Beagle dogs have long been employed in toxicology studies and as skin disease models. Compared with other experimental animal species, they are known to be susceptible to skin responses, such as rashes, from exposure to various chemical compounds. Here, a unique dog phenotype was identified that showed no skin response to compound 48/80, a mast cell degranulating agent. Although the skin responses to intradermal injection of polyoxyethylene castor oil derivative (HCO-60, a nonionic detergent), histamine dihydrochloride, concanavalin A (IgE receptor-mediated stimuli), or calcium ionophore A23187 were comparable in wild-type (WT) dogs and these nonresponder (NR) dogs, only the response to compound 48/80 was entirely absent from NR dogs. The skin mast cell density and histamine content per mast cell were histologically comparable between WT and NR dogs. By checking for skin responses to compound 48/80, NR dogs were found to exist at the proportion of 17–20% among four animal breeders. From retrospective analysis of in-house breeding histories, the NR phenotype appears to conform to the Mendelian pattern of recessive inheritance. The standard skin response in WT dogs developed at 2–4 months of age. In conclusion, this unique phenotype, typified by insensitivity in the compound 48/80-induced degranulation pathway in mast cells, has been widely retained by recessive inheritance in beagle dogs among general experimental animal breeders. The knowledge concerning this phenotype could lead to better utilization of dogs in studies and aid in model development.
Previously we demonstrated coxsackievirus B3 (CVB3) infection during early gestation as a cause of pregnancy loss. Here, we investigated the impacts of CVB3 infection on female mouse fertility. Coxsackievirus-adenovirus receptor (CAR) expression and CVB3 replication in the ovary were evaluated by immunohistochemistry or reverse transcription-polymerase chain reaction (RT-PCR). CAR was highly expressed in granulosa cells (GCs) and CVB3 replicated in the ovary. Histological analysis showed a significant increase in the number of atretic follicles in the ovaries of CVB3-infected mice (CVBM). Estrous cycle evaluation demonstrated that a higher number of CVBM were in proestrus compared to mock mice (CVBM vs. mock; 61.5%, 28.5%, respectively). Estradiol concentration in GC culture supernatant and serum were measured by an enzyme-linked immunosorbent assay. Baseline and stimulated levels of estradiol in GC were decreased in CVBM, consistent with significantly reduced serum levels in these animals. In addition, aromatase transcript levels in GCs from CVBM were also decreased by 40% relative to the mock. Bone mineral density evaluated by micro-computed tomography was significantly decreased in the CVBM. Moreover, the fertility rate was also significantly decreased for the CVBM compared to the mock (CVBM vs. mock; 20%, 94.7%, respectively). This study suggests that CVB3 infection could interfere with reproduction by disturbing ovarian function and cyclic changes of the uterus.
Encapsulating peritoneal sclerosis is one of the most serious complications of long-term peritoneal dialysis. The pathogenesis of encapsulating peritoneal sclerosis has not been elucidated, but several putative factors necessary for the development of peritoneum fibrosis (PF) have been reported. However, the roles of T helper (Th) cells in the progression of PF are unknown. The purpose of this study was to clarify the roles of Th1, Th2, and Th17 cells in the progression of PF. T-bet, GATA-3, and RORγt are Th1, Th2, and Th17 lineage commitment transcription factors, respectively. We previously generated Th1-biased (T-bet transgenic (Tg)) mice, Th2-biased (GATA-3 Tg) mice, and Th17-biased (RORγt Tg) mice. In this study, Th1, Th2, Th17-biased, and wild-type mice were administered chlorhexidine gluconate (CG) intraperitoneally and analyzed on day 21. CG-injected GATA-3 Tg mice showed a distended intestinal tract and developed marked thickening of the submesothelial space compared with the other groups. CG-injected GATA-3 Tg mice also showed significant expression of α-SMA positive cells, macrophages, and collagen III in the submesothelium. In contrast, CG-injected T-bet Tg mice only developed mild peritoneal fibrosis. Cytokines analysis in peritoneal fluid showed that IFN-γ was significantly increased in CG-injected T-bet Tg mice and that IL-13 was significantly increased in CG-injected GATA-3 Tg mice. Moreover, intraperitoneal administration of IFN-γ improved PF in GC-injected wild-type mice. Our results suggest that Th2 cells may play roles in the development of experimental PF and that Th1 cells may alleviate the severity of experimental PF.
Trichomonadid protozoa have been found in the intestinal tracts of common marmosets (Callithrix jacchus). However, there is little information available on species identification and the pathogenicity of these trichomonads. In this study, we conducted a fecal survey of a common marmoset colony maintained as laboratory animals in Japan and identified the trichomonad species. Screening using a fecal smear examination revealed that 66% (58/88) of the marmosets had trichomonadid trophozoites in their feces. The trichomonads were found in both normal feces (31/49, 63%) and diarrhea (27/39, 69%), with no significant difference in frequency. The protozoa were identified as Pentatrichomonas hominis using morphological characters and the 100% identity of the nucleotide sequence of the partial 18S rRNA gene (297 bp). The intraspecific genetic variability between P. hominis from the marmosets in this study and P. hominis from other reported mammal hosts was ≤1% in the nucleotide sequence, including the internal transcribed spacer (ITS)-1, 5.8S rRNA gene, and ITS-2 (293 bp). P. hominis inhabits the large intestine of various mammalian hosts, including primates, and is considered nonpathogenic. These results suggest that P. hominis is transmitted among marmosets and other mammals but is not a primary cause of bowel disease in marmosets.
To confirm our hypothesis that the sex and age of cynomolgus monkeys influences the effect of training, we employed a new training technique designed to increase the animal’s affinity for animal care personnel. During 151 days of training, monkeys aged 2 to 10 years accepted each 3 raisins/3 times/day, and communicated with animal care personnel (5 times/day). Behavior was scored using integers between −1 and 5. Before training, 35 of the 61 monkeys refused raisins offered directly by animal care personnel (Score −1, 0 and 1). After training, 28 of these 35 monkeys (80%) accepted raisins offered directly by animal care personnel (>Score 2). The mean score of monkeys increased from 1.2 ± 0.1 to 4.3 ± 0.2. The minimum training period required for monkeys to reach Score 2 was longer for females than for males. After 151 days, 6 of the 31 females and 1 of the 30 males still refused raisins offered directly by animal care personnel. Beneficial effects of training were obtained in both young and adult monkeys. These results indicate that our new training technique markedly improves the affinity of monkeys for animal care personnel, and that these effects tend to vary by sex but not age. In addition, abnormal behavior and symptoms of monkeys were improved by this training.
Helicobacter sp. MIT 01-6451 has been detected in SPF mice kept in Japan. To characterize strain MIT 01-6451, its infection route during fetal and neonatal life and effects on pregnancy were investigated using immunocompetent and immunodeficient mouse strains (BALB/c, C57BL/6, and SCID). MIT 01-6451 was detected in the uterus, vagina, and mammary glands of 50% of infected SCID mice, whereas these tissues were all negative in immunocompetent mice. No fetal infections with MIT 01-6451 were detected at 16–18 days after pregnancy in any mouse strain. In newborn mice, MIT 01-6451 was detected in intestinal tissue of C57BL/6 and SCID mice at 9–11 days after birth, but not in BALB/c mice. The IgA and IgG titers to MIT 01-6451 in sera of C57BL/6 female mice were significantly lower than those of BALB/c mice. Although no significant differences in the number of newborns per litter were observed between MIT 01-6451-infected and MIT 01-6451-free dams, the birth rate was lower in infected SCID mice than in control SCID mice. The present results indicated that MIT 01-6451 infects newborn mice after birth rather than by vertical transmission to the fetus via the placenta and that MIT 01-6451 infection shows opportunistically negative effects on the birth rate. In addition, the maternal immune response may affect infection of newborn mice with MIT 01-6451 through breast milk.
Traumatic and stressful events during childhood are associated with the development of eating disorders. We conducted an animal study to test if association stress in childhood affects ingestive behavior later in life by using female rats that have an adjusted estrous cycle. First, electrical impedance of the vagina was conducted to test estrous cycle adjustment. Second, the effects of 6 h per day maternal separation from birth to weaning, which models a psychologically stressful experience in childhood, was used to test feeding behavior during an ovarian cycle in female adult rats with matched estrous cycles. Food and water intake in maternal separated and non-separated rats was measured in each estrous phase. Non-separated rats showed periodical changes, but maternal separated rats showed no significant changes in food and water intake during an estrous cycle. An opposing tendency for food and water intake was seen between maternal separated and non-separated rats. These observations suggest that electrical impedance of the vagina showed the highest value in the estrous phase of rats housed in a reversed light-dark cycle, and maternal separation was found to disturb changes in feeding behavior during the estrous cycle.
Thiopental sodium (TPS) needs to be applied together with adrenalin in order to establish its analgesic effect in general anesthesia. We aimed to investigate the effect of TPS on the claw pain threshold in rats and evaluated its relationship with endogenous adrenalin (ADR), noradrenalin (NDR), and dopamine (DOP) levels. Intact and adrenalectomized rats were used in the experiment. Intact animals were divided into the following groups: 15 mg/kg TPS (TS), 0.3 mg/kg ADR+15 mg/kg TPS (ATS) and 0.3 mg/kg ADR alone (ADR). Adrenalectomized animals were divided into the following groups: 15 mg/kg TPS (A-TS), 0.3 mg/kg ADR+15 mg/kg TPS (A-ATS) and 0.3 mg/kg ADR alone (A-ADR). Claw pain threshold and blood ADR, NDR, and DOP levels were measured. The TS group’s claw pain threshold was found low. However, the claw pain thresholds of the ATS and ADR groups increased significantly. In the A-TS group, the pain threshold decreased compared with normal, and in the A-ATS and A-ADR groups, the pain threshold increased. TPS reduced the blood ADR levels in intact rats; however, no significant changes were observed in the NDR and DOP levels. #TPS provides hyperalgesia by reducing the production of ADR in rats. The present study shows that to achieve analgesic activity, TPS needs to be applied together with ADR.
Mammalian tissues are always exposed to diverse threats from pathological conditions and aging. Therefore, the molecular systems that protect the cells from these threats are indispensable for cell survival. A variety of diseases, including neurodegenerative diseases, cause intracellular damage and disturb homeostasis. Heat shock transcription factor 1 (HSF1) positively regulates heat shock protein (Hsp) and maintains the precise folding of proteins. Moreover, HSF1 induces the non-Hsp genes expression, and degrades damaged/misfolded protein. Recently, my colleagues and I revealed non-Hsp genes have more protective roles than Hsps at the cellular level. However, whether these protective systems are similarly important to cellular defense in each tissue is still elusive. In this study, I compared polyglutamine (polyQ) protein aggregations/inclusion development in each tissue of WT- and HSF1KO-Huntington’s disease (HD) mice, and examined the expression of the eight non-Hsp HSF1 target genes that have a strong suppressive effect on polyQ protein aggregation. Of these genes, Nfatc2, Pdzk3, Cryab, Csrp2, and Prame were detected in most tissues, but the other genes were not. Surprisingly, the obvious effect of HSF1 deficiency on the expression of these five genes was detected in only heart, spleen, and stomach. In addition, polyQ protein aggregations/inclusion was not detected in any tissues of WT-HD and HSF1KO-HD mice, but higher level of pre-aggregative polyQ protein was detected in HSF1KO-HD tissues. These results indicate non-Hsp genes are indispensable for the maintenance of intracellular homeostasis in mammalian tissues, resulting in whole body homeostasis.
We previously reported that LMO3 and HEN2 act as oncogenes in neuroblastoma development through up-regulating MASH1 transcription by interfering with HES1. To confirm these results in vivo, we generated transgenic mice of these genes. Lmo3 or Hen2 was expressed under the control of Wnt1 promoter, which is expressed in the central nervous system and neural crest of the sympathoadrenal lineage from which neuroblastoma develops. Heterozygous Lmo3 and Hen2 transgenic mice (Tg (Lmo3) and Tg (Hen2)) developed hydrocephalus at higher frequency than for the wild type mice, and all heterozygous double-transgenic mice (Tg (Lmo3; Hen2)) developed hydrocephalus. Therefore, Lmo3 and Hen2 may be involved in and have synergistic effects on hydrocephalus development. Although aqueduct stenosis occurred in all genotypes, it was mild in Tg (Lmo3; Hen2) mice. Furthermore, hydrocephalus was detected at E18.5 in Tg (Lmo3; Hen2). These results suggest that the causes of hydrocephalus are not only aqueduct stenosis but also disorder of neocortical development. A similar phenotype was reported in Robo1/2−/− mice, in which Hes1 expression level was decreased in ventricular zone progenitors. Thus, it is suggested that the expression levels of Lmo3 and/or Hen2 could determine the fate of stem cells by inhibiting Hes1 function during nervous system development and might be a trigger of aberrant neurogenesis in vivo.
Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.
Sarcopenia is an age-related systemic syndrome with progressive deterioration in skeletal muscle functions and loss in mass. Although the senescence-accelerated mouse P8 (SAMP8) was reported valid for muscular ageing research, there was no report on the details such as sarcopenia onset time. Therefore, this study was to investigate the change of muscle mass, structure and functions during the development of sarcopenia. Besides the average life span, muscle mass, structural and functional measurements were also studied. Male SAMP8 animals were examined at month 6, 7, 8, 9, and 10, in which the right gastrocnemius was isolated and tested for ex vivo contractile properties and fatigability while the contralateral one was harvested for muscle fiber cross-sectional area (FCSA) and typing assessments. Results showed that the peak of muscle mass appeared at month 7 and the onset of contractility decline was observed from month 8. Compared with month 8, most of the functional parameters at month 10 decreased significantly. Structurally, muscle fiber type IIA made up the largest proportion of the gastrocnemius, and the fiber size was found to peak at month 8. Based on the altered muscle mass, structural and functional outcomes, it was concluded that the onset of sarcopenia in SAMP8 animals was at month 8. SAMP8 animals at month 8 should be at pre-sarcopenia stage while month 10 at sarcopenia stage. It is confirmed that SAMP8 mouse can be used in sarcopenia research with established time line in this study.
Currently, the metabolic syndrome (MS) is occurring at growing rates worldwide, raising extensive concerns on the mechanisms and therapeutic interventions for this disorder. Herein, we described a novel method of establishing MS model in rodents. Male Institute of Cancer Research (ICR) mice were fed with high-fat-high-fructose (HFHF) diet or normal chow (NC) respectively for 12 weeks. Metabolic phenotypes were assessed by glucose tolerance test, insulin tolerance test and hyperinsulinemic-euglycemic clamp. Blood pressure was measured by a tail-cuff system. At the end of the experiment, mice were sacrificed, and blood and tissues were harvested for subsequent analysis. Serum insulin levels were measured by ELISA, and lipid profiles were determined biochemically. The HFHF diet-fed ICR mice exhibited obvious characteristics of the components of MS, including obvious obesity, severe insulin resistance, hyperinsulinemia, dislipidemia, significant hypertension and hyperuricemia. Our data suggest that HFHF diet-fed ICR mice may be a robust and efficient animal model that could well mimic the basic pathogenesis of human MS.