The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.
Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.
Pazopanib is a tyrosine kinase inhibitor that is generally used for the treatment of metastatic renal cell cancer and advanced soft tissue sarcoma. It can cause various degrees of hepatotoxicity. Our study aimed to investigate the effect of taxifolin on pazopanib-induced liver toxicity. A total of 18 rats were divided into three groups: the pazopanib (PP), pazopanib plus taxifolin (TPP), and control (C) group. Taxifolin was administered to the TPP (n=6) group with a dose of 50 mg/kg. Distilled water was orally admnistered to the C (n=6) and PP (n=6) groups as a solvent. Subsequently, pazopanib 200 mg/kg was administered to the TPP and PP groups via the stomach. This procedure was repeated once a day for four weeks. Then, all rats were sacrificed, and their livers were removed. Malondialdehyde (MDA), total glutathione (tGSH), total oxidant status (TOS), and total antioxidant status (TAS) levels were evaluated. MDA and TOS levels were higher in the PP group compared with the levels of the other parameters (P<0.001). tGSH and TAS levels were lower in the PP group than in the TPP and C groups (P<0.001), and the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were higher. Furthermore, liver tissue damage, including hemorrhage, hydropic degeneration, and necrosis was observed in the PP group. Administration of taxifolin before pazopanib significantly improved degenerative changes. Our study demonstrated that the administration of taxifolin is significantly effective in preventing pazopanib-induced hepatotoxicity in rats.
To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.
Despite decades-long existence of the Philippine stingless bee industry, the biological activity of propolis from this native bee species (Tetragonula biroi Friese) remains poorly understood and sparingly investigated. Herein, we examined the potential anti-inflammatory efficacy of Philippine stingless bee propolis using the lambda (λ)-carrageenan-induced mice model of hind paw edema. Thirty (30), six-week-old, male ICR mice were randomly assigned into three treatment groups (n=10/group) as follows: distilled water group, diclofenac sodium group (10 mg/kg), and propolis group (100 mg/kg). All treatment were administered an hour prior to the injection of the phlogistic agent. As observed at 3 h post-injection, λ-carrageenan remarkably evoked the classical signs of hind paw edema exemplified grossly by swelling and hyperemia. The ameliorative effect of propolis became apparent at the onset of 6 h post-injection with a statistically significant finding evident at the 24-h period. This gross attenuation histologically correlated to a considerable and specific reduction of the dermal edema, which mirrored those of the diclofenac sodium group. Furthermore, both propolis and diclofenac sodium significantly attenuated the λ-carrageenan-induced increase in the protein expression levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) depicting more than two-fold decrement relative to the distilled water group. Altogether, these suggest that Philippine stingless bee propolis also exhibited a promising in vivo anti-inflammatory property, which can be partly mediated through the inhibition of TNF-α.
One of the leading risk factors for atherosclerosis is obesity, which is commonly caused by a nutrient-rich Western-style diet, sedentary behaviors, and shift work. Time-restricted (TR) feeding and intermittent fasting are both known to prevent overweight and adiposity, improve glucose tolerance, and decrease plasma cholesterol in high-fat diet-induced obese mice. Here we examined the overall effects of TR feeding of a Western diet (fat, 40.5 Kcal%; cholesterol, 0.21 g%) using 8-week-old Apoe−/− mice. Mice were assigned into three groups: (1) an ad libitum (AL) group fed an AL Western diet, (2) a TR group with restricted access to a Western diet (15 h/day, 12:00 to 3:00 Zeitgeber time [ZT]); and (3) an Ex/TR group fed a TR Western diet and subjected to physical exercise at 12:00 ZT. Mice in the AL group gained body weight rapidly during the 14-week observation period. With TR feeding, excessive weight gain, liver adiposity, visceral fat, and brown adipose tissue volume were effectively suppressed. Although TR feeding failed to decrease Oil Red O-stained aortic plaques in Apoe−/− mice, physical exercise significantly decreased them. Neither TR feeding with exercise nor that without exercise decreased the mean area under the curve of the plasma cholesterol level or the fasting plasma glucose. Collectively, TR feeding of a Western diet prevented the development of obesity but failed to ameliorate atherosclerosis in Apoe−/− mice.
This study aimed to investigate the association between microbiota found in the maternal gut and placenta, and whether ceftriaxone exposure during pregnancy could alter these microbiota, and consequently affect the immunity of the mothers and their offspring. The microbiota in the feces and placenta of the dams were comprehensively analyzed using16S rRNA sequencing. Furthermore, viable bacteria in the placentas and blood of pups were also isolated by plate cultivation then taxonomically identified in detail by clone sequencing. Serum cytokines collected from dams and pups were quantitatively profiled using Luminex. The spleen organ index of dams was significantly lower and the offspring serum interleukin-6 levels were significantly higher in ceftriaxone-treated mice compared with the control group. The maternal fecal microbiota community was drastically altered in ceftriaxone-treated mice with significantly decreased diversity, depletion of Bacteroidetes and the blooming of Tenericutes. However, the placenta microbiota was dominated by Proteobacteria especially characteristically by Ralstonia, which was distinct from the maternal gut microbiota, regardless of whether ceftriaxone treatment or not. Viable bacteria have been found in placenta and blood cultures. These results indicated that ceftriaxone exposure in pregnancy could dramatically alter maternal intestinal microbiota, which affected the immunity of the mothers and their offspring at least partly, characteristically by enhanced pro-inflammatory responses. This study also indicated that the placenta might harbor its own microbes and the microbes were distinct from maternal gut microbiota, which may not be affected by oral administration of ceftriaxone during pregnancy.
Both cholinergic and adrenergic stimulation can induce sweat secretion in human eccrine sweat glands, but whether cholinergic and adrenergic stimulation play same roles in rat eccrine sweat glands is still controversial. To explore the innervations, and adrenergic- and cholinergic-induced secretory response in developing and developed rat eccrine sweat glands, rat hind footpads from embryonic day (E) 15.5–20.5, postanal day (P) 1–14, P21 and adult were fixed, embedded, sectioned and subjected to immunofluorescence staining for general fiber marker protein gene product 9.5 (PGP 9.5), adrenergic fiber marker tyrosine hydroxylase (TH) and cholinergic fiber marker vasoactive intestinal peptide (VIP), and cholinergic- and adrenergic-induced sweat secretion was detected at P1–P21 and adult rats by starch-iodine test. The results showed that eccrine sweat gland placodes of SD rats were first appeared at E19.5, and the expression of PGP 9.5 was detected surrounding the sweat gland placodes at E19.5, TH at P7, and VIP at P11. Pilocarpine-induced sweat secretion was first detected at P16 in hind footpads by starch-iodine test. There was no measurable sweating when stimulated by alpha- or beta-adrenergic agonists at all the examined time points. We conclude that rat eccrine sweat glands, just as human eccrine sweat glands, co-express adrenergic and cholinergic fibers, but different from human eccrine sweat glands, cholinergic- rather than adrenergic-induced sweating plays a role in the developing and developed rat eccrine sweat glands.
Malignant peritoneal mesothelioma (MPM) is a rare malignancy with few experimental models. This study used the human surgical specimen to establish MPM patient-derived xenograft (PDX) models and primary cell lines to provide a study platform for MPM in vitro and in vivo, and conducted histopathological analysis. Our study used the experimental peritoneal cancer index (ePCI) score to evaluate gross pathology, and the results showed that the ePCI score of the female and male nude mice were 8.80 ± 1.75 and 9.20 ± 1.81 (P=0.6219), respectively. The Hematoxylin and eosin (HE) staining of animal models showed that the tumor was epithelioid mesothelioma and invaded multiple organs. Immunohistochemistry (IHC) staining showed that Calretinin, Cytokeratin 5/6, WT-1 and Ki-67 were all positive. The Swiss-Giemsa and Immunofluorescence (IF) staining of primary cell lines were also consistent with the pathological characteristics of mesothelioma. We also performed the whole-exome sequencing (WES) to identify the mutant genes between models and the patient. And the results showed that 21 mutant genes were shared between the two groups, and the genes related to tumorigenesis and development including BAP1, NF2, MTBP, NECTIN2, CDC23, LRPPRC, TRIM25, and DHRS2. In conclusion, the PDX models and primary cell lines of MPM were successfully established with the epithelioid mesothelioma identity confirmed by histopathological evidence. Moreover, our study has also illustrated the shared genomic profile between models and the patient.
Clarification of the criteria for managing animal health is essential to increase the reliability of experiments and ensure transparency in animal welfare. For experiments performed in space, there is no consensus on how to care for animals owing to technical issues, launch mass limitation, and human resources. Some biological processes in mammals, such as musculoskeletal or immune processes, are altered in the space environment, and mice in space can be used to simulate morbid states, such as senescence acceleration. Thus, there is a need to establish a novel evaluation method and evaluation criteria to monitor animal health. Here, we report a novel method to evaluate the health of mice in space through a video downlink in a series of space experiments using the Multiple Artificial-gravity Research System (MARS). This method was found to be more useful in evaluating animal health in space than observations and body weight changes of the same live mice following their return to Earth. We also developed criteria to evaluate health status via a video downlink. These criteria, with “Fur condition” and “Respiratory” as key items, provided information on the daily changes in the health status of mice and helped to identify malfunctions at an early stage. Our method and criteria led to the success of our missions, and they will help establish appropriate rules for space experiments in the future.
Reproducibility in animal research is crucial for its reliance and translational relevance. The 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced model of inflammatory bowel disease (IBD) is widely used but inconsistently and incompletely characterized throughout the literature. This hinders comparisons between studies and influences the low rate of translation of effective preclinical molecules. The purpose of this study was to categorize TNBS-induced colitis, based on macroscopic and microscopic scoring systems, and to identify basic routine parameters that could anticipate those categories. We retrospectively analysed male Wistar Rattus norvegicus (n=28 for the control group and n=87 for the TNBS group) and categorized TNBS-induced colitis in three phenotypes: Mild, Moderate and Severe colitis, as for human IBD. Also, we showed that the time course of food intake and fecal excretion (but not body weight, fluid intake or welfare scores) could foresee those categories. So, routine evaluation of food intake and fecal excretion may guide researchers in planning their experiments, selecting the animals with the severity of colitis that better matches their aims, or applying early humane endpoints to animals that will not be used in the experiments. In conclusion, categorizing TNBS-induced colitis enhances the reproducibility of data gathered with this experimental model and strengths its translational relevance.
A reproducible swine thoracic aortic aneurysm (TAA) model is useful for investigating new therapeutic interventions. We report a surgical method for creating a reproducible swine saccular TAA model. We used eight female swine weighing 20–25 kg (LWD; ternary species). All procedures were performed under general anesthesia and involved left thoracotomy. Following aortic cross-clamping, the thoracic aorta was surgically dissected and the media and intima were resected, and the dissection plane was extended by spreading the outer layer for aneurysmal space. Subsequently, only the adventitial layer of the aorta was sutured. At 2 weeks after these procedures, angiography and computed tomography were performed. After follow-up imaging, the model animals were euthanized. Macroscopic, histological, and immunohistological examinations were performed. All model animals survived, and a saccular TAA was confirmed by follow-up imaging in all cases. The mean length of the shorter and the longer aortic diameter after the procedure were 14.01 ± 1.0 mm and 18.35 ± 1.4 mm, respectively (P<0.001). The rate of increase in the aortic diameter was 131.7 ± 13.8%, and the mean length of aneurysmal change at thoracic aorta was 22.4 ± 1.9 mm. Histological examination revealed intimal tears and defects of elastic fibers in the media. Immunostaining revealed MMP-2 and MMP-9 expressions at the aneurysm site. We report our surgical method for creating a swine saccular TAA model. Our model animal may be useful to investigate new therapeutic interventions for aortic disease.