Using In Vivo Label-Free Multiphoton Microscopy To Monitor Wound Metabolism
Jake D. Jones, Hallie E. Ramser, Alan Woessner, Kyle P. Quinn.
University of Arkansas, Fayetteville, AR, USA.
BACKGROUND- Non-healing wounds, such as diabetic foot ulcers, are challenging to diagnose and treat due to their numerous etiologies and the variable efficacy of wound care products. There is a critical need to develop new diagnostic technologies and quantitative biomarkers that are sensitive to specific wound characteristics. Multiphoton microscopy (MPM) techniques are well-suited for 3D skin imaging and are capable of non-invasively detecting autofluorescence from metabolic cofactors (NADH and FAD) without the need for exogenous dyes. The objective of this study was to evaluate the utility of label-free MPM for characterizing wound healing in vivo.
METHODS- Full-thickness excisional wounds were produced in diabetic (streptozotocin-induced) and control C57BL/6J mice (n=7 mice/group). Using MPM, we isolated and measured an optical redox ratio of FAD/(NADH+FAD) autofluorescence at the wound edge to provide 3D maps of cellular metabolism over a 10-day period from each mouse.
RESULTS- A significant decrease in the optical redox ratio of the epidermis in both groups was observed between days 1 and 3 (p<0.003) and days 1 and 5 (p<0.005). By day 10, the redox ratio at the epithelial edge in the nondiabetic group had significantly increased (p<0.03) relative to days 3 and 5, while the diabetic mice displayed no significant temporal change. Ki-67 staining and wound closure rates indicate our optical measurements of cell metabolism are sensitive to the relative rates of keratinocyte proliferation and migration during healing. These findings demonstrate that keratinocytes at the edge of diabetic wounds remain in a proliferative state at later time points compared to control wounds.
CONCLUSIONS- Our work demonstrates label-free MPM offers potential to provide non-invasive optical biomarkers associated with different stages of skin wound healing, which may be used to detect impaired healing and guide treatment.
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