Development of a Simple ImageJ-Based Method for Dynamic Blood Flow Tracking in Zebrafish Embryos and Its Application in Drug Toxicity Evaluation

Abstract

This study aimed to develop a simple and cost-effective method to measure blood flow in zebrafish by using an image-based approach. Three days post fertilization (dpf) zebrafish embryos were mounted with methylcellulose and subjected to video recording for tracking blood flow under an inverted microscope equipped with a high-speed CCD camera. In addition, Hoffman lens was used to enhance the blood cell contrast. The red blood cell movement was tracked by using the TrackMate plug-in in the ImageJ image processing program. Moreover, Stack Difference and Time Series Analyzer plug-in were used to detect dynamic pixel changes over time to calculate the blood flow rate. In addition to blood flow velocity and heart rate, the effect of drug treatments on other cardiovascular function parameters, such as stroke volume and cardiac output remains to be explored. Therefore, by using this method, the potential side effects on the cardiovascular performance of ethyl 3-aminobenzoate methanesulfonate (MS222) and 3-isobutyl-1-methylxanthine (IBMX) were evaluated. MS222 is a common anesthetic, while IBMX is a naturally occurring methylxanthine. Compared to normal embryos, MS222- and IBMX-treated embryos had a reduced blood flow velocity by approximately 72% and 58%, respectively. This study showed that MS222 significantly decreased the heart rate, whereas IBMX increased the heart rate. Moreover, it also demonstrated that MS222 treatment reduced 50% of the stroke volume and cardiac output. While IBMX decreased the stroke volume only. The results are in line with previous studies that used expensive instruments and complicated software analysis to assess cardiovascular function. In conclusion, a simple and low-cost method can be used to study blood flow in zebrafish embryos for compound screening. Furthermore, it could provide a precise measurement of clinically relevant cardiac functions, specifically heart rate, stroke volume, and cardiac output.

Cardiovascular Performance Measurement in Water Fleas by Utilizing High-Speed Videography and ImageJ Software and Its Application for Pesticide Toxicity Assessment

“…Subsequently, water fleas mounted with 3% methylcellulose on Petri dishes were transferred to record the heartbeat and blood flow using an inverted microscope (ICX41, Sunny Optical Technology, China) equipped with a high-speed digital charged coupling device (CCD) (Zgenebio, Taipei, Taiwan). HiBestViewer (AZ Instrument, Taiwan) software was used to conduct high-speed video recording at 200 fps (frames per second) for 10 s. This study was performed according to our previously established method [28].…”

“…The heart rate was defined as beats per minute (bpm) and was obtained by dividing one minute (60 s) by the time interval. The heartbeat time interval was calculated by subtracting two consecutive time points [28,29] (detailed method can be found in the File S1).…”

An Overview of Methods for Cardiac Rhythm Detection in Zebrafish

“…Normally, heart rate assays in zebrafish embryo are performed on one embryo per video/imaging field [9,27]. The high-throughput method from multiple zebrafish embryos per imaging field was established [32].…”

“…On the other hand, another study proved that the heart rhythm calculation from blood flow velocity could be done using the ImageJ-based method [27]. The dorsal aorta (DA) vessels are directly connected to the heart chamber, making the DA regional blood flow pattern following the ventricle's pumping.…”

“…Heart rate measurement could also be done indirectly based on the recorded blood flow video. However, heart rate measurement calculated from blood flow patterns may lead to biased results [27]. According to Grimm and colleagues (1996), the variability of heartbeat interval could be determined not only from ECG but also from the blood pressure.…”