Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.

Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23+/+ and Cdh23v2J/+ mice, but failed to detect GTTR uptake in Cdh23v2J/v2J hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23v2J/v2J and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.

ORAL PRESENTATIONSdecompression surgery are low risk of hearing impediment and long effective period after onset of paralysis.Objective: Test the hypothesis that open mechanotransducer (MET) channels are required for aminoglycoside entry and toxicity in auditory hair cells.Method: Cochlear cultures from postnatal 4-day-old rats were treated with gentamicin with and without MET channel blockers. The effects of MET channel blockers on hair cell survival were assessed by cell counts after immunohistochemistry. Two-photon imaging was used to quantify uptake of TexasRed-conjugated gentamicin (GTTR) into live hair cells.Results: Gentamicinalone (0.1mM) caused 76.5% hair cell loss in the basal cochlear turn. Cotreatment with MET channel blockers, 1.0 mM curare, and 0.5 mM quinine conferred protection (0.4% and 7.4% hair cell loss, both P < .001); whereas treatment with endocytosis inhibitors, 2.6 µM conconavalin A and 80 µM dynosore, did not (63.7% and 76.0% hair cell loss). Two-photon imaging showed that GTTR uptake (3 µM) into live hair cells was rapid and selective. Quinine and curare, but not concanavalin A, reduced GTTR uptake. Furthermore, as low calcium increases the open probability of MET channels, hypocalcemia showed facilitated GTTR uptake.Conclusion: Functional MET channels are required for aminoglycoside uptake into hair cells and its toxicity. Results suggest that limiting permeation of aminoglycosides through MET channels is a potential therapeutic target for preventing hair cell death. Otology/Neurotology Habituation following Tinnitus Retraining Therapy in Tinnitus SufferersJiun Fong Thong (presenter); Mei Ching Wong; S. Junaidah; Yew Meng Chan, FRCS Objective: Evaluate the efficacy of Tinnitus Retraining Therapy (TRT) in habituating patients with tinnitus.Method: Prospective study of patients presenting to the tinnitus clinic at a tertiary referral otorhinolaryngology unit in Singapore over a 13-year period (1997 to 2010). Patients who underwent Tinnitus Retraining therapy were followed up with structured interviews with the aid of questionnaire forms. Habituation following TRT was evaluated.Results: A total of 702 patients were studied (55% male, 45% female). Average age was 51 years. Habituation of reaction to tinnitus and habituation of perception were analyzed. Average duration of follow-up was 33 months (range, 0.25-151 months). A total of 68% of patients described improvement in annoyance following TRT. Of these, 80% described habituation of perception as well. There was no statistical difference in gender and age between patients who did and did not respond to TRT. However, duration of treatment was significantly longer in patients who habituated. Conclusion:The goal of TRT is to achieve habituation of reaction to tinnitus. Habituation of perception is often a secondary result of sufficiently habituated response. From our study, more than two-thirds of patients with tinnitus achieved habituation of reaction, and of these, the majority also habituated to awareness of the tinnitus. Otology/Neuroto...

Presentation schedule is subject to change. For the most up-to-date information, visit www.entannualmeeting.org. intervention. The objective of this report is to describe a case of osteoradionecrosis (ORN) of the temporal bone that was managed medically with PENTOCLO (pentoxifylline-tocopherol-clodronate). The use of PENTOCLO in the treatment of ORN of other sites in the head and neck will also be described.Methods: We report the case of a patient treated at our institution and present a review of literature. A 52-year-old woman presented with ORN of the temporal bone 20 years following radiation therapy for an ipsilateral parotid tumor. The patient had chronic infections and otorrhea.Results: She failed conservative treatment including antibioticsteroid ear drops and aural lavage. As an alternative to hyperbaric oxygen and/or temporal bone resection, treatment with PENTOCLO was pursued and her condition improved dramatically. A literature review of ORN of the temporal bone and the use of PENTOCOLO in the management of ORN of other sites of the head and neck are described.Conclusions: PENTOCLO was effectively used in the management of our patient and may represent a valuable management option for other patients with ORN of the temporal bone. Further research is required to determine the effectiveness of PENTOCLO.Objectives: (1) Demonstrate gentamicin uptake from perilymph into hair cells via basolateral channels such as the postsynaptic acetylcholine receptor (nAChR) and the TRPA1 in neonatal mice in vivo and in vitro. (2) Recognize alternative gentamicin trafficking pathways to the well established endolymphatic mechanoelectrotransducer channel underlying aminoglycoside ototoxicity.Methods: Neonatal mice were intraperitoneally injected with fluorescently-conjugated Gentamicin-Texas Red (GTTR), prior to fixation 30 minutes later. Cochlear explants were treated with GTTR 1 µg/mL, with or without 100 µM acetylcholine or cinnamaldehyde then fixed, and analyzed. Fluorescence intensities were measured by calculating the mean gray value of each cell with Image J. Student t test was used to determine any significant differences in fluorescence values between treatment groups.Results: Application of acetylcholine increased GTTR uptake by wildtype outer hair cells (OHC) in vivo and in vitro. These results were also replicated in Myo7a8J/8J mice, which lack functional mechanoelectrotransducer channels. Application of cinnamaldehyde (TRPA1 agonist) also increased GTTR uptake in OHCs in wildtype, myo7a8J/8J mice and guinea pigs.Conclusions: Endolymphatic gentamicin trafficking via the mechanoelectrotransducer (MET) channel is a well established mechanism of ototoxicity. Noise-induced threshold shifts (NITS) have been shown to damage the mechanically gated tip link, thus closing MET channel. However, aminoglycoside hair cell uptake is enhanced by NITS. This suggests one or more aminoglycoside entry route(s) into hair cells in addition to, and independent of, the MET channel. The results of this study support perilymphatic ...