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

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.

Automated high-throughput heartbeat quantification in medaka and zebrafish embryos under physiological conditions

“…To date, several methods have been used to assess heartbeat in zebrafish hatchlings, medaka embryos, fish species, Drosophila, cardiomyocytes or mouse embryos 21,22,24,25,27,28,[32][33][34][35][36] . Some of these methods require transgenic embryos with fluorescent cardiomyocytes or erythrocytes to measure heartbeats 26,32 .…”

“…Readouts of heart rate and rhythm have been employed increasingly in zebrafish-and medaka-based screens [21][22][23][24][25][26][27][28] . Challenges of current methods to capture heart rate in high-throughput include: (i) immobilisation of dechorionated embryos or hatchlings, which in the majority of cases is achieved by anaesthesia with tricaine leading to severe adverse cardiovascular effects 29 , (ii) imaging of larvae in standardized orientations, involving labour-intensive manual mounting on dedicated sample carriers or embedding in low-melting agarose, (iii) fluorescent reporter readouts, which preclude studies of specific genetic backgrounds and (iv) lack of user-friendly software for efficient analysis of multiple hearts simultaneously.…”

Automated high-throughput heart rate measurement in medaka and zebrafish embryos under physiological conditions

“…To date, several methods have been used to assess heartbeat in zebrafish hatchlings, medaka embryos, fish species, Drosophila, cardiomyocytes or mouse embryos 23,24,26,27,29,30,[39][40][41][42][43] . Some of these methods require transgenic embryos with fluorescent cardiomyocytes or erythrocytes to measure heartbeats 28,39 .…”

“…Another caveat is immobilization of fish larvae in low-melting agarose to achieve specific orientations, which is time-consuming and complicates genuine high-throughput assays. Some methods were restricted to a narrow time window of newly hatched zebrafish prior to swim bladder inflation to avoid movements of the larvae during sampling 30,39 . In addition, larvae were acclimatized to the illumination of the well scans.…”

“…Readouts of heart rate and rhythm have been employed increasingly in zebrafish-and medaka-based screens [23][24][25][26][27][28][29][30] . Challenges of current methods to capture heart rate in high-throughput include: (i) immobilization of dechorionated embryos or hatchlings, which in the majority of cases is achieved by anesthesia with tricaine leading to severe adverse cardiovascular effects 31 , (ii) imaging of larvae in standardized orientations, involving labor-intensive manual mounting on dedicated sample carriers or embedding in low-melting agarose, (iii) fluorescent reporter readouts, which preclude studies of specific genetic backgrounds and (iv) lack of user-friendly software for efficient analysis of multiple hearts simultaneously.…”