Acute traumatic coagulopathy (ATC) is an acute and endogenous mechanism triggered by the association of trauma and hemorrhage. Several animal models have been developed, but some major biases have not yet been identified. Our aim was to develop a robust and clinically relevant murine model to study this condition. Anesthetized adult Sprague Dawley rats were randomized into 4 groups: C, control; T, trauma; H, hemorrhage; TH, trauma and hemorrhage (n = 7 each). Trauma consisted of laparotomy associated with four-limb and splenic fractures. Clinical variables, ionograms, arterial and hemostasis blood tests were compared at 0 and 90 min. ATC and un-compensated shock were observed in group TH. In this group, the rise in prothrombin time and activated partial thromboplastin was 29 and 40%, respectively. Shock markers, compensation mechanisms and coagulation pathways were all consistent with human pathophysiology. The absence of confounding factors, such as trauma-related bleeding or dilution due to trans-capillary refill was verified. This ethic, cost effective and bias-controlled model reproduced the specific and endogenous mechanism of ATC and will allow to identify potential targets for therapeutics in case of trauma-related hemorrhage.

A method for oxygenating and mixing suspensions of turbot Psetta maxima red blood cells (RBC) was tested in (31)P nuclear magnetic resonance (NMR) spectroscopy. In normoxia, the levels of inorganic phosphate (Pi) and nucleoside triphosphates (NTP) were stable up to 140 min and intracellular pH (pHi) was maintained and decreased oxygen partial pressure (P(O ( 2) )) from 30 to 15 and 600 Pa produced a significant fall in the intensity of NTP resonance, balanced by an increase in the Pi signal. Treatment of RBC with 0. 5 M isoproterenol during hypoxia exposure did not affect the pattern of changes in NTP or pHi induced by hypoxia and the effect was manifest only on Pi levels.

Regulatory volume decrease (RVD) following hyposmotic stimulation was studied in isolated turbot, Scophthalmus maximus, hepatocytes. Exposed to a reduced osmolality (from 320 to 240 mosm kg−1), cells first swelled and then exhibited a RVD. Volume regulation was significantly inhibited in presence of NPPB, 9-AC, acetazolamide, DIDS and barium. Taken together, these results could suggest that RVD operated via separate K+ and Cl- channels and probably Cl-/HCO 3 − exchanger in turbot hepatocytes. The K+/Cl- cotransporter could also be involved as furosemide and DIOA strongly inhibited the process whereas NEM, a K+/Cl- cotransporter activator, added under isosmotic conditions, led to cell shrinkage. RVD in turbot hepatocytes appeared also to depend on proteins p38 MAP kinase and tyrosine kinase but not on proteins ERK 1/2. Arachidonic acid and leukotrienes could also be involved since inhibition of synthesis of both these compounds by quinacrine and NDGA, respectively, inhibited the volume regulation. Likewise, Ca2+ has been proved to be an essential messenger as RVD was prevented in absence of Ca2+. Finally, this work provides bases for novel studies on cell volume regulation in marine teleosteans.

The molecular response to hypoxia stress in aquatic invertebrates remains relatively unknown. In this study, we investigated the response of the Pacific oyster Crassostrea gigas to hypoxia under experimental conditions and focused on the analysis of the differential expression patterns of specific genes associated with hypoxia response. A suppression subtractive hybridization method was used to identify specific hypoxia up-and downregulated genes, in gills, mantle and digestive gland, after 7-10 days and 24 days of exposure. This method revealed 616 different sequences corresponding to 12 major physiological functions. The expression of eight potentially regulated genes was analysed by RT-PCR in different tissues at different sampling times over the time course of hypoxia. These genes are implicated in different physiological pathways such as respiration (carbonic anhydrase), carbohydrate metabolism (glycogen phosphorylase), lipid metabolism (delta-9 desaturase), oxidative metabolism and the immune system (glutathione peroxidase), protein regulation (BTF3, transcription factor), nucleic acid regulation (myc homologue), metal sequestration (putative metallothionein) and stress response (heat shock protein 70). Stress proteins (metallothioneins and heat shock proteins) were also quantified. This study contributes to the characterization of many potential genetic markers that could be used in future environmental monitoring, and could lead to explore new mechanisms of stress tolerance in marine mollusc species.

A comparative study of blood oxygen binding and carrying capacities of turbot Scophthalmus maximus and sea bass Dicentrarchus labrax, two fish species differing in their demand for oxygen, was carried out under three levels of chronic hypoxia (Po2 = 93, 65 and 40 mmHg) for 40 days. Blood O2 affinity in normoxia was moderately high in both species (P50 was c. 12–13 mmHg at pH 7·7). The Bohr factor was significantly lower in turbot (−0·52) than in sea bass (−0·85). In both species, blood O2 affinity was not significantly affected by oxygen depletion whatever its level and duration. In turbot, however, P50 appeared to slightly decrease at the two more severe levels of hypoxia. In both species, blood O2 carrying capacity was not affected by hypoxia and remained twice as high in sea bass than in turbot.

Turbot juveniles (45 g) were exposed for 41 d (17 C, 34parts per thousand salinity) to constant normoxic (100-100% air saturation, 100-100) or moderate hypoxic (75-75% air saturation, 75-75) conditions and to repeated hypoxic shocks (20% saturation for 1 h, 5 d per week) from normoxic (100-20% air saturation. 100-20) or moderate hypoxic (75-20% air saturation, 75-20) conditions. A normoxic group was feed restricted (100-FR). Mass increase of 100-100 and 75-75 groups fed to satiation was not significantly different. In comparison, it was significantly lower in the 100-20 and 75-20 groups (NS between the two hypoxic shocks groups). Intermediate results were obtained in the 100-100-FR group. The lowest mass increase under hypoxic shocks was explained by a significant decrease in both feed intake and food conversion efficiency (FCE). FCE was lower in the two hypoxic groups, but only the 75-20 group was significantly different from all the other groups. There was no sign of stress and no change in the physiological status of fish in any group. When challenged, pre-conditioning of turbot to regular hypoxic shocks extended survival time, slightly but significantly, for 50% of the population. It was 8 h longer in starved than in fed fish. When reared for 1 year in normoxic water, the growth rate of post-challenged survivors was dependent on pre-conditioning: day 0-375 specific growth rate was significantly higher in the two groups acclimated to repeated hypoxic shocks. In the second experiment, it was shown that exposure to 20% air saturation for 12 h led to major physiological changes within 4 h: a significant decrease in plasma total CO2 and increase in plasma lactate contributing in maintaining blood pH stable, and a significant increase in osmolarity and chloride concentration. When returned to normoxic water, the recovery capacity of the fish was high: plasma osmolarity and total CO2 returned to pre-exposure levels within 1 h. The results are discussed in terms of turbot capacity to cope with repeated hypoxic shocks and to acclimate. (C) 2003 Editions scientifiques et medicates Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Hormonal changes, substrate mobilization and energy metabolism were studied in turbot Scophthalmus maximus exposed to 3 hypoxic conditions (oxygen partial pressure in water, PwO(2) = 90, 60 and 30 mm Hg) followed by recovery under normoxia. Measurements of the blood pH, total CO2 concentration, arterial oxygen partial pressure, hematocrit, glucose, lactate, and 'stress' hormones (cortisol, adrenaline and noradrenaline) plasmatic concentrations were performed. High-energy phosphorylated compounds, glycogen, glucose and lactate concentrations were also determined in liver and white muscle tissues. Exposure to 90 or 60 mm Hg did not induce any major physiological change, as hyperventilation by itself could compensate for the decrease in water oxygen tension. At 30 mm Hg, marked increases in cortisol, adrenaline and noradrenaline concentrations, associated with a decrease in blood arterial oxygen partial pressure, were observed. During exposure to 30 nun Hg, turbot resorted to anaerobic metabolism, resulting in liver glycogen depletion and lactate production. This mechanism appeared to be efficient enough to produce energy, as no significant change in phosphorylated compounds and adenylate energy charges in muscle and liver could be observed. These results indicate an absence of metabolic depression in turbot down to 30 mm Hg and confirm the high capacity of this species to cope with low water oxygen tension.

Effects of O-2 supersaturation on metabolism and growth were studied in juvenile turbot (Scophthalmus maximus L.). When fish were reared for 30 days in water containing O-2 at 147% or 223% air saturation, there were no significant differences in food intake, growth, food conversion or protein utilization compared to fish exposed to normoxia (100% air saturation in water outlet). Exposure to hyperoxia resulted in increased body fat deposition. Daily rates of O-2 consumption of resting fish were not affected by O-2-concentrations, and there were no significant differences in rates of nitrogenous excretion among fish exposed to the different O-2-concentrations. Turbot tolerated severe hyperoxia, 350% air saturation, for 10 days.There were changes in acid-base balance that compensated for the respiratory acidosis resulting from O-2 supersaturation. Blood pH was regulated within 24 h (it averaged 7.69 over the 30-day experiment) by significant increases in plasma CO2 content and pCO(2). Plasma CO2 was dose dependent averaging 11.3 and 18.9 mmol l(-1) under 147% and 224% O-2 saturation, respectively, compared to 6.7 mmol l(-1) under notmoxia. Over the 30-day experiment, the only change in hydromineral balance was a slight, but non-significant decrease in plasma chloride content in fish exposed to hyperoxia (137 mmol l(-1) compared to 139 under normoxia). There were no changes in haematocrit, haemoglobin and red blood cell counts (they averaged 18.3%, 3.7 g dl(-1) and 1.37 x 10(6) mm(-3). respectively) and no signs of stress (plasma cortisol averaged 3.8 ng ml(-1)) related to exposure to O-2-supersaturation for 30 days. (C) 2002 Elsevier Science B.v. All rights reserved.

In mammals, growth hormone (GH) is under a dual hypothalamic control exerted by growth hormone-releasing hormone (GHRH) and somatostatin (SRIH). We investigated GH release in a pleuronectiform teleost, the turbot (Psetta maxima), using a serum-free primary culture of dispersed pituitary cells. Cells released GH for up to 12 days in culture, indicating that turbot somatotropes do not require releasing hormone for their regulation. SRIH dose-dependently inhibited GH release up to a maximal inhibitory effect of 95%. None of the potential stimulators tested induced any change in basal GH release. Also, neither forskolin, an activator of adenylate cyclase, nor phorbol ester (TPA), an activator of protein kinase C, were able to modify GH release, suggesting that spontaneous basal release already represents the maximal secretory capacity of turbot somatotropes. In contrast, forskolin and TPA were able to increase GH release in the presence of SRIH. In this condition (coincubation with SRIH), pituitary adenylate cyclase-activating polypeptide (PACAP) stimulated GH release, whereas none of the other neuropeptides tested (GHRHs; sea bream or salmon or chicken II GnRHs; TRH; CRH) had any significant effect. These data indicate that inhibitory control by SRIH may be the basic control of GH production in teleosts and lower vertebrates, while PACAP may represent the ancestral growth hormone-releasing factor in teleosts, a role taken over in higher vertebrates by GHRH.

Variations in respiratory and acid-base status were studied in turbot (Scophthalmus maximus) during progressive severe hypoxia followed by recovery under normoxic conditions. The first behavioural strategy of turbot under hypoxia was an increase in amplitude and frequency of ventilation. Consequently, standard O2 consumption remained unchanged over a broad range of O2 tensions, until a low critical level of 30 mmHg. The hyperventilation induced a moderate blood alkalosis, compensated by a lactic acidosis. The fact that blood pH did not decrease below control values could be explained by the retention in white muscle of most of the lactate produced and by a high capacity for H+ excretion. During the recovery period, the marked increase in O2 uptake corresponding to an oxygen debt repayment, was partly related to the lactate elimination. When total energy contributions of aerobic and anaerobic processes were assessed in terms of ATP, the anaerobic contribution, estimated at the deepest hypoxia level, was higher than 20% of the total energy budget and appeared to totally compensate for the decline in aerobic metabolism. Moreover, the high value of O2 tension in arterial blood in normoxia and during recovery from hypoxia showed high diffusing capacity of gills in turbot. Our results explain the high tolerance of turbot for O2 deficient waters.

Des turbots d'un poids moyen initial de 32 g ont été exposés pendant 60 jours à 4 photopériodes constantes (8L:160, 12L:120, 16L:80, 24L:0O) et à 2 photophases variables : l'une croissante (de 12 à 16 h) et l'autre décroissante (de 12 à 8 h). Les autres paramètres du milieu ont été maintenus constants pendant les 2 mois de l'expérimentation (température : 17 ± 0,5 °C, salinité : 34,5 ppm, intensité lumineuse : 2 W.m-2 , concentration en oxygène supérieure à 6 mg.l-1). La survie, la croissance pondérale et différents indicateurs alimentaires (taux de conversion apparent, coefficients d'utilisation et d'efficacité protéiques) ont été déterminés. L'excrétion azotée (niveaux journaliers et profils horaires de l'excrétion d'Azote Ammoniacal Total, AAT, et d'azote uréique) et certains paramètres plasmatiques (osmolarité, natrémie, kaliémie, chlorémie, niveaux circulants des hormones thyroïdiennes) ont été mesurés. Au cours de l'expérience, aucune mortalité n'est apparue. Les croissances pondérales et les taux de croissance spécifique ont été identiques pour tous les traitements (poids moyens multipliés par trois en 60 jours). De même, la prise alimentaire (1,5-1,7 % de la biomasse par jour), le taux de conversion alimentaire (0,70-0,75) et les coefficients d'utilisation (36-39 %) et d'efficacité (2,5-2,7) protéiques ne sont pas influencés par la photopériode. La composition corporelle des animaux en protéines, lipides totaux, cendres et eau n'a pas subi de modification au cours de l'expérimentation. Osmolarité et electrolytes plasmatiques sont stables et identiques pour les différentes conditions. La thyroxine est stable quelle que soit la photopériode, les niveaux circulants sont compris entre 2 et 4 ng.ml-1. Les concentrations plasmatiques en T3 apparaissent significativement différentes en fin d'expérience : elles sont plus basses chez les animaux en photophase décroissante et chez ceux maintenus en photophase courte (8 h). Les taux journaliers d'excrétion azotée sous la forme d'AAT (200-230 mg.kg-1.j-1) et d'urée (65-75 mg.kg-1.j-1) sont identiques pour tous les traitements. Les profils d'excrétion azotée ont présenté des différences notables selon la photopériode. L'excrétion d'AAT est caractérisée par un pic post-prandial chez les animaux soumis à une alternance jour/nuit. Par contre, en éclairage continu, les taux horaires d'excrétion d'AAT restent stables tout au long du nycthémère. L'urée présente un pic nocturne dont l'amplitude, l'étalement et l'heure d'apparition dépendent de la photopériode. En éclairage continu, cette augmentation de l'excrétion d'urée est observée à un moment durant lequel les autres poissons sont en phase nocturne. A l'instar d'autres espèces de poissons plats, le turbot est peu sensible à la photopériode du moins lorsque les conditions d'alimentation sont ajustées à la durée de la phase éclairée.