Prolonged Anoxia Research Articles

Behaviour and mortality of benthic crustaceans in response to experimentally induced hypoxia and anoxia in situ

Crustaceans are sensitive to hypoxia and are therefore useful indicator organisms for oxygen depletions. We used the experimental anoxia generating unit in the northern Adriatic Sea to artificially induce and document hypoxia and anoxia on a small scale (0.25 m 2 per deployment). Behavioural responses and mortalities were documented for 9 crustacean species typical for the sub- littoral soft bottom of the Gulf of Trieste. All species showed a similar succession of atypical responses, albeit at different thresholds. The first reaction to declining dissolved oxygen (DO) was avoidance by climbing to more oxygenated (higher) positions. The animals left their shelters, altered their activity patterns, and exposed themselves to a higher risk of predation at mild hypoxia (2 to 1 ml l -1 DO). Moderate hypoxia (1 to 0.5 ml l -1 DO) triggered changes in inter- and intraspecific interac- tions, resulting in aggregations of up to 27 individuals at the highest elevations. At severe hypoxia (0.5 to 0.01 ml l -1 DO), sublethal responses such as discarding of camouflage (Ethusa mascarone) were recorded, and 54% of all crustaceans died. Anoxia left most remaining individuals immobile and moribund until death. Almost all responses were related to DO thresholds—hydrogen sulphide (H2S) had not yet evolved. Pisidia longimana, Galathea spp., and Macropodia spp. were the most sen- sitive; Pilumnus spinifer and Ebalia tuberosa were the somewhat more tolerant species. Only Nepin- notheres pinnotheres survived prolonged anoxia as well as high H2S concentrations.

Effects of cadmium on anaerobic energy metabolism and mRNA expression during air exposure and recovery of an intertidal mollusk Crassostrea virginica

Marine organisms are exposed to periodical oxygen deficiency and pollution stress in estuarine and coastal zones which may strongly affect their performance and survival. We studied the combined effects of exposure to a common pollutant, cadmium (Cd), and intermittent anoxia on anaerobic metabolism, energy status and mRNA expression of 13 genes involved in and/or controlled by the hypoxia inducible factor-1 (HIF-1) pathway in hepatopancreas of an intertidal bivalve, the eastern oyster Crassostrea virginica. In control oysters, prolonged anoxia resulted in a selective suppression of nitric oxide synthase (NOS) and upregulation of cytochrome c oxidase subunit IV (COX4) while the levels of other transcripts remained unchanged. During post-anoxic recovery, mRNA expression of hypoxia inducible factor-1α (HIF-1α) was elevated, phosphoenolpyruvate carboxykinase (PEPCK), NOS and LON protease suppressed, and mRNA expression of other studied genes not changed. Notably, most of the key glycolytic genes that are stimulated by HIF-1 in mammals, either remained unchanged or were downregulated in anoxic oysters suggesting a different mechanism of molecular response to oxygen deficiency. Patterns of transcriptional response during anoxia and reoxygenation were significantly altered by Cd exposure in a gene-specific manner. Anaerobic metabolism (indicated by accumulation of l-alanine, succinate and acetate during anoxia) was also suppressed in Cd-exposed oysters. In control oysters, ATP turnover rate ( M ATP) during anoxia was mostly sustained by anaerobic glycolysis with negligible contributions from ATP and PLA breakdown. In contrast, in Cd-exposed oysters ATP breakdown contributed significantly to anaerobic M ATP. Thus, while control oysters could efficiently defend the ATP levels and tissue energy status during prolonged anoxia, Cd-exposed oysters experienced a disturbance in tissue energy balance indicated by the depletion of ATP, a rapid decline in adenylate energy charge and increase in ADP/ATP ratios. This energy deficiency combined with suppression of anaerobic metabolism may strongly affect performance and survival of oysters in polluted estuaries where metal pollution may co-occur with “dead zones”.

Cadmium affects metabolic responses to prolonged anoxia and reoxygenation in eastern oysters (Crassostrea virginica)

Benthic marine organisms such as mollusks are often exposed to periodic oxygen deficiency (due to the tidal exposure and/or seasonal expansion of the oxygen-deficient dead zones) and pollution by metals [e.g., cadmium, (Cd)]. These stressors can strongly affect mollusks' survival; however, physiological mechanisms of their combined effects are not fully understood. We studied the effects of Cd exposure on metabolic responses to prolonged anoxia and subsequent recovery in anoxia-tolerant intertidal mollusks Crassostrea virginica (eastern oysters). Anoxia led to an onset of anaerobiosis indicated by accumulation of l-alanine, acetate, and succinate. Prolonged anoxia (for 6 days) caused a decline in the maximum activity of electron transport chain and ADP-stimulated (state 3) oxygen uptake by mitochondria (MO(2)), but no change in the resting (state 4) MO(2) of oyster mitochondria, along with a slight but significant reduction of mitochondrial respiratory control ratio. During reoxygenation, there was a significant overshoot of mitochondrial MO(2) (by up to 70% above the normoxic steady-state values) in control oysters. Mild mitochondrial uncoupling during prolonged shutdown in anoxic tissues and a subsequent strong stimulation of mitochondrial flux during recovery may help to rapidly restore redox status and protect against elevated reactive oxygen species formation in oysters. Exposure to Cd inhibits anaerobic metabolism, abolishes reoxygenation-induced stimulation of mitochondrial MO(2), and leads to oxidative stress (indicated by accumulation of DNA lesions) and a loss of mitochondrial capacity during postanoxic recovery. This may result in increased sensitivity to intermittent hypoxia and anoxia in Cd-exposed mollusks and will have implications for their survival in polluted estuaries and coastal zones.

Metabolic depression is delayed and mitochondrial impairment averted during prolonged anoxia in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935)

Lepidophthalmus louisianensis burrows deeply into oxygen-limited estuarine sediments and is subjected to extended anoxia at low tides. Large specimens (> 2 g) have a lethal time for 50% mortality (LT 50) of 64 h under anoxia at 25 °C. Small specimens (< 1 g) have a significantly higher LT 50 of 113 h, which is the longest ever reported for a crustacean. Whole body lactate levels rise dramatically under anoxia and exceed 120 μmol g.f.w. − 1 by 72 h. ATP, ADP, and AMP do not change during 48 h of anoxia, but arginine phosphate declines by over 50%. Thus arginine phosphate may help stabilize the ATP pool. Surprisingly, when compared to the aerobic resting rate, ATP production under anoxia declines only moderately during the first 12 h, and drops to only about 30% between 12 and 48 h. Finally, after 48 h of anoxia, a major metabolic depression to less than 5% occurs. Downregulation of metabolism is delayed in L. louisianensis compared to many invertebrates that exhibit facultative anaerobiosis. Bioenergetic constraints as a result of eventual metabolic depression lead to ionic disturbances like calcium overload and compromised membrane potential of mitochondria. Because these phenomena trigger apoptosis in mammalian species, we evaluated the susceptibility of ghost shrimp mitochondria to opening of the mitochondrial permeability transition pore (MPTP) and associated damage. Energized mitochondria isolated from hepatopancreas possess a pronounced capacity for calcium uptake. Exogenous calcium does not stimulate opening of the MPTP, which potentially could reduce cell death during prolonged anoxia.

Efficacy of exclusion fencing to protect ephemeral floodplain lagoon habitats from feral pigs (Sus scrofa)

Foraging by feral pigs can strongly affect wetland vegetation assemblages and so too wider ecological processes, although their effects on freshwater ecosystems have seldom been studied. We assessed the ecological effects of pig foraging in replicate fenced and unfenced ephemeral floodplain lagoons in tropical north-eastern Australia. Pig foraging activities in unfenced lagoons caused major changes to aquatic macrophyte communities and as a consequence, to the proportional amounts of open water and bare ground. The destruction of macrophyte communities and upheaval of wetland sediments significantly affected wetland turbidity, and caused prolonged anoxia and pH imbalances in the unfenced treatments. Whilst fencing of floodplain lagoons will protect against feral pig foraging activities, our repeated measures of many biological, physical and chemical parameters inferred that natural seasonal (i.e. temporal) effects had a greater influence on these variables than did pigs. To validate this observation requires measuring how these effects are influenced by the seemingly greater annual disturbance regime of variable flooding and drying in this tropical climate.

Terminal Ediacaran anoxia in deep-ocean: Trace element evidence from cherts of the Liuchapo Formation, South China

Here we report a detailed trace element study of the cherts from Liuchapo Formation, which is a terminal Ediacaran (551-542 Ma) succession in South China deposited in deep-water basinal setting. The REE of Liuchapo cherts shows similar features as observed for anoxic modern seawater (but not for hydrothermal fluids), characterized by positive La anomaly (LaN/CeN = 0.83–1.91, average 1.37), moderately negative Ce anomaly (0.53–1.1, average 0.73), positive Gd anomaly (average 1.08), positive Y anomaly (average 1.21), and depleted LREE and MREE. In addition, the Liuchapo cherts have low ΣREE (3.36–56.13 ppm, average 20.6 ppm), low Al2O3, Ti, Th and Zr concentrations, and high Y/Ho ratios (up to 43.9). The redox-sensitive trace elements concentrations in the cherts do not correlate with detrital input proxies. All of these features suggest that the redox-sensitive trace elements in the cherts were authigenically concentrated in water column and their concentrations thus are excellent indicators of ancient redox conditions. Very low Th/U ratios, high V/(V+Ni) and Fe⊤/Al ratios, enrichments of redox-sensitive trace elements (U, V, Mo), and low concentration of Mn in the cherts imply anoxia in the deep seawater. Our data reveal that the terminal Ediacaran ocean was not completely oxidized and the deep ocean was still anoxic, at least in South China. We propose that although the oxidative events existed in the terminal Ediacaran oceans, decomposition of organic matter prolonged anoxia in the deep ocean.

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.

Ametabolic embryos of Artemia franciscana accumulate DNA damage during prolonged anoxia

Encysted embryos of the brine shrimp Artemia franciscana are able to survive prolonged periods of anoxia even when fully hydrated. During this time there is no metabolism, raising the question of how embryos tolerate spontaneous, hydrolytic DNA damage such as depurination. When incubated at 28 degrees C and 40 degrees C for several weeks, hydrated anoxic embryos were found to accumulate abasic sites in their DNA with k=5.8x10(-11) s(-1) and 2.8x10(-10) s(-1), respectively. In both cases this is about 3-fold slower than expected from published observations on purified DNA. However, purified calf thymus DNA incubated under similar anoxic conditions at pH 6.3, the intracellular pH of anoxic cysts, also depurinated more slowly than predicted (about 1.7-fold), suggesting that cysts may in fact accumulate abasic sites only slightly more slowly than purified DNA. Upon reoxygenation of cysts stored under N(2) for 30 weeks at 28 degrees C, the number of abasic sites per 10(4) bp DNA fell from 21.1+/-4.0 to 9.8+/-2.0 by 12 h and to 6.2+/-2.1 by 24 h. Larvae hatched after 48 h and 72 h had only 0.59+/-0.17 and 0.48+/-0.07 abasic sites per 10(4) bp, respectively, suggesting that repair of these lesions had largely taken place before hatching commenced. Thus, unlike bacterial spores, Artemia cysts appear to have no specific protective mechanism beyond what might be afforded by chromatin structure to limit spontaneous depurination, and rely on the repair of accumulated lesions during the period between reoxygenation and hatching.

Proteomic analysis of anoxia tolerance in the developing zebrafish embryo

While some species and tissue types are injured by oxygen deprivation, anoxia tolerant organisms display a protective response that has not been fully elucidated and is well-suited to genomic and proteomic analysis. However, such methodologies have focused on transcriptional responses, prolonged anoxia, or have used cultured cells or isolated tissues. In this study of intact zebrafish embryos, a species capable of > 24 h survival in anoxia, we have utilized 2D difference in gel electrophoresis to identify changes in the proteomic profile caused by near-lethal anoxic durations as well as acute anoxia (1 h), a timeframe relevant to ischemic events in human disease when response mechanisms are largely limited to post-transcriptional and post-translational processes. We observed a general stabilization of the proteome in anoxia. Proteins involved in oxidative phosphorylation, antioxidant defense, transcription, and translation changed over this time period. Among the largest proteomic alterations was that of muscle cofilin 2, implicating the regulation of the cytoskeleton and actin assembly in the adaptation to acute anoxia. These studies in an intact embryo highlight proteomic components of an adaptive response to anoxia in a model organism amenable to genetic analysis to permit further mechanistic insight into the phenomenon of anoxia tolerance.

Effects of prolonged anoxia on glycogen dynamics of the crucian carp

Effects of prolonged anoxia on glycogen dynamics of the crucian carp

Does anoxia tolerance involve altering the energy currency towards PPi?

Oxygen deficit is an important abiotic stress influencing plants, because this condition results in an 'energy crisis'. Most species only survive short periods of anoxia, but several wetland species tolerate prolonged anoxia. Transcriptomic and proteomic studies, using anoxia-tolerant rice and anoxia-intolerant Arabidopsis, have provided evidence for the selective adoption of pyrophosphate (PPi) over ATP as high-energy donor molecules, which may contribute to anoxia tolerance. The use of PPi in some tolerant plant species is similar to that observed in many anaerobic prokaryotes. Investigations are being performed to better understand the origin and regulation of reversible PPi-dependent glycolytic enzymes such as cytosolic pyruvate phosphate dikinase, as well as PPi-consuming enzymes, which are engaged during the anoxic energy crisis. This will be crucial in unraveling this currency switch and its contribution to anoxia tolerance.

Commercially Available Gas-Permeable Cell Culture Bags May Not Prevent Anoxia in Cultured or Shipped Islets

Prolonged anoxia has deleterious effects on islets. Gas-permeable cell culture devices can be used to minimize anoxia during islet culture and especially during shipment when elimination of gas-liquid interfaces is required to prevent the formation of damaging gas bubbles. Gas-permeable bags may have several drawbacks, such as propensity for puncture and contamination, difficult islet retrieval, and significantly lower oxygen permeability than silicone rubber membranes (SRM). We hypothesized that oxygen permeability of bags may be insufficient for islet oxygenation. We measured oxygen transmission rates through the membrane walls of three different types of commercially available bags and through SRM currently used for islet shipment. We found that the bag membranes have oxygen transmission rates per unit area about 100-fold lower than SRM. We solved the oxygen diffusion-reaction equation for 150-μm diameter islets seeded at 3000 islet equivalents per cm 2, a density adequate to culture and ship an entire human or porcine islet preparation in a single gas-permeable device, predicting that about 40% of the islet volume would be anoxic at 22°C and about 70% would be anoxic at 37°C. Islets of larger size or islets accumulated during shipment would be even more anoxic. The model predicted no anoxia in islets similarly seeded in devices with SRM bottoms. We concluded that commercially available bags may not prevent anoxia during islet culture or shipment; devices with SRM bottoms are more suitable alternatives.

Mitochondrial ATP‐sensitive K+ channels regulate NMDAR activity in the cortex of the anoxic western painted turtle

Hypoxic mammalian neurons undergo excitotoxic cell death, whereas painted turtle neurons survive prolonged anoxia without apparent injury. Anoxic survival is possibly mediated by a decrease in N-methyl-d-aspartate receptor (NMDAR) activity and maintenance of cellular calcium concentrations ([Ca(2+)](c)) within a narrow range during anoxia. In mammalian ischaemic models, activation of mitochondrial ATP-sensitive K(+) (mK(ATP)) channels partially uncouples mitochondria resulting in a moderate increase in [Ca(2+)](c) and neuroprotection. The aim of this study was to determine the role of mK(ATP) channels in anoxic turtle NMDAR regulation and if mitochondrial uncoupling and [Ca(2+)](c) changes underlie this regulation. In isolated mitochondria, the K(ATP) channel activators diazoxide and levcromakalim increased mitochondrial respiration and decreased ATP production rates, indicating mitochondria were 'mildly' uncoupled by 10-20%. These changes were blocked by the mK(ATP) antagonist 5-hydroxydecanoic acid (5HD). During anoxia, [Ca(2+)](c) increased 9.3 +/- 0.3% and NMDAR currents decreased 48.9 +/- 4.1%. These changes were abolished by K(ATP) channel blockade with 5HD or glibenclamide, Ca(2+)(c) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by activation of the mitochondrial Ca(2+) uniporter with spermine. Similar to anoxia, diazoxide or levcromakalim increased [Ca(2+)](c) 8.9 +/- 0.7% and 3.8 +/- 0.3%, while decreasing normoxic whole-cell NMDAR currents by 41.1 +/- 6.7% and 55.4 +/- 10.2%, respectively. These changes were also blocked by 5HD or glibenclamide, BAPTA, or spermine. Blockade of mitochondrial Ca(2+)-uptake decreased normoxic NMDAR currents 47.0 +/- 3.1% and this change was blocked by BAPTA but not by 5HD. Taken together, these data suggest mK(ATP) channel activation in the anoxic turtle cortex uncouples mitochondria and reduces mitochondrial Ca(2+) uptake via the uniporter, subsequently increasing [Ca(2+)](c) and decreasing NMDAR activity.

Effects of anoxia, aglycemia, and acidosis on cytosolic Mg2+, ATP, and pH in rat sensory neurons

Sensory neurons can detect ischemia and transmit pain from various organs. Whereas the primary stimulus in ischemia is assumed to be acidosis, little is known about how the inevitable metabolic challenge influences neuron function. In this study we have investigated the effects of anoxia, aglycemia, and acidosis upon intracellular Mg(2+) concentration [Mg(2+)](i) and intracellular pH (pH(i)) in isolated sensory neurons. Anoxia, anoxic aglycemia, and acidosis all caused a rise in [Mg(2+)](i) and a fall in pH(i). The rise in [Mg(2+)](i) in response to acidosis appears to be due to H(+) competing for intracellular Mg(2+) binding sites. The effects of anoxia and aglycemia were mimicked by metabolic inhibition and, in a dorsal root ganglia (DRG)-derived cell line, the rise in [Mg(2+)](i) during metabolic blockade was closely correlated with fall in intracellular ATP concentration ([ATP](i)). Increase in [Mg(2+)](i) during anoxia and aglycemia were therefore assumed to be due to MgATP hydrolysis. Even brief periods of anoxia (<3 min) resulted in rapid internal acidosis and a rise in [Mg(2+)](i) equivalent to a decline in MgATP levels of 15-20%. With more prolonged anoxia (20 min) MgATP depletion is estimated to be around 40%. With anoxic aglycemia, the [Mg(2+)](i) rise occurs in two phases: the first beginning almost immediately and the second after an 8- to 10-min delay. Within 20 min of anoxic aglycemia [Mg(2+)](i) was comparable to that observed following complete metabolic inhibition (dinitrophenol + 2-deoxyglucose, DNP + 2-DOG) indicating a near total loss of MgATP. The consequences of these events therefore need to be considered in the context of sensory neuron function in ischemia.

Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis

Articular cartilage is an avascular tissue dependent on diffusion mainly from synovial fluid to service its metabolic requirements. Levels of oxygen (O(2)) in the tissue are low, with estimates of between 1 and 6%. Metabolism is largely, if not entirely, glycolytic, with little capacity for oxidative phosphorylation. Notwithstanding, the tissue requires O(2) and consumes it, albeit at low rates. Changes in O(2) tension also have profound effects on chondrocytes affecting phenotype, gene expression, and morphology, as well as response to, and production of, cytokines. Although chondrocytes can survive prolonged anoxia, low O(2) levels have significant metabolic effects, inhibiting glycolysis (the negative Pasteur effect), and also notably matrix production. Why this tissue should respond so markedly to reduction in O(2) tension remains a paradox. Ion homeostasis in articular chondrocytes is also markedly affected by the extracellular matrix in which the cells reside. Recent work has shown that ion homeostasis also responds to changes in O(2) tension, in such a way as to produce significant effects on cell function. For this purpose, O(2) probably acts via alteration in levels of reactive oxygen species. We discuss the possibility that O(2) consumption by this tissue is required to maintain levels of ROS, which are then used physiologically as an intracellular signalling device. This postulate may go some way towards explaining why the tissue is dependent on O(2) and why its removal has such marked effects. Understanding the role of oxygen has implications for disease states in which O(2) or ROS levels may be perturbed.

Regulation of extracellular glutamate levels in the long-term anoxic turtle striatum: coordinated activity of glutamate transporters, adenosine, KATP+ channels and GABA

Early in anoxia the mammalian brain experiences an uncontrolled release of glutamate, which combined with the failure of glutamate reuptake mechanisms, leads to massive neurotoxic increases in extracellular glutamate. By contrast, the anoxia tolerant turtle (Trachemys scripta) shows no increase in extracellular glutamate levels over many hours of anoxia. During the first hours of anoxia extracellular glutamate levels are maintained by a reduction in glutamate release (mainly due to the inhibition of neuronal vesicular glutamate release), combined with continued uptake by still active glutamate transporters. The early down-regulation in glutamate release is modulated by adenosine receptors and K (ATP) (+) channels, but is not affected by GABA(A )receptors. During long-term anoxia there is a further reduction in the rate of glutamate release, reaching 30% of normoxic control values at 5 h of anoxia. Adenosine and GABA(A) receptors but not K (ATP) (+) channels regulate this reduction in glutamate release. We conclude that the reduction in glutamate release during progressive anoxia is a dynamic process requiring continuous but changing synergistic activity of K (ATP) (+) channels, adenosine and GABA(A) receptors. The fact that there is a still active glutamate release and uptake in prolonged anoxia suggests that extracellular glutamate has a vital function in the deeply hypometabolic brain.

Tribute to P. L. Lutz: cardiac performance and cardiovascular regulation during anoxia/hypoxia in freshwater turtles

Freshwater turtles overwintering in ice-covered ponds in North America may be exposed to prolonged anoxia, and survive this hostile environment by metabolic depression. Here, we review their cardiovascular function and regulation, with particular emphasis on the factors limiting cardiac performance. The pronounced anoxia tolerance of the turtle heart is based on the ability to match energy consumption with the low anaerobic ATP production during anoxia. Together with a well-developed temporal and spatial energy buffering by creatine kinase, this allows for cellular energy charge to remain high during anoxia. Furthermore, the turtle heart is well adapted to handle the adverse effects of free phosphate arising when phosphocreatine stores are used. Anoxia causes tenfold reductions in heart rate and blood flows that match the metabolic depression, and blood pressure is largely maintained through increased systemic vascular resistance. Depression of the heart rate is not driven by the autonomic nervous system and seems to arise from direct effects of oxygen lack and the associated hyperkalaemia and acidosis on the cardiac pacemaker. These intra- and extracellular changes also affect cardiac contractility, and both acidosis and hyperkalaemia severely depress cardiac contractility. However, increased levels of adrenaline and calcium may, at least partially, salvage cardiac function under prolonged periods of anoxia.

Anoxia or oxygen and glucose deprivation in SH-SY5Y cells: A step closer to the unraveling of neuroglobin and cytoglobin functions

Several studies support the hypothesis that neuroglobin and cytoglobin play a protective role against cell death when cellular oxygen supply is critical. Although the underlying molecular mechanisms are unknown, previous reports suggest that this protection can be realised by the fact that they act as ROS scavengers. In this study, expression of neuroglobin and cytoglobin was evaluated in a human neuroblastoma cell line (SH-SY5Y) under conditions of anoxia or oxygen and glucose deprivation (OGD). The cells could survive prolonged anoxia without significant loss of viability. They became anoxia sensitive when deprived of glucose. OGD induced significant cell death after 16 h resulting in 54% dead cells after 32 h. Necrosis was the main process involved in OGD-induced cell death. After reoxygenation, apoptotic neurons became more abundant. Real-time quantitative PCR and Western blotting revealed that neuroglobin and cytoglobin were upregulated, the former under OGD and the latter under anoxic conditions. Under OGD, cell survival was significantly reduced after inhibiting cytoglobin expression by transfection with antisense ODN. Moreover, cell survival was significantly enhanced by neuroglobin or cytoglobin overexpression. When neuroglobin or cytoglobin protein expression increased or decreased, the H 2O 2 level was found to be lower or higher, respectively. We conclude that neuroglobin or cytoglobin act as ROS scavengers under ischemic conditions.

Effect of temperature and prolonged anoxia exposure on electrophysiological properties of the turtle (Trachemys scripta) heart

Cardiac activity of the turtle (Trachemys scripta) is greatly depressed with cold acclimation and anoxia. We examined what electrophysiological modifications accompany and perhaps facilitate this depression of cardiac activity. Turtles were first acclimated to 21 degrees C or 5 degrees C and held under either normoxic or anoxic (6 h at 21 degrees C; 14 days at 5 degrees C) conditions. We then measured cardiac action potentials (APs) using spontaneously contracting whole heart preparations and whole cell current densities of sarcolemmal ion channels using isolated ventricular myocytes under appropriate normoxic and anoxic conditions. Compared with 21 degrees C-acclimated turtles, 5 degrees C-acclimated turtles exhibited a less negative resting membrane potential (by 18-29 mV), a 4.7- to 6.8-fold slower AP upstroke rate, and a 4.2- to 4.9-fold greater AP duration. Correspondingly, peak densities of ventricular voltage-gated Na(+) (I(Na)) and L-type Ca(2+) currents and inward slope conductances of inward rectifier K(+) (I(K1)) channel current were approximately 1/7th (Q(10) = 3.4), 1/13th (Q(10) = 5.0), and one-half (Q(10) = 1.4) of those of 21 degrees C-acclimated ventricular myocytes, respectively. With anoxia at 21 degrees C, peak I(Na) density doubled and ventricular AP duration increased by 47%, a change proportional to the reported approximately 30% reduction of intrinsic heart rate. In contrast, with anoxia at 5 degrees C, ventricular AP characteristics were unaffected; of the ion currents investigated, only the inward conductance via I(K1) changed significantly (reduced by 46%). The present findings indicate that cold temperature, more so than prolonged anoxia, results in substantial modifications of cardiac APs and reduction of ventricular ion current densities. These changes likely prepare cardiac muscle for winter anoxia conditions.

Adenosine does not save the heart of anoxia-tolerant vertebrates during prolonged oxygen deprivation

Despite adenosine being regarded as an important signaling molecule capable of coordinating ATP supply and demand during periods of oxygen deprivation in anoxia-intolerant mammals, the importance of adenosinergic cardiovascular control in anoxia-tolerant vertebrates is poorly understood. Here, we report on adenosinergic cardiovascular control during normoxia and prolonged (hours to days) oxygen deprivation for three vertebrate species tolerant of severe hypoxia/anoxia, the closely related common (Cyprinus carpio) and crucian (Carassius carassius) carp, and the freshwater turtle (Trachemys scripta). Using an intra-arterial injection of the non-specific adenosine receptor antagonist aminophylline while measuring cardiac output (Q), heart rate (f(H)) and arterial blood pressure, we establish that adenosinergic cardiovascular control is unimportant during prolonged anoxia in the freshwater turtle (6 h at 21 degrees C and 14 d at 5 degrees C) and the crucian carp (5 d at 8 degrees C). In contrast, adenosinergic control contributes to the down-regulation of cardiac activity exhibited by 5 degrees C-acclimated common carp during a 12.5 h severe hypoxia (<0.3 mg O2 l(-1)) exposure. Specifically, aminophylline injection resulted in significant increases in f(H) and Q, and a decrease in total peripheral resistance. These species-specific differences in adenosinergic cardiovascular control during prolonged periods of oxygen deprivation may be related to differences displayed by these three species in their anoxia tolerance and survival strategies.