Tuesday, October 10, 2006 - 2:04 PM

Continuous Live Imaging of TGF- Activity Using a Novel Transgenic Mouse Wound-Healing Model

Thomas S. Satterwhite, BS, Alphonsus Chong, MD, Jian Luo, PhD, Hung Pham, BS, Melinda Costa, MD, Michael T. Longaker, MD, MBA, Tony Wyss-Coray, PhD, and James Chang, MD.

Purpose: Scar formation is a fundamental plastic surgery problem. TGF-1 expression correlates with increased scarring. We used a novel transgenic mouse system that allows real-time in vivo tracking of TGF-1 activity by measuring bioluminescence after skin wounding. Methods: The transgenic mouse model includes a Smad2/3-responsive luciferase (SBE-luc) construct. In vivo: Two dorsal excisional skin wounds were made using a skin biopsy punch. Bioluminescence was detected with the In Vivo Imaging System at fixed time points following surgery. In vitro: A TGF-1 dose response was conducted on dermal fibroblasts cultured from SBE-luc mice. Fibroblasts were then co-treated with TGF-1 and increasing doses of either neutralizing antibody to TGF- (NA-TGF) or SB-431542, an inhibitor of the TGF receptor. Bioluminescence was measured with an automatic luminometer. Statistical analysis was performed with Student's t-test and ANOVA. Results: In vivo: Bioluminescence peaked on Day 17 and was 4-fold higher than baseline (p<0.05). In vitro: Maximal luciferase activity was seen with 0.1 ng/ml of TGF-1 (p<0.05). Both NA-TGF and SB-431542 produced dose response inhibition, with complete inhibition achieved by 1 ug/ml and 1 uM, respectively (p<0.05). Conclusion: The in vivo data suggest that TGF activity can be measured through real-time imaging. The in vitro data suggest that the stably transfected SBE-luc cell line is responsive to TGF-1, and it can be inhibited in a Smad2/3-dependent fashion. The use of biophotonics and real-time imaging will translate to a more thorough understanding of the healing process, leading to the development of better treatments to prevent post-wound scarring.
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