Experimental Animals最新文献

Translation regulation is crucial for cellular homeostasis. Recent studies have demonstrated that, in addition to the conventional AUG start codon, eukaryotic mRNA can also possess non-canonical start codons. These non-canonical start codons, including non-AUG codons, can be found both upstream and downstream of the conventional AUG start codon. Translation of these non-canonical open reading frames (ORFs) has been implicated in the development of diseases, such as cardiac diseases, neurodegeneration and cancer development. Non-AUG translation initiation is regulated by eukaryotic initiation factor (eIF) 2A and eIF2D; however, their target non-canonical ORFs, roles in disease development, and the underlying precise mechanisms of translation regulation remain poorly understood. To address these gaps, we generated mice lacking either or both of Eif2a and Eif2d genes on an ICR background and investigated their cardiac function using echocardiography. The results indicated that simultaneous disruption of both Eif2a and Eif2d led to perinatal cardiac impairment, as evidenced by a significant reduction in cardiac contractility as measured by ejection fraction. Furthermore, the absence of phenotypic changes in single knockouts of either Eif2a or Eif2d suggests that eIF2A and eIF2D function redundantly in their molecular roles. These findings underscore the importance of non-AUG translation initiation in maintaining cardiac function and suggest its broader implications in other physiological and pathological processes. We propose the Eif2a and Eif2d double-knockout mice as a novel stress-sensitive animal model to investigate the molecular mechanisms of translation regulation and their contribution to disease pathogenesis.

Injectable anesthesia is widely used in laboratory animals because of its ease of administration and minimal equipment requirements. However, it necessitates careful monitoring as well as thermal and oxygen support. This study evaluated the efficacy of medetomidine-alfaxalone-butorphanol (MAB) anesthesia in male rabbits using a dual-route administration protocol. The anesthetic doses were as follows: medetomidine, 0.2 mg/kg; alfaxalone, 2.0 mg/kg; and butorphanol, 2.0 mg/kg. MAB anesthesia, administered via intravenous and subcutaneous routes, induced rapid and smooth induction, achieving anesthetic scores comparable to those of medetomidine-midazolam-butorphanol (MMB) anesthesia. MAB anesthesia resulted in mild hypothermia during the procedure. Upon atipamezole administration, rabbits under MAB anesthesia exhibited faster recovery of the righting reflex and respiration rate than those under MMB. Importantly, no abnormal behaviors, such as jumping or agitation, were observed during induction or recovery, as reported with alfaxalone use in other species. Both protocols maintained spontaneous breathing, although transient hypoxemia was observed in all rabbits. The dual-route MAB protocol provided effective anesthesia while addressing the limitations of conventional MMB anesthesia in rabbits, suggesting its potential as a refined anesthetic method for this species. Unlike mice, which showed weaker anesthetic effects with MAB compared to MMB, MAB demonstrated superior anesthetic properties in rabbits. This study highlights the importance of species-specific anesthetic protocols and the potential benefits of MAB anesthesia in rabbits, particularly its smooth induction and recovery profile, without adverse behaviors often associated with alfaxalone in other species.

The Lepr gene encodes a receptor for leptin, a hormone instrumental in the regulation of appetite and metabolism. Mutations in the Lepr gene impair leptin signaling, leading to metabolic dysfunctions and facilitating the development of non-alcoholic fatty liver disease (NAFLD). In this study, we compared the NAFLD-associated phenotypes of two mutant strains of mice, C57BL/6J-Lepr^em1hwl/Korl (Lepr^em1hwl) and C57BLKS/J-Lepr^db/J (Lepr^db/db), carrying different alleles of the Lepr gene. Although both Lepr^em1hwl and Lepr^db/db mice were characterized by similar obesity phenotypes, leptin resistance, insulin resistance, and glucose intolerance, comparatively, Lepr^em1hwl mice were found to have relatively more severe hepatic steatosis, along with the upregulated expression of enzymes associated with lipogenesis and triglyceride synthesis, and, notably, the histological characteristics of steatohepatitis were observed only in these mice. In addition, compared with the Lepr^db/db mice, Lepr^em1hwl mice developed hepatic fibrosis characterized by elevated levels of collagen deposition and expression of profibrotic factors. Moreover, we detected elevated levels of pro-inflammatory mediators and increases in classically activated macrophage markers in the serum and liver, respectively, of Lepr^em1hwl mice. These findings highlight the distinct NAFLD-associated phenotypic differences between Lepr^em1hwl and Lepr^db/db mice, and thereby indicate that Lepr^em1hwl mice could serve as a valuable model for studying NAFLD, including steatohepatitis and fibrosis.

Archiving and sharing cryopreserved rat sperm can improve animal experiments' reliability, reproducibility, and sustainability in the scientific community. When sharing cryopreserved sperm from genetically engineered rats, a shipment system is required. Generally, a dry shipper, which can maintain at below -150°C, is the most widely used for sperm transport. However, using it for shipping cryopreserved sperm faces some difficulties, such as the risk of transporting hazardous materials (liquid nitrogen), its high cost, and the round-trip fee. Recently, the shipment of cryopreserved mouse sperm with dry ice at -79°C has been alternatively accepted in the scientific community. However, its outcome in terms of the fertilization and developmental abilities of the cryopreserved rat sperm was not examined. Therefore, this study aimed to examine the fertilization and developmental abilities of cryopreserved rat sperm after being stored in a deep freezer (-80°C) and dry ice (-79°C). We also demonstrated the transport of cryopreserved rat sperm in a Styrofoam box with dry ice. The fertilization rate of cryopreserved sperm stored in a deep freezer or dry ice was comparable to that in liquid nitrogen. In the transport experiment, the rat sperm transported between Kumamoto and Hokkaido maintained a high fertilization rate, and live pups were obtained from the embryos derived from the transported sperm. Fertilization and developmental abilities of cryopreserved rat sperm were maintained after shipment using a Styrofoam box with dry ice for storage.

Euthanasia agents should induce a rapid and painless loss of consciousness, followed by cardiopulmonary arrest and subsequent brain death. Injectable drugs such as pentobarbital sodium are commonly used for laboratory rodents due to their quick and smooth action. However, the discontinuation of pharmaceutical-grade pentobarbital sodium and secobarbital sodium in Japan, along with a global shortage of pentobarbital in late 2020, has increased the demand for new injectable euthanasia drugs. In Japan, the combination of medetomidine, midazolam, and butorphanol (MMB), as well as a newer formulation in which midazolam is replaced with alfaxalone (MAB), have been widely used as balanced anesthesia for rodents. To evaluate their potential as alternative euthanasia agents in mice, we compared mortality rates and the time intervals to the loss of the righting reflex, respiratory arrest, and cardiac arrest following anesthetic administration. An intraperitoneal injection of MAB at five times the anesthetic dose induced death within 10 min with the loss of the righting reflex, respiratory arrest, and cardiac arrest occurring at 1.5 min, 4 min, and 9 min respectively, in all mice, which was comparable to those observed with 300 mg/kg of secobarbital. In contrast, none of the mice administered MMB at five times the anesthetic dose experienced cardiopulmonary arrest within 30 min. Intraperitoneal overdose of MAB induces rapid and irreversible death, supporting its potential use as an effective euthanasia agent in mice.

The pathophysiology of endometriosis remains incompletely understood, necessitating the development of effective animal models for research. We generated and characterized a luminescent endometriosis mouse model utilizing luminescent B6-CAG-ELuc transgenic mice as uterine tissue donors and B6.Cg-c/c-hr/hr mice as recipients, enabling non-invasive in vivo imaging. Following transplantation of minced uterine tissue fragments into the peritoneal cavity of recipients, we monitored lesion growth via in vivo imaging system on 0, 14, 28, 42 days post transplantation. Morphology of the lesion was observed by dissecting microscopy, X-ray micro-computed tomography, and conventional histology. Inflammation-related serum cytokines were quantified using multiplex immunobeads assay. The growth of endometriotic lesions was efficiently observed by bioluminescence from day 0 through 42 days post transplantation. Comprehensive morphological observations revealed typical endometriotic lesions consisted of multiple fluid-filled cysts lined with single-layered epithelium, associated with glandular epithelial tissues and interstitial stroma. The level of IL-1β, IL-2, IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α was quantified simultaneously in each serum sample to evaluate the temporal changes of each cytokine, showing four distinct patterns: IFN-γ and TNF-α showed continuous increase, IL-12p70 and IL-1β demonstrated gradual increase followed by marked elevation, IL-6 and IL-2 exhibited dramatic increase in later stages, while IL-10 showed transient increase followed by gradual decrease. In conclusion, this luminescent endometriosis mouse model using B6 luminescent transgenic mice as uterine tissue donor and B6.Cg-c/c-hr/hr recipient could be used to investigate comprehensive cytokine profiling in the development of endometriosis.

Rats (Rattus norvegicus) have been widely utilized as model animals due to their physiological characteristics, making them suitable for surgical and long-term studies. They have played a crucial role in biomedical research, complementing studies conducted in mice. The advent of genome editing technologies has facilitated the generation of genetically modified rat strains, advancing studies in experimental animals. Among these innovations, Cre-driver rat models have emerged as powerful tools for spatiotemporal control of gene expression. However, their development and characterization remain less advanced compared to mouse models. In this study, we developed liver-targeting Cre knock-in rats and reporter knock-in rats to evaluate Cre recombinase expression profiles in different genetic contexts. Our results revealed that insertion orientation and promoter origin significantly influence Cre expression patterns. Notably, forward insertion of the Albumin (Alb) promoter-driven Cre sequence at the ROSA26 locus resulted in ubiquitous Cre expression, while reverse insertion confined Cre expression predominantly to the liver. Interestingly, Cre expression under an endogenous Alb promoter unexpectedly induced expression in non-liver tissues, which may suggest a potential link to the in vivo dynamics of albumin. These findings underscore the importance of rigorous characterization in Cre-based transgenic systems. By elucidating the roles of promoter origin, insertion site, and orientation, our study provides valuable insights for optimizing Cre-driver rat models. These findings pave the way for refining genetic strategies to enhance tissue specificity and reliability in functional genomics and disease modeling.

In mammals, blastocyst-stage trophectoderm (TE) contacts the maternal body at the time of implantation and forms the placenta after implantation, which supports the development of the fetus. Studying gene function in TE and placenta is important to understand normal implantation and pregnancy processes and their dysfunction. However, genetically modified mice are commonly generated by manipulating pronuclear-stage zygotes, which modify both the genome of the fetus and the placenta. Therefore, we previously developed TE/placenta-specific gene expression technology by transducing blastocysts with lentiviral vectors. However, the zona pellucida (ZP) needed to be removed before transduction. In this study, we examined various adeno-associated viral (AAV) vectors to develop a new TE/placenta-specific gene transduction method. As AAV1 can path through ZP, we succeeded in trophoblast-specific gene expression without ZP removal. Furthermore, TE cells genetically modified by AAV1-Cre contributed uniformly to the placenta. Our new technology contributes to advances in implantation and placenta research and leads to the development of new assisted reproductive technology.

Unbalanced redox homeostasis leads to the production of reactive oxygen species and exacerbates inflammatory bowel disease. To investigate the role of the transcription factor Nrf2, a major antioxidative stress sensor, in intestinal epithelial cells (IECs), we generated IEC-specific Nrf2 gene knock-in mice (Nrf2-vRes), which express Nrf2 only in IECs, using the cre/loxp system. Colitis was induced in wild-type (WT) mice, whole-body Nrf2-knockout (Nrf2-KO) mice, and Nrf2-vRes mice by administering dextran sulfate sodium (DSS) for 1 week (acute model) or intermittently for 5 weeks (chronic model). The mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), which is involved in the oxidative stress response in a manner regulated by Nrf2, were reduced in Nrf2-KO compared with those in WT, while these decreases were reversed in Nrf2-vRes at all timepoints. Nrf2-KO mice administered DSS developed more severe colitis with higher disease activity index, higher leucine-rich α2 glycoprotein in serum, shorter colon length, and more severe epithelial damage and infiltration of inflammatory cells histopathologically than did WT mice in the acute model; moreover, these exacerbations of colitis were ameliorated in Nrf2-vRes mice. However, these differences were not observed among the three sets of mice in the chronic model. IEC-specific expression of Nrf2 ameliorated DSS-induced acute colitis. These results suggest that Nrf2 expression in IECs plays a protective role against early-stage colitis and undertakes important regulatory functions during intestinal inflammation.

In most cases, the diagnosis of diabetes in animal models is based solely on blood glucose levels. While hemoglobin A1c (HbA1c) is widely used in the diagnosis of diabetes in humans, it is rarely measured in mice in diabetes research. This is thought to be because there are no established reference values for mouse HbA1c, as well as the fact that there are very few reports on the variability and reproducibility of measurements taken using different devices. In this study, we measured HbA1c levels in diabetic mouse models using different devices based on different principles, including capillary electrophoresis, high-performance liquid chromatography, and enzymatic methods, and compared the results. A positive correlation was observed between blood glucose and HbA1c levels in all measurement methods, and high reproducibility was confirmed in the measurement of HbA1c. However, HbA1c levels measured using the enzymatic method were slightly higher than those measured using the other two methods. In addition, an examination of diabetic mice given a sodium-glucose cotransporter 2 inhibitor, which is used to treat diabetes, revealed that there was a 2-week difference in the fluctuation of mouse HbA1c levels compared with the fluctuation of blood glucose levels. Based on these results, it is thought that HbA1c can be a reliable indicator in diabetic mouse models, and it is expected to make the evaluation of abnormal glucose metabolism in mice more reliable.