2.7. Vascular Response of the Transgenic Zebrafish Tg(flk:EGFP) Model

The transgenic zebrafish Tg(flk:EGFP) was used to perform an in vivo investigation [22]. Fish were housed in 3 L tanks (aquatic habitats). The zebrafish facility contained buffered water (pH 7.5) at 28.5 °C. Fertilized eggs were collected from the bottom of the tank in an automatic circulation culture system (ESEN, Beijing, China), maintained at a temperature of 28.5 °C, pH 7.5, dissolved oxygen 7.0, conductivity 800 µS, containing methylene blue. The eggs were placed in Petri dishes after thorough washing in the system water and then transferred to the incubator. For experiments, the larvae were first maintained in 12-well plates containing egg water (reverse osmosis water containing 60 mg sea salt per liter of water (pH 7.5)). After sample treatment, the number of larvae was checked daily. For DK, an in vivo toxicity test was performed using the zebrafish model as follows. The test was based on the exposure of newly fertilized zebrafish eggs to the test sample for up to 120 h; 15 eggs per treatment (three replicates) were selected and distributed in 12-well microplates. The test was initiated with newly fertilized eggs exposed to 4, 13, 40, and 134 µM of DK and run for 144 h. Embryos were observed for up to 144 h under a stereomicroscope (magnification used in the stereomicroscope for observations was 4×). The vasodilatory effect of DK in larvae was demonstrated as follows: Larvae were treated with 4, 13, 40, and 134 µM of DK at three days post-fertilization (3 dpf). After six days of treatment, the larvae were photographed using a fluorescence microscope; 4× magnification was used to capture vessels in the whole body. The fluorescence intensities of whole-body vessels were determined using Gen5 3.04 (Synergy HT, BioTek Instruments, Winooski, VT, USA) and then averaged. The fluorescence intensities of the images (4× magnification) were analyzed using ImageJ software.

The dorsal aorta (DA) was used to measure cardiac parameters, including heartbeat, mean linear flow, arterial pulse, and diameter. Images of vessel diameter were captured using a fluorescence microscope (Gen5 v.3.04 software, Synergy HT, BioTek Instruments, Winooski, VT, USA), and the mean value was analyzed and calculated using ImageJ software. Moreover, the arterial pulse (beats per minute), mean blood flow velocity (μm/s), and blood flow (nL/s) were determined using a pre-recorded video at 120 frames per second (fps) for 1 min using the Gen5 v.3.04 software (Synergy HT, BioTek Instruments, Winooski, VT, USA). Then, the MicoZebraLab application from ViewPoint (v.3.4.4, Lyon, France) was used to evaluate the cardiovascular parameters mentioned above.