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

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.

A Reliable Preclinical Model to Study the Impact of Cigarette Smoke in Development and Disease

“…To regulate vacuum intensity, and thus the time it takes for a cigarette to be smoked, it is necessary to build an air pressure regulator into the system (plastic stopcock) and then empirically estimate its optimal setting for proper smoking conditions. To confirm the reproducibility of the extraction method, it is possible to measure the pH and tar content of the CSE obtained, as we previously described (Alvarez et al, 2018). Finally, it is highly recommended to perform the extraction under an air-extraction fume hood to avoid exposure.…”

“…Alvarez et al. () evaluated angiogenesis in the caudal fin amputation model in adult fish that were pretreated with CSE. In this model, CSE exposure had no effect on the revascularization capacity of exposed zebrafish.…”

“…Current Protocols in Toxicology Data analysis 11. To evaluate fin regeneration, use appropriate image analysis software (e.g., ImageJ; Alvarez et al, 2018) to measure the initial and regenerating caudal fin area using the amputation plane as a reference.…”

“…This method allows for selectively trapping the watersoluble components of cigarette smoke in a stock solution, but the protocol may be slightly modified to extract the lipophilic molecules if an organic solvent is chosen. Once this cigarette smoke extract (CSE) is generated, it can be easily handled and used for further toxicity-related tests, both in vitro (Wahl, Schenck, Machens, & Egaña, 2016) and in vivo (Alvarez et al, 2018).…”

“…In this article, we describe a protocol to study the toxic effects of water-soluble compounds from cigarette smoke in larvae and adult zebrafish, as we have previously described (Alvarez et al, 2018). Given the relevance of angiogenesis and wound healing, with this protocol we aimed to evaluate how cigarette smoke impacts vascular development and tissue regeneration.…”