Protocol for the induction of arthritis in C57BL/6 mice

Collagen-induced arthritis is a well-validated, but strain-dependent mouse model of rheumatoid arthritis, with H-2(q) and H-2(r) strains showing the greatest degree of susceptibility. This protocol describes the induction of arthritis in the C57BL/6 strain (H-2(b)), which forms the genetic background of the majority of genetically modified strains. This protocol involves purification of type II collagen from chicken sternums, immunization of mice, clinical assessment of arthritis and analysis of T- and B-cell responses to type II collagen. Key aspects of the protocol are the need to use chicken collagen for immunization and the importance of avoiding aggressive behavior in males. The incidence of arthritis varies from 50 to 80% and is milder than the classical collagen-induced arthritis model. This procedure takes approximately 3 months to complete.     

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.     

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.     

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.     

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.     

Abnormal head posture associated with induction of cleft palate by methylmercury in C57BL/6J mice

Maternal treatment with methylmercury (MeHg) has been shown to induce a high frequency of cleft palate and produce growth retardation in rat and mouse fetuses, but the relation between these effects is unknown. The objective of this study was to determine if mandibular growth retardation was a factor that contributed to induction of cleft palate in C57BL/6J mice. Two doses of MeHg (10 mg/kg maternal body weight) were given subcutaneously on days 10 and 11 of gestation, and the fetuses were morphometrically studied on days 14, 15, and 18. Full clefts of the secondary palate were present in approximately half of the treated day 15 and 18 fetuses; therefore, the cleft palate (CP) and noncleft palate (NCP) groups were analyzed separately to facilitate identification of morphologic changes associated with the clefting. The results showed that, compared with controls, the day 14 MeHg-treated fetuses had significantly smaller placental weights, but only half of the fetuses had delayed palatal shelf elevation, reduced body weight, and delayed morphological development. However on day 15, the CP and the NCP groups had similar reductions in body weight and placental weight. A striking downward and forward positioning of the head was present in the MeHg-treated fetuses with the CP group more severely affected than the NCP group. Significant differences between the three groups (control, NCP, and CP) were present with mean head-to-body angles of 67 degrees, 60 degrees and 51 degrees, respectively. The absence of normal head lifting resulted in a relative mandibular retrognathia that when combined with a decrease in mandibular length produced alterations in spatial relations that were most severe in the CP fetuses. The results suggest that after exposure to MeHg, palatal closure is affected by altered tongue posture associated with the abnormal head positioning and shortening of the mandible that develop following placental and embryonic growth retardation.     

Epstein-Barr virus nuclear antigen 1 does not cause lymphoma in C57BL/6J mice

To test whether transgenic Epstein-Barr virus nuclear antigen 1 (EBNA1) expression in C57BL/6 mouse lymphocytes causes lymphoma, EBNA1 expressed in three FVB lineages at two or three times the level of latent infection was crossed up to six successive times into C57BL/6J mice. After five or six crosses, 14/36, (38%) EBNA1 transgenic mice, 11/31 (36%) littermate EBNA1-negative controls, and 9/25 (36%) inbred C57BL/6J mice housed in the same facility had lymphoma. These data indicate that EBNA1 does not significantly increase lymphoma prevalence in C57BL/6J mice.     

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.     

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.     

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.     

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.     

Fractionated, low-dose-rate ionizing radiation exposure and chronic ulcerative dermatitis in normal and Trp53 heterozygous C57BL/6 mice

The influence of low-dose-rate chronic radiation exposure and adaptive responses on non-cancer diseases is largely unknown. We examined the effect of low-dose/low-dose-rate fractionated or single exposures on spontaneous chronic ulcerative dermatitis in Trp53 normal or heterozygous female C57BL/6 mice. From 6 weeks of age, mice were exposed 5 days/week to single daily doses (0.33 mGy, 0.7 mGy/h) totaling 48, 97 or 146 mGy over 30, 60 or 90 weeks, and other Trp53+/- mice were exposed to a single dose of 10 mGy (0.5 mGy/min) at 20 weeks of age. The 90-week exposure produced an adaptive response, decreasing both disease frequency and severity in Trp53+/+ mice and extending the life span of older animals euthanized due to severe disease. The 30- or 60-week exposures had no significant protective or detrimental effect. In contrast, the chronic, fractionated exposure for 30 or 60 weeks significantly increased the frequency and severity of the disease in older Trp53+/- mice, significantly decreasing the life span of the animals required to be euthanized for disease. Similarly, the single 10-mGy exposure also increased disease frequency in older animals. However, the chronic, fractionated exposure for 90 weeks prevented these detrimental effects, with disease frequency and severity not different from unexposed controls. We conclude that very low-dose fractionated exposures can induce a protective adaptive response in both Trp53 normal and heterozygous mice, but that a lower threshold level of exposure, similar in both cases, must first be passed. In mice with reduced Trp53 functionality, doses below the threshold can produce detrimental effects.     

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.     

Inhibition of premature death by isothiocyanates through immune restoration in LP-BM5 leukemia retrovirus-infected C57BL/6 mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.