Quantification of the vascular area of retina at 4 dpf using Fiji-ImageJ software. An example of a retinal vascular image: use Fiji-ImageJ “area measurement” function under “Analyze” to measure the relative vascular vessel area over the retinal area. (a) Total retinal area by drawing a uniform circle of 120 pixel diameter = 11304 pixel area. (b) Draw and measure areas outside the vascular vessels = 2393 + 1256 + 638 + 1640 + 259 + 366 = 6552. Then, calculate % vascular vessel area of the retina = (11304 − 6552)/11304 × 100% = 42.0%. ^∗Branch point of retinal vessels.

Effects of MG and glucose treatment on retinal angiogenesis in 4 dpf zebrafish. Zebrafish embryos were incubated with solution containing MG with or without glucose from 10 hpf to 4 dpf. (a) Fluorescence microscope observations of the retinal vessels of drug-treated embryos are shown at 4 dpf. The 1000 µM MG treatment with or without 30 mM glucose group induced retinal vessel branching and vascular area compared with the untreated control in zebrafish embryos. As the positive control, zebrafish embryos treated with VEGF inducer GS4012 (1 µM and 3 µM) showed severe branching and disorganization in the retinal vasculature. Quantification of the percentage of vascular area (b) and the number of branch points (c) in midperiphral retina by MG, glucose, and GS4012 treatment in zebrafish embryos at 4 dpf. n = 30 eyes of 15 zebrafish embryos per group; mean ± SEM. ^∗p < 0.05 and ^∗∗p < 0.01 vs control group. Data were analyzed using the one-way ANOVA and then Student's t-test. Glu, glucose. Ctl, control.

Effect of VEGF receptor inhibitor (PTK787) in MG-induced zebrafish. (a) Fluorescence microscope observations shown added 0.5 µM PTK787 in 1 dpf in the 1000 µM MG and 30 mM glucose treatment group reverted blood vessel hyperbranching in zebrafish embryos at 4 dpf. The proangiogenic effect induced by MG treatment was inhibited and exhibited no significant difference compared with the no-treatment control. Quantification of the percentage of vascular area (b) and number of branch points (c) in midperiphral retina by PTK787, MG, and glucose treatment in zebrafish embryos at 4 dpf. n = 30 eyes of 15 zebrafish embryos per group; mean ± SEM. ^∗p  <  0.05, ^∗∗p < 0.01 vs control group. p > 0.05 nonsignificant (NS). Data were analyzed using the one-way ANOVA and then Student's t-test. Glu, glucose. CTL, control.