Delayed Neuronal Death After Brief Histotoxic Hypoxia In Vitro

Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 m M ) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 m M . These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.     

Primary cortical neuronal cultures reduce cellular energy utilization during anoxic energy deprivation

It has been widely hypothesized that neurons reduce cellular energy use in response to periods of energy deprivation. To test this hypothesis, we measured rates of energy use under normoxia and anoxia in immature (6 days in vitro) and mature (13 days in vitro) neuronal cultures. During anoxic incubation immature and mature cultures reduced cellular energy use by 80% and 45%, respectively. Reduced cellular energy use dramatically affected ATP depletion in neuronal cultures under anoxia. Intracellular ATP stores were expected to deplete within 3 min of anoxia. However, ATP was maintained at decreased but stabilized concentrations for at least 3 h. The capacity of neuronal cultures to reduce cellular energy use during anoxia correlated with their sensitivity towards simulated ischemia. Immature cultures, with the largest capacity to reduce cellular energy use, survived simulated ischemia 2.5 times longer than mature cultures. The addition of glutamate receptor antagonists to mature cultures further decreased cellular energy use during anoxia and significantly extended their survival time under simulated ischemia. This study verifies that primary cortical neuronal cultures reduce cellular energy use during energy deprivation. Additionally, we show that maturation of glutamate receptor activity increases non-depressible energy demand in neuronal cultures.     

Conditioning Heat Stress Reduces Excitotoxic and Apoptotic Components of Oxygen-Glucose Deprivation-Induced Neuronal Death In Vitro

Abstract: We investigated the effects of sublethal heat stress in murine cortical cell cultures exposed to combined oxygen and glucose deprivation. Pretreatment with sublethal heat stress mildly attenuated the widespread neuronal death induced a day later by 30–60 min of oxygen-glucose deprivation. Heat stress also blunted the increase in extracellular glutamate concentrations induced by the oxygen-glucose deprivation, as well as the neuronal death and ⁴⁵Ca²⁺ uptake induced by exogenous addition of NMDA, although no reduction was seen in neuronal death caused by exogenous kainate or in NMDA-induced whole-cell currents. However, arguing against the idea that the neuroprotective effect of heat stress against neuronal death was exclusively due to reduction of excitotoxicity was the finding that heat stress also reduced the neuronal apoptosis induced by oxygen-glucose deprivation in the presence of glutamate antagonists. This antiapoptotic effect was specific in that heat stress did not reduce neuronal vulnerability to staurosporine-induced apoptosis. Whereas heat stress transiently suppressed protein synthesis, achieving comparable protein synthesis inhibition with cycloheximide did not reproduce the neuroprotective effects of heat stress. These studies suggest that a conditioning heat stress is able to attenuate both the excitotoxic and the apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro, by mechanisms independent of protein synthesis reduction.     

Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen-glucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective KATP channel blocker glibenclamide did not. The non-selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide-pre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.     

21-Aminosteroids attenuate excitotoxic neuronal injury in cortical cell cultures

We studied the protective efficacy of novel 21-aminosteroids against several forms of neuronal injury in murine cortical cell cultures. Concentrations of 200 nM to 20 microM partially attenuated the damage induced by glucose deprivation, combined oxygen-glucose deprivation, or exposure to NMDA; maximal protection was less than that produced by NMDA antagonists, but the combination of a 21-aminosteroid plus an NMDA antagonist produced a greater benefit than either drug alone. 21-Aminosteroid addition did not attenuate NMDA-induced whole-cell current, but did block almost all of the damage induced by exposure to iron, a protective action consistent with inhibition of free radical-mediated lipid peroxidation. Lipid peroxidation may be a downstream event mediating a portion of the injury triggered by excess stimulation of NMDA receptors.     

Nitric Oxide Produced During Ischemia Is Toxic but Crucial to Preconditioning-Induced Ischemic Tolerance of Neurons in Culture

The present study investigated the roles of nitric oxide (NO) in preconditioning (PC)-induced neuronal ischemic tolerance in cortical cultures. Ischemia in vitro was simulated by subjecting cultures to both oxygen and glucose deprivation (OGD). A sublethal OGD (PC) significantly increased the survival rate of neurons when cultures were exposed to a lethal OGD 24 h later. Both the inhibition of nitric oxide synthase (NOS) and scavenging of NO during PC significantly attenuated the PC-induced neuronal tolerance. In addition, exposure to an NO donor emulated the PC. In contrast, the inhibition of NOS and the scavenging of NO during lethal OGD tended to increase the survival rate of neurons. This study suggested that NO produced during ischemia was fundamentally toxic, but critical to the development of PC-induced neuronal tolerance.     

Neuroprotective role of delta-opioid receptors in cortical neurons

We recently demonstrated that delta-opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons (days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([D-Ala2,D-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) mu- or kappa-opioid receptor inhibition (beta-funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.     

A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons

In order to investigate the relationship between nitric oxide-mediated regulation of mitochondrial function and excitotoxicity, the role of mitochondrial ATP synthesis and intracellular redox status on the mode of neuronal cell death was studied. Brief (5 min) glutamate (100 microM) receptor stimulation in primary cortical neurons collapsed the mitochondrial membrane potential (psi(m)) and transiently (30 min) inhibited mitochondrial ATP synthesis, causing early (1 h) necrosis or delayed (24 h) apoptosis. The transient inhibition of ATP synthesis was paralleled to a loss of NADH, which was fully recovered shortly after the insult. In contrast, NADPH and the GSH/GSSG ratio were maintained, but progressively decreased thereafter. Twenty-four hours after glutamate treatment, ATP was depleted, a phenomenon associated with a persistent inhibition of mitochondrial succinate-cytochrome c reductase activity and delayed necrosis. Blockade of either nitric oxide synthase (NOS) activity or the mitochondrial permeability transition (MPT) pore prevented psi(m) collapse, the transient inhibition of mitochondrial ATP synthesis, early necrosis and delayed apoptosis. However, blockade of NOS activity, but not the MPT pore, prevented the inhibition of succinate-cytochrome c reductase activity and delayed ATP depletion and necrosis. From these results, we suggest that glutamate receptor-mediated NOS activation would trigger MPT pore opening and transient inhibition of ATP synthesis leading to apoptosis in a neuronal subpopulation, whereas other groups of neurons would undergo oxidative stress and persistent inhibition of ATP synthesis leading to necrosis.     

Neuroprotective Effect of Hypothermia in Cortical Cultures Exposed to Oxygen-Glucose Deprivation or Excitatory Amino Acids

Abstract: We examined the effect of moderate hypothermia (30°C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen-glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor-mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.     

Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling

Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-D-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca²⁺-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], but not by a less active analog, Mn(III)tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)tetra(N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A₁ receptors, nitric oxide synthase, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase fail to suppress OGD preconditioning despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although it induces mitochondrial Ca²⁺ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 2,4-dinitrophenol. Un-couplers will prevent production of mitochondrial reactive oxygen species, implying nonmitochondrial targets by MnTBAP, Zn(II)TBAP, and DIDS. Emphasizing the importance of an increase in cytosolic Ca²⁺ during preconditioning, a Ca²⁺/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways that have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular glutamate is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning models. N-methyl-D-aspartate; Ca²⁺; antioxidants; mitochondria     

Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures.

To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investigated the efficacy of melatonin in serum-free primary neuronal cultures of rat cortex by using three different models of caspase-dependent apoptotic, excitotoxin-independent neurodegeneration and compared it to that in necrotic neuronal damage. Neuronal apoptosis was induced by either staurosporine or the neurotoxin ethylcholine aziridinium (AF64A) with a delayed occurrence of apoptotic cell death (within 72 h). The apoptotic component of oxygen-glucose deprivation (OGD) unmasked by glutamate antagonists served as a third model. As a model for necrotic cell death, OGD was applied. Neuronal injury was quantified by LDH release and loss of metabolic activity. Although melatonin (0.5 mM) partly protected cortical neurons from OGD-induced necrosis, as measured by a significant reduction in LDH release, it was not effective in all three models of apoptotic cell death. In contrast, exaggeration of neuronal damage by melatonin was observed in native cultures as well as after induction of apoptosis. The present data suggest that the neuroprotectiveness of melatonin strongly depends on the model of neuronal cell death applied. As demonstrated in three different models of neuronal apoptosis, the progression of the apoptotic type of neuronal cell death cannot be withhold or is even exaggerated by melatonin, in contrast to its beneficial effect in the necrotic type of cell death.     

GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation

Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.     

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.     

Ginsenoside Rd attenuates early oxidative damage and sequential inflammatory response after transient focal ischemia in rats

We previously found that ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, attenuates neuronal oxidative damage in vitro induced by hydrogen peroxide and oxygen-glucose deprivation. In this study, we sought to investigate the potential protective effects and associated mechanisms of Rd in a rat model of focal cerebral ischemia. Rats administered with Rd (0.1-200mg/kg) or vehicle was subjected to transient middle cerebral artery occlusion. Rd at the dose of 10-50mg/kg significantly reduced the infarct volume and improved the long-term neurological outcome up to 6 weeks after ischemia. To evaluate the underlying mechanisms, in vivo free radical generation was monitored using microdialysis, oxidative DNA damage was identified by 8-hydroxy-deoxyguanosine immunostaining, oxidative protein damage was identified by the assessment of protein carbonyl and advanced glycosylation end products, and lipid peroxidation was estimated by determining the malondialdehyde and 4-hydroxynonenal formations. Microdialysis results displayed a prominent inhibitory effect of Rd on the hydroxy radical formation trapped as 2,3- and 2,5-DHBA. Early accumulations of DNA, protein and lipid peroxidation products were also suppressed by Rd treatment. Although Rd partly preserved endogenous antioxidant activities in the ischemic penumbra, in sham rats without stroke, endogenous antioxidant activities were not affected by Rd. Furthermore, we assayed sequential inflammatory response in a later phase after ischemia. Rd significantly eliminated inflammatory injury as indicated by the suppression of microglial activation, inducible nitric oxide synthase and cyclooxygenase-2 expression. Collectively, these findings demonstrated that Rd exerts neuroprotection in transient focal ischemia, which may involve early free radicals scavenging pathway and a late anti-inflammatory effect.     

Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture

The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells. Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h. Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium. At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA. During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia. t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated. Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect. Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity. These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals. Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.     

Amyloid β Peptide (25–35) Inhibits Na+-Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l -glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l -glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.     

Intraischaemic hypothermia reduces free radical production and protects against ischaemic insults in cultured hippocampal slices

Hypothermia has been demonstrated to be an effective neuroprotective strategy in a number of models of ischaemic and excitotoxic neurodegeneration in vitro and in vivo. Reduced glutamate release and free radical production have been postulated as potential mechanisms underlying this effect but no definitive mechanism has yet been reported. In the current study, we have used oxygen-glucose deprivation in organotypic hippocampal slice cultures as an in vitro model of cerebral ischaemia. When assessed by propidium iodide fluorescence, reducing the temperature during oxygen-glucose deprivation to 31-33 degrees C was significantly neuroprotective but this effect was lost if the initiation of hypothermia was delayed until the post-insult recovery period. The neuroprotective effects of hypothermia were associated with a significant decrease in both nitric oxide production, as assessed by 3-amino-4-aminomethyl-2',7'-difluorofluorescein fluorescence, and superoxide formation. Further, hypothermia significantly attenuated NMDA-induced nitric oxide formation in the absence of hypoxia/hypoglycaemia. We conclude that the neuroprotective effects of hypothermia are mediated through a reduction in nitric oxide and superoxide formation and that this effect is likely to be downstream of NMDA receptor activation.     

Implication of PTEN in production of reactive oxygen species and neuronal death in in vitro models of stroke and Parkinson's disease

Oxidative stress plays crucial role in the pathogenesis of neurodegenerative diseases. However, the precise mechanism for an increased production of reactive oxygen species (ROS) under pathological conditions is not yet fully understood. We have recently demonstrated an implication of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a tumor suppressor, in ROS generation and neuronal apoptosis induced by staurosporine. These findings raised further interest whether PTEN functions as a common mediator of oxidative stress in neurodegenerative processes. To address this issue, neural cells were exposed to oxygen-glucose deprivation (OGD) and to the neurotoxin 1-methyl-4-phenylpyridinium iodide (MPP(+)), which mimic cerebral ischemia and Parkinson's disease, respectively. OGD for 4 h followed by 16 h of reoxygenation or incubation with MPP(+) (250 microM) for 48 h induced 33% and 45% neuronal death in rat hippocampal and in human dopaminergic SH-SY5Y neurons, respectively, accompanied by a gradual increase in the intracellular level of ROS. The increase in ROS by OGD and by MPP(+) did not cause oxidative inactivation of PTEN and thus, PTEN remains constitutively active. In support, the protein level of PTEN was not reduced in both cell cultures after challenging with OGD or MPP(+). Importantly, the elevated intracellular ROS levels and the neuronal death caused by OGD or by MPP(+) toxicity were significantly inhibited when PTEN was downregulated by a specific antisense oligonucleotide or by siRNA. Because SOD2 protein level is not altered either by knockdown of PTEN nor by an inhibition of the PI3K/Akt signalling, we suggest that SOD2 do not contribute to the pathomechanism of oxidative stress induced by PTEN or by inhibiting the related Akt signalling. The present study highlights PTEN as a crucial and common mediator of ROS generation and neuronal death and suggests that PTEN could become a potential therapeutic target for interfering with neurodegeneration.     

Ornithine Decarboxylase Activity in In Vivo and In Vitro Models of Cerebral Ischemia

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis, and an increase in putrescine after central nervous system (CNS) injury appears to be involved in neuronal death. Cerebral ischemia and reperfusion trigger an active series of metabolic events, which eventually lead to neuronal death. In the present study, ODC activity was evaluated following transient focal cerebral ischemia and reperfusion in rat. The middle cerebral artery (MCA) was occluded for 2 h in male rats with an intraluminal suture technique. Animals were sacrificed between 3 and 48 h of reperfusion following MCA occlusion, and ODC activity was assayed in cortex and striatum. ODC activity was also estimated in an in vitro ischemia model using primary rat cortical neuron cultures, at 6–24 h reoxygenation following 1 h oxygen-glucose deprivation (OGD). In cortex, following ischemia, ODC activity was increased at 3 h (P < .05), reached peak levels by 6–9 h (P < .001) and returned to sham levels by 48 h reperfusion. In striatum the ODC activity followed a similar time course, but returned to basal levels by 24 h. This suggests that ODC activity is upregulated in rat CNS following transient focal ischemia and its time course of activation is region specific. In vitro, ODC activity showed a significant rise only at 24 h reoxygenation following ischemic insult. The release of lactate dehydrogenase (LDH), an indicator for cell damage, was also significantly elevated after OGD. 0.25 mM -difluoromethylornithine (DFMO) inhibited ischemia-induced ODC activity, whereas a 10-mM dose of DFMO appears to provide some neuroprotection by suppressing both ODC activity and LDH release in neuronal cultures, suggesting the involvement of polyamines in the development of neuronal cell death.