Anoxia Exposure Research Articles

Pushing the limit: examining factors that affect anoxia tolerance in a single genotype of adult D. melanogaster.

Drosophila melanogaster is a promiscuous species that inhabits a large range of harsh environments including flooded habitats and varying temperature changes. To survive these environments, fruit flies have adapted mechanisms of tolerance that allow them to thrive. During exposure to anoxic stress, fruit flies and other poikilotherms enter into a reversible, protective coma. This coma can be manipulated based on controlled environmental conditions inside the laboratory. Here we utilize a common laboratory raised strain of D. melanogaster to characterize adaptation abilities to better understand coma recovery and survival limitations. Our goal is to mimic the fly's natural environments (wet anoxia) and relate findings to a typical gas induced environment (dry anoxia) that is commonly used in a laboratory. Despite the abundance of research regarding acute and chronic anoxic exposure and cold stress, the literature is lacking evidence linking anoxic stress with variable environmental conditions such as animal age and stress duration. We present novel ways to assess coma recovery and survival using readily available laboratory tools. Our findings suggest that younger age, exposure to colder temperatures and wet environments increase resistance to anoxic stress.

Characterization of mitochondrial bioenergetics in neonatal anoxic model of rats.

Neonatal anoxia at the time of birth can lead to mitochondrial dysfunction and further neurodevelopmental abnormalities. The present study investigated the mitochondrial bioenergetics and associated sensorimotor changes in the anoxic neonatal rats. Rat pups after 30h to birth (2days) were subjected to anoxia of two episodes (10min in each) at a time interval of 24h by passing 100%N2 into an enclosed chamber. Brain mitochondrial respiration was measured using clark type oxygen electrode. A significant decrease in brain respiratory control ratio (RCR; State III/IV respiration) at all-time points, complex I (24h) and complex II (30min, 6 and 24h) enzyme activities indicated loss of mitochondrial integrity and function A significant increase in levels of nitric oxide was observed after second anoxic episode at all-time points. A significant change in sensorimotor activity in terms of increased reflex latency was observed 24h after second episode in this model, which is an indication of loss of subcortical maturation. All the above changes were observed after second but not after the first anoxic exposure. Therefore, this anoxic model shows significant changes in mitochondrial bioenergetics, nitric oxide levels and sensorimotor effects after second episode of anoxia. This model may be helpful to evaluate mitochondrial targeted pharmacological intervention for the treatment of anoxia.

Diurnal effects of anoxia on the metabolome of the seagrass Zostera marina

We investigated the response, adaptation and tolerance mechanisms of the temperate seagrass Zostera marina to water column anoxia. We exposed Z. marina to a diurnal light/dark cycle under anoxia and assessed the metabolic response by measuring the metabolome with gas chromatography coupled to mass spectrometry (GC–MS). During anoxia and light exposure the roots showed an altered metabolome whereas the leaves were only marginally affected, indicating that photosynthetically derived oxygen could satisfy the oxygen demand in the leaves but not in the roots. Nocturnal anoxia caused a biphasic shift in the metabolome of roots and leaves. The first phase, after 15 h under anoxia and 3 h of darkness showed a fast increase of lactate, pyruvate, GABA (γ-aminobutyric acid), succinate, alanine and a decrease in glutamate and glutamine. The second phase, after 21 h under anoxia and 9 h of darkness showed a decrease in lactate and pyruvate and an increase in alanine, GABA and succinate. This reprogramming of the metabolome after 21 h under anoxia indicates a possible mitigation mechanism to avoid the toxic effects of anoxia. A pathway enrichment analysis proposes the alanine shunt, the GABA shunt and the 2-oxoglutarate shunt as such mitigation mechanisms that alleviate pyruvate levels and lead to carbon and nitrogen storage during anoxia. This work demonstrates the applicability of metabolomics to assess low oxygen stress responses of Z. marina and allows us to propose an anoxia recovery model.

Adaptation of Actin Cytoskeleton during Suspended Animation

Confronted with limited oxygen, numerous organisms demonstrate evolutionarily conserved responses, arresting fundamental biological processes to achieve a physiological state termed suspended animation. An example of direct relevance to human biology and disease is the vertebrate zebrafish, Danio rerio. Physiologically active embryos with heartbeat, circulation, and a functional central nervous system arrest all of these dynamic processes, enter a state of suspended animation during anoxic exposure and then recover with normal developmental program upon return to normoxia. The same response occurs with inhibitors of the oxidative phosphorylation, indicating that inhibition of the mitochondrial production of ATP, rather than absence of molecular oxygen, is the proximate signal inducing suspended animation. Although this unique defense strategy has been extensively studied for more than a century, the biophysical mechanisms remain largely unknown. Our proteomic analysis during the first hour of anoxia implicated rapid regulation of the actin cytoskeleton.We have now discovered a rapid and reversible adaptation of actin cytoskeleton during anoxia. Using fluorescent labels that target monomeric and filamentous actin, we have quantified changes in the distribution of muscle fibers in somites, an increase in the abundance of monomeric actin in pronephric duct, and thickening of filamentous actin bundles at the cortex of keratinocytes. Consistent with these observations, we observe rapid arrest in growth of muscle fibers during anoxia and rapid recovery during normoxia. The time course of these events suggests that suspended animation requires rapid and dynamic adaptation of the existing cytoskeleton and our data suggest that this process may be critical to tissue preservation and survival.

Characterizing the metabolic capacity of the anoxic hagfish heart

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia at 10°C. Such anoxia tolerance demands the mobilization of anaerobic fuels and the removal of metabolic wastes--processes that require a functional heart. The purpose of this study was to measure the metabolic response of the excised, cannulated hagfish heart to anoxia using direct calorimetry. These experiments were coupled with measurements of cardiac pH and metabolite concentrations, at multiple time points, to monitor acid-base balance and anaerobic ATP production. We also exposed hagfish to anoxia to compare the in vitro responses of the excised hearts with the in vivo responses. The calorimetry results revealed a significant reduction in the rate of metabolic heat production over the first hour of anoxia exposure, and a recovery over the subsequent 6 h. This response is likely attributable to a rapid anoxia-induced depression of aerobic ATP-production pathways followed by an upregulation of anaerobic ATP-production pathways such that the ATP production rate was restored to that measured in normoxia. Glycogen-depletion measurements suggest that metabolic processes were initially supported by glycolysis but that an alternative fuel source was used to support the sustained rates of ATP production. The maintenance of intracellular pH during anoxia indicates a remarkable ability of the myocytes to buffer/regulate protons and thus protect cardiac function. Altogether, these results illustrate that the low metabolic demand of the hagfish heart allows for near-routine levels of cardiac metabolism to be supported anaerobically. This is probably a significant contributor to the hagfish's exceptional anoxia tolerance.

Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence

Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development.

Oxygen radical formation in anoxic transgression and anoxia-reoxygenation: Foe or phantom? Experiments with a hypoxia tolerant bivalve

Intertidal blue mussels, Mytilus edulis, experience hypoxia reoxygenation during tidal emersion and resubmersion cycles, and this is often suggested to represent a major stress for the animals, especially for their respiratory tissues, the gills. We exposed mussels to experimental short and prolonged anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on reactive oxygen species (mainly ROS: superoxide anion, O2·− and hydrogen peroxide, H2O2), formation using live imaging techniques and confocal microscopy. Our aim was to understand if this “natural stress” would indeed produce oxidative damage and whether antioxidant defenses are induced under anoxia, to prevent oxidative damage during reoxygenation. Exposure to declining pO2 in the respiration chamber caused an increase of gill metabolic rate between 21 and 10 kPa, a pO2 range in which whole animal respiration is reported to be oxyregulating. Exposure of the animals to severe anoxia caused an onset of anaerobiosis (succinate accumulation) and shifted high and low critical pc values (pc1: onset of oxyregulation in gills, pc2: switch from oxyregulation to oxyconformity) to higher pO2. Concentrations of both ROS decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS burst induced lipid peroxidation in the mantle, but neither were protein carbonyl levels increased (oxidative damage in the protein fraction), nor did the tissue glutathione concentration change in the gills. Further, analysis of apoptosis markers indicated no induction of cell death in the gills. To our knowledge, this is the first paper that directly measures ROS formation during anoxia reoxygenation in mussels. We conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under these experimental conditions.

Avoidance of apoptosis in embryonic cells of the annual killifish Austrofundulus limnaeus exposed to anoxia.

Embryos of the annual killifish Austrofundulus limnaeus have unequalled ability among vertebrates to survive long-term anoxia. Surprisingly, these embryos can survive for months in anoxia despite a large-scale decrease in ATP levels during the initial hours of anoxic exposure. These conditions are known to trigger apoptotic cell death in mammalian cells as a result of ischemia or anoxia. Anoxia-induced induction of apoptosis was investigated in four developmental stages of A . limnaeus that differ in their tolerance of anoxia, and thus may respond to anoxia uniquely. Exposure to staurosporine was used to determine if A . limnaeus cells were competent to enter apoptosis via cues other than anoxia. Apoptotic cells were identified by TUNEL assays and by measuring caspase 3/7 activity. Exposure to 48 hr of anoxia did not induce an increase in TUNEL-positive cells and generally did not lead to an increase in caspase 3/7 activity. However, treatment of anoxic embryos with 10 μM staurosporine resulted in a significant increase in caspase 3/7 activity in both normoxic and anoxic embryos. These results suggest that apoptosis is avoided in embryos of A . limnaeus following exposure to anoxia at least in part by mechanisms that prevent the activation of caspase 3/7 activity. While this mechanism remains unknown, it may be triggered by a protein kinase that can be experimentally inhibited by staurosporine.

Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart

The freshwater turtle Trachemys scripta can survive in the complete absence of O2 (anoxia) for periods lasting several months. In mammals, anoxia leads to mitochondrial dysfunction, which culminates in cellular necrosis and apoptosis. Despite the obvious clinical benefits of understanding anoxia tolerance, little is known about the effects of chronic oxygen deprivation on the function of turtle mitochondria. In this study, we compared mitochondrial function in hearts of T. scripta exposed to either normoxia or 2 weeks of complete anoxia at 5°C and during simulated acute anoxia/reoxygenation. Mitochondrial respiration, electron transport chain activities, enzyme activities, proton conductance and membrane potential were measured in permeabilised cardiac fibres and isolated mitochondria. Two weeks of anoxia exposure at 5°C resulted in an increase in lactate, and decreases in ATP, glycogen, pH and phosphocreatine in the heart. Mitochondrial proton conductance and membrane potential were similar between experimental groups, while aerobic capacity was dramatically reduced. The reduced aerobic capacity was the result of a severe downregulation of the F1FO-ATPase (Complex V), which we assessed as a decrease in enzyme activity. Furthermore, in stark contrast to mammalian paradigms, isolated turtle heart mitochondria endured 20 min of anoxia followed by reoxygenation without any impact on subsequent ADP-stimulated O2 consumption (State III respiration) or State IV respiration. Results from this study demonstrate that turtle mitochondria remodel in response to chronic anoxia exposure and a reduction in Complex V activity is a fundamental component of mitochondrial and cellular anoxia survival.

PGE2- metabolite levels in CSF correlate to HIE score and outcome after perinatal asphyxia

Acute anoxic exposure rapidly increases prostaglandin E2 (PGE2 ) production and release in neonatal mice brains. We hypothesize that PGE2 is released in human cerebrospinal fluid (CSF) during perinatal asphyxia and that it might be used as a biomarker for perinatal asphyxia. In full-term infants with lumbar puncture performed within 72h of birth (n=35), CSF was analysed for prostaglandin E2 metabolite (PGEM) using an enzyme immunoassay. Term infants with suspected but unverified infections were used as controls (n=11). Hypoxic-ischaemic encephalopathy (HIE) was classified as mild, moderate or severe (HIE I-III). Neurological assessment of surviving patients was performed at 18months of age. Prostaglandin E2 metabolite levels correlated to a low Apgar score at 5min (p<0.01) and 10min (p<0.01), a low pH (p<0.001) and HIE score (p<0.05). The HIE-III cases (n=7) had significantly higher PGEM levels compared with both controls and the HIE-I group (n=8). Irrespective of HIE grade, patients with adverse or fatal outcome had higher PGEM values compared with controls and asphyxiated infants with normal outcome (p<0.05). PGE2 is released during anoxic events in newborn infants, and PGEM may be useful as a biomarker for estimating degree of insult and predicting long-term outcome after perinatal asphyxia.

Genetic and Dietary Affects on Oxygen Deprivation Response and Survival in C. elegans

Oxygen deprivation, which is central to many human‐health related issues (Ex: stroke, cardiac and pulmonary disorders) occurs frequently in the elderly and can have a more detrimental affect on individuals who are diabetic or obese. We are using Caenorhabditis elegans to examine how environment, diet, age and genotype influences oxygen deprivation response and survival. C. elegans fed the standard OP50 diet and grown at 20C will survive severe oxygen deprivation (hypoxia and anoxia). However, survival rate is severely decreased if anoxia exposure time is increased or if genetic mutations lead to sensitivity to hypoxia. We are examining the genetic and dietary affects on oxygen deprivation response and survival. Factors associated with long‐term anoxia tolerance include age (3–5 day old adults hermaphrodites survive long‐term anoxia), a decrease in ovulation rate, development at 25C instead of 20C, a diet of HT115 E. coli or pretreatment with the diabetic drug metformin. Long‐term anoxia survival rate is associated with an increase in carbohydrate stores (glycogen, treholose) in the intestine however a glucose‐supplemented diet leads to sensitivity to anoxia exposure, suggesting that a homeostatic balance of carbohydrate stores is important for oxygen deprivation survival. Analysis of mutants identified the following signaling pathways as influencing anoxia survival: insulin‐like signaling (daf‐2/daf‐16), developmental pathways that alter germline function (glp‐1), carbohydrate metabolism (aak‐2), hexosamine signaling (oga‐1) and methyglycoxal protein modifications. Insulin‐signaling, carbohydrate homeostasis, oxidative stress and hexosamine signaling impact diabetes in humans suggesting that C. elegans can be used as a cellular and genetic model to understand the relationship between diet, metabolism, oxygen deprivation stress and disease states such as type II diabetes.

Excess post-hypoxic oxygen consumption is independent from lactate accumulation in two cyprinid fishes

Carassius carassius responds to hypoxic conditions by conversion of lactate into ethanol, which is excreted over the gills. However, a closely related species, Cyprinus carpio, does not possess the ability to produce ethanol and would be expected to accumulate lactate during hypoxic exposure. While the increase in oxygen consumption in fish required following strenuous exercise or low environmental oxygen availability has been frequently considered, the primary contributing mechanism remains unknown. This study utilized the close relationship but strongly divergent physiology between C. carpio and C. carassius to examine the possible correlation between excess post-hypoxic oxygen consumption (EPHOC) and lactate accumulation. No difference in the EPHOC:O2 deficit ratio was observed between the two species after 2.5h anoxia, with ratios of 2.0±0.6 (C. carpio) and 1.3±0.3 (C. carassius). As predicted, lactate accumulation dynamics did significantly differ between the species in both plasma and white muscle following anoxic exposure. Significant lactate accumulation was seen in both plasma and muscle in C. carpio, but there was no accumulation of lactate in white muscle tissue of C. carassius. These findings indicate that lactate accumulated as a consequence of 2.5h anoxic exposure is not a major determinant of the resulting EPHOC.

Isolation technique and proteomic analysis of the erythrocyte ghosts of red‐eared turtle (Trachemys scripta)

To proceed proteomic analysis of erythrocyte of the red-eared turtle Trachemys scripta, a method for obtaining turtle erythrocyte ghosts (TEG) was first developed. After hypotonic lysis using a special buffer, forcing the erythrocyte through the syringe with an "N"-shaped needle, applying low speed homogenizing and differential centrifugation, highly purified TEG fractions were obtained. The isolated TEG proteins were treated with in-gel digestion separated by SDS-PAGE or in-solution digestion followed by HPLC predissociation, and then identified by nano-ESI-LC MS/MS techniques. A total of 169 TEG proteins was identified, validated, and categorized. Among these proteins, tubulins, and cell-surface-located F-type ATP synthase revealed important information into the super tolerance of Trachemys scripta in anoxia and low temperature exposure. Altogether, this study not only provided a method to isolate high quality TEG and a dataset of comprehensive characterization of TEG proteins, but also provides a tool for proteomic research of all nucleated red blood cells, and thus opened a new research field for exploring the mechanisms of super tolerance of turtles in harsh environment.

Activation of the unfolded protein response during anoxia exposure in the turtle Trachemys scripta elegans

Red-eared slider turtles, Trachemys scripta elegans, can survive for several weeks without oxygen when submerged in cold water. We hypothesized that anaerobiosis is aided by adaptive up-regulation of the unfolded protein response (UPR), a stress-responsive pathway that is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER) and functions to restore ER homeostasis. RT-PCR, western immunoblotting and DNA-binding assays were used to quantify the responses and/or activation status of UPR-responsive genes and proteins in turtle tissues after animal exposure to 5 or 20 h of anoxic submergence at 4 °C. The phosphorylation state of protein kinase-like ER kinase (PERK) (a UPR-regulated kinase) and eukaryotic initiation factor 2 (eIF2α) increased by 1.43-2.50 fold in response to anoxia in turtle heart, kidney, and liver. Activation of the PERK-regulated transcription factor, activating transcription factor 4 (ATF4), during anoxia was documented by elevated atf4 transcripts and total ATF4 protein (1.60-2.43 fold), increased nuclear ATF4 content, and increased DNA-binding activity (1.44-2.32 fold). ATF3 and GADD34 (downstream targets of ATF4) also increased by 1.38-3.32 fold in heart and liver under anoxia, and atf3 transcripts were also elevated in heart. Two characteristic chaperones of the UPR, GRP78, and GRP94, also responded positively to anoxia with strong increases in both the transcript and protein levels. The data demonstrate that the UPR is activated in turtle heart, kidney, and liver in response to anoxia, suggesting that this pathway mediates an integrated stress response to protect tissues during oxygen deprivation.

Regulation of p53 by reversible post-transcriptional and post-translational mechanisms in liver and skeletal muscle of an anoxia tolerant turtle, Trachemys scripta elegans

The red-eared slider turtle (Trachemys scripta elegans) exhibits well-developed natural anoxia tolerance that depends on multiple biochemical adaptations, including anoxia-induced hypometabolism. We hypothesized that signaling by the p53 protein could aid in establishing the hypometabolic state by arresting the cell cycle, protecting against DNA damage as well as altering pathways of energy metabolism. Immunoblotting was used to evaluate the regulation and post-transcriptional modifications of p53 in liver and skeletal muscle of red-eared slider turtles subjected to 5h or 20h of anoxic submergence. Tissue specific regulation of p53 was observed with the liver showing a more rapid activation of p53 in response to anoxia as well as differential expression of seven serine phosphorylation and two lysine acetylation sites when compared with skeletal muscle. Protein expression of MDM2, a major p53 inhibitor, was also examined but did not change during anoxia. Reverse-transcriptase PCR was used to assess transcript levels of selected p53 target genes (14-3-3σ, Gadd45α and Pgm) and one microRNA (miR-34a); results showed down-regulation of Pgm and up-regulation of the other three. These findings show an activation of p53 in response to anoxia exposure and suggest an important role for the p53 stress response pathway in regulating natural anoxia tolerance and hypometabolism in a vertebrate facultative anaerobe.

MicroRNA Regulation in Extreme Environments: Differential Expression of MicroRNAs in the Intertidal Snail Littorina littorea During Extended Periods of Freezing and Anoxia

Several recent studies of vertebrate adaptation to environmental stress have suggested roles for microRNAs (miRNAs) in regulating global suppression of protein synthesis and/or restructuring protein expression patterns. The present study is the first to characterize stress-responsive alterations in the expression of miRNAs during natural freezing or anoxia exposures in an invertebrate species, the intertidal gastropod Littorina littorea. These snails are exposed to anoxia and freezing conditions as their environment constantly fluctuates on both a tidal and seasonal basis. The expression of selected miRNAs that are known to influence the cell cycle, cellular signaling pathways, carbohydrate metabolism and apoptosis was evaluated using RT-PCR. Compared to controls, significant changes in expression were observed for miR-1a-1, miR-34a and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-125b, miR-29b and miR-2a in foot muscle after freezing exposure at −6°C for 24h (P<0.05). In addition, in response to anoxia stress for 24h, significant changes in expression were also observed for miR-1a-1, miR-210 and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-29b and miR-2a in foot muscle (P<0.05). Moreover, protein expression of Dicer, an enzyme responsible for mature microRNA processing, was increased in foot muscle during freezing and anoxia and in hepatopancreas during freezing. Alterations in expression of these miRNAs in L. littorea tissues may contribute to organismal survival under freezing and anoxia.

Gene Expression and Physiological Changes of Different Populations of the Long-Lived Bivalve Arctica islandica under Low Oxygen Conditions

The bivalve Arctica islandica is extremely long lived (>400 years) and can tolerate long periods of hypoxia and anoxia. European populations differ in maximum life spans (MLSP) from 40 years in the Baltic to >400 years around Iceland. Characteristic behavior of A. islandica involves phases of metabolic rate depression (MRD) during which the animals burry into the sediment for several days. During these phases the shell water oxygen concentrations reaches hypoxic to anoxic levels, which possibly support the long life span of some populations. We investigated gene regulation in A. islandica from a long-lived (MLSP 150 years) German Bight population and the short-lived Baltic Sea population, experimentally exposed to different oxygen levels. A new A. islandica transcriptome enabled the identification of genes important during hypoxia/anoxia events and, more generally, gene mining for putative stress response and (anti-) aging genes. Expression changes of a) antioxidant defense: Catalase, Glutathione peroxidase, manganese and copper-zinc Superoxide dismutase; b) oxygen sensing and general stress response: Hypoxia inducible factor alpha, Prolyl hydroxylase and Heat-shock protein 70; and c) anaerobic capacity: Malate dehydrogenase and Octopine dehydrogenase, related transcripts were investigated. Exposed to low oxygen, German Bight individuals suppressed transcription of all investigated genes, whereas Baltic Sea bivalves enhanced gene transcription under anoxic incubation (0 kPa) and, further, decreased these transcription levels again during 6 h of re-oxygenation. Hypoxic and anoxic exposure and subsequent re-oxygenation in Baltic Sea animals did not lead to increased protein oxidation or induction of apoptosis, emphasizing considerable hypoxia/re-oxygenation tolerance in this species. The data suggest that the energy saving effect of MRD may not be an attribute of Baltic Sea A. islandica chronically exposed to high environmental variability (oxygenation, temperature, salinity). Contrary, higher physiological flexibility and stress hardening may predispose these animals to perform a pronounced stress response at the expense of life span.

Tolerance of benthic macrofauna to hypoxia and anoxia in shallow coastal seas: a realistic scenario

Oxygen depletion can lead to the collapse of benthic ecosystems, i.e. to dead zones, and large-scale biodiversity loss. Based on mortality and survival, we evaluated ranges of sensi- tivity and tolerance to hypoxia levels, duration of anoxia and H2S exposure across taxa and key life habits. Experiments were conducted on a sublittoral soft-bottom under realistic in situ conditions in a community setting featuring both a well-developed epi- and infauna. Overall, 495 individuals representing 40 species were examined over almost 1000 h (using time-lapse camera and sensor arrays). Mortality started at dissolved oxygen (DO) concentrations <0.5 ml l �1 and centred at the transition from severe hypoxia to early anoxia. A total of 58% of the individuals belonging to 27 species died. Thirteen species (39 individuals) died exclusively during anoxia. All of the individu- als that died during hypoxia, and most of those that died during anoxia, did so before the onset of hydrogen sulphide (H2S). In 11 species, all individuals survived: survival rates were highest among molluscs, anthozoans and ascidians. In contrast, most polychaetes, decapods and echino- derms died. Epifauna was more vulnerable than infauna, mobile forms were more vulnerable than sessile forms, and predators more vulnerable than deposit-feeders and suspension-feeders. While hypoxia primarily affected total abundance, anoxia significantly reduced the number of species. The former represents a quantitative, the latter a qualitative decline in ecosystem function. Most of the macrofauna may initially survive shorter-term (day-long) or intermittent hypoxia, but the onset of anoxia marks community collapse and biodiversity loss.

Evidence for cell cycle suppression and microRNA regulation of cyclin D1 during anoxia exposure in turtles

The red-eared slider turtle (Trachemys scripta elegans) has a well-developed natural tolerance for oxygen deprivation that derives from biochemical adaptations, including anoxia-induced suppression of metabolic rate. We hypothesized that mechanisms that suppress ATP-expensive cell cycle activity would contribute significantly to establishing the hypometabolic state during anaerobiosis. Cyclin D1 is a critical regulator of the G1 phase of the cell cycle and is regarded as key to initiating cell proliferation. The relative protein expression of cyclin D1 was analyzed in both whole-cell and nuclear fractions of liver, kidney and skeletal muscle from turtles exposed to 5 or 20 h of submergence anoxia. Expression of cyclin D1 in both total and nuclear fractions decreased significantly under anoxia in liver and kidney as compared with aerobic controls, but no significant change occurred in muscle. The relative phosphorylation state of cyclin D1 (threonine 286) was also unchanged during anoxia in all tissues. Since phosphorylation of threonine 286 is necessary for proteasomal degradation of cyclin D1, this implies that an alternative mechanism is responsible for cyclin D1 suppression in anoxia. Levels of cyclin D1 mRNA transcripts did not change under anoxia in any tissue, so a post-transcriptional method of regulation was implicated. Analysis of the 3’UTR of cyclin D1 showed the presence of both an AU-rich region and a conserved binding site for microRNA-16-1 and microRNA-15a. Levels of both microRNAs increased in liver and kidney (but not in muscle) under anoxic conditions, implicating microRNA inhibition of mRNA translation as the mechanism underlying the suppression of cyclin D1 protein levels in the anoxic turtle.

Postnatal loss of brainstem serotonin neurones compromises the ability of neonatal rats to survive episodic severe hypoxia

Pet-1(-/-) mice with a prenatal, genetically induced loss of 5-hydroxytryptamine (5-HT, serotonin) neurones are compromised in their ability to withstand episodic environmental anoxia via autoresuscitation. Given the prenatal role of 5-HT neurones in the development of neural networks, here we ask if a postnatal loss of 5-HT neurones also compromises autoresuscitation. We treated neonatal rat pups at postnatal day (P)2-3 with an intra-cisternal injection of 5,7-dihydroxytryptamine (5,7-DHT; ~40 μg; n = 8) to pharmacologically lesion the 5-HT system, or vehicle (control; n = 14). At P7-10 we exposed unanaesthetized treated and control pups to 15 episodes of environmental anoxia (97% N(2), 3% CO(2)). Medullary 5-HT content was reduced 80% by 5,7-DHT treatment (P < 0.001). Baseline ventilation (V(E)), metabolic rate (V(O(2))), ventilatory equivalent (V(E)/V(O(2))), heart rate (HR), heart rate variability (HRV) and arterial haemoglobin saturation (S(aO(2))) were no different in 5-HT-deficient pups compared to controls. However, only 25% of 5-HT-deficient pups survived all 15 episodes of environmental anoxia, compared to 79% of control littermates (P = 0.007). High mortality of 5,7-DHT-treated pups was associated with delayed onset of gasping (P < 0.001), delayed recovery of HR from hypoxic-induced bradycardia (P < 0.001), and delayed recovery of eupnoea from hypoxic-induced apnoea (P < 0.001). Treatment with 5,7-DHT affected neither the gasping pattern once initiated, nor HR, V(E)/V(O(2)) or S(aO(2)) during the intervening episodes of room air. A significant increase in HRV occurred in all animals with repeated exposure, and in 5-HT-deficient pups this increase occurred immediately prior to death. We conclude that a postnatal loss of brainstem 5-HT content compromises autoresuscitation in response to environmental anoxia. This report provides new evidence in rat pups that 5-HT neurones serve a physiological role in autoresuscitation. Our data may be relevant to understanding the aetiology of the sudden infant death syndrome (SIDS), in which there is medullary 5-HT deficiency and in some cases evidence of severe hypoxia and failed autoresuscitation.