Cyclosporine A (CsA) is used in hematopoietic stem cell transplantations (HSCT) to prevent graft-versus-host disease (GvHD). GvHD is the most severe side effect of allogeneic HSCT and efficient therapies are lacking. Mouse models are an essential tool for assessing potential new therapeutic strategies. Our aim is to mimic a clinical setting as close as possible using CsA treatment after sublethal irradiation in NSG mice and thereby evaluate the feasibility of this mouse model for GvHD studies. The effect of CsA (7.5 mg/kg body weight) on sublethally X-ray irradiated (2 Gy) and non-irradiated NSG mice was tested. CsA was administered orally every twelve hours for nine days. Animals irradiated and treated with CsA showed a shorter survival (n=3/10) than irradiated animals treated with NaCl (n=10/10). Furthermore, combined therapy resulted in severe weight loss (82 ± 6% of initial weight, n=7, day 8), with weight recovery after the CsA application was ceased. A high number of apoptotic events in the liver was observed in these mice (0.431 ± 0.371 apoptotic cells/cm2, n=2, compared to 0.027 ± 0.034 apoptotic cells/cm2, n=5, in the non-irradiated group). Other adverse effects, including a decrease in white blood cell counts were non-CsA-specific manifestations of irradiation. The combination of CsA treatment with irradiation has a hepatotoxic and lethal effect on NSG mice, whereas the treatment without irradiation is tolerated. Therefore, when using in vivo models of GvHD in NSG mice, a combined treatment with CsA and X-ray irradiation should be avoided or carefully evaluated.
Use of transponders, small electronic identification devices, in experimental swine is expected to be more reliable than the current common use of ear tags. However, it is necessary to determine the optimal implantation site for transponders with high readability, retentionability (i.e., long-term retention in tissues without detachment or loss), and biocompatibility, as this has not yet been investigated. Thus, we aimed to determine the optimal implantation site. Two types of transponders were subcutaneously implanted into four different sites (ear base, ear auricle, ventral neck, and back) in 3 domestic swine each. The transponders were scanned at 1, 2, 3, and 84 days after implantation. The location of the transponders was examined by X-ray and echography at 84 days. Histopathological examinations were performed at 84 days. The transponders in the back were successfully scanned in a shorter time than those in other implantation sites, without any re-scanning procedures. X-ray examination revealed one transponder in the ventral neck was lost, whereas those in the other sites were retained in their original location for 84 days. Echography indicated that the transponders in the back were retained more deeply than those in other implantation sites, suggesting better retentionability. Acceptable biocompatibility was confirmed in all implantation sites, as evidenced by the finding that all transponders were covered by a connective tissue capsule without severe inflammation. In conclusion, the present results demonstrated that the back is the optimal implantation site for transponders in experimental swine.
The CRISPR/Cas9 system can efficiently introduce biallelic mutations in ES cells (ESCs), and its application with fluorescently-tagged ESCs enables phenotype analysis in chimeric mice. We have utilized ESCs that express EGFP in the cytosol and acrosome [EGR-G101 129S2 × (CAG/Acr-EGFP) B6] in previous studies; however, the EGFP signal in the sperm cytosol is weak and the signal in the acrosome is lost after the acrosome reaction, precluding analysis between wild type and ESC derived spermatozoa. In this study, we established an ESC line from RBGS (Red Body Green Sperm) transgenic mice [B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb] whose spermatozoa exhibit green fluorescence in the acrosome and red fluorescence in the mitochondria within the flagellar midpiece that is retained after the acrosome reaction. We utilized these new ESCs to analyze HYDIN, which is reported to function in sperm motility in humans. Analysis of Hydin-disrupted spermatozoa in mice is difficult as Hydin-mutant mice (hy3) die within 3 weeks, before sexual maturation, due to hydrocephaly. To circumvent the early lethality of the whole-body knockout, we disrupted Hydin in RBGS-ESCs and generated chimeric mice, which survived into sexual maturity. Hydin-disrupted spermatozoa obtained from the chimeric mice possessed short tails and were immotile. When we injected Hydin-disrupted spermatozoa into oocytes, heterozygous pups were obtained, which suggests that the genome of Hydin-disrupted spermatozoa can produce viable pups. Consequently, RBGS-ESCs can be a useful tool for screening and analysis of male-fertility related genes in chimeric mice.
Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells (iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT-3/4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body (EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51 ± 1.48%) on day 5 and cardiac troponin-T protein (10.29 ± 1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2), α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.
A relationship between coat color and behavioral characteristics has been reported for numerous species. We previously indicated that particular behavioral traits contributing to the genotype at the agouti locus manifest only when possessing a wild-type allele at the albino (i.e., tyrosinase: Tyr) locus. The present study was performed to investigate tyrosinase expression with marked activity in central nervous systems. The whole brain of male B10 and B10-c mice, a B10 congenic strain of the albino locus from BALB/c, at 8 to 9 weeks of age was removed, and obtained several regions of brain, especially catecholaminergic. Comparatively large amounts of Tyr mRNA and its translation products of approximately 68 kDa were found in the regions obtained, and definitely possessed the enzyme activity for the oxidation of L-tyrosine. The present results indicate the possibility that the amount of catecholamines produced in albino mice is higher than that of colored mice due to the deficit in tyrosinase heritably.
Basic research on obesity is becoming more important due to an increasing number of obese people. Experiments using obesity-model animals often require surgical interventions, such as gastric operation, and proper selection of anesthesia is important. Avertin, an agent mainly composed of 2,2,2-Tribromoethanol, has been used as general anesthesia for a long time, without the use of narcotic drugs. In the current study, we found that a single injection of avertin can decrease body weight (BW) in male and female C57BL/6J and ICR mice with high fat-diet (HFD)-induced obesity, but not in standard diet-fed nonobese males and females. Because the BW-reducing effect was more prominent in the female mice, we compared the effects of avertin and a mixture of three types of anesthetic agents (3MIX), which was developed in 2011, on BW reduction in HFD-induced obese female mice. Although both avertin and 3MIX decreased food intake and BW, the effects of avertin were significantly more potent than those of 3MIX. C-Fos expression, a neural activation marker, was dramatically increased in the brain regions related to the regulation of both food intake and the autonomic nervous system after avertin injection, but not after 3MIX injection. This suggests that avertin strongly stimulates the center of feeding regulation and the autonomic nervous system and therefore decreases BW. The current study suggests the advantages of using 3MIX for surgical interventions in mice in obesity research, as it is ideal to prevent anesthesia-induced BW decline.
This article presents an experimental preparation for establishing conditioned food aversion (CFA) by voluntary wheel running in rats with laboratory chow and water freely available. In Experiment 1, unfamiliar food (raisins) was avoided by rats when they first encountered it. This neophobic food avoidance was habituated by repeated tests; the rats gradually increased their raisin consumption. However, the consumption remained suppressed in rats that accessed the raisins after wheel running. This finding implies that running yielded CFA, which suppressed consumption of the unfamiliar food rather than increasing it. Because running generated kaolin clay ingestion, which is a behavioral marker of nausea, it is suggested that the running-based CFA was mediated by weak gastrointestinal discomfort. Experiment 2 supported the claim that the suppressed consumption is due to running-based CFA by showing the specificity of food suppression. Demonstration of CFA based on voluntary activity in non-deprived rats will contribute to basic research on learning and memory as an alternative technique for studying aversive conditioning with minimized discomfort in animals.
Percutaneous coronary intervention (PCI) is main treatment for acute coronary syndrome (ACS). However, restenosis caused by PCI-induced injury influences the outcome of patients. Linagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has been reported to ameliorate intimal hyperplasia post vascular injury. The underlying mechanisms by which linagliptin protects against balloon injury are unclear and require to be explored. Herein, Wistar rats with carotid artery balloon injury were given 1, 2 or 3 mg/kg/day linagliprin for 6 weeks. We found that linagliptin attenuated vascular injury-mediated neointima formation in rats without affecting body weight and blood glucose levels. ELISA results indicated that linagliptin significantly reduced overproduction of cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 post balloon injury. By detecting the level of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), we found that linagliptin prevented balloon injury-induced oxidative stress. Additionally, linagliptin decreased the level of Kelch ECH-associating protein 1 (KEAP1) compared with injury group. Results of Western blots and electrophoretic mobility shift assay (EMSA) demonstrated that linagliptin augmented nuclear accumulation of nuclear factor-E2-related factor 2 (NRF2) and its binding ability to target genes in rats with balloon injury. Moreover, heme oxygenase-1 (HO-1) and NAD (P) H quinine oxidoreductase 1 (NQO1), two downstream targets of NRF2, were further up-regulated after linagliptin treatment compared with injury group. In conclusion, our data suggest that linagliptin protects carotid artery from balloon injury-induced neointima formation and activates the NRF2 antioxidant pathway.
The present study was conducted to clarify multiple cardiohemodynamic and electrophysiological properties including inotropic/lusitropic effects of nifekalant, a class III antiarrhythmic drug, in an isoflurane-anesthetized monkey. Nifekalant was administered intravenously at the therapeutic dose of 0.3 mg/kg over 10 min to male cynomolgus monkeys (n=4), followed by higher dose of 1 (n=3) or 3 mg/kg (n=1) that was limited due to arrythmogenicity. Left ventricular (LV) pressure-volume (PV) analysis revealed that the 0.3 mg/kg dose of nifekalant induced a negative lusitropic effect, recognized as a decrease in maximal rate of reduction in LV pressure and a prolonged isovolumic relaxation time. Nifekalant also decreased heart rate and increased LV end-diastolic pressure, but had no effects on the other cardiohemodynamic parameters examined. Electrophysiological analysis showed nifekalant at 0.3 mg/kg prolonged QT/QTc intervals with no evidence of arrhythmia. Higher doses of nifekalant induced ventricular arrhythmia in 3 out of 4 animals, in which both the short-term and long-term variability of the QT interval increased just before the occurrence of arrhythmia. In conclusion, a therapeutic dose of nifekalant had no effect on inotropic activity or cardiac compliance, whereas it showed negative lusitropic properties and QT/QTc prolongation in isoflurane-anesthetized monkeys. In addition, higher doses of nifekalant showed remarkable QT/QTc prolongation leading to arrhythmogenicity, which showed good accordance with clinical findings. Caution should be paid to negative lusitropic properties as well as arrhythmogenisity for the safe use of nifekalant.
Multicentric carpotarsal osteolysis (MCTO) is a condition involving progressive osteolysis of the carpal and tarsal bones that is associated with glomerular sclerosis and renal failure (MCTO nephropathy). Previous work identified an autosomal dominant missense mutation in the transactivation domain of the transcription factor MAFB as the cause of MCTO. Several methods are currently used for MCTO nephropathy treatment, but these methods are invasive and lead to severe side effects, limiting their use. Therefore, the development of alternative treatments for MCTO nephropathy is required; however, the pathogenesis of MCTO in vivo is unclear without access to a mouse model. Here, we report the generation of an MCTO mouse model using the CRISPR/Cas9 system. These mice exhibit nephropathy symptoms that are similar to those observed in MCTO patients. MafbMCTO/MCTO mice show developmental defects in body weight from postnatal day 0, which persist as they age. They also exhibit high urine albumin creatinine levels from a young age, mimicking the nephropathic symptoms of MCTO patients. Characteristics of glomerular sclerosis reported in human patients are also observed, such as histological evidence of focal segmental glomerulosclerosis (FSGS), podocyte foot process microvillus transformation and podocyte foot process effacement. Therefore, this study contributes to the development of an alternative treatment for MCTO nephropathy by providing a viable mouse model.
Chronic stress has been associated with impairment of memory, learning, and social cognition. In animal studies, chronic stress has been shown to impair rodent sociability behaviour which mimics social withdrawal as observed in depression patients. The effect of chronic stress on social recognition, however, is uncertain. Moreover, with reference to spatial learning and memory, the effect of chronic stress is dependent on the type of behavioural task: an appetitively or aversively motivated tasks. The effect of chronic stress was consistent in impairing spatial learning and memory in the appetitive task; however, the effect was inconsistent in an aversive task like the Morris water maze. Thus, we aimed to investigate the effect of chronic restraint stress on sociability and social recognition by using a modified protocol of the three-chamber paradigm and the effect of chronic restraint stress on spatial learning and memory by using the Morris water maze test in young adult C57BL/6J male mice. The present report also describes a modified protocol of the three-chamber paradigm. Our modification is based on measurement of sniffing behaviour, which is a direct social interaction that represents sociability. We used the chronic restraint stress paradigm for 6 h/day for 21 days to induce depression-like symptoms in male C57BL/6J mice which were validated by forced-swim test. We observed that the stressed group had impairments in their sociability behaviour but that social recognition was not affected. Furthermore, we confirmed that chronic stress produced no significant impairment in spatial learning and memory of the mice in the water maze.
In the article “Investigation of the synergistic effects of haloperidol combined with Calculus Bovis Sativus in treating MK-801-induced schizophrenia in rats”, the statement of equal contributions of Kai Lei and Guo-Fang He was mistakenly omitted. The publisher regrets this error.