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.     

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/).     

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.     

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.     

Restraint increases prolactin and REM sleep in C57BL/6J mice but not in BALB/cJ mice

Sleep is generally considered to be a recovery from prior wakefulness. The architecture of sleep not only depends on the duration of wakefulness but also on its quality in terms of specific experiences. In the present experiment, we studied the effects of restraint stress on sleep architecture and sleep electroencephalography (EEG) in different strains of mice (C57BL/6J and BALB/cJ). One objective was to determine if the rapid eye movement (REM) sleep-promoting effects of restraint stress previously reported for rats would also occur in mice. In addition, we examined whether the effects of restraint stress on sleep are different from effects of social defeat stress, which was found to have a non-REM (NREM) sleep-promoting effect. We further measured corticosterone and prolactin levels as possible mediators of restraint stress-induced changes in sleep. Adult male C57BL/6J and BALB/cJ mice were subjected to 1 h of restraint stress in the middle of the light phase. To control for possible effects of sleep loss per se, the animals were also kept awake for 1 h by gentle handling. Restraint stress resulted in a mild increase in NREM sleep compared with baseline, but, overall, this effect was not significantly different from sleep deprivation by gentle handling. In contrast, restraint stress caused a significant increase in REM sleep compared with handling in the C57BL/6J mice but not in BALB/cJ mice. Corticosterone levels were significantly and similarly elevated after restraint in both strains, but prolactin was increased only in the C57BL/6J mice. In conclusion, this study shows that the restraint stress-induced increase in REM sleep in mice is strongly strain dependent. The concomitant increases in prolactin and REM sleep in the C57BL/6J mice, but not in BALB/cJ mice, suggest prolactin may be involved in the mechanism underlying restraint stress-induced REM sleep. Furthermore, this study confirms that different stressors differentially affect NREM and REM sleep. Whereas restraint stress promotes REM sleep in C57BL/6J mice, we previously found that in the same strain, social defeat stress promotes NREM sleep. As such, studying the consequences of specific stressful stimuli may be an important tool to unravel both the mechanism and function of different sleep stages.     

Neospora caninum: high susceptibility to the parasite in C57BL/10ScCr mice

C57BL/10ScCr mice, lack Toll-like receptor 4 and a functional Interleukin-12 receptor. Taking this into account, susceptibility of these mice to Neospora caninum infection was assessed comparatively to that of immunocompetent C57BL/10ScSn mice. C57BL/10ScCr mice inoculated intraperitoneally with 5x10(5)N. caninum tachyzoites showed a high susceptibility to this parasite. All infected C57BL/10ScCr mice were dead by day 8 post-infection whereas all control C57BL/10ScSn mice survived this parasitic challenge. Immunohistochemical analysis of infected C57BL/10ScCr mice showed N. caninum tachyzoites spread in the pancreas, liver, lung, intestine, heart and brain whereas no parasites were detected in similarly infected C57BL/10ScSn controls. The higher susceptibility of C57BL/10ScCr mice to neosporosis correlates with reduced interferon-gamma mRNA expression and increased IL-4 mRNA expression, comparatively to C57BL/10ScSn controls, detected in the spleen after the parasitic challenge. C57BL/10ScCr mice could thus be used as a new experimental model where to study immunobiological mechanisms associated with host susceptibility to neosporosis.     

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.     

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.     

Discordant susceptibility of inbred C57BL/6 versus outbred CD1 mice to experimental fungal sepsis

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response‐related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan‐induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)‐γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN‐γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN‐γ doses (0.2 μg/kg) either alone or in combination with the ß‐glucan‐binding CD5 protein (0.7 mg/kg) leading to improved post zymosan‐induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 10⁴ CFU/gr) were ameliorated by low‐dose IFN‐γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs.     

Macrophage cholesterol efflux to free apoprotein A-I in C3H and C57BL/6 mice

Cholesterol efflux from peritoneal macrophages of mice C57BL/6 susceptible and C3H resistant to atherosclerosis was compared, using apoprotein A-I as acceptor. The elicited macrophages were labeled with 3H-cholesterol and cholesterol enriched by incubation for 24 h with acetylated LDL. After incubation for 6 or 24 h, 3H-cholesterol efflux to free apoA-I (10 microg/ml) was significantly higher with macrophages derived from C3H mice compared to C57BL/6 mice. The cells were also pretreated with 0.3-0.45 mM cyclic AMP, 10 microM 9-cis-retinoic acid or 10 microM 22(R)-hydroxycholesterol, RXR and LXR ligands. Treatment with cyclic AMP, RXR, or LXR ligands, resulted in enhancement of 3H-cholesterol efflux in both strains. Under all conditions, 3H-cholesterol efflux was significantly higher in C3H compared to C57BL/6 macrophages. In conclusion, the higher cholesterol efflux from C3H macrophages could contribute toward the resistance of this strain to diet-induced atherosclerosis despite hypercholesterolemia.     

Streptobacillus moniliformis epizootic in barrier-maintained C57BL/6J mice and susceptibility to infection of different strains of mice

We report a Streptobacillus moniliformis epizootic in barrier-maintained SPF mice. Although various inbred and F1 hybrid strains of mice have been kept in this animal facility, only C57BL/6J Han [corrected] mice showed clinical signs of disease. During the course of the epizootic, 825 breeding animals (approximately 36% of the breeders) died or had to be killed because of severe clinical signs. Although sequential treatment with ampicillin and chlortetracycline gave good therapeutic results, the animal facility was vacated in order to exclude any risk of cross-contamination of the other rodent colonies in our institute. The source and route of transmission of S. moniliformis could not be elucidated. To investigate strain dependent differences experimental infection of different strains of mice with our S. moniliformis isolate was performed. After oral infection only C57BL/6J showed the typical signs of a cervical lymphadenitis and gave an immunological response. BALB/cJ, C3H/He, DBA/2J, CB6F1 and B6D2F1 mice were not affected except in two cases of DBA/2J and B6D2F1 mice where seroconversion was observed. After intravenous infection of C57BL/6J, DBA/2J [corrected] and BALB/cJ all animals showed positive titers in the indirect immunofluorescence test (IIF). One hundred percent of the C57BL/6J, forty percent of the DBA/2J, and none of the BALB/cJ mice developed severe symptoms. The results demonstrate that the susceptibility to streptobacillosis is predominantly influenced by genetic factors.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.     

Age-related fibrillar deposits in brains of C57BL/6 mice

The present article reviews findings regarding the age-related occurrence of clusters of unusual granules in the brains of C57BL/6 (B6) mice and discusses the potential relevance of this phenomenon as a model of specific aspects of brain aging in humans. The granules occur predominatly in the hippocampus of B6 mice and represent aggregations of fibrillar material that are mostly associated with astrocytes. The deposits become evident at about 4 to 6 mo of age, and increase markedly in both number and size thereafter. Similar structures have been observed in adult senescence accelerated mice (SAM) and have been noted, although very rarely, in older mice from other strains. The deposits appear to manifest dominant genetic heritability. Heparan sulfate proteoglycan and laminin or related molecules have been identified as components of the granular material. Although the deposits do not represent senile plaques with β-amyloid deposition, they might mimic the deposition of extracellular matrix molecules that is thought to be an early event in amyloidogenesis in the aged brain and in Alzheimer's disease.     

Berberine attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice

Background

Berberine, an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-inflammatory and neuroprotective effects. However, there are no reports about the effects and mechanisms of berberine in experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (MS).

Methodology/Principal Findings

Female C57 BL/6 mice immunized with myelin oligodendrocyte glycoprotein 35–55 amino acid peptide were treated with berberine at the day of disease onset and medication was administered daily until mice were sacrificed. Blood–brain barrier (BBB) permeability and the alteration of matrix metalloproteinase-2 (MMP-2, 72 kDa) and matrix metalloproteinase-9 (MMP-9, 92 kDa) in the brain and cerebrospinal fluid (CSF) of EAE mice were detected by quantitative measurement for Evan''s blue (EB) content, Western blot and gelatin zymography respectively. The results showed that berberine attenuated clinical and pathological parameters of EAE, reduced the permeability of BBB, inhibited the activity and expression of MMP-9 but not MMP-2 in the CSF and brain of EAE mice.

Conclusions/Significance

These findings suggest that berberine is effective to attenuate the clinical severity of EAE in C57 BL/6 mice by reducing the permeability of BBB, decreasing the expression and activity of MMP-9, and decreasing the inflammatory infiltration. We think that berberine might be a potential therapeutic agent for MS.     

The superior olivary complex in C57BL/6 mice.

The cellular and cytoarchitectural features of the lateral superior olive, the medial superior olive, the superior paraolivary nucleus and the medial, lateral and ventral nuclei of the trapezoid body are described in C57BL/6 mice using Nissl, Bodian and Golgi techniques. Principal, spindle and marginal cells are present in a well-defined lateral superior olive. The dendrites of these cells run primarily within rostrocaudal sheets as in the cat. The principal cells of the medial nucleus of the trapezoid body are similar to the principal cells in the cat. Large multipolar cells characterize the lateral nucleus of the trapezoid body and bipolar cells with a medial-lateral orientation are found in the medial superior olive. The largest neurons are found in the superior paraolivary nucleus and the lateral superior olive, and the medial and ventral nuclei of the trapezoid body. While brain weight and neuronal packing density change with development, the characteristic location of cell groups and the shape and Nissl-staining pattern of neurons in the youngest brains examined were essentially unchanged in the adult mice, although dendritic maturation had occurred. The homologies of the C57BL/6 superior olivary complex nuclei with the same areas described in other mouse strains, rat and cat are discussed. This study expands our understanding of the organization of the superior olivary complex in an inbred strain of Mus musculus and relates it to other species. The data about changes occurring during postnatal maturation may aid in the interpretation of behavioral and physiological studies of neonatal plasticity of the auditory system.     

Self-mutilation of the penis in C57BL/6N mice

A major cause of male reproductive failure in a C57BL/6N mouse production colony is self-inflicted mutilation of the penis. The extent of the damage ranged from loss of the distal end to loss of the entire penis. From January 1974 to August 1976, 645 adult male mice with mutilated penis were removed from this colony--where the monthly census was 9500 mice, mated 1 male to 4 females using a continuous mating system. On necropsy, it was observed that the substance blocking the urethra in the penile stump resulted in urine retention, grossly distending the urinary bladder. Proteus mirabilis and other bacteria isolated from the urethral plugs were considered secondary invaders.     

Plasma and pituitary concentrations of LH, FSH and prolactin in aged female C57BL/6 mice

Plasma and pituitary concentrations of LH, FSH and prolactin were determined by radioimmunoassay in 2-month-old (young) and 16-20-month-old (old) C56BL/6 mice. There were no statistical differences in hormonal levels between aged females in oestrus (those exhibiting a copulatory plug) and those in constant dioestrus. In the old females plasma levels of LH (P < 0.002) and FSH (P < 0.001) were significantly elevated, while levels of prolactin (P < 0.001) were significantly depressed when compared with those from young animals. Pituitary homogenates from old females also contained more gonadotrophins (P < 0.001) and less prolactin (P < 0.001) than those of the young females. A radioreceptor assay utilizing a plasma membrane of luteinized rat or mouse ovaries indicated that LH from 2-month-old animals bound better to ovarian receptors (P < 0.05) than did LH from old mice, although radioimmunoassay of the same samples gave higher (P < 0.01) plasma LH levels for the old mice. Since the radioreceptor assay is considered to be a more sensitive test for biologically active LH, the results from these two types of assays suggest that there may be an alteration in the mouse LH molecule with age.