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. 2018 Jul 19;8(1):10926.
doi: 10.1038/s41598-018-28687-1.

A Novel In Vivo Model to Study Impaired Tissue Regeneration Mediated by Cigarette Smoke

Marjorie Alvarez  1 , Myra N Chávez  1   2 , Miguel Miranda  1   3 , Geraldine Aedo  1 , Miguel L Allende  4 , José T Egaña  5
Affiliations

A Novel In Vivo Model to Study Impaired Tissue Regeneration Mediated by Cigarette Smoke

Marjorie Alvarez et al. Sci Rep. .

Abstract

Cigarette smoke is associated with several pathologies including chronic respiratory diseases and cancer. In addition, exposure to cigarette smoke is correlated with impaired wound healing, where a significant decrease in the regenerative capacity of smokers is well documented and broadly considered a negative risk factor after trauma or surgery. So far, some in vitro and in vivo models have been described to study how exposure to cigarette smoke diminishes the regenerative potential in different organisms. However, although useful, many of these models are difficult and expensive to implement and do not allow high-throughput screening approaches. In order to establish a reliable and accessible model, we have evaluated the effects of cigarette smoke extract (CSE) on zebrafish development and regeneration. In this work, zebrafish embryos and larvae were exposed to low doses of aqueous CSE showing severe developmental abnormalities in a dose-dependent manner. Furthermore, when adult zebrafish were subjected to caudal fin amputation, we observed a significant decrease in the regenerative capacity of animals exposed to CSE. The effect was exacerbated in male and aged fish compared to female or young organisms. The establishment of a zebrafish model to assess the consequences of cigarette smoke and its effects on animal physiology could provide a new tool to study the underlying mechanisms involved in impaired tissue regeneration, and aid the development of novel approaches to treat complications associated with cigarette smoke toxicity.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
Effects of cigarette smoke on zebrafish embryonic and larval development. The effect of increasing CSE-concentrations on zebrafish development at different embryonic and larval stages was evaluated and compared to a non-treated control (0.00% CSE) and based on the following parameters: normal head-trunk angle (A), correct somite development (B), normal otic vesicle development (C), normal eye development (D), normal pigmentation (E), heart edema formation (F), normal heart rate (G), normal blood circulation (H), swim bladder inflation (I), spontaneous movement inside the chorion (J), eclosion (K), response to mechanic stimulus (L). The percentage of embryos/larvae showing the abnormal phenotype, as described in the materials and methods section, is shown. Results were obtained from three independent experiments, using a total of N ≥ 120 embryos for each condition. *p < 0.05.
Figure 3
Figure 3
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.
Figure 4
Figure 4
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.
Figure 5
Figure 5
Effect of cigarette smoke in the regeneration potential of zebrafish of different gender and age. Tail fin regeneration outcome was evaluated between control and CSE-treated zebrafish of different ages and gender. While CSE affects regeneration in all conditions, note (in C) that male fish are more susceptible to the effect than female fish, and (in F) that aged fish are more susceptible than young ones. Results represent three independent experiments using a total of N ≥ 10 zebrafish for each condition. *p < 0.05.
Figure 6
Figure 6
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.
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