Female CBA Mice Research Articles

1,2-Dimethylhydrazine carcinogenesis in cBA male mice prenatally treated with diethylstilbestrol

CBA female mice, at the 17th day of pregnancy, received single intraperitoneal injection of diethylstilbestrol (DES) at the dose of 1 mg/kg body weight (b. w.). Their descentands at the age of 2 months started receiving subcutaneous injections of 1,2-dimethylhydrazine (DMH) at the dose level of 8 mg(base)/kg b. w., total 15 weekly injections. Prenatal DES treatment of male mice considerably accelerated the development and increased the incidence of DMH-induced androgen-dependent renal adenomas and, notably, renal capsule angiosarcomas (RCA). In males receiving prenatal DES (without subsequent DMH-treatment), there was a statistically significant increase of renal adenomas and 9% of RCA were observed which are exceedinly rare in untreated mice. The enhancement of androgen-dependent tumourigenesis may be considered as an indication, even though indirect, of the hyperandrogenization produced in male mice by the prenatal DES treatment.

Incidence and spectrum of spontaneous neoplasms in male and female CBA/J mice

Although CBA mice are occasionally used in biomedical research, little is known about their life-data and diseases ("background pathology"). Therefore, it was the aim of this study to determine the life expectancy, spectrum and incidence of spontaneous neoplasms of the inbred CBA/J mouse strain. A total of 631 untreated mice (293 females; 338 males) were kept from birth to death under standard laboratory conditions. A complete histological examination was performed on all organs/tissues. In female CBA mice, the average lifespan was 94 weeks, while the mean age in males was 85 weeks. Neoplastic lesions were observed in 70% (238/338) of the males and 51% (150/293) of the female CBA/J mice. Tumours of the liver, lung and haematopoietic tissue were common in males, while tumours of the lung, ovary and haematopoietic tissue were the most frequent neoplasms in female CBA/J mice.

Hematopoietic reconstitution of syngeneic mice with a peripheral blood-derived, monoclonal CD34-, Sca-1+, Thy-1(low), c-kit+ stem cell line.

Previous work had revealed that a CD34- fibroblast-like cell is the earliest hematopoietic progenitor population. This cell type is able to differentiate into hematopoietic progeny of all lineages and circulates in the peripheral blood, from where it can be isolated by IL-6-mediated plastic adherence. We isolated peripheral blood-derived mononuclear cells (MNC) from male CBA mice and established in vitro a fibroblast-like, adherent growing cell layer. Cells were immortalized by SV-40 transfection for cellular cloning. Monoclonal fibroblast-like cell clones were established, and the surface expression of early stem cell markers was determined by flow cytometry. Clones were CD34-, Sca-1+, Thy-1(low), and c-kit+. Lethally irradiated female CBA mice were successfully transplanted with a fibroblast-like cell clone, R-M26/2-1. After syngeneic transplantation, peripheral blood counts were back to normal in transplanted mice on days 15-20, and fluorescence in situ hybridization (FISH) revealed the sole presence of male hematopoietic cells in the BM of female recipients at weeks 7, 9, 11, and 16 after transplantation. Immunohistochemistry for the expression of CD34, Sca-1, Thy-1, and c-kit showed the presence of the phenotype of the transplanted stem cell clone along the bone spicules in the marrow cavity, giving rise to HPC of all lineages. In summary, we have shown that a CD34-, Sca-1+, Thy-1(low), and c-kit+ fibroblast-like cell is consistent with the phenotype of the earliest hematopoietic and repopulating stem cell and can be isolated from peripheral blood cells.

Estrogenic effect of DDT in CBA female mice

Dichlorodiphenyltrichloroethane (DDT) administered to CBA female mice provoked a significant uterine weight increase and pseudoestrus comparable to that produced by diethylstilbestrol (DES) used in a positive control.

1,2-dimethylhydrazine carcinogenesis in C3HA and CBA female mice prenatally treated with diethylstilbestrol

C3HA and CBA female mice received a single intraperitoneal (i.p.) injection of 0.1 or 0.3 mg/kg body weight (b.w.) of diethylstilbestrol (DES) at day 17 of pregnancy. The descendants, starting from the age of 2-3 months, were receiving weekly subcutaneous (s.c.) injections of 1,2-dimethylhydrazine (DMH) (8 mg/kg b.w.), total 20 injections. The survival of C3HA mice treated with DMH together with prenatal DES was considerably better than in mice treated with DMH alone, this being due to the strong inhibiting effect of DES on the induction by DMH of the hemorrhagic ovarian lesions (78.4% in DMH alone vs. 53.3 and 43.7% in groups with DES plus DMH), which frequently were the cause of animal death. Prenatal DES also inhibited the induction by DMH of clitoral gland tumors: 51.4% in the group with DMH alone vs. 26.6 and 28.1% in two groups of DES plus DMH, respectively. DES treatment, at the above doses, did not influence significantly the DMH carcinogenesis in CBA mice. Prenatal DES given to CBA mice at the dose of 1 mg/kg b.w. significantly increased the incidence of DMH-induced uterine sarcomas (42.8% vs. 73.3% in the group with DMH alone and the group receiving DMH together with prenatal DES, respectively) and accelerated their growth. The effects of prenatal DES on DMH-induced carcinogenesis correlated with the degree of hyperestrogenization produced in both strains of mice by DES.

1,2‐dimethylhydrazine carcinogenesis in C3HA and CBA female mice prenatally treated with diethylstilbestrol

C3HA and CBA female mice received a single intraperitoneal (i.p.) Injection of 0.1 or 0.3 mg/kg body weight (b.w.) Of diethylstilbestrol (DES) at day 17 of pregnancy. The descendants, starting from the age of 2–3 months, were receiving weekly subcutaneous (s.c.) Injections of 1,2-dimethylhydrazine (DMH) (8 mg/kg b.w.), total 20 injections. The survival of C3HA mice treated with DMH together with prenatal DES was considerably better than in mice treated with DMH alone, this being due to the strong inhibiting effect of DES on the induction by DMH of the hemorrhagic ovarian lesions (78.4% in DMH alone vs. 53.3 and 43.7% in groups with DES plus DMH), which frequently were the cause of animal death. Prenatal DES also inhibited the induction by DMH of clitoral gland tumors: 51.4% in the group with DMH alone vs. 26.6 and 28.1% in two groups of DES plus DMH, respectively. DES treatment, at the above doses, did not influence significantly the DMH carcinogenesis in CBA mice. Prenatal DES given to CBA mice at the dose of 1 mg/kg b.w. significantly increased the incidence of DMH-induced uterine sarcomas (42.8% vs. 73.3% in the group with DMH alone and the group receiving DMH together with prenatal DES, respectively) and accelerated their growth. The effects of prenatal DES on DMH-induced carcinogenesis correlated with the degree of hyperestrogenization produced in both strains of mice by DES. Teratogenesis Carcinog. Mutagen. 17:19–28, 1997. © 1997 Wiley-Liss, Inc.

The autoimmune response induced by immunising female mice with recombinant human thyrotropin receptor varies with the genetic background

In a previous study, we have described the induction of thyroid blocking (TBAB) and thyrotropin binding inhibiting antibodies accompanied by thyroiditis in female BALBc mice (H2d) immunised with the extra-cellular domain (ECD) of the human thyrotropin receptor (TSHR) expressed as a maltose binding protein (MBP) fusion. In the present study we have investigated the response induced in mice of varying MHC haplotype. Two groups of female NOD (H2g), CBA (H2k) and C57 (H2b) mice were immunised intra-peritoneally with MBP-ECD or MBP on days 0 (100 μg), 15, 30 and 43 (50 μg). Blood samples from individual mice were obtained on days 0, 22, 36 and 50 and assessed for thyroid binding inhibiting immunoglobulins (TBII), thyroid stimulating (TSAB) and TBAB. On day 50 the treated mice and five age/sex matched NOD mice were sacrificed, their thyroids removed, examined histologically and any infiltrate characterised. Induction of antibodies to the ECD was tested by ELISA in which plates had been coated with either MBP-ECD or an ECD-protein A fusion. All of the mice developed a strong antibody response to the relevant immunogen but none of them contained TBII, TSAB or TBAB activities. No lymphocytic infiltration of the thyroid glands of the CBA or C57 mice was observed. In contrast, all of the NOD mice displayed severe thyroiditis, whilst one of seven MBP-treated mice had moderate infiltration and none of five untreated controls. Immunohistochemical analysis revealed that the infiltrate was predominantly activated T helper cells with little evidence of B cells or the cytokines IL-10 or IL-4, indicating that a Th1 response had been induced, contrary to our findings in BALBc mice which mount a Th2 response. In conclusion we have shown that the type and extent of response induced by immunising with the TSHR varies in mice of differing genetic background. H2d mice develop thyroiditis and TBAB TBII , H2g mice develop thyroiditis in the absence of functional TSHR antibodies, whilst H2b and H2k mice are resistant.

Metastatic spreading of induced uterine sarcomas

A description is given of the frequency and morphology of metastases of uterine sarcomas induced by dimethylhydrazine combined with estrogen in female CBA mice. The data on the frequency and location of metastases are taken from an experiment carried out to study the effect of ascorbic acid on the induction of uterine sarcomas. Experimental uterine sarcomas in mice metastasize in 20–30% of cases. More than 70% of metastases are to the stomach, and thence to the intestine, whereas metastases in the liver, kidneys, and lungs are less frequent. It is shown that the weight of uterine sarcomas with or without metastases is markedly lower in mice treated with ascorbic acid. Dimethylhydrazine-induced uterine sarcomas may be used as a model to study the effects of different factors on metastatic spreading.

Sarcomatous lesions in CBA female mice treated with 1,2-dimethylhydrazine: independent primaries or metastases?

CBA female mice treated with 1,2-dimethylhydrazine (DMH) alone or in combination with oestradiol dipropionate (EP) or ascorbic acid (AA) developed, as expected, a high incidence of uterine sarcomas. In addition, sarcomatous lesions at unusual sites (mainly in the forestomach) were evident. The incidence of sarcomatous lesions at other sites was 53/220 in mice having uterine sarcomas and 0/186 in mice treated with DMH but without uterine sarcomas. The difference between the two groups was highly statistically significant (P < 0.001) and demonstrates non-coincidental association of the above sarcomatous lesions with uterine sarcomas. Uterine sarcomas which presented in association with lesions at other sites were of a larger size than those found in isolation, and the difference in weights in three out of four groups was statistically significant (P = 0.008, 0.035 and 0.011). Histologically, sarcomatous lesions were similar in structure to those of uterine sarcomas, i.e. were of a fibroblastic-histiocytic nature with admixture of giant cells. On the basis of the above data the sarcomatous lesions described appear to represent uterine sarcoma metastases rather than independent primary tumours. AA did not have any influence on carcinogenesis induced by DMH alone but inhibited the growth of uterine sarcomas (whether or not they were associated with other sarcomatous lesions) induced by DMH combined with oestradiol dipropionate.

Evaluation of the role of exogenous pathogens on the incidence of embryo loss during early pregnancy in mice

The mating of CBA/j♀ mice (H2 k) by DBA/2j♂ mice (H2 d) typically results in an elevated incidence of spontaneous embryo loss thus providing an ideal genetically controlled laboratory model for the study of the factors causing early embryo loss during pregnancy. There is now considerable data on the cells and factors involved in fetal resorption but little is known about the events which activate this process. While the activation of the maternal response to the fetal implant could have endogenous or genetic origins, a role for exogenous factors including microbial pathogens could also be involved. In order to investigate these possibilities, the reproductive success of CBA/j♀ × DBA/2j♂ matings in a conventional animal care facility were compared with matings in a specific pathogen free (SPF) animal facility. All animals housed under these conditions were routinely screened by immunoassay and culture, for the presence of a number of viral and bacterial pathogens of mice. The incidence of spontaneous embryo loss in specific pathogen free CBA female mice mated by DBA and other male strains was found to be virtually identical to that of CBA female mice infected with multiple viral pathogens and housed under otherwise identical conditions (non-SPF). However, the numbers of implantation per pregnancy was significantly greater in an SPF facility. Therefore, exposure of mating mice to exogenous viral and bacterial pathogens did not appear to alter the overall incidence of spontaneous embryo resorption. It was concluded that the immunomodulatory effects of infection by common murine pathogens neither augmented nor reduced post-implantation embryo losses.

Collagen Metabolism in the Murine Colon Following X Irradiation

Female CBA mice, aged 16 weeks, were irradiated to the total pelvic region with either single doses (5-20 Gy) or two equal fractions (10- to 30-Gy total dose, 24-h interval) of 240 kV X rays. Total protein and collagen synthesis rates, collagen breakdown, and net collagen content of the colon were measured at various times postirradiation using a radioisotope incorporation method and HPLC analysis. Immunohistochemical staining and computerized image analysis were used to assess the relative amounts of collagen types I and III at various times postirradiation, in various regions of the colon. Total protein and collagen synthesis rates were elevated above control levels at 4 and 8 weeks postirradiation, as was collagen degradation. Values had returned to control levels by 16 weeks postirradiation, and there were no further changes up to 71 weeks postirradiation. The net amount of collagen in the colon did not change relative to controls at any time during the investigation. There was, however, increased immunohistochemical staining for collagen type I 52 weeks postirradiation in all regions of the colon and decreased staining of type III in the circular muscle layer and villi. Altered ratios of these two collagen isotypes are consistent with changes in mechanical properties of the tissue.

Effect of α-difluoromethylornithine on the polyamine levels and proliferation in two transplantable tumours

The effect of inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) on the growth of two murine transplantable tumours was studied. Female CBA mice were implanted with either the sarcoma F (SaF) or an anaplastic mammary carcinoma (CaNT), and 3% DFMO in the drinking water was provided once the tumours were established. Over a 10-day period control SaF tumours increased exponentially from 20 mm3 to over 800 mm3, whereas DFMO-treated SaF reached only 300 mm3. CaNT grew more slowly, requiring 22 days to achieve a similar volume increase, and DFMO was as effective in retarding growth as it had been in SaF. DFMO depleted tumour tissues of putrescine and spermidine, but did not reduce spermine levels. Metaphase arrest experiments with vincristine demonstrated that DFMO could substantially reduce the rates of tumour cell production, but there was no indication the DFMO accelerated the rate of cell loss from the tumours. Despite reduced rates of cell production, labelling studies with bromodeoxyuridine failed to detect differences between control and treated tumours: an increase in transit time through the S-phase was suspected. The number of nuclear organizer regions, detected by the argyrophilia of their associated proteins, was less in DFMO-treated tumours, and within a tumour the degree of silver deposition unequivocally reflected the proliferative heterogeneity. Ultrastructural studies revealed no differences between DFMO-treated and untreated tumours.

Polyclonal Stimulation of Lymphocytes Induced by Beta Blockers in the Mouse: Influences of Age, Sex and Strain

The immune effects of acebutolol, a beta-blocker, are investigated in various strains of mice. In 6-12 weeks old C57Bl/6 female mice, an increase in the number of immunoglobulin secreting cells is observed only at the age of 9 weeks. At that age, such an effect is seen neither in C57Bl/6 male mice nor in female mice from 6 other strains including OF1 outbred mice. An increase in anti-trinitrophenol plaque forming cells is found in 9 week old C57Bl/6, Balb/c and CBA female mice.

Rheumatoid factor induction in the mouse: sex differences and the effect of the sex steroids

Female CBA mice produced a significantly higher plasma rheumatoid factor (RF) response to Salmonella typhosa lipopolysaccharide than did male mice. The peak level in females was observed on day 5–6 after injection and in males on day 7–8. Elevated RF levels continued to be detected more than 30 days later. A second injection of LPS, 38 days after the first, to assess the secondary response, had no more than an additive effect on plasma RF concentration, although the day of peak response was earlier by two days in both sexes. Administration of oestradiol-17β by Silastic implant brought forward the day of peak response by two days in both sexes although it reduced its amplitude considerably. Testosterone had little effect on the peak concentrations achieved in both sexes, but did produce a slower decay in plasma RF level. This investigation indicates that the sex hormones can influence the response to LPS, a polyclonal B cell activator. This may have implications for the sex differences seen in autoimmune diseases.

Sudden outbreak of a leukemia-like lesion in female CBA mice after repeated injections of 5-azacytidine

A total of 11 i.p. injections of 1 mg/kg 5-azacytidine given within 21 weeks induced a sudden outbreak of a leukemia-like disease in female CBA mice about 2 weeks after the last injection. The outbreak of disease was highly synchronous; 68% of animals who had survived the treatment period succumbed within a period of 5 days, 2/3 of them on a single day. The lesion had a characteristic form commonly presenting as a result of hemorrhagic effusion in the thoracic and/or abdominal cavities, associated with diffuse infiltration of lymphatic and fatty tissue by lymphoblasts. It appears that a critical combination of factors was responsible for the unexpected appearance of the lesion. Female BALB/c mice treated identically were not affected. The factors indicating that the lesion was induced by an epigenetic mechanism are discussed.

1,2-Dimethylhydrazine carcinogenesis in neonatally androgenized CBA mice.

The aim of this study was to investigate the possible influence of exposure to steroid hormones early in life on the susceptibility of animals as adults to chemical carcinogens. CBA male and female mice received a single subcutaneous injection of 0.5 mg testosterone propionate (TP) in olive oil within 24 h after birth. At the age of 2 months, neonatally androgenized and control mice started receiving weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH). By the end of the experiment, 90% of neonatally androgenized females treated with DMH developed uterine sarcoma against 9% in control females treated with DMH, this difference being attributed to the hyperoestrogenization of androgenized females. In neonatally androgenized males treated with DMH 79% developed pararenal sarcoma and 71% colon tumours versus 25 and 32% respectively of control males treated with DMH.

Biomarkers of aging: Tissue markers. Future research needs, strategies, directions and priorities

Objective tests that allow early detection of deleterious changes with age are necessary to develop treatments enhancing the health span — the length of healthy life. Here we report tests of eight biological systems that can be performed in mice with no harm to the subjects. Male and female B6, CBA and F1 mice were used. While most test results correlated with chronological age in most genotypes, none predicted subsequent longevities in more than two genotypes. Surprisingly, the open field activity test that most consistently predicted longevities, did not correlate with chronological age. Six tests predicted beneficial effects of food restriction in F1 males, but only one correctly predicted the deleterious effects of the same food restriction regimen in B6 males. These results suggest that different biological systems age at different rates, that rates are affected by genotype and that an antiaging treatment beneficial in one genotype may be harmful in another.

The age dependence of radiation sensitivity of the gonads of female mice.

Female CBA-mice were continuously gamma-irradiated with 0.9 or 2.4 Gy. The dose was given during a 4-day period with start at 50, 90, 135 or 190 days of age respectively. Fifty-six days after the irradiation the females were killed and the number of germ cells in their ovaries were compared with that from unirradiated control females of corresponding age. The total number of germ cells was, compared with the controls, reduced by about 70% in all age groups after the dose of 0.9 Gy and to between 3 and 8% after 2.4 Gy. Seven stages of oocyte development were observed as well as 2 stages in degeneration. The observations suggest a changing radiation sensitivity by age during an initial period of about 90 days followed by a period of fatal radiation effects.

Modulation of the natural killer cell activity in pregnant mice alters the spontaneous abortion rate

Effector cells associated with an aborting fetus appear to be both thymus derived (T) and natural killer (NK) cells. In order to test the hypothesis that NK cells are a major effector mediating early spontaneous abortion (<day 8–10), CBA female mice mated by DBA/2 males were treated with either polyinosinic/cytidylic acid (poly I:C) to boost NK activity, or rabbit anti-asialo GM1 (RaASGM1) to decrease NK activity. The results of the NK assays of the spleens of treated mice confirmed that the reagents had the expected effect on NK activity and an inspection of the uteri indicated a significant increase in aborted embryos after poly I:C and a marked decrease in spontaneous abortions after RaASGM1 treatment. Therefore, spontaneous abortions may be mediated in part by the cytotoxic activity of unregulated NK cells.

Quantitative analysis of neuronal mosaic formation in the mouse neocortex and hippocampus

The mosaic pattern of neurogenesis, reflected in the arrangement of concentrations of neurons intensely labeled with 3H-thymidine, was described as a result of the writers' investigations with injection of 3H-thymidine into mice at different times of embryogenesis, followed by analysis of the arrangement of the labeled cells in the neocortex and hippocampus of day-old mice [4, 5, 13]. It was concluded that the mosaic pattern of neurogenesis is determined by the nonsynchronized course of differentiation of cell groups in the ventricular zone of the embryonic brain, i.e., evidence was obtained of its discrete organization in the form of loci of neurogenesis. The aim of the present investigation was to obtain mathematical confirmation of the nonrandomness of mosaic formation of neuronal groups in the cerebral cortex, to characterize this process quantitatively, and to compare its scale with the formation of the modular organization of the cortex. EXPERIMENTAL METHOD Pregnant female CBA mice were given a single intraperitoneal injection of 3H-thymidine (i0 ~Ci/g) on the 13th-19th day of pregnancy (EI3-EI9). At the age of i day, two or three mice from each mother were killed by decapitation. The cerebral hemispheres of mice receiving 3H-thymidine from El5 to El9 were fixed in Karnovsky's fixative and embedded in Durcupan. When the isotope was injected during the period E13-19 the cerebral hemispheres were fixed in Carnoy's mixture and embedded in paraffin wax. One hemisphere from each animal was cut into frontal sections, the other into sagittal. Preparations with glued semithin (i u) or paraffin (6 B) sections were covered with type M emulsion and, after standard autoradiographic processing, were stained with 1% toluidine blue solution in 2.5% sodium carbonate solution or with 0.1% cresyl violet. The arrangement of the centers of the nuclei of the intensely labeled neurons in area 6 of the frontal region of the neocortex and in area CAI of the dorsal hippocampus was mapped on an NU-2E microscope with projection screen. Mapping in the neocortex was carried out on frontal semithin brain sections from mice receiving 3H-thymidine from El5 through EfT, whereas mapping in the hippocampus was carried out on paraffin and semithin frontal brain sections from animals receiving the isotope from El3 through El7. Cells were recorded as intensely labeled if the number of grains of silver above the nucleus varied from maximal to half that number of grains per section. Maps of areas 6 and CAI from two animals at each time of the experiment studied were analyzed mathematically to discover nonrandom groups of intensely labeled neurons. For this purpose an approach was used which enables the regularity of neurogenesis to be assessed in brain structures organized on the principle of a rec