Endogenous Differences in Cochlear Sensory and Supporting Cell Mitochondrial Metabolism Bias Free Radical Production during Ototoxin Exposure

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Aminoglycosides, including gentamicin (GM), are the most frequently used antibiotics in the world despite irreversible cochlear damage and hearing loss associated with their use. Although there are numerous causes of deafness, reactive oxygen species (ROS) are key regulators of multiple pathologies including: ototoxicity, noise-induced and age-related hearing loss. Unfortunately, the source of these cell-damaging ROS remains controversial. Given that ROS are normal byproducts of ATP synthesis, intrinsic differences in cochlear sensory (inner and outer hair cell, I/OHC) and supporting (pillar) cell mitochondrial metabolism may explain why high-frequency OHCs are profoundly sensitive to a host of challenges. Mitochondrial metabolism was compared in low-frequency and high-frequency IHCs, OHCs and pillar cells from acutely cultured cochlear explants. Intensity-based changes in the metabolic intermediate, nicotinamide adenine dinucleotide (NADH), were used to measure endogenous, mitochondrial toxin and GM-induced differences in sensory and supporting cell mitochondrial metabolism. Sensory cell mitochondrial metabolism was significantly enhanced relative to supporting cells. Despite similar amounts of mitochondria in IHCs and OHCs, endogenous levels of NADH were greatest in high-frequency OHCs. Metabolic profiling of NADH metabolism revealed basal turn, high-frequency OHCs to be metabolically responsive to a number of changes in their microenvironment including metabolic toxins and GM, while high-frequency IHCs, low-frequency I/OHCs and, pillar cells are substantially less sensitive. DHR-123 was used to detect sensory and supporting cell ROS production during GM exposure. Within 30 minutes of GM application, ROS are dramatically and specifically increased in high-frequency sensory cells. GM-induced changes in mitochondrial metabolism and cell-damaging free radical production were greatest in high-frequency OHCs. This metabolic predisposition biases basal turn OHC responses to a variety of cochlear insults, particularly those involving energy metabolism and ROS production.