Re: Examining the contribution of surrounding intact skin during cutaneous healing

We would like to thank the authors of the letter for their concerns. Taking into account the nature of our experiment, anesthesia was inevitable for ethical and feasibility reasons. Anesthesia minimizes discomfort in animals during surgery and prevents movements during the procedure. It is important to note that the use of isoflurane is an acceptable and established procedure in various types of animal models for wound healing models (Singer & McClain, 2003; Dorsett-Martin, 2004; Dunn et al. 2013; Ansell et al. 2014). We acknowledge that murine models do not fully recapitulate the human body, and therefore more experiments are required to confirm our findings in humans. However, our experiment provides a first step towards understanding the effect of the intact skin surrounding the wound on wound healing. Furthermore, the issue of the potential effect of isoflurane on granulation tissue formation through attenuating inflammation was raised by the authors. Whether isoflurane had an effect on granulation tissue formation was not in the scope of our study, but it is important to note that by using proper controls, we were able to show the effect of the surrounding intact skin on granulation tissue formation despite both groups receiving isoflurane prior to the experiment. Thus, by having both groups receive similar isoflurane treatments, this potential confounding factor was eliminated through proper controls. By focusing on the independent variable (i.e. perforated vs nonperforated dome) our experiments are properly controlled to minimize the effect of all other variables (including anesthesia). In general, this increased the validity of the results by holding all of the other factors constant for both groups, while having the only variable between the groups as the difference in perforation. Thus, these conditions suggest that any differences seen between the two groups in the results are due to the perforation or non-perforation of the dome. We have to emphasize that although proper controls minimize the effect of other variables, we usually aim to minimize the variables as much as possible. For instance, the papers that have been cited by the author either used long exposure times to isoflurane which could have resulted in a greater effect or the experiments were performed after a short period of time from the isoflurane treatment (Du et al. 2017; Liu et al. 2017; Zhang et al. 2019). In our study, the mice were exposed to isoflurane for a few minutes (5 min), and the wounds were harvested after 11 days. Upon researching exposure times in the literature, no studies were found indicating an effect of 5-min exposure of isoflurane on wound healing or inflammatory response. Nonetheless, as discussed, we believe that having the right controls as previously mentioned, minimize potential confounding factors, if any. We acknowledge the authors’ concern that ‘isoflurane inhalation can inhibit the activation of nuclear factor (NF)-κB’ and the effect of NF-κB p65 translocation on NF-κB activation to induce inflammation. Although the cited paper used 12 min of isoflurane exposure, which was closer to our exposure time, the paper did not address changes in NF-κB p65 in response to isoflurane (Thiele et al. 2019). Instead, they have discussed the effect of anesthesia on NF-κB inhibitor alpha (also known as IκBα). There are two important factors to note in this study. The first, the increase in NF-κB inhibitor alpha in response to anesthesia was only found to be in response to propofol, not isoflurane. The second, both anesthesia groups showed an increase in other proinflammatory factors, such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α. In fact, showing an increase in NF-κB inhibitor alpha does not necessarily indicate an anti-inflammatory effect, as increases in the levels of this protein are a normal response to inflammation and one of the main upregulated genes in reaction to NF-κB signaling pathway as a negative feedback loop (Nelson et al. 2004). Thus, more studies would be required to examine the direct effect of isoflurane on NF-κB levels. In conclusion, although it is possible that granulation tissue was compromised by isoflurane, using proper control and minimizing variables in experiments allowed us to draw scientific conclusions as we have shown that granulation tissue formation increased in thickness which highlights the role of the surrounding intact skin. This does not mean that our ongoing effort to use variables with less effect (e.g. a less comprising effect than isoflurane) is not warranted.

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