Mechanisms of neuroprotection during ischemic preconditioning: lessons from anoxic tolerance

Different physiological adaptations for anoxia resistance have been described in the animal kingdom. These adaptations are particularly important in organs that are highly susceptible to energy deprivation such as the heart and brain. Among vertebrates, turtles are one of the species that are highly tolerant to anoxia. In mammals however, insults such as anoxia, ischemia and hypoglycemia, all cause major histopathological events to the brain. However, in mammals even ischemic or anoxic tolerance is found when a sublethal ischemic/anoxic insult is induced sometime before a lethal ischemic/anoxic insult is induced. This phenomenon is defined as ischemic preconditioning. Better understanding of the mechanisms inducing both anoxic tolerance in turtles or ischemic preconditioning in mammals may provide novel therapeutic interventions that may aide mammalian brain to resist the ravages of cerebral ischemia. In this review, we will summarize some of the mechanisms implemented in both models of tolerance, emphasizing physiological and biochemical similarities.     

Cultivation of Arabidopsis cell cultures in a stirred bioreactor at variable oxygen levels: Influence on tocopherol production

Abstract

In plants, an increased production of toxic oxygen species is commonly observed under low oxygen stress, but cellular responses still have to be fully investigated. Plant cell cultures can be a valuable tool to study plant metabolic responses to various environmental stresses including low oxygen condition. Arabidopsis suspension cultures growing in shake flasks were subjected to hypoxia by stopping shaking for different intervals, showing an increase of the antioxidant metabolite α‐tocopherol. In order to obtain a more controlled condition, cultivation of Arabidopsis suspension cultures was established in a 5‐l stirred bioreactor. A constant aeration of 20% dissolved oxygen was found to be the most suitable for cell growth. A 4‐h anoxic shock was induced by suspending the aeration and flushing into the vessel with nitrogen. During the anoxic stress, tocopherol levels resulted increased at the end of the treatment, indicating that the complete oxygen deprivation, indeed, induced a defence response involving antioxidant metabolism. The presence of an oxidative stress as a consequence of anoxic condition was also confirmed by the increased levels of H₂O₂. Overall, these results indicate that Arabidopsis suspension cultures grown in a stirred bioreactor can be a useful in vitro system for investigating low oxygen stress.     

Preconditioning with hyperbaric oxygen induces tolerance against oxidative injury via increased expression of heme oxygenase-1 in primary cultured spinal cord neurons

Hyperbaric oxygen (HBO) preconditioning can induce ischemic tolerance in the spinal cord. The effect can be attenuated by the administration of an oxygen free radical scavenger or by inhibition of antioxidant enzymes. However, the mechanism underlying HBO preconditioning of neurons against ischemic injury remains enigmatic. Therefore, in the present study primary cultured spinal cord neurons were treated with HBO and then subjected to a hydrogen peroxide (H(2)O(2)) insult. The results show that H(2)O(2) stimulation of the cultured spinal neurons caused severe DNA damage and decreased cell viability, and that these neurons were well protected against damage after a single exposure to HBO preconditioning (0.35 MPa, 98% O(2), 37 degrees C, 2 h). The protective effect started 4 h after pretreatment and lasted for at least 24 h. The cultured neurons after HBO treatment also exhibited increased heme oxygenase-1 (HO-1) expression at both the protein and mRNA levels, which paralleled the protective effect of HBO. Treatment with tin-mesoporphyrin IX (SnMP), a specific HO-1 inhibitor, before HBO pretreatment abolished the HBO-induced adaptive protection noted in the cultured spinal neurons. In conclusion, HBO preconditioning can protect primary cultured spinal cord neurons against oxidative stress, and the upregulation of HO-1 expression plays an essential role in HBO induced preconditioning effect.     

Preconditioning stimuli do not benefit the myocardium of hypoxia-tolerant rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss)</Emphasis>

In many vertebrates, a short episode of oxygen lack protects against myocardial necrosis during a subsequent, longer period of oxygen deprivation. This protective effect, termed preconditioning, also improves the functional recovery. Improved functional recovery has been reported for hypoxia-sensitive, in situ perfused rainbow trout hearts, but appears absent in another strain of rainbow trout that has a more hypoxia-tolerant heart. The results for the hypoxia-tolerant rainbow trout heart, however, might have occurred because the preconditioning stimuli were insufficient in either intensity or type to elicit cardioprotective effects. In the present study, we attempted to induce preconditioning in in situ perfused hearts from hypoxia-tolerant rainbow trout (Oncorhynchus mykiss), acclimated and tested at 10 °C, by either doubling the anoxic preconditioning stimulus (PO₂ of the perfusate <0.5 kPa) relative to earlier studies or by using short exposures to high concentrations of adrenaline. In addition, anoxic-preconditioning experiments were conducted at an acutely elevated temperature (15 °C) to increase myocardial sensitivity to oxygen lack. The effect of preconditioning stimuli was assessed by measuring cardiac performance before and after exposure to a 20-min anoxic challenge. In addition, myocardial condition was evaluated at the termination of the experiment by measuring myocardial concentrations of glycogen, high energy phosphates and lactate, as well as activities of pyruvate kinase and lactate dehydrogenase. Maximal cardiac performance in oxygenated control hearts was unchanged by the 2-h experimental protocol, whereas inclusion of a 20-min period of anoxia led to 25 and 35% reductions in maximal cardiac performance at 10 and 15 °C, respectively. Reduced contractility, however, could not be ascribed to myocardial necrosis, as the biochemical and energy state of the hearts was unaffected. Hence, anoxic exposure merely stunned the myocardium. At 10 °C, neither the anoxic nor adrenergic preconditioning protocols improved post-anoxic cardiac performance. Further, the preconditioning protocols did not reduce post-anoxic myocardial dysfunction at 15 °C, despite the increased cardiac sensitivity to anoxia at this temperature. Thus, despite using strong and different preconditioning stimuli compared with earlier studies, the cardio-protective effect of preconditioning seems to be absent in rainbow trout hearts that are inherently more hypoxia-tolerant.     

Astrocyte responses to injury: VEGF simultaneously modulates cell death and proliferation

Hypoxia is linked to changes in blood-brain barrier (BBB) permeability, and loss of BBB integrity is characteristic of many pathological brain diseases including stroke. In particular, astrocytes play a central role in brain homeostasis and BBB function. We investigated how hypoxia affects astrocyte survival and assessed whether VEGF release through hypoxia-inducible factor-1alpha (HIF-1alpha) induction plays a role in tolerance of these cells to insult. Thus primary astrocytes were subjected to normoxic (21% O(2)), hypoxic (1% O(2)), or near-anoxic (<0.1% O(2)) conditions in the presence or absence of glucose. Cell death was significantly initiated after combined oxygen glucose deprivation, and, surprisingly, astrocyte proliferation increased concomitantly. Near anoxic, but not hypoxic, conditions stabilized HIF-1alpha protein and provoked DNA binding activity, whereas oxygen and glucose deprivation accelerated HIF-1alpha accumulation. Unexpectedly, Hif-1alpha knockdown studies showed that elevated VEGF levels following increased insult was only partially due to HIF-1alpha induction, suggesting alternative mechanisms of VEGF regulation. Notably, endogenous VEGF signaling during insult was essential for cell fate since VEGF inhibition appreciably augmented cell death and reduced proliferation. These data suggest Hif-1 only partially contributes to VEGF-mediated astrocyte responses during chronic injury (as occurs in clinical hypoxic/ischemic insults) that may ultimately be responsible for disrupting BBB integrity.     

Induction of stress proteins in anoxic and hyperthermicSpodoptera frugiperda cells

In this study, we compare stress protein induction in anoxic and hyperthermicSpodoptera frugiperda cells. Anoxia transiently induces a cluster of heat shock proteins at 71 and 72 kDa. This is a subset of a larger group of stress proteins induced by heat shock. Several heat shock proteins reported in this study were previously undetected inS. frugiperda. With these additional proteins, the stress response of hyperthermicS. frugiperda closely resembles that ofDrosophila melanogaster. Prior investigations of stress protein induction during oxygen deprivation focused on mammalian cells. In sharp contrast to these cells, anoxicS. frugiperda cells neither induce glucose-regulated proteins nor suppress the heat shock family of 71/72 kDa proteins. These findings provide insight into the virtually unexplored area of stress protein induction in anoxic insect cells. In addition, they help to explain the effects of oxygen deprivation on heterologous protein yield from virally infected insect cells and to develop an oxygenregulated promoter for stably transformed insect cells.Abbreviations DO dissolved oxygen concentration - GRP's glucose-regulated proteins - HSP's heat shock proteins - ORP's oxygen-regulated proteins - PAGE polyacrylamide gel electrophoresis - Sf9 Spodoptera frugiperda cells     

Oxidative stress regulated expression of Ubiquitin Carboxyl-terminal Hydrolase-L1: Role in cell survival

The ubiquitin Carboxyl-terminal Hydrolase-L1 gene (UCHL1) is a key enzyme in the protein degradation pathway; however, its precise role in protecting cells under stress conditions is unclear. In the present study we investigated the activity of this gene in human NT2/D1 embryonal carcinoma cells subjected to oxygen-glucose deprivation (OGD) and reoxygenation. OGD/reoxygenation cause global metabolic changes due to energy withdrawal and the subsequent generation of reactive oxygen species which initiates either a stress-adaptation-survival response or cell death, depending on the severity of the insult. A bi-phasic change in UCHL1 expression was observed by Q-PCR, Western blotting and flow cytometry. Down regulation of UCHL1 was detected immediately after OGD treatment and its expression was subsequently restored and increased 6 h after OGD treatment as well as during reoxygenation. Furthermore, flow cytometry analysis detected a lower level of UCHL1 only in apoptotic cells that had severe loss of mitochondrial membrane potential. Accordingly, down-regulation of endogenous UCHL1 by antisense cDNA in mouse N2a neuroblastoma cells increased the cell’s sensitivity to OGD treatment. This down-regulation of endogenous UCHL1 led to the accumulation of p27, suggesting that UCHL1 is an essential gene to maintain cell homeostasis under normal growth and oxidative stress conditions.     

Overexpression of human copper/zinc-superoxide dismutase in transgenic animals attenuates the reduction of apurinic/apyrimidinic endonuclease expression in neurons after in vitro ischemia and after transient global cerebral ischemia

Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen-glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc-superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia.     

Survival and Recovery of Methanotrophic Bacteria Starved under Oxic and Anoxic Conditions

The effects of carbon deprivation on survival of methanotrophic bacteria were compared in cultures incubated in the presence and absence of oxygen in the starvation medium. Survival and recovery of the examined methanotrophs were generally highest for cultures starved under anoxic conditions as indicated by poststarvation measurements of methane oxidation, tetrazolium salt reduction, plate counts, and protein synthesis. Methylosinus trichosporium OB3b survived up to 6 weeks of carbon deprivation under anoxic conditions while maintaining a physiological state that allowed relatively rapid (hours) methane oxidation after substrate addition. A small fraction of cells starved under oxic and anoxic conditions (4 and 10%, respectively) survived more than 10 weeks but required several days for recovery on plates and in liquid medium. A non-spore-forming methanotroph, strain WP 12, displayed 36 to 118% of its initial methane oxidation capacity after 5 days of carbon deprivation. Oxidation rates varied with growth history prior to the experiments as well as with starvation conditions. Strain WP 12 starved under anoxic conditions showed up to 90% higher methane oxidation activity and 46% higher protein production after starvation than did cultures starved under oxic conditions. Only minor changes in biomass and morphology were seen for methanotrophic bacteria starved under anoxic conditions. In contrast, starvation under oxic conditions resulted in morphology changes and an initial 28 to 35% loss of cell protein. These data suggest that methanotrophic bacteria can survive carbon deprivation under anoxic conditions by using maintenance energy derived solely from an anaerobic endogenous metabolism. This capability could partly explain a significant potential for methane oxidation in environments not continuously supporting aerobic methanotrophic growth.     

Functional Significance of the Preconditioning-induced Down-regulation of Glutamate Transporter GLT-1 in Neuron/astrocyte Co-cultures

In the brain, prior sublethal ischemia (preconditioning, PC) is known to produce tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating what alterations were induced in neurons and/or astrocytes by PC treatment. The rise in the extracellular concentration of glutamate during ischemia was markedly suppressed by the prior PC treatment. Immunocytochemical and Western blot analyses demonstrated that the expression of the astrocytic glutamate transporter GLT-1 was transiently down-regulated after the PC insult. The PC insult possibly suppressed the neuron-derived factors up-regulating GLT-1. Here we show that PC-induced down-regulation of GLT-1 is crucial for the increased neuronal resistance to subsequent severe ischemic insult.     

Exogenous Hsc70, but not thermal preconditioning, confers protection to motoneurons subjected to oxidative stress

Proper sensing of stress and the initiation of the stress response are critical to maintaining cell viability in response to noxious stimuli. Induction of the stress response prior to the exposure of a lethal stress (preconditioning) can be protective. Heat shock proteins (Hsps), the main products of the stress response, are considered to be responsible for this protective effect. Most cells readily initiate a stress response, but some neuronal phenotypes, including motoneurons (MNs), have a diminished capacity to do so. We have found that, given a proper stimulus, MNs can execute a heat stress response; but, it does not protect them from death caused by hydrogen peroxide (H(2)O(2)) induced oxidative stress, despite inhibiting H(2)O(2)-induced caspase activation. Conversely, we demonstrate that incubation with the heat shock cognate 70 (Hsc70) protein prior to oxidative insult can protect MNs from oxidative stress. This survival promoting effect may be mediated through the substrate binding domain (SBD) of Hsc70. Our data suggest that stress preconditioning may not be beneficial to MNs, but that pharmacological interventions and alternative means of acquiring components of the stress response are an effective means of ameliorating lethal stress in MNs and may be potentially useful therapeutically in preventing pathological MN loss.     

Effect of sublethal 6-hydroxydopamine on the response to subsequent oxidative stress in dopaminergic cells: evidence for preconditioning

Exposure to sublethal stress can trigger endogenous protection against subsequent, higher levels of stress. We tested for this preconditioning phenomenon in a model of Parkinson's disease by applying 6-hydroxydopamine to the dopaminergic MN9D cell line. Exposure to sublethal concentrations of 6-hydroxydopamine (5-10 microM) protected against the toxic effects of a subsequent exposure to a higher concentration (50 microM), as measured by the Hoechst assay for nuclear viability. This was accompanied by little or no protection against 6-hydroxydopamine-induced lactate dehydrogenase release, decline in ATP, or reduction in (3)H-dopamine uptake. The antioxidant, N-acetyl cysteine (20 mM), when applied during preconditioning, abolished protection, as did the protein synthesis inhibitor, cycloheximide (0.2 microM). Preconditioning did not affect superoxide dismutase or glutathione peroxidase enzymes, or levels of heat shock protein-72. However, Bcl-2 protein levels rose with preconditioning. Preconditioning rapidly increased phosphorylation of kinases ERK1/2, Akt and JNK, and was abolished by pharmacological inhibitors of their activity. Finally, sublethal 6-hydroxydopamine preconditioned against the toxicity of proteasome inhibitor, MG-132 (1 microM). Thus, exposure of a dopaminergic cell line to sublethal oxidative stress can protect against additional oxidative stress due to translational and post-translational modifications, as well as confer 'cross-tolerance' against a different insult, proteasome inhibition.     

Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation

Prior exposure to sub toxic insults can induce a powerful endogenous neuroprotective program known as ischemic preconditioning. Current models typically rely on a single stress episode to induce neuroprotection whereas the clinical reality is that patients may experience multiple transient ischemic attacks (TIAs) prior to suffering a stroke. We sought to develop a neuron-enriched preconditioning model using multiple oxygen glucose deprivation (OGD) episodes to assess the endogenous protective mechanisms neurons implement at the metabolic and cellular level. We found that neurons exposed to a five minute period of glucose deprivation recovered oxygen utilization and lactate production using novel microphysiometry techniques. Using the non-toxic and energetically favorable five minute exposure, we developed a preconditioning paradigm where neurons are exposed to this brief OGD for three consecutive days. These cells experienced a 45% greater survival following an otherwise lethal event and exhibited a longer lasting window of protection in comparison to our previous in vitro preconditioning model using a single stress. As in other models, preconditioned cells exhibited mild caspase activation, an increase in oxidized proteins and a requirement for reactive oxygen species for neuroprotection. Heat shock protein 70 was upregulated during preconditioning, yet the majority of this protein was released extracellularly. We believe coupling this neuron-enriched multi-day model with microphysiometry will allow us to assess neuronal specific real-time metabolic adaptations necessary for preconditioning.     

Glucose deprivation increases nuclear DNA repair protein Ku and resistance to radiation induced oxidative stress in human cancer cells

Recent studies have indicated that nutrient deprivation particularly glucose may play a major role in tumor cell tolerance to a generally oxidative stress environment in solid tumors. Here, we studied the impact of glucose deprivation on the response of human colon (HT29) and prostate (DU145) cancer cells to γ radiation. A significant decrease in intracellular glucose level was observed in glucose deprived cells as measured by bioreductive assay. The survival of HT29 and DU145 were increased by 30 and 100% respectively when these cells were exposed to γ radiation in the absence of glucose compared to that in the presence of glucose. In glucose depleted medium, glutathione (GSH), a free radical scavenger, content remained the same, and showed no correlation with the radiation resistance induced by glucose deprivation. Glucose regulated protein78 (GRP78), a stress response survival protein, was not significantly increased in cells deprived of glucose for 4 h compared to those cells in glucose. DNA repair protein Ku, which is known to play a major role in cellular resistance to radiation, was significantly increased in glucose deprived cancer cells that showed enhanced radiation resistance. These results have demonstrated, for the first time, that glucose deprivation mediated stress increased the expression of nuclear Ku and resistance to radiation induced oxidative stress in human cancer cells. The additional resistance caused by glucose deprivation in cancer cells has clinical significance since solid tumors are known to have low level of glucose due to diffusion limited blood supply and higher metabolic activity. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo stress preconditioning

The heat shock or stress protein response is a highly conserved defense mechanism. Activation of the stress protein response by mild hyperthermia or by pharmacological agents allows cells to withstand a subsequent metabolic insult that would otherwise be lethal, a phenomenon referred as "thermotolerance" or "preconditioning." Heat shock response is characterized by increased expression of stress proteins that provide cellular protection, e.g., via increased chaperoning activity in all organisms, from bacteria to animals and humans. Indeed, there is experimental evidence that overexpression of specific heat shock proteins or heat shock factors produce protective effects similar to those observed after stress preconditioning. The purpose of this review is first to discuss the methods used to induce in vivo thermotolerance with mild hyperthermia or pharmacological agents. Then, as an example of the organ protection provided by in vivo stress preconditioning, the second part of this paper will examine how the induction of thermotolerance modulates the lung inflammatory response associated with acute lung injury, thus providing broad organ and tissue protection against oxidative stress associated this syndrome.     

Pharmacological induction of ischemic tolerance in hippocampal slices by sarcosine preconditioning

Brain ischemic tolerance is a protective mechanism induced by a preconditioning stimulus, which prepare the tissue against harmful insults. Preconditioning with N-methyl-d-aspartate (NMDA) agonists induces brain tolerance and protects it against glutamate excitotoxicity. Recently, the glycine transporters type 1 (GlyT-1) have been shown to potentiate glutamate neurotransmission through NMDA receptors suggesting an alternative strategy to protect against glutamate excitotoxicity. Here, we evaluated the preconditioning effect of sarcosine pre-treatment, a GlyT-1 inhibitor, in rat hippocampal slices exposed to ischemic insult. Sarcosine (300mg/kg per day, i.p.) was administered during seven consecutive days before induction of ischemia in hippocampus by oxygen/glucose deprivation (OGD). To access the damage caused by an ischemic insult, we evaluated cells viability, glutamate release, nitric oxide (NO) production, lactate dehydrogenase (LDH) levels, production of reactive oxygen species (ROS), and antioxidant enzymes as well as the impact of oxidative stress in the tissue. We observed that sarcosine reduced cell death in hippocampus submitted to OGD, which was confirmed by reduction on LDH levels in the supernatant. Cell death, glutamate release, LDH levels and NO production were reduced in sarcosine hippocampal slices submitted to OGD when compared to OGD controls (without sarcosine). ROS production was reduced in sarcosine hippocampal slices exposed to OGD, although no changes were found in antioxidant enzymes activities. This study demonstrates that preconditioning with sarcosine induces ischemic tolerance in rat hippocampal slices submitted to OGD.     

Intermittent Hypoxia Preconditioning-Induced Epileptic Tolerance by Upregulation of Monocarboxylate Transporter 4 Expression in Rat Hippocampal Astrocytes

Noxious stimuli applied at doses close to but below the threshold of cell injury induce adaptive responses that provide a defense against additional stress. Epileptic preconditioning protects neurons against status epilepticus and ischemia; however, it is not known if the converse is true. During hypoxia/ischemia (H/I), lactate released from astrocytes is taken up by neurons and is stored for energy, a process mediated by monocarboxylate transporter 4 (MCT4) in astroglia. The present study investigated whether H/I preconditioning can provide protection to neurons against epilepsy through upregulation of MCT4 expression in astrocytes in vitro and in vivo. An oxygen/glucose deprivation protocol was used in primary astrocyte cultures, while rats were subjected to an intermittent hypoxia preconditioning (IHP) paradigm followed by lithium-pilocarpine-induced epilepsy as well as lactate transportation inhibitor injection, with a subsequent evaluation of protein expression as well as behavior. H/I induced an upregulation of MCT4 expression, while an IHP time course of 5 days provided the greatest protection against epileptic seizures, which was most apparent by 3 days after IHP. However, lactate transport function disturbances can block the protective effect induced by IHP. These findings provide a potential basis for the clinical treatment of epilepsy.