Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice

We utilized variations in caloric availability and ambient temperature (T(a)) to examine interrelationships between energy expenditure and cardiovascular function in mice. Male C57BL/6J mice (n = 6) were implanted with telemetry devices and housed in metabolic chambers for measurement of mean arterial pressure (MAP), heart rate (HR), O(2) consumption (VO(2)), and locomotor activity. Fasting (T(a) = 23 degrees C), initiated at the onset of the dark phase, resulted in large and transient depressions in MAP, HR, VO(2), and locomotor activity that occurred during hours 6-17, which suggests torporlike episodes. Food restriction (14 days, 60% of baseline intake) at T(a) = 23 degrees C resulted in progressive reductions in MAP and HR across days that were coupled with an increasing occurrence of episodic torporlike reductions in HR (<300 beats/min) and VO(2) (<1.0 ml/min). Exposure to thermoneutrality (T(a) = 30 degrees C, n = 6) reduced baseline light-period MAP (-14 +/- 2 mmHg) and HR (-184 +/- 12 beats/min). Caloric restriction at thermoneutrality produced further reductions in MAP and HR, but indications of torporlike episodes were absent. The results reveal that mice exhibit robust cardiovascular responses to both acute and chronic negative energy balance. Furthermore, we conclude that T(a) is a very important consideration when assessing cardiovascular function in mice.     

Glutathione deficient C57BL/6J mice are not sensitized to ozone-induced lung injury

In this study we examined the role of the antioxidant glutathione (GSH) in pulmonary susceptibility to ozone toxicity, utilizing GSH deficient C57BL/6J mice that lack the expression of glutamate-cysteine ligase modifier subunit (GCLM). Gclm(−/−) knockout mice had 70% GSH depletion in the lung. Gclm(+/+) wild-type and Gclm(−/−) mice were exposed to either 0.3 ppm ozone or filtered air for 48 h. Ozone-induced lung hyperpermeability, as measured by total protein concentration in bronchoalveolar lavage fluid, was surprisingly lower in Gclm(−/−) mice than in wild-type mice. Lung hyperpermeability did not correlate with the degree of neutrophilia or with inflammatory gene expression. Pulmonary antioxidant response to ozone, assessed by increased mRNA levels of metallothionein 1 and 2, α-tocopherol transporter protein, and solute carrier family 23 member 2 (sodium-dependent vitamin C transporter) was greater in Gclm(−/−) mice than in Gclm(+/+) mice. These results suggest that compensatory augmentation of antioxidant defenses in Gclm(−/−) mice may confer increased resistance to ozone-induced lung injury.     

Restoration of gallstone susceptibility by leptin in C57BL/6J ob/ob mice

The absence of leptin due to the ob mutation leads to obesity and confers resistance to diet-induced cholesterol gallstone formation in otherwise susceptible C57BL/6J mice. To investigate contributions of obesity and leptin to gallstone susceptibility, C57BL/6J ob/ob mice were treated daily with i.p. saline or recombinant murine leptin at low (1 microgram/g bw) or high (10 microgram/g bw) doses and were pair-fed a lithogenic diet (15% dairy fat, 1.25% cholesterol, 0.5% cholic acid). Weight loss in ob/ob mice increased in proportion to leptin dose, indicating that the lithogenic diet did not impair leptin sensitivity. In a dose-dependent manner, leptin promoted cholesterol crystallization and gallstone formation, which did not occur in saline-treated mice. Notwithstanding, leptin decreased biliary lipid secretion rates without enriching cholesterol in bile. Leptin did not affect bile salt hydrophobicity, but did increase the biliary content of the most abundant molecular species of phosphatidylcholine, 16:0-18:2. Treatment with leptin down-regulated 3-hydroxy-3-methylglutaryl CoA reductase and prevented cholesterol from accumulating in liver. Consistent with increased hepatic clearance, leptin decreased plasma HDL cholesterol concentrations. This was accommodated in liver without up-regulation of cholesterol 7alpha-hydroxylase or Acat. These data suggest that despite the lithogenic diet, endogenous sources constitute a significant proportion of biliary cholesterol during leptin-induced weight loss. Kinetic factors related to cholesterol nucleation, gallbladder contractility, or mucin secretion may have accounted for leptin-induced gallstone formation.     

Interferon-gamma deficiency reveals that 129Sv mice are inherently more susceptible to Anaplasma phagocytophilum than C57BL/6 mice

Immunocompetent mice 129Sv (129) and C57BL/6 (B6) mice are similarly susceptible to Anaplasma phagocytophilum. We now show that 129 mice lacking interferon-gamma (IFN-gamma) develop more severe infection with A. phagocytophilum than IFN-gamma deficient B6 mice. These data demonstrate that there is an inherent increased susceptibility of 129 mice, compared with B6 mice, to A. phagocytophilum that can only be discerned in the absence of IFN-gamma.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Estrogen and progesterone affect responses to malaria infection in female C57BL/6 mice

Background: Previous data from our laboratory suggest that gonadally intact C57BL/6 male mice are more likely than their female counterparts to die from Plasmodium chabaudi infection, to recover more slowly from weight loss and hematocrit loss, and to have reduced interferon-γ (IFN-γ) and interleukin-10 (IL-10) responses. Removal of the ovaries, and hence, the primary production of sex steroids in females, reverses these differences.Objective: We hypothesized that sex differences in response to P chabaudi may be mediated by differential synthesis of IFN-γ and IL-10 that is influenced by estrogen, progesterone, or both.Methods: C57BL/6 female mice (n = 200; n = 10/time point/treatment/experiment) were ovariectomized and implanted with a 21-day controlled-release pellet containing either 0.1 mg of 17β-estradiol (E₂), 10 mg of progesterone (P₄), 0.1 mg of E₂ plus 10 mg of P₄, or cholesterol (placebo). Females were inoculated with 10⁶P chabaudi-infected erythrocytes. Body mass, body temperature, hematocrit, parasitemia, cytokine production, and antibody responses were monitored 0, 3, 5, 7, 10, 14, and 21 days postinoculation.Results: Administration of E₂, either alone or in combination with P₄, mitigated infection-induced weight loss, hematocrit loss, and hypothermia, compared with females receiving placebo pellets (P < 0.05 in each case). Hormone treatment did not affect levels of parasitemia. Females administered E₂ alone or in combination with P₄ produced 4 to 7 times higher IFN-γ and IL-10 during peak parasitemia than did females implanted with pellets containing either P₄ alone or placebo (P < 0.05 in each case). Exposure to E₂, either alone or in combination with P₄, increased anti-P chabaudi immunoglobulin G (IgG1) responses and the ratio of IgG1 to IgG2c (P < 0.05 in each case).Conclusion: This animal study suggests that physiological levels of estrogen, rather than progesterone, enhance immunity and, possibly, protect females from disease symptoms during malaria infection.     

Lifelong calorie restriction affects indicators of colonic health in aging C57Bl/6J mice

Diminished colonic health is associated with various age-related pathologies. Calorie restriction (CR) is an effective strategy to increase healthy lifespan, although underlying mechanisms are not fully elucidated. Here, we report the effects of lifelong CR on indicators of colonic health in aging C57Bl/6J mice. Compared to an ad libitum control and moderate-fat diet, 30% energy reduction was associated with attenuated immune- and inflammation-related gene expression in the colon. Furthermore, expression of genes involved in lipid metabolism was higher upon CR, which may point towards efficient regulation of energy metabolism. The relative abundance of bacteria considered beneficial to colonic health, such as Bifidobacterium and Lactobacillus, increased in the mice exposed to CR for 28 months as compared to the other diet groups. We found lower plasma levels of interleukin-6 and lower levels of various metabolites, among which are bile acids, in the colonic luminal content of CR-exposed mice as compared to the other diet groups. Switching from CR to an ad libitum moderate-fat diet at old age (24 months) revealed remarkable phenotypic plasticity in terms of gene expression, microbiota composition and metabolite levels, although expression of a subset of genes remained CR-associated. This study demonstrated in a comprehensive way that CR affects indicators of colonic health in aging mice. Our findings provide unique leads for further studies that need to address optimal and feasible strategies for prolonged energy deprivation, which may contribute to healthy aging.     

Substrain specific behavioral responses in male C57BL/6N and C57BL/6J mice to a shortened 21-hour day and high-fat diet

ABSTRACT

Altered circadian rhythms have negative consequences on health and behavior. Emerging evidence suggests genetics influences the physiological and behavioral responses to circadian disruption. We investigated the effects of a 21 h day (T = 21 cycle), with high-fat diet consumption, on locomotor activity, explorative behaviors, and health in male C57BL/6J and C57BL/6N mice. Mice were exposed to either a T = 24 or T = 21 cycle and given standard rodent chow (RC) or a 60% high-fat diet (HFD) followed by behavioral assays and physiological measures. We uncovered numerous strain differences within the behavioral and physiological assays, mainly that C57BL/6J mice exhibit reduced susceptibility to the obesogenic effects of (HFD) and anxiety-like behavior as well as increased circadian and novelty-induced locomotor activity compared to C57BL/6N mice. There were also substrain-specific differences in behavioral responses to the T = 21 cycle, including exploratory behaviors and circadian locomotor activity. Under the 21-h day, mice consuming RC displayed entrainment, while mice exposed to HFD exhibited a lengthening of activity rhythms. In the open-field and light-dark box, mice exposed to the T = 21 cycle had increased novelty-induced locomotor activity with no further effects of diet, suggesting daylength may affect mood-related behaviors. These results indicate that different circadian cycles impact metabolic and behavioral responses depending on genetic background, and despite circadian entrainment.     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.     

Nanoemulsified green tea extract shows improved hypocholesterolemic effects in C57BL/6 mice

Nanoemulsification of nutrients could improve bioavailability by enhancing intestinal uptake. We investigated the antioxidant and hypolipidemic effects of nanoemulsified green tea extract (NGTE). Antioxidant effect was measured by 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging assay and dichlorofluorescein diacetate (DCFH-DA) assay. C57BL/6 mice were fed a control high-fat diet, green tea extract (GTE), or NGTE diet for 4 weeks. In composition analysis, GTE and NGTE contained similar total catechin concentrations. The antioxidative effect of GTE was comparable with that of NGTE. In the ABTS assay, GTE had a marked effect, although NGTE was more effective than GTE in the DCFH-DA assay. In the mouse feeding experiment, total and low-density lipoprotein (LDL) cholesterol concentrations were significantly reduced after NGTE treatment in comparison with GTE treatment in high-fat-fed C57BL/6J mice over the course of 4 weeks. The hypocholesterolemic effects were greater in the NGTE group compared with the GTE group (24% vs. 15.4% LDL cholesterol reduction compared with the control). Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase was significantly down-regulated. Protein expression of LDL receptor was significantly increased in the livers of both the GTE- and NGTE-treated groups (+234.1%, P<.01 and +274.7%, P<.001), with a greater effect in the NGTE than in the GTE group. Cholesterol 7α-hydroxylase gene expression was similarly increased in both the GTE and NGTE groups. These results suggest that nanoemulsification significantly increased hypocholesterolemic effects of GTE in vivo due to increased bioavailability.     

Immune responses against allogeneic and syngeneic tumors in aged C57BL/6 mice

Aged C57BL/6 (B6) mice could reject allogeneic BALB/c RL male 1 tumor as efficiently as young B6 mice. However, in vitro analysis showed impaired generation of cytotoxic T cell response in aged B6 mice against allogeneic tumor. The reaction could be augmented by the addition of recombinant interleukin-2 (rIL-2). Enzyme-linked immunospots (ELISPOT) produced by CD8+ T cells purified from spleen cells showed no reduction in aged mice. The findings suggested that the number of CD8+ T cells capable of reacting against allogeneic H-2 antigens was similar in young and aged B6 mice. Low cytotoxic T lymphocyte (CTL) responsiveness in aged B6 mice appeared to have resulted from low responsiveness of CD4+ T cells producing IL-2. Although CTL generation was apparently impaired, strong multiple antigenicity of allogeneic tumor evoked a rejection response in aged B6 mice. On the other hand, no rejection response was observed against syngeneic EL4 tumor in aged B6 mice even after depletion of CD4+ CD25+ immunoregulatory cells. Depletion of CD4+ CD25+ cells caused rejection of EL4 tumor in young B6 mice. The findings suggested that aged B6 mice were incapable of inducing effector cells against weak tumor antigens. Only marginal CTL response and small number of ELISPOTs were generated in young but not aged B6 mice against EL4. Addition of rIL-2 to the culture augmented EL4 killing and ELISPOTs in spleen cells from young and aged B6 mice.     

Enhanced susceptibility to lupus contributed from the nonautoimmune C57BL/10, but not C57BL/6, genome

Genes from New Zealand Black and New Zealand White mice have been implicated in the development of a disease similar to human systemic lupus erythematosus. In an attempt to define the MHC class II genes involved in disease, we previously studied similarly designed backcrosses of New Zealand Black mice with C57BL/6 (B6) mice transgenic for Ez genes or with C57BL/10 (B10) mice transgenic for Az genes. Although the transgenes showed no effect on the development of autoantibody production or lupus nephritis in either backcross, surprisingly, there was greatly increased expression of these disease traits in the backcrosses involving B10 compared with B6 mice. These studies therefore implicated genetic contributions in B10 vs B6 backgrounds, despite their 98% identity. A genome-wide linkage analysis uncovered a B10 locus on mid-chromosome 13, which enhanced nephritis and was strongly linked with the production of pathogenic retroviral gp70-anti-gp70 immune complexes when contributed by B10, but not B6, mice. The subsequent identification of a single marker polymorphic between B10 and B6, along with the extreme genetic similarity between the two strains in this region, is likely to permit expedited identification of the lupus-susceptibility gene from this nonautoimmune strain.     

Autoimmune hair loss induced by alloantigen in C57BL/6 mice

Exponentially growing Meth-A cells expressing H-2K(d).D (d) antigen were found to induce alopecia when injected intraperitoneally into normal C57BL/6 mice, which express the H-2K(b).D (b) antigen. However, the capacity to induce alopecia disappeared when Meth-A cells were treated with K252a, which inhibits protein kinases. Histologically, skin in affected areas showed dense mononuclear cell infiltration and a focal foreign-body giant-cell reaction in hair follicles. The subtyping of lymphocytes in peripheral blood demonstrated a significant difference between normal mice and Meth-A cell-injected mice. To further examine the mechanism by which the alloantigen induces alopecia, lymphocytes isolated from the peripheral blood of normal C57BL/6 mice were cultured in medium containing Meth-A cell homogenate, phytohemagglutinin (PHA) and recombinant mouse interleukin-2 (rm IL-2), and intravenously injected into normal C57BL/6 mice. The adoptive transfer of the lymphocytes induced alopecia in a similar way. These findings suggest that the protein kinase-modulated alloantigen induces alopecia by disturbing the immunological homeostasis, and that lymphokine-activated killer cells play an important role in induction of alopecia by cross-reacting with hair follicles.     

NPY ablation in C57BL/6 mice leads to mild obesity and to an impaired refeeding response to fasting

Neuropeptide Y (NPY) is an orexigenic (appetite-stimulating) peptide that plays an important role in regulating energy balance. When administered directly into the central nervous system, animals exhibit an immediate increase in feeding behavior, and repetitive injections or chronic infusions lead to obesity. Surprisingly, initial studies of Npy(-/-) mice on a mixed genetic background did not reveal deficits in energy balance, with the exception of an attenuation in obesity seen in ob/ob mice in which the NPY gene was also deleted. Here, we show that, on a C57BL/6 background, NPY ablation is associated with an increase in body weight and adiposity and a significant defect in refeeding after a fast. This impaired refeeding response in Npy(-/-) mice resulted in a deficit in weight gain in these animals after 24 h of refeeding. These data indicate that genetic background must be taken into account when the biological role of NPY is evaluated. When examined on a C57BL/6 background, NPY is important for the normal refeeding response after starvation, and its absence promotes mild obesity.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

C57BL/6JRj mice are protected against diet induced obesity (DIO)

The C57BL/6 (B6) is one of the oldest inbred mouse strains. It has been widely used as control strain in metabolism research for many decades. Preliminary data from our lab indicated that C57BL/6JRj mice are not responding to diet induced obesity. Therefore, the aim of this study was to compare the two different B6 substrains, C57BL/6NTac and C57BL/6JRj, in regard to their response to diet induced obesity (DIO) and to investigate genetic differences which may explain such phenotypic differences. Sixteen male mice of C57BL/6NTac and C57BL/6JRj were fed a high fat diet (HFD) or standard chow diet (SD) for 10 weeks. Phenotypic characterization included measurements of bodyweight, physical activity, food intake and relative epigonadal fat mass. Genetic differences between both substrains were analyzed using a panel of 1449 single nucleotide polymorphism (SNP) markers. Our study revealed that C57BL/6JRj mice are protected against DIO independently from food intake and physical activity. Genetic SNP analysis among C57BL/6 mice identified genetic differences in at least 11 SNPs. Our data strongly support the importance of attention on the genetic background in obesity research.