Negative hearing effects of a single course of IV aminoglycoside therapy in cystic fibrosis patients.

OBJECTIVE: Identify hearing effects of a single course of intravenous (IV) aminoglycoside antibiotics (AGs) therapy in adult cystic fibrosis (CF) patients. Determine whether the change is large enough to enable a proof-of-concept study of a new drug preventing AG-associated hearing loss. DESIGN: Retrospective case review of CF patients with sequential audiograms ± an intervening course of IV AG therapy. STUDY SAMPLE: 84 patients with no intervening IV AG treatment, 38 patients undergoing a single course of IV AGs. RESULTS: Using ASHA ototoxicity metrics, 45% of adult CF patients in the Single-IV group met the criteria for ototoxicity compared to 23% of the No-IV patients. Other hearing metrics including the average maximal threshold shift (TS) and average high frequency TS showed highly significant differences between groups. Testing only participants with mild or greater pre-therapy high frequency hearing loss further increased the differences between the two groups by every metric tested. CONCLUSION: Adult CF patients exposed to a single course of IV AGs have significantly greater TS than patients without IV AG exposure. Patients with mild to moderate hearing loss prior to AG-IVs are at increased risk of developing ototoxicity from subsequent parenteral AG therapy.

Activity of coenzyme Q 10 (Q-Ter multicomposite) on recovery time in noise-induced hearing loss.

A potential consequence of exposure to noise is a temporary reduction in auditory sensitivity known as temporary threshold shift (TTS), which mainly depends on the intensity and duration of exposure to the noise. Recovery time is related to the amount of initial hearing loss, and the most recovery takes place during the first 15 min following exposure. This study evaluated the efficacy in otoprotection against noise-induced hearing loss of an orally administrated food supplement containing coenzyme Q 10 -Ter. This water-soluble formulation of coenzyme Q 10 shows better bioavailability than the native form and has been found to have a protective effect on outer hair cells after exposure to noise in animal models. Thirty volunteers were enrolled, and the right ear of each subject was exposed to a narrow-band noise centered at 3 kHz for 10 min at the intensity of 90 dB HL. In the 30 subjects enrolled, TTS was evaluated after 2, 15, and 30 min and the recovery time was recorded in each subject. The longest recovery time was 45 min. Among the 18 subjects who underwent a second test after treatment with Q-Ter, the mean recovery time was 31.43 min. The results of the present study show that 30 days' treatment with Q-Ter can aid faster recovery after exposure to noise (P < 0.0001). The reduction in the recovery time following treatment can be explained by Q-Ter-mediated improvement of the outer hair cells' response to oxidative stress.

Evaluating D-methionine dose to attenuate oxidative stress-mediated hearing loss following overexposure to noise.

Noise exposure causes an excessive reactive oxygen species (ROS) generation as an unwanted byproduct of high metabolic activity. Oxidative stress and antioxidative protective mechanisms have been therefore proposed as the most interesting issues in the development of noise-induced hearing loss. The aim of this study was to examine changes in superoxide dismutase (SOD), catalase (CAT) and the auditory brainstem response (ABR) in the cochlea of C57BL/6 mice 1, 7 and 14 days after exposure to 4 kHz octave band noise at the intensity of 110 dB SPL for 8 h. The evaluation of three D-methionine (D-met) doses (100, 200 and 400 mg/kg) has been performed in order to choose an optimal concentration displaying most effectively its antioxidant and thereby otoprotective functions. Administering D-met at the dose of 400 mg/kg resulted in a significant decrease in threshold shift (TS) independently of the evaluation time after exposure to noise. SOD activity was strongly supported by the same concentration (400 mg/kg) of D-met. This effect was seen not shortly, but 7 and 14 days after exposure to noise. CAT activity was induced only by noise and it reached the peak levels 7 days after exposure. D-Met at the doses of 200 and 400 mg/kg significantly decreased noise-induced changes in CAT activity. The findings of this study indicate that the protective effect depends on the concentration of D-met and can be fully expressed only when the drug is administered in the dose 400 mg/kg.

Inheritance patterns of noise vulnerability and "protectability" in (C57BL/6J × CBA/J) F1 hybrid mice.

BACKGROUND: Interindividual variation in cochlear vulnerability to noise and ototoxins must in part reflect allelic variation in genes that largely remain unknown. Work in our laboratory has shown that young adult CBA/J mice are more vulnerable to cochlear noise injury than are similar-aged mice of other well-studied strains such as C57BL/6J (B6). Conversely, young CBA/J mice are dramatically protected against noise exposure by low-dose kanamycin (KM) treatment, while B6 mice are not. Genetic differences that distinguish these two strains may include genes that help establish the early "sensitive period" in mammals, as well as genes that shape innate protective responses to stress. These genes may have human homologs that exert similar influences and thereby partly govern individual risk of acquired hearing loss. PURPOSE: We hypothesize that young CBA/J and B6 mice carry different alleles at unknown loci that mediate their characteristic sensitivities to noise and responses to kanamycin. The first step in any experimental genetic analysis of two divergent populations is to examine F1 hybrids formed from these. Accordingly, we evaluated both noise vulnerability and the extent of protection from noise by low-dose KM in 6-wk-old F1 hybrids derived from a B6 × CBA/J cross. STUDY SAMPLE: The study included 52 CBA/J, 59 C57BL/6J (B6), and 45 (B6 × CBA/J) F1 hybrid mice, aged 6 wk at time of noise exposure. Both genders were included. INTERVENTION: For experiments aimed at noise vulnerability, B6 and F1 mice were exposed to loud broadband noise (4-45 kHz, 110 dB SPL) for varying durations, and the resulting noise-induced permanent threshold shifts (NIPTSs, measured 2 wk postnoise) were compared with previous data from CBA/J mice. For experiments aimed at KM-based "protectability," CBA/J, B6, and F1 mice received either kanamycin (300 mg/kg, sc) or saline twice daily for 10 days and then were noise exposed for 30 min, followed by measurement of NIPTS at 2 wk postnoise. DATA COLLECTION AND ANALYSIS: Data comprised auditory brainstem response (ABR) thresholds examined by two-way ANOVA (threshold × frequency, group) and derived metrics for NIPTS, plotted versus noise duration. RESULTS: The "threshold" noise exposure duration for NIPTS in F1 hybrid mice was similar to that in CBA/J. Like CBA/J mice, F1 mice were also significantly protected from noise by KM although the protection appeared less robust than in the CBA/J parent strain. B6 mice appeared harmed by KM alone, even without noise exposure. None of the experimental groups provided any evidence for synergistic interactions between noise and KM. CONCLUSIONS: Our data support the hypothesis that young CBA/J and B6 mice carry different alleles that underlie their divergent responses to KM and sensitivities to noise exposure. While the number and type of genes remain unknown, they are worth pursuing because they establish completely novel hearing phenotypes with potential relevance to humans. Our results lay the foundation for mapping of the underlying genes, and ultimately gene identification.

Enhancing autophagy by down-regulating GSK-3ß alleviates cisplatin-induced ototoxicity in vivo and in vitro.

Previous study reported that either selective GSK-3ß inhibitor or up-regulating autophagy can alleviate cisplatin-induced ototoxicity. Other studies indicate that the activity of GSK-3ß is closely associated with the autophagy level. The purpose of this study is to primarily explore the role of autophagy in the alleviation effect of GSK-3ß inhibition on cisplatin-induced ototoxicity in vivo and in vitro. We observed the autophagy changes induced by GSK-3ß inhibitor in outer hair cells (OHCs) in a cisplatin-induced ototoxicity rat model. In addition, autophagy inhibitor 3-MA was used in vitro experiments to observe the influence of autophagy inhibition on the cell protection effect due to GSK-3ß inactivation. The relationship among autophagy, GSK-3ß and cell damage were inferred. Negative regulation of GSK-3ß significantly enhanced autophagy and alleviated cisplatin-induced hearing loss, OHC death in vivo and apoptosis in vitro. The autophagy inhibitor 3-MA inverted the protective effect of negative regulation of GSK-3ß. These results indicated that enhancing autophagy may be a key downstream effect of GSK-3ß inhibition in the alleviation of cisplatin-induced ototoxicity both in vivo and in vitro.

Hearing preservation at low frequencies by insulin-like growth factor 1 in a guinea pig model of cochlear implantation.

The hybrid or electric-acoustic stimulation cochlear implant is indicated in patients with a residual hearing at low frequencies. It provides electric and acoustic stimulation for compensating for high- and low-frequency sounds, respectively. However, the implantation procedure damages the cochlea, resulting in loss of the residual-hearing and diminished effects of the acoustic-hearing in several patients. To prevent hearing loss after implantation, corticosteroids have been used clinically although their effects are limited. As an alternative to corticosteroids, insulin-like growth factor 1 (IGF1) has shown potent effects in various types of cochlear injury. In this study, the effects of IGF1 on hearing preservation were examined after cochlear implantation to a normal-hearing guinea pig model. The electrode was inserted in an atraumatic way through the round window membrane of guinea pigs with the application of a gelatin-sponge soaked with IGF1 or saline. The auditory brainstem response (ABR) was recorded pre-operatively, immediately after cochlear implantation, and 7, 14, 28, and 56 days after electrode insertion. In comparison to the control group, the IGF1-treated group showed better hearing preservation at low frequencies, 7 days after surgery. IGF1 application was effective at low frequencies (2 and 4 kHz) throughout the period of examination. Histological studies revealed that outer hair cell numbers, in the IGF1-treated group, were maintained in the cochlear region responsible for low-frequency hearing (upper midbasal turn) and that there was less fibrous tissue formation around the electrode. Both the outer hair cell counts and the extent of fibrosis significantly correlated with the ABR threshold shifts at low frequencies. These results indicate that IGF1 might attenuate loss of low-frequency hearing after cochlear implantation, suggesting its possible clinical use in soft surgeries involving cochlear implants with electric-acoustic stimulation for hearing preservation.

Chronotolerance for cisplatin ototoxicity in the rat.

Cisplatin is a potent chemotherapeutic compound for which ototoxicity is a significant side effect. Cisplatin has shown sensitivity to circadian time, in that cisplatin is most effective as an anti-tumor compound, and least nephrotoxic, when given in the active (dark) period of the light-dark cycle in rodents. The objective of the study was to determine the sensitivity of cisplatin ototoxicity to circadian time. Fifty-seven Fischer 344/NHsd rats were exposed to 12 mg/kg cisplatin by intra-peritoneal injection at one of six time points on a 12 h light-12 h dark cycle: 2, 6, or 10 h after light onset or 2, 6, or 10 h after light offset. Cochlear injury was evaluated using auditory brainstem response threshold shifts and postmortem outer hair cell counts. All animals experienced threshold shift in the highest frequencies tested (30 and 40 kHz). The animals exposed to cisplatin at 6 h after light onset (the inactive period) had significantly higher mid-frequency threshold shifts and outer hair cell losses than the groups exposed during the dark hours. The results indicate that cisplatin is less likely to cause ototoxicity in the Fischer 344/NHsd rat when given during the active period. This finding is consistent with the lower nephrotoxicity that has been detected in cisplatin-exposed animals treated during the dark hours, and the magnitude of differences in threshold shifts between the light and dark exposure indicates that circadian timing has a significant impact on susceptibility to cisplatin ototoxicity.

The expression of oxidative stress response genes is modulated by a combination of resveratrol and N-acetylcysteine to ameliorate ototoxicity in the rat cochlea.

Aminoglycoside antibiotics are used widely in medicine despite their ototoxic side-effects. Oxidative stress and inflammation are key mechanisms determining the extent and severity of the damage. Here we evaluate the protective effect of a treatment with resveratrol plus N-acetylcysteine on the ototoxic actions of kanamycin and furosemide in the rat. Resveratrol (10 mg/kg) and N-acetylcysteine (400 mg/kg) were administered together to Wistar rats on 5 consecutive days. The second day, a concentrated solution of kanamycin and furosemide was placed on the round window to induce ototoxicity. Hearing was assessed by recording auditory brainstem responses before and 5, 16 and 23 days after the beginning of the treatment. Cochlear samples were taken at day 5 (end of the treatment) and at day 23, and targeted PCR arrays or RT-qPCR were performed to analyze oxidative balance and inflammation related genes, respectively. In addition, the cytoarchitecture and the presence of apoptosis, oxidative stress and inflammation markers were evaluated in cochlear sections. Results indicate that administration of resveratrol plus N-acetylcysteine reduced the threshold shifts induced by ototoxic drugs at high frequencies (≈10 dB), although this protective effect fades after the cessation of the treatment. Gene expression analysis showed that the treatment modulated the expression of genes involved in the cellular oxidative (Gpx1, Sod1, Ccs and Noxa1) and inflammatory (Il1b, Il4, Mpo and Ncf) responses to injury. Thus, co-administration of resveratrol and NAC, routinely used individually in patients, could reduce the ototoxic secondary effects of aminoglycosides.

Differential gene expression profiles in salicylate ototoxicity of the mouse.

CONCLUSION: This study demonstrated differential gene expression profiles in salicylate ototoxicity with oligonucleotide microarray. This study may also provide basic information on candidate genes associated with hearing loss and/or tinnitus or recovery after salicylate-induced cochlear dysfunction. OBJECTIVES: Salicylate ototoxicity is accompanied by temporary hearing loss and tinnitus. The purpose of the present study was to evaluate the gene expression profiles in the mouse cochlea with salicylate ototoxicity using DNA microarray. MATERIALS AND METHODS: The subject mice were injected intraperitoneally with 400 mg/kg of sodium salicylate; an approximate 30 dB threshold shift that was observed by auditory brainstem response was achieved 3 h after an injection of sodium salicylate and the hearing threshold returned to within normal range at 3 days. Differential gene expression profiles at 3 h after salicylate injection in comparison to the normal cochlea were analyzed with DNA microarray technology. RESULTS: No ultrastructural changes in the mice cochlea were observed by TEM at 3 h after salicylate injection. Microarray revealed that 87 genes were up-regulated twofold or more in the mouse cochlea with salicylate ototoxicity in comparison to the normal cochlea. Among these genes, increased expression levels of 30 functional genes were confirmed by semi-quantitative RT-PCR.

Cannula-based drug delivery to the guinea pig round window causes a lasting hearing loss that may be temporarily mitigated by BDNF.

Sustained local delivery of drugs to the inner ear may be required for future regenerative and protective strategies. The round window is surgically accessible and a promising delivery route. To be viable, a delivery system should not cause hearing loss. This study determined the effect on hearing of placing a drug-delivery microcatheter on to the round window, and delivering either artificial perilymph (AP) or brain-derived neurotrophic factor (BDNF) via this catheter with a mini-osmotic pump. Auditory brainstem responses (ABRs) were monitored for 4 months after surgery, while the AP or BDNF was administered for the first month. The presence of the microcatheter - whether dry or when delivering AP or BDNF for 4 weeks - was associated with an increase in ABR thresholds of up to 15 dB, 16 weeks after implantation. This threshold shift was, in part, delayed by the delivery of BDNF. We conclude that the chronic presence of a microcatheter in the round window niche causes hearing loss, and that this is exacerbated by delivery of AP, and ameliorated temporarily by delivery of BDNF.

Low-dose, long-term caroverine administration attenuates impulse noise-induced hearing loss in the rat.

CONCLUSION: Physiological and morphological assessments indicated that low-dose and long-term caroverine delivery might be a new approach to protect against impulse noise-induced hearing loss. BACKGROUND: Although the exact mechanisms by which impulse noise causes hearing loss are still unclear, there is accumulating evidence that increased reactive oxygen species (ROS) production and excessive glutamate released from the inner hair cells lead to hair cell loss and consequently hearing loss. Caroverine is an antagonist of two glutamate receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the inner ear, as well as an antioxidant. MATERIALS AND METHODS: In this study, caroverine was delivered subcutaneously using an osmotic pump. This kind of delivery has the advantage, via continuous, long-term and low dose drug administration, of avoiding systemic side effects. RESULTS: It was shown that caroverine could significantly protect the cochlea against impulse noise trauma.

The role of extended preoperative steroids in hearing preservation cochlear implantation.

INTRODUCTION: Steroids have been shown to reduce the hearing threshold shifts associated with cochlear implantation. Previous studies have examined only the administration of steroids just prior to surgery. The aim of this study is to examine the role of extended preoperative systemic steroids in hearing preservation cochlear implantation. METHODS: An animal model of cochlear implantation was used. 24 Hartley strain guinea pigs with a mean weight of 768 g and normal hearing were randomised into a control group, a second group receiving a single dose of systemic dexamethasone one day prior to surgery, and a third group receiving a daily dose of systemic dexamethasone for 5 days prior to surgery. A specially designed cochlear implant electrode by Med-EL (Innsbruck) was inserted through a dorsolateral approach to an insertion depth of 5 mm and left in-situ. Auditory brain stem responses at 8 kHz, 16 kHz and 32 kHz were measured preoperatively, and 1 week, 1 month and 2 months postoperatively. Cochlear histopathology was examined at the conclusion of the study. RESULTS: At 1-week post operative, both groups receiving dexamethasone prior to implantation had smaller threshold shifts across all frequencies and which was significant at 32 kHz (p < 0.05). There were no differences among the three groups in the area of electrode related fibrosis. Spiral ganglion neuron (SGN) density was significantly higher in the group receiving steroids for 5 days, but only in the basal cochlear turn. DISCUSSION: This is study demonstrates the benefits of extended preoperative systemic steroids on hearing outcomes and SGN density in an animal model of cochlear implantation surgery.

Aspirin can potentiate the temporary hearing loss induced by intense sounds.

UNLABELLED: Aspirin is known to produce a reversible loss of hearing that can be as great as 40 dB, depending upon the dose and the individual subject. Here we show that aspirin-induced losses exacerbate the temporary hearing loss induced by exposure to intense sound. EXPOSUREs that ordinarily produce about 14 dB of temporary threshold shift (TTS) will produce about 18-27 dB of TTS if the listener has been taking 3.9 g of aspirin for the past two days or more. A lesser dose or a shorter duration of use produces a smaller, or no, increment in temporary hearing loss. This greater TTS, and an apparent prolongation of recovery from exposure, make chronic aspirin use ill-advised for people routinely exposed to intense sounds. EXPOSURE: 2500 Hz, 10 min, varying intensity. TTS frequency: 3550 Hz. Psychophysical method: 2IFC, adaptive.

Electrical stimulation of cochlear efferents at the round window reduces auditory desensitization in guinea pigs. I. Dependence on electrical stimulation parameters.

Electrical stimulation at the round window with pulsed short trains has been shown to elicit classical efferent effects on N1 amplitudes at the cochlea. This report demonstrates that round window stimulation as a continuous burst can reduce temporary threshold shifts (TTS) caused by a simultaneous monaural loud sound exposure. This result is similar to recent reports that stimulation of the crossed olivocochlear bundle (COCB) at the floor of the fourth ventricle can reduce TTS. Like COCB stimulation at the brainstem, the effect of round window stimulation could be abolished by strychnine, with a time course paralleling the blocking action of strychnine on the traditional COCB effects of pulsed short trains on N1 amplitudes. This report also established parameters for optimal effects of the round window stimulus and found them to be similar to the optimal parameters for the effects of brainstem stimulation on TTS. Tonic effects on TTS were also observed, with reductions in TTS being obtained as much as 7 min after a 1 min-long round window stimulus. Such tonic effects did not appear to be due to persistent effects at the cochlea but were suggested to be due to a long term resetting of some central site activated by antidromic stimulation from the round window.

Crossed cochlear influences on monaural temporary threshold shifts.

Temporary threshold shifts (TTS) of the cochlear action potential in one (ipsilateral) ear, caused by a brief intense pure tone, were reduced either by (1) contralateral acoustic stimulation at the same frequency, or (2) destruction of the contralateral cochlea. The effect of contralateral cochlear destruction persisted, though slightly reduced in effect, after a delay of one hour between the destruction and the ipsilateral exposure. Contralateral acoustic stimulation had no effect after a delay of 10 min. The effect of contralateral cochlear destruction could be blocked by strychnine, a known antagonist of auditory efferent activity. However, contralateral cochlear destruction resulted in none of the effects upon normal ipsilateral thresholds or input-output curves for the action potential classically seen when the efferent pathways are stimulated. The results suggest that the crossed effects reported here are due to a complex interaction between the activity in both cochleas, possibly resulting in a reduction in a central inhibitory influence on an efferent feedback pathway that is then expressed during the ipsilateral exposure.

Diltiazem does not protect the ear from noise-induced hearing loss in mongolian gerbils.

Recent data suggest that diltiazem reduces noise-induced hearing loss. Our study was designed to replicate and extend the results of Maurer et al. by using the gerbil as a model. In experiment A, subjects received diltiazem (30 mg/kg/day intraperitoneally) or saline for 3 days. After peripheral thresholds were measured, each subject was exposed to a 4-kHz tone (90-dB sound pressure level) for 20 minutes. Similar amounts of temporary threshold shifts (ITS) were measured in the saline and diltiazem groups. In experiment B, subjects were given saline or diltiazem (30 mg/kg/day intraperitoneally) for 3 days and then exposed to an octave band of noise centered at 4 kHz for 5 days, during which time the subjects continued to receive the drug or saline. The TTS and permanent threshold shifts were similar in the two groups. Measures of cochlear nonlinearities also showed no effect of diltiazem, suggesting that diltiazem does not protect the ear from the effects of noise.

Combined effects of noise and cisplatin: short- and long-term follow-up.

The combined effects of noise exposure and intravenous cisplatin injection on electrophysiologic hearing thresholds in guinea pigs were studied with short-term and long-term follow-up. The combined effects on the permanent threshold shift were dependent on the order of exposure. A potentiation was achieved when noise exposure preceded cisplatin injection by 30 minutes or by 3 days. Cisplatin injection 2 or 3 days before noise exposure produced no significant potentiation or inhibition. The combined effects on the temporary threshold shift were not influenced by the sequence of exposure.

The interaction of smoking and noise on temporary threshold shifts.

Four separate experiments examined the effects of cigarette smoking on temporary threshold shifts (TTS) following noise exposure. One experiment compared smokers and non-smokers after the subjects had abstained from smoking for at least 6 h. A second experiment tested only smokers who smoked a cigarette just before noise exposure, during the noise exposure and in a control condition during which they did not smoke. A third experiment tested only non-smokers who were exposed to noise after chewing nicotine gum or after a control condition in which they rested without chewing the gum. A fourth experiment tested smokers and non-smokers in one condition which required them to smoke a cigarette just prior to noise exposure and in another condition which prevented them from smoking. The results indicate that smokers consistently evidence slightly smaller TTS than do non-smokers. Non-smokers evidenced significantly greater TTS at one frequency after they had chewed nicotine gum than when they had not. These results suggest that the smaller TTS associated with cigarette smoking is related to both the chronic and the acute effects of smoking and that these effects may be more attributable to carbon monoxide than to nicotine.

The value of methylprednisolone in the treatment of an experimental sensorineural hearing loss following drill-induced ossicular chain injury: a randomized, blinded study in guinea-pigs.

In a previously established animal model a standardized drill-induced injury to the body of the incus was applied, and the effects on hearing were characterized by electrocochleography. In a placebo-controlled, randomized, blinded study methylprednisolone showed no protective effect in reducing or improving the auditory threshold shifts, which occurred within seconds after drilling and remained stable throughout the 5-week observation period. Therefore the otologic surgeon must pay close attention to avoiding any contact of a rotating burr with an ossicle in an intact ossicular chain.

Protective effect of brain-derived neurotrophic factor against the ototoxicity of Pseudomonas aeruginosa exotoxin A.

OBJECTIVE: The protective effect of brain-derived neurotrophic factor (BDNF) against the ototoxicity resulting from exposure of Pseudomonas aeruginosa exotoxin A (PaExoA) to the middle ear was analyzed. The combined effect of BDNF and N(G)-nitro-L-arginine methyl ester (L-NAME) was also investigated. MATERIAL AND METHODS: Six groups of albino rats were instilled through the tympanic membrane into the round window niche with the following solutions: saline; PaExoA; BDNF; L-NAME; PaExoA + BDNF; and PaExoA + BDNF + L-NAME. Frequency-specific (2-31.5 kHz) auditory brainstem responses were used to obtain the hearing thresholds before and 2, 5 and 15 days after instillation. RESULTS: PaExoA penetrated from the middle ear into the cochlea, causing initially mixed hearing loss, followed by persistent sensorineural hearing loss. This impairment was blocked by BDNF at 6, 8 and 10 kHz on Day 2 and at 8 kHz on Day 5. L-NAME given in combination with BDNF did not show any additional protective effect. There were no significant differences in the thickness of the round window membrane between control ears and those in each instillation group. CONCLUSION: Our results suggest that BDNF may protect against cochlear damage caused by PaExoA in the middle turns of the ear.