(WHS-L1.02) Quantifying The Interaction Between Age And Diabetes On Skin Wound Metabolism Using In Vivo Multiphoton Microscopy
Thursday, May 16, 2024
10:30 AM – 11:30 AM East Coast USA Time
Diabetic foot ulcers are a significant health care burden. Current diagnostic approaches are primarily limited to symptomatic assessment and wound size monitoring, and new diagnostic tools are lacking. Therefore, there is a need for non-destructive imaging methods capable of characterizing and predicting healing outcomes. We have previously demonstrated that label-free multiphoton microscopy (MPM) can serve as a non-invasive diagnostic tool for skin wounds. Through MPM, we can visualize the autofluorescence of metabolic cofactors NADH and FAD, as well as collagen organization in 3D at a high-resolution. We have shown an optical redox ratio of FAD/(NADH+FAD) fluorescence intensities of the wound epithelium is sensitive to both diabetes- and age-related delays in healing. However, the interaction of these comorbidities on the epithelial redox ratio has not been elucidated. The goal of this study was to quantify the interaction of diabetes and age effects on wound closure and metabolism using in vivo label-free MPM. Young (5 mo.) and aged (24 mo.) streptozotocin-induced diabetic (n=17; 50/50 sex split) and control (n=17; 50/50 sex split) C57BL/6J mice received full-thickness, excisional wounds on their dorsum. Wounds were imaged longitudinally over 10 days using a multiphoton microscope that collected NADH (755nm excitation/460nm emission) and FAD (855nm excitation/525nm emission) autofluorescence. Image z-stacks were acquired at three wound edge locations using a 20x, 1.0 NA objective. Wound boundaries were manually traced to evaluate the rate of closure. Changes in the optical redox ratio within the epithelium were assessed using a multi-factor ANOVA and posthoc Tukey’s HSD tests. Wound size measurements indicated that closure was most delayed in aged diabetic mice, while closure was fastest in young non-diabetic controls. The epithelial redox ratio at the wound edge changed over 10 days similar to previous studies. The epithelial redox ratio of young non-diabetic control mice significantly decreased (p < 0.0001) by day 3 during the initial inflammatory phase, which then increased during re-epithelialization, allowing the redox ratio to return to baseline levels by day 10. Aged control mice, however, did not exhibit a significant change in wound metabolism until day 7 (p < 0.0001), suggesting delays during the inflammatory phase. The epithelial redox ratio of young and aged diabetic mice significantly decreased by day 3 (p < 0.0029) like young control mice, but did not increase at later time points. While not statistically different from other groups, aged diabetic mice had the lowest redox ratio at day 10. In conclusion, these findings suggest that in vivo label-free MPM is sensitive to diabetes-induced delays in healing, independent of the metabolic effects associated with advanced age.