Abstract
Heart rate assays in wild-type zebrafish embryos have been limited to analysis of one embryo per video/imaging field. Here we present for the first time a platform for high-throughput derivation of heart rate from multiple zebrafish (Danio rerio) embryos per imaging field, which is capable of quickly processing thousands of videos and ideal for multi-well platforms with multiple fish/well. This approach relies on use of 2-day post fertilization wild-type embryos, and uses only bright-field imaging, circumventing requirement for anesthesia or restraint, costly software/hardware, or fluorescently-labeled animals. Our original scripts (1) locate the heart and record pixel intensity fluctuations generated by each cardiac cycle using a robust image processing routine, and (2) process intensity data to derive heart rate. To demonstrate assay utility, we exposed embryos to the drugs epinephrine and clonidine, which increased or decreased heart rate, respectively. Exposure to organic extracts of air pollution-derived particulate matter, including diesel or biodiesel exhausts, or wood smoke, all complex environmental mixtures, decreased heart rate to varying degrees. Comparison against an established lower-throughput method indicated robust assay fidelity. As all code and executable files are publicly available, this approach may expedite cardiotoxicity screening of compounds as diverse as small molecule drugs and complex chemical mixtures.
Highlights
Zebrafish have a functional two-chambered heart within the first two days of life[16], and their transparent skin during early embryonic development allows for unimpeded visualization of the heart using light microscopy, making heart rate a widely measured endpoint in embryonic zebrafish
Video files of each well were processed to acquire heart rate in two steps: (1) use of FisHRateZ, an original algorithm, that capitalizes on the anatomical features of the embryo to automatically locate the heart and derive average pixel intensity vs. time data generated from the cardiac cycle of each embryo, and (2) use of a custom fast Fourier Transform script written and run in open-source software that enables batch derivation of heart rate from FisHRateZ data
Following the programmed illumination time, light was interrupted via microprocessor-controlled shutter (Sutter Instruments, Novato, CA), and the motorized stage moved the well into view and the process was repeated until completion of the plate
Summary
Zebrafish have a functional two-chambered heart within the first two days of life[16], and their transparent skin during early embryonic development allows for unimpeded visualization of the heart using light microscopy, making heart rate a widely measured endpoint in embryonic zebrafish. Some combination of attributes that limit reliability, throughput, and/or transferability to other researchers These limitations include the use of transgenic lines that express fluorescent proteins in cardiac tissue requiring fluorescence microscopy for visualization[12,17], the use of costly software and video microscopy systems or robotics, and/ or the use of software-hardware combinations that are not commonplace or transferable[18,19,20]. The main objective of this study was to develop a distributable, simplified, and automated video processing platform that facilitates analysis of single videos or entire directories of videos to measure heart rate of multiple zebrafish embryos per imaging field. Given the linkage of respirable particulate matter (PM), a complex mixture of environmental contaminants with adverse cardiovascular outcomes[2,23,24,25], we compared the effects of organic extracts from different sources of air pollution-derived PM, including petroleum diesel and soy biodiesel exhausts, and red oak biomass smoke
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