Experimental Animals最新文献

In 2016, an outbreak of Rattus norvegicus polyomavirus 2 (RatPyV2) infection was reported in a colony of X-linked severe combined immunodeficiency (XSCID) rats in the United States. While RatPyV2 infection persists asymptomatically in immunocompetent rats, immunodeficient XSCID rats develop variable respiratory symptoms, emaciation, impaired breeding performance, and systemic deteriorating condition. RatPyV2 is an epitheliotropic virus targeting epithelial cells of the salivary glands, Harderian glands, extraorbital lacrimal glands, respiratory system, and reproductive or accessory reproductive organs. Histopathologically, the formation of large basophilic nuclear inclusion bodies in the infected epithelial cells is a characteristic feature, along with hyperplasia or dysplasia. Glandular atrophy and loss, accompanied by fibrosis and mononuclear cell infiltration, are also observed in the salivary glands, Harderian glands, and extraocular lacrimal glands. In particular, the parotid salivary glands are prone to be severely and extensively affected with relatively severe and diffuse lesions even at one month of age. Severely affected animals also develop interstitial pneumonia. Among target tissues, the parotid salivary glands appear to be higher susceptible to RatPyV2, therefore pathological examination and PCR examination of the salivary glands, including the parotid salivary glands, are essential for the diagnosis of RatPyV2 infection. This review paper provides a comprehensive summary of the features (clinical signs, pathological findings, and transmission), diagnostic methods, and prevalence of RatPyV2 infection, based on our research and reports from research groups in the United States.This is a secondary publication of "Rat polyomavirus 2 infection" [25] published in JALAS Newsletter (in Japanese) 2021, 70 (1): 17-25. It also includes updated content.

Alternative educational tools, such as training videos and simulators, are recommended in the education of laboratory animal science. However, evidence supporting their educational utility in the training of rodent experimental techniques remains limited. In this study, we assessed the utility of alternative educational tools in the practice of laboratory animal science for novice veterinary students. 149 students participated in a stepwise program beginning with lectures, followed by preparatory learning sessions using training videos and two types of mouse simulators (a silicone-based model and fabric toy mouse), and then hands-on training with live mice. The program covered basic techniques: habituation, restraint, and vaginal smear sampling for estrous cycle determination. A survey-based evaluation was conducted to assess the educational utility of alternative educational tools. The contribution of each preparatory resource (videos, lectures, simulators, printed materials, and notes) to skill acquisition was evaluated, showing that videos, lectures, and printed materials highly contributed. The training videos were rated as more necessary than the simulators for skill acquisition. Psychological evaluation showed that 84% of students experienced anxiety before practice. A positive correlation was found between anxiety levels and frequency of use for all three tools, and students reported that all tools were effective in reducing anxiety during practice. All techniques showed high proficiency rates. Our findings suggest that integrating alternative tools with live-animal training promotes technical skill acquisition, enhances psychological readiness, and supports 3Rs-based laboratory animal practice.

Chronic kidney disease (CKD) is a complicated systemic disease displaying various pathophysiological symptoms including mineral bone disorder (CKD-MBD). Ideally, early intervention for CKD-MBD would be desirable, however, there is not enough evidence regarding treatment of CKD-MBD, especially in its early stages, due to its multifactorial pathophysiology and the difficulty in generating adequate animal models. In this study, we evaluated the efficacy of a tissue nonspecific alkaline phosphatase (TNAP) inhibitor, SBI-425 in a CKD-MBD animal model, produced by a combination of nephrectomy and high inorganic phosphate (P_i) diet. This combination induced renal damage, and significantly elevated blood urea nitrogen (BUN). Plasma levels of fibroblast growing factor 23 (FGF-23), parathyroid hormone (PTH) and phosphate were also elevated, leading to ectopic calcification in the kidneys, particularly in the renal tubules. We orally administered SBI-425 twice daily for 12 weeks at doses of 1 and 10 mg/kg, and this treatment significantly inhibited the progression of calcium deposition in the renal tubules. Furthermore, SBI-425 effectively prevented the deterioration of plasma parameters, BUN, FGF-23, PTH, and phosphate. In conclusion, our findings suggest that TNAP inhibition can effectively slow the progression of CKD-MBD by inhibiting the calcification in the renal tubules. These results may have implications for better clinical care of patients with CKD.

Heart failure (HF) is a clinical syndrome related to multiple causes, including oxidative stress. Acyl-CoA thioesterase 1 (ACOT1) is an enzyme in fatty acids metabolism, but it remains unclear in HF. Transverse aortic coarctation induced HF mouse model and hypoxia-stimulated cardiomyocyte (HL-1) model were established. ACOT1 expression was down-regulated in heart tissues of HF mice. Adeno-associated virus serotype 9 (AAV9)-mediated ACOT1 overexpression improved cardiac function and pathological injury of heart tissues in transverse aortic coarctation (TAC)-induced HF mice. ACOT1 overexpression ameliorated oxidative stress in heart tissues of HF mice and hypoxia-stimulated HL-1 cells, as indicated by reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels and elevated superoxide dismutase (SOD) and glutathione (GSH) levels. We found that ACOT1 overexpression inhibited apoptosis both in vivo and in vitro, with decreased protein levels of cleaved PARP, cleaved CASPASE-3, and cleaved CASPASE-9. Mechanically, ACOT1 activated Kelch-like ECH-associated protein1-NF-E2-related factor2 (KEAP1-NRF2) pathway, leading to the nuclear translocation of NRF2 and increased NRF2-regulated gene Nqo1 expression. Rescue experiment indicated that ML385 (NRF2 inhibitor) abolished the effect of ACOT1 overexpression on oxidative stress. Collectively, these results suggested that ACOT1 overexpression protects heart from injury by inhibiting oxidative stress and apoptosis, possibly through activating KEAP1-NRF2 pathway.

In vivo imaging enables real-time detection of excitation and emission signals and is useful for the noninvasive evaluation of temporal changes in biological tissues. The near-infrared fluorescent protein iRFP can be used for deep-tissue imaging because it emits light at wavelengths that are less attenuated by biological tissues. However, autofluorescence originating from diet, tissues, and the imaging environment can interfere with fluorescence detection; therefore, appropriate animal pretreatment and optimization of imaging conditions are essential. We generated two mouse strains: Alb^eGiR reporter mice, in which enhanced green fluorescent protein (eGFP) and iRFP713 genes were tandemly inserted downstream of the Albumin gene, and hairless mice (Hr^Δ164/Δ164), carrying a mutation in the hairless gene. Their offspring were used in in vivo imaging experiments to investigate: (i) the localization of eGFP and iRFP713 fluorescence, (ii) the influence of hair on fluorescence detection, and (iii) suitable filter combinations for fluorescence detection. In the resulting mice, liver-specific expression of both eGFP and iRFP713 was observed at the same anatomical location. Although autofluorescence was more prominent in hairless mice than in furred mice, signal detection was improved either by using longer-wavelength excitation/emission filters or by applying spectral unmixing to separate the target signal. These findings provide practical guidance for optimizing in vivo fluorescence imaging conditions using standard IVIS platforms.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that causes the selective loss of motor neurons. A histopathological hallmark of ALS is the cytoplasmic aggregation of TDP-43, a ubiquitously expressed RNA-binding protein involved in transcription and splicing regulation. To prevent abnormal accumulation, TDP-43 controls its expression levels through an autoregulatory feedback loop. While most ALS studies have focused on pathogenic variants that impair the protein function of TDP-43, the mechanisms underlying endogenous TDP-43 dysregulation mediated by non-coding elements, including the 3' untranslated region (3'UTR), remain incompletely understood. In this study, we generated a mouse model carrying a targeted deletion of the Tardbp 3'UTR that encompasses the TDP-binding region, polyadenylation signals, and alternative intronic sequences. Our findings demonstrate that the Tardbp 3'UTR is essential for normal mouse development. Loss of this region led to decreased Tardbp mRNA expression and embryonic lethality after gastrulation. Young heterozygous mice were phenotypically normal with no overt disruption in TDP-43 autoregulation. However, aged heterozygous mice displayed mild locomotor dysfunction accompanied by a modest increase in spinal cord TDP-43 protein levels and a reduction in motor neuron numbers. These findings indicate that regulatory elements within the Tardbp 3'UTR play a pivotal role in normal development and contribute to TDP-43 pathology relevant to ALS.

Emerging evidence indicates that oxidative stress in skeletal muscle is a prerequisite for sarcopenia in diabetic patients. In this study, we show that ubiquitin-specific protease (USP) 2 mitigates the accumulation of reactive oxygen species (ROS) in mature muscle cells. Treatment with ML364, a canonical USP2 inhibitor, robustly increased mitochondrial ROS in mouse C2C12 myotubes and caused an accompanying increase in the glutathione disulfide (GSSG)/glutathione (GSH) ratio. ML364 also caused mitochondrial damage in C2C12 myotubes, resulting in a reduction in intracellular adenosine triphosphate levels. Correspondingly, under diabetic condition, the muscle-specific Usp2-knockout (msUsp2KO) C57BL/6N mice exhibited a significantly higher lipid peroxide level and GSSG/GSH ratio in skeletal muscle than the control mice. The msUsp2KO mice also exhibited augmented insulin resistance and glucose intolerance, but showed no obvious deterioration in muscle weight or histology relative to the control mice. However, damaged mitochondria in the soleus muscle were more frequently observed in msUsp2KO mice than in the control mice. Together, these data suggest that USP2 mitigates ROS accumulation and subsequent mitochondrial damage in muscle cells in mice.

As aging affects the appearance of the skin, anti-aging research has intensified in dermatology, skincare, and aesthetic medicine. Because natural aging takes a very long time, one essential anti-aging approach is to pharmacologically mimic aging, such as with D-galactose treatment. Hairless mice (HR-1) have been extensively used in skin research because of their lack of body hair and ease of animal care. In the present study, HR-1 mice were treated with D-galactose to determine whether detrimental effects were induced in the skin. After 3 months of D-galactose treatment, AGEs in the skin significantly increased. On the other hand, no signs of skin disorders (dermal thickness, type I collagen content, expression of various genes, collagen synthesis, and degradation signals) were observed. Even when the concentration of D-galactose increased, no apparent changes in dermal thickness were observed. These findings suggest that D-galactose treatment induces AGEs accumulation but no further detrimental effects in the HR-1 skin.

The ε-containing GABA(A) receptors (GABAARs), a lesser-studied subtype within the GABAAR family, have garnered attention due to their distinct pharmacological properties and potential involvement in brain injury. Zolpidem (ZPM), a widely used Z-drug, is known to induce paradoxical effects in patients with brain injury, although the underlying molecular mechanisms remain unclear. In this study, a chronic cerebral hypoperfusion (CCH) rat model was established using Permanent Bilateral Occlusion of the Common Carotid Arteries (PBOCCA), followed by administration of ZPM at doses of 1.0, 2.0, and 4.0 mg/kg. Behavioral assessments demonstrated that the 1.0 mg/kg dose of ZPM significantly improved spatial learning and memory acquisition (P<0.01) and enhanced memory retention (P<0.001), whereas higher doses resulted in sedation and cognitive impairment. Immunohistochemical analysis revealed an upregulation of the ε subunit expression in the hippocampal CA1 and CA3 regions of CCH rats (P<0.05), suggesting alterations in receptor composition in response to cerebral hypoperfusion. Further investigation of ZPM's interaction with ε-containing GABAARs (specifically the α1β2ε subtype) was conducted using in silico techniques. Molecular docking identified the α1+/ε- binding interface as a favorable ZPM binding site, with key residues being either conserved or suitably replaced. Molecular dynamics simulations demonstrated that ZPM stabilizes the receptor while permitting conformational flexibility, consistent with its role as a positive allosteric modulator. These findings provide evidence that ZPM interacts with ε-containing GABAARs, potentially explaining its paradoxical effects observed in brain injury models.

The cynomolgus monkey (Macaca fascicularis) is an important experimental animal; however, its menstrual patterns, lifespan, and age-related changes in anti-Müllerian hormone (AMH) levels remain poorly characterized. This study aimed to analyze these factors and evaluate the usefulness of Cynomolgus monkeys in ovarian function research. The age at menarche was examined in 21 cynomolgus monkeys, and the age at menopause and age at death were tracked in another 22 postmenopausal monkeys. In addition, AMH levels were analyzed in 74 cynomolgus monkeys aged 0 to 33 years to evaluate ovarian reserve throughout their lives. Results showed a mean age at menarche of 3.69 ± 2.51 years, menopause at 27.00 ± 2.50 years, and a mean age at death of 32.04 ± 5.33 years. AMH levels throughout life showed a weak negative correlation with age. These findings suggest that changes in ovarian reserve throughout the life span of cynomolgus monkeys are similar to those in humans. To our knowledge, this study is the first to analyze menstruation, lifespan, and lifelong AMH levels in cynomolgus monkeys. Ovarian function throughout life, including childhood and postmenopause, was similar to that in humans, suggesting cynomolgus monkeys may be a useful experimental model.