Quantifying Age-Related Changes in Skin Wound Metabolism Using Multiphoton Microscopy.

The elderly are at high risk for developing chronic skin wounds, but the effects of intrinsic aging on skin healing are difficult to isolate due to common comorbidities like diabetes. Our objective is to use multiphoton microscopy (MPM) to find endogenous, noninvasive biomarkers to differentiate changes in skin wound healing metabolism between young and aged mice . We utilized MPM to monitor skin metabolism at the edge of full-thickness, excisional wounds in 24- and 4-month-old mice of both sexes for 10 days. MPM can assess quantitative biomarkers of cellular metabolism by utilizing autofluorescence from the cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). An optical redox ratio of FAD/(NADH+FAD) autofluorescence and NADH fluorescence lifetime imaging revealed dynamic changes in keratinocyte function during healing. Aged female mice demonstrated an attenuation of keratinocyte proliferation during wound healing detectable optically through a higher redox ratio and longer NADH fluorescence lifetime. By measuring the correlation between NADH lifetime and the optical redox ratio at each day, we also demonstrate sensitivity to the proliferative phase of wound healing. Label-free MPM was used to longitudinally monitor individual wounds , which revealed age-dependent differences in wound metabolism. These results indicate MPM can provide quantitative biomarkers of age-related delays in healing, which can be used in the future to provide patient-specific wound care.