Multiphoton NAD(P)H FLIM reveals metabolic changes in individual cell types of the intact cochlea upon sensorineural hearing loss

An increasing volume of data suggests that changes in cellular metabolism have a major impact on the health of tissues and organs, including in the auditory system where metabolic alterations are implicated in both age-related and noise-induced hearing loss. However, the difficulty of access and the complex cyto-architecture of the organ of Corti has made interrogating the individual metabolic states of the diverse cell types present a major challenge. Multiphoton fluorescence lifetime imaging microscopy (FLIM) allows label-free measurements of the biochemical status of the intrinsically fluorescent metabolic cofactors NADH and NADPH with subcellular spatial resolution. However, the interpretation of NAD(P)H FLIM measurements in terms of the metabolic state of the sample are not completely understood. We have used this technique to explore changes in metabolism associated with hearing onset and with acquired (age-related and noise-induced) hearing loss. We show that these conditions are associated with altered NAD(P)H fluorescence lifetimes, use a simple cell model to confirm an inverse relationship between τbound and oxidative stress, and propose such changes as a potential index of oxidative stress applicable to all mammalian cell types.