Evaluation of cigarette smoke toxicity in zebrafish. 3–5 hpf zebrafish embryos were subjected to increasing concentrations of CSE for 96 hours. Representative pictures of the larvae after 48 hours of treatment are shown (A). Zebrafish survival was recorded daily, showing a dose-dependent lethal effect of continuous CSE-exposure in zebrafish larvae (B,B’). Results were acquired from three independent experiments, using a total of N ≥ 120 embryos for each condition. *p < 0.05. Scale bar represents 200 μm.

Effect of cigarette smoke on zebrafish vascular development. An impairment of intersegmental vessel (ISVs) formation and extension in one-day old transgenic Tg(fli1a:EGFP)^y1 embryos was observed upon exposure to increasing CSE-concentrations for 24 hours. The number of anastomosed ISVs was quantified as a parameter for vessel maturation for the five anterior vessels proximal to the urogenital opening for each CSE-condition. Results were obtained from three independent experiments, using a total of N ≥ 90 larvae for each condition. *p < 0.05. Scale bars represent 100 μm.

Effect of cigarette smoke on adult zebrafish survival and regeneration potential. The lethal effect of CSE was studied in 6–12 month adult zebrafish exposed to increasing dilutions of cigarette smoke extract for 10 days. Results represent three independent experiments, using a total of N ≥ 15 zebrafish for each condition (A). Zebrafish, which underwent tail fin amputation showed diminished regeneration capacity, when exposed to a pre- and post-conditioning treatment with non-lethal concentrations of CSE (0.25%, 20-day treatment in total), as shown by the diminished regenerated fin tissue compared to non-treated condition. Results were obtained from three independent experiments, using a total of N ≥ 25 adult zebrafish for each condition (B,C). *p < 0.05. Scale bar represents 1 mm.

Effect of cigarette smoke extract in adult zebrafish vascular regeneration. Adult Tg(fli1a:EGFP)^y1 zebrafish were exposed to a 0.25% dilution of CSE for ten days before, and 10 days after tail fin amputation. The rate of revascularization was calculated using the signal of the fin fluorescent endothelial cells and normalized to the total regenerated area. While exposure to CSE limited fin regrowth in the treated group, re-vascularization of the fin was not affected. Results were obtained from three independent experiments using a total of N ≥ 20 zebrafish for each condition. *p < 0.05. Scale bars represent 1 mm.