Short Periods Of Hypoxia Research Articles

The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

Enumeration and Characterization of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Undergoing Transarterial Chemoembolization.

Circulating tumor cells (CTCs), and particularly circulating cancer stem cells (cCSC), are prognostic biomarkers for different malignancies and may be detected using liquid biopsies. The ex vivo culture of cCSCs would provide valuable information regarding biological aggressiveness and would allow monitoring the adaptive changes acquired by the tumor in real time. In this prospective pilot study, we analyzed the presence of EpCAM+ CTCs using the IsoFlux system in the peripheral blood of 37 patients with hepatocellular carcinoma undergoing transarterial chemoembolization (TACE). The average patient age was 63.5 ± 7.9 years and 91.9% of the patients were men. All patients had detectable CTCs at baseline and 20 patients (54.1%) showed CTC aggregates or clusters in their peripheral blood. The increased total tumor diameter (OR: 2.5 (95% CI: 1.3-4.8), p = 0.006) and the absence of clusters of CTCs at baseline (OR: 0.2 (95% CI: 0.0-1.0), p = 0.049) were independent predictors of a diminished response to TACE. Culture of cCSC was successful in five out of thirty-three patients, mostly using negative enrichment of CD45- cells, ultra-low adherence, high glucose, and a short period of hypoxia followed by normoxia. In conclusion, the identification of clusters of CTCs before TACE and the implementation of standardized approaches for cCSC culture could aid to predict outcomes and to define the optimal adjuvant therapeutic strategy for a true personalized medicine in hepatocellular carcinoma.

Transcriptomes Suggest That Pinniped and Cetacean Brains Have a High Capacity for Aerobic Metabolism While Reducing Energy-Intensive Processes Such as Synaptic Transmission.

The mammalian brain is characterized by high energy expenditure and small energy reserves, making it dependent on continuous vascular oxygen and nutritional supply. The brain is therefore extremely vulnerable to hypoxia. While neurons of most terrestrial mammals suffer from irreversible damage after only short periods of hypoxia, neurons of the deep-diving hooded seal (Cystophora cristata) show a remarkable hypoxia-tolerance. To identify the molecular mechanisms underlying the intrinsic hypoxia-tolerance, we excised neurons from the visual cortices of hooded seals and mice (Mus musculus) by laser capture microdissection. A comparison of the neuronal transcriptomes suggests that, compared to mice, hooded seal neurons are endowed with an enhanced aerobic metabolic capacity, a reduced synaptic transmission and an elevated antioxidant defense. Publicly available whole-tissue brain transcriptomes of the bowhead whale (Balaena mysticetus), long-finned pilot whale (Globicephala melas), minke whale (Balaenoptera acutorostrata) and killer whale (Orcinus orca), supplemented with 2 newly sequenced long-finned pilot whales, suggest that, compared to cattle (Bos taurus), the cetacean brain also displays elevated aerobic capacity and reduced synaptic transmission. We conclude that the brain energy balance of diving mammals is preserved during diving, due to reduced synaptic transmission that limits energy expenditure, while the elevated aerobic capacity allows efficient use of oxygen to restore energy balance during surfacing between dives.

Short-term acclimation in adults does not predict offspring acclimation potential to hypoxia

The prevalence of hypoxic areas in coastal waters is predicted to increase and lead to reduced biodiversity. While the adult stages of many estuarine invertebrates can cope with short periods of hypoxia, it remains unclear whether that ability is present if animals are bred and reared under chronic hypoxia. We firstly investigated the effect of moderate, short-term environmental hypoxia (40% air saturation for one week) on metabolic performance in adults of an estuarine amphipod, and the fitness consequences of prolonged exposure. We then reared the offspring of hypoxia-exposed parents under hypoxia, and assessed their oxyregulatory ability under declining oxygen tensions as juveniles and adults. Adults from the parental generation were able to acclimate their metabolism to hypoxia after one week, employing mechanisms typically associated with prolonged exposure. Their progeny, however, did not develop the adult pattern of respiratory regulation when reared under chronic hypoxia, but instead exhibited a poorer oxyregulatory ability than their parents. We conclude that species apparently hypoxia-tolerant when tested in short-term experiments, could be physiologically compromised as adults if they develop under hypoxia. Consequently, we propose that the increased prevalence of hypoxia in coastal regions will have marked effects in some species currently considered hypoxia tolerant.

Progression of Neuronal Damage in an In Vitro Model of the Ischemic Penumbra

Improvement of neuronal recovery in the ischemic penumbra around a brain infarct has a large potential to advance clinical recovery of patients with acute ischemic stroke. However, pathophysiological mechanisms leading to either recovery or secondary damage in the penumbra are not completely understood. We studied neuronal dynamics in a model system of the penumbra consisting of networks of cultured cortical neurons exposed to controlled levels and durations of hypoxia. Short periods of hypoxia (pO2≈20mmHg) reduced spontaneous activity, due to impeded synaptic function. After ≈6 hours, activity and connectivity partially recovered, even during continuing hypoxia. If the oxygen supply was restored within 12 hours, changes in network connectivity were completely reversible. For longer periods of hypoxia (12–30 h), activity levels initially increased, but eventually decreased and connectivity changes became partially irreversible. After ≈30 hours, all functional connections disappeared and no activity remained. Since this complete silence seemed unrelated to hypoxic depths, but always followed an extended period of low activity, we speculate that irreversible damage (at least partly) results from insufficient neuronal activation. This opens avenues for therapies to improve recovery by neuronal activation.

Morphofunctional changes in the myocardium of the rat heart ventricles with noncoronarogenic necrosis in different stages of ischemic precondition.

Background. . The incidents of the cardiac ischemic disease and it’s complications for the last years increase in all over the world. Some authors think that short periods of ischemia make myocardium more resist to long coronary occlusion. This cardioprotective mechanism was named ischemic preconditioning(IPC). Some authors believe that short periods of hypoxia contribute to the resistance to hypoxic myocardial injury . Today, however, poorly understood mechanisms of cardioprotective action IPC. Objective. The purpose of the study was to determine the effectiveness of ischemic preconditioning to increase the body resistance and morphofunctional adaptation of the heart to noncoronary necrosis of myocardium Methods and results. The study was carried out on the white Wistar rats. Exposition dynamics of sorption in the myocardium of heart ventricules was studied. This score was studied by the intracardiac entering 0,5% water solution neutral red with next getting alcoholic extract from myocardium tissue. This extract was studied with concentration photocolorimeter. Sorption index was calculated in micrograms / mg by dividing the quantitative indicator of the concentration of neutral red on the weight of solids piece myocardium. Also histological sections of the myocardium of ventricls were studied at different stages of ischemic precondition and noncoronarogenic necrosis. Conclusion. Positive dynamics of functional scores manifested in the fact that the exposition dynamics of sorption in the myocardium of the heart ventricles was increased in all the experimental series, compared to the control and to a greater extent after the modeling of the necrosis of myocardium in ischemic precondition first step. In second step this score was lower. Changes in the functional parameter correlated with the morphological changes in the myocardium.

Effects of a Single Bout of Interval Hypoxia on Cardiorespiratory Control in Patients With Type 1 Diabetes

Hypoxemia is common in diabetes, and reflex responses to hypoxia are blunted. These abnormalities could lead to cardiovascular/renal complications. Interval hypoxia (IH) (5–6 short periods of hypoxia each day over 1–3 weeks) was successfully used to improve the adaptation to hypoxia in patients with chronic obstructive pulmonary disease. We tested whether IH over 1 day could initiate a long-lasting response potentially leading to better adaptation to hypoxia. In 15 patients with type 1 diabetes, we measured hypoxic and hypercapnic ventilatory responses (HCVRs), ventilatory recruitment threshold (VRT-CO2), baroreflex sensitivity (BRS), blood pressure, and blood lactate before and after 0, 3, and 6 h of a 1-h single bout of IH. All measurements were repeated on a placebo day (single-blind protocol, randomized sequence). After IH (immediately and after 3 h), hypoxic and HCVR increased, whereas the VRT-CO2 dropped. No such changes were observed on the placebo day. Systolic and diastolic blood pressure increased, whereas blood lactate decreased after IH. Despite exposure to hypoxia, BRS remained unchanged. Repeated exposures to hypoxia over 1 day induced an initial adaptation to hypoxia, with improvement in respiratory reflexes. Prolonging the exposure to IH (>2 weeks) in type 1 diabetic patients will be a matter for further studies.

Regional genome transcriptional response of adult mouse brain to hypoxia

BackgroundSince normal brain function depends upon continuous oxygen delivery and short periods of hypoxia can precondition the brain against subsequent ischemia, this study examined the effects of brief hypoxia on the whole genome transcriptional response in adult mouse brain.ResultPronounced changes of gene expression occurred after 3 hours of hypoxia (8% O2) and after 1 hour of re-oxygenation in all brain regions. The hypoxia-responsive genes were predominantly up-regulated in hindbrain and predominantly down-regulated in forebrain - possibly to support hindbrain survival functions at the expense of forebrain cognitive functions. The up-regulated genes had a significant role in cell survival and involved both shared and unshared signaling pathways among different brain regions. Up-regulation of transcriptional signaling including hypoxia inducible factor, insulin growth factor (IGF), the vitamin D3 receptor/retinoid X nuclear receptor, and glucocorticoid signaling was common to many brain regions. However, many of the hypoxia-regulated target genes were specific for one or a few brain regions. Cerebellum, for example, had 1241 transcripts regulated by hypoxia only in cerebellum but not in hippocampus; and, 642 (54%) had at least one hepatic nuclear receptor 4A (HNF4A) binding site and 381 had at least two HNF4A binding sites in their promoters. The data point to HNF4A as a major hypoxia-responsive transcription factor in cerebellum in addition to its known role in regulating erythropoietin transcription. The genes unique to hindbrain may play critical roles in survival during hypoxia.ConclusionDifferences of forebrain and hindbrain hypoxia-responsive genes may relate to suppression of forebrain cognitive functions and activation of hindbrain survival functions, which may coordinately mediate the neuroprotection afforded by hypoxia preconditioning.

Convulsion and cardiorespiratory collapse with first epidural test dose

To the Editor: Accidental intravascular injection of local anesthetic can cause central nervous system (CNS) toxicity, presenting as tremors, convulsions, dysrhythmias, and cardiorespiratory arrest. During epidural anesthesia, a test dose is used to diagnose inadvertent intravascular or intrathecal administration of drugs. We present a case where the test dose itself caused central neural toxicity. A 38-year-old woman, weighing 50 kg and 159 cm tall, with chondrosarcoma in the left femur, presented for tumor resection, irradiation, and reconstruction under a combined epidural and general anesthesia. She had no significant past or family medical history, with no associated comorbid illnesses or evidence of metastasis. She was positioned in the right lateral position for insertion of an epidural catheter under monitoring of heart rate (HR), blood pressure (BP), and oxygen saturation (SpO2). The baseline HR was 100 bpm, BP 124/75 mmHg, and room-air SpO2 98%. After inserting an epidural catheter in L2‐3 interspace and carefully confirming negative aspiration, a 3-ml test dose of 1.5% lidocaine with epinephrine 1:200,000 was injected slowly, with no change in HR. Approximately 1 min later, the patient had perioral paresthesia and twitching followed by loss of consciousness and generalized convulsions. She was immediately turned to the supine position and midazolam 2 mg was given intravenously, followed by thiopentone 250 mg to control the convulsions. After mask ventilation with 100% oxygen, the trachea was intubated and lungs were ventilated. The electrocardiograph (ECG) showed sinus bradycardia with HR 34 bpm during convulsion, (before institution of mask ventilation), which failed to respond to 0.6 mg atropine administered intravenously. A short period of hypoxia may have caused failure to respond to the atropine. As the radial pulse was not palpable, cardiopulmonary resuscitation was started, and 1 mg adrenaline was given intravenously. Radial pulse was noted immediately after resuscitation (ECG showing sinus rhythm), and the patient regained consciousness within 5 min. Aspiration of the epidural catheter revealed blood, and a diagnosis of probable intravascular injection was made. Subsequently, brain magnetic resonance imaging (MRI) was normal, and the patient underwent the planned operation the following week under combined epidural and general anaesthesia without any complication.

Scanning Electron Microscopy and Energy-Dispersive X-Ray Microanalysis: A Valuable Tool for Studying Cell Surface Antigen Expression on Tissue-Engineered Scaffolds

The influence of an acellular porcine matrix on proinflammatory activation of endothelial cells (EC) during normoxia and hypoxia was investigated by a newly established semi-quantitative electron microscopic procedure. As a model, three adhesion molecules (E-selectin, ICAM-1, and VCAM-1) were localized by silver-enhanced immunogold staining and energy dispersive X-ray microanalysis after normoxic or hypoxic pretreatment of the cells and subsequent stimulation with IL-1beta. Morphology of EC grown on porcine matrix or coverslips was recorded simultaneously using secondary electron imaging. EC appeared tightly attached to the underlying surfaces with their typical cobblestone-like morphology. Statistically significant upregulations upon stimulation with IL-1beta were observed in both groups for all three adhesion molecules. Hypoxic pretreatment of the specimens with subsequent reoxygenation neither induced morphological changes nor caused an upregulation of adhesion molecule expression in cells grown on acellular porcine tissue. Unexpectedly, in cells seeded onto the acellular matrix, IL-1beta failed to upregulate ICAM-1 expression after a short period of hypoxia. The surface expression of VCAM-1 was also significantly lower even under normoxic conditions, which might indicate the development of functional impairment of cells in contact with acellular porcine tissue. The method presented in this study has proven valuable for the determination of antigen expression on scaffold materials in parallel with the characterization of surface morphology.

Role of microphytobenthos in recovery of functions in a shallow-water sediment system after hypoxic events

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 357:1-16 (2008) - DOI: https://doi.org/10.3354/meps07426 Role of microphytobenthos in recovery of functions in a shallow-water sediment system after hypoxic events Fredrik Larson1,2,*, Kristina Sundbäck1 1Department of Marine Ecology, University of Gothenburg, PO Box 461, Göteborg 405 30, Sweden 2Present address: SERG-group, Gatty Marine Laboratory, University of St Andrews, East Sands, St Andrews, Fife KY16 8LB, Scotland, UK *Email: fredrik.larson@marecol.gu.se ABSTRACT: Several studies in shallow-water sediments have followed the recovery from hypoxia on a macro- or meiobenthic level, but on the microbenthic level such studies are rare. Outdoor flow-through mesocosms were used to monitor the resilience of a microbenthic community and nutrient turnover after hypoxia in natural, previously undisturbed sediment. Variables included oxygen and nutrient fluxes, denitrification and biomass (chl a) of microphytobenthos (MPB). Results showed that the community recovered rapidly after hypoxia (<15% of oxygen saturation). In a 7 d experiment preceded by 2 d of hypoxia, the initially changed oxygen and nutrient fluxes remained affected only during the first days after hypoxia. In a 50 d experiment, starting with 17 d of hypoxia, only nitrate under dark conditions remained significantly different at the termination of the experiment after a recovery period of 4.5 wk. The microbenthic community, as well as the basal functions, of shallow-water illuminated sediments possess a high resilience, at least after single events with short periods of hypoxia. This resilience appears to be related to the high resistance of MPB (especially diatoms) to hypoxia, which implies a rapid restoration of the oxygenation of the sediment surface and the base of the food web, securing food supply for new colonising grazers. The recovery was faster for functions under light conditions, indicating that the functions related to microalgal activity are less sensitive to hypoxic events than are heterotrophic processes. KEY WORDS: Resilience · Recovery · Hypoxia · Sediment · Microphytobenthos · Denitrification · Oxygen · Nutrient fluxes Full text in pdf format NextCite this article as: Larson F, Sundbäck K (2008) Role of microphytobenthos in recovery of functions in a shallow-water sediment system after hypoxic events. Mar Ecol Prog Ser 357:1-16. https://doi.org/10.3354/meps07426 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 357. Online publication date: April 07, 2008 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2008 Inter-Research.

A1 adenosine receptors play an essential role in protecting the embryo against hypoxia

Embryos can be exposed to environmental factors that induce hypoxia. Currently, our understanding of the effects of hypoxia on early mammalian development is modest. Potential mediators of hypoxia action include the nucleoside adenosine, which acts through A(1) adenosine receptors (A(1)ARs) and mediates adverse effects of hypoxia on the neonatal brain. We hypothesized that A(1)ARs may also play a role in mediating effects of hypoxia on the embryo. When pregnant dams were exposed to hypoxia (10% O(2)) beginning at embryonic day (E) 7.5 or 8.5 and continued for 24-96 h, A(1)AR+/+ embryos manifested growth inhibition and a disproportionate reduction in heart size, including thinner ventricular walls. Yet, when dams were exposed to hypoxia, embryos lacking A(1)ARs (A(1)AR-/-) had much more severe growth retardation than A(1)AR+/+ or +/- embryos. When levels of hypoxia-inducible factor 1alpha (HIF1alpha) were examined, A(1)AR-/- embryos had less stabilized HIF1alpha protein than A(1)AR+/- littermates. Normal patterns of cardiac gene expression were also disturbed in A(1)AR-/- embryos exposed to hypoxia. These results show that short periods of hypoxia during early embryogenesis can result in intrauterine growth retardation. We identify adenosine and A(1)ARs as playing an essential role in protecting the embryo from hypoxia.

Chronic hypoxia promotes hypoxia-inducible factor-1α–dependent resistance to etoposide and vincristine in neuroblastoma cells

Hypoxia is widespread in solid tumors as a consequence of poorly structured tumor-derived neovasculature. Direct measurement of low oxygen levels in a range of adult tumor types has correlated tumor hypoxia with advanced stage, poor response to chemotherapy and radiotherapy, and poor prognosis. Little is known about the importance of hypoxia in pediatric tumors; therefore, we evaluated the effects of hypoxia on the response of the neuroblastoma cell lines SH-EP1 and SH-SY5Y to the clinically relevant drugs, vincristine, etoposide, and cisplatin. Short periods of hypoxia (1% O2) of up to 16 hours had no effect on drug-induced apoptosis or clonogenic survival. Prolonged hypoxia of 1 to 7 days leads to reduction in vincristine- and etoposide-induced apoptosis in SH-SY5Y and SH-EP1 cells, and this was reflected in increased clonogenic survival under these conditions. Neither short-term nor prolonged hypoxia had any effect on the clonogenic response to cisplatin in SH-SY5Y cells. Hypoxia-inducible factor-1 (HIF-1) alpha was stabilized in these cell lines within 2 hours of hypoxia but was no longer detectable beyond 48 hours of hypoxia. Up-regulation of carbonic anhydrase IX showed HIF-1alpha to be transcriptionally active. Down-regulation of HIF-1alpha by short hairpin RNA interference and the small-molecule 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole reduced hypoxia-induced drug resistance. These results suggest that prolonged hypoxia leads to resistance to clinically relevant drugs in neuroblastoma and that therapies aimed at inhibiting HIF-1alpha function may be useful in overcoming drug resistance in this tumor.

TNFα-induced cytoprotection requires the production of free radicals within mitochondria in C 2C 12 myotubes

We previously reported that tumour necrosis factor alpha (TNFα) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNFα-induced reactive oxygen species (ROS). We hypothesized that TNFα cytoprotection occurs through the generation of ROS which originate within the mitochondria. C 2C 12 myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFα (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNFα-induced ROS was examined with Mitotracker Red CM-H 2XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFα improved cell viability compared with the simulated ischemic control (TNFα: 75 ± 1% versus 34 ± 1% for the control: p < 0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFα-stimulated cells to 15 ± 1% ( p < 0.001 versus TNFα). Similar results were obtained with IPC. TNFα stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFα to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFα-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFα.

Endothelial hypoxic preconditioning in rat hypoxic isolated aortic segments

Our aim was to analyse endothelial hypoxic preconditioning after hypoxia-reperfusion (HR). Endothelial functionality was analysed through the vasorelaxation responses to acetylcholine (Ach) and the level of serine1177 phosphorylated endothelial nitric oxide synthase (eNOS) (ser1177-eNOS) measured by Western blot in in vitro hypoxic preconditioned (P + HR) isolated rat aortic segments. Relaxation in response to Ach was reduced in phenylephrine-precontracted aortic segments after HR (control: IC50, 5 +/- 2.5 x 10(-8) mol l(-1); HR: IC50, 3 +/- 1.2 x 10(-7) mol l(-1); P < 0.05). Ach-dependent vasodilatation was improved by P + HR. The content of ser1177-eNOS in the HR segments was 1.5-fold lower than in P + HR. Confocal microscopy showed an increased content of both superoxide anion and peroxynitrite in the vascular wall of HR aortic segments, which it was reduced by P + HR. Geldanamycin (10 microg ml(-1)), an agent known to inhibit heat shock protein 90 (hsp90), reduced the level of ser1177-eNOS in P + HR aortic segments. However in the presence of geldanamycin, endothelial hypoxic preconditioning persisted. We conclude that short periods of hypoxia induced endothelial hypoxic preconditioning that was accompanied by enhanced levels of ser1177-eNOS in the vascular wall. The fact that endothelial hypoxic preconditioning persisted in the presence of geldanamycin suggests that other molecular mechanisms are involved in the endothelial adaptation to HR injury.

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels.

In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri- to diphosphate ratios progressively declined for ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxia was increased from 5 to 30 min. Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassium current failed to indicate an allosteric regulation of a plasmalemmal K(ATP) channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in combination with a hypoxic stimulus; it is suggested that both plasmalemmal and mitochondrial K(ATP) channels are involved under these conditions. The hypoxic depolarization may be due to blockade of K+,Na+-ATPase by limitation of energy supplying substrate.

Nest temperature and respiratory gases during natural incubation in the broad-shelled river turtle, Chelodina expansa (Testudinata : Chelidae)

Temperature was monitored in three natural nests, and oxygen and carbon dioxide partial pressure monitored in one natural nest of the broad-shelled river turtle, Chelodina expansa, throughout incubation. Nest temperature decreased after nest construction in autumn, remained low during winter and gradually increased in spring to a maximum in summer. In a nest where temperature was recorded every hour, temperature typically fluctuated through a 2°C cycle on a daily basis throughout the entire incubation period, and the nest always heated faster than it cooled. Oxygen and carbon dioxide partial pressures in this nest were similar to soil oxygen and carbon dioxide partial pressures for the first 5 months of incubation, but nest respiratory gas tensions deviated from the surrounding soil over the last three months of incubation. Nest respiratory gas tensions were not greatly different from those in the atmosphere above the ground except after periods of rain. After heavy rain during the last 3 months of incubation the nest became moderately hypoxic (PO2 ~ 100 Torr) and hypercapnic (PCO 2 ~ 50 Torr) for several successive days. These short periods of hypoxia and hypercapnia were not lethal.

Michaelis-Menten kinetics model of oxygen consumption by rat brain slices following hypoxia.

In the present study, we have measured partial pressure of oxygen (pO2) profiles through rat brain slices before and after periods of hypoxia (5 and 10 min) to determine its effect on tissue oxygen demand. Tissue pO2 profiles were measured through rat cerebral cortex slices superfused with phosphate buffer using oxygen (O2)-sensitive microelectrodes at different times in controls [40% O2 balance nitrogen (N2)], and at different times before and after 5 or 10 min of hypoxia (100% N2). A one-dimensional, steady-state model of ordinary diffusion with a Michaelis-Menten model of O2 consumption where the maximal O2 consumption (Vmax) and the rate at half-maximal O2 consumption (Km) were allowed to vary was used to determine the kinetics of O2 consumption. Actual pO2 profiles through tissue were fitted to theoretical profiles by a least-squares method. Vmax varied among penetrations in a control slice and among slices. Vmax seemed to decrease after hypoxic insult, but the change was not statistically significant. The Km value measured before hypoxia was lower than the first Km value measured after the end of hypoxia, indicating that hypoxia induced a compensatory change in the metabolic state of the tissue. Km increased immediately after both 5- and 10-min hypoxic insults and returned to normal after recovery for each case. It seems that during and immediately after short periods of hypoxia, Km increases from near zero but returns to normal values within a few minutes.

Cerebral blood oxygenation in rat brain during hypoxic hypoxia. Quantitative MRI of effective transverse relaxation rates

Oxygenation-sensitive MRI of respiratory challenges in the brain of experimental animals will considerably benefit from a quantitative relationship between cerebral blood oxygenation and MRI parameters. Here, a multi-echo gradient-echo MRI technique was used to determine effective transverse relaxation rates R2* = 1/T2* of rat brain in vivo during short periods of hypoxia and interleaved normoxic phases. The differential contribution delta R2* observed during hypoxia was found to increase linearly with arterial blood deoxygenation for mild to moderate conditions. Severe deoxygenation resulted in a plateau most likely due to enhanced cerebral blood flow.

Isolation of a novel inducible rat heat-shock protein (HSP70) gene and its expression during ischaemia/hypoxia and heat shock

Most of the members of the mammalian heat-shock protein (HSP) gene family have been studied and isolated from human and mouse cells. Few studies have concentrated on the HSPs of rat, a commonly used experimental animal. We have isolated and characterized a novel inducible rat HSP70 gene using an HSP70 cDNA sequence obtained from an ischaemic rat heart cDNA library. The isolated rat HSP70 gene was found to be a functional gene, as indicated by RNAase-protection and Northern-blot analysis. The deduced amino acid sequence of the inducible rat HSP70 exhibits a high degree of similarity to previously isolated mammalian inducible HSP70 gene products. Expression of the inducible HSP70 gene in rat myogenic cells (H9c2) is markedly increased after relatively short periods of hypoxia as well as by heat shock. Two heat-shock elements (HSE) are present in the rat HSP70 promoter. Transient transfection of rat HSP70 promoter/chloramphenicol acetyltransferase constructs into H9c2 cells shows that the presence of either of the two HSEs is sufficient for heat-shock inducibility. In contrast, induction of the rat HSP70/chloramphenicol acetyltransferase constructs by hypoxia is only detectable when both HSEs are present. This leads us to conclude that the induction of HSP70 by hypoxia and heat shock occurs through the same regulatory HSEs but the activation of the inducible HSP70 gene by heat shock is several-fold higher than by hypoxia.