Retired Breeder Mice Research Articles

Normal tissue and tumor microenvironment adaptations to aerobic exercise enhance doxorubicin anti-tumor efficacy and ameliorate its cardiotoxicity in retired breeder mice.

Aerobic exercise is receiving increased recognition in oncology for its multiple purported benefits. Exercise is known to induce physiologic adaptations that improve patient quality-of-life parameters as well as all-cause mortality. There also is a growing body of evidence that exercise may directly alter the tumor microenvironment to influence tumor growth, metastasis, and response to anticancer therapies. Furthermore, the physiologic adaptations to exercise in normal tissues may protect against treatment-associated toxicity and allow for greater treatment tolerance. However, the exercise prescription required to induce these beneficial tumor-related outcomes remains unclear. This study characterized the aerobic adaptations to voluntary wheel running in normal tissues and the tumor microenvironment. Female, retired breeder BALB/c mice and syngeneic breast adenocarcinoma cells were utilized in primary tumor and metastasis models. Aerobic exercise was found to induce numerous adaptations across various tissues in these mice, although primary tumor growth and metastasis were largely unaffected. However, intratumoral hypoxia and global metabolism were altered in the tumors of exercising hosts relative to non-wheel running controls. Doxorubicin chemotherapy also was found to be more efficacious at delaying tumor growth with adjuvant aerobic exercise. Additionally, doxorubicin-induced cardiac toxicity was ameliorated in exercising hosts relative to non-wheel running controls. Taken together, these data suggest that the normal tissue and tumor microenvironment adaptations to aerobic exercise can improve doxorubicin efficacy while simultaneously limiting its toxicity.

Activation of AMP-Activated Protein Kinase by A769662 Ameliorates Sepsis-Induced Acute Lung Injury in Adult Mice.

A serious consequence of sepsis is acute lung injury, whose severity is particularly impacted by the age of the patient. AMP-activated protein kinase (AMPK) is a crucial regulator of cellular metabolism, which controls mitochondrial biogenesis and autophagy. Here, we investigated the effect of pharmacological activation of AMPK with A769662 on lung injury by using a model that would preferably mimic the clinical condition of adult patients. Male C57BL/6 retired breeder mice (7-9 months old) were subjected to sepsis by cecal ligation and puncture (CLP). Mice received vehicle or A769662 (10 mg/kg) intraperitoneally at 1 h after CLP. At 6 h after CLP, vehicle-treated mice exhibited severe lung injury and elevation of plasma pro-inflammatory cytokines when compared with control mice. At molecular analysis, lung injury was associated with downregulation of AMPKα1/α2 catalytic subunits and reduced phosphorylation of AMPKβ1 regulatory subunit. Treatment with A769662 ameliorated lung architecture, reduced bacterial load in lung and blood, and attenuated plasma levels of interleukin-6. This protective effect was associated with nuclear phosphorylation of AMPKα1/α2 and AMPKβ1, increased nuclear expression of peroxisome proliferator-activated receptor γ co-activator-α and increased autophagy, as evaluated by the light-chain (LC)3B-I and LC3B-II content, without changes in sirtuin-1 cellular dynamics. Treatment with A769662 alone or in combination with the antimicrobial agent imipenem (25 mg/kg) increased survival rate (29% and 51%, respectively) when compared with vehicle treatment (10%) at 7 days after CLP. These data suggest that pharmacological activation of AMPK might be a beneficial approach for the treatment of sepsis in adult population.

N‐3 fatty acids prevent trabecular bone loss

N‐3 fatty acids have been implicated in preventing bone loss. Whether n‐3 fatty acids can restore bone loss is not established. We conducted a study using the ovariectomized mouse model to determine if n‐3 fatty acids can restore bone.Ten months old, C57BL/6, retired breeder mice were either sham operated or ovariectomized and left for 3 months. The different groups were: Group 1. LC S; Group 2. LC O; Group 3. 18/12 S; Group 4. 18/12 O Group 5. 30/20 S Group 6. 30/20 O. Mice were fed either LC or FO (10%). The LC and FO fed groups were kept in the respective diet regimens for the next 3 months, at the end of which they were sacrificed and long bones were subjected to pQCT analyses.In the distal femoral metaphysis, trabecular bone mineral content (Tb BMC) was decreased significantly in the LC O mice while it was increased in the 18/12 O group. Similarly in the proximal tibial metaphysis, the Tb CNT and cortical bone mineral content were significantly higher in the 18/12 O and 30/20 O groups.In summary, the 18/12 n‐3 fatty acids increased trabecular content in both the femur and tibia and the cortical BMC was increased only in the tibia, suggesting that 18/12 is more beneficial.Support or Funding InformationFunding: Engaged Scholarship for Undergraduate Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Incidence of spontaneous lymphomas in non-experimental NOD/Shi-scid, IL-2Rγnull (NOG) mice.

Severely immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice provide an in vivo model for human cell/tissue transplantation studies. NOG mice were established by combining interleukin-2 receptor-γ chain knockout mice and NOD/Shi-scid mice. They exhibit a high incidence of thymic lymphomas and immunoglobulin (Ig) leakiness. In this study, we assessed the incidence of malignant lymphomas and the occurrence of leakiness in 2,184 non-experimental NOG retired breeder mice aged 16–40 weeks. We established that the total incidence of lymphomas was only 0.60% (13/2,184). Most lymphomas (10/13) occurred in female mice by the age of around 25 weeks. No mice developed Ig leakiness. All lymphomas were derived from the thymus, and consisted mainly of CD3-positive and CD45R-negative lymphoblastic-like cells. Therefore, based on the absence of Ig leakiness and a very low incidence of lymphomas, including thymic lymphomas, NOG mice may be useful in regeneration medicine for xenotransplantation of human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, and in transplantation experiments involving tumor cells.

Systemic Administration of Proteoglycan Protects BALB/c Retired Breeder Mice from Experimental Arthritis.

This study was undertaken to evaluate the prophylactic potential of proteoglycan (PG) administration in experimental arthritis. Female BALB/c retired breeder mice received two (2xPG50 and 2xPG100 groups) or three (3xPG50 group) intraperitoneal doses of bovine PG (50 μg or 100 μg) every three days. A week later the animals were submitted to arthritis induction by immunization with three i.p. doses of bovine PG associated with dimethyldioctadecylammonium bromide adjuvant at intervals of 21 days. Disease severity was daily assessed after the third dose by score evaluation. The 3xPG50 group showed significant reduction in prevalence and clinical scores. This protective effect was associated with lower production of IFN-γ and IL-17 and increased production of IL-5 and IL-10 by spleen cells restimulated in vitro with PG. Even though previous PG administration restrained dendritic cells maturation this procedure did not alter the frequency of regulatory Foxp3+ T cells. Lower TNF-α and IL-6 levels and higher expression of ROR-γ and GATA-3 were detected in the paws of protected animals. A delayed-type hypersensitivity reaction confirmed specific tolerance induction. Taken together, these results indicate that previous PG inoculation determines a specific tolerogenic effect that is able to decrease severity of subsequently induced arthritis.

Commercial bovine proteoglycan is highly arthritogenic and can be used as an alternative antigen source for PGIA model.

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease. It affects mainly the joints, causing synovitis, cartilage destruction, and bone erosion. Many experimental models are used to study the mechanisms involved in immunopathogenesis and new therapies for this disease. Proteoglycan-induced arthritis (PGIA) is a widely used model based on the cross-reactivity of injected foreign (usually human) PG and mice self-PG. Considering the complexity of the extraction and purification of human PG, in this study we evaluated the arthritogenicity of bovine PG that is commercially available. Bovine PG was highly arthritogenic, triggering 100% incidence of arthritis in female BALB/c retired breeder mice. Animals immunized with bovine PG presented clinical symptoms and histopathological features similar to human RA and other experimental models. Moreover, bovine PG immunization determined higher levels of proinflammatory and anti-inflammatory cytokines in arthritic mice compared to healthy ones. As expected, only the arthritic group produced IgG1 and IgG2a antibodies against PG. Thus, commercial bovine PG can be used as an alternative antigenic source to PGIA for the study of many RA aspects, including the immunopathogenesis of the disease and also the development of new therapies.

Consequences of manganese administration for striatal dopamine and motor behavior in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-exposed C57BL/6 mice

Environmental compounds may be important contributors to Parkinson's disease etiology. Epidemiological and experimental evidence for the facilitation of parkinsonism by manganese is equivocal. This work addressed methodological concerns in the few studies of manganese modulation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in C57BL/6 mice. Male, retired breeder mice received 0 or 100 mg/kg of manganese chloride (MnCl₂; subcutaneously on days 1, 4 and 7) and 0 or 20 mg/kg of MPTP (intraperitoneally on day 8) and survived up to day 15 or 22. On the day of sacrificing, horizontal (grid crossing) and vertical (rearing) open field movement, swimming, grip strength and grip fatigue were examined. Striata were analyzed for dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) using high-performance liquid chromatography. MPTP produced a main effect decrease in striatal dopamine (48.8%) and DOPAC (38.1%), but there was no main effect of MnCl₂ or MnCl₂ x MPTP interaction. However, modulatory interactions were observed between the effects of MnCl₂ and MPTP for grid crossing, rearing and grip strength. Interestingly, these interactions reduced the severity of behavioral deficits attributable to either of these compounds alone. For rearing and grip strength, the MnCl₂ x MPTP interaction was dependent upon survival time. The mechanistic nature of the MnCl₂ x MPTP interaction upon these behaviors, in the absence of such an interaction for striatal dopamine and DOPAC, remains to be clarified.

Chronic social defeat stress disrupts regulation of lipid synthesis

Several psychiatric disorders increase the risk of cardiovascular disease, including posttraumatic stress disorder and major depression. While the precise mechanism for this association has not yet been established, it has been shown that certain disorders promote an unfavorable lipid profile. To study the interaction of stress and lipid dysregulation, we utilized chronic social defeat stress (CSDS), a mouse model of chronic stress with features of posttraumatic stress disorder and major depression. Following exposure to CSDS, mice were given access to either regular chow or a Western-style diet high in fat and cholesterol (HFD). The combination of social stress and HFD resulted in significant perturbations in lipid regulation, including two key features of the metabolic syndrome: increased plasma levels of non-HDL cholesterol and intrahepatic accumulation of triglycerides. These effects were accompanied by a number of changes in the expression of hepatic genes involved in lipid regulation. Transcriptional activity of LXR, SREBP1c, and ChREBP were significantly affected by exposure to HFD and CSDS. We present CSDS as a model of social stress induced lipid dysregulation and propose that social stress alters lipid metabolism by increasing transcriptional activity of genes involved in lipid synthesis.

Effects of triglycerides, obesity, and starvation on ghrelin transport across the blood–brain barrier

Human ghrelin is transported across the blood–brain barrier (BBB) of normal mice. Here, we studied the effects of triglycerides, obesity, and starvation in retired breeder mice maintained on a high fat diet, mice age-matched to the retired breeders but maintained on normal chow, and 8-week-old mice maintained on breeder chow. The rate of ghrelin transport across the BBB was studied by both the intravenous administration method of multiple-time regression analysis and by the brain perfusion method. We found that (1) obese, aged mice lost the ability to transport intravenously administered ghrelin across the BBB, resulting in an inverse relation between body weight and ghrelin BBB permeability; (2) serum triglycerides promoted transport of intravenously administered ghrelin across the BBB, whereas epinephrine had no effect; (3) fasting tended to promote ghrelin transport across the BBB as most readily shown in brain perfusion studies; (4) evidence suggested that a serum factor promoted ghrelin transport in 8-week-old mice. Overall, these results show that serum factors and physiological states influence the rate at which ghrelin is transported across the blood–brain barrier.

Basal Ganglia Accumulation and Motor Assessment Following Manganese Chloride Exposure in the C57BL/6 Mouse

Equivocal clinical evidence for involvement of manganese in development of Parkinson's disease necessitates experimental studies on this issue. The aged, 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine-treated C57BL/6 mouse is one of the most common models for Parkinson's disease. However, there is little information on brain bioaccumulation of manganese, and little or no information on clinical/behavioral manifestations of manganese neurotoxicity, in this strain. Male C57BL/6 retired breeder mice were given a single subcutaneous injection of either 0, 50, or 100 mg/kg of MnCl(2) (single-dose regimen) or three injections of either of these doses over 7 days (multiple-dose regimen). Behavioral assessment was performed 24 h after final injection, followed by sacrifice, and body weight was recorded each day. There was a 105% increase in striatal manganese concentration 1 day after a single 100 mg/kg injection, and 421% and 647% increases, respectively, 1 day after multiple doses of 50 or 100 mg/kg of MnCl(2). One day after a single injection, there were respective 30.9% and 38.9% decreases in horizontal movement (grid crossing) for the 50 and 100 mg/kg doses and a 43.2% decrease for the multiple dose of 100 mg/kg. There was no significant main effect of dose level on rearing, swimming, grip strength, or grip fatigue. Unlike previous work with the C57BL/6 strain using smaller intraperitoneal doses, this study established dosing regimens that produced significant increases in basal ganglia manganese concentration reminiscent of brain increases in the CD-1 mouse following subcutaneous doses close to our lowest. A decrease in locomotor behavior, significant but not severe in this study, has been reported following manganese exposure in other mouse strains. These data, particularly the significant increase in basal ganglia manganese concentration, provide guidance for designing studies of the potential role of manganese in Parkinson's disease using the most common animal model for the disorder.

Increased numbers of microchimeric cells of fetal origin are associated with dermal fibrosis in mice following injection of vinyl chloride.

To develop a murine model for use in examining the role of microchimeric cells and certain chemical exposures in the pathogenesis of systemic sclerosis (SSc). Female BALB/cJ retired breeder mice were bled before and after vinyl chloride injection. The DNA from their white blood cells was obtained, and the number of microchimeric cell equivalents was determined by quantitative polymerase chain reaction using DNA primers specific for the H-2Kb gene, a sequence not found in BALB/cJ mice. Skin was obtained at autopsy, embedded in paraffin, sectioned, and stained with Masson's trichrome. Hydroxyproline analyses were performed on 4-mm skin biopsy samples. Microchimeric cells were identified and quantitated before and after 20 daily intraperitoneal injections of vinyl chloride. The number of microchimeric cells in the peripheral blood increased an average of 48-fold after treatment with vinyl chloride. Histologic examination of the skin of these same mice (which had an increased number of microchimeric cells) showed inflammation, with abundant fibroblasts and a heavy mononuclear infiltration in the dermis. The collagen fibers appeared densely packed and disorganized. Histologic examination of the skin of untreated retired breeder mice and treated virgin mice appeared normal. Quantitative assays to determine the collagen content of skin biopsy samples obtained from treated microchimeric mice compared with nontreated microchimeric or with treated nonmicrochimeric mice showed a 2-3-fold increase in collagen content in the treated microchimeric mice. Extraordinary splenomegaly was present in the vinyl chloride-treated microchimeric mice, accompanied by cellular infiltration and fibrosis. The results suggest that vinyl chloride injections into BALB/cJ retired breeder mice lead to activation of microchimeric cells, which causes the cells to divide and multiply. The correlation between the 48-fold increase in microchimeric cells and the appearance of dermal inflammation and fibrosis similar to that of graft-versus-host disease suggests that activated microchimeric cells may be a necessary factor in the pathogenesis of autoimmune diseases such as SSc.

Effects of chronic indomethacin therapy on the development and progression of spontaneous mammary tumors in C3H/HEJ mice.

The present study was designed to test the hypothesis that endogenous prostaglandin E (PGE) promotes the development, growth and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The effect of chronic oral indomethacin (indo) therapy starting at 6 months of age was tested on these parameters as well as on animal survival, in comparison with control mice placed on 0.2% ethanol in drinking water for up to 25 months of age. Indo treatment delayed the initial (up to 27 weeks) development of primary tumors by 11-12 weeks; however, the subsequent rate of tumor appearance was unaffected (totaling 82% in indo-treated vs. 90% in controls by 25 months of age). Spontaneous regression of primary tumors (26% in controls) increased 2-fold (53%) with indo therapy. While the apparent reduction in the growth rate of primary tumors and the overall prolongation of animal survival were not significant, the lifespan of mice bearing multiple tumors was significantly prolonged by therapy. There was also a 2-fold reduction in the incidence of lung metastases in mice bearing detectable primary tumors, and this was more pronounced during the earlier phase of tumor development. Positive immunostaining for cyclooxygenase-2 enzyme (indicative of the cellular source of PGE) was exhibited by tumor cells, stromal cells and macrophages within the primary tumors. Tumors in indo-treated mice exhibited histological evidence of increased differentiation (acinar architecture), significant tumor cell death, mononuclear cell infiltration and reduction in vascularity, indicating that the beneficial effects of indo were due to multiple mechanisms, including improved immune response and reduced angiogenesis.

Comments

Alving and WassefThe authors state that “stealth” properties have been studied only in models in which the tested animals consisted of relatively young mice. We have reported results with GM1-containing liposomes in retired breeder mice, as compared to 6–8 week mice. We found that the blood/RES ratios were reduced by approximately 2-fold in the older mice lending some confirmation to their hypothesis that circulation half-lives may be dependent on the presence of antibodies or other age-related factors [1]. Stealth liposomes have also been studied in rats, with very similar results to those found in mice [2,3].

The effect of hypophysectomy on thymic aging in mice.

Abstract Surgical removal of the pituitary (hypophysectomy) followed by endocrine supplementation in middle-aged rats has been reported to reverse immunologic decline with age. We attempted to confirm and extend these reports by using a well-defined and readily available mouse model system. Hypophysectomy and endocrine supplementation in 8- to 9-mo-old C57BL/6J (B6) male retired breeder mice improved some, but not all, T dependent immune functions tested at 15 mo of age. In hypoxed mice, spleen cell proliferation in response to phytohemagglutinin (PHA) in vitro, and delayed type hypersensitivity (DTH) responses to sheep red blood cells (SRBC) measured by footpad swelling improved to levels shown by young controls. Direct anti-SRBC plaque-forming cell (PFC) responses by spleen cells, and serum agglutination responses against SRBC were not improved. Hypoxed mice had larger thymuses and much higher ratios of cortex-medulla areas than did age-matched controls. Hair regrowth after shaving was much faster in hypoxed mice. Nevertheless, hypophysectomy reduced mean and maximum longevities. These results conflict in several ways with the previously reported studies in rats, in which direct PFC responses and maximum longevities were improved by this treatment. There have been no previous studies of the effect of hypophysectomy and endocrine supplementation on thymic aging in mice, nor has it previously been reported that this treatment causes improvements in PHA and DTH responses and in thymic morphology. These effects show that at least some aging processes are reversible in aging individuals. They also suggest that hypophysectomy of middle-aged mice will be useful for studying neuroendocrine and thymic interactions that occur during the aging process.

Age-related changes in passive avoidance retention: modulation with dietary choline.

Two studies were performed to evaluate the effects of dietary choline manipulation on behavior in mice. Retired breeder mice (8.5 months old) were placed on purified diets that were either deficient in free choline or choline-enriched. After 4.5 months, the mice were trained in a single-trial, passive avoidance task and tested for retention either 24 hours or 5 days later. Their performance was compared with that of mice, of various ages, that were maintained on a control diet. The two salient findings were (i) a dramatic decrease in retention of the task in senescent mice (23 months and older) and (ii) marked behavioral differences between choline-deficient and choline-enriched mice (13 months old). In fact, the choline-enriched mice performed as well as 3-month-old mice, whereas the choline-deficient mice performed as poorly as the senescent mice. In a replication, three groups of retired breeder mice were placed on the same choline-deficient diet; control and enriched groups were given choline through their drinking water. Again, retention of learning was superior in the choline-enriched mice and inferior in the choline-deficient mice. These studies demonstrate that dietary manipulation of choline can significantly alter behavior in ways that are qualitatively and quantitatively similar to those occurring across the life-span of the mouse. Thus certain behavioral changes that occur with age might be modulated through appropriate precursor control.

Respiratory infection in mice with sialodacryoadenitis virus, a coronavirus of rats

Sialodacryoadenitis virus (SDAV), a coronavirus of rats, evoked both serum neutralization and complement fixation antibody responses when inoculated intranasally in mice. Weanling gnotobiotic CD-1 mice inoculated intranasally with 10(3.0) mean tissue culture infective doses of SDAV remained asymptomatic. Virus was recovered from the nasopharynx, trachea, and lung from day 2 to day 7. Viral antigen was readily detected by indirect immunofluorescence in the lung but rarely in the nasopharynx. Infected mice developed interstitial pneumonia. Susceptible mice contact exposed to experimentally infected mice developed antibody to SDAV. Epizootiological studies indicated that retired breeder mice can have complement-fixing antibody to SDAV and mouse hepatitis virus (MHV) in the absence of MHV infection. These studies show that SDAV is infectious for mice and can be a pathogen for the respiratory system. Thus, SDAV infection of mice may be responsible for spurious seroconversions to MHV.

Plasmodium berghei NK65 in the inbred A-J mouse: age immunity in the female retired breeder A-J mouse.

SYNOPSIS. Death rates of A/J and CF1 female mice 4 weeks and 6 months of age were compared after the mice were infected with Plasmodium berghei NK65C deme (population) and NK65RR deme. Death rates were compared also when female A/J retired breeder mice were infected with blood passages 18 and 40 of NK65C.NK65C was found to be less virulent than NK65RR. The 40th blood passage of NK65C was more virulent than the 18th passage, but still not as virulent at the NK65RR deme. A/J retired breeders were clearly more resistant to infection than 4 week old A/J mice, while little difference was found in the different age groups of the CF1 mice.

In vitro stimulation of antibody formation by peritoneal cells. II. Cell interactions and effects of immunochemical or metabolic inhibitors.

Peritoneal cells (PC) from normal, unimmunized mice were placed in ultra-thin monolayer cultures containing carboxymethylcellulose (CMC), sheep red blood cells (SRBC), and complement, and tested for the appearance of plaques of lysis. The behavior of PC from young male mice and from female mice that had given birth to several litters (retired breeder mice) was studied. It was found that cells from spleen, mesenteric lymph node, thymus, bone marrow, thoracic duct lymph, or Peyer's patches could not form plaques in the CMC microcultures. Also, various combinations of these cells did not lead to plaque formation. When cells from any of these sources were mixed with PC, there was either no effect or an actual inhibition of plaque formation, the plaque counts being lower than would have been expected from the number of PC present in the mixture. Optimal plaque formation by peritoneal cells was found to be dependent on an optimal cell concentration, this optimum being around 5 x 10(6)/ml for young male mice and 0.5 x 10(6)/ml for retired breeders. Inhibition of plaque formation was found with either supra- or suboptimal cell concentrations. The inhibition by excess cell concentration may have been a simple nutritional or nonspecific overcrowding effect, as it could also be induced by an addition of an excess of spleen or lymph node cells. The failure of more dilute PC preparations to give adequate numbers of plaques appeared to be more specific, as plaque numbers could not be restored to normal by addition of spleen cells. The suggestion was that some cell to cell interaction between PC was involved. This dependence on cell concentration was not seen with immunized spleen PFC. Plaque appearance could be specifically and reversibly suppressed by placing PC in a medium containing rabbit anti-mouse IgM serum. Anti-IgG serum had no such effect. These experiments strengthened our view, expressed in the accompanying paper, that plaque formation was due to the formation of IgM, hemolytic antibody to SRBC by the PC. Metabolic inhibitors were incorporated into monolayer cultures and had different effects with the different types of PFC used. In the case of spleen cells from mice actively immunized against SRBC 4 days before killing, actinomycin D had no effect on plaque counts and puromycin reduced plaque numbers by a factor of 2. In the case of PC from young male mice, actinomycin D in concentrations above 0.01 microg/ml caused reductions down to < 2% of control values in plaque counts, and puromycin (10 microg/ml) had a similar effect. The PC from retired breeder mice occupied an intermediate position between the two cases just discussed. A compartment of cells, equal to about one-fifth of the total normal PFC compartment, was identified as resistant to high concentrations of either actinomycin D or puromycin, being similar in these respects to PFC from spleens of intentionally preimmunized mice. The mitotic poison, Colcemid, did not affect plaque counts in any situation tested. The theoretical implications of these results are briefly discussed.