18. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. Here, we demonstrate that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation. Abstract:Over the past decade, zebrafish are being increasingly used in assessing the … (C) Dystrophin-null fish. (A) Genotyping of each of 40 fish treated with each of the seven potential candidate chemicals. Aminophylline is a nonselective PDE inhibitor, and it is among a group of PDE inhibitors with different specificities (Table S1). Two fish models of DMD with dystrophin deficiency used for chemical screening were the sapje (stop codon in exon 4) and sapje-like (splice site mutation in exon 62) (12, 15) mutants. The fish also develop rapidly, and researchers can simply add drugs to the water to test the effects. These results suggest that treatment with chemical 4 restored the muscle structure of these dystrophin-null fish, because they had abnormal structure when selected at 4 dpf before chemical treatment. Researchers are still trying to understand what causes this strong correlation between neural and social networks. The Molecular Genetics Core Facility at Children’s Hospital Boston sequenced PCR products after PCR purification using a standard kit (Qiagen). Bars show the ratio of phosphorylated peptides and nonphosphorylated peptide. For all surviving fish treated with each of the seven compounds and showing apparent corrective results in the second screen, genotypes were determined using the genomic DNA extracted from heads of the individual fish (40 treated fish with each chemical). Here we describe the use of zebrafish bioassays for assessing … Using a two-tiered screening strategy, first, pooling compounds and then, screening individual compounds, resulted in identification of seven individual chemicals that decreased the percentage of phenotypically affected fish, which suggests that these seven chemicals might rescue the muscle phenotype found in the dystrophin-null fish. Zebrafish were introduced as a drug screening platform in the context of LQTS almost 20 years ago (Langheinrich et al., 2003; Milan et al., 2003; see Table 1). (A) Treatment with 0.1% DMSO had no e ect on vessel formation or embryo morphology. was an investigator with The Howard Hughes Medical Institute. These studies recognized the … - "Zebrafish angiogenesis: A new model for drug screening" Figure 3. Thank you for your interest in spreading the word on PNAS. Error bars indicate SDs. Copyright © 2021 Elsevier B.V. or its licensors or contributors. Recently, a number of chemical and drug screens have been published using zebrafish embryos (7–11). All restored fish with chemicals 1–7 showed no expression of dystrophin, similar to those of untreated dystrophin-null fish (C). The orthologs of many dystrophin–glycoprotein complex (DGC) components are expressed in zebrafish (19). The immunohistochemical results of the dystrophin expression showed that the chemical treatment did not restore dystrophin expression. This work was supported by National Institute of Neurological Disorders and Stroke Grant 5P50NS040828-09 and the Muscular Dystrophy Association. 1A). S3). S1). 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Zebrafish xenograft assays are ideal for testing drug sensitivity directly from patient biopsies in a personalized medicine context. Currently, prednisone is the only treatment option available for muscular dystrophy patients in the United States, although there are currently other options through approved clinical trials. As such, our laboratory has two available fish models of DMD with dystrophin deficiency, sapje (12) and sapje-like fish (15). We examine key findings and clinical implications of notable screens, and discuss limitations as well as recent technological improvements that offer potential solutions. This disorder results in a muscle pathology that can be detected by birefringence under polarizing light and usually results in death by 7–9 dpf. (D–J) Dystrophin immunostaining of fish with restored muscle structure with each of the seven chemicals (chemicals 1–7 in Table 2). analyzed data; and G.K. and L.M.K. Enoximone (PDE3 inhibitor; ◆), milrinone (PDE3 inhibitor; ◇), ibudilast (PDE4 inhibitor; ▲), rolipram (PDE4 inhibitor; △), sildenafil citrate salt (PDE5 inhibitor; ●), dipyridamole (PDE5 inhibitor; ○), aminophylline (nonselective PDE inhibitor; ■), and DMSO (vehicle; X). 4 C and D, P < 0.05). Adult Zebrafish Model for Screening Drug-Induced Kidney Injury Yuki Kato, Yuki Kato Drug Safety Evaluation, Research Laboratory for Development, Shionogi and . Approved drug could make radiation therapy more effective for head, neck cancer: study 4 hours ago Nearly pain-free microneedle patch can test for antibodies and more in the fluid between cells Because zebrafish offspring can survive in multiwell plates, if high-throughput phenotypes can be developed that represent the disease endpoints, a chemical suppressor screen can be undertaken to identify tool compounds or drug leads. The results are plotted (Fig. The six chemical pools (total of 48 chemicals) that had less than 7.5% of fish showing abnormal birefringence were selected as therapeutic candidates for restoring normal muscle function and were tested further in a secondary screening. Online ISSN 1091-6490. Abstract. These pathways may also have additional compounds that influence their expression. Dear Editor, Here we report findings of zebrafish behavior and development during drug screening. Blotted proteins were incubated with primary antibody, anti-PKA C (1:100; Cell Signaling Technology), anti-phospho-PKA C (Thr197, 1:100; Cell Signaling Technology), or anti–β-actin (1:500; Sigma). These embryos also showed markedly reduced expression of dystrophin in their myosepta. By April 21, 2020, the official number of Covid-19 cases comprised of 2,397,217 confirmed cases and 162,956 fatalities worldwide (W. H. O., 2020). For long-term treatment of dystrophin-deficient fish, mutant fish showing abnormal birefringence were identified under the dissection scope at 4 dpf and placed in a new plate to be treated with candidate chemicals 1–7 from 4 to 30 dpf; 10 affected and 10 unaffected embryos were arrayed in 24-well plates and cultured in 1 mL fish water containing individual chemicals at 28.5 °C starting at 4 dpf. Three chemicals proved toxic to zebrafish after prolonged incubation. Because of these advantages, zebrafish bioassays are cheaper and faster than mouse assays, and are suitable for large-scale drug screening. After excising the negatively charged phosphorylated and positively charged nonphosphorylated bands from the gel, the gels were melted at 95 °C. Both aminophylline and sildenafil citrate clearly decreased the percentage of dystrophin-null fish showing abnormal birefringence. Co., Ltd., Toyonaka, Osaka 561-0825, Japan. The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD), representing more than 90% of the diagnosed cases. Collectively, this work demonstrates that zebrafish embryos can be used to screen systematically for complex drug effects. Biology, Husbandry, Diseases, and Research Applications, American College of Laboratory Animal Medicine, Zebrafish as a Platform for Drug Screening. S1. 5): enoximone (PDE3 inhibitor), milrinone (PDE3 inhibitor), ibudilast (PDE4 inhibitor), rolipram (PDE4 inhibitor), sildenafil citrate salt (PDE5 inhibitor), dipyridamole (PDE5 inhibitor), aminophylline (nonselective PDE inhibitor), and DMSO (vehicle). In the nontreated fish used as controls, the distribution of mutant fish showing abnormal birefringence ranged between 17.5% and 27.5% (Fig. The Zebrafish Disease and Drug Screening Model: A Strong Ally Against Covid-19. Similarly, the zebrafish is also an attractive screening tool for cardiovascular risk assessment after treatment with atypical antipsychotic drugs, as it facilitates the evaluation of the … It is generally known that PDE inhibitors cause an increase in intracellular cAMP and/or cGMP. In the experimental groups tested with the chemical pools, the distribution of the affected fish ranged between 2.5% and 27.5%; 6 groups had less than 7.5% of mutant fish showing abnormal birefringence, and 16 groups had less than 10% (Fig. Muscular dystrophy is a disease in which the muscle forms normally at first but then degenerates faster than it can be repaired. Drug screening in zebrafish. These results indicate that action of these chemicals may be able to restore dystrophin-null muscle to normal muscle without restoring the dystrophin expression. In contrast to severing the tail fin, our assay using larval zebrafish … The active compounds in the library were selected for their high chemical and pharmacological diversity, as well as their known bioavailability and safety in humans. The treated dystrophin-null fish with aminophylline have normal structure in skeletal muscle. Here we describe the use of zebrafish bioassays for assessing … Keywords:Zebrafish, drug, toxicity screening, high throughput assay, neurotoxicity, hepatotoxicity, cardiotoxicity. 3). Frozen sections were obtained as for immunohistochemistry and stained with H&E. Keywords:Zebrafish, drug discovery, drug testing, large-scale screen, chemical genetics, pre-clinical studies. Zebrafish embryos were collected and raised at 28.5 °C according to standard procedures and criteria. 1E). To test whether other PDE inhibitors might also ameliorate dystrophic symptoms in sapje fish, 20 embryos from matings of heterozygous sapje fish were treated with a series of PDE inhibitors from 1 to 4 dpf (in triplicate). Imaging and behavioral assays can be used to gather information vital to drug development or toxicity testing. Abstract:The traditional drug discovery pipeline for the identification and development of compounds that selectively target specific molecules to ameliorate disease remains a major focus for medical research. Nontreated dystrophin-null fish have broken and disturbed structure (arrows) of skeletal muscle fibers. All sequencing was accomplished in the Intellectual and Developmental Disabilities Research Center Molecular Core Laboratory supported by National Institute of Child Health and Human Development Grant 2P30HD018655-26.