Noriaki Miyoshi

Microminipig, a Non-rodent Experimental Animal Optimized for Life Science Research: Novel Atherosclerosis Model Induced by High Fat and Cholesterol Diet

Atherosclerotic lesions were observed in male and ovariectomized female Microminipig (MMP) fed a high fat and cholesterol diet with sodium cholate (HFCD/SC) for 3 months. HFCD/SC induced hypercholesterolemia accompanied by an increase in serum total cholesterol (T-Cho), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and cholesterol ester (CE). Unlike the mouse or rabbit, a dominant LDL-C fraction in the intact MMP, similar to that in humans, was observed by serum lipoprotein analysis. HFCD/SC increased body weight gain. At the end of the experiment, computed tomography scans of conscious animals showed that HFCD/SC had decreased liver attenuation values (Hounsfield unit) and increased subcutaneous and abdominal fat, suggesting the induction of fatty liver and obesity. HFCD/SC induced atherosclerotic lesions in systemic arteries, including the external and internal iliac arteries, abdominal aorta, coronary artery, and cerebral arterial circle. Atherosclerosis and pathological findings induced by HFCD/SC in MMP were similar to those in humans. The MMP is a potentially suitable tool for investigating human atherosclerosis.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

SUMMARY Ticks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis.

Comparative study of chronic kidney disease in dogs and cats: Induction of myofibroblasts

We investigated the kidneys of dogs and cats to clarify whether renal myofibroblasts induction is associated with the severity of chronic kidney disease (CKD). Immunohistochemical expression of myofibroblast markers, alpha-smooth muscle actin (SMA) and vimentin, were evaluated quantitatively. The degrees of glomerulosclerosis, glomerular hypertrophy, interstitial cell infiltration, and interstitial fibrosis were also evaluated quantitatively. The plasma creatinine (pCre) concentrations correlated with glomerulosclerosis, cell infiltration, and fibrosis in dogs, and only with fibrosis in cats. The alpha-SMA expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, and with pCre and fibrosis in cats. Tubular vimentin expression correlated with fibrosis in cats, but not in dogs. Interstitial vimentin expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, but only with pCre in cats. In conclusion, it was suggested that the severity of CKD in dogs and cats was mediated by different pathways associated with myofibroblasts expression.

A Dermal Phototoxicity Study Following Intravenous Infusion Administration of Ciprofloxacin Hydrochloride in the Novel Microminipigs

The authors evaluated dermal phototoxicity using the world smallest minipig (MMPig: Microminipig). MMPigs were administered 100 mg/kg ciprofloxacin hydrochloride with an infusion pump. The dorsal area of each animal was irradiated with ultraviolet-A irradiation. The left dorsal skin was irradiated at intensities of 5, 10, 15, and 20 J/cm2, and the right dorsal back skin was set as a nonirradiated site. Gross and histopathological examinations were conducted before irradiation and from 1 to 72 hr after irradiation. Initial changes in the skin were necrosis of the basal and/or prickle cell layer and cellular infiltration from 24 hr after irradiation. Vesicle formation observed from 48 hr after irradiation was considered similar to bullous eruptions, a known side effect of fluoroquinolones in humans. Therefore, the authors suggest that the MMPig may be a useful experimental animal model for dermal phototoxicity studies.

Development of a Medium-term Animal Model Using gpt Delta Rats to Evaluate Chemical Carcinogenicity and Genotoxicity.

In this study, the potential for development of an animal model (GPG46) capable of rapidly detecting chemical carcinogenicity and the underlying mechanisms of action were examined in gpt delta rats using a reporter gene assay to detect mutations and a medium-term rat liver bioassay to detect tumor promotion. The tentative protocol for the GPG46 model was developed based on the results of dose-response exposure to diethylnitrosamine (DEN) and treatment with phenobarbital over time following DEN administration. Briefly, gpt delta rats were exposed to various chemicals for 4 weeks, followed by a partial hepatectomy (PH) to collect samples for an in vivo mutation assay. The mutant frequencies (MFs) of the reporter genes were examined as an indication of tumor initiation. A single intraperitoneal (ip) injection of 10 mg/kg DEN was administered to rats 18 h after the PH to initiate hepatocytes. Tumor-promoting activity was evaluated based on the development of glutathione S-transferase placental form (GST-P)-positive foci at week 10. The genotoxic carcinogens 2-acetylaminofluorene (2-AAF), 2-amino-3-methylimidazo [4,5-f] quinolone (IQ) and safrole (SF), the non-genotoxic carcinogens piperonyl butoxide (PBO) and phenytoin (PHE), the non-carcinogen acetaminophen (APAP) and the genotoxic non-hepatocarcinogen aristolochic acid (AA) were tested to validate the GPG46 model. The validation results indicate that the GPG46 model could be a powerful tool in understanding chemical carcinogenesis and provide valuable information regarding human risk hazards.

Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1-3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P<0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.

A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100 protein in veterinary diagnostic cytology.

The objective of this study was to establish a simple and rapid immunocytochemical technique that can be used in veterinary diagnostic cytology. Air-dried impression smears were collected from canine tumors. Samples of epithelial tumors, mesenchymal tumors, and malignant peripheral nerve sheath tumors and melanomas were used for detection of cytokeratin, vimentin, and S-100 protein, respectively. The labeled streptavidin-biotin system was used in the present study. Optimal fixation was determined using standard immunocytochemical procedures, and acetone fixation was found to be the most effective. Optimal concentrations of primary and secondary antibodies were determined at a preset 5-min incubation. Omission of H2O2 treatment, shortening the time for blocking and labeled-streptavidin incubation, and simplifying washing did not decrease immunopositive intensities or enhance false-positive reactions. The described rapid protocol requires approximately 45 min without the use of any special equipment.

GM2 Gangliosidosis Variant 0 (Sandhoff Disease) in a Mixed-Breed Dog

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive, neurodegenerative lysosomal storage disease caused by simultaneous deficiencies of acid β-hexosaminidases A and B. Canine SD has so far been identified only in two purebreeds. In this article, we present the case of a 10 mo old, male dog of mixed breed that developed progressive neurological signs including ataxia, postural deficit, and visual deficits and finally died at the age of 21 mo. The dog was diagnosed with SD on the basis of the results of biochemical and histopathological analyses. This is the third report of canine SD and the first time it has been identified in a mixed breed.

A medium-term gpt delta rat model as an in vivo system for analysis of renal carcinogenesis and the underlying mode of action

The kidney is a major target site of chemical carcinogenesis. However, a reliable in vivo assay for rapid identification of renal carcinogens has not been established. The purpose of this study was to develop a new medium-term gpt delta rat model (the GNP model) to facilitate identification of renal carcinogens. In this model, we carried out an in vivo mutation assay using unilaterally nephrectomized kidney tissue and a tumor-promoting assay using residual kidney tissue, with diethylnitrosamine (DEN) as the renal tumor initiator. To clarify the optimal time of DEN injection after nephrectomy, time-dependent changes in bromodeoxyuridine-labeling indices in the tubular epithelium of nephrectomized rats were examined. The optimal dose of DEN injection and sufficient duration of subsequent nitrilotriacetic acid treatment were determined for detection of renal preneoplastic lesions. The standard protocol for the GNP model was determined as follows. Six-week-old female gpt delta rats were treated with test chemicals for 4 weeks, followed by a 2-week washout period, and 40 mg/kg DEN was administered intraperitoneally to initiate renal carcinogenesis. Unilateral nephrectomy was performed 48 h before DEN injection, followed by gpt assays using excised kidney tissues. One week after DEN injection, rats were further exposed to test chemicals for 12 weeks, and histopathological analysis of renal preneoplastic lesions was performed as an indicator of tumor-promoting activity in residual kidney tissue. Validation studies using aristolochic acid, potassium dibasic phosphate, phenylbutazone, and d-limonene indicated the reliability of the GNP model for predicting renal carcinogens and the underlying mode of action.

Interferon-β response is impaired by hepatitis B virus infection in Tupaia belangeri

To date, the chimpanzee has been used as the natural infection model for hepatitis B virus (HBV). However, as this model is very costly and difficult to use because of ethical and animal welfare issues, we aimed to establish the tupaia (Tupaia belangeri) as a new model for HBV infection and characterized its intrahepatic innate immune response upon HBV infection. First, we compared the propagation of HBV genotypes A2 and C in vivo in tupaia hepatocytes. At 8-10days post infection (dpi), the level of HBV-A2 propagation in the tupaia liver was found to be higher than that of HBV-C. Abnormal architecture of liver cell cords and mitotic figures were also observed at 8 dpi with HBV-A2. Moreover, we found that HBV-A2 established chronic infection in some tupaias. We then aimed to characterize the intrahepatic innate immune response in this model. First, we infected six tupaias with HBV-A2 (strains JP1 and JP4). At 28 dpi, intrahepatic HBV-DNA and serum hepatitis B surface antigens (HBsAg) were detected in all tupaias. The levels of interferon (IFN)-β were found to be significantly suppressed in the three tupaias infected with HBV A2_JP4, while no significant change was observed in the three infected with HBV A2_JP1. Expression of toll-like receptor (TLR) 1 was suppressed, while that of TLR3 and TLR9 were induced, in HBV A2_JP1-infected tupaias. Expression of TLR8 was induced in all tupaias. Next, we infected nine tupaias with HBV-A2 (JP1, JP2, and JP4), and characterized the infected animals after 31 weeks. Serum HBsAg levels were detected at 31 weeks post-infection (wpi) and IFN-β was found to be significantly suppressed in all tupaias. TLR3 was not induced, except in tupaia #93 and #96. Suppression of TLR9 was observed in all tupaias, except tupaia #93. Also, we investigated the expression levels of cyclic GMP-AMP synthase, which was found to be induced in all tupaias at 28 dpi and in four tupaias at 31 wpi. Additionally, we evaluated the expression levels of sodium-taurocholate cotransporting polypeptide, which was found to be suppressed during chronic HBV infection. Thus, the tupaia infection model of HBV clearly indicated the suppression of IFN-β at 31 wpi, which might have contributed to the establishment of chronic HBV infection.