Fluorescence Lifetime Imaging Microscopy Reveals Metabolic Changes in Aging Lacrimal Gland Suggestive of Increased Inflammation and Oxidative Stress

Although dry eye disease (DED) is common with age, the cellular and metabolic changes in the lacrimal gland (LG) that contribute to this condition are not fully un-derstood. Here, we applied fluorescence lifetime imaging microscopy (FLIM) to ex-amine metabolic alterations in LG tissue from aged (20-22 months) female C57BL/6J mice, a model of age-related DED, versus young (~3 months) C57BL/6J mice. Phasor analysis of NAD(P)H fluorescence revealed a shift in aged LG toward more glycolytic metabolism and reduced oxidative phosphorylation. We recently identified a novel subpopulation of F4/80-enriched multinucleated macrophages rich in lipids and lipid metabolizing enzymes in aged female mice. Using FLIM combined with immuno-labeling enabled isolation of the metabolic signature of these macrophages, confirming their increased NADPH oxidase 2 (NOX2) activity, an enzyme which generates reactive oxygen species which are characteristically expressed in M1-type macrophages. In-creased phosphorylation of P47phox, associated with NOX2 activation, was also ob-served in these macrophages, supporting their classification as M1-like cells. FLIM thus provides a valuable tool both to capture metabolic changes in the LG overall, and in defining metabolic features of specific cell populations that may be important in diseases such as DED.