Live imaging of Smad2/3 signaling in mouse skin wound healing

Authors

  • Alphonsus K.S. Chong MD,

    1. Divisions of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California,
    2. VA Palo Alto Health Care System, Palo Alto, California, and
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  • Thomas Satterwhite BS,

    1. Divisions of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California,
    2. VA Palo Alto Health Care System, Palo Alto, California, and
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  • Hung M. Pham BS,

    1. Divisions of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California,
    2. VA Palo Alto Health Care System, Palo Alto, California, and
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  • Melinda A. Costa MD,

    1. Divisions of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California,
    2. VA Palo Alto Health Care System, Palo Alto, California, and
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  • Jian Luo PhD,

    1. Department of Neurology and Neurological Sciences, Stanford School of Medicine, Stanford, California
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  • Michael T. Longaker MD,

    1. Divisions of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California,
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  • Tony Wyss-Coray PhD,

    1. Department of Neurology and Neurological Sciences, Stanford School of Medicine, Stanford, California
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  • James Chang MD

    1. Divisions of Plastic and Reconstructive Surgery, Stanford University Medical Center, Stanford, California,
    2. VA Palo Alto Health Care System, Palo Alto, California, and
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Reprint requests:
James Chang, MD, Division of Plastic and Reconstructive Surgery, Stanford University Medical Center, 770 Welch Road, 4th Floor, Stanford, CA 94305.
Tel: +1 650 723 5824;
Email: Changhand@aol.com

ABSTRACT

Biophotonics and real-time imaging are novel technologies that can greatly enhance the study of complex biological processes. We applied this technology in a transgenic mouse with a luciferase reporter gene fused to a transforming growth factor-β (TGF-β) responsive Smad2/3-binding element to study bioluminescence after skin wounding. Two dorsal midline excisional skin wounds were made using a biopsy punch. One wound was randomized to suture closure and the other allowed to heal by secondary intention (n=8 each wound). Bioluminescence was measured at fixed time points following surgery. Phospho-Smad2/3 immunohistochemistry was performed to localize expression in skin wound samples. In vivo bioluminescence increased following skin wounding. Peak activity occurred on day 17 and was fourfold that of baseline (p<0.05). Subgroup analysis of primary and secondary healing showed that primarily sutured wounds had peak activities earlier than those with secondary healing, although this did not reach statistical significance. Intense phospho-Smad2/3 staining was found in the hair follicles. In vivo bioluminescence tracks Smad2/3-dependent TGF-β signaling in the in vivo wound healing process. Our findings suggest that signaling increases after wound healing, which contrasts with other studies that show raised TGF-β signaling in the initial days following wounding.

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