Release of mitochondrial Opa1 following oxidative stress in HT22 cells

Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.     

Early release of cytochrome C and activation of caspase-3 in hyperglycemic rats subjected to transient forebrain ischemia

The mechanisms underlying the aggravating effect of hyperglycemia on brain damage are still elusive. The present study was designed to test our hypothesis that hyperglycemia-mediated damage is caused by mitochondrial dysfunction with mitochondrial release of cytochrome c (cyt c) to the cytoplasm, which leads to activation of caspase-3, the executioner of cell death. We induced 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of recirculation in sham, normoglycemic and hyperglycemic rats. Release of cyt c was observed in the neocortex and CA3 in hyperglycemic rats after only 0.5 h of reperfusion, when no obvious neuronal damage was observed. The release of cyt c persisted after 1 and 3 h of reperfusion. Activation of caspase-3 was observed after 1 and 3 h of recovery in hyperglycemic animals. No cyt c release or caspase-3 activation was observed in sham-operated controls while a mild increase of cyt c was observed in normoglycemic ischemic animals after 1 and 3 h of reperfusion. The findings that there is caspase activation and cyt c relocation support a notion that the biochemical changes that constitute programmed cell death occur after ischemia and contribute, at least in part, to hyperglycemia-aggravated ischemic neuronal death.     

Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats

Postconditioning mitigates ischemia‐induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen‐glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10‐min reoxygenation/10‐min rehypoxia after OGD. The MPT inhibitor N‐methyl‐4‐isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC‐1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψ_m). Transient global cerebral ischemia (tGCI) was induced via the two‐vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10‐sec reperfusion/10‐sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase‐3 activity and maintained △Ψ_m of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase‐9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia. © 2014 Wiley Periodicals, Inc.     

Estrogen-receptor-mediated protection of cerebral endothelial cell viability and mitochondrial function after ischemic insult in vitro

Protective effects of estrogen against experimental stroke and neuronal ischemic insult are well-documented, but it is not known whether estrogen prevents ischemic injury to brain endothelium, a key component of the neurovascular unit. Increasing evidence indicates that estrogen exerts protective effects through mitochondrial mechanisms. We previously found 17β-estradiol (E2) to improve mitochondrial efficiency and reduce mitochondrial superoxide in brain blood vessels and endothelial cells. Thus we hypothesized E2 will preserve mitochondrial function and protect brain endothelial cells against ischemic damage. To test this, an in vitro ischemic model, oxygen-glucose deprivation (OGD)/reperfusion, was applied to immortalized mouse brain endothelial cells (bEnd.3). OGD/reperfusion-induced cell death was prevented by long-term (24, 48 h), but not short-term (0.5, 12 h), pretreatment with 10 nmol/L E2. Protective effects of E2 on endothelial cell viability were mimicked by an estrogen-receptor (ER) agonist selective for ERα (PPT), but not by one selective for ERβ (DPN). In addition, E2 significantly decreased mitochondrial superoxide and preserved mitochondrial membrane potential and ATP levels in early stages of OGD/reperfusion. All of the E2 effects were blocked by the ER antagonist, ICI-182,780. These findings indicate that E2 can preserve endothelial mitochondrial function and provide protection against ischemic injury through ER-mediated mechanisms.     

Survival- and death-promoting events after transient cerebral ischemia: phosphorylation of Akt, release of cytochrome C and Activation of caspase-like proteases.

Release of cytochrome c (cyt c) into cytoplasm initiates caspase-mediated apoptosis, whereas activation of Akt kinase by phosphorylation at serine-473 prevents apoptosis in several cell systems. To investigate cell death and cell survival pathways, the authors studied release of cyt c, activation of caspase, and changes in Akt phosphorylation in rat brains subjected to 15 minutes of ischemia followed by varying periods of reperfusion. The authors found by electron microscopic study that a portion of mitochondria was swollen and structurally altered, whereas the cell membrane and nuclei were intact in hippocampal CA1 neurons after 36 hours of reperfusion. In some neurons, the pattern of immunostaining for cyt c changed from a punctuate pattern, likely representing mitochondria, to a more diffuse cytoplasmic localization at 36 and 48 hours of reperfusion as examined by laser-scanning confocal microscopic study. Western blot analysis showed that cyt c was increased in the cytosolic fraction in the hippocampus after 36 and 48 hours of reperfusion. Consistently, caspase-3-like activity was increased in these hippocampal samples. As demonstrated by Western blot using phosphospecific Akt antibody, phosphorylation of Akt at serine-473 in the hippocampal region was highly increased during the first 24 hours but not at 48 hours of reperfusion. The authors conclude that transient cerebral ischemia activates both cell death and cell survival pathways after ischemia. The activation of Akt during the first 24 hours conceivably may be one of the factors responsible for the delay in neuronal death after global ischemia.     

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.     

Translocation of apoptosis-inducing factor in vulnerable neurons after transient cerebral ischemia and in neuronal cultures after oxygen-glucose deprivation.

Loss of mitochondrial membrane integrity and the resulting release of apoptogenic factors may play a critical role in mediating hippocampal neurodegeneration after transient global ischemia. In the present study, the authors have cloned and characterized the rat cDNA encoding apoptosis-inducing factor (AIF), an intramitochondrial protein that promotes cell death in a caspase-independent manner upon release into nonmitochondrial compartments. In contrast to the expression patterns of a number of apoptosis-regulatory gene products during brain development, the expression of AIF protein increases gradually with brain maturation and peaks in adulthood. In a rat model of transient global ischemia, AIF was found to translocate from mitochondria to the nucleus in the hippocampal CA1 neurons after ischemia and to manifest a DNA-degrading activity that mimicked the purified AIF protein and was inhibitable by AIF immunodepletion. The temporal profile of AIF translocation after ischemia (24 to 72 hours) coincided with the induction of large-scale DNA fragmentation at the size of 50 kbp, a well-characterized hallmark of AIF-like activity but preceded the formation of internucleosomal DNA fragmentation (72 hours), a DNA degradation associated with the terminal stage of cell death. Further, the nuclear translocation of AIF after ischemia was not blocked by inhibiting caspase-3/-7 activities, but, as shown in neuronal cultures that were challenged with transient oxygen-glucose deprivation, it can be prevented by intracellular delivery of the mitochondria-associated antiapoptotic protein Bcl-xL. The results presented here strongly suggest that mitochondrial release of AIF may be an important factor, in addition to the previously reported cytochrome c and Smac, which could contribute to the selective vulnerability of CA1 neurons to transient global ischemic injury.     

14, 15-EET alleviates neurological impairment through maintaining mitochondrial dynamics equilibrium via AMPK/SIRT1/FoxO1 signal pathways in mice with cerebral ischemia reperfusion

Aim

To explore whether 14, 15-EET regulates mitochondrial dynamics to exert neuroprotective effects after cerebral ischemia–reperfusion and its underlying mechanisms.

Methods

The mouse middle cerebral artery occlusion reperfusion model was used to observe brain infarct volume and neuronal apoptosis by TTC staining and Tunel assay, modified neurological severity score to detect neurological impairment, HE staining and Nissl staining to observe neuron damage, western blot and immunofluorescence methods to detect the expression of mitochondrial dynamics-related proteins, transmission electron microscopy, and Golgi-Cox staining to detect mitochondrial morphology and neuronal dendritic spines.

Results

14, 15-EET reduced the neuronal apoptosis and cerebral infarction volume induced by middle cerebral artery occlusion reperfusion (MCAO/R), inhibited the degradation of dendritic spines, maintained the structural integrity of neurons, and alleviated neurological impairment. Cerebral ischemia–reperfusion induces mitochondrial dynamics disorders, upregulates the expression of the mitochondrial division protein Fis 1, and inhibits the expression of mitochondrial fusion proteins MFN1, MFN2, and OPA1, while 14, 15-EET treatment reverses this process. Mechanistic studies have shown that 14, 15-EET promotes the phosphorylation of AMPK, upregulates the expression of SIRT1 and phosphorylation of FoxO1, thereby inhibiting mitochondrial division and promoting mitochondrial fusion, preserving mitochondrial dynamics, maintaining neuronal morphological and structural integrity, and alleviating neurological impairment induced by middle cerebral artery occlusion reperfusion. Compound C treatment diminishes the neuroprotective effect of 14, 15-EET following MCAO/R in mice.

Conclusion

This study elucidates the novel neuroprotective mechanism of 14, 15-EET, providing a novel approach for the development of drugs based on mitochondrial dynamics.     

Effect of S-Nitrosylation of RIP3 Induced by Cerebral Ischemia on its Downstream Signaling Pathway

ObjectivesOur preliminary experiments indicate that receptor-interacting protein 3 (RIP3) is S-nitrosylated and contributes to its autophosphorylation (activation) after 3 h of rat brain ischemia/reperfusion mediated by activation of the N-methyl-D-aspartate receptor (NMDAR)-dependent neuronal NO synthase (nNOS) and is involved in the process of neuronal injury. Here, we will to demonstrate whether S-nitrosylation of RIP3 facilitates the activation of the downstream signaling pathway and finally exacerbates ischemic neuron death.Materials and methodsAdult male Sprague-Dawley rat transient brain ischemia/reperfusion and cortical neurons oxygen and glucose deprivation (OGD)/reoxygenation models were performed. The hippocampal CA1 regions or cultured cells were homogenized and the cytosolic fraction were collected as tissue samples. Coimmunoprecipitation and western blot analysis were carried out for detecting phosphorylation of RIP1 and mixed lineage kinase-like domains (MLKL) and the Cleaved-Caspase8 (Cl-Caspase8). The activities of Glycogen phosphorylase (PYGL), Glutamate-ammonia ligase (GLUL) and Glutamate dehydrogenase (GLUD1) were detected with ultraviolet absorption method.ResultsThis study showed that active RIP3 could phosphorylate RIP1 and MLKL through its kinase activity, promote the conversion of Caspase8 to active Cl-Caspase8, enhance the activities of PYGL, GLUL and GLUD1, and finally aggravate neuronal injury in cerebral ischemia/reperfusion. The inhibition of RIP3 S-nitrosylation inhibited the phosphorylation of RIP1 and MLKL, inhibited the activities of Caspase8, PYGL, GLUL, and GLUD1, and alleviated neuronal damage in cerebral ischemia/reperfusion.ConclusionsS-nitrosylation of RIP3 increased RIP1 and MLKL phosphorylation levels, Cl-Caspase8 content and PYGL, GLUL and GLUD1 activities and aggravated neuronal damage during cerebral ischemia/reperfusion and regulating the S-nitrosylation of RIP3 and its downstream signaling pathway might be a therapeutic target for stroke.     

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

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

MicroRNA-124-3p alleviates cerebral ischaemia-induced neuroaxonal damage by enhancing Nrep expression

Objective: Ischaemic stroke has a high death rate and frequently results in long-term and severe brain damage in survivors. miRNA-124-3p (miR-124-3p) treatment has been suggested to reduce ischaemia and play a vital function in avoiding neuron death. An investigation of the role of miR-124-3p, in the ischaemia damage repair or protection in the middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation/reperfusion (OGD/R) model, was the purpose of this research. Methods: The expression of miRNA and mRNA in the MCAO model was predicted using bioinformatics analysis. The OGD/R neuronal model was developed. We examined the influence of a number of compounds on the OGD/R model in vitro using gain- and loss-of-function approaches. Results: For starters, miR-124-3p and Nrep level in the MCAO model were found to be lower in the model predicted by bioinformatics than in the sham-operated group. And then in the OGD/R model, miR-124-3p treatment reduced OGD/R neuronal damage, increased neuronal survival, and reduced apoptosis in cell lines. Moreover, we further looked at the impact of miR-124-3p on downstream Rnf38 and Nrep using the OGD/R model. Western blot analysis and dual-luciferase reporter assays indicated that miR-124-3p binds and inhibits Rnf38. Finally, although Nrep expression was reduced in the OGD/R model neuronal model, it was shown that miR-124-3p administration reduced apoptosis and increased neuronal activity, particularly with regard to axon regeneration-related proteins. Conclusion: Our studies have shown that miR-124-3p may reduce neuronal injury by preventing Rnf38-mediated effects on the Nrep axis.     

Insulin activates the PI3K-Akt survival pathway in vulnerable neurons following global brain ischemia

Abstract

Insulin is neuroprotective following transient global brain ischemia; however, the mechanisms by which insulin exerts its salutary effects remain unclear.

Objective: We assessed insulin's effect on the PI3K-Akt survival system and consequent modulation of the pro-apoptotic proteins Bim, Bad and FoxO3a.

Methods: We utilized rats subjected to 10 minutes of global brain ischemia, with or without insulin administered at the onset of reperfusion.

Results: In sham-operated animals, minimal pAkt immunofluorescence was detected in the CA1. Moreover, at 30 minute reperfusion, there was no change in pAkt in CA1 neurons. Single bolus high-dose insulin treatment resulted in an early increase in pAkt after 30 minutes, preservation of CA1 neurons to 14 days of reperfusion and preservation of spatial learning ability. Insulin treatment increased cytoplasmic and nuclear staining for pAkt in both CA1 and cortex. Insulin-induced Akt phosphorylation was suppressed by the PI3K inhibitor wortmannin. Neither reperfusion nor insulin induced any change in the phosphorylation or subcellular localization of FoxO3a, Bim or Bad. A single bolus of high-dose insulin reduced CA1 neuronal cell death and thus represents a potential therapeutic intervention for global brain ischemia.

Discussion: These results reveal that proximal elements of a known cell-survival pathway are triggered by high-dose insulin during early reperfusion. Insulin induces robust PI3K-dependent phosphorylation of Akt by 30 minute reperfusion and results in improvement of hippocampal structure and function. However, the Akt substrates FoxO3a, Bim and Bad do not undergo corresponding changes in phosphorylation or subcellular localization in this model of global brain ischemia. The downstream components of insulin-induced Akt survival signaling after transient global brain ischemia remain to be identified.     

Trans-anethole protects cortical neuronal cells against oxygen–glucose deprivation/reoxygenation

Trans-anethole has been studied on pharmacological properties such as anti-inflammation, anti-oxidative stress, antifungal and anticancer. However, to date, the anti-ischemic effects of trans-anethole have not been assessed. Therefore, we investigated the neuroprotection of trans-anethole against oxygen–glucose deprivation/reoxygenation (OGD/R)-induced cortical neuronal cell injury, an in vitro model of ischemia. The abilities of trans-anethole to block excitotoxicity, oxidative stress and mitochondrial dysfunction were evaluated in OGD/R-induced neurons. Trans-anethole significantly ameliorated OGD/R-induced neuronal cell injury by attenuating the intracellular calcium overload via the activation of NMDA receptors. Trans-anethole also inhibited OGD/R-induced reactive oxygen species overproduction, which may be derived from the scavenging activity in peroxyl radicals, assessed in an oxygen radical absorbance capacity assay. Furthermore, trans-anethole was shown to attenuate the depolarization of mitochondrial transmembrane. These results indicated that the neuroprotective effect of trans-anethole on OGD/R-induced neuronal injury might be due to its ability to inhibit excitotoxicity, oxidative stress and mitochondrial dysfunction. Considering these multiple pathways causing ischemic neuronal damage, the multi-functional effect of trans-anethole suggested that it may be effective in treating ischemic stroke.     

Zn2+‐induced ERK activation mediates PARP‐1‐dependent ischemic‐reoxygenation damage to oligodendrocytes

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca²⁺]_i, loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia‐like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia‐induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O₂ (reperfusion‐like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high‐affinity Zn²⁺ chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high‐affinity Zn²⁺ probe, we showed that ischemia induced an intracellular Zn²⁺ rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia‐induced oligodendrocyte death. Moreover, UO126 blocked the ischemia‐induced increase in poly‐[ADP]‐ribosylation of proteins, and the poly[ADP]‐ribose polymerase 1 (PARP‐1) inhibitor DPQ significantly inhibited ischemia‐induced oligodendroglial cell death—demonstrating that PARP‐1 was required downstream in the Zn²⁺‐ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn²⁺, blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia‐reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult. © 2012 Wiley Periodicals, Inc.     

Nuclear translocation of apoptosis-inducing factor after focal cerebral ischemia.

Signaling cascades associated with apoptosis contribute to cell death after focal cerebral ischemia. Cytochrome c release from mitochondria and the subsequent activation of caspases 9 and 3 are critical steps. Recently, a novel mitochondrial protein, apoptosis-inducing factor (AIF), has been implicated in caspase-independent programmed cell death following its translocation to the nucleus. We, therefore, addressed the question whether AIF also plays a role in cell death after focal cerebral ischemia. We detected AIF relocation from mitochondria to nucleus in primary cultured rat neurons 4 and 8 hours after 4 hours of oxygen/glucose deprivation. In ischemic mouse brain, AIF was detected within the nucleus 1 hour after reperfusion after 45 minutes occlusion of the middle cerebral artery. AIF translocation preceded cell death, occurred before or at the time when cytochrome c was released from mitochondria, and was evident within cells showing apoptosis-related DNA fragmentation. From these findings, we infer that AIF may be involved in neuronal cell death after focal cerebral ischemia and that caspase-independent signaling pathways downstream of mitochondria may play a role in apoptotic-like cell death after experimental stroke.     

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.     

Reduction of copper, zinc-superoxide dismutase in knockout mice does not affect edema or infarction volumes and the early release of mitochondrial cytochrome c after permanent focal cerebral ischemia

Copper,zinc-superoxide dismutase (SOD1) was shown to be highly protective against ischemia/reperfusion injury in the brain. We have recently reported that SOD1 prevents the release of mitochondrial cytochrome c and subsequent apoptosis after ischemia/reperfusion in mice. To investigate its dose dependent effect on permanent focal cerebral ischemia, we examined neurological deficit scores, infarction volume, and the amount of hemisphere enlargement after 24 h of focal cerebral ischemia in both knockout mutants of SOD1 (Sod1 -/+ and Sod1 -/-) and wild-type littermates. We also examined the release of cytochrome c and subsequent DNA fragmentation after ischemia. There were no differences in the neurological deficit scores, infarction volumes and edema formation. There was also no difference of the amount cytosolic cytochrome c at 2 h and of the amount of DNA fragmentation at 24 h after focal cerebral ischemia. The results indicate that the SOD1 enzyme does not appear to affect cerebral infarction, cerebral edema nor the mitochondrial signaling pathway for apoptosis following permanent focal cerebral ischemia where there is no reperfusion injury.     

Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death.

Activation of terminal caspases such as caspase-3 plays an important role in the execution of neuronal cell death after transient cerebral ischemia. Although the precise mechanism by which terminal caspases are activated in ischemic neurons remains elusive, recent studies have postulated that the mitochondrial cell death-signaling pathway may participate in this process. The bcl-2 family member protein Bax is a potent proapoptotic molecule that, on translocation from cytosol to mitochondria, triggers the activation of terminal caspases by increasing mitochondrial membrane permeability and resulting in the release of apoptosis-promoting factors, including cytochrome c. In the present study, the role of intracellular Bax translocation in ischemic brain injury was investigated in a rat model of transient focal ischemia (30 minutes) and reperfusion (1 to 72 hours). Immunochemical studies revealed that transient ischemia induced a rapid translocation of Bax from cytosol to mitochondria in caudate neurons, with a temporal profile and regional distribution coinciding with the mitochondrial release of cytochrome c and caspase-9. Further, in postischemic caudate putamen in vivo and in isolated brain mitochondria in vitro, the authors found enhanced heterodimerization between Bax and the mitochondrial membrane permeabilization-related proteins adenine nucleotide translocator (ANT) and voltage-dependent anion channel. The ANT inhibitor bongkrekic acid prevented Bax and ANT interactions and inhibited Bax-triggered caspase-9 release from isolated brain mitochondria in vitro. Bongkrekic acid also offered significant neuroprotection against ischemia-induced caspase-3 and caspase-9 activation and cell death in the brain. These results strongly suggest that the Bax-mediated mitochondrial apoptotic signaling pathway may play an important role in ischemic neuronal injury.     

Excessive Autophagy Contributes to Neuron Death in Cerebral Ischemia

Aims: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. Methods: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo. Results: In both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3–7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. Conclusion: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.