The effect of cold acclimation and exercise training on cold tolerance in aged C57BL/6J mice

We investigated whether intermittent cold acclimation and low intensity exercise training, two interventions known to improve thermoregulatory function in young rodents, enhanced cold tolerance in aged C57BL/6J mice. Mice, aged 18 to 20 months, were randomly assigned to four treatment conditions: (a) intermittent cold acclimation (CA) (50 min per day, 5 times per week for 3 weeks at 5 degrees C), (b) submaximal treadmill exercise (EX) (15 m min-1 for 30 min, 5 times per week for 8 weeks, (c) sequential treatment of CA + EX, and (d) control group. Mice were exposed to a 3-hr cold stress test (15 degrees C) prior to and following treatment. CA aged mice maintained normothermia and demonstrated significant increases in oxygen consumption and brown adipose tissue protein concentration compared with controls; enhanced cold tolerance was probably due to increased utilization of nonshivering thermogenesis. EX aged mice had elevated O2 consumption and increased skeletal muscle enzyme activity compared with controls; however, cold tolerance was not enhanced compared with CA mice. The data suggest that intermittent CA effectively reduces hypothermia during cold challenge in aged mice.     

Metabolic heat production and cold tolerance during cold stress of different intensity of adult and aged male C57BL/6J mice

Adult and aged male C57BL/6J mice were subjected to a 3-h cold stress test at either 24°C, 18°C, 12°C, or 6°C. Body mass was measured before the test, and colonic temperature, O₂ consumption, and CO₂ production were measured during the test. The slopes of colonic temperature over time of test and the mean metabolic heat production were calculated for each animal. While adult mice had a relatively small reduction in colonic temperature during the test at all four ambient temperatures, in the aged mice ambient temperatures resulted in steeper reductions of colonic temperature. In adult mice, an increase in metabolic heat production was proportional to ambient cold. The thermogenic response of aged mice at 24°C and at 18°C was similar to adult mice, suggesting that the ability of aged mice to respond to cold by increasing heat production does not diminish with age. However, in aged mice metabolic heat production at 12°C and 6°C was significantly below that of adult mice, which indicated a reduced capacity for thermogenesis.     

Hybrid C57BL/6J x FVB/NJ mice drink more alcohol than do C57BL/6J mice

BACKGROUND: From several recent strain surveys (28 strains: Bachmanov et al., personal communication; 22 strains: Finn et al., unpublished), and from data in >100 other published studies of 24-hr two-bottle ethanol preference, it is known that male C57BL/6 (B6) mice self-administer about 10-14 g/kg/day and that female B6 mice self-administer about 12-18 g/kg/day. No strain has been found to consume more ethanol than B6. In one of our laboratories (Texas), we noted a markedly greater intake of ethanol in an F1 hybrid of B6 and FVB/NJ (FVB) mice. METHODS: To confirm and extend this finding, we repeated the study at another site (Portland) using concentrations up to 30% ethanol and also tested B6xFVB F1 mice in restricted access drinking procedures that produce high levels of alcohol intake. RESULTS: At both sites, we found that B6xFVB F1 mice self-administered high levels of ethanol during two-bottle preference tests (females averaging from 20 to 35 g/kg/day, males 7-25 g/kg/day, depending on concentration). F1 hybrids of both sexes drank significantly more 20% ethanol than both the B6 and FVB strains. Female F1 hybrids also drank more 30% ethanol. In the restricted access tests, ethanol consumption in the F1 hybrids was equivalent to that in B6 mice. CONCLUSIONS: These data show that this new genetic model has some significant advantages when compared to existing inbred strains, and could be used to explore the genetic basis of high ethanol drinking in mice.     

Heat loss during cold exposure in ADULT and AGED C57BL/6J mice

Metabolic heat production (MHP), colonic temperature (Tco), and nonevaporative (dry) heat loss were measured in ADULT and AGED C57BL/6J male mice during cold exposure. Dry heat loss was assessed as a differential temperature (Td) between incoming and outgoing air through the chamber for indirect calorimetry. The average Td during cold exposure normalized to surface area for ADULT mice was significantly higher than that for the AGED animals (0.0618 ± 0.0003°C/cm² and 0.0553 ± 0.0005°C/cm², respectively). Linear regression analysis showed that at the same Tco AGED mice showed lower values of Td normalized to surface area, indicating that at the same body temperature they were losing less heat than ADULT animals. It was concluded that age-related decline in cold tolerance in mice is not due to a lack of ability to reduce heat loss during cold exposure. On the contrary, AGED animals had lower heat loss in comparison with ADULT. We suggest that augmentation of heat conservation mechanisms is an adaptive response to diminishing cold-induced heat production.     

Body temperature of C57BL/6J mice with age

On the basis of a study of rectal temperature in a group of 180, C57BL/6J male mice, ranging in age from 3 months to 30 months, the following conclusions were drawn: 1) The positive correlation between body weight and body temperature typical for rodents was found only for young adults of the C57BL/6J strain; 2) body temperature of male C57BL/6J did not appear to decline until about 23.5 months, after which there was a significant negative correlation (r = -0.53) between age and temperature.     

Nonshivering thermogenesis during acute cold exposure in adult and aged C57BL/6J mice

In C57BL/6J adult and aged mice, housed at room temperature (22.5 ± 1°C), we measured O₂ consumption and CO₂ production and calculated metabolic heat production under conditions of anesthesia and myorelaxation during acute cold stimulation when body temperature was lowered 7.5°C below control level. An independent group of mice was subjected to a three hour partial physical restraint at 6°C and concentration of uncoupling protein (thermogenin) was measured in interscapular brown adipose tissue mitochondria at different times after cold exposure. Heat production under anesthesia and myorelaxation was about 57–66% lower than in nonanesthetized conditions, but increased significantly during cold stimulation in both age groups. Under anesthesia and myorelaxation before and during cold stimulation aged mice produced about 20% more heat than adult mice. Because in these experiments all sources of facultative thermogenesis, except nonshivering, were suppressed by anesthesia and myorelaxation, and because brown adipose tissue is the major source of nonshivering thermoproduction, we concluded that aged mice housed at room temperature have an increased thermogenesis in brown adipose tissue. This conclusion was also supported by the finding that the concentration of uncoupling protein measured in the mitochondria of brown adipose tissue after single cold exposure was significantly higher in aged than in adult mice. Therefore, we propose that the lower, cold-induced, heat production typically observed in nonanesthetized aged mice may reflect reduced thermogenic capacity of skeletal muscles. While aged mice have less brown adipose tissue than adult animals, the remaining brown adipose tissue may compensate by increasing the concentration of uncoupling protein.     

A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice

Aims/hypothesis C57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes.Methods Glucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis.Results C57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close K_ATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver.Conclusions/interpretation The C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.Electronic Supplementary Material Supplementary material is available for this article at .     

Schedule-induced ethanol self-administration in DBA/2J and C57BL/6J mice

BACKGROUND: The purpose of these experiments was to provide an initial investigation into ethanol self-administration elicited in the schedule-induced polydipsia (SIP) paradigm. METHODS: Mature male mice were food deprived to between 80 and 85% of their baseline weight and received 20 daily 1 hr SIP test sessions in which a food pellet (20 mg) was delivered on a fixed-time 60 sec schedule. In different groups, the acquisition of drinking 5% (v/v) ethanol solution (experiment 1) or water (experiment 2) was recorded along with other behaviors that occurred in the test chambers. RESULTS: Results indicated that C57BL/6J mice drank significantly more ethanol than DBA/2J mice and that C57 mice achieved blood alcohol concentrations as high as 300 mg/dl. Blood alcohol concentrations were consistently correlated with g/kg ethanol intake. The groups did not differ in consumption of water. SIP test sessions using higher concentrations of ethanol (10-20% v/v, experiment 1) or sucrose solutions (0.1-2% w/v, experiment 2) then were performed. Group differences in ethanol consumption were maintained at all ethanol concentrations. Although DBAs drank more of a low concentration of sucrose (0.1%), when expressed as g/kg, sucrose intake was equivalent in the two strains at all concentrations. Analysis of the time course of drinking clearly showed that this behavior was adjunctive in nature. CONCLUSION: These results demonstrate the effectiveness of this procedure in inducing ethanol self-administration and its utility for investigating the genetic bases of vulnerability toward excessive ethanol consumption.     

Experimental scoliosis in melatonin-deficient C57BL/6J mice without pinealectomy

The etiology of idiopathic scoliosis is unknown. Scoliosis with many characteristics closely resembling those seen in idiopathic scoliosis has been produced in young chickens and bipedal rats after pinealectomy. In this study, we induced experimental scoliosis in C57BL/6J mice without pinealectomy and melatonin treatment suppressed the development of scoliosis. A total of 100 mice were divided into four groups: 20 quadrupedal mice served as controls; 30 mice underwent resection of two forelegs and tail at 3 wk of age (bipedal mice); the remaining 20 quadrupedal and 30 bipedal mice received intraperitoneal melatonin (8 mg/kg BW) at 19:00 hr daily. Before killing, blood samples were collected in the middle of dark cycle and melatonin levels were measured by radioimmunoassay. Spine X-ray and helical 3D-CT were examined after killing at 5 months of age. The bipedal mice without a tail were able to walk with standing posture, whereas the quadrupedal mice did not walk with standing posture. In C57BL/6J mice, the serum melatonin was reduced to nearly zero; however, the normal level was restored in both bipedal and quadrupedal mice after the injection of melatonin. Scoliosis with rib humps developed in 29 of 30 bipedal and in five quadrupedal mice. None of mice with melatonin treatment developed scoliosis. The results suggest that melatonin deficiency in bipedal mice appears to play crucial role for development of scoliosis. Also the restoration of melatonin levels prevents the development of scoliosis.     

Survival and disease patterns in C57BL/6J mice subjected to undernutrition

This study reports survival and disease patterns in a long-lived mouse strain subjected to undernutrition. Four cohorts were studied, each composed of two or more groups of mice, each normally-fed or restricted either pre- and/or postweaning. Restriction prior to weaning was effected by limiting access to the mother. animals restricted postweaning received a nutritionally complete diet, including a normal complement of vitamins and salts, but were fed only 4 portions/week vs 7 portions/week for those animals normally fed—hence the term under-nutrition to differentiate between this and malnutrition.Comparisons of disease patterns among groups revealed that the incidence of lymphoma, the most prevalent tumor, was uniformly decreased in the groups restricted postweaning, with or without preweaning restriction. In the last cohort, deaths of animals with lymphoma were shifted to later ages in the restricted groups, compared with the normally-fed controls.Whereas the lymphoma pattern was considerably modified by undernutrition, the effect on overall survival did not seem as dramatic. Gompertzian parameters for survival past 120 weeks were not statistically different, although with one exception, maximum survival and one of the Gompertzian parameters was consistently greater in groups restricted postweaning, compared with those restricted preweaning only, or not at all. Maximum survival is a parameter not unduly influenced by environmental factors such as infectious disease; consequently, this represents a meaningful effect of undernutrition.Statistically, more significant differences in tumor patterns than in survival suggests that the former are more sensitive to undernutrition than is the latter—at least in this strain of mouse. Greater lifespan prolongation in the restricted animals may be possible through better “fine tuning” of the diet, including improved portion control, particularly in the early postweaning period, to prevent rapid weight gain, and possibly through changes in dietary composition. Finally, it is suggested that undernutrition may exert its effects through an alteration in gene expression.     

Temporal decrease of body temperature in middle-aged C57BL/6J mice

Colonic temperature of C57BL/6J mice of both sexes between 12 and 19 months was measured at normal ambient temperature in four independent studies. One study was cross-sectional and the others were longitudinal. In all samples, colonic temperature at about 14 months was always lower than temperature at earlier ages. Furthermore, temperature always returned to earlier levels within 2 weeks; however, it remained unstable up to the age of 18 months after which it returned to the pre-14-month level until finally it fell late in life (after 23 months). Additionally, parametric studies showed that there were differences in the reliability of temperature probes made by different manufacturers to measure absolute temperature. These differences, however, did not affect relative differences among temperatures. Further parametric studies showed that colonic temperature in restrained mice rises 1.2 degrees C within the first 5 min after restraint indicating that duration of restraint is an important factor in studies such as these.     

Calcium and calmodulin changes with ageing in C57BL/6J mice

Male C57BL/6J mice ranging in age from 50 to 1186 days were used to measure total calcium and calmodulin concentrations. The increase in calcium between 0 and 1,000 days of age was 260% for kidney, followed by brain (189%), heart (173.5%), lung (106.5%) and liver (78.5%). Calcium in femur declined by 28.2%. The calmodulin content of liver increased with ageing. Both liver and kidney calmodulin concentrations declined early in life followed by ageing-related increases. Brain, lung and heart calmodulin concentrations did not change significantly with ageing. We conclude that changes in calcium homeostasis are not reflected in calmodulin changes. The loss of calcium in bone is consistent with the occurrence of osteoporosis in ageing C57 mice.     

Aging and uterine growth during implantation in C57BL/6J mice

Uterine growth during implantation was compared in C57BL/6J mice aged 3–7 mo vs. 11–12 mo. All mice had given birth to at least one previous litter. Older mice had smaller uteri during early gestation (3 to 10 days post coitum) as measured by DNA content, although wet weight and protein were generally similar in both age groups. However, there were no age effects on uterine growth during implantation, as measured by the increments of DNA, as well as protein and wet weight per implantation site (decidual swelling) on days 6–10. These data are discussed in terms of the major increase in fetal death and resorptions in older mice subsequently observed by day 12–13. Larger age-related impairment of the artificially-induced decidual response is also considered. We conclude that impairments of the artificially induced decidual response do not predict the extent of decidual responses during pregnancy in aging mice.     

Iron accumulation and lipid peroxidation in aging C57BL/6J mice

Total iron concentrations in organs from C57BL/6J male mice increased with age. In animals ranging 45 to 900 days of age liver iron increased by 216%, heart by 66%, kidney by 54% and brain by 27%. Two separate phases of iron accumulation were found in brain, kidney and liver. Between 45-355 days of age brain iron increased by 33% and after 355 days there was no change. For both kidney and liver no change was found until after 355 days of age when liver increased by 140% and kidney by 44%. The kidney and liver results suggest a true aging phenomenon. Liver peroxidation potential as measured by the thiobarbituric acid test tended to increase with age but the differences were not significant. The addition of metal ion chelators greatly reduced lipid peroxide values for all organs but no significant age-related trend was evident. We conclude that the large aging-related increases in tissue iron are not correlated with increased concentrations of lipid peroxides.     

Characterization of T-cell immune responses of Echinococcus multilocularis-infected C57BL/6J mice

Specific and non-specific parasite-induced changes in lymphocyte responses were analysed in C57BL/6J mice after intrahepatic infection with Echinococcus multilocularis. Spleen cells harvested at selected times after infection were in vitro stimulated with mitogens or a crude soluble parasitic extract (EmAg) at an optimized dose. Cell proliferative responses to Con-A were not modified by the infection over the first 22 weeks. In contrast, LPS-induced responses were decreased from the 13th week. A strong CD4 ⁺ proliferative T-cell response to the parasitic extract of infected mouse spleen cells was observed at the early stage of infection. This response then progressively decreased but remained significantly higher than that of control mice until the 19th week of infection. Cytokine production was investigated after in vitro EmAg stimulation of spleen cells. IFN-γ, IL-2. IL-5 were produced within the first weeks after infection whereas the detection of IL-10 was slightly delayed. Thus, the promotion of the disease does not appear associated with the expansion of one rather than another T-cell subset in C57BL/6J mice. A general immunosuppression affecting both mitogenic and parasite-specific T-cell responses was observed at the end of the infection.     

Varying susceptibility to myocardial infarction among C57BL/6 mice of different genetic background

Genetically manipulated mouse lines are invaluable to investigate the effects of a single gene on sensitivity to ischemia. When choosing appropriate controls, we were concerned that intrinsic, strain-independent but colony-dependent differences may influence the susceptibility to ischemia. We, therefore, compared the infarct:risk volume ratio (I:R%) after 30-min global ischemia in Langendorff-perfused hearts from outbred C57BL/6 mice with that in wild-type mice derived from heterozygote x heterozygote crosses of two different in-house C57BL/6 mouse lines with targeted disruption of an MKK3 or MAPKAPK2 allele. Despite similar hemodynamic characteristics, I:R% in outbred C57BL/6 hearts was significantlysmaller (40.8 +/- 2.8%) than in C57BL/6 MAPKAPK2 wild types (65.8 +/- 4.5%, P = 0.0003) and significantly larger than in C57BL/6 MKK3 wild types (23.7 +/- 2.9%, P = 0.002). Therefore, inherent colony substrain-dependent differences appear to influence the susceptibility to infarction in response to global ischemia, underscoring the importance of using colony-matched wild-type controls in murine studies of myocardial ischemia.     

Age-related changes in hypothalamic-pituitary-adrenal axis activity of male C57BL/6J mice

As there is little known about age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis of mice, we determined the daily patterns of corticosterone secretion every 2 h, together with adrenocorticotropic hormone (ACTH) release and central HPA axis markers in the morning and evening of 3-, 9- and 16-month-old male C57BL/6J mice. We observed that: (i) corticosterone secretion showed a distinct age-related circadian pattern. During the light period this was expressed by relative hypercorticism in 9-month-old mice and relative hypocorticism in 16-month-old mice. ACTH was elevated at 16 months of age; (ii) mineralocorticoid (MR) and glucocorticoid receptor (GR) mRNA expression in the hippocampus was significantly decreased in 9-month-old mice, whereas in 16-month-old mice, expression was similar to young animals. Circadian variation was modest in all age groups; (iii) the parvocellular hypothalamic paraventricular nucleus (PVN) expressed very high vasopressin mRNA, which was subject to circadian variation in 3- and 9-month-old mice. Furthermore, significant levels of MR mRNA were expressed in the PVN. In conclusion, basal HPA axis activity and expression of its central regulatory markers are age-dependent in mice. This suggests that the capacity to adjust to environmental demands is either a function of age, or depends on different dynamics of the HPA axis.     

Physiological and behavioral correlates of lifespan in aged C57BL/6J mice

Physiological and behavioral measurements were made in a cohort of 29-month-old male C57BL/6J mice to determine whether any correlated significantly with lifespan. Significant linear relationships with lifespan were found among the physiological measures, including hematocrit and hemoglobin levels and collagen denaturation rate; however, body weight failed to be a significant predictor of survival. Among the behavioral variables studied, significant quadratic relationships with lifespan were found for exploratory activity and passive avoidance learning, while performance on a rotorod and a tightwire showed no significant relationships with lifespan. Through the use of multiple regression techniques, about one-third of the variance in lifespan could be explained by a combination of physiological variables, and about two-fifths could be explained by a combination of behavioral variables.