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.

Water fleas are a good model for ecotoxicity studies, and were proposed for this purpose by the United States Environmental Protection Agency, due to their easy culture, body transparency, and high sensitivity to chemical pollution. Cardiovascular function parameters are usually used as an indicator of toxicity evaluation. However, due to the nature of the heart and blood flow, and the speed of the heartbeat, it is difficult to perform precise heartbeat and blood flow measurements with a low level of bias. In addition, the other cardiovascular parameters, including stroke volume, cardiac output, fractional shortening, and ejection fraction, have seldom been carefully addressed in previous studies. In this paper, high-speed videography and ImageJ-based methods were adopted to analyze cardiovascular function in water fleas. The heartbeat and blood flow for three water flea species, Daphnia magna, Daphnia silimis, and Moina sp., were captured by high-speed videography and analyzed using open-source ImageJ software. We found the heartbeat is species-dependent but not size-dependent in water fleas. Among the three water fleas tested, D. magna was identified as having the most robust heartbeat and blood flow rate, and is therefore suitable for the ecotoxicity test. Moreover, by calculating the diameter of the heart, we succeeded in measuring other cardiovascular parameters. D. magna were challenged with temperature changes and a pesticide (imidacloprid) to analyze variations in its cardiovascular function. We found that the heartbeat of D. magna was temperature-dependent, since the heartbeat was increasing with temperature. A similar result was shown in the cardiac output parameter. We also observed that the heartbeat, cardiac output, and heartbeat regularity are significantly reduced when exposed to imidacloprid at a low dose of 1 ppb (parts per billion). The blood flow rate, stroke volume, ejection fraction, and fractional shortening, on the contrary, did not display significant changes. In conclusion, in this study, we report a simple, highly accurate, and cost-effective method to perform physiological and toxicological assessments in water fleas.

The heart is the most important muscular organ of the cardiovascular system, which pumps blood and circulates, supplying oxygen and nutrients to peripheral tissues. Zebrafish have been widely explored in cardiotoxicity research. For example, the zebrafish embryo has been used as a human heart model due to its body transparency, surviving several days without circulation, and facilitating mutant identification to recapitulate human diseases. On the other hand, adult zebrafish can exhibit the amazing regenerative heart muscle capacity, while adult mammalian hearts lack this potential. This review paper offers a brief description of the major methodologies used to detect zebrafish cardiac rhythm at both embryonic and adult stages. The dynamic pixel change method was mostly performed for the embryonic stage. Other techniques, such as kymography, laser confocal microscopy, artificial intelligence, and electrocardiography (ECG) have also been applied to study heartbeat in zebrafish embryos. Nevertheless, ECG is widely used for heartbeat detection in adult zebrafish since ECG waveforms’ similarity between zebrafish and humans is prominent. High-frequency ultrasound imaging (echocardiography) and modern electronic sensor tag also have been proposed. Despite the fact that each method has its benefits and limitations, it is proved that zebrafish have become a promising animal model for human cardiovascular disease, drug pharmaceutical, and toxicological research. Using those tools, we conclude that zebrafish behaviors as an excellent small animal model to perform real-time monitoring for the developmental heart process with transparent body appearance, to conduct the in vivo cardiovascular performance and gene function assays, as well as to perform high-throughput/high content drug screening.

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.

As a nicotinoid neurotoxic insecticide, imidacloprid (IMI) works by disrupting nerve transmission via nicotinic acetylcholine receptor (nAChR). Although IMI is specifically targeting insects, nontarget animals such as the freshwater shrimp, Neocaridina denticulata, could also be affected, thus causing adverse effects on the aquatic environment. To investigate IMI toxicity on nontarget organisms like N. denticulata, their physiology (locomotor activity, heartbeat, and gill ventilation) and biochemical factors (oxidative stress, energy metabolism) after IMI exposure were examined. IMI exposure at various concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 ppm) to shrimp after 24, 48, 72 h led to dramatic reduction of locomotor activity even at low concentrations. Meanwhile, IMI exposure after 92 h caused reduced heartbeat and gill ventilation at high concentrations. Biochemical assays were performed to investigate oxidative stress and energy metabolism. Interestingly, locomotion immobilization and cardiac activity were rescued after acetylcholine administration. Through molecular docking, IMI demonstrated high binding affinity to nAChR. Thus, locomotor activity and heartbeat in shrimp after IMI exposure may be caused by nAChR blockade and not alterations caused by oxidative stress and energy metabolism. To summarize, N. denticulata serves as an excellent and sensitive aquatic invertebrate model to conduct pesticide toxicity assays that encompass physiologic and biochemical examinations.

Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.

Safety is one of the most important and critical issues in drug development. Many drugs were abandoned in clinical trials and retracted from the market because of unknown side effects. Cardiotoxicity is one of the most common reasons for drug retraction due to its potential side effects, i.e., inducing either tachycardia, bradycardia or arrhythmia. The zebrafish model could be used to screen drug libraries with potential cardiotoxicity in a high-throughput manner. In addition, the fundamental principles of replacement, reduction, and refinement of laboratory animal usage, 3R, could be achieved by using zebrafish as an alternative to animal models. In this study, we used a simple ImageJ-based method to evaluate and screen 70 ion channel ligands and successfully identify six compounds with strong cardiotoxicity in vivo. Next, we conducted an in silico-based molecular docking simulation to elucidate five identified compounds that might interact with domain III or domain IV of the Danio rerio L-type calcium channel (LTCC), a known pharmaceutically important target for arrhythmia. In conclusion, in this study, we provide a web lab and dry lab combinatorial approach to perform in vivo cardiotoxicity drug screening and in silico mechanistic studies.

Tail coiling is a reflection response in fish embryos that can be used as a model for neurotoxic analysis. The previous method to analyze fish tail coiling is largely based on third-party software. In this study, we aim to develop a simple and cost-effective method called TCMacro by using ImageJ macro to reduce the operational complexity. The basic principle of the current method is based on the dynamic change of pixel intensity in the region of interest (ROI). When the fish tail is moving, the average intensity is increasing. In time when the fish freeze, the peak of mean intensity is maintaining at a relatively low level. By using the optimized macro settings and excel VBA scripts, all the tail coiling measurement processes can be archived with few operation steps with high precision. Three major endpoints of tail coiling counts, tail coiling duration and tail coiling intervals can be obtained in batch. To validate this established method, we tested the potential neurotoxic activity of Tricaine (methanesulfonate, MS-222) and psychoactive compound of caffeine. Zebrafish embryos after Tricaine exposure displayed significantly less tail coiling activity in a dose-dependent manner, and were comparable to manual counting through the Wilcoxon test and Pearson correlation double validation. Zebrafish embryos after caffeine exposure displayed significantly high tail coiling activity. In conclusion, the TCMacro method presented in this study provides a simple and robust method that is able to measure the relative tail coiling activities in zebrafish embryos in a high-throughput manner.