Ischaemia and tissue pyruvate dehydrogenase activities in the rat: a comparison of the effects of cervical dislocation and pentobarbital anaesthesia

The work defined the relationship between [long-chain acylcarnitine] and PDHa activities in hearts, kidneys and livers of rats sampled after cervical dislocation or pentobarbital anaesthesia. Although tissue [long-chain acylcarnitine] correlated with fatty acid availability or its mitochondrial oxidation in anaesthetized rats, this was not the case for hearts or kidneys of rats sampled after cervical dislocation. Cardiac [long-chain acylcarnitine] and PDHa activities were higher in rats killed by cervical dislocation. Metabolite changes within the hearts were consistent with tissue hypoxia and the effects of cervical dislocation were mimicked in hearts of pentobarbital-anaesthetized rats by 20s ischaemia. Renal and hepatic PDHa activities were unaffected by this short period of ischaemia. The susceptibility of cardiac PDHa to hypoxia or ischaemia may explain the variability in activities often observed within or between laboratories.     

Loss of cortical function in mice after decapitation, cervical dislocation, potassium chloride injection, and CO2 inhalation

Electroencephalograms (EEG) and visual evoked potentials (VEP) in mice were recorded to evaluate loss of cortical function during the first 30 s after euthanasia by various methods. Tracheal cannulae (for positive-pressure ventilation, PPV) and cortical surface electrodes were placed in mice anesthetized with inhaled halothane. Succinylcholine was used to block spontaneous breathing in the mice, which then underwent continuous EEG recording. Photic stimuli (1 Hz) were presented to produce VEPs superimposed on the EEG. Anesthesia was discontinued immediately before euthanasia. Compared with that obtained before euthanasia, EEG activity during the 30-s study period immediately after euthanasia was significantly decreased after cervical dislocation (at 5 to 10 s), 100% PPV-CO2 (at 10 to 15 s), decapitation (at 15 to 20 s), and cardiac arrest due to KCl injection (at 20 to 25 s) but not after administration of 70% PPV-CO2. Similarly, these euthanasia methods also reduced VEP amplitude, although 100% PPV-CO2 treatment affected VEP amplitude more than it did EEG activity. Thus, 100% PPV-CO2 treatment significantly decreased VEP beginning 5 to 10 s after administration, with near abolition of VEP by 30 s. VEP amplitude was significantly reduced at 5 to 10 s after cervical dislocation and at 10 to 15 s after decapitation but not after either KCl or 70% PPV-CO2 administration. The data demonstrate that 100% PPV-CO2, decapitation, and cervical dislocation lead to rapid disruption of cortical function as measured by 2 different methods. In comparison, 70% PPV-CO2 and cardiac arrest due to intracardiac KCl injection had less rapid effects on cortical function.     

POST-MORTEM CHANGES IN HIGH AFFINITY CHOLINE UPTAKE

Abstract— When animals were killed by decapitation and the heads kept refrigerated at 2–4°C. high affinity choline uptake was maintained up to 3 days post-mortem. At 5 days post-mortem, there was a significant reduction in uptake. In tissues kept at 2–4°C for 1 day, the ionic dependence, drug sensitivity and kinetic parameters of uptake were identical to that of control tissues. At 3 days post-mortem, intact synaptosomal profiles, although with features characteristic of degenerating neuronal tissues, were observed in electronmicroscopic studies. In tissues maintained at room temperature, however, the uptake activity was nearly completely gone by 1 day. It is concluded that high affinity choline uptake is maintained for days, a surprisingly long time, in tissues kept in the cold immediately after death.
When pentylenetetrazol or pentobarbital were administered to rats in order to activate or depress the choline uptake, it was found that the activity-related changes in choline uptake undergo a reversal in post-mortem tissues. The changes in uptake were significantly lost by 10min post-mortem and totally absent by 30min post-mortem. In vitro studies with whole hippocampi indicate that the postmortem reversal in activity-related changes in uptake is temperature-dependent. It is concluded that because of post-mortem reversals in activity-related states of uptake the true magnitude of these activity-related changes in uptake may be underestimated by existing methods of assay. Acetylcholine levels in synaptosomal preparations did not clearly correlate with levels of choline uptake.     

Maternal proximity and infant CO2 environment during bedsharing and possible implications for SIDS research

Sudden infant death syndrome (SIDS) is the leading cause of human infant mortality after the neonatal period in Western countries. Recently, child care practices have been shown to be important in determining infant vulnerability to SIDS. However, very little is known about the impact of parent-infant cosleeping on infant sleep physiology and behavior and SIDS risk. This reflects the failure of Western societal research paradigms to appreciate the human infant's evolutionary history of cosleeping, the recency of the emergence of solitary infant sleeping as a practice and the fact that parent-infant cosleeping is still the preferred sleeping arrangement for the majority of contemporary societies. Incorporating current hypotheses on the mechanisms of SIDS, we have hypothesized that the comparatively sensory-rich cosleeping environment might be protective against SIDS in some contexts. As a first step to characterize cosleeping environments, this investigation is aimed at assessing, in routinely bedsharing mothers and infants, their relative sleeping positions and the potential for sleeping in close face-to-face proximity and for infant exposure to increased environmental CO2 produced by maternal respiration. The latter is important in that breathing elevated levels of CO2 can have diverse effects, ranging from respiratory stimulation at low levels to suffocation at very high levels. Two related laboratory studies were performed. In the first, all-night videotapes of 12 healthy, routinely bedsharing mother-infant pairs were analyzed for sleeping positions and time spent in face-to-face orientation and distances separating their faces. Infants were 11–15 wk old. Mothers predominantly positioned themselves on their sides facing their infants, with the infants placed either supine or on their sides. Mothers and infants slept oriented face-to-face for 64 ± 27% (S.D.) of non-movement time, with distances less than 20 cm commonly separating their faces. In the second study, concentrations of CO2 in air were measured in six young women at distances of up to 21 cm from their nares. Peak expiratory CO2 concentrations remained above 1.0% at distances up to 9 cm and above 0.5% at 18 cm. Both baseline and peak CO2 levels were further increased at all distances when measured within a partial air pocket created to simulate a bedding environment sometimes seen during bedsharing. We conclude that during bedsharing there is potential for 1) a high degree of face-to-face orientation and close proximity and consequently 2) increased environmental CO2, as a result of maternal respiration, to non-lethal levels that might stimulate infant respiration. The close proximity would also maximize the sensory impact of the mother on the infant through other modalities. We also suggest that bedsharing may minimize prone infant positioning, a known risk factor for SIDS. Am J Phys Anthropol 103:315–328, 1997. © 1997 Wiley-Liss, Inc.     

Microbiology in sudden infant death syndrome (SIDS) and other childhood deaths

The usefulness of post-mortem microbiology in the assessment of sudden unexpected deaths in infants and children has been debated by many pathologists. In our centre, microbiological investigations have been part of the post-mortem protocol for investigation of sudden deaths in infants and children for the past 12 years. The objective of this study was to review the microbiological findings for infants and children examined by our unit during the past 4 years in relation to gross and histological findings of the autopsy and the medical and social histories of the children. We reviewed 57 consecutive sudden deaths in infants and children examined by our Referral Centre between November 1994 and October 1998. These 57 sudden deaths were aged from 1 day to 4 years and 9 months including 40 cases of sudden infant death syndrome (SIDS) and 17 non-SIDS deaths. Results of the microbiological investigations of tissues and body fluids were assessed during the case review with reference to histological shock signs, severe gastric aspiration, and signs of acute thymic involution. Bacteria alone or in association with viruses were identified in 45/57 (79%) cases including 34/40 (85%) SIDS. The most frequent bacterial isolate was Escherichia coli (27), and the virus identified most frequently was enterovirus (8). C-reactive protein was increased in 10 out of the 42 cases tested including 8/32 (25%) SIDS. Significant gastric content aspiration was found in 17/57 (29.8%) including 13/40 (32.5%) SIDS. Histological signs of shock were present in 33/55 (60%) cases including 22/39 SIDS (56.4%). The microbiological findings were positive for 27/33 (81.8%). We conclude that post-mortem microbiology is essential in sudden death investigation. The conclusion that a death is unexplained if no microbiology was done is not valid, even if in some cases it may be difficult to know precisely in what way the pathogen contributed to the death.     

Pretreatment with 3-O-methyl-d-glucose or 2-deoxy-d-glucose attenuates the post-mortem rise in rat brain lactate

The present investigation examined the effects of pretreatment with 3-O-methyl-d-glucose (3OMG) or 2-deoxy-d-glucose (2DOG) on post-mortem rise in rat brain lactate to evaluate their potential use for minimizing ischemia-induced rise in brain lactate. The results showed that iv administration of either glucose analogue (2 g/kg) at 2.5 min prior to sacrifice significantly attenuated (to 0.61 of control levels) post-mortem brain lactate rise. Pretreating rats with 2-deoxy-d-glucose (2 g/kg) 15 min prior to sacrifice resulted in a greater inhibition (to 0.52 of control) of the post-mortem lactate rise. The effects of these two analogues (3OMG and 2DOG) can be accounted for by their inhibition of brain glucose transport and inhibition of brain glucose metabolism by 2DOG. The present results suggest that intervention with either of these glucose analogues under the proper experimental procedures may minimize the cytopathological consequences of ischemia related to the rise in brain lactate.     

Extraintestinal Escherichia coli isolations from SIDS cases and other cases of sudden death in Victoria, Australia

This investigation is an extension of previous studies on the possible role of intestinal Escherichia coli in sudden infant death syndrome (SIDS) to include the isolation of extraintestinal E. coli. The lungs of 52 and the blood of 144 SIDS infants were cultured and isolates were investigated. E. coli was isolated from about a quarter of post-mortem lung samples and about 15% of blood samples from SIDS infants. The isolates were subjected to microbiological studies, including serotyping and haemolysin assays. The majority were found to belong to serogroups commonly associated with bacteraemia. These results may indicate that extraintestinal E. coli plays a role in SIDS.     

Evidence for infection, inflammation and shock in sudden infant death: parallels between a neonatal rat model of sudden death and infants who died of sudden infant death syndrome

This study compared pathological findings from a neonatal rat model of sudden death with those from 40 sudden infant death syndrome (SIDS) infants collected at autopsy. In the rat model, influenza A virus was administered intranasally on postnatal day 10, and on day 12 a sublethal, intraperitoneal dose of Escherichia coli endotoxin; mortality was 80%. Tissue samples from the animals and infants were fixed in formaldehyde, embedded in paraffin, and sections stained with hematoxylin and eosin. Tissues from the SIDS specimens were additionally cultured for bacteria and viruses; post-mortem blood samples were evaluated for signs of inflammation. All sections were examined by a pediatric forensic pathologist familiar with SIDS pathology. Comparisons between the rat model and the human SIDS cases revealed that both exhibited gross and microscopic pathology related to organ shock, possibly associated with the presence of endotoxin. Uncompensated shock appeared to be a likely factor that caused death in both infants and rat pups. Response to a shock-inducing event might have played an important role in the events leading to death. The similarities between the neonatal rats and the human cases indicate that further research with the model might elucidate additional aspects of SIDS pathology.     

Assessment of in vivo and post-mortem mechanical behavior of brain tissue using magnetic resonance elastography

The knowledge of in vivo brain tissue mechanical properties is essential in several biomedical engineering fields, such as injury biomechanics and neurosurgery simulation. Almost all existing available data have been obtained in vitro by invasive experimental protocols. However, the difference between in vivo and post-mortem mechanical properties remains poorly known, essentially due to the lack of a common method that could measure them both in vivo and ex vivo. In this study, we report the use of magnetic resonance elastography (MRE) for the non-invasive assessment of in vivo brain tissue viscoelastic properties and for the investigation of their evolution after the death. Experiments were performed on seven adult male rats. Shear storage and loss moduli were measured in vivo, just after death and at post-mortem time of approximately 24h. A significant increase in shear storage modulus G(') of approximately 100% was found to occur just after death (p=0.002), whereas no significant difference was found between in vivoG(') and G(') at 24h post-mortem time. No significant difference was found between shear loss modulus G(')in vivo and just after death, whereas a decrease of about 50% was found to occur after 24h (p=0.02). These results illustrate the ability of MRE to investigate some of the critical soft tissue biomechanics-related issues, as it can be used as a non-invasive tool for measuring soft tissue viscoelastic properties.     

Sudden infant death syndrome and Canadian Aboriginals: bacteria and infections

Aboriginal populations in Canada, America and Australia have higher incidences of sudden infant death syndrome (SIDS) than non-Aboriginal groups. Canadian Aboriginal populations (known also as first nation, native or Indian) experience infant morbidity/mortality rates 3-7 times that of non-Aboriginals, with upper track respiratory infection and SIDS recorded as the leading causes. The aim of this investigation was to examine the home environment of Aboriginal infants, particularly during winter months when respiratory tract infections and SIDS are more common. Environmental bacteria, fungi and air particulates were examined in the residences of Aboriginal infants during visits to individual homes on an Aboriginal reserve. The physical histories of SIDS victims were gathered from medical files. Air and surfaces were sampled by agar strips which were processed by a commercial laboratory. The levels of fungi, bacteria and air particulate rates recorded in the reserve homes of Aboriginal infants registered levels considered to be detrimental to the health of the inhabitants. Such extreme levels could contribute to the high incidence of respiratory disease and SIDS experienced by Canadian Aboriginal infants.     

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.     

Dopamine transport function is elevated in cocaine users

Dopaminergic transmission has been suggested to be a primary mechanism mediating reinforcement, withdrawal and craving associated with psychostimulant addiction. Pyscho-stimulants attenuate dopamine transporter (DAT) clearance efficiency, resulting in a net increase in synaptic dopamine levels. Re-uptake rate is determined by the number of functional DAT molecules at the membrane surface. Previous in vivo imaging studies in humans and in vitro studies in post-mortem human brain have demonstrated that chronic cocaine abuse results in a neuroadaptive increase in DAT-binding site density in the limbic striatum. Whether this increase in DAT availability represents an increase in the functional activity of the transporter is unknown. Here, we present evidence that DAT function is elevated by chronic cocaine abuse. The effect of increasing post-mortem interval on the functional viability of synaptosomes was modeled in the baboon brain. Baboon brains sampled under conditions similar to human brain autopsies yielded synaptosomal preparations that were viable up to 24 h post-mortem. Dopamine (DA) uptake was elevated twofold in the ventral striatum from cocaine users as compared to age-matched drug-free control subjects. The levels of [3H]DA uptake were not elevated in victims of excited cocaine delirium, who experienced paranoia and marked agitation prior to death. In keeping with the increase in DAT function, [3H]WIN 35,428 binding was increased in the cocaine users, but not in the victims of excited delirium. These results demonstrate that DA uptake function assayed in cryopreserved human brain synaptosomes is a suitable approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of addictive drugs in man.     

Postmortem Stability of Brain 3-Methoxy-4-Hydroxyphenylethyleneglycol and 3,4-Dihydroxyphenylethyleneglycol in the Rat and Mouse

Abstract: To assess the postmortem stability of brain 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and 3,4-dihydroxyphenylethyleneglycol (DHPG) levels, groups of rats and mice were killed by cervical dislocation and left at either 21° or 4°C for intervals of up to 24 h until removal and freezing of whole brain. Whole brain free and total MHPG and DHPG levels were determined simultaneously by gas chromatography-mass fragmentography (GC-MF). By 2 h after death, statistically significant decrements occurred in rat brain free DHPG (20%), total MHPG (21%), and total DHPG (11%) at 4°C, but free MHPG increased significantly (50%) compared with controls. At 21°C, rat brain total MHPG increased compared with controls at 2 h (15%) but decreased at 4 h (15%) and 8 h (15%), whereas free MHPG levels were increased at these times. Although brain total and conjugated DHPG levels showed little change, free DHPG levels were reduced at all times. In mouse brain no significant changes occurred in free MHPG and DHPG by 24 h at 4°C. At 21°C, mouse brain DHPG levels decreased whereas MHPG concentrations increased over the 8-h period of study. These findings demonstrate the occurrence of significant postmortem time- and temperature-dependent changes in brain MHPG and DHPG concentrations and indicate caution in the interpretation of changes in these metabolites in studies employing human postmortem brain tissue.     

Elevated body temperature enhances the laryngeal chemoreflex in decerebrate piglets.

Hyperthermia and reflex apnea may both contribute to sudden infant death syndrome (SIDS). Therefore, we investigated the effect of increased body temperature on the inhibition of breathing produced by water injected into the larynx, which elicits the laryngeal chemoreflex (LCR). We studied decerebrated, vagotomized, neonatal piglets aged 3-15 days. Blood pressure, end-tidal CO(2), body temperature, and phrenic nerve activity were recorded. To elicit the LCR, we infused 0.1 ml of distilled water through a polyethylene tube passed through the nose and positioned just rostral to the larynx. Three to five LCR trials were performed with the piglet at normal body temperature. The animal's core body temperature was raised by approximately 2.5 degrees C, and three to five LCR trials were performed before the animal was cooled, and three to five LCR trials were repeated. The respiratory inhibition associated with the LCR was substantially prolonged when body temperature was elevated. Thus elevated body temperature may contribute to the pathogenesis of SIDS by increasing the inhibitory effects of the LCR.     

Survival and distribution of Echinostoma caproni in the small intestine of ICR mice up to 36 hours after the death of the host

This study examined survival and distribution of Echinostoma caproni in the small intestine of ICR mice at various times up to 36 hr following the death of the host. Adult worms were obtained at 2-wk postinfection of 21 ICR mice each infected with 50 metacercarial cysts. Mice were killed with light ether anesthetization and cervical dislocation and maintained at room temperature (22 +/- 1C) until examination at 0 (controls), 2, 4, 6, 12, 24, and 36 hr postmortem. Survival was based on worm activity and distribution was assessed on the basis of worm location in 1 of 5 equal intestinal segments numbered from the pylorus to the ileocecal valve. Worms were alive up to 36 hr post-mortem and were distributed mainly in segments 3 and 4 at all times postmortem. Histochemical Oil Red O studies on whole control and experimental worms showed neutral lipids localized in the protone-phridial tubules and the excretory bladder. Eggs from experimental worms at all times produced miracidia that infected Biomphlaria glabrata snails.     

Hormonal changes during the early development of ovarian cysts in the rat

The daily administration of human chorionic gonadotropin (hCG) to rats with thiouracil-induced hypothyroidism results in the development of cystic ovaries. This study was undertaken to delineate hormonal changes during the first 48 h of hCG treatment. Groups of euthyroid and hypothyroid rats were injected daily with hCG or saline for up to two days and killed at 0, 12, 24, or 48 h after the initial hCG injection. Sera were analyzed for progesterone (P), testosterone (T), 17 beta-estradiol (E2), and prolactin (Prl) by specific radioimmunoassay (RIA). Serum levels of these hormones were not significantly different in the euthyroid and hypothyroid rats. However, P was significantly elevated at 12, 24, and 48 h in the hypothyroid/hCG rats. T and Prl were significantly elevated at 12 and 48 h in the hypothyroid/hCG rats. T levels were also elevated at 12 and 48 h in the euthyroid rats receiving hCG. In contrast, hCG had no effect on P and Prl levels in the euthyroid rats. E2 levels were undetectable in the euthyroid and hypothyroid rats. The administration of hCG increased E2 in both the euthyroid and hypothyroid rats at 48 h with significantly more E2 detected in the hypothyroid rats. These results show that ovarian steroids and Prl levels increase during the early stages of cyst induction and suggest they may be important in triggering ovarian cyst formation.     

Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome

Background

Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated.

Methodology/Principal Findings

Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [³H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled B_max value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls).

Conclusions

In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism.     

Assessment of genomic instability in normal and diabetic rats treated with metformin

To examine if a single or multiple oral administration of metformin, a member of the biguanide class of anti-diabetic agents, has any genotoxic and cytotoxic potential in normal and diabetic rats, a mammalian model, cytogenetic assays through several endpoints such as induction of micronuclei, chromosome aberrations, mitotic activity of bone marrow cells, sperm-head anomaly and assays of some oxidative stress markers have been conducted by the use of standard techniques. Diabetes was induced by streptozotocin injection. Metformin was administrated to both diabetic and non-diabetic rats in single doses of 100, 500 or 2500 mg/kg along with vehicle control groups for diabetic and non-diabetic rats. The animals were killed by cervical dislocation at 24 h after treatment, and then bone marrow cells were sampled. Also, a multiple dose study has done in which diabetic and non-diabetic animals were treated with 100 or 500 mg/kg of metformin daily for 4 or 8 weeks after which the animals were killed by cervical dislocation, and then bone marrow and sperm cells were collected. Concurrent control groups were also included in each experiment. The obtained results revealed that metformin was neither genotoxic nor cytotoxic for the rats in all groups at all tested doses. Moreover, metformin significantly reduced the diabetes-induced genomic instability and cell proliferation changes in somatic and germinal cells in a dose-dependent manner (2500, 500, >100 mg/kg). In addition, diabetes induced marked biochemical alterations characteristic of oxidative stress including, enhanced lipid peroxidation and reduction in the reduced glutathione level. Treatment with metformin ameliorated these biochemical markers. In conclusion, metformin is a non-genotoxic or cytotoxic compound and may protect from genomic instability induced by hyperglycemia. Apart from its well-known anti-diabetic effect, the antigenotoxic effect of metformin could be possibly ascribed to its radical scavenger effect that modulated the genomic instability responses and cell proliferation changes induced by hyperglycemia.     

Beta-endorphin and neurotensin in brainstem and cerebrospinal fluid in the sudden infant death syndrome.

Beta-endorphin (BE) and neurotensin (NT) are two neuropeptides which induce apneas. In infants who died of Sudden Infant Death Syndrome (SIDS) we measured, in brainstem and CSF, BE and NT by IRMA and RIA respectively. BE and NT levels are compared to same aged infant and adult controls. CSF BE level was significantly higher in SIDS than in the two control groups (86 +/- 14 vs 33 +/- 13 and 16 +/- 5 pmol/l). In 6 SIDS victims NT and BE were assayed in 5 brainstem sections, each of them divided in median, intermediate and lateral parts. We found high levels of BE in every fragment (3-11 pmol/mg protein) while NT elevated values were restricted to the mesencephalic regions (1.4-12 pmol/mg), the medial pons (6 pmol/mg) and the intermediate parts of the medulla (including the olive: 1.3-1.6 pmol/mg). These results support the hypothesis that NT and/or BE could induce or participate to the fetal issue of SIDS.     

Effects of lethal levels of environmental hypercapnia on cardiovascular and blood-gas status in yellowtail,Seriola quinqueradiata

The cardiorespiratory responses were examined in yellowtail, Seriola quinqueradiata exposed to two levels of hypercapnia (seawater equilibrated with a gas mixture containing 1% CO(2) (water PCO(2) = 7 mmHg) or 5% CO(2) (38 mmHg)) for 72 hr at 20 degrees C. Mortality was 100% within 8 hr at 5% CO(2), while no fish died at 1% CO(2). No cardiovascular variables (cardiac output, Q; heart rate, HR; stroke volume, SV and arterial blood pressure, BP) significantly changed from pre-exposure values during exposure to 1% CO(2). Arterial CO(2) partial pressure (PaCO(2)) significantly increased (P < 0.05), reaching a new steady-state level after 3 hr. Arterial blood pH (pHa) decreased initially (P < 0.05), but was subsequently restored by elevation of plasma bicarbonate ([HCO(3)(-)]). Arterial O(2) partial pressure (PaO(2)), oxygen content (CaO(2)), and hematocrit (Hct) were maintained throughout the exposure period. In contrast, exposure to 5% CO(2) dramatically reduced Q (P < 0.05) through decreasing SV (P < 0.05), although HR did not change. BP was transiently elevated (P < 0.05), followed by a precipitous fall before death. The pHa was restored incompletely despite a significant increase in [HCO(3)(-)]. PaO(2) decreased only shortly before death, whereas CaO(2) kept elevated due to a large increase in Hct (P < 0.05). We tentatively conclude that cardiac failure is a primary physiological disorder that would lead to death of fish subjected to high environmental CO(2) pressures.