Two-Photon Fluorescence Lifetime Imaging for Metabolic Profiling of Cochlear Dysfunction

Abstract

More than 120,000 individuals treated with lifesaving antibiotics develop hearing or balance disorders annually. Although research has shown that of the two cochlear cell types, sensory and supporting cells, sensory cells are readily damaged due to age-related hearing loss, acoustic trauma and ototoxins, the reason for this remains unknown. Furthermore, cochlear sensory hair cells can be divided into two types, inner and outer hair cells (IHCs, OHCs). OHCs in the high-frequency region of the cochlea exhibit the greatest sensitivity to the above conditions. To determine if variations in sensory and supporting mitochondrial metabolism account for these differences, two-photon fluorescence lifetime microscopy (FLIM) was used to measure changes in the metabolic reporter molecule NADH in sensory and supporting cells from explanted murine cochleae. Mitochondrial uncouplers, inhibitors and an ototoxic antibiotic, gentamicin (GM), were used to assess high- and low-frequency IHC, OHC and supporting cell mitochondrial metabolism. Chemically induced changes in metabolic state resulted in a reorganization of specific NADH lifetimes into altered subcellular fluorescence lifetime pools. Variations in NADH intensity and average NADH lifetime were greatest in high-frequency OHCs. Pretreatment with GM significantly increased NADH intensity in high-frequency sensory cells but not supporting cells. Treatment with GM significantly increased the average NADH fluorescence lifetime within IHCs but not OHCs. GM also caused a significant increase of NADH concentration in OHCs, not IHCs. These results demonstrate: differences between sensory and supporting cell metabolism; GM alters mitochondrial metabolism; and IHCs and OHCs display differing metabolic effects when exposed to GM. Such fundamental differences between sensory and supporting cell mitochondrial metabolism indicate differing metabolic changes during antibiotic exposure. Understanding these antibiotic-induced metabolic changes may explain the ototoxic effects of these drugs which is crucial for preventing and treating numerous auditory deficits and diseases.