The Center for Animal Resources and Development (CARD), Kumamoto University was established in 1998. We provide advanced research support services for the mouse-based biomedical research community via an official and a premium mouse bank system. To efficiently manage these mouse banks, we have actively developed and modified basic mouse reproductive techniques. We shall introduce these techniques in this paper.
Visual impairment leads to a decrease in quality of life. Cataract is the most commonly observed ocular disease in humans that causes vision disorders. The risk factors associated with cataract development include aging, infections, eye injuries, environmental causes, such as radiation and exposure to ultraviolet rays in sunlight, and genetic mutations. Additionally, several cataract patients display phenotypic heterogeneity, suggesting the role of genetic modifiers in the modulation of severity and onset time of cataractogenesis. However, the genetic modifiers associated with cataract have not been identified in humans yet. In contrast, the identification and mapping of genetic modifiers have been successfully carried out in mice and rats. In this review, we focus on the genetic modifiers of cataract in the rodent models.
Metabolic syndrome is a cluster including hyperglycaemia, obesity, hypertension, and hypertriglyceridaemia as a result of biochemical and physiological alterations and can increase the risk of cardiovascular disease and diabetes. Fundamental research on this disease requires validated animal models. One potential animal model that is rapidly gaining in popularity is zebrafish (Danio rerio). The use of zebrafish as an animal model conveys several advantages, including high human genetic homology, transparent embryos and larvae that allow easier visualization. This review discusses how zebrafish models contribute to the development of metabolic syndrome studies. Different diseases in the cluster of metabolic syndrome, such as hyperglycaemia, obesity, diabetes, and hypertriglyceridaemia, have been successfully studied using zebrafish; and the model is promising for hypertension and cardiovascular metabolic-related diseases due to its genetic similarity to mammals. Genetic mutation, chemical induction, and dietary alteration are among the tools used to improve zebrafish models. This field is expanding, and thus, more effective and efficient techniques are currently developed to fulfil the increasing demand for thorough investigations.
The changes in free amino acid (AA) levels in blood during the progression from non-alcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) are unclear. We investigated serum AA levels, along with biochemical and histological events, in a mouse model of NASH. We induced NASH in male C57BL/6J mice with a streptozotocin injection and high-fat diet after 4 weeks of age (STAM group). We chronologically (6, 8, 10, 12, and 16 weeks, n=4–12 mice/group) evaluated the progression from steatohepatitis to HCC by biochemical and histological analyses. The serum AA levels were determined using an AA analyzer. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in the STAM group than in the normal group (non-NASH-induced mice). Histological analysis revealed that STAM mice had fatty liver, NASH, and fibrosis at 6, 8, and 10 weeks, respectively. Moreover, the mice exhibited fibrosis and HCC at 16 weeks. The serum branched-chain AA levels were higher in the STAM group than in the normal group, especially at 8 and 10 weeks. The Fischer ratio decreased at 16 weeks in the STAM group, with increasing aromatic AA levels. These results suggested that this model sequentially depicts the development of fatty liver, NASH, cirrhosis, HCC, and AA metabolism disorders within a short experimental period. Additionally, serum amyloid A was suggested to be a useful inflammation biomarker associated with NASH. We believe that the STAM model will be useful for studying AA metabolism and/or pharmacological effects in NASH.
The body surface area (BSA) of animals has generally been estimated by multiplying the k value by the measured body weight (BW) raised to the power of 2/3 (Meeh’s formula). In mathematical terms, the assumption that the density and body shape of animals are essentially constant means that the BSA is proportional to BW2/3. In this study, we measured the BSA and volume of 50 male Japanese White rabbits (JW) at 10 to 54 weeks of age using a computed tomography scanner, then calculated the k value, density, and sphericity. The variations in these parameters were then analyzed in relation to growth. The obtained results indicated that the k value was negatively correlated to BW, and that this negative correlation was mainly due to the variation in density. Based on the regression analysis, we propose the following linear regression equation for calculating the k value in male JW at 10–54 weeks of age: the k value = 14.602 − 0.959 × BW [kg]. The calculated values ranged from 10.9 to 12.7 (working BW range: 1.98 to 3.81 kg). The k value of male New Zealand White rabbits (NZW) determined in our previous study and that of male JW in the present study were compared. It was revealed that the k value of male JW was larger than that of male NZW. We concluded that different breeds of rabbits express different k values.
Mammalian postnatal growth is regulated primarily by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. MafB is a basic leucine zipper (bZip) transcription factor that has pleiotropic functions. Although MafB plays a critical role in fetal brain development, such as in guidance for hindbrain segmentation, its postnatal role in neurons remains to be elucidated. To investigate this, we used neuron-specific Mafb conditional knockout (cKO) mice. In addition to an approximately 50% neonatal viability, the Mafb cKO mice exhibited growth retardation without apparent signs of low energy intake. Notably, serum IGF-I levels of these mice in the postnatal stage were lower than those of control mice. They seemed to have a neuroendocrine dysregulation, as shown by the upregulation of serum GH levels in the resting state and an inconsistent secretory response of GH upon administration of growth hormone-releasing hormone. These findings reveal that neuronal MafB plays an important role in postnatal development regulated by the GH/IGF-I axis.
Medetomidine (MED), midazolam (MID), and butorphanol (BUT) mixed anesthetic (MMB) has been used in laboratory animals since ketamine (KET) was designated as a narcotic in Japan in 2007. We previously reported that MMB produced anesthetic effects in mice and rats. We also demonstrated the efficacy of atipamezole (ATI), an antagonist of MED produced a quick recovery from anesthesia. Anesthetics have various anesthetic effects among different animal species. However, there is little information regarding its effects in rabbits. In the present study, we examined anesthetic effects of MMB compared to KET and xylazine mixed anesthetic (KX). We examined the antagonistic effects of ATI by intramuscular (IM) or intravenous (IV) injection in rabbits. We used the anesthetic score to measure surgical anesthetic duration and recovery time from anesthesia. During the experiments, we measured heart rate, respiratory rate, O2-saturation, and blood pressure. We found there were no significant differences in anesthetic duration and recovery time between MMB and KX. There were no significant differences in heart rate after administration of MMB or KX. Systolic blood pressure at 10 min after administration of MMB was higher than that of KX. The antagonistic effect of ATI by IV injection worked faster than that by IM injection. Overall, MMB is a useful drug that can induce similar anesthetic effects to KX and has an antagonist of ATI that makes rabbits quickly recover from anesthesia. These results may contribute to the welfare of laboratory animals, especially rabbits.
Osteoporosis is a common skeletal disorder resulting in elevated fracture risk. Improvement of osteogenic differentiation is thought to be the top priority in osteoporosis treatment projects. Significant characteristics of bone marrow mesenchymal stem cells (BMMSCs), especially attractive ability to differentiate into osteoblasts, have made them alternatives for osteoporosis treatment. However, therapeutic effect with BMMSCs remains to be improved. Here, osthole, a bioactive simple coumarin derivative extracted from many medicinal plants, was introduced to pre-stimulate BMMSCs and then applied in osteoporosis therapy. The results showed that osthole-treated-BMMSCs (OBMMSCs) brought a better outcome than BMMSCs alone in estrogen deficiency-induced osteoporosis model. And elevated autophagy level was suggested to be the underlying mechanism of the ability of osthole to promote osteoblast differentiation, which is indicated by the upregulation of protein and mRNA expression level of autophagy-associated genes, Beclin1 and LC3. We concluded from these experiments that OBMMSCs are more effective than BMMSCs in osteoporosis treatment maybe through upregulation level of autophagy level induced by osthole.
The formation of the caudal vena cava is a complex process involving development, regression, and anastomosis. In mammals, the normal caudal vena cava runs to the right side of the abdominal aorta, while duplication of the caudal vena cava has been identified as a congenital abnormality in both companion animals and humans. The present study demonstrates that Slc:Hartley guinea pigs frequently possess asymptomatic duplicated caudal vena cava. The prevalence was 30% and 24% for males and females, respectively, with no sex-related differences. In accordance with Saad et al. (2012)’s criteria, duplicated caudal vena cava were classified into two distinct variations. The dominant variation was a complete duplication without iliac anastomosis where the left caudal vena cava continued from the left common iliac vein and joined the left renal vein; the left renal vein ran to the right to join the right caudal vena cava. The alternative variation was an incomplete duplication where the left caudal vena cava joined the right infrarenal caudal vena cava at a more cranial point than in normal cases; the renal segment was unchanged. Iliac anastomosis was not found in any cases. Duplicated caudal vena cava neither affected the body weight nor the kidney weight. In conclusion, Slc:Hartley guinea pigs frequently possess asymptomatic duplicated caudal vena cava in the absence of iliac anastomosis and appear to be a novel and useful animal model for duplicated caudal vena cava in animals and humans.
Biological background data up to 11 weeks of age and tumorigenic susceptibility to xenotransplantation with HeLa cells were compared between severely immuno-deficient NOG and NSG mice. The body weight was lower in NOG mice than in NSG mice. Severe depletion of peripheral blood lymphocytes and lymphoid hypoplasia that are well-known characteristics of these mice were equally observed. No lymphoproliferative lesions developed in any mouse of either strain. The occurrence of ectopic exocrine gland and cyst was a common finding in the thymus of both strains. In addition, minimal spongiotic change was observed in the medulla oblongata and spinal cord in both strains, and its incidence in female NOG mice was a little higher than that in NSG mice. In the adrenal, subcapsular cell hyperplasia that is known as an age-related change in non-genetically modified mice developed earlier and its incidence was higher in NSG mice than in NOG mice. The development of female genital organs of NOG mice was slightly retarded in comparison with that of NSG mice. To evaluate tumorigenic susceptibility to xenotransplantation, female mice were implanted in the dorsal subcutis with 1×103 to 1×106 cells/head of HeLa cells, and were checked up to 16 weeks after implantation. As a result, there was no significant strain difference on tumor formation rate and tumor volume. In conclusion, the present study clearly demonstrated that NOG and NSG mice showed no distinct strain differences in either biological features or biological disadvantages.
IL-17-producing T helper cells (Th17) are attracting attention as a new CD4-positive subset of T cells, reported to be responsible for various autoimmune diseases through stimulation of the release of inflammatory cytokines from target cells. However, most investigations of Th17 mediation of autoimmune diseases have focused on the experimental autoimmune models derived from young animals, with few studies that have analyzed physiological factors such as aging. The present study analyzed autoreactive T cells established in a syngeneic mixed lymphocyte culture (sMLC) from aged mice and examined their similarity with Th17. IL-17-producing autoreactive CD4-intermediate T cells were observed in the sMLC; these expressed several stem cell markers or an immunosuppressive receptor PD-1 on the cell surface and so seemed to be different to typical Th17 cells. RT-PCR analysis revealed that purified Th17-like cells also expressed Il17a, Il17f, Il23r, Rorc and Tdt mRNA, but not Rag1 or Rag2 mRNA. These findings that it is likely that Th17-like cells are involved in autoimmune responses in aged mice.
T helper 17 (Th17) cells have been suggested to play a crucial role in various complications during pregnancy by participating in maternal immune activation (MIA). To test a possible role for Th17 cells in MIA-mediated abortion, we analyzed transgenic mice overexpressing retinoic acid receptor-related orphan receptor gamma-t (RORγt), a master regulator of IL-17 producing cell development. These mutant mice (RORγt Tg mice) exhibited a constitutive upregulation of serum IL-17A and decreased E-cadherin expression in cell–cell junctions of placental tissues. Abortion after the administration of a viral-mimicking synthetic double-stranded RNA polyinosinic–polycytidylic acid was more frequent in RORγt Tg mice than wild-type mice. These results suggest that excessive Th17 cell activity alters immune responsiveness and increases the rate of abortion during gestation.
Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5–8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.
The senescence-accelerated mouse (SAM) strain has been established as an inbred strain with an accelerated aging phenotype. SAM prone-8 (SAMP8), one of the SAM strain, exhibits learning disability, immune deficiency, and circadian rhythm loss at a relatively young age. However, it has not been clarified whether aging affects the autonomic nervous activity in SAMP8. The aim of this study was to clarify the utility of SAMP8 in age-related studies of autonomic nervous function. Electrocardiogram (ECG), body temperature, and locomotor activity were recorded to evaluate bio-behavioral activities. Autonomic nervous activity was evaluated via power spectral analysis of heart rate variability from ECG recordings. SAMP8 significantly decreased both biological and autonomic nervous functions, and the animals exhibited circadian rhythm loss of locomotive activity at as early as 40 weeks of age compared with a control strain at the same age. We concluded that the SAMP8 strain can be used as an animal model for age-related studies of autonomic nervous function.
The nuclear receptor corepressor Hairless (HR) interacts with nuclear receptors and controls expression of specific target genes involved in hair morphogenesis and hair follicle cycling. Patients with HR gene mutations exhibit atrichia, and in rare cases, immunodeficiency. Pigs with HR gene mutations may provide a useful model for developing therapeutic strategies because pigs are highly similar to humans in terms of anatomy, genetics, and physiology. The present study aimed to knockout the HR gene in pigs using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated-9 (Cas9) system and to investigate the molecular and structural alterations in the skin and thymus. We introduced a biallelic mutation into the HR gene in porcine fetal fibroblasts and generated nine piglets via somatic cell nuclear transfer. These piglets exhibited a lack of hair on the eyelids, abnormalities in the thymus and peripheral blood, and altered expression of several signaling factors regulated by HR. Our results indicate that introduction of the biallelic mutation successfully knocked out the HR gene, resulting in several molecular and structural changes in the skin and thymus. These pigs will provide a useful model for studying human hair disorders associated with HR gene mutations and the underlying molecular mechanisms.
The Asian house shrew, Suncus murinus, is an insectivore (Eulipotyphla, Mammalia) and an important laboratory animal for life-science studies. The gastrointestinal tract of Suncus is simple: the length of the entire intestine is very short relative to body size, the large intestine is quite short, and there are no fermentative chambers such as the forestomach or cecum. These features imply that Suncus has a different nutritional physiology from those of humans and mice, but little is known about whether Suncus utilizes microbial fermentation in the large (LI) or small (SI) intestine. In addition, domestication may affect the gastrointestinal microbial diversity of Suncus. Therefore, we compared the gastrointestinal microbial diversity of Suncus between laboratory and wild Suncus and between the SI and LI (i.e., four groups: Lab-LI, Lab-SI, Wild-LI, and Wild-SI) using bacterial 16S rRNA gene library sequencing analyses with a sub-cloning method. We obtained 759 cloned sequences (176, 174, 195, and 214 from the Lab-LI, Lab-SI, Wild-LI, and Wild-SI samples, respectively), which revealed that the gastrointestinal microbiota of Suncus is rich in Firmicutes (mostly lactic acid bacteria), with few Bacteroidetes. We observed different bacterial communities according to intestinal region in laboratory Suncus, but not in wild Suncus. Furthermore, the gastrointestinal microbial diversity estimates were lower in laboratory Suncus than in wild Suncus. These results imply that Suncus uses lactic acid fermentation in the gut, and that the domestication process altered the gastrointestinal bacterial diversity.
Dietary antioxidant supplements such as L-glutathione have gained considerable attention in dermatology and cosmeceutical fields. L-glutathione possesses antiaging, antimelanogenic, antioxidant, and anticancer properties. This study aimed to investigate the inhibitory effects of L-glutathione on melanogenesis activity and oxidative stress in ultraviolet B (UVB)-irradiated BALB/c mice. Eighteen female BALB/c mice were randomly divided into 3 groups: a control group (n=6), a group without UVB irradiation and L-glutathione administration; a UVB irradiated group (n=6), a group irradiated with a UVB dose of 250 mJ/cm2 for 3 min; and a treatment group (n=6), a group irradiated with UVB and treated with 100 mg/kg of L-glutathione by oral gavage. Treatment was given for 14 days, and UVB irradiation was given on days 9, 11, and 13. Oral L-glutathione significantly (P<0.05) reduced lipid peroxidation and elevated superoxide dismutase activity the and glutathione level. L-glutathione also inhibited melanin content and tyrosinase activity significantly (P<0.05) as compared with the UVB-irradiated group. Histopathological examination also showed that L-glutathione reduced the deposition of melanin pigment in the basal layer of the epidermis as compared with that in UVB-irradiated mice. All in all, the present study demonstrated that L-glutathione has the potential to be developed as a photoprotection agent against UVB-induced oxidative stress and melanogenesis.
Apoptosis plays a crucial role in regulating cardiomyopathy and injuries of coxsackievirus B3 (CVB3)-induced viral myocarditis (VM). It has been reported that Astragaloside IV (AST-IV) from Astragalus membranaceus could inhibit apoptosis under a variety of pathological conditions in vivo or in vitro. However, the functional roles of AST-IV in CVB3-induced VM still remain unknown. Here, we found that AST-IV significantly enhanced survival for CVB3-induced mice. AST-IV protected the mice against CVB3-induced virus myocarditis characterized by the increased body weight, decreased serum level of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), supressed expression of Ifn-γ, Il-6 in heart, enhanced systolic and diastolic function of left ventricle. At the pathological level, AST-IV ameliorated the mice against CVB3-induced myocardial damage and myocardial fibrosis. In vitro, the results from flow cytometry showed that AST-IV significantly suppressed CVB3-induced cardiomyocytes apoptosis, which also were verified in vivo. Moreover, an increased expression of pro-apoptotic genes including FAS, FASL, cleaved caspase-8 and cleaved caspase-3 was found in CVB3-induced cardiomyocytes, while those was inhibited in cardiomyocytes treated with AST-IV. Taken together, the data suggest that AST-IV protected against CVB3-induced myocardial damage and fibrosis, which may partly attribute to supress activation of FAS/FASL signaling pathway.
An uncontrolled inflammation induced critical health problems with serious morbidity and death, which namely acute lung injury (ALI). Recently researchs have found the anti-inflammatory effects of emodin. Here, we investigated the potential effects of emodin on a mouse model with a lethal dose of the potential mechanisms and lipopolysaccharide (LPS)-induced inflammatory lung injury in mice. The pulmonary histological abnormalities, the Evans blue’s leakage, the myeloperoxidase (MPO) activity, the grades of TNF-α, IL-6, nitric oxide (NO), lactic acid (LA) in lung tissues were determined 18 h post exposure of LPS. Based on the expression of LC3-II with BECN1 was determined using Western blotting. Besides, the LPS-exposed mice for survival rate was monitored. The results indicated that intervention with emodin was important for mitigating LPS-induced pulmonary histological change and LPS-induced leakage of Evans blue, which were associated with suppressed elevation of MPO activity and inhibited up-regulation of TNF-α, IL-6, NO with LA in lung tissues. Moreover, intervention with emodin enhanced the survival rate of LPS-exposed mice. Finally, therapy with emodin increased the LC3 and BECN1 in lungs of LPS-exposed mice. Treatment with 3-MA (the autophagy inhibitor) reversed the beneficial effects of emodin. In conclusion, emodin might provide pharmacological benefits in LPS-induced inflammatory lung injury, and the mechanisms might be related to the restoration of autophagy.
Studies of environmental enrichment are progressing in the fields of nervous system, stress and exercise. Recently, housing in enriched environment have shown to influence to carcinogenesis and life span. However, the study for antitumor effect of environmental enrichment are difficult to reproduce due to the complexity of the experimental technique. Thus, a simpler experiment system is needed for antitumor study using environmental enrichment. In this research, we propose a minimum environmental enrichment, which is an experimental system by placing one mouse igloo which is normally used as a mouse shelter in the rearing environment. The experimental system of minimum environmental enrichment is not only easy to reproduce but also have enhanced activity to suppress the growth of transplanted tumor significantly. It was found that the activation of NK cells is involved also in the immune system related to tumor immunity of minimum environmental enrichment. Because minimum environmental enrichment is effective in activating antitumor immunity to transplanted tumor cells in mice, we believe this will be useful for promoting antitumor studies using environmental enrichment.