Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed (e.g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47^phox and p67^phox, but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs.

Destruction of auditory hair cells results in a subsequent loss of auditory neurons. In situ hybridization and neuronal cell culture studies as well as analyses of the inner ears of neurotrophin and neurotrophin receptor gene knockout mice have shown that NT-3 and BDNF mediate both the development and survival of auditory neurons. In this study guinea pigs were exposed to the ototoxic combination of an aminoglycoside antibiotic and a loop diuretic and then received 8 weeks of intracochlear infusion of either NT-3, BDNF or NT-3 + BDNF to determine whether site-specific application of these neurotrophins could prevent the loss of auditory neurons that follows a loss of auditory hair cells. Infusion of either NT-3 or NT-3 + BDNF into the scala tympani resulted in a > 90% survival of auditory neurons while BDNF infusion yielded a 78% survival rate, compared with a 14–24% neuronal survival rate in untreated ototoxin-exposed cochleae. These results show that loss of auditory neurons that occurs subsequent to a loss of auditory hair cells can be prevented by in vivo neurotrophin therapy with either NT-3 or BDNF.