R. M. Clark1, B. Coffman1, D. A. Ramirez2, S. Godoy2, S. A. Meyers2, T. McGregor2, S. Krishna2, P. G. McGuire3, T. R. Howdieshell1 1University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 2Skinfrared LLC,Albuquerque, NM, USA 3University Of New Mexico HSC,Cell Biology And Physiology,Albuquerque, NM, USA
Introduction: Murine models have provided valuable insight into the pathogenesis of both diabetes and chronic wounds. The db/db mouse possesses a spontaneous mutation in the leptin receptor gene resulting in obesity, hyperglycemia, hyperinsulinemia, hypercholesterolemia, and insulin resistance. This strain is characterized by impaired epithelialization of excisional wounds, with little known about wound neovascularization.
Methods: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of C57BL6 (wild-type) and db/db mice (n=16 total; 5 mice per operative group and 3 unoperated mice per group as controls). Planimetric analysis of serial digital photographic images was utilized to determine flap viability in wild-type and db/db mice. Real-time myocutaneous flap perfusion and surface temperature were determined by laser speckle contrast and thermal infrared imaging respectively. Mice with surgical flaps were sacrificed on postoperative day 10. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Quantitative RT-PCR was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression.
Results: Day 10 planimetric analysis revealed a mean flap viability of 95% in db/db mice compared to 79% in wild-type mice, and 60% of wild-type mice developed distal flap dehiscence not evident in db/db mice. Over 10 days, laser speckle contrast imaging documented markedly increased perfusion at all times points (p<0.001) with functional revascularization to supranormal perfusion in db/db flaps. In contrast, wild-type flaps displayed expected graded flap ischemia with failure to return to baseline perfusion during the postoperative period. Thermal infrared imaging documented complementary spatiotemporal assessment of thermal-metabolic tissue state. Immunostaining confirmed significant differences in preoperative and postoperative microvascular density (mean post-op distal vessel count 60 ± 8 versus 80 ± 15 vessels/mm2 for wild-type and db/db mice respectively, p=0.018; mean post-op distal vascular surface area 4108 ± 95 versus 8250 ± 795 µm2/mm2 for wild-type and db/db mice respectively, p<0.001). Finally, quantitative RT-PCR demonstrated statistically significant differences in angiogenic gene expression between wild-type and db/db mice at baseline (unoperated) and at day 10.
Conclusion: In a graded-ischemia wound healing model, accelerated myocutaneous revascularization and improved wound healing were evident in db/db mice compared to wild-type controls. Gene expression analysis reveals the db/db mouse may be “primed” for wound neovascularization and warrants further investigation into the role of the leptin-leptin receptor axis in wound angiogenesis.