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Automated measurement of the spontaneous tail coiling of zebrafish embryos as a sensitive behavior endpoint using a workflow in KNIME.

ABSTRACT: Neuroactive substances are the largest group of chemicals detected in European surface waters. Mixtures of neuroactive substances occurring at low concentrations can induce adverse neurological effects in humans and organisms in the environment. Therefore, there is a need to develop new screening tools to detect these chemicals. Measurement of behavior or motor effects in rodents and fish are usually performed to assess potential neurotoxicity for risk assessment. However, due to pain and stress inflicted on these animals, the scientific community is advocating for new alternative methods based on the 3R principle (reduce, replace and refine). As a result, the behavior measurement of early stages of zebrafish embryos such as locomotor response, photomotor response and spontaneous tail coiling are considered as a valid alternative to adult animal testing. In this study, we developed a workflow to investigate the spontaneous tail coiling (STC) of zebrafish embryos and to accurately measure the STC effect in the KNIME software. We validated the STC protocol with 3 substances (abamectin, chlorpyrifos-oxon and pyracostrobin) which have different mechanisms of action. The KNIME workflow combined with easy and cost-effective method of video acquisition makes this STC protocol a valuable method for neurotoxicity testing.•Video acquisition duration of 60 s at 25 ± 1 hpf was used•20 embryos exposed per dish and acclimatized for 30 min before video acquisition•Capability to inspect and correct errors for high accuracy.

SUBMITTER: Ogungbemi AO

PROVIDER: S-EPMC8374338 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

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Automated measurement of the spontaneous tail coiling of zebrafish embryos as a sensitive behavior endpoint using a workflow in KNIME.

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