Hypoxia tolerance in Atlantic cod

Oxygen saturation levels that killed 50 and 5% of cod Gadus morhua over 96 h averaged 21·2 and 27·7%, respectively. No fish survived at 10% saturation and only a few survived at 16% saturation, whereas no mortality occurred at 34 and 40% oxygen saturation. Since metabolic rate and oxygen consumption increase with increasing temperature, we hypothesized that cod would be less tolerant to hypoxic conditions at 6 than at 2° C. However, temperature (2 and 6° C) had no measurable impact on cod survival. Small (mean & S.D.; 45·2 ± 4·2 cm) and large (57·5 ± 3·8 cm) cod had the same tolerance to hypoxia. At the end of the experiments, hypoxia had a significant effect on blood haematocrit, mean cellular haemoglobin content, liver lactate, plasma glucose and plasma lactate, but accounted for only a small fraction (< 10%) of the variation, except for plasma lactate which exhibited a strong response with concentrations increasing progressively with decreasing levels of oxygen saturation. Temperature had a significant effect on most variates in normoxia and hypoxia. Variates also affected by oxygen level showed significant interactions between oxygen and size or temperature effects. However, these interactions accounted for only a small proportion of the variation. Physiological parameters indicated that extending the duration of our tests beyond 96 h would not have changed our estimates of the lethal thresholds. Hypoxic conditions are a permanent feature of the deep waters of the Gulf of St Lawrence. This study shows that a significant portion of the benthic habitats in the Gulf are uninhabitable for cod which would be expected to avoid waters below 28% oxygen saturation.     

Effects of acute hypoxia and hyperoxia on respiratory rate in albino rats chronically exposed to hypoxia

Changes in respiratory frequencies with hypoxic or hyperoxic exposure were studied in: 12 normoxic control rats (N) born and raised in normoxic environment at sea level; 12 rats (A) born and raised in normoxic environment at sea level exposed to normobaric hypoxia (10% O2 in N2) as adults; 12 rats of first generation (G1) raised in the above mentioned hypoxic environment since a few hours after birth; 12 rats of third generation (G3) conceived and born in the hypoxic environment of hypoxic parents of second generation and maintained continuously under hypoxic conditions until their utilization. The response of A rats to 10% O2 and 7% O2 breathing was elevated (57% and 86% over air breathing). The mean respiratory frequency of A rats exposed to 7% O2 rose to a greater extent than did that of N rats. The G1 and G3 rats were less responsive to 7% O2 (64% and 37% over air breathing, respectively) than N and A rats; however, in G1 rats the exposure to 7% O2 produced a greater rise of frequency than in G3 rats. Furthermore A rats, G1 rats and G3 rats were less responsive to 97% O2 breathing (19%, 19% and 11% below air breathing, respectively). Comparing these data with previous findings we suggest that, with chronic exposure to hypoxia, changes in ventilatory response to hypoxia and hyperoxia occur in the following manner: I) loss of response to hypoxia if chronic exposure is begun in the immediate postnatal period; 2) degree of response to hypoxia or hyperoxia influenced by duration of chronic exposure.     

Physiological biomarkers of hypoxic stress in red swamp crayfish Procambarus clarkii from field and laboratory experiments

The crayfish industry in Louisiana is the largest in the United States, with crayfish frequently harvested from waters that experience episodic or chronic hypoxia (dissolved oxygen [DO]≤ 2 mg/l). We examined physiological biomarkers (hemolymph lactate, glucose, and protein concentrations) of hypoxic stress in the red swamp crayfish Procambarus clarkii from chronically hypoxic natural habitats and laboratory hypoxia experiments. P. clarkii from normoxic and hypoxic areas in the Atchafalaya River Basin were sampled monthly from April to July 2010. Laboratory experiments subjected P. clarkii to severe hypoxia (1 mg/l DO), moderate hypoxia (2 mg/l DO), or normoxic conditions (control: DO>7.5 mg/l) for 12, 24, and 48 h. P. clarkii from normoxic and hypoxic natural habitats did not display significantly different hemolymph lactate or glucose concentrations; however, mean hemolymph protein concentration was significantly lower in crayfish from hypoxic areas. P. clarkii exposed to severe hypoxia in laboratory experiments had significantly higher hemolymph lactate and glucose concentrations for all three exposure times, whereas large differences in protein concentrations were not observed. These results suggest that elevated hemolymph lactate and glucose concentrations are responses to acute hypoxia in P. clarkii, while differences in protein concentrations are the result of chronic hypoxic exposure.     

The occurrence of aerial respiration in Rhinelepis strigosa during progressive hypoxia

Rhinelepis strigosa did not surface for air breathing in normoxic or moderate hypoxic water. This species initiated air breathing when the P _io₂ in the water reached 22 ± 1 mmHg. Once begun, the air-breathing frequency increased with decreasing P _io₂. Aquatic oxygen consumption was 21·0 ± 1·9ml O₂ kg⁻¹h⁻¹ in normoxic water, and was almost constant during progressive hypoxia until the P _io₂ reached 23·9 mmHg, considered the critical oxygen tension (P_co₂). Gill ventilation increased until close to the P _co₂ (7·9-fold) as a consequence of a greater increase in ventilatory volume than in breathing frequency. Gill oxygen extraction was 42 ± 5% and decreased with hypoxia, but under severe hypoxia returned to values characteristic of normoxic. The critical threshold for air breathing was coincident with the P_co₂ during aquatic respiration. This suggests that the air-breathing response is evoked by the aquatic oxygen tension at which the respiratory mechanisms fail to compensate for environmental hypoxia, and the gill O₂ uptake becomes insufficient to meet O₂ requirements.     

The behavioural and physiological response of Atlantic cod Gadus morhua L. to short-term acute hypoxia

The average rate of swimming speed and the physiological status or stress of individual Atlantic cod Gadus morhua was monitored in response to short-term acute (STA) hypoxia ( i.e. partial pressure of oxygen,     , reduced from 20·9 to 4·3 kPa within 1 h at 10° C). The STA hypoxic response of Atlantic cod was associated with a large primary increase (+29%) and a large secondary decrease (−54%) in swimming speed as well as major physiological stress ( e.g. plasma cortisol = 214·7 ng ml⁻¹ and blood lactate = 2·41 mmol l⁻¹).     

Stiffened erythrocytes augment the pulmonary hemodynamic response to hypoxia

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) during normoxic and hypoxic ventilation to assess the effect of reduced RBC deformability on the hypoxic pressor response. RBC suspensions were prepared with cells previously incubated in isotonic phosphate-buffered saline with or without 0.0125% glutaraldehyde. The washed RBCs were resuspended in isotonic bicarbonate-buffered saline (with 4% albumin) to hematocrits of approximately 35%. The lungs were perfused with control and experimental cell suspensions in succession while pulmonary arterial pressure was measured during normoxic (21% O2) and hypoxic (3% O2) ventilation. On the attainment of a peak hypoxic pressor response, flow rate was changed so that pressure-flow curves could be constructed for each suspension. RBC deformability was quantified by a filtration technique using 4.7-microns-pore filters. Glutaraldehyde treatment produced a 10% decrease in RBC deformability (P less than 0.05). Over the range of flow rates, Ppa was increased by 15-17% (P less than 0.05) and 26-31% (P less than 0.05) during normoxic and hypoxic ventilation, respectively, when stiffened cells were suspended in the perfusate. The magnitude of the hypoxic pressor response was 50-54% greater with stiffened cells over the three flow rates. In a separate set of experiments, normoxic and hypoxic arterial blood samples from conscious unrestrained rats were used to investigate the effects of acute hypoxia on RBC deformability. Deformability was measured with the same filtration technique. There was no difference in the deformability of hypoxic compared with normoxic RBCs. We conclude that the presence of stiffened RBCs enhances the hemodynamic response to hypoxia but acute hypoxia does not affect RBC deformability.     

Decreased genetic variation in metabolic variables of Litopenaeus vannamei shrimp after exposure to acute hypoxia

Concentration of proteins, carbohydrates, lactate, total lipids, acylglycerides, and carotenoids in shrimp were evaluated for their changes under acute hypoxia, and for their genetic variation under normoxic and hypoxic conditions. Proteins and lactate concentrations in muscle and hepatopancreas were significantly higher and carbohydrates in hepatopancreas were decreased in the hypoxic group. Family variances were significantly different only for proteins and carbohydrates in hepatopancreas in the normoxic group, indicating the existence of genetic variation for these traits. When family variances for each biochemical component were compared between normoxic and hypoxic groups, it was seen that most decreased. However, total variance was not significantly changed in response to hypoxia except for lactate (increased) and carotenoids (decreased) in hepatopancreas. The decrease in genetic variance without an increase in phenotypic variances in an acute response to hypoxia might be related to the known suppression of metabolic pathways that either use or produce ATP, which could result in a decreased expression of additive genes.     

Neuroprotective role of delta-opioid receptors in cortical neurons

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

Effects of hypoxia on the energy status and nitrogen metabolism of African lungfish during aestivation in a mucus cocoon

We examined the energy status, nitrogen metabolism and hepatic glutamate dehydrogenase activity in the African lungfish Protopterus annectens during aestivation in normoxia (air) or hypoxia (2% O(2) in N(2)), with tissues sampled on day 3 (aerial exposure with preparation for aestivation), day 6 (entering into aestivation) or day 12 (undergoing aestivation). There was no accumulation of ammonia in tissues of fish exposed to normoxia or hypoxia throughout the 12-day period. Ammonia toxicity was avoided by increased urea synthesis and/or decreased endogenous N production (as ammonia), but the dependency on these two mechanisms differed between the normoxic and the hypoxic fish. The rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the normoxic fish. By contrast, the rate of N production in the hypoxic fish decreased by 58%, with no increase in the rate of urea synthesis. Using in vivo (31)P NMR spectroscopy, it was demonstrated that hypoxia led to significantly lower ATP concentration on day 12 and significantly lower creatine phosphate concentration on days 1, 6, 9 and 12 in the anterior region of the fish as compared with normoxia. Additionally, the hypoxic fish had lower creatine phosphate concentration in the middle region than the normoxic fish on day 9. Hence, lowering the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing N production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase (GDH) amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, our results indicate for the first time that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia.     

Expression levels of genes associated with oxygen utilization,glucose transport and glucose phosphorylation in hypoxia exposed Atlantic cod (Gadus morhua)

Expression level of genes associated with oxygen [cytochrome oxidase 1 (COX1) and myoglobin (Mb)] and glucose utilization [glucose transporters (GLUTs) and hexokinases (HKs)] along with metabolic indices were determined in Atlantic cod (Gadus morhua) subjected to an hypoxic challenge of < 45% oxygen saturation for 24 days. There were two closely related HKs considered to be homologues of mammalian HKIs. HKIa and HKIb share 86% sequence identity and are both ubiquitously expressed. Mb was also expressed in many tissues with highest levels occurring in heart. Over the first 15 days of hypoxia there were transient increases in plasma lactate in hypoxic relative to normoxic fish associated with a significant decrease in liver glycogen. Over days 1–6, there were in ten of eleven cases, increased average (with a number of conditions being statistically significant) expression levels of GLUTs (1, 2, 4) and HKs (1a and b) in gill, heart, liver, and white muscle in hypoxic relative to normoxic fish. There were significant increases in COX1 and Mb expression levels in gill by day 24 but no changes in these aerobic indicators in heart or liver. Overall the data suggest a transient increase in genes associated with glucose utilization during the early part of the hypoxic challenge followed by alterations in gene expression in gill.     

Effects of graded hypoxia on Atlantic salmon infected with amoebic gill disease

Atlantic salmon Salmo salar with amoebic gill disease (AGD) were exposed to a graded hypoxia (135–40 mmHg water P O₂) and blood samples analysed for respiratory gases and pH at 119, 79·5 and 40 mmHg water P O₂. There were no differences in the rate of oxygen uptake between infected and control fish. However, arterial P O₂, and pH were significantly lower in the infected fish whereas P CO₂ was significantly higher in infected fish compared with controls prior to hypoxia and at 119 mmHg water P O₂. At 79·5 and 40 mmHg water P O₂ saturation, there were no significant differences in blood P O₂ or pH although blood P CO₂ was elevated in AGD affected fish at 50% hypoxia (79·5 mmHg water P O₂). The elevated levels of P CO₂ in fish affected by AGD resulted in a persistent respiratory acidosis even during hypoxic challenge. These data suggest that even though the fish were severely affected by AGD, the presence of AGD while impairing gas transfer under normoxic conditions, did not contribute to respiratory failure during hypoxia.     

Differences in response to hypoxia in the three‐spined stickleback from lotic and lentic localities: dominance and an anaerobic metabolite

Dominance hierarchies of the three‐spined stickleback Gasterosteus aculeatus from river and pond populations were subjected to hypoxia (20%, range ± 1%). Under hypoxia, the hierarchies were less stable in terms of rank position and tissue L‐lactate was higher in river fish than pond fish under normoxia and hypoxia. Dominant fish gained mass under normoxia but lost mass under hypoxic conditions possibly due to them maintaining high levels of aggression.     

Juvenile atlantic and shortnose sturgeons (family: Acipenseridae) have different hematological responses to acute environmental hypoxia

Experiments were conducted to determine the behavioral and physiological responses to acute hypoxic challenges in Atlantic (Acipenser oxyrinchus) and shortnose (Acipenser brevirostrum) sturgeons. We measured the ventilatory rate following a 45-mmHg hypoxic challenge, as well as a variety of hematological parameters, including O2 transport and hormonal, ionic, and metabolic variables, following a 1-h exposure to either 75- or 30-mmHg hypoxic challenges. Compared to fish in normoxic conditions (Pwo2 150 mmHg), juveniles of both species increased their ventilatory rate by approximately 40% when exposed to a 1-h challenge at 45 mmHg Pwo2. Hematological variables (e.g., hematocrit, hemoglobin, and Na+ and Cl- levels) did not change substantially following a 1-h challenge at 75 mmHg Pwo2. Conversely, a severe hypoxic challenge of 30 mmHg caused changes in several hematological variables (e.g., whole blood glucose and plasma cortisol and lactate levels). Most of these hematological parameters returned to prehypoxic levels within 2 h. Severe environmental hypoxia elicited the same basic pattern of response in both species; however, maximal plasma lactate levels were higher in Atlantic sturgeons, and maximal cortisol levels were higher in shortnose sturgeons. Whether these species differences are related to dissimilar hypoxia-tolerance, ecological, and/or endocrinological characteristics between these two species is not entirely clear.     

Hypoxia and interdemic variation in Poecilia latipinna

Variation in respiratory traits was quantified between two populations of the sailfin molly Poecilia latipinna (one from a periodically hypoxic salt marsh, Cedar Key, and one from a chronically normoxic river site, Santa Fe River). Two suites of characters were selected: traits that may show both short‐term acclimation response and interdemic variation in acclimation response (metabolic rate, critical oxygen tension and respiratory behaviour), and those that are not likely to respond to short‐term acclimation but may vary among populations (gill morphometric characters). Sailfin mollies from the salt marsh, acclimated to hypoxia (1 mg l⁻¹, c . 20 mmHg) for 6 weeks, spent less time conducting aquatic surface respiration and had lower gill ventilation rates than hypoxia‐acclimated conspecifics from the well‐oxygenated river site. Poecilia latipinna acclimated to hypoxia exhibited a lower critical oxygen tension ( P _c) than fish acclimated to normoxia; however, there was also a significant population effect. Poecilia latipinna from Cedar Key exhibited a lower P _c than fish from the Santa Fe River, regardless of acclimation. Cedar Key fish had a 14% higher mean gill surface area relative to fish from the Santa Fe River, a character that could account, at least in part, for their greater tolerance to hypoxia.     

Metabolic and endocrine responses to graded exercise under acute hypoxia

Eight male subjects (24 +/- 1 years old) performed graded ergocycle exercises in normoxic (N) and acute hypoxic (H) conditions (14.5% O2). VO2max decreased from 55.5 +/- 1.3 to 45.8 +/- 1.4 ml . kg-1 . min-1 in H condition. Plasma glucose and free fatty acid concentrations remained unchanged throughout exercise in both conditions. Increase in blood lactate concentration was associated with relative workload in both conditions. At VO2max lactate concentrations were similar in the two conditions, plasma insulin, glucagon, and LH concentrations did not significantly change in either. Plasma delta 4-androstenedione and testosterone increased in a similar manner in both conditions. Finally plasma norepinephrine concentration reached at VO2max was significantly lower in hypoxia. These results suggest that acute moderate hypoxia does not affect metabolic and hormonal responses to short exercise performed at similar relative workloads, i.e. when the reduction of VO2max due to hypoxia is taken into consideration. The lower catecholamine response to maximal exercise under acute hypoxia might suggest that the sympathetic response could be related to relative as well as absolute workloads.     

Chronic hypoxia abolishes posthypoxia frequency decline in the anesthetized rat

In anesthetized rats, increases in phrenic nerve amplitude and frequency during brief periods of hypoxia are followed by a reduction in phrenic nerve burst frequency [posthypoxia frequency decline (PHFD)]. We investigated the effects of chronic exposure to hypoxia on PHFD and on peripheral and central O2-sensing mechanisms. In Inactin-anesthetized (100 mg/kg) Sprague-Dawley rats, phrenic nerve discharge and arterial pressure responses to 10 s N2 inhalation were recorded after exposure to hypoxia (10 +/- 0.5% O2) for 6-14 days. Compared with rats maintained at normoxia, PHFD was abolished in chronic hypoxic rats. Because of inhibition of PHFD, the increased phrenic burst frequency and amplitude after N2 inhalation persisted for 1.8-2.8 times longer in chronic hypoxic (70 s) compared with normoxic (25-40 s) rats (P < 0.05). After acute bilateral carotid body denervation, N2 inhalation produced a short depression of phrenic nerve discharge in both chronic hypoxic and normoxic rats. However, the degree and duration of depression of phrenic nerve discharge was smaller in chronic hypoxic compared with normoxic rats (P < 0.05). We conclude that after exposure to chronic hypoxia, a reduction in PHFD contributes to an increased duration of the acute hypoxic ventilatory response in anesthetized rats. Furthermore, after exposure to chronic hypoxia, the central network responsible for respiration is more resistant to the depressant effects of acute hypoxia in anesthetized rats.     

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO₂ levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO₂. Increased incubation temperature caused an increase in the developmental rate at high PO₂s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO₂. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO₂s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.     

Ultrastructure and metabolism of skeletal muscle fibres in the tench: Effects of long-term acclimation to hypoxia

Summary Tench (Tinca tinca) were acclimated to either aerated (P _{O 2} 17.6 KPa) or hypoxic (P _{O 2} 1.5 KPa) water for 6 weeks.Acclimation to hypoxia resulted in a decrease in mitochondrial volume fraction in both slow (22.9 to 15.0 %) and fast glycolytic (4.5 to 1.8 %) myotomal muscles fibres (P<0.01).Intermyofibrillar mitochondrial populations (4.4 to 1.2% slow; 0.6 to 0.04% fast fibres) were affected to a greater extent than those in the subsarcolemmal zone (18.5 to 13.8% slow; 3.9 to 1.8% fast fibres). After acclimation to hypoxia, cytochrome-oxidase activities decreased by 31 and 33 % in slow and fast fibres, respectively, but were maintained in the liver.Fibre size remained unchanged and actively differentiating fibres were observed in muscles from both groups of fish. Hypoxia resulted in a significant increase in myofibrillar volume fraction in both slow (43.1 to 56.1 %) and fast glycolytic fibres (73.1 to 82.7%) (P<0.05).Glycogen concentrations (mg/100g tissue) for liver (6616) slow muscle (1892) and fast muscle (334) were similar for fish acclimated to aerated or hypoxic water. Acclimation to hypoxia increased carnitine palmitoyl transferase activity (moles substrate utilised g·dry wt_-1 min^-1) in slow (0.42 to 1.1), fast glycolytic muscle (<0.01 to 0.15) and liver (1.1 to 3.7) indicating an enhanced capacity for fatty acid oxidation.Phosphofructokinase activities of fast glycolytic fibres were similar in fish acclimated to either aerated or hypoxic water, consistent with an unaltered capacity for anaerobic glycogenolysis. Hexokinase activities (moles substate utilised, g·dry wt^-1 min^-1) decreased in fast fibres (1.2 to 0.4) but were maintained in the slow muslce (2.1 to 2.5) and liver (4.5 to 4.8) of hypoxic fish. The activities of phosphofructokinase in slow muscle and phosphofructokinase, pyruvate kinase and lactate dehydrogenase in liver were two times higher in fish acclimated to hypoxia. An enhanced capacity for glycolysis in these tissues may reflect a reduced threshold for anaerobic metabolism during activity and/or an adaptation for acute exposure to anoxia in fish acclimated to hypoxia.Abbreviations/Glossary CO cytochrome oxidase activity - CPT carnitine palmitoyltransferase activity - HK hexokinase activity - LDH lactate dehydrogenase activity - PFK phosphofructokinase activity - PK pyruvate kinase activity - Vv volume fractions of cell components - normoxic fish acclimated to aerated water - hypoxic fish acclimated to reduced oxygen tensions - P _{O 2} partial pressure of oxygen tension A preliminary account of part of this work was presented at theXth European Meeting on Muscle and Cell Motility held at Galway, Ireland, in September 1981     

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.     

Chronic hypoxia in development selectively alters the activities of key enzymes of glucose oxidative metabolism in brain regions

The immature brain is more resistant to hypoxia/ischemia than the mature brain. Although chronic hypoxia can induce adaptive-changes on the developing brain, the mechanisms underlying such adaptive changes are poorly understood. To further elucidate some of the adaptive changes during postnatal hypoxia, we determined the activities of four enzymes of glucose oxidative metabolism in eight brain regions of hypoxic and normoxic rats. Litters of Sprague-Dawley rats were put into the hypoxic chamber (oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal (P3). Age-matched normoxic rats were use as control animals. In P10 hypoxic rats, lactate dehydrogenase (LDH) activity in cerebral cortex, striatum, olfactory bulb, hippocampus, hypothalamus, pons and medulla, and cerebellum was significantly increased (by 100%–370%) compared to those in P10 normoxic rats. In P10 hypoxic rats, hexokinase (HK) activity in hypothalamus, hippocampus, olfactory bulb, midbrain, and cerebral cortex was significantly decreased (by 15%–30%). Neither -ketoglutarate dehydrogenase complex (KGDHC, which is believed to have an important role in the regulation of the tricarboxylic acid [TCA] cycle flux) nor citrate synthase (CS) activity was significantly decreased in the eight regions of P10 hypoxic rats compared to those in P10 normoxic rats. In P30 hypoxic rats, LDH activity was only increased in striatum (by 19%), whereas HK activity was only significantly decreased (by 30%) in this region. However, KGDHC activity was significantly decreased in olfactory bulb, hippocampus, hypothalamus, cerebral cortex, and cerebellum (by 20%–40%) in P30 hypoxic rats compared to those in P30 normoxic rats. Similarly, CS activity was decreased, but only in olfactory bulb, hypothalamus, and midbrain (by 9%–21%) in P30 hypoxic rats. Our results suggest that at least some of the mechanisms underlying the hypoxia-induced changes in activities of glycolytic enzymes implicate the upregulation of HIF-1. Moreover, our observation that chronic postnatal hypoxia induces differential effects on brain glycolytic and TCA cycle enzymes may have pathophysiological implications (e.g., decreased in energy metabolism) in childhood diseases (e.g., sudden infant death syndrome) in which hypoxia plays a role.