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Development of a Quantitative Morphological Assessment of Toxicant-Treated Zebrafish Larvae Using Brightfield Imaging and High Content Analysis
Citation:
Deal, S., J. Wambaugh, R. Judson, S. Moser, N. Radio, K. Houck, AND S. Padilla. Development of a Quantitative Morphological Assessment of Toxicant-Treated Zebrafish Larvae Using Brightfield Imaging and High Content Analysis. JOURNAL OF APPLIED TOXICOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, 36:1214-1222, (2016). https://doi.org/10.1002/jat.3290
Impact/Purpose:
This study was done to have a better way to measure the degree of malformations in zebrafish larvae. We wanted a method that was objective, automated as much as possible, and gave us a permanent record of each larvae. This approach should provide data that is more objective and defensible than in the past.
Description:
One of the rate-limiting procedures in a general toxicity screen using a developmental zebrafish (Danio rerio) assay is the morphological assessment of each larva. The present studies were designed to develop an objective, accurate, and rapid method for screening zebrafish larvae for dysmorphology. Most researchers opt for a structured visual assessment by trained human observer(s). In an effort to streamline our own testing paradigm, we have developed the Computational Malformation Index which combines the use of high-content imaging with a very brief human visual assessment instead of using the very detailed structured human assessment. At the end of exposure each larva was briefly assessed by a human observer (Basic Visual Assessment), euthanized, fixed, and assessed for dysmorphology with the Zebratox V4 BioApplication (Thermo Scientific) using the Cellomics® ArrayScan® VTI high content image analysis platform. The Basic Visual Assessment adds in-life parameters, and the high-content analysis assesses each individual larva for various features (e.g., total area, width, spine length, head-to-tail length, length-to-width ratio, perimeter-to-area ratio). In developing the Computational Malformation Index, a training set of hundreds of embryos treated with hundreds of different chemicals were visually assessed using a basic or detailed method. In the second phase we assessed both the stability of these high-content measurements as well as its performance using a test set of zebrafish treated with doses ranging from inactive to teratogenic for two reference chemicals. We found that the measures were stable for at least one week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our Computational Malformation Index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay.
URLs/Downloads:
DOI: Development of a Quantitative Morphological Assessment of Toxicant-Treated Zebrafish Larvae Using Brightfield Imaging and High Content Analysis
https://onlinelibrary.wiley.com/doi/epdf/10.1002/jat.3290