A Simple ImageJ-Based Method to Measure Cardiac Rhythm in Zebrafish Embryos

Sampurna, Bonifasius Putera; Audira, Gilbert; Juniardi, Stevhen; Lai, Yu‐Heng; Hsiao, Chung-Der

doi:10.3390/inventions3020021

Cited by 36 publications

(57 citation statements)

References 35 publications

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“…Heart rate is defined as beats per minute (bpm) and can be obtained by multiplying the time interval (in seconds) between two peaks by a factor of 60. Furthermore, to validate the result of the heartbeat, the heart rate was measured directly from the heart by following a previously established ImageJ-based method [34].…”

Section: Blood Flow Pattern and Heart Rate Calculationmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Blood Flow Pattern and Heart Rate Calculationmentioning

confidence: 99%

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

et al. 2019

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“…The calculation principles and mathematical formulae for these cardiophysiological parameters are summarized in Figure 3 C–E. The use of a high-speed digital charged coupling device (CCD) and a Hoffman lens enabled the ventricle volume and other parameters to be measured with high precision in concordance with our previously published method [ 63 ]. A significant decrease in heart rate at the ventricle was observed after incubation at a sublethal dose of CuSO 4 at 10 nM, but this bradycardia phenotype was rescued if the Cu 2+ -exposed embryos were cotreated with a recombinant GHK tripeptide, even at the lowest concentration of 1 nM.…”

Section: Resultsmentioning

confidence: 70%

Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish

Hsiao

Malhotra

et al. 2020

Biomolecules

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In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO4-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time.

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“…Furthermore, we also validate the result by recording at a different ambient temperature as D. magna used in this research have the ectothermic property [17,18]. We also uses astemizole to induce arrhythmia in zebrafish [10] and test whether our kymograph method is able to detect the corresponding cardiac performance changes.…”

Section: Introductionmentioning

confidence: 78%

“…This study aimed to develop a kymographbased method built in ImageJ platform [16] to provide a fast, easy-to-use and inexpensive one-stop method to obtain multiple cardiac performance endpoints with high accuracy for both Daphnia neonates and zebrafish embryos. To validate our method's accuracy, we compared the overall performance with a previously established method [10]. Furthermore, we also validate the result by recording at a different ambient temperature as D. magna used in this research have the ectothermic property [17,18].…”

Section: Introductionmentioning

confidence: 94%

“…The current method for heartbeat measurement in Zebrafish and Daphnia involves record the dynamic pixel changes in the region of interest (ROI) by using either third-party or custom-designed software. Previously, we proposed a simple ImageJ-based method to measure cardiac rhythm in zebrafish embryos [10]. Heartbeat was recorded using a digital charged coupling device (CCD) mounted on a dissecting microscope, and the dynamic pixel changes were extracted from two consecutive frames.…”

Section: Introductionmentioning

confidence: 99%

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One-Stop Method to Measure Multiple Cardiac Performance Endpoints in Daphnia and Zebrafish by Kymograph

Kurnia¹,

Saputra²,

Roldan³

et al. 2020

Preprint

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Cardiovascular disease (CVD) is the number one cause of death worldwide. This condition resulted in huge research on CVD increasing the need for animal models suitable for the in vivo research. Daphnia and Zebrafish are good animal models for cardiovascular research due to their relative body transparency and easy culture property. Several methods have been developed to conduct cardiac performance measurement in Daphnia and Zebrafish. However, most of the methods only able to obtain heartbeat rate. The other important cardiac endpoints like stroke volume, ejection fraction, fraction shortening, cardiac output and heartbeat regularity must use other programs for measurement. To overcome this limitation, in this study, we successfully developed a one-stop ImageJ-based method using kymograph macros language that able to obtain multiple cardiac performance endpoints simultaneously for the first time. To validate its utility, we incubate Daphnia magna at different ambient temperatures and exposed zebrafish with astemizole to detect the corresponding cardiac performance alterations. In summary, the kymograph method reported in this study provides a new, easy to use, and inexpensive one-stop method on obtaining multiple cardiac performance endpoints with high accuracy and convenience.

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A Simple ImageJ-Based Method to Measure Cardiac Rhythm in Zebrafish Embryos

Cited by 36 publications

References 35 publications

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

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

Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish

One-Stop Method to Measure Multiple Cardiac Performance Endpoints in Daphnia and Zebrafish by Kymograph

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