Two-Photon Imaging of NADH in SKH1 Mice Reveals Changes in Keratinocyte Metabolism with Chronic UVA Exposure

Abstract

Cancer development is characterized by altered cell metabolism. Alterations of growth-factor signaling pathways adjust the metabolic phenotype to better support tumorigenesis and disease progression, which affects the concentration and binding characteristics of endogenous coenzymes such as nicotinamide adenine dinucleotide (NADH) and flavoproteins. To characterize metabolic changes induced by chronic UVA exposure in vivo, we designed and constructed a multiphoton laser scanning imaging platform specifically for in situ metabolic imaging of NADH within the skin of anesthetized mice. Age matched SKH1 hairless mice were divided equally into sham and UV treatment groups, with the treatment group receiving daily illumination of 15 kJ/m2 UVA. NADH phasor FLIM was used to characterize metabolism of keratinocytes as a function of cell layer within the epidermis in both groups over a period of months. Early changes in the enzyme-bound-to-free ratio of NADH were clearly evident prior to the onset of disease, with UVA- treated mice showing significantly greater proportion of enzyme-bound NADH. Basal keratinocytes generally had a lower bound-to-free ratio than keratinocytes from stratum spinosum or granulosa layers. Keratinocytes in vivo had a relatively greater fraction of bound NADH compared to squamous cell carcinoma cells in vitro. Changes in skin metabolism with normal aging was also clearly evident. This study demonstrates the feasibility and challenges of phasor FLIM metabolic imaging for long term longitudinal studies. This study was made possible by grants from the National Institute for General Medical Science (NIGMS) (5P20GM103427), a component of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.