Development of a Zebrafish Larvae Model for Diabetic Heart Failure With Reduced Ejection Fraction

Background and Objectives@#Diabetes mellitus (DM)-associated heart failure (HF) causes high morbidity and mortality. In this study, we established a zebrafish larvae model for in vivo research on diabetic HF. @*Methods@#DM-like phenotypes were induced by treating zebrafish larvae with a combination of D-glucose (GLU) and streptozotocin (STZ). HF was induced by treatment with terfenadine (TER), a potassium channel blocker. Additionally, myocardial contractility, motility, and viability were evaluated. @*Results@#The zebrafish larvae treated with a combination of GLU and STZ showed significantly higher whole-body glucose concentrations, lower insulin levels, and higher phosphoenolpyruvate carboxykinase levels, which are markers of abnormal glucose homeostasis, than the group treated with only GLU, with no effect on viability. When treated with TER, DM zebrafish showed significantly less myocardial fractional shortening and more irregular contractions than the non-DM zebrafish. Furthermore, in DM-HF with reduced ejection fraction (rEF) zebrafish, a significant increase in the levels of natriuretic peptide B, a HF biomarker, markedly reduced motility, and reduced survival rates were observed. @*Conclusions@#We established a DM-HFrEF zebrafish model by sequentially treating zebrafish larvae with GLU, STZ, and TER. Our findings indicate the potential utility of the developed zebrafish larvae model not only in screening studies of new drug candidates for DM-HFrEF but also in mechanistic studies to understand the pathophysiology of DM-HFrEF.

Fimasartan, an angiotensin II receptor antagonist, ameliorates an in vivo zebrafish model of heart failure

Background/Aims@#Angiotensin II in the failing heart initially helps to maintain cardiac output and blood pressure, but ultimately accelerates its deterioration. In this study, we established a model of arrhythmia-induced heart failure (HF) in zebrafish and investigated the role of renin-angiotensin-aldosterone system (RAAS) modulation by using an angiotensin II type 1 receptor blocker, fimasartan, through the assessment of cellular and physiologic responses, morbidity, and mortality. @*Methods@#HF was induced in zebrafish larvae by exposure to 20 μM terfenadine. Morphologic, physiologic, and functional parameters were assessed in the presence or absence of fimasartan treatment. @*Results@#Zebrafish exposed to terfenadine showed marked dilatation of the ventricle and reduced systolic function. Treatment with terfenadine was associated with 10-fold higher expression of atrial natriuretic peptide (p < 0.001 vs. vehicle), increased p53 mRNA expression, and chromatin fragmentation in the TUNEL assay, all of which were significantly reduced by fimasartan treatment. Moreover, fimasartan improved fractional shortening (terfenadine + fimasartan 16.9% ± 3.1% vs. terfenadine + vehicle 11.4% ± 5.6%, p < 0.05) and blood flow (terfenadine + fimasartan 479.1 ± 124.1 nL/sec vs. terfenadine + vehicle 273.0 ± 109.0 nL/sec, p < 0.05). Finally, treatment with fimasartan remarkably reduced mortality (terfenadine + fimasartan 36.0% vs. terfenadine + vehicle 96.0%, p < 0.001). @*Conclusions@#Fimasartan effectively protected against the progression of HF in zebrafish by improving hemodynamic indices, which improved survival. A reduction in apoptotic cell death and an improvement in hemodynamics may be the mechanisms behind these effects. Further human studies are warranted to evaluate the possible role of fimasartan in the treatment of HF.

Fasiglifam (TAK-875), a G Protein-Coupled Receptor 40 (GPR40) Agonist, May Induce Hepatotoxicity through Reactive Oxygen Species Generation in a GPR40-Dependent Manner

Fasiglifam (TAK-875) a G-protein coupled receptor 40 (GPR40) agonist, significantly improves hyperglycemia without hypoglycemia and weight gain, the major side effects of conventional anti-diabetics. Unfortunately, during multi-center Phase 3 clinical trials, unexpected liver toxicity resulted in premature termination of its development. Here, we investigated whether TAK-875 directly inflicts toxicity on hepatocytes and explored its underlying mechanism of toxicity. TAK-875 decreased viability of 2D and 3D cultures of HepG2, a human hepatocarcinoma cell line, in concentration- (>50 μM) and time-dependent manners, both of which corresponded with ROS generation. An antioxidant, N-acetylcysteine, attenuated TAK-875-mediated hepatotoxicity, which confirmed the role of ROS generation. Of note, knockdown of GPR40 using siRNA abolished the hepatotoxicity of TAK-875 and attenuated ROS generation. In contrast, TAK-875 induced no cytotoxicity in fibroblasts up to 500 μM. Supporting the hepatotoxic potential of TAK-875, exposure to TAK-875 resulted in increased mortality of zebrafish larvae at 25 μM. Histopathological examination of zebrafish exposed to TAK-875 revealed severe hepatotoxicity as manifested by degenerated hypertrophic hepatocytes with cytoplasmic vacuolation and acentric nuclei, confirming that TAK-875 may induce direct hepatotoxicity and that ROS generation may be involved in a GPR40-dependent manner.

Zebrafish Larvae Model of Dilated Cardiomyopathy Induced by Terfenadine

BACKGROUND AND OBJECTIVES: Dilated cardiomyopathy can be the end-stage of severe cardiac disorders and directly affects the cardiac muscle, inducing cardiomegaly and heart failure (HF). Although a wide variety of animal models are available to study dilated cardiomyopathy, there is no model to assess dilated cardiomyopathy with non-invasive, simple, and large screening methods. METHODS: We developed a dilated cardiomyopathy model in zebrafish larvae using short duration terfenadine, a known cardiotoxic drug that induces ventricular size dilation. Fractional shortening of zebrafish hearts was calculated. RESULTS: We treated zebrafish with 5 to 10 µM terfenadine for 24 hours. In terfenadine-treated zebrafish, blood frequently pooled and clotted in the chamber, and circulation was remarkably reduced. Atria and ventricles were swollen, and fluid was deposited around the heart, mimicking edema. Cardiac contractility was significantly reduced, and ventricular area was significantly enlarged. Heart rate was markedly reduced even after terfenadine withdrawal. Acridine orange staining also showed that terfenadine increased cardiomyocyte apoptosis. A significant increase of natriuretic peptide B (NPPB) mRNA was found in terfenadine-treated zebrafish. A low dose of terfenadine (5–10 µM) did not show mortality in short-term treatment (24 hours). However, moderate dose (35–45 µM) terfenadine treatment reduced zebrafish survival within 1 hour. CONCLUSION: With advantages of rapid sample preparation procedure and transparent observation of the live heart, this model can potentially be applied to large-scale drug screening and toxicity assays for non-ischemic HF.

Epidemiological Characteristics and Risk Factors of Dengue Infection in Korean Travelers

Dengue viral infection has rapidly spread around the world in recent decades. In Korea, autochthonous cases of dengue fever have not been confirmed yet. However, imported dengue cases have been increased since 2001. The risk of developing severe dengue in Korean has been increased by the accumulation of past-infected persons with residual antibodies to dengue virus and the remarkable growth of traveling to endemic countries in Southeast Asia. Notably, most of imported dengue cases were identified from July to December, suggesting that traveling during rainy season of Southeast Asia is considered a risk factor for dengue infection. Analyzing national surveillance data from 2011 to 2015, males aged 20–29 years are considered as the highest risk group. But considering the age and gender distribution of travelers, age groups 10–49 except 20–29 years old males have similar risks for infection. To minimize a risk of dengue fever and severe dengue, travelers should consider regional and seasonal dengue situation. It is recommended to prevent from mosquito bites or to abstain from repetitive visit to endemic countries. In addition, more active surveillance system and monitoring the prevalence asymptomatic infection and virus serotypes are required to prevent severe dengue and indigenous dengue outbreak.

The Preparedness for Re-emerged and Emerging Infectious Diseases: A Lesson Through Outbreak of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in South Korea

The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease and systemic dysfunction that may eventually lead to the death of the patients. After MERS-CoV was first diagnosed in the South Korea, in May 2015, it affected 186 individuals and claimed 37 lives in short span of time (case fatality rate = 19.9%). Compared to MERS-CoV in the Middle East, MERS-CoV in South Korea appeared to be more transmissible, and induced multiple human-to-human transmission. These knowledge gaps caused the failure of early prevention, and disseminated MERS-CoV brought out a great loss of lives and economy. The MERS-CoV outbreak revealed the potential weakness of public health system in South Korea, and promoted the reestablishment of preventive strategies for imported infectious diseases. In these regards, we analyzed the potential for additional import of re-emerged and emerging infectious diseases, such as dengue fever, malaria, chikungunya fever and hepatitis A, from Africa or South-East Asia. Then we suggest the investment expansion and the administration of global networks for effective research and control for newly or re-emerged infectious diseases. In conclusion, it is required to expect and prepare for the surveillance of the importation of foreign pathogens, and constitute the internal collaborative systems for rapid detection and risk communication. In addition, we should take an active part in the global networks to perform rapid preparedness and control for re-emerged or emerging infectious diseases.

Detection of Corynebacterium bovis infection in athymic nude mice from a research animal facility in Korea

Corynebacterium (C.) bovis infection in nude mice causes hyperkeratosis and weight loss and has been reported worldwide but not in Korea. In 2011, nude mice from an animal facility in Korea were found to have white flakes on their dorsal skin. Histopathological testing revealed that the mice had hyperkeratosis and Gram-positive bacteria were found in the skin. We identified isolated bacteria from the skin lesions as C. bovis using PCR and 16S rRNA sequencing. To the best of our knowledge, this is the first report of C. bovis infection in nude mice from Korea.

Effects of dietary high fat on prostate intraepithelial neoplasia in TRAMP mice / 한국실험동물학회지

Increased fat intake is known to be a major cause of prostate cancer. In this study, we investigated the effect of dietary high fat on prostate intraepithelial neoplasia using transgenic adenocarcinoma mouse prostate (TRAMP) mice. Six-week-old male TRAMP mice were fed AIN93G (control group, 4.0 kcal/kg, n=6) and AIN93G-HFD (experimental group, 4.8 kcal/kg, n=7) for 10 weeks. Prostate histopathology, urogenital tract (UGT) weight, epididymal white adipose tissue weight, argyrophilic nucleolar organizer regions (AgNORs) counts, and serum leptin levels were examined. AIN93G-HFD fed group showed progressed neoplastic lesions in the prostate (P<0.05) compared to AIN93G fed group. AIN93G-HFD intake resulted in a increase in the weight of UGT (P<0.05) and epididymal white adipose tissue. The number of Ag-NOR positive dots significantly increased in each prostate lobe and final serum leptin levels in AIN93G-HFD fed group were about twice those of AIN93G fed group (P<0.05). Dietary high fat was related to the prostate cancer progression in the early stage of TRAMP mice and increased serum leptin levels, suggesting that the regulation of dietary components could delay the progression of prostate cancer.

Ethical Guidelines for Use of Experimental Animals in Biomedical Research

Increasing concerns on animal welfare, the discussion about the necessity and the ethical acceptability of animal experiments is getting important. In this article, we describe ethical issues between the benefits of the animal experiments and the suffering of the animals. If there are no alternatives for animal experiments, the ethical guidelines, 3Rs (Reduction, Replacement, Refinement) by Russell and Burch for humane animal experiments should be considered. Moreover, Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee (IBC) assist researchers to perform animal testing humanely and safely. We point out three important aspects in support of humane animal experiments; 1) the development of diverse scientific alternatives on the animal testing, 2) the ethical education of animal experiments for researchers, 3) the productive interactions among various social groups on ethics and safety of animal experiments.

Adiaspiromycosis of an Apodemus agrarius captured wild rodent in Korea / 한국실험동물학회지

Adiaspiromycosis is caused by pulmonary infection with Emmonsia. Inhalated spores of Emmonsia cause asymptomatic infection to necrogranulomatous pneumonia, depending on the burden of adiaspore and host immunity. For disease monitoring of wild rodents captured on Jeju Island in Korea, we examined the lung tissue of wild rodents histopathologically. Spores composed of thick three-layered walls were found following histopathological examination and were diagnosed as adiaspiromycosis. Adiaspiromycosis has been found in mammals in many parts of the world. To our knowledge, this is the first report of adiaspiromycosis of an Apodemus agrarius captured in Korea.

Detection of sarcocystic infection in a wild rodent (Apodemus agrarius chejuensis) captured on Jeju island / 한국실험동물학회지

Sarcocystis spp is a causative agent of sarcocystosis. They have a characteristic life cycle infecting both prey and predator. Sarcocystis can cause myositis, atrophy of the adjacent cells and abortion in cattle. In mice, sarcocystosis causes mild cellular reactions without clinical disease. Severe haemorrhage and abortion were also reported. For monitoring the disease in wild rodents of the Korean peninsula, we captured Apodemus agrarius chejuensis on Jeju island and examined the specimen histopathologically. Intramuscular cysts were found and diagnosed as Sarcocystis. Sarcocystic infection has been reported in worldwide. There have been many reported infections in cattle and pigs in Korea. To our knowledge, this is the first report of Sarcocystis in Apodemus agrarius chejuensis captured in Korea.

Production of specific antibodies against SARS-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229E and OC43

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.

NaCl plus chitosan as a dietary salt to prevent the development of hypertension in spontaneously hypertensive rats

The effect of NaCl plus 3% chitosan on the systolic blood pressure of spontaneously hypertensive rats (SHR) were evaluated and compared with NaCl plus KCl (NaCl, 49.36% + KCl 49.36%) and chitosan or NaCl treatment alone. In SHR, administration of NaCl plus chitosan (44 mM Na/day) for two months significantly decreased the systolic blood pressure greater than of NaCl plus KCl and NaCl alone. NaCl plus chitosan resulted, though not statistically significant, in decreased urinary Na+ excretion and decreased blood urea nitrogen levels. Urinary creatinine of NaCl plus chitosan was slightly decreased compared to 3 treated groups. Serum electrolytes levels, however, remained unchanged. The combination of NaCl and chitosan may be superior to the conventional use of NaCl plus KCl or NaCl alone in the prevention of hypertension. Even though these supplementary diets have demonstrated potential anti-hypertensive effects in the experimental animal model, further research is needed before any recommendations can be made.

In vivo alternative testing with zebrafish in ecotoxicology

Although rodents have previously been used in ecotoxicological studies, they are expensive, time-consuming, and are limited by strict legal restrictions. The present study used a zebrafish (Danio rerio) model and generated data that was useful for extrapolating toxicant effects in this system to that of humans. Here we treated embryos of the naive-type as well as a transiently transfected zebrafish liver cell line carrying a plasmid (phAhREEGFP), for comparing toxicity levels with the well-known aryl hydrocarbon receptor (AhR)-binding toxicants: 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene. These toxicants induced a concentration-dependent increase in morphological disruption, indicating toxicity at early life-stages. The transient transgenic zebrafish liver cell line was sensitive enough to these toxicants to express the CYP1A1 regulated enhanced green fluorescent protein. The findings of this study demonstrated that the zebrafish in vivo model might allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development. We have also shown that the transient transgenic zebrafish liver cell line can be used for research on AhR mechanism studies.

Protective and therapeutic effects of an extract mixture of alder tree, labiate herb, milk thistle green bean-rice bran fermentation, and turnip against ethanol-induced toxicity in the rat

An herbal extract mixture and yogurt added to the herbal extract mixture were tested for their protective and therapeutic effects on ethanol-induced liver injury. The herbal extract mixture, yogurt and commercial drugs were used for treatment for two weeks prior to administering a single oral dose of ethanol (3 g/kg body weight). The herbal extract mixture and yogurt added to the herbal extract mixture were found to provide protection against ethanolinduced toxicity comparable to the commercial drug treatment, according to the serum and histopathological analysis. It was also shown that co-treatment with herbal extract mixture and yogurt against a triple oral dose of ethanol (2 g/kg body weight, over one week) provided protection against ethanol toxicity. After the initial set of experiments, the herbal extract mixture and yogurt treatments were extended for three more weeks. When compared to the positive control, further treatment with both the herbal extract and yogurt significantly reduced liver injury and resulted in a lower grade of lipid deposition.

Idiopathic intranuclear inclusion bodies in the renal tubular epithelia of Japanese quail (Coturnix coturnix japonica)

We report idiopathic intranuclear inclusion bodies in the renal tubular epithelia of two cases of among the 960 Japanese quail (Coturnix coturnix japonica) in the course of the acute oral toxicity and dietary toxicity test. Basophilic inclusion bodies were seen only in the nuclei of renal tubular epithelia. We could not classify our case into any adenovirus infection by clinical signs and lesions. The inclusion bodies were only identified as adenovirus-like particles based upon the electronmicroscopical features.