Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Mie University

[Genomic drug discovery of vascular remodeling].

GPRC5A is a member of G-protein-coupled receptors, which was originally identified as an all-trans-retinoic acid-induced gene. Although recent studies reported that this gene was highly expressed in the cancer cell lines and that GPRC5A might positively regulate cell proliferation, its mechanism remains unknown. We investigated the upstream and downstream signaling of GPRC5A and its biological function, and found that cAMP signaling is the novel GPRC5A induction pathway. When GPRC5A gene was overexpressed, intracellular cAMP concentration was decreased, and Gsalpha gene expression was downregulated. On the other hand, RNA interference of GPRC5A increased mRNA levels of Gsalpha and intracellular cAMP, reduced cell number, and induced apoptosis. Conversely, cell number was increased by GPRC5A overexpression. We first report the novel negative feedback model of cAMP signaling through GPRC5A gene expression. This evidence explains one of the mechanisms of the GPRC5A-regulated cell growth in some cancer cell lines.

Novel reciprocal regulation of cAMP signaling and apoptosis by orphan G-protein-coupled receptor GPRC5A gene expression.

We cloned and pharmacologically characterized the guinea pig cysteinyl leukotriene (CysLT) 2 receptor (gpCysLT2). gpCysLT2 consists of 317 amino acids with 75.3%, 75.2%, 73.3% identity to those of humans, mice and rats, respectively. The gpCysLT2 gene is highly expressed in the lung, moderately in eosinophils, skin, spleen, stomach, colon, and modestly in the small intestine. CysLTs accelerated the proliferation of gpCysLT2-expressing HEK293. Leukotriene C4 (LTC4) and Leukotriene D4 (LTD4) enhanced the cell proliferation higher than Bay-u9773, a CysLT2 selective partial agonist and a nonselective antagonist for CysLT receptors. Bay-u9773 did not antagonize the cell proliferation by LTC4 and LTD4. Despite the equipotency of the mitogenic effect among these chemicals, calcium mobilization (CM) levels were variable (LTC4> LTD4> Bay-u9773), and Bay-u9773 antagonized the CM by LTC4. Moreover, the Gi/o inhibitor pertussis toxin perfectly inhibited agonist-induced cell proliferation. These results reveal that cell proliferation via CysLT2 signaling was mediated by Gi/o signaling but independent of calcium mobilization.

Guinea pig cysteinyl leukotriene receptor 2 (gpCysLT2) mediates cell proliferation and intracellular calcium mobilization by LTC4 and LTD4.

The most important strategies in pharmacogenomics are gene expression profiling and the network analysis of human disease models. We have previously discovered novel drug target candidates in cardiovascular diseases through investigations of these pharmacogenomics. The significant induction of S100C mRNA and protein expression was detected in the rat pulmonary hypertension and myocardial infarction model. We also found increased taurine in hypoxia, a calcium-associated cytoprotective compound, to suppress the hypoxia-induced S100C gene expression and vascular remodeling. These results suggest that S100C may be one of the potential novel drug targets in hypoxic or ischemic diseases. Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage causes cerebral ischemia and infarction. Using a DNA microarray, a prominant upregulation of heme oxygenase-1 (HO-1) and heat shock protein (HSP) 72 mRNAs were observed in the basilar artery of a murine vasospasm model. Antisense HO-1 and HSP 72 oligodeoxynucleotide inhibited HO-1 and HSP 72 induction, respectively, and significantly aggravated cerebral vasospasm. Moreover, we have also developed a unique heart failure model in zebrafish and identified several candidate genes as novel drug targets. These results suggest that pharmacogenomic network analysis has the potential to bridge the gap between in vitro and in vivo studies and could define strategies for identifying novel drug targets in various cardiovascular diseases.

Pharmacogenomics of cardiovascular pharmacology: pharmacogenomic network of cardiovascular disease models.

Beta adrenergic receptors (beta-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of beta-ARs in zebrafish are, however, largely unknown. We have identified zebrafish beta-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these beta-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.

Zebrafish beta-adrenergic receptor mRNA expression and control of pigmentation.

Cerebral vasospasm remains a major cause of morbidity and mortality in patients with subarachnoid hemorrhage. Heme oxygenase-1 (HO-1) is an oxidative stress-inducible enzyme with multiple protective functions against vascular and neurological diseases, including delayed cerebral vasospasm. In the present study, intravenous administration (i.v.) of nicaraven (1 mg/kg/min, for 2 days after subarachnoid hemorrhage) ameliorated delayed cerebral vasospasm in rat subarachnoid hemorrhage models, marked synergistic induction of HO-1 protein (> 2.5-fold than 'subarachnoid hemorrhage with saline i.v.'), and elicited a rapid increase of cGMP accumulation in the basilar arteries. In the sham-operated rats, nicaraven could not induce HO-1 expression. Antisense HO-1 oligodeoxynucleotides abrogated this HO-1 induction and the antivasospastic effect of nicaraven. In vitro study using Hela cells, nicaraven enhanced the human HO-1 promoter (-4.5 kbp) activity, which was pre-activated with the blood component oxyhemoglobin to mimic the ability of subarachnoid hemorrhage. These results suggest that this enhanced HO-1 expression through a combination of pathological state and pharmacological agent could be an effective strategy to improve the prognosis of heme- and oxidative stress-induced diseases, such as delayed cerebral vasospasm.

Synergistic induction of heme oxygenase-1 by nicaraven after subarachnoid hemorrhage to prevent delayed cerebral vasospasm.

Obesity is a multifactorial disorder influenced by genetic and environmental factors. Animal models of obesity are required to help us understand the signaling pathways underlying this condition. Zebrafish possess many structural and functional similarities with humans and have been used to model various human diseases, including a genetic model of obesity. The purpose of this study was to establish a zebrafish model of diet-induced obesity (DIO).

Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.

High dietary intake of vegetable products is beneficial against obesity and its related diseases such as dyslipidemia, nonalcoholic fatty liver disease, and cancer. We previously developed a diet-induced obesity model of zebrafish (DIO-zebrafish) that develops visceral adiposity, dyslipidemia, and liver steatosis. Zebrafish is a polyphagous animal; thus we hypothesized that DIO-zebrafish could be used for transcriptome analysis of anti-obesity effects of vegetables.

Transcriptome analysis of anti-fatty liver action by Campari tomato using a zebrafish diet-induced obesity model.

A novel protocol using gluten as a carrier material was developed to administer chemicals to adult zebrafish, per os (p.o.). To evaluate the capacity of gluten to retain chemicals, we prepared gluten granules containing eight types of chemicals with different Log P(ow) values and immersed them in water. Less than 5% of chemicals were eluted from gluten granules within 5 min, a standard feeding time for zebrafish. Although retention capability was dependent on the hydrophilicity and hydrophobicity of the chemicals, the gluten granules retained 62%-99% of the total amount of chemical, even after immersion in water for 60 min. Vital staining dyes, such as 4-Di-2-Asp and Nile red, administered p.o., were delivered into the gastrointestinal tract where they were digested and secreted. Subsequently, we conducted a pharmacokinetic study of oral administration of felbinac and confirmed that it was successfully delivered into the blood of zebrafish. This indicates that chemicals administered using gluten granules are satisfactorily absorbed from the digestive tract and delivered into the metabolic system. The absorption, distribution, and pharmacokinetics of chemicals given by oral administration were also compared with those of chemicals given by alternative administration routes such as intraperitoneal injection and exposure to chemical solution.

A novel protocol for the oral administration of test chemicals to adult zebrafish.

Collecting blood from laboratory animals is necessary for a wide variety of scientific studies, but the small size of the zebrafish makes this common procedure challenging. We developed a novel, minimally invasive method to collect repeated blood samples from adult zebrafish. This method minimizes trauma to the zebrafish and yields a low mortality rate of 2.3%. The maximum volume of blood that can be collected using this technique is approximately 2% of body weight. To avoid blood loss anemia and hemorrhagic death, we recommend that the total blood sample volume collected over repeat bleeds should be ≤0.4% of body weight per week, and ≤1% of body weight per 2 weeks. Additionally, we applied this method to the study of zebrafish glycolipid metabolism by measuring blood glucose and plasma triacylglyceride levels weekly over a 5-week period in both control and overfed zebrafish. The overfed fish developed significantly increased fasting blood glucose levels compared with normally fed fish. This new method of blood collection is essential for zebrafish or other small aquarium fish research requiring repeated blood samples, and increases the utility of the zebrafish as a model animal in hematological studies of human diseases.

A novel, reliable method for repeated blood collection from aquarium fish.

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.

Fluorescent-based methods for gene knockdown and functional cardiac imaging in zebrafish.

Lemon (Citrus limon) contains various bioactive flavonoids, and prevents obesity and obesity-associated metabolic diseases. We focused on eriocitrin (eriodictyol 7-rutinoside), a powerful antioxidative flavonoid in lemon with lipid-lowering effects in a rat model of high-fat diet. To investigate the mechanism of action of eriocitrin, we conducted feeding experiments on zebrafish with diet-induced obesity. Oral administration of eriocitrin (32 mg/kg/day for 28 days) improved dyslipidaemia and decreased lipid droplets in the liver. DNA microarray analysis revealed that eriocitrin increased mRNA of mitochondrial biogenesis genes, such as mitochondria transcription factor, nuclear respiratory factor 1, cytochrome c oxidase subunit 4, and ATP synthase. In HepG2 cells, eriocitrin also induced the corresponding orthologues, and reduced lipid accumulation under conditions of lipid loading. Eriocitrin increased mitochondrial size and mtDNA content, which resulted in ATP production in HepG2 cells and zebrafish. In summary, dietary eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.

Liqing Zang

Department of Translational Medical Science,
Graduate School of Medicine

Mie University

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Liqing Zang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Translational Medical Science,Graduate School of Medicine

Mie University

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Department of Translational Medical Science,
Graduate School of Medicine