High-Throughput Video Processing of Heart Rate Responses in Multiple Wild-type Embryonic Zebrafish per Imaging Field

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.