. MLCDP treatment when compared to free or LCDP treatment prevented global moderate cerebral ischemia-reperfusion induced mitochondrial damage as evident ultra structurally and release of cytochrome c (cyt c) from mitochondria into cytosol and protected mitochondria to restore its normal structure and functions.

Topics: Aging; Analysis of Variance; Animals; Brain; Brain Ischemia; Catalase; Cytidine Diphosphate Choline; Cytochromes c; Disease Models, Animal; Glutathione; Lipid Peroxidation; Liposomes; Male; Mannose; Microscopy, Electron, Transmission; Mitochondria; Nootropic Agents; Rats; Rats, Wistar; Reperfusion; Statistics as Topic; Superoxide Dismutase

We simultaneously measured the diffuse reflectance spectra and transmittance spectra of in vitro rat cerebral cortical tissue slices perfused with artificial cerebrospinal fluid (aCSF) in the wavelength range from 500 to 900 nm. An ischemia-like condition in the cortical tissue was induced by oxygen/glucose deprivation (OGD) of the aCSF. Diffuse reflectance and transmittance of the cortical slices were decreased and increased, respectively, during OGD. Spectral data of reduced scattering coefficients and absorption coefficients were estimated by the inverse Monte Carlo simulation for light transport in tissue. As with OGD, significant decrease of the reduced scattering coefficients and alteration of the absorption coefficient spectrum were observed over the measured wavelength range. The mean maximum amplitudes of change in the absorption coefficient at 520, 550, 605, and 830 nm were 0.33 ± 0.14, 0.30 ± 0.12, 0.30 ± 0.14, and -0.04 ± 0.16, respectively, whereas those in the reduced scattering coefficient at 520, 550, 605, and 830 nm were -0.37 ± 0.08, -0.38 ± 0.08, -0.38 ± 0.08, and -0.39 ± 0.08. Variations in the reduced scattering coefficients implied cell deformation mainly due to cell swelling, whereas those in the absorption spectra indicated reductions in heme aa(3) and CuA in cytochrome c oxidase and cytochrome c.

Topics: Absorption; Animals; Brain Ischemia; Cerebral Cortex; Cerebrospinal Fluid; Cytochromes c; Electron Transport Complex IV; Glucose; Heme; In Vitro Techniques; Light; Male; Models, Biological; Monte Carlo Method; Oxygen; Rats; Rats, Wistar; Scattering, Radiation; Spectrophotometry

Ischemic brain is particularly susceptible to free radicals mediated secondary neuronal damage, especially mitochondrial dysfunctions. Chinese Herbal Medicine with antioxidant properties is believed to have potential therapeutic effect. Leonurine, an alkaloid present in Herba Leonuri (HL), has shown biological effects such as antioxidant, anticoagulant, anti-apoptosis and protection against ischemic heart disease. In this study, neuroprotective effects of leonurine against cerebral ischemia/reperfusion-induced mitochondrial dysfunctions in cortex were evaluated. We used transient rat middle cerebral artery occlusion (MCAO) model of brain ischemia. The rats were treated with their respective treatments for 1 week prior to the MCAO. We found that leonurine significantly improved neurological outcome and reduced ischemia/reperfusion (I/R)-induced cerebral infarction 24 h after MCAO. Leonurine decreased reactive oxygen species (ROS) level in mitochondria isolated from ischemic cortex, which was increased by MCAO. Terminal deoxyuridine triphosphate (dUTP) Nick-End Labeling (TUNEL) staining showed anti-apoptotic effect of leonurine on ischemic cortex. Western blot analysis showed a marked decrease in the expression of Bax and an increase of Bcl-2 as a result of leonurine treatment. The attenuation of mitochondrial membrane swelling, restore of mitochondrial membrane potential and content of cytochrome c (Cyt-C) in mitochondria isolated from ischemic cortex could also be observed in leonurine treated group. The findings of this study suggest that leonurine has promising therapeutic effect for ischemic stroke treatment through antioxidant and anti-apoptotic mechanisms.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Brain Ischemia; Cerebral Cortex; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Drugs, Chinese Herbal; Gallic Acid; In Situ Nick-End Labeling; Leonurus; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Neuroprotective Agents; Oxidative Stress; Phytotherapy; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

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

Global ischemia was induced in gerbil by bilateral occlusion of the common carotid arteries for 5 min. Sodium ionophore monensin or sodium channel blocker tetrodotoxin (TTX) was administered at doses of 10 micorg/kg, i.p., 30 min before ischemia induction; the dose was repeated after 22 hr. Subsequently, brain infarct occurred, determined at 24 hr after occlusion. Large, well-demarcated infarcts were observed in both hemispheres, an important observation because it critically influences the interpretation of the data. Because nitric oxide (NO) production is thought to be related to ischemic neuronal damage, we examined increases in Ca(2+) influx, which lead to the activation of nitric oxide synthase (NOS). Then we evaluated the contributions of neuronal NOS, endothelial NOS, and inducible NOS to NO production in brain cryosections. The cytosolic release of apoptogenic molecules like cytochrome c and p53 were confirmed after 24 hr of reflow. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) labeling detected the apoptotic cells, which were confirmed in neuron-rich cell populations. After 24 hr, all the ischemic changes were amplified by monensin and significantly attenuated by TTX treatment. Additionally, the nesting behavior and histological outcomes were examined after 7 day of reflow. The neuronal damage in the hippocampal area and significant decrease in nesting scores were observed with monensin treatment and reduced by TTX pretreatment after day 7 of reflow. To our knowledge, this report is the first to highlight the involvement of the voltage-sensitive Na(+) channel in possibly regulating in part NO system and apoptosis in a cytochrome c-dependent manner in global ischemia in the gerbil, and thus warrants further investigation.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Calcium; Cytochromes c; DNA Fragmentation; Endothelial Cells; Gerbillinae; Hippocampus; In Situ Nick-End Labeling; Ionophores; Male; Monensin; Neurons; Nitric Oxide Synthase; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Tumor Suppressor Protein p53

Astaxanthin (ATX) is a dietary carotenoid of crustaceans and fish that contributes to their coloration. Dietary ATX is important for development and survival of salmonids and crustaceans and has been shown to reduce cardiac ischemic injury in rodents. The purpose of this study was to examine whether ATX can protect against ischemic injury in the mammalian brain. Adult rats were injected intracerebroventricularly with ATX or vehicle prior to a 60-min middle cerebral artery occlusion (MCAo). ATX was present in the infarction area at 70-75 min after onset of MCAo. Treatment with ATX, compared to vehicle, increased locomotor activity in stroke rats and reduced cerebral infarction at 2 d after MCAo. To evaluate the protective mechanisms of ATX against stroke, brain tissues were assayed for free radical damage, apoptosis, and excitoxicity. ATX antagonized ischemia-mediated loss of aconitase activity and reduced glutamate release, lipid peroxidation, translocation of cytochrome c, and TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as body temperature, brain temperature, cerebral blood flow, blood gases, blood pressure, and pH. Collectively, our data suggest that ATX can reduce ischemia-related injury in brain tissue through the inhibition of oxidative stress, reduction of glutamate release, and antiapoptosis. ATX may be clinically useful for patients vulnerable or prone to ischemic events.

Topics: Aconitate Hydratase; Animals; Behavior, Animal; Brain Injuries; Brain Ischemia; Cerebrovascular Circulation; Crustacea; Cytochromes c; Diet; Glutamic Acid; Humans; In Situ Nick-End Labeling; Lipid Peroxidation; Male; Molecular Structure; Motor Activity; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Xanthophylls

The identification of a neuroprotective drug for stroke remains elusive. Given that mitochondria play a key role both in maintaining cellular energetic homeostasis and in triggering the activation of cell death pathways, we evaluated the efficacy of newly identified inhibitors of cytochrome c release in hypoxia/ischemia induced cell death. We demonstrate that methazolamide and melatonin are protective in cellular and in vivo models of neuronal hypoxia.. The effects of methazolamide and melatonin were tested in oxygen/glucose deprivation-induced death of primary cerebrocortical neurons. Mitochondrial membrane potential, release of apoptogenic mitochondrial factors, pro-IL-1beta processing, and activation of caspase -1 and -3 were evaluated. Methazolamide and melatonin were also studied in a middle cerebral artery occlusion mouse model. Infarct volume, neurological function, and biochemical events were examined in the absence or presence of the 2 drugs.. Methazolamide and melatonin inhibit oxygen/glucose deprivation-induced cell death, loss of mitochondrial membrane potential, release of mitochondrial factors, pro-IL-1beta processing, and activation of caspase-1 and -3 in primary cerebrocortical neurons. Furthermore, they decrease infarct size and improve neurological scores after middle cerebral artery occlusion in mice.. We demonstrate that methazolamide and melatonin are neuroprotective against cerebral ischemia and provide evidence of the effectiveness of a mitochondrial-based drug screen in identifying neuroprotective drugs. Given the proven human safety of melatonin and methazolamide, and their ability to cross the blood-brain-barrier, these drugs are attractive as potential novel therapies for ischemic injury.

Topics: Animals; Antioxidants; Blotting, Western; Brain Ischemia; Carbonic Anhydrase Inhibitors; Caspase 1; Caspase 3; Cell Death; Cytochromes c; Enzyme Activation; In Situ Nick-End Labeling; Interleukin-1beta; L-Lactate Dehydrogenase; Melatonin; Membrane Potentials; Methazolamide; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Membranes; Neurodegenerative Diseases; Neurons; Neuroprotective Agents

Our previous studies showed that the assembly of the GluR6-PSD95-mixed lineage kinase 3 (MLK3) signaling module played an important role in rat ischemic brain injury. In this study, we aimed to elucidate whether ischemic preconditioning could downregulate the assembly of the GluR6-PSD95-MLK3 signaling module and suppress the activation of MLK3, MKK4/7, and c-Jun N-terminal kinase (JNK). As a result, ischemic preconditioning could not only inhibit the assembly of the GluR6-PSD95-MLK3 signaling module, diminish the phosphorylation of the transcription factor c-Jun, downregulate Fas ligand expression, attenuate the phosphorylation of 14-3-3 and Bcl-2 and the translocation of Bax to mitochondria, but also increase the release of cytochrome c and the activation of caspase-3. In contrast, both GluR6 antisense ODNs (oligodeoxynucleotides) and 6,7,8,9-tetrahydro-5-nitro-1 H-benz[g]indole-2,3-dione-3-oxime (NS102), an antagonist of GluR6 receptor, prevented the above effects of preconditioning, which shows that suppressing the expression of GluR6 or inhibiting GluR6 activity contributes negatively to preconditioning-induced ischemia tolerance. Taken together, our results indicate that preconditioning can inhibit the over-assembly of the GluR6-PSD95-MLK3 signaling module and the JNK3 activation. GluR6 subunit-containing kainite receptors play an important role in the preconditioning-induced neuronal survival and provide new insight into stroke therapy.

Topics: 14-3-3 Proteins; Animals; Apoptosis; Brain Ischemia; Caspase 3; Cell Nucleus; Cytochromes c; Disks Large Homolog 4 Protein; Enzyme Activation; Fas Ligand Protein; GluK2 Kainate Receptor; Hippocampus; Intracellular Signaling Peptides and Proteins; Ischemic Preconditioning; Male; MAP Kinase Kinase Kinases; Membrane Proteins; Mitogen-Activated Protein Kinase 10; Mitogen-Activated Protein Kinase Kinase Kinase 11; Mitogen-Activated Protein Kinase Kinases; Neurons; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid; Reperfusion Injury; Signal Transduction

Ghrelin is known to promote neuronal defense and survival against ischemic injury by inhibiting apoptotic processes. In the present study, we investigated the role of prostate apoptosis response-4 (Par-4), a proapoptotic gene the expression of which is increased after ischemic injury, in ghrelin-mediated neuroprotection during middle cerebral artery occlusion (MCAO). Both ghrelin and des-acyl ghrelin protected cortical neurons from ischemic injury. Ghrelin receptor specific antagonist abolished the protective effects of ghrelin, whereas those of des-acyl ghrelin were preserved, suggesting the involvement of a receptor that is distinct from GHS-R1a. The expression of Par-4 was increased by MCAO, which was attenuated by ghrelin and des-acyl ghrelin treatments. Both ghrelin and des-acyl ghrelin increased the Bcl-2/Bax ratio, prevented cytochrome c release, and inhibited caspase-3 activation. Our data indicate that des-acyl ghrelin, as well as ghrelin, protect cortical neurons against ischemic injury through the inhibition of Par-4 expression and apoptotic molecules in mitochondrial pathway.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Brain Ischemia; Caspase 3; Cytochromes c; Ghrelin; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, Ghrelin; Reperfusion Injury

Asiatic acid, a triterpenoid derivative from Centella asiatica, has shown biological effects such as antioxidant, antiinflammatory, and protection against glutamate- or beta-amyloid-induced neurotoxicity. We investigated the neuroprotective effect of asiatic acid in a mouse model of permanent cerebral ischemia. Various doses of asiatic acid (30, 75, or 165 mg/kg) were administered orally at 1 hr pre- and 3, 10, and 20 hr postischemia, and infarct volume and behavioral deficits were evaluated at day 1 or 7 postischemia. IgG (blood-brain barrier integrity) and cytochrome c (apoptosis) immunostaining was carried out at 24 hr postischemia. The effect of asiatic acid on stress-induced cytochrome c release was examined in isolated mitochondrial fractions. Furthermore, its effects on cell viability and mitochondrial membrane potential were studied in HT-22 cells exposed to oxygen-glucose deprivation. Asiatic acid significantly reduced the infarct volume by 60% at day 1 and by 26% at day 7 postischemia and improved neurological outcome at 24 hr postischemia. Our studies also showed that the neuroprotective properties of asiatic acid might be mediated in part through decreased blood-brain barrier permeability and reduction in mitochondrial injury. The present study suggests that asiatic acid may be useful in the treatment of cerebral ischemia.

Topics: Animals; Brain; Brain Ischemia; Cell Hypoxia; Cell Line; Cell Survival; Cytochromes c; Disease Models, Animal; Glucose; Immunoglobulin G; Infarction, Middle Cerebral Artery; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mitochondria; Neuroprotective Agents; Pentacyclic Triterpenes; Severity of Illness Index; Time Factors; Treatment Outcome; Triterpenes

Apoptosis arises from neuronal damage following an ischemic insult. Apoptosis-inducing factor (AIF) is a protein released from mitochondria in response to pro-apoptotic signals which then translocates to the nucleus and triggers DNA fragmentation. In parallel with this, pro-apoptotic signals cause the release of cytochrome c from mitochondria, activating caspase-dependent apoptosis. During post-ischemic reperfusion, reactive oxygen species (ROS) are formed in excess in mitochondria and can play a role in initiating apoptosis. In cultures, ROS are formed during post oxygen glucose deprivation (OGD) normoxia/normoglycemia that is used as a model for ischemia. In this study, we delivered viral vectors to overexpress antioxidants (GPX, catalase, CuZnSOD, or MnSOD) in mixed cortical cultures, in order to investigate the effects of ROS-reduction on the release of cytochrome c and AIF. Overexpression of MnSOD, CuZnSOD, catalase or GPX all prevented AIF translocation from mitochondria to the nucleus. Potentially, this could reflect broadly non-specific protection due to reducing ROS load. Arguing against this, overexpression of the same antioxidants did not inhibit cytochrome c release. These findings suggest a specific interaction between ROS formation and the caspase-independent route of apoptosis.

Topics: Animals; Apoptosis Inducing Factor; Brain Ischemia; Catalase; Cell Nucleus; Cells, Cultured; Coculture Techniques; Copper; Cytochromes c; Genetic Vectors; Glutathione Peroxidase; Herpesvirus 1, Human; Manganese; Mitochondria; Protein Transport; Rats; Reactive Oxygen Species; Superoxide Dismutase; Zinc

Cerebral ischemia and reperfusion increase superoxide anions (O(2)(*-)) in brain mitochondria. Manganese superoxide dismutase (Mn-SOD; SOD2), a primary mitochondrial antioxidant enzyme, scavenges superoxide radicals and its overexpression provides neuroprotection. However, the regulatory mechanism of Mn-SOD expression during cerebral ischemia and reperfusion is still unclear. In this study, we identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse Mn-SOD gene, and elucidated the mechanism of O(2)(*-) overproduction after transient focal cerebral ischemia (tFCI). We found that Mn-SOD expression is significantly reduced by reperfusion in the cerebral ischemic brain. We also found that activated STAT3 is usually recruited into the mouse Mn-SOD promoter and upregulates transcription of the mouse Mn-SOD gene in the normal brain. However, at early postreperfusion periods after tFCI, STAT3 was rapidly downregulated, and its recruitment into the Mn-SOD promoter was completely blocked. In addition, transcriptional activity of the mouse Mn-SOD gene was significantly reduced by STAT3 inhibition in primary cortical neurons. Moreover, we found that STAT3 deactivated by reperfusion induces accumulation of O(2)(*-) in mitochondria. The loss of STAT3 activity induced neuronal cell death by reducing Mn-SOD expression. Using SOD2-/+ heterozygous knock-out mice, we found that Mn-SOD is a direct target of STAT3 in reperfusion-induced neuronal cell death. Our study demonstrates that STAT3 is a novel transcription factor of the mouse Mn-SOD gene and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the mouse brain.

Topics: Animals; Brain; Brain Infarction; Brain Ischemia; Cells, Cultured; Chromatin Immunoprecipitation; Cytochromes c; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; Glucose; Humans; Hypoxia; Interleukin-6; Male; Mice; Mice, Knockout; Neurons; Neuroprotective Agents; Reperfusion; RNA, Small Interfering; STAT3 Transcription Factor; Superoxide Dismutase; Time Factors; Transfection; Tyrphostins; Up-Regulation

The critical event of the intrinsic pathway of apoptosis following transient global brain ischemia is the release of cytochrome c from the mitochondria. In vitro studies have shown that insulin can signal specifically via phosphatidylinositol-3-OH-kinase (PI3-K) and Akt to prevent cytochrome c release. Therefore, insulin may exert its neuroprotective effects during brain reperfusion by blocking cytochrome c release. We hypothesized that insulin acts through PI3-K, Akt, and Bcl-2 family proteins to inhibit cytochrome c release following transient global brain ischemia. We found that a single bolus of insulin given immediately upon reperfusion inhibited cytochrome c release for at least 24 h, and produced a fivefold improvement in neuronal survival at 14 days. Moreover, insulin's ability to inhibit cytochrome c release was completely dependent on PI3-K signaling and insulin induces phosphorylation of Akt through PI3-K. In untreated animals, there was an increase in mitochondrial Bax at 6 h of reperfusion, and Bax binding to Bcl-X(L) was disrupted at the mitochondria. Insulin prevented both these events in a PI3-K-dependent manner. In summary, insulin regulates cytochrome c release through PI3-K likely by activating Akt, promoting the binding between Bax and Bcl-X(L), and by preventing Bax translocation to the mitochondria.

Topics: Animals; bcl-2-Associated X Protein; bcl-X Protein; Brain Ischemia; Cytochromes c; Insulin; Male; Phosphatidylinositol 3-Kinases; Protein Binding; Rats; Rats, Long-Evans; Reperfusion Injury; Signal Transduction

Ischemic postconditioning (Postcond) is defined as a series of intermittent interruptions of blood flow in the early phase of reperfusion that mechanically alters the hydrodynamics of reperfusion. A recent study showed that Postcond reduced infarct size in cerebral ischemia/reperfusion (I/R) injury. However, little is known about the mechanisms of Postcond in cerebral I/R injury. In the present study, we investigated the effects of Postcond in focal cerebral I/R injury in the rat middle cerebral artery occlusion model.. Adult male Sprague-Dawley rats were treated with Postcond after 60 minutes of occlusion (beginning of reperfusion). Neurologic scores and infarct volumes were assessed at 24 and 72 hours. Oxidative stress was evaluated by malondialdehyde assay, and apoptosis-related molecules were studied by Western blotting.. Postcond treatment upregulated Bcl-2 and heat-shock protein 70 expression and downregulated cytochrome c release to the cytosol, Bax translocation to the mitochondria, and caspase-3 activity. Postcond treatment also reduced infarct volumes and oxidative stress levels and improved neurologic scores compared with the I/R-only group.. These findings indicate that Postcond inhibits focal cerebral I/R injury. This neuroprotective effect is likely achieved by antiapoptotic mechanisms.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspase 3; Cytochromes c; Disease Models, Animal; Down-Regulation; HSP70 Heat-Shock Proteins; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Ischemic Preconditioning; Male; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Up-Regulation

We investigated mechanisms underlying the Na+/H+ exchanger isoform 1 (NHE1)-mediated neuronal damage in transient focal ischemia. Physiological parameters, body and tympanic temperatures, and regional cerebral blood flow during 30 min of middle cerebral artery occlusion were similar in wild-type NHE1 (NHE1+/+) and NHE1 heterozygous (NHE1+/-) mice. NHE1+/+ mice developed infarct volume of 57.3 +/- 8.8 mm(3) at 24 h reperfusion (Rp), which progressed to 86.1 +/- 10.0 mm(3) at 72 h Rp. This delayed cell death was preceded by release of mitochondrial cytochrome c (Cyt. C), nuclear translocation of apoptosis-inducing factor (AIF), activation of caspase-3, and TUNEL-positive staining and chromatin condensation in the ipsilateral hemispheres of NHE1+/+ brains. In contrast, NHE1+/- mice had a significantly smaller infarct volume and improved neurological function. A similar neuroprotection was obtained with NHE1 inhibitor HOE 642. The number of apoptotic cells, release of AIF and Cyt. C or levels of active caspase-3 was significantly reduced in NHE1+/- brains. These data imply that NHE1 activity may contribute to ischemic apoptosis. Ischemic brains did not exhibit changes of NHE1 protein expression. In contrast, up-regulation of NHE1 expression was found in NHE1+/+ neurons after in vitro ischemia. These data suggest that NHE1 activation following cerebral ischemia contributes to mitochondrial damage and ischemic apoptosis.

Topics: Animals; Apoptosis; Astrocytes; Brain; Brain Ischemia; Caspase 3; Cation Transport Proteins; Cell Nucleus; Cells, Cultured; Cerebrovascular Circulation; Cytochromes c; Gene Silencing; Hypoxia-Inducible Factor 1, alpha Subunit; In Situ Nick-End Labeling; Mice; Mice, Transgenic; Mitochondria; Neurons; Protein Isoforms; Regional Blood Flow; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers

Ischemic postconditioning has been found to decrease brain infarct area and spinal cord ischemic injury. In this study, we tested the hypothesis that ischemic postconditioning reduces global cerebral ischemia/reperfusion-induced structural and functional injury in rats.. Ten-minute global ischemia was induced by 4-vessel occlusion in male Sprague-Dawley rats. The animals underwent postconditioning consisting of 3 cycles of 15-second/15-second (Post-15/15), 30-second/30-second (Post-30/30), or 60-second/15-second (Post-60/15) reperfusion/reocclusion or 15-second/15-second reperfusion/reocclusion applied after a 45-second reperfusion (Post-45-15/15).. Ten minutes of ischemia and 7 days of reperfusion destroyed 85.8% of CA1 hippocampal neurons and 64.1% of parietal cortical neurons. Three cycles of Post-15/15, Post-30/30, and Post-45-15/15 reperfusion/reocclusion markedly reduced neuronal loss after 7 days or 3 weeks of reperfusion and diminished the deficiency in spatial learning and memory. After reperfusion, a period of hyperperfusion followed by hypoperfusion was observed, both of which were blocked by postconditioning. The cytosolic level of cytochrome c increased significantly after 48 hours of reperfusion, and this was inhibited by Post-15/15, Post-30/30, and Post-45-15/15. However, 3 cycles of 60-second/15-second reperfusion/reocclusion failed to protect against neuronal damage, behavioral deficit, or cytochrome c translocation.. Our data provide the first evidence that an appropriate ischemic postconditioning strategy has neuroprotective effects against global cerebral ischemia/reperfusion injury and a consequent behavioral deficit and that these protective effects are associated with its ability to improve disturbed cerebral blood flow and prevent cytochrome c translocation.

Topics: Animals; Brain Ischemia; Cell Count; Cerebrovascular Circulation; Cytochromes c; Cytoprotection; Cytosol; Hippocampus; Ischemic Preconditioning; Male; Maze Learning; Memory; Neurons; Parietal Lobe; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Swimming; Time Factors

We and others have shown that prior exposure to the volatile anesthetic isoflurane induces ischemic tolerance in the brain. Our results also suggest that isoflurane preconditioning reduces cell apoptosis in the penumbral region of rat brain. We designed this study to determine whether isoflurane preconditioning decreased mitochondria-dependent cell apoptosis. Adult male Sprague-Dawley rats were exposed to or not exposed to 2% isoflurane for 30 min at 24 h before the permanent middle cerebral arterial occlusion. Western blotting was used to quantify protein expression in the cytosolic and mitochondrial fractions of non-ischemic brain cortex and brain cortex in the ischemic core and penumbra. Isoflurane preconditioning significantly decreased the infarct volume of cerebral cortex and improved neurological outcome. Isoflurane increased the expression of the antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins in the cerebral cortex of rats without brain ischemia. Rats preconditioned with isoflurane before brain ischemia had increased Bcl-2 expression in the penumbra. Isoflurane preconditioning reduced the release of cytochrome c from the mitochondria and the activation of caspase 3 in the penumbra. However, isoflurane preconditioning did not alter the translocation of Bid and Bax from the cytosol to the mitochondria, identified mechanisms for Bcl-2 to block the release of cytochrome c from the mitochondria. Our results suggest that isoflurane preconditioning increases Bcl-2 expression to block the release of cytochrome c from the mitochondria to decrease the cell apoptosis in the penumbra.

Topics: Anesthetics, Inhalation; Animals; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Brain; Brain Ischemia; Caspase 3; Cell Line, Tumor; Cytochromes c; Cytosol; Enzyme Activation; Gene Expression; Genes, bcl-2; Humans; Infarction, Middle Cerebral Artery; Isoflurane; Male; Mitochondria; Neuroprotective Agents; Psychomotor Performance; Rats; Rats, Sprague-Dawley

Although cap-dependent translation initiation is the prevalent mode of ribosome binding to mRNAs in eukaryotes, some mRNAs exhibit the ability to bypass the requirement for the cap structure. The translation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA is controlled by an internal ribosome entry site (IRES) element, which requires the interaction of the heterogeneous nuclear ribonucleoprotein C1-C2 (hnRNP-C1/C2). We analyze, at the protein level, the time course and distribution of XIAP and hnRNP-C1/C2 upon ischemia in mice or staurosporine (STP)-induced apoptosis in HT22 cells. Both ischemia and STP induced a parallel upregulation of XIAP and hnRNP-C1/C2 protein levels in the penumbra and in HT22 cells. These results suggest that the increased levels of hnRNP C1/C2 may modulate XIAP translation, probably by interacting with the XIAP-IRES. The up-regulation of hnRNP-C1/C2 may foster the synthesis of XIAP as a protective pathway by which neurons try to counteract the initial deleterious effects of apoptosis.

Topics: Animals; Apoptosis; Brain Ischemia; Caspase 3; Cell Line; Cell Survival; Cytochromes c; Enzyme Inhibitors; Gene Expression Regulation; Heterogeneous-Nuclear Ribonucleoprotein Group C; Male; Mice; Mice, Inbred C57BL; Neurons; Staurosporine; X-Linked Inhibitor of Apoptosis Protein

Oxidative stress is believed to contribute to neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. The present study was undertaken to evaluate the possible antioxidant neuroprotective effect of genistein against neuronal death in hippocampal CA1 neurons following transient global cerebral ischemia in the rat. Transient global cerebral ischemia was induced in male Sprague-Dawley rats by four-vessel-occlusion for 10min. At various times of reperfusion, the histopathological changes and the levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA), cytosolic cytochrome c and caspase-3 activity in hippocampus were measured. We found extensive neuronal death in the CA1 region at day 5 after I/R. The ischemic changes were preceded by increases in ROS generation and MDA concentration and followed by increased cytosolic cytochrome c, and subsequently caspase-3 activation and apoptosis. Treatment with genistein (15mg/kg, i.p.) significantly attenuated ischemia-induced neuronal death. Genistein administration also decreased ROS generation, MDA concentration and the apoptotic indices. These results suggest that genistein protects neurons from transient global cerebral I/R injury in rat hippocampus by attenuating oxidative stress, lipid peroxidation and the signaling cascade leading to apoptotic cell death.

Topics: Animals; Antioxidants; Apoptosis; Brain Ischemia; Caspase 3; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Down-Regulation; Genistein; Lipid Peroxidation; Male; Malondialdehyde; Nerve Degeneration; Neuroprotective Agents; Oxidative Stress; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Treatment Outcome

Ghrelin is an endogenous ligand for the GH secretagogue receptor, produced and secreted mainly from the stomach. Ghrelin stimulates GH release and induces positive energy balances. Previous studies have reported that ghrelin inhibits apoptosis in several cell types, but its antiapoptotic effect in neuronal cells is unknown. Therefore, we investigated the role of ghrelin in ischemic neuronal injury using primary hypothalamic neurons exposed to oxygen-glucose deprivation (OGD). Here we report that treatment of hypothalamic neurons with ghrelin inhibited OGD-induced cell death and apoptosis. Exposure of neurons to ghrelin caused rapid activation of ERK1/2. Ghrelin-induced activation of ERK1/2 and the antiapoptotic effect of ghrelin were blocked by chemical inhibition of MAPK, phosphatidylinositol 3 kinase, protein kinase C, and protein kinase A. Ghrelin attenuated OGD-induced activation of c-Jun NH2-terminal kinase and p-38 but not ERK1/2. We also investigated ghrelin regulation of apoptosis at the mitochondrial level. Ghrelin protected cells from OGD insult by inhibiting reactive oxygen species generation and stabilizing mitochondrial transmembrane potential. In addition, ghrelin-treated cells showed an increased Bcl-2/Bax ratio, prevention of cytochrome c release, and inhibition of caspase-3 activation. Finally, in vivo administration of ghrelin significantly reduced infarct volume in an animal model of ischemia. Our data indicate that ghrelin may act as a survival factor that preserves mitochondrial integrity and inhibits apoptotic pathways.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspase 3; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cytochromes c; Disease Models, Animal; Ghrelin; Glucose; Hypothalamus; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Oxygen; Peptide Hormones; Phosphatidylinositol 3-Kinases; Protein Kinase C; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, G-Protein-Coupled; Receptors, Ghrelin; RNA, Messenger

The proapoptotic B-cell lymphoma-2 family protein Bax is a key regulatory point in the intrinsic apoptotic pathway. However, the factors controlling the process of Bax activation and translocation to mitochondria have yet to be fully identified and characterized. We performed affinity chromatography using peptides corresponding to the mitochondrial-targeting region of Bax, which is normally sequestered within the inactive structure. The molecular chaperone nucleophosmin was identified as a novel Bax-binding protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Reciprocal co-immunoprecipitation and proximity assays confirmed the Bax-nucleophosmin protein-protein interaction and verified that nucleophosmin only bound to activated conformationally altered Bax. Confocal microscopy in a cell-based apoptosis model, demonstrated that nucleophosmin translocation from nucleolus to cytosol preceded Bax movement. Specific knockdown of nucleophosmin expression using RNAi attenuated apoptosis as measured by mitochondrial cytochrome c release and activation of the caspase cascade. In a mouse model of ischaemic stroke, subcellular fractionation studies verified that nucleophosmin translocation occurred within 3 h, at a time before Bax translocation but after Bax conformational changes have occurred. Thus, we have elucidated a novel molecular mechanism whereby Bax becomes activated and translocates to the mitochondria to orchestrate mitochondrial dysfunction and apoptotic cell death, which opens new avenues for therapeutic intervention.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspases; Cell Nucleolus; Chromatography, Affinity; Cytochromes c; Cytosol; Humans; Immunoprecipitation; Male; Mice; Mitochondria; Molecular Chaperones; Neuroblastoma; Nuclear Proteins; Nucleophosmin; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tumor Cells, Cultured

JNK signaling pathway is activated and involved in the selective neuronal death in the hippocampal CA1 subfield following cerebral ischemia. However, little is known about upstream partner controlling the pathway. Here we reported that ischemia/reperfusion significantly elevated Cdc42 activity, enhanced assembly of the Cdc42-MLK3 complex and activation of JNK pathway. Most importantly, knock-down endogenous Cdc42 selectively suppressed the MLK3/MKK7/JNK3 cascade, and subsequently blocked the phosphorylation of c-Jun and FasL expression. Meanwhile, Bcl-2 was inactivated and the release of cytochrome c was diminished. These alterations eventually perturbed the caspase-3 activation as well as post-ischemic neuronal cell death. Taken together, our findings strongly suggest that Cdc42 serves as an upstream activator and modulates JNK-mediated apoptosis machinery in vivo, which ultimately results in neuronal apoptosis via nuclear and non-nuclear pathways. Thus, Cdc42 may be a potential therapeutic target in ischemic brain injury.

Topics: Animals; Apoptosis; Brain Ischemia; Caspase 3; cdc42 GTP-Binding Protein; Cells, Cultured; Cytochromes c; Down-Regulation; Enzyme Activation; Hippocampus; Male; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 10; Mitogen-Activated Protein Kinase Kinase Kinase 11; Neurons; Oligonucleotides, Antisense; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion

Mild hypothermia is a robust neuroprotective treatment for stroke. Understanding the mechanisms underlying hypothermia's benefits will lead to more effective treatments to prevent stroke damage. Delta protein kinase C (deltaPKC) is a kinase that has been strongly implicated in executing ischemic damage. We investigated the effects of hypothermia on deltaPKC activation, as determined by its subcellular translocation, proteolytic cleavage, and phosphorylation in a focal cerebral ischemia model. The amount of constitutively activated C-terminal catalytic fragment of deltaPKC (CF-deltaPKC) increased after stroke. Both hypothermia (30 degrees C) and the caspase-3-specific inhibitor, Z-DQMD-FMK, blocked the accumulation of activated deltaPKC in the penumbra. Other hallmarks of deltaPKC activation, its translocation to the mitochondria, and nucleus were observed in the penumbra as early as 10 mins after reperfusion. These events were blocked by hypothermia. Hypothermia also blocked CF-deltaPKC increases in the mitochondria and nuclei. Conversely, a specific deltaPKC activator, psideltaRACK, decreased the neuroprotective effect of hypothermia. Finally, deltaPKC activity may lead to mitochondrial injury and cytochrome c release, as the timing of cytochrome c release corresponded to the time course of deltaPKC translocation. Both cytochrome c release and deltaPKC translocation were blocked by hypothermia. In conclusion, hypothermia protects against ischemic damage in part by suppressing deltaPKC activation after stroke.

Topics: Active Transport, Cell Nucleus; Animals; Brain Ischemia; Caspase Inhibitors; Cell Membrane; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Hypothermia; Isoenzymes; Male; Mitochondria; Phosphorylation; Protein Kinase C-delta; Rats; Rats, Sprague-Dawley; Receptors for Activated C Kinase; Receptors, Cell Surface

The actin-modulating protein Wiskott-Aldrich syndrome protein verprolin homologous-1 (WAVE1) and a novel CNS-specific protein, pancortin, are highly enriched in adult cerebral cortex, but their functions are unknown. Here we show that WAVE1 and pancortin-2 interact in a novel cell death cascade in adult, but not embryonic, cerebral cortical neurons. Focal ischemic stroke induces the formation of a protein complex that includes pancortin-2, WAVE1, and the anti-apoptotic protein Bcl-xL. The three-protein complex is associated with mitochondria resulting in increased association of Bax with mitochondria, cytochrome c release, and neuronal apoptosis. In pancortin null mice generated using a Cre-loxP system, ischemia-induced WAVE1-Bcl-xL interaction is diminished, and cortical neurons in these mice are protected against ischemic injury. Thus, pancortin-2 is a mediator of ischemia-induced apoptosis of neurons in the adult cerebral cortex and functions in a novel mitochondrial/actin-associated protein complex that sequesters Bcl-xL.

Topics: Animals; bcl-X Protein; Blotting, Western; Brain Ischemia; Cell Death; Cells, Cultured; Cerebral Cortex; Cytochromes c; Embryo, Mammalian; Extracellular Matrix Proteins; Functional Laterality; Glycoproteins; Green Fluorescent Proteins; Immunohistochemistry; Immunoprecipitation; Male; Mice; Mice, Knockout; Mitochondria; Neurons; Rats; Rats, Wistar; Time Factors; Wiskott-Aldrich Syndrome Protein Family

Cerebral ischemia followed by reperfusion activates numerous pathways that lead to cell death. One such pathway involves the release of large quantities of the excitatory amino acid glutamate into the synapse and activation of N-methyl-D-aspartate receptors. This causes an increase in mitochondrial calcium levels ([Ca(2+)](m)) and a production of reactive oxygen species (ROS), both of which may induce the mitochondrial permeability transition (MPT). As a consequence, there is eventual mitochondrial failure culminating in either apoptotic or necrotic cell death. Thus, agents that inhibit MPT might prove useful as therapeutic interventions in cerebral ischemia. In this study, we have investigated the neuroprotective efficacy of the novel compound NIM811. Similar in structure of its parent compound cyclosporin A, NIM811 is a potent inhibitor of the MPT. Unlike cyclosporin A, however, it is essentially void of immunosuppressive actions, allowing the role of MPT to be clarified in ischemia/reperfusion injury. The results of these studies demonstrate that NIM811 provides almost 40% protection in a model of transient focal cerebral ischemia. This was associated with a nearly 10% reduction in mitochondrial reactive species formation and 34% and 38% reduction of cytochrome c release in core and penumbra, respectively. Treatment with NIM811 also increased calcium retention capacity by approximately 20%. Interestingly, NIM811 failed to improve ischemia-induced impairment of bioenergetics. The neuroprotective effects of NIM811 were not due to drug-induced alterations in cerebral perfusion after ischemia. Activation of MPT appears to be an important process in ischemia/reperfusion injury and may be a therapeutic target.

Topics: Animals; Brain Infarction; Brain Ischemia; Calcium Signaling; Cell Death; Cyclosporine; Cytochromes c; Disease Models, Animal; Energy Metabolism; Male; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Inbred SHR; Reactive Oxygen Species; Treatment Outcome

Neuroprotection exerted by 17beta-estradiol (17beta-E(2)) has been widely investigated in animal models of acute cerebral ischemia. Estrogens interact with intracellular receptors (ERalpha and ERbeta) to modulate the transcription of target genes, including those implicated in neuronal survival. Neuroprotection may also occur via interaction with ER-like membrane receptors mediating rapid, non-genomic, actions or via receptor-independent mechanisms. There is also evidence that blockade of inflammatory factors may represent an important mechanism involved in estrogenic neuroprotection. Here we investigate whether reduced brain damage by acute pharmacological treatment with 17beta-E(2) in male rats subjected to transient (2h) middle cerebral artery occlusion (tMCAo) involves modulation of interleukin-1beta (IL-1beta), a proinflammatory cytokine strongly implicated in the pathophysiology of ischemic stroke. Administration of 17beta-E(2) (0.2mg/kg, i.p., 1h before tMCAo) results in significant reduction of brain infarct volume, and this is reverted by the ER antagonist ICI 182,780 (0.25mg/kg, i.p.) administered 1h before 17beta-E(2). Two hours MCAo followed by 2-h reperfusion results in a significant, threefold increase of IL-1beta levels in the cortical tissue ipsilateral to the ischemic damage. Interestingly, a pretreatment with a neuroprotective dose of 17beta-E(2) attenuates the cytokine elevation and this appears to occur through ER activation. In addition, neuroprotection by 17beta-E(2) is accompanied by reduced cytochrome c translocation both in the striatum and in the cortex as revealed by Western blotting 3h after reperfusion. In conclusion, we report the original observation that neuroprotection exerted by 17beta-E(2) in a rat model of transient focal brain ischemia is accompanied by reduced cytochrome c translocation to the cytosol and involves early modulation of IL-1beta production.

Topics: Animals; Blotting, Western; Brain Ischemia; Cytochromes c; Cytosol; Enzyme-Linked Immunosorbent Assay; Estradiol; Estrogen Antagonists; Fulvestrant; Infarction, Middle Cerebral Artery; Interleukin-1beta; Male; Neuroprotective Agents; Rats; Rats, Wistar; Receptors, Estrogen; Subcellular Fractions

We demonstrate that X chromosome-linked inhibitor of apoptosis protein (XIAP) counteracts oxidative stress in two essentially different disease-related models of brain injury, hypoxia-ischemia and irradiation, as judged by lower expression of nitrotyrosine (5-fold) and 4-hydroxy-2-nonenal (10-fold) in XIAP-overexpressing compared with wild-type mice. XIAP overexpression induced up-regulation of at least three antioxidants residing in mitochondria, superoxide dismutase 2, thioredoxin 2 and lysine oxoglutarate reductase. Cytochrome c release from mitochondria was reduced in XIAP-overexpressing mice. Hence, in addition to blocking caspases, XIAP can regulate reactive oxygen species in the brain, at least partly through up-regulation of mitochondrial antioxidants. XIAP-induced prevention of oxidative stress was not secondary to tissue protection because although XIAP overexpression provides tissue protection after hypoxia-ischemia, it does not prevent tissue loss after irradiation. This is a previously unknown role of XIAP and may provide the basis for development of novel protective strategies for both acute and chronic neurodegenerative diseases, where oxidative stress is an integral component of the injury mechanisms involved.

Topics: Aldehydes; Animals; Antioxidants; Brain; Brain Ischemia; Cytochromes c; Female; Gene Expression; Hypoxia, Brain; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria; Oxidative Stress; Tyrosine; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

Na+/H+ exchanger isoform 1 (NHE1) is a major acid extrusion mechanism following intracellular acidosis. We hypothesized that stimulation of NHE1 after cerebral ischemia contributes to disruption of Na+ homeostasis and neuronal death. In the present study, expression of NHE1 was detected in cultured mouse cortical neurons. Oxygen and glucose deprivation (OGD) for 3 hours followed by 21 hours of reoxygenation (REOX) led to 68 +/- 10% cell death. Inhibition of NHE1 with the potent inhibitor HOE 642 or genetic ablation of NHE1 reduced OGD-induced cell death by approximately 40% to 50% (p < 0.05). In NHE1 +/+ neurons, OGD/REOX triggered significant increases in Na+ and Ca(i)2+. Genetic ablation of NHE1 and HOE 642 treatment reduced the rise of Na(i)+ by approximately 40% to 50% and abolished the OGD/REOX-mediated Ca2+ accumulation. Moreover, mitochondrial cytochrome C release was significantly attenuated by inhibition of NHE1 activity. These results imply that NHE1 activity disrupts Na+ and Ca2+ homeostasis and contributes to ischemic neuronal damage.

Topics: Animals; Brain Ischemia; Cation Transport Proteins; Cells, Cultured; Cerebral Cortex; Cytochromes c; Guanidines; Membrane Proteins; Mice; Mitochondrial Proteins; Neurons; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sulfones

Our previous studies and the others have strongly suggested that JNK signaling pathway plays a critical role in ischemic brain injury. Here, we reported that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. As a result, SP600125 diminished the increased phosphorylation of c-Jun and the increased expression of FasL induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 and the release of Bax from Bcl-2/Bax dimers, SP600125 attenuated Bax translocation to mitochondria and the release of cytochrome c induced by ischemia/reperfusion (I/R). Furthermore, the activation of caspase-3 induced by ischemia/reperfusion was also significantly suppressed by preinfusion of SP600125. Importantly, the same neuropotective effect was showed by administration of SP600125 both before and after ischemia. Thus, our findings imply that SP600125 can inhibit the activation of JNK signaling pathway and induce neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via suppressing the extrinsic and intrinsic pathways of apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury.

Topics: Animals; Anthracenes; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspase 3; Caspases; Cerebral Infarction; Cytochromes c; Enzyme Inhibitors; Fas Ligand Protein; Hippocampus; JNK Mitogen-Activated Protein Kinases; Male; Membrane Glycoproteins; Nerve Degeneration; Neuroprotective Agents; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Signal Transduction; Tumor Necrosis Factors

The overall goal of this study was to determine the molecular basis by which mixed-lineage kinase 3 (MLK3) kinase and its signaling pathways are negatively regulated by the pro-survival Akt pathway in cerebral ischemia. We demonstrated that tyrosine phosphorylation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) underlies the increased Akt-Ser473 phosphorylation by orthovanadate. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacts with Rac1 in the hippocampal CA1 region, and this interaction is promoted on tyrosine phosphatase inhibition. The elevated Akt activation can deactivate MLK3 by phosphorylation at the Ser71 residue of Rac1, a small Rho family of guanidine triphosphatases required for MLK3 autophosphorylation. Subsequently, inhibition of c-Jun N-terminal kinase 3 (JNK3) results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c and activation of caspase 3. At the same time, the expression of Fas-ligand decreases in the CA1 region after inhibition of c-Jun activation. The neuroprotective effect of Akt activation is significant in the CA1 region after global cerebral ischemia. Our results suggest that the activation of the pro-apoptotic MLK3/JNK3 cascade induced by ischemic stress can be suppressed through activation of the anti-apoptotic phosphatidylinositol 3-kinase/Akt pathway, which provides a direct link between Akt and the family of stress-activated kinases.

Topics: 14-3-3 Proteins; Animals; bcl-2-Associated X Protein; Biological Transport; Brain Ischemia; Caspase 3; Caspases; Cytochromes c; Cytosol; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; Hippocampus; Male; MAP Kinase Kinase Kinases; Membrane Glycoproteins; Mitochondria; Mitogen-Activated Protein Kinase 10; Mitogen-Activated Protein Kinase Kinase Kinase 11; Neuroprotective Agents; Phosphorylation; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; rac1 GTP-Binding Protein; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Tumor Necrosis Factor Inhibitors; Tyrosine; Vanadates

Protein kinase C (PKC) isozymes have been known to mediate a variety of complex and diverse cellular functions. deltaPKC has been implicated in mediating apoptosis. Using two models of cerebral ischemia, cardiac arrest in rats and oxygen glucose deprivation (OGD) in organotypic hippocampal slices, we tested whether an ischemic insult promoted deltaPKC cleavage during the reperfusion and whether the upstream pathway involved release of cytochrome c and caspase 3 cleavage. We showed that cardiac arrest/OGD significantly enhanced deltaPKC translocation and increased its cleavage at 3 h of reperfusion. Since deltaPKC is one of the substrates for caspase 3, we next determined caspase 3 activation after cardiac arrest and OGD. The maximum decrease in levels of procaspase 3 was observed at 3 h of reperfusion after cardiac arrest and OGD. We also determined cytochrome c release, since it is upstream of caspase 3 activation. Cytochrome c in cytosol increased at 1 h of reperfusion after cardiac arrest/OGD. Inhibition of either deltaPKC/caspase 3 during OGD and early reperfusion resulted in neuroprotection in CA1 region of hippocampus. Our results support the deleterious role of deltaPKC in reperfusion injury. We propose that early cytochrome c release and caspase 3 activation promote deltaPKC translocation/cleavage.

Topics: Animals; Blood Pressure; Brain Ischemia; Caspase 3; Caspases; Cell Death; Cytochromes c; Electrocardiography; Glucose; Heart Arrest; Hippocampus; Nerve Degeneration; Organ Culture Techniques; Oxygen; Protein Kinase C; Protein Kinase C-delta; Rats; Rats, Sprague-Dawley; Signal Transduction

Mitochondrial release of cytochrome c (cyt-c) plays a critical role in initiating cell death after cerebral ischemia. The objective of this study was to determine whether bongkrekic acid (BKA) ameliorates ischemic neuronal damage by inhibiting the release of cyt-c. These results showed that a 10min period of global ischemia caused neuronal death, increased the release of cyt-c and activated astrocytes in the cortex and CA1. BKA treatment reduced ischemic-induced neuronal death, prevented cyt-c release and inhibited astrocyte activation in the cortex, but not in the CA1. These results suggest that the neuroprotective effect of BKA is associated with its ability to prevent cyt-c release and to inhibit astrocyte activation.

Topics: Animals; Apoptosis; Astrocytes; Bongkrekic Acid; Brain Ischemia; Cytochromes c; Male; Neurons; Neuroprotective Agents; Rats; Rats, Wistar

Increased oxidative stress has been regarded as an important underlying cause for neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. In recent years, there has been increasing interest in investigating polyphenols from botanical source for possible neuroprotective effects against neurodegenerative diseases. In this study, we investigated the mechanisms underlying the neuroprotective effects of curcumin, a potent polyphenol antioxidant enriched in tumeric. Global cerebral ischemia was induced in Mongolian gerbils by transient occlusion of the common carotid arteries. Histochemical analysis indicated extensive neuronal death together with increased reactive astrocytes and microglial cells in the hippocampal CA1 area at 4 days after I/R. These ischemic changes were preceded by a rapid increase in lipid peroxidation and followed by decrease in mitochondrial membrane potential, increased cytochrome c release, and subsequently caspase-3 activation and apoptosis. Administration of curcumin by i.p. injections (30 mg/kg body wt) or by supplementation to the AIN76 diet (2.0 g/kg diet) for 2 months significantly attenuated ischemia-induced neuronal death as well as glial activation. Curcumin administration also decreased lipid peroxidation, mitochondrial dysfunction, and the apoptotic indices. The biochemical changes resulting from curcumin also correlated well with its ability to ameliorate the changes in locomotor activity induced by I/R. Bioavailability study indicated a rapid increase in curcumin in plasma and brain within 1 hr after treatment. Together, these findings attribute the neuroprotective effect of curcumin against I/R-induced neuronal damage to its antioxidant capacity in reducing oxidative stress and the signaling cascade leading to apoptotic cell death.

Topics: Analysis of Variance; Animals; Apoptosis; Astrocytes; Behavior, Animal; Behavioral Symptoms; Brain; Brain Ischemia; Caspase 3; Caspases; CD13 Antigens; Cell Count; Curcumin; Cytochromes c; Disease Models, Animal; Gerbillinae; Glial Fibrillary Acidic Protein; Histocytochemistry; Immunohistochemistry; In Situ Nick-End Labeling; Indoles; Lectins; Lipid Peroxidation; Liver; Male; Microglia; Mitochondria; Motor Activity; Neurons; Neuroprotective Agents; Time Factors

An in vitro ischemia model was used to determine the molecular mechanisms responsible for the ischemia-induced neuronal cell death. Additionally, the neuronal protective mechanisms of anti-apoptotic drugs against ischemia were also evaluated. In this study, the primary neuronal cultures were incubated in an anoxic chamber with 95% of N2 and 5% of CO2 for various times. The death rate, degree of the apoptotic damage, reduction of mitochondrial membrane potential, translocation of Bax, release of cytochrome C and activation of caspase-9 and -3 were determined at each time point. Results showed that a Bax-regulated mitochondria- mediated apoptosis is responsible for the in vitro ischemia-induced neuronal death. Reduction in mitochondrial membrane potential plays no role in triggering this apoptosis. Furthermore, the anti-apoptotic drugs: furosemide (a Bax blocker) and ZVAD-fmk (caspase inhibitor) but not cyclosporine A (a MPT pore blocker), significantly protected the neurons against ischemia-induced damage. This provides an additional consideration in the future selection of new anti-ischemic drugs.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspases; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Intracellular Membranes; Membrane Potentials; Mitochondria; Nerve Degeneration; Protein Synthesis Inhibitors; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Signal Transduction

The involvement of matrix metalloproteinases (MMPs) in cerebral ischemia-induced apoptosis was investigated in a model of transient focal cerebral ischemia in rats treated intracerebroventricularly (i.c.v.) with 4-((3-(4-phenoxylphenoxy)propylsulfonyl)methyl)-tetrahydropyran-4-carboxylic acid N-hydroxy amide, a broad spectrum non-peptidic hydroxamic acid MMP inhibitor, and in MMP-9-deficient mice. Our results showed that MMP inhibition reduced DNA fragmentation by 51% (P < 0.001) and cerebral infarct by 60% (P < 0.05) after ischemia. This protection was concomitant with a 29% reduction of cytochrome c release into the cytosol (P < 0.005) and a 54% reduction of calpain-related alpha-spectrin degradation (P < 0.05), as well as with an 84% increase in the immunoreactive signal of the native form of poly(ADP) ribose polymerase (P < 0.01). By contrast, specific targeting of the mmp9 gene in mice did reduce cerebral damage by 34% (P < 0.05) but did not modify the apoptotic response after cerebral ischemia. However, i.c.v. injection of MMP-9-deficient mice with the same broad-spectrum inhibitor used in rats significantly reduced DNA degradation by 32% (P < 0.05) and contributed even further to the protection of the ischemic brain. Together, our pharmacological and genetic results indicate that MMPs other than MMP-9 are actively involved in cerebral ischemia-induced apoptosis.

Topics: Animals; Apoptosis; Blotting, Western; Brain Ischemia; Calpain; Caspase 3; Caspases; Cell Count; Cytochromes c; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Functional Laterality; Hydroxamic Acids; In Situ Nick-End Labeling; Injections, Intraventricular; Interleukin-1; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Membrane Glycoproteins; Mice; Mice, Knockout; Rats; Rats, Sprague-Dawley; Staining and Labeling; Subcellular Fractions; Sulfonamides

We showed previously that Bcl-2 overexpression with the use of herpes simplex viral (HSV) vectors improved striatal neuron survival when delivered 1.5 hours after stroke but not when delivered 5 hours after stroke onset. Here we determine whether hypothermia prolongs the therapeutic window for gene therapy.. Rats were subjected to focal ischemia for 1 hour. Hypothermia (33 degrees C) was induced 2 hours after insult and maintained for 3 hours. Five hours after ischemia onset, HSV vectors expressing Bcl-2 plus beta-gal or beta-gal alone were injected into each striatum. Rats were killed 2 days later.. Striatal neuron survival of Bcl-2-treated, hypothermic animals was improved 2- to 3-fold over control-treated, hypothermic animals and Bcl-2-treated, normothermic animals. Neuron survival among normothermic, Bcl-2-treated animals was not different from control normothermics or control hypothermics. Double immunostaining of cytochrome c and beta-gal demonstrated that Bcl-2 plus hypothermia significantly reduced cytochrome c release.. Postischemic mild hypothermia extended the time window for gene therapy neuroprotection using Bcl-2 and reduced cytochrome c release.

Topics: Animals; beta-Galactosidase; Brain Ischemia; Cell Survival; Corpus Striatum; Cytochromes c; Disease Models, Animal; Genes, Reporter; Genetic Therapy; Genetic Vectors; Hypothermia, Induced; Neurons; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Simplexvirus; Stroke; Time Factors

The efflux of mitochondrial protein cytochrome C to cytoplasm is one of the key events of mitochondrial dysfunction observed in post-ischemic pathology. We investigated the effect of intra-carotid infusion of 5-10 mg/kg of cyclosporin A (CsA) on the neuronal survival in CA1 sector of hippocampus and on the subcellular localization of cytochrome C in the model of 5 min gerbil brain ischemia. To discriminate between the immunosuppressive and the mitochondria protecting component of CsA action, we compared the effect of CsA with one other immunosuppressant FK506. Almost 75% of neurons in ischemia-affected brain area were saved after CsA but not after FK506 treatment. This protective effect was only observed when the drug was infused immediately upon reperfusion. Early CsA treatment was able to block an initial phase of cytochrome C release, occurring transiently at 30 min post-ischemia, an effect never observed after FK506 administration. We assessed the neuroprotective potency of CsA vs. FK506 in rat cortical primary culture treated with compounds that mimic destructive signals induced by brain ischemia. In all cases, neuronal death and cytochrome C release were evidently suppressed by CsA applied not later than 30 min after the initial insult. Thus, early treatment with CsA in vitro and after bolus intra-carotid injection in vivo can save neurons by inhibition of cytochrome C efflux to cytoplasm.

Topics: Analysis of Variance; Animals; Biological Transport; Blotting, Western; Brain Ischemia; Cell Count; Cell Death; Cerebral Cortex; Cyclosporine; Cytochromes c; Cytoplasm; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Gerbillinae; Glutamic Acid; Hippocampus; Hydrogen Peroxide; Immunosuppressive Agents; In Vitro Techniques; Ischemic Attack, Transient; Male; Microscopy, Confocal; Rats; Rats, Wistar; Staurosporine; Tacrolimus; Time Factors; Tubulin

To determine the effect of immunophilin ligand GPI-1046 on ischemic brain injury, 90 min of transient middle cerebral artery occlusion (MCAO) was carried out in rat brains. In contrast to cases treated with vehicle, the infarct volume was reduced greatly and rotamase activity was inhibited significantly at 24 hr of reperfusion by treatment with GPI-1046. Immunoreactivity and the number of cells stained positively for FKBP12, FKBP52, caspase-8, cytochrome c, and caspase-3 were also reduced markedly in the brain after GPI-1046 treatment. The present results suggest that GPI-1046 significantly decreased infarct volume and provided neuroprotective effect on rats after transient focal cerebral ischemia by inhibiting the increase of rotamase activity and of the number of FKBP12-, FKBP52-, caspase-8-, cytochrome c-, and caspase-3-positive cells in the ischemic area.

Topics: Analysis of Variance; Animals; Brain; Brain Ischemia; Caspases; Cytochromes c; Immunophilins; Male; Neuroprotective Agents; Pyrrolidines; Rats; Rats, Wistar; Tacrolimus Binding Proteins

To evaluate the effects of different antagonists on the release of cytochrome c from mitochondria to cytosol and the expression of Bcl-2 in mitochondria in rat hippocampus after ischemia, we examined Bcl-2 and cytochrome c expression by immunoblotting using 4-vessel occlusion (4-VO) as brain ischemia model. The results showed that after 24 h ischemia/reperfusion (I/R) cytochrome c decreased markedly in mitochondria, which was correspondingly increased in the cytosolic fraction. Bcl-2 expression was time-dependent, reaching its peak level after 6 h I/R. In all those samples, there were no alterations in the subcellular distribution of cytochrome oxidase, a mitochondrial respiratory chain protein. The decreases in Bcl-2 and cytochrome c in mitochondria were restored by pretreatment with non-competitive NMDA receptor antagonist ketamine or L-type voltage-gated Ca(2+) channel (L-VGCC) antagonist nifedipine at 20 min prior to ischemia. The results demonstrate that the release of cytochrome c from mitochondria to cytosol and the up-regulation of Bcl-2 are possibly mediated by NMDA receptors or L-VGCC following brain ischemia. Cytochrome c release may be injurious while Bcl-2 up-regulation may be protective to ischemic hippocampus.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Calcium Channels, L-Type; Cytochromes c; Cytosol; Hippocampus; Ketamine; Male; Mitochondria; Nifedipine; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Up-Regulation

Uncoupling protein 2 (UCP2) is suggested to be a regulator of reactive oxygen species production in mitochondria. We performed a detailed study of brain injury, including regional and cellular distribution of UCP2 mRNA, as well as measures of oxidative stress markers following permanent middle cerebral artery occlusion in UCP2 knockout (KO) and wild-type (WT) mice. Three days post ischemia, there was a massive induction of UCP2 mRNA confined to microglia in the peri-infarct area of WT mice. KO mice were less sensitive to ischemia as assessed by reduced brain infarct size, decreased densities of deoxyuridine triphosphate nick end-labelling (TUNEL)-labelled cells in the peri-infact area and lower levels of lipid peroxidation compared with WT mice. This resistance may be related to the substantial increase of basal manganese superoxide dismutase levels in neurons of KO mice. Importantly, we found a specific decrease of mitochondrial glutathione (GSH) levels in UCP2 expressing microglia of WT, but not in KO mice after ischemia. This specific association between UCP2 and mitochondrial GSH levels regulation was further confirmed using lipopolysaccharide models of peripheral inflammation, and in purified peritoneal macrophages. Moreover, our data imply that UCP2 is not directly involved in the regulation of ROS production but acts by regulating mitochondrial GSH levels in microglia.

Topics: Animals; Antioxidants; Brain Ischemia; Cell Count; Cytochromes c; Disease Models, Animal; Genetic Predisposition to Disease; Glutathione; In Situ Nick-End Labeling; Ion Channels; Lipopolysaccharides; Macrophages, Peritoneal; Male; Membrane Transport Proteins; Mice; Mice, Knockout; Microglia; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Protein Transport; RNA, Messenger; Superoxide Dismutase; Uncoupling Protein 2

Apoptosis plays a critical role in many neurologic diseases, including stroke. Cytochrome c release and activation of various caspases are known to occur after focal and global ischemia. However, recent reports indicate that caspase-independent pathways may also be involved in ischemic damage. Apoptosis-inducing factor (AIF) is a novel flavoprotein that helps mediate caspase-independent apoptotic cell death. AIF translocates from mitochondria to nuclei where it induces caspase-independent DNA fragmentation. Bcl-2, a mitochondrial membrane protein, protects against apoptotic and necrotic death induced by different insults, including cerebral ischemia. In the present study, Western blots confirmed that AIF was normally confined to mitochondria but translocated to nuclei or cytosol 8, 24, and 48 hours after onset of ischemia. Overall, AIF protein levels also increased after stroke. Confocal microscopy further demonstrated that nuclear AIF translocation occurred in the peri-infarct region but not in the ischemic core where only some cytosolic AIF release was observed. Our data also suggest that AIF translocated into nuclei after cytochrome c was released into the cytosol. Bcl-2 transfection in the peri-infarct region blocked nuclear AIF translocation and improved cortical neuron survival.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Brain Ischemia; Cell Nucleus; Cytochromes c; Flavoproteins; Fluorescent Dyes; Humans; Membrane Proteins; Mitochondria; Neurons; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Simplexvirus; Subcellular Fractions; Transfection

Cerebral ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro are characterized by major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na(+), Ca(2+), and Cl(-) and extracellular K(+). Recently, considerable attention has been focused on the cation-chloride cotransporters Na-K-Cl cotransporter isoform I (NKCC-1) and K-Cl cotransporter isoform II (KCC2), as they may play an important role in the disruption of ion gradients and subsequent ischemic damage. In this study, we examined the ability of cation-chloride transport inhibitors to influence the biochemical (i.e. ATP) and histological recovery of neurons in adult hippocampal slices exposed to OGD. In the hippocampus, 7 min of OGD caused a loss of ATP that recovered partially (approximately 50%) during 3 h of reoxygenation. Furosemide, which inhibits the NKCC-1 and KCC2 cotransporters, and bumetanide, a more specific NKCC-1 inhibitor, enhanced ATP recovery when measured 3 h after OGD. Furosemide and bumetanide also attenuated area CA1 neuronal injury after OGD. However, higher concentrations of these compounds appear to have additional non-specific toxic effects, limiting ATP recovery following OGD and promoting neuronal injury. The KCC2 cotransporter inhibitor DIOA and the Cl(-) ATPase inhibitor ethacrynic acid caused neuronal death even in the absence of OGD and promoted cytochrome c release from isolated mitochondria, indicating non-specific toxicities of these compounds.

Topics: Adenosine Triphosphate; Animals; Brain Ischemia; Bumetanide; Carrier Proteins; Cell Survival; Chlorides; Cytochromes c; Diuretics; Energy Metabolism; Ethacrynic Acid; Furosemide; Glucose; Hippocampus; Hypoxia; In Vitro Techniques; Male; Neurons; Rats; Rats, Sprague-Dawley; Sodium Potassium Chloride Symporter Inhibitors; Solute Carrier Family 12, Member 1; Solute Carrier Family 12, Member 2

Oxidative stress is one of the major pathological factors in the cascade that leads to cell death in cerebral ischemia. Here, we investigated the neuroprotective effect of a naturally occurring antioxidant, oxyresveratrol, to reduce brain injury after cerebral stroke. We used the transient rat middle cerebral artery occlusion (MCAO) model of brain ischemia to induce a defined brain infarction. Oxyresveratrol was given twice intraperitoneally: immediately after occlusion and at the time of reperfusion. Oxyresveratrol (10 or 20 mg/kg) significantly reduced the brain infarct volume by approximately 54% and 63%, respectively, when compared to vehicle-treated MCAO rats. Also, the neurological deficits as assessed by different scoring methods improved in oxyresveratrol-treated MCAO rats. Histological analysis of apoptotic markers in the ischemic brain area revealed that oxyresveratrol treatment diminished cytochrome c release and decreased caspase-3 activation in MCAO rats. Also, staining for apoptotic DNA showed that the number of apoptotic nuclei in ischemic brain was reduced after oxyresveratrol treatment as compared to the vehicle-treated MCAO rats. This dose-dependent neuroprotective effect of oxyresveratrol in an in vivo stroke model demonstrates that this drug may prove to be beneficial for a therapeutic strategy to limit brain injury in acute brain ischemia.

Topics: Analysis of Variance; Animals; Brain Ischemia; Cell Death; Cerebral Cortex; Cerebral Infarction; Cytochromes c; Disease Models, Animal; DNA Fragmentation; Dose-Response Relationship, Drug; Epoprostenol; Immunohistochemistry; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Microtubule-Associated Proteins; Mitochondria; Neurologic Examination; Neurons; Neuroprotective Agents; Phosphopyruvate Hydratase; Plant Extracts; Rats; Rats, Wistar; Stilbenes; Time Factors

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemic cell death with respect to cytochrome c translocation and DNA fragmentation, which are pivotal events in the necrotic and apoptotic signaling pathway, using permanent focal cerebral ischemia in rats. Immunohistochemically, cytochrome c was observed in the cytoplasm as early as 1 h after middle cerebral artery (MCA) occlusion in the infarcted hemisphere. Cytosolic release of cytochrome c after MCA occlusion was also confirmed by Western blot analysis and enzyme immunoassay. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) showed DNA fragmentation evolving in the ipsilateral cortex and the caudate putamen after 3 and 6 h, respectively, following MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, significantly attenuated the release of cytochrome c in the ischemic region, the number of TUNEL-positive cells in the ischemic penumbra zone, and the size of cortical ischemic lesions. This study demonstrated that tacrolimus ameliorated the accumulation of cytochrome c in the cytosol and the increase of TUNEL-positive cells induced by cerebral ischemia, indicating that the neuroprotective action of tacrolimus on ischemic brain injury caused by permanent focal cerebral ischemia could partially be attributed to the attenuation of the activation of the apoptotic execution machinery.

Topics: Animals; Brain Ischemia; Cerebral Infarction; Cytochromes c; DNA Fragmentation; Immunosuppressive Agents; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tacrolimus

To investigate the effects of beta-aescin on apoptosis induced by transient focal brain ischemia in rats.. Rats were pretreated with beta-aescin for 7 d and then subjected to brain ischemia/reperfusion (I/R) injury induced by a middle cerebral artery occlusion. After 2 h ischemia and 24 h reperfusion, Hematoxylin-Eosin (HE) staining, in situ end-labeling of nuclear DNA fragmentation (TUNEL) were employed to determine the level of apoptosis. The expressions of caspase-3 and Bcl-2 in the cortex were determined by immunohistochemistry and Western blot. The release of cytochrome c was analyzed by Western blot.. The increased numbers of HE- and TUNEL-positive staining cells were significantly observed at 24 h after reperfusion. The immunoreactivity was inhibited by beta-aescin (30, 60 mg/kg) (P<0.01 or P<0.05 vs vehicle-treated). After cerebral I/R, cytochrome c was released into the cytosol and caspase-3 was activated, whereas Bcl-2 expression was inhibited. beta-Aescin (30, 60 mg/kg) markedly inhibited the expression of caspase-3 and the release of cytochrome c, and up-regulated the expression of Bcl-2 (P<0.05, P<0.01 vs vehicle-treated).. beta-Aescin could potently inhibit caspase-3 activation and the release of cytochrome c, increasing the expression of Bcl-2 after cerebral I/R in rats. These findings on the inhibitory effects of beta-aescin on brain ischemic injury-induced apoptosis might have important theoretical basis for the treatment on ischemic cerebrovascular diseases.

Topics: Animals; Apoptosis; Brain Ischemia; Caspase 3; Caspases; Cerebral Cortex; Cytochromes c; Escin; Infarction, Middle Cerebral Artery; Male; Mitochondria; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via glutamate receptor 2 (GluR2)-lacking AMPA receptors (AMPARs), and selective, delayed death of hippocampal CA1 neurons. The molecular mechanisms underlying Zn2+ toxicity in vivo are not well delineated. Here we show the striking finding that intraventricular injection of the high-affinity Zn2+ chelator calcium EDTA (CaEDTA) at 30 min before ischemia (early CaEDTA) or at 48-60 hr (late CaEDTA), but not 3-6 hr, after ischemia, afforded robust protection of CA1 neurons in approximately 50% (late CaEDTA) to 75% (early CaEDTA) of animals. We also show that Zn2+ acts via temporally distinct mechanisms to promote neuronal death. Early CaEDTA attenuated ischemia-induced GluR2 mRNA and protein downregulation (and, by inference, formation of Zn2+-permeable AMPARs), the delayed rise in Zn2+, and neuronal death. These findings suggest that Zn2+ acts at step(s) upstream from GluR2 gene downregulation and implicate Zn2+ in transcriptional regulation and/or GluR2 mRNA stability. Early CaEDTA also blocked mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein-binding protein with low pI), caspase-3 activity (but not procaspase-3 cleavage), p75NTR induction, and DNA fragmentation. These findings indicate that CaEDTA preserves the functional integrity of the mitochondrial outer membrane and arrests the caspase death cascade. Late injection of CaEDTA at a time when GluR2 is downregulated and caspase is activated inhibited the delayed rise in Zn2+, p75NTR induction, DNA fragmentation, and cell death. The finding of neuroprotection by late CaEDTA administration has striking implications for intervention in the delayed neuronal death associated with global ischemia.

Topics: Animals; Apoptosis Regulatory Proteins; Brain Ischemia; Carrier Proteins; Caspase 3; Caspases; Cell Death; Chelating Agents; Cytochromes c; DNA Fragmentation; Edetic Acid; Gerbillinae; Hippocampus; Male; Mitochondria; Mitochondrial Proteins; Neurons; Rats; Rats, Sprague-Dawley; Receptor, Nerve Growth Factor; Receptors, AMPA; Receptors, Nerve Growth Factor; Time Factors; Zinc

Transient focal ischaemia by middle cerebral artery occlusion (MCAO) may produce cell death, but the mechanisms leading to cell death differ in the infarct core and in the penumbra, the immediate zone surrounding the infarct core. In the present study, transient focal ischaemia to adult rats was produced by intraluminal occlusion of the middle cerebral artery for 1 h followed by 0 h (n=6), 1 h (n=10), 4 h (n=8), 6 h (n=2) and 12 h (n=3) of reperfusion. The present model of ischaemia causes a large cortico-striatal infarct extending through the mediolateral cortex and dorsolateral striatum at 12 h. The expression and subcellular distribution of several proteins involved in apoptosis have been examined in the penumbra and in the infarct core by using combined methods of immunohistochemistry, cell subfractionation and Western blotting. Transient focal ischaemia by MCAO results in activation of complex signal pathways for cell death in the penumbra. Increased expression of Bcl-2 and Bax, but not of Bcl-x, occurs in the penumbra at the time when Bax translocates from the cytosol to the mitochondria, cytochrome c is released to the cytoplasm and active caspase-3 is expressed. Bax translocation, cytochrome c release and active caspase-3 are observed at 4 h, but not at 1 h, following reperfusion, and together indicate activation of the caspase-dependent pathway of apoptosis in the penumbra. In contrast, reduced Bax expression but not Bax translocation and cytochrome c release occurs in the infarct core, thus suggesting apoptosis signals restricted to the penumbra. In addition, increased expression of an apoptosis-inducing factor in the cytoplasm and nuclei of selected cells shows, for the first time, activation of the caspase-independent mitochondrial pathway in the penumbra following transient focal ischaemia and reperfusion.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Brain Ischemia; Caspases; Cytochromes c; Cytosol; Enzyme Activation; Flavoproteins; Immunohistochemistry; Infarction, Middle Cerebral Artery; Male; Membrane Proteins; Mitochondria; Neuroglia; Neurons; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Reperfusion Injury; Time Factors

Caspase-1 plays a key role in inflammatory pathways by processing pro-IL-1beta into the active cytokine mature IL-1beta. Given its sequence similarity with the Caenorhabditis elegans cell death gene ced-3,it has long been speculated that caspase-1 may also play a role in cell death. However, an unequivocal role for caspase-1 in cell death has been questioned, and not definitively demonstrated. Furthermore, if caspase-1 does play a role in cell death, its position in the apoptotic hierarchy has not been clearly defined. Previous studies have shown that caspase-1 knockout (KO) mice and transgenic mice expressing a dominant-negative caspase-1 construct are resistant to ischemic brain injury. We provide direct evidence that caspase-1 plays a key role in neuronal cell death and that caspase-1 is an apical activator of the cell death pathway in the premitochondrial collapse stage. Furthermore, we demonstrate that Rip2/Cardiak/Rick is a stress-inducible upstream modulator of pro-caspase-1 apoptotic activation. We provide evidence that Bid cleavage appears to be an important downstream effector of caspase-1-mediated cell death. Our data demonstrate that caspase-1 is an apical mediator of neuronal cell death during in vitro hypoxia, and confirmed in vivo in ischemia, and provide insights into the sequence of events involved in this pathological cell death process.

Topics: Animals; Apoptosis; Brain Ischemia; Caspase 1; Caspases; Cell Death; Cell Hypoxia; Cerebral Cortex; Cytochromes c; Enzyme Activation; L-Lactate Dehydrogenase; Mice; Mice, Knockout; Neurons; Protein Serine-Threonine Kinases; Receptor-Interacting Protein Serine-Threonine Kinase 2; Receptor-Interacting Protein Serine-Threonine Kinases

The search for an effective treatment for global ischemia following cardiac arrest has proved to be very difficult. However, studies by Uchino et al. show that the immunosuppressant cyclosporin A (CsA), when administered in such a way that the drug can bypass the blood brain barrier (BBB), dramatically reduces ischemic damage in rat forebrain preparations. An alternative immunosuppressant, FK506, is apparently less efficacious. Both CsA and FK506 are specific inhibitors of immunophilins, (CsA inhibits cyclophilins, FK506 inhibits FKBPs), and of calcineurin, a type 2B Ser/Thr phosphatase that is abundant in the central nervous system. The superiority of CsA may be partly attributable to its selective amelioration of mitochondrial damage, as assayed in vivo and in vitro. Our results suggest that pathways involving calcineurin and cyclophilins, particularly mitochondrial cyclophilin D, play pivotal roles in the development of ischemic brain damage. The present findings may inform the search for new drugs in the treatment of global ischemic damage to the brain, and in other organs.

Topics: Animals; bcl-Associated Death Protein; Brain; Brain Ischemia; Calcineurin; Calcineurin Inhibitors; Calcium; Carrier Proteins; Cyclophilins; Cyclosporine; Cytochromes c; Immunosuppressive Agents; Mitochondrial Swelling; Neuroprotective Agents; Peptidyl-Prolyl Isomerase F; Phosphorylation; Prosencephalon; Rats; Rats, Wistar; Tacrolimus