Nd₂O₃ Nanoparticles Induce Toxicity and Cardiac/Cerebrovascular Abnormality in Zebrafish Embryos via the Apoptosis Pathway

Figures & data

Figure 1 Nd₂O₃ nanoparticles can be ingested by zebrafish. Representative confocal images are shown. (A–E) and (F–J) show Nd₂O₃ trajectory images in the digestive tract of two zebrafish embryos. Black arrows indicate the Nd₂O₃ particles. Whole time-lapse imaging can be seen in Supplementary Movie S1.

Figure 2 Effects of Nd₂O₃ on the mortality and malformation of larvae at 120 hpf. (A) The mortality of the larvae increased in a dose-dependent manner. Compared with the 0 ug/mL group. ***P≤0.001 (Student t test). (B) The percentage of abnormal embryos treated with Nd₂O₃. These experiments were repeated at least 3 times. (C) Light microscope image of zebrafish wild-type (WT) embryos at 5 dpf. (D–G) Light microscope images of embryos treated with Nd₂O₃ at 5 dpf. Black arrows indicate bent tail. White arrows indicate cardiac oedema.

Figure 3 Nd₂O₃ contributed to reduced heart rate and arrhythmia in zebrafish embryos. (A) Quantitative analysis of heart rate at 5 dpf. Compared with the 0 ug/mL group. **P≤0.01. (B) Representative results of heart rate analysis. (C) Representative results of arrhythmia analysis of Nd₂O₃ exposed embryo. Whole time-lapse imaging can be seen in Supplementary Movies S2–S5.

Figure 4 Nd₂O₃ disturbed the development of cerebrovascular at 5 dpf. (A, B, B’) The cerebrovascular images of wild-type embryo. (C, D, D’) The cerebrovascular images of embryos exposed to Nd₂O₃ (100 μg/mL). (E, F, F’) The cerebrovascular images of embryos exposed to Nd₂O₃ (200 μg/mL). Whole time-lapse imaging can be seen in Supplementary Movies S6–7. Scale bars, 100 μm.

Abbreviations: PrA, prosencephalic artery (white arrow); ACeV, anterior (rostral) cerebral vein (white arrowhead); MCeV, middle cerebral vein (yellow arrowhead); CtA, central artery (red arrow); PCeV, posterior (caudal) cerebral vein (red arrowhead); PHBC, primordial hindbrain channel (red arrowhead); MMCtA, middle mesencephalic central artery (yellow arrow); MtA, metencephalic artery (yellow arrow).

Figure 5 Nd₂O₃ disturbed vascular sprouting at 3 dpf. (A–C) The cerebrovascular images of wild-type embryo. (D–F) The cerebrovascular images of embryos exposed to Nd₂O₃ (100 μg/mL). (G–I) The cerebrovascular images of embryos exposed to Nd₂O₃ (200 μg/mL). Whole Z-steps lapse imaging can be seen in Supplementary Movies S8, S9 and S10. Scale bars, 100 μm.

Figure 6 The autophagy level was checked in Nd₂O₃ treated embryos. (A) Immunoblot analysis with autophagy marker Lc3. (B–J) Representative confocal images of mCherry–Lc3 puncta (autophagosomes) in Tg(Flk1:EGFP) live embryos. (K) Quantitative results were summarised in (B–J). mCherry–Lc3 puncta were counted in EGFP+ cells in 7–10 individual embryos. Error bars in (K) represent standard error of the mean (SEM). **P≤0.01 (Student’s t-test). Scale bars, 100 μm.

Figure 7 The apoptosis pathway was activated in Nd₂O₃-treated embryos. (A–I) The TUNEL staining of Nd₂O₃-treated embryos. (A–C) Representative images of WT embryos are shown. (D–F) Representative images of 100 μg/mL Nd₂O₃ treated embryos are shown. (G–I) Representative images of 200μg/mL Nd₂O₃-treated embryos are shown. (J) Immunoblot analysis with apoptosis marker Bax and Bcl-2. (K) Immunoblot analysis with apoptosis marker p53. (L) Immunoblot analysis with apoptosis marker cleaved caspase 3.