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Reproducible strategy for excisional skin-wound-healing studies in mice

Nature Protocols volume 19, pages 184–206 (2024)Cite this article

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Abstract

Wound healing is a complex physiological process involving various cell types and signaling pathways. The capability to observe the dynamics of wound repair offers valuable insights into the effects of genetic modifications, pharmaceutical interventions or other experimental manipulations on the skin-repair process. Here, we provide a comprehensive protocol for a full-thickness, excisional skin-wound-healing assay in mice, which can easily be performed by any scientist who has received an animal welfare course certificate and can be completed within ~3 h, depending on the number of animals. Crucially, we highlight the importance of considering key aspects of the assay that can dramatically contribute to the reliability and reproducibility of these experiments. We thoroughly discuss the experimental design, necessary preparations, wounding technique and analysis. In addition, we discuss the use of lineage-tracing techniques to monitor cell migration, differentiation and the contribution of different cell populations to the repair process. Overall, we explore key aspects of the skin-wound-healing assay, supplying a detailed procedure and guidelines essential for decreasing variability and obtaining reliable and reproducible results.

Key points

  • The protocol provides an accurate method for performing a full-thickness excisional wound-healing (WH) assay in murine dorsal skin with high reproducibility.

  • This method can improve WH studies by decreasing deviations and improving reliability, eventually reducing the number of animals required per experiment. In addition, the assay can be combined with lineage-tracing experiments to investigate the contribution of different cell populations to the repair process.

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Fig. 1: Classical models of skin wound healing.
Fig. 2: Schematic outlines of the bulge-oriented WH assay.
Fig. 3: Detailed stages of the WH experiment.
Fig. 4: Visualization of lineage-traced scarred epidermal whole mounts.

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Data availability

All raw data, including measurements for Supplementary Fig. 1b and original images for Fig. 4, are available in Supplementary Data 2. Source data are provided with this paper.

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Acknowledgements

We thank current and former members of the Fuchs group for their insights and experience. We thank M. Yosopuva, R. Ankawa and N. Goldberger, for their technical assistance; V. Zlobin and Y. Nissan for animal house support; and N. Dahan and Y. Lupu-haber for insights regarding microscopy. We extend special thanks to D. Kulinsky and A. Hezi-Yamit for valuable input. Y.F. is supported by the EMBO Young Investigator program.

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Authors and Affiliations

  1. Augmanity, Rehovot, Israel

    Matan Yampolsky, Ido Bachelet & Yaron Fuchs

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  1. Matan Yampolsky
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  2. Ido Bachelet
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  3. Yaron Fuchs
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Contributions

M.Y. and Y.F. designed, analyzed and conceived the project. M.Y. performed imaging. M.Y, I.B. and Y.F. wrote the manuscript.

Corresponding author

Correspondence to Yaron Fuchs.

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Competing interests

M.Y., I.B. and Y.F. are employees and/or shareholders of Augmanity, a privately funded research institute in Rehovot, Israel. The authors declare no competing interests.

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Related links

Key references using this protocol

Koren, E. et al. Nat. Commun. 13, 4628 (2022): https://doi.org/10.1038/s41467-022-31629-1

Ankawa, R. et al. Dev. Cell 56, 1900–1916.e5 (2021): https://doi.org/10.1016/j.devcel.2021.06.008

Extended data

Extended Data Fig. 1 Whole-mount preparation of a sealed wound.

a, HFs in the vicinity of the wound edge enter the anagen phase, extending to the hypodermis and hindering epidermis-dermis separation. b, Scar tissue (30 d after wound inflection) is scrapped from fat and immersed entirely in EDTA to weaken the epidermal-dermal connection. c, Under a stereomicroscope, using two pairs of forceps, the epidermis is gently separated from the dermis. d, The dermis is initially fixed with a low volume of fixative to prevent the tissue from folding.

Extended Data Fig. 2 Calculating the wound area by using the Fiji (Image J) Software.

a–c, Open high-resolution scan in Fiji (a) and select the ‘Make Binary’ command to transform the image to white & black (b and c). The red square in c defines the selected zoomed-in area in D. d–g, Zoom-in to detect small gaps in the outlines (the purple-dashed square marks the gapped area). Use the hand-free selection tool (d, blue square) and the ‘Clear’ command to fill the gaps. Use the ‘Wand’ tool (g, blue square) to select the area you wish to calculate and press ‘M’ on your keyboard to perform the measurements. The values will be shown in a pop-up window and can be copied directly to an Excel file.

Supplementary information

Supplementary Information

Supplementary Protocols 1–3, Discussion and Figs. 1 and 2

Supplementary Data 1

Theoretical data analysis template

Supplementary Data 2

Source data for Supplementary Fig. 1B (graph)

Source data

Source Data Fig. 4

Original images

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Yampolsky, M., Bachelet, I. & Fuchs, Y. Reproducible strategy for excisional skin-wound-healing studies in mice. Nat Protoc 19, 184–206 (2024). https://doi.org/10.1038/s41596-023-00899-4

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