Mechanisms underlying induced autoresuscitation failure in BALB/c and SWR mice.

Mechanisms underlying failure of autoresuscitation from hypoxic apnea were investigated. Failure was induced by repeated exposure to hypoxia. The influence of maturation was studied in adults, weanlings, and 10- and 5-day-old mice. Mice successful at autoresuscitation (BALB/c) as well as those prone to autoresuscitation failure (SWR weanlings) were studied. Hypoxic apnea was induced with 97% N2-3% CO2, and 21% O2 was given at its onset; electrocardiogram and ventilation were recorded. Hypoxic exposure was repeated if autoresuscitation (recovery of eupnea) occurred. Autoresuscitation failure (death) was induced in all mice. Young BALB/c mice tolerated more trials than older mice. SWR weanlings frequently failed to autoresuscitate on the initial exposure and tolerated fewer repeat trials overall than age-matched BALB/c mice. Induced autoresuscitation failure in all mice appeared to be unrelated to gasping regulation, because both gasp number and amplitude were similar during the failed trial and the previous successful trial. In most mice, failure was associated with absent recovery of heart rate during gasping. In BALB/c mice in particular, this persistent bradycardia was usually due to heart block, which occurred in 95% of failed trials. In addition, heart block occurred with increasing frequency on later successful trials, but conversion to sinus rhythm always preceded successful autoresuscitation. Heart block was also frequent in SWR mice and had similar consequences. BALB/c mice exposed to continuous anoxia survived longer than SWR mice, indicating increased endurance of components of the autoresuscitation mechanism not directly related to the ventilatory function of gasping (e.g., cardiovascular components).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of hyperthermia on mechanisms involved in autoresuscitation from hypoxic apnea in mice: a model for thermal stress causing SIDS.

The physiological mechanisms that might be involved in an association between heat stress and sudden infant death syndrome (SIDS) are obscure. We tested the hypothesis that a combination of acute hypoxia and elevated body temperature (T(B)) might prevent autoresuscitation from hypoxic apnea (AR). We exposed 21-day-old mice (total = 216) to hyperthermia (40.5-43.5 degrees C), hypoxia, or a combination of the two. Neither hyperthermia alone (40.5-42.5 degrees C) nor hypoxia alone was found to be lethal, but the combination produced failure to AR during the first hypoxic exposure with increasing frequency as T(B) increased. The ability to withstand multiple hypoxic exposures was also reduced as T(B) increased. In contrast, heat stress causing moderate T(B) increase (40.5 degrees C) had no effect on survival. Increased T(B) (43.5 degrees C) reduced gasping duration and number of gasps. It increased heart rate during anoxia but did not alter gasping rate. Furthermore, the oxygen-independent increase in heart rate observed before gasping failure was usually delayed until after the last gasp in hyperthermic animals. Mild dehydration occurred during T(B) elevation, but this did not appear to be a primary factor in AR failure. We conclude that a thermal stress, which by itself is nonlethal, frequently prevents AR from hypoxic apnea. This may be due, at least in part, to decreased gasp number and duration as well as to hyperthermia-related asynchrony of reflexes regulating heart and gasping frequencies during attempted AR.     

Mechanism of failure of recovery from hypoxic apnea by gasping in 17- to 23-day-old mice.

The mechanism of failure of autoresuscitation from hypoxic apnea in 17- to 23-day-old (weanling) Swiss Webster related/J mice was investigated by recording electrocardiogram (ECG) and ventilation in adult, weanling, and 11-day-old mice. Hypoxic apnea was induced with 97% N2-3% CO2. O2 (21% or 50% O2) or 97% N2-3% CO2 was given at the onset of apnea. The ECG showed no arrhythmias predictive of failure of autoresuscitation. The first indication of failure was a progressive fall in gasp volume ("run down"). This pattern also occurred in animals given continuous 97% N2-3% CO2 and was significantly different from that in mice that survived. Gasping duration in 97% N2 was longer in weanlings than adults but shorter than in 11 day olds. Respiratory and heart rate recovery were more rapid in adults than in weanlings. Although recovery in high O2 was more rapid, the survival rate was not increased. The lack of effect of high O2 on survival and the virtually identical pattern of gasping in mice dying in 97% N2 and air leads us to conclude that in mice that fail to autoresuscitate little or no O2 reaches the medullary respiratory centers. We speculate that this may be due to increased vulnerability of cardiac muscle to anoxia in 17- to 23-day-old mice, resulting in early and severe heart failure.     

Postnatal age influences the ability of rats to autoresuscitate from hypoxic-induced apnea

Failure to autoresuscitate from apnea by gasping has been suggested to have a role in sudden infant death. Little is known, however, about the factors that influence the ability of gasping to sustain life during acute hypoxia in the newborn. The present experiments were carried out on 105 rat pups to investigate the influence of postnatal age on the time to last gasp during a single hypoxic exposure and on the ability to autoresuscitate from primary apnea during repeated hypoxic exposures. On days 1-2, 5-6, 10-11, 15-16, and 19-20 postpartum, each pup was placed into a temperature-controlled chamber regulated to 37 +/- 1 degrees C and was exposed either to a single period of hypoxia produced by breathing an anoxic gas mixture (97% N(2)-3% CO(2)), and the time to last gasp was determined, or repeated exposure to hypoxia was performed, and the ability to autoresuscitate from primary apnea was determined. Increases in postnatal age decreased the time to last gasp following a single hypoxic exposure and decreased the number of successful autoresuscitations following repeated hypoxic exposures. Thus our data provide evidence that postnatal age influences protective responses that may prevent death during hypoxia as may occur during episodes of prolonged sleep apnea.     

Genetic control of heat sensitivity and activity level of murine arylsulfatase B

BALB/c male mice possess twofold higher kidney p-nitrocatechol-SO4 arylsulfatase B than do A/HeJ male mice; however, their liver arylsulfatase activities are comparable. Twentyfold-purified kidney arylsulfatases B from these two strains have similar Michaelis constants, electrophoretic mobilities, pH optima, and inhibitor profiles; however, the BALB/c enzyme is more heat stable than the A/HeJ enzyme. BALB/c, C3H/HeJ, DBA/2J, and SWR/J mice share an autosomal allele, As-1a, which apparently determines the heat-stable arylsulfatase B, while A/HeJ and C57BL/6J mice possess the As-1b allele, which determines the heat-sensitive enzyme. A second autosomal locus, Asr-1, determines liver arylsulfatase B activity. C57BL/6J mice carry the Asr-1a allele, which results in high liver activities, while C3H/HeJ mice are homozygous for the low-activity allele, Asr-1b. Male mice generally have 30-40% higher kidney activities than females; however, female kidney arylsulfatase activities rise and actually surpass those of males during late pregnancy and lactation.     

HYPOXIC SURVIVAL OF NORMOGLYCAEMIC YOUNG ADULT AND ADULT MICE IN RELATION TO CEREBRAL METABOLIC RATES12

The hypoxic tolerance and the cerebral metabolic rates (CMR) of young adult mice (20 to 25 g, 4 to 5 weeks old) and adult mice (30 g and above, 6 to 7 weeks old), respectively, were determined and their interrelationship was evaluated. CMRs increased from 25 mmol - P/kg.min to 38 mmol/kg.min as the animals grew older from young to full adulthood. Concurrently the tolerance to aerogcnic hypoxia (5% O₂-95%j N₂) declined. The effects of hypoxia on the cerebral energy metabolism were greater in adult than in young adult animals. It is concluded that the full metabolic maturation of the brain is reached in adult animals only. They become more dependent on an adequate oxygen supply as the aerobic activity of the energy metabolism of the brain is further increasing. Hypoxic gasping occurred while the pool of cerebral energy reserves was still far from being depleted. A failure to utilize energy reserves rather than their exhaustion is suggested as the ultimate cause of death from hypoxia. An acid-soluble form of glycogen or related polyglucan was found in addition to the usual amounts of insoluble glycogen. It was utilizcd rapidly during hypoxia and ischaemic anoxia and it may, therefore, constitute an additional source of carbohydrate substrates in thc brain.     

Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

Different sensitivity of inbred mice to hepatocarcinogen ortho-aminoazotoluene may be due to differences in the negative control mechanisms of hepatocyte proliferation

A most convenient model to study mechanisms of live organism response to chemical carcinogens is tumor induction in murine liver by aminoazodyes, in particular by ortho-aminoazotoluene (OAT). We studied both early and late stages of hepatocarcinogenesis on several lines of inbred mice differing in sensibility to OAT. By means of autoradiography, we examined proliferative activity of hepatocytes obtained from the liver of sensitive (A/He, DD, SWR) and resistant to OAT AKR, CC57Br, BALB/c lines of mice, which were injected carcinogen. The level of p53, p21Cip1, bax, mdm2, cyclin G, gadd45 genes expression in the liver of mice of different lines given OAT injection was studied by multiplex PCR method. Carcinogen caused a decrease of hepatocyte proliferative activity induced by partial hypatectomy (PHE), and an increase in p53, p21Cip, bax, mdm2, and cyclin G genes within mice of A/He, DD and SWR lines. Cell fusion experiments on hepatocytes obtained from regenerating murine liver sensitive to A/He line carcinogen and given long-time OAT administrations with resting and proliferating fibroblasts of NIH 3T3 mice revealed no obvious suppression of DNA synthesis in heterokaryons. Unlike, in fusion experiments on serum-stimulated fibroblasts with hepatocytes obtained from the liver of BALB/c line mice also given OAT suppression of DNA synthesis in stimulated fibroblasts in heterokaryons was observed 15 days following PHE. These results enable us to conclude that OAT administrations break negative endogenous mechanisms of hepatocyte proliferation control in the liver of mice sensitive to carcinogenes.     

Adipose triglyceride lipase plays a key role in the supply of the working muscle with fatty acids

FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice deficient for adipose triglyceride lipase (ATGL-ko) exhibit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL deficiency affects energy availability and substrate utilization in working muscle. In this study, we investigated the effect of moderate treadmill exercise on blood energy metabolites and liver glycogen stores in mice lacking ATGL. Because ATGL-ko mice exhibit massive accumulation of TG in the heart and cardiomyopathy, we also investigated a mouse model lacking ATGL in all tissues except cardiac muscle (ATGL-ko/CM). In contrast to ATGL-ko mice, these mice did not accumulate TG in the heart and had normal life expectancy. Exercise experiments revealed that ATGL-ko and ATGL-ko/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. Together, our studies suggest that ATGL-ko mice cannot adjust circulating FA levels to the increased energy requirements of the working muscle, resulting in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.     

Metabolic adjustments of an air-breathing teleost, Channa maculata, to acute and prolonged exposure to hypoxic water

Plasma and tissue metabolite levels were measured in the air-breathing Channa maculata during acute and prolonged exposure to normoxic and hypoxic water. Exposure of the fish to hypoxic water (water oxygen partial pressure, PwO ₂= 50 mmHg) for 1 h caused increases in plasma glucose and lactate, liver and brain lactate, liver a-amino acid, heart and brain alanine and brain succinate levels. The metabolic changes in heart, brain and muscle could only be detected when Pw O₂ was 30 or 10 mmHg. Heart glycogen and liver lipid decreased during acute exposure. Prolonged exposure to hypoxic water ( Pw O₂= 30 mmHg) for 3 days caused an increase in plasma glycerol and liver lactate dehydrogenase activity, and a depletion of glycogen store in all tissues investigated. However, metabolite levels which had been elevated during acute hypoxic exposure were observed to return to their normoxic values after prolonged exposure. It was concluded that anaerobic metabolism was triggered by acute exposure to hypoxic water. Prolonged exposure to hypoxic water induced a metabolic readjustment involving mobilisation of lipid and glycogen stores, which is probably a reflection of the high metabolic load of aerial respiration imposed on the fish during exposure to hypoxic water.     

Spontaneous heart failure in BALB/c mice

The hearts of BALB/c mice are known to acquire pronounced greyish white spots (cardiac white spots). BALB/c male mice were examined for the relationship between the incidence of cardiac white spots and weekly age, and compared with DDY male mice. During the observation period of 0.4-30 weeks, cardiac white spots on the right ventricle of BALB/c mice were first detected at three weeks (6 of 20 mice; 30%), and the maximal incidence of cardiac white spots was obtained at nine weeks (39 of 44 mice; 88%). In contrast, DDY mice were completely devoid of cardiac spots. Histopathologically, the cardiac spots were dystrophic calcinosis. There were significant increases in the relative organ weights of the heart and kidney of BALB/c mice compared with those of DDY mice. However, there was no significant difference between BALB/c and DDY mice in serum calcium concentration or histological characteristics of the parathyroid gland or bone marrow. The cardiac white spots of BALB/c mice were considered to be controlled by genetic susceptibility that occurred spontaneously with aging. The results described here suggest that BALB/c mice are adequate experimental animals for the study of myocardial disease that occurs spontaneously.     

Lesions of experimentally induced Tyzzer's disease in Syrian hamsters, guineapigs, mice and rats

The relative susceptibilities of C57BL/6NCR and BALB/cANNCR mice, F344/NCR rats, 2/NCR guineapigs and CR:RGH Syrian hamsters to Bacillus piliformis infection were determined by orally inoculating 20 weanling females from each species with suspensions of B. piliformis spores. Animals from each group were sequentially necropsied over 2 week periods to document the lesions produced. No significant gross or microscopic lesions were observed in the BALB mice or the Fischer rats. Gross and microscopic lesions were observed in the livers and intestines of many guineapigs and hamsters killed 3-14 days after inoculation. A large lesion was observed in the left cardiac ventricle of one C57BL mouse 10 days after inoculation. Warthin-Starry silver-stained tissue sections revealed clusters of B. piliformis within the cytoplasm of intestinal epithelial cells, smooth muscle cells, hepatocytes and myocytes bordering foci of necrosis in the intestines, liver and heart.     

Strain differences in the immune response of mice to homologous sperm-specific lactate dehydrogenase (LDH-C4).

The sperm-specific isozyme of murine lactate dehydrogenase (LDH-C4) was injected into female mice of various strains. Two regulatory phenotypes characterize the resultant immunity to LDH-C4: one is manifested by high, intermediate or low levels of response, the other by the immediate or delayed maturation of peak titer. The response of several strains can be classified as high (SWR, SJL, BABL/c, C3H/He) and intermediate to low (A, CBA, DBA/2, DBA/1, C57BL/6) according to the level of antibody production and cell mediated immunity. BALB/c, SJL and SWR strains are immediate responders while DBA/2 and C3H/He mice are clearly delayed responders. Maturation and magnitude of response do not appear to be related. Both the antibody and cell mediated responses are T-dependent, but are not obviously associated with Ig allotype or H-2 regulation.     

Cardiomyocyte Aldose Reductase Causes Heart Failure and Impairs Recovery from Ischemia

Aldose reductase (AR), an enzyme mediating the first step in the polyol pathway of glucose metabolism, is associated with complications of diabetes mellitus and increased cardiac ischemic injury. We investigated whether deleterious effects of AR are due to its actions specifically in cardiomyocytes. We created mice with cardiac specific expression of human AR (hAR) using the α–myosin heavy chain (MHC) promoter and studied these animals during aging and with reduced fatty acid (FA) oxidation. hAR transgenic expression did not alter cardiac function or glucose and FA oxidation gene expression in young mice. However, cardiac overexpression of hAR caused cardiac dysfunction in older mice. We then assessed whether hAR altered heart function during ischemia reperfusion. hAR transgenic mice had greater infarct area and reduced functional recovery than non-transgenic littermates. When the hAR transgene was crossed onto the PPAR alpha knockout background, another example of greater heart glucose oxidation, hAR expressing mice had increased heart fructose content, cardiac fibrosis, ROS, and apoptosis. In conclusion, overexpression of hAR in cardiomyocytes leads to cardiac dysfunction with aging and in the setting of reduced FA and increased glucose metabolism. These results suggest that pharmacological inhibition of AR will be beneficial during ischemia and in some forms of heart failure.     

Heart rate variability responses to hypoxic and hypercapnic exposures in different mouse strains.

Heart rate variability (HRV) is a well-characterized, noninvasive means of assessing cardiac autonomic nervous system activity. This study examines the basic cardiac responses to hypoxic and hypercapnic challenges in seven strains of commonly used inbred mice (A/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J, and FVB/J). Adult male mice, 8-12 wk of age, were chronically instrumented to a femoral artery catheter for the continuous measurement of systemic arterial blood pressure and heart rate. Mice were exposed to multiple 4-min periods of hypoxia (10% O2), hypercapnia (5% CO2), and combined hypoxia/hypercapnia (10% O2 + 5% CO2). HRV was derived from pulse intervals of the blood pressure tracings. Hypoxia induced increases in high-frequency HRV power and decreased low-frequency (LF) HRV power in most strains. Hypercapnia led to decreased high-frequency HRV power and increased LF HRV power in most strains. Strain differences were most notable in regard to the concomitant exposures of hypoxia and hypercapnia, with FVB/J mice mirroring their own response to hypercapnia alone, whereas CBA/J mice mirrored their own responses to hypoxia. As blood pressure is most likely the driving factor for heart rate changes via the baroreflex pathway, it is interesting that LF, considered to reflect cardiac sympathetic activity, was negatively correlated with heart rate, suggesting that LF changes are driven by baroreflex oscillation and not necessarily by absolute sympathetic or parasympathetic activity to the heart. These findings suggest that genetic background can influence the centrally mediated cardiovascular responses to basic hypoxic and hypercapnic challenges.     

Pas1 is a common lung cancer susceptibility locus in three mouse strains

Inherited predisposition to lung cancer is a phenotypic trait shared by different mouse inbred strains that show either a high or an intermediate predisposition. Other strains are instead genetically resistant. The Pas1 locus is the major determinant of lung cancer predisposition in the A/J strain (Gariboldi et al. 1993). To define the determinants of susceptibility to lung tumorigenesis in the highly susceptible SWR/J and in the intermediately susceptible BALB/c mice, we analyzed (BALB/c × SWR/J)F₂ and (BALB/c × C3H/He)F₂ crosses by genetic linkage experiments. The present results provide unequivocal evidence that the same Pas1/+ allele that leads to lung cancer predisposition is shared by A/J, SWR/J, and BALB/c strains. The intermediate susceptibility of the BALB/c strain would result by interaction of Pas1 locus with lung cancer resistance loci. Received: 18 April 1997 / Accepted: 15 June 1997     

Effects of hypoxia on megakaryocyte size and number of C3H and BALB/c mice.

In an effort to explain the different platelet production capabilities of both normal and hypoxic male and female C3H and BALB/c mice, megakaryocyte size and number were determined utilizing bone marrow from both normal and hypoxic mice. The results indicate that normal BALB/c female mice have increased numbers of megakaryocytes, but of smaller size compared with either BALB/c male mice or to both sexes of C3H mice. An inverse relationship between the size and number of megakaryocytes was found in both normal and hypoxic mice; therefore, to evaluate total megakaryocyte characteristics, we calculated total megakaryocyte masses (TMM). With hypoxia, megakaryocyte number decreased, whereas megakaryocyte size increased. Despite the increase in megakaryocyte size, hypoxia caused a significant decrease in TMM (P less than 0.005) in all mice, but female C3H mice had higher TMM (P less than 0.05) than did female BALB/c mice. These data show that hypoxia decreases TMM in mice, and that the effect is greater in C3H mice than in BALB/c mice.     

Ovariectomy and overeating palatable, energy-dense food increase subcutaneous adipose tissue more than intra-abdominal adipose tissue in rats

Background

Men are at an increased risk of dying from heart failure caused by inflammatory heart diseases such as atherosclerosis, myocarditis and dilated cardiomyopathy (DCM). We previously showed that macrophages in the spleen are phenotypically distinct in male compared to female mice at 12 h after infection. This innate immune profile mirrors and predicts the cardiac immune response during acute myocarditis.

Methods

In order to study sex differences in the innate immune response, five male and female BALB/c mice were infected intraperitoneally with coxsackievirus B3 (CVB3) or phosphate buffered saline and their spleens were harvested 12 h later for microarray analysis. Gene expression was determined using an Affymetrix Mouse Gene 1.0 ST Array. Significant gene changes were verified by quantitative real-time polymerase chain reaction or ELISA.

Results

During the innate immune response to CVB3 infection, infected males had higher splenic expression of genes which are important in regulating the influx of cholesterol into macrophages, such as phospholipase A₂ (PLA₂) and the macrophage scavenger receptor compared to the infected females. We also observed a higher expression in infected males compared to infected females of squalene synthase, an enzyme used to generate cholesterol within cells, and Cyp2e1, an enzyme important in metabolizing cholesterol and steroids. Infected males also had decreased levels of the translocator protein 18 kDa (TSPO), which binds PLA₂ and is the rate-limiting step for steroidogenesis, as well as decreased expression of the androgen receptor (AR), which indicates receptor activation. Gene differences were not due to increased viral replication, which was unaltered between sexes.

Conclusions

We found that, compared to females, male mice had a greater splenic expression of genes which are important for cholesterol metabolism and activation of the AR at 12 h after infection. Activation of the AR has been linked to increased cardiac hypertrophy, atherosclerosis, myocarditis/DCM and heart failure in male mice and humans.