Testing for Tinnitus in Animals
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1 Testing for Tinnitus in Animals Henry E. Heffner, Ph.D., Department of Psychology, University of Toledo 2015 SHAV Convention, March 26, Session 25, 3:45-4:45 PM Introduction characteristics of the tinnitus are (e.g., noise or tonal and, if tonal, what pitch), and how long it will persist. A procedure for determining if an animal has tinnitus is Thus, one cannot always be certain that a particular essential for testing drug treatments for tinnitus as well treatment will induce tinnitus in an animal or what the as for discovering its neurological basis. However, characteristics of the tinnitus will be. testing an animal for tinnitus is complicated because the procedures used to induce tinnitus also cause other The second point is that procedures used to induce auditory problems, such as hearing loss and tinnitus will affect hearing in other ways (e.g., Davis et hyperacusis, that can confound the results. The tests al., 1950; Cazals, 2000). Specifically, loud sound and developed so far can be classified into three types. ototoxic drugs sufficient to cause tinnitus also cause a significant hearing loss. Another effect of such First are tests in which animals are trained to treatments is that they may cause physical sounds to be discriminate between the presence and absence of a perceived as distorted. Thus, it is necessary to rule out physical sound and then tested to determine if a the possibility that an animal’s responding is affected tinnitus-inducing agent causes the animals to respond as by changes to its hearing other than tinnitus. if a sound is present. Second are tests that look for an interaction between tinnitus and physical sounds such as determining if a Tinnitus produced by exposure to loud sound tinnitus-inducing agent enhances the perception of a There have been three relatively comprehensive studies sound or degrades the ability to detect a temporal gap in on tinnitus caused by exposure to loud sound (Davis et an ongoing sound. al., 1950; Loeb & Smith, 1967; Atherley et al., 1968; Third are tests in which animals are trained in a sound these studies are discussed in Heffner & Heffner, 2012). localization task to indicate whether a sound comes Their results can be summarized as follows: from their left or right side. Tinnitus is then induced in 1. Exposure to loud sound sufficient to cause tinnitus one ear by exposing it to a loud sound and the animals produces an immediate hearing loss; if the exposure tested to determine which side they go to when no does not cause permanent damage, both the hearing sound is presented. loss and tinnitus subsides in a few days. These procedures can be compared on whether they 2. The tinnitus is more likely to have a definite and would reliably detect tinnitus in humans and whether constant pitch if it is produced by exposure to tones the results would be confounded by hearing loss and or narrowband noise rather than broadband noise. hyperacusis. Some tentative conclusions on the neural 3. The exposure may affect the perception of physical mechanisms underlying tinnitus can be made. stimuli, causing them to sound distorted, at least In evaluating tinnitus in animals, the first step is to during recovery from the temporary portion of the determine if the results are similar to what we know hearing loss. (Davis et al, 1950, noted that exposure about tinnitus in humans. Therefore, we will begin with to loud sound could cause a pure tone to sound what is known from human studies in which subjects “rough”, “noisy”, or “buzzing”, or to cause a single were either exposed to loud sound or given salicylate tone to sound like two tones presented in and their resulting tinnitus evaluated. combination, which they referred to as “doubles”.) 4. It is likely that exposing one ear to loud sound will Tinnitus in humans cause any resulting tinnitus to be lateralized to that ear. However, there are rare reports of exposing one Let us begin with two general points. First, although it ear causing tinnitus to be reported in the other ear. is well established that loud sound and ototoxic drugs, However, this apparently occurs when the other ear such as salicylate, cause tinnitus in humans, there is has recently been exposed, suggesting that presenting significant variability between subjects (McFadden, sound to the opposite ear may reinstate tinnitus in a 1982). Specifically, humans differ in whether a previously exposed ear. particular level or dosage induces tinnitus, what the
2 5. There is considerable individual variation in both the 3. The degree of hearing loss varies with the amount of hearing loss and the pitch of the tinnitus induced by a salicylate, but the relationship between plasma loud sound. salicylate levels and hearing loss is not perfect and 6. Pitch of the tinnitus: there is much individual variation (Cazals, 2000). Some studies indicate that the hearing loss is equal at Tone-induced tinnitus: The median pitch tinnitus all frequencies whereas others have found that the induced by a loud tone is higher than the frequency of loss is greater at high frequencies (cf., McCabe & the exposing tone; it is also higher than the frequency Dey, 1965; Myers & Bernstein, 1965). of the maximum hearing loss. 4. No relationship between the pitch of the tinnitus and Noise-induced tinnitus: The median pitch of tinnitus the hearing loss has been observed, possibly because induced by a loud noise is usually near or slightly the audiograms have not been sufficiently detailed, as higher than the center frequency of the exposing they are typically conducted in octave steps, or noise; on the other hand, it is lower than the because they did not extend to frequencies above 8 frequency of maximum hearing loss. kHz (McFadden, 1982). 5. Besides inducing tinnitus and hearing loss, salicylate Side Note: An interesting observation made by Davis has been found to affect the perception of sound, the et al. (1950) is that while the hearing loss caused by a most prominent example being a hypersensitivity that particular sound varies between individuals, it does not causes some sounds to be especially irritating, a vary much within an individual. In other words, a phenomenon also referred to as hyperacusis. particular sound will cause the same hearing loss in Salicylate also affects other auditory functions such both ears of a subject. This leads to a research topic: as frequency selectivity, temporal integration, and Specifically, what are the anatomical, physiological, gap detection (Cazals, 2000). histological, and biochemical characteristics of the ears 6. Two final points are especially noteworthy. of individual animals that are resistant to noise-induced hearing loss as opposed to those that are susceptible. First, the effect of salicylate is highly variable; not Might there be some treatment that would make ears only do the hearing loss and tinnitus vary between more resistant to noise-induced hearing loss? individuals with the same blood levels of salicylate, but the blood levels of salicylate among individuals given the same dosage may also differ noticeably Tinnitus produced by salicylate (Cazals, 2000). Thus, animals given the same dose A number of studies have examined the effect of of salicylate would be expected to vary in their salicylate on hearing and the auditory system; for tinnitus. reviews, see McFadden, 1982; Cazals, 2000 (also Second, salicylate crosses the blood brain barrier reviewed in Heffner & Heffner, 2012). giving it the potential to cause tinnitus by acting 1. The most noticeable effects of high doses of directly on the central auditory system. However, salicylate, usually administered orally, are tinnitus elderly people with hearing loss resulting from loss and hearing loss, both of which increase during the of hair cells in the cochlea that encode high initial days of treatment and then level off, fluctuate, frequencies (presbycusis) do not develop tinnitus or decrease. The effects are reversible and typically when given salicylate (Schuknect & Gacek, 1993; disappear a few days after treatment is stopped Mongan et al., 1973). This suggests that it is the (Cazals, 2000). (However, a college student once told effect of salicylate on hair cells that causes tinnitus. me that she has a permanent hearing loss caused by high doses of aspirin that were prescribed by her Animal Tests of Tinnitus physician, although this could have been caused by Nearly a dozen behavioral procedures have been used exposure to loud sound while she was being treated.) to test animals for tinnitus and their designs are often 2. The pitch of the tinnitus is usually described as a complex. This talk will cover the general types of high-frequency tone or noise, although it is procedures with a few specific examples, comparing occasionally lower in frequency. One study found them on the following points: pitch matches ranging from 14.5 kHz down to 900 Hz Validity with the loudness of the tinnitus matched to external tones of over 60 dB (Day et al., 1989). However, 1. Would the tinnitus-inducing agent used cause untrained subjects are not always good at making tinnitus in humans? pitch matches. 2. Would the procedure detect tinnitus in humans?
3 3. Has the procedure been tested by simulating tinnitus likely to stop drinking when the noise was turned off with physical sounds? because they would still hear their tinnitus. 4. Would the test be affected by an accompanying The results showed that animals given salicylate were hearing loss? less likely to stop drinking when the noise was turned 5. Would the test be affected by hyperacusis or other off than were control animals that were not given side effects such as diplacusis? salicylate, a result suggesting that the treatment animals had developed tinnitus (Fig. 1). Utility With regard to the criteria: 6. Can the procedure be used to determine the pitch of tinnitus? 1. The salicylate dosage would be expected to cause tinnitus in humans. 7. Does the procedure rely on group averages or can tinnitus be assessed in individual animals? 2. Training subjects to make a response when they hear a sound is similar to asking human subjects to report 8. Can the procedure follow an animal’s tinnitus over when they hear a sound. time? 3. Animals show a positive response when presented with sounds that simulate tinnitus. 1. Tests requiring animals to discriminate sound 4. Although the test could be affected by hearing loss if from silence the animals could no longer tell when the sound was The first tinnitus test to be developed for animals on, control tests in which the amplitude of the noise consisted of training rats to discriminate the presence of was reduced to simulate the hearing loss ruled out sound from absence of sound; this was done by having that possibility. In addition some studies exposed one them drink from a water spout in the presence of ear of the animals, leaving the other ear with normal broadband noise and training them to stop drinking hearing. when the noise was turned off by administering foot 5. There is no reason to think that hyperacusis would shock at the end of a 60-sec silent interval (e.g., affect the results. Jastreboff et al., 1988; Jastreboff & Brennan, 1994). 6. The procedure has been used to determine the pitch The rats were then given salicylate to induce tinnitus of tinnitus by administering salicylate during training and tested with the idea that the animals would be less so that the perception of an animal’s tinnitus would Fig. 1. Effect of exposing one ear of hamsters to10 kHz, 124 dB for .5 to 4 hours. Shaded area indicates range of unexposed animals. Scores below the range of the unexposed animals suggest that an exposed animal perceived a sound when the physical sound was turned off, i.e., tinnitus. The effect increased with the duration of the exposure (From Heffner & Harrington, 2002.)
4 become associated with foot shock. Testing consisted exposed in one ear to octave noise centered at 16 kHz of presenting tones during the silent intervals in (110-120 dB, 1 hr). They were then tested with a 10- which it was found that the rats were more likely to kHz tone which the rats treated as a safe signal, stop drinking when high-frequency tones were especially if it was sufficiently loud (e.g., 80 dB). At 8- presented, suggested that their tinnitus was 10 kHz or 9 weeks post-exposure, the exposed animals showed higher in pitch. slightly better performance than unexposed control 7. The test requires groups of animals as the results animals to the 10-kHz tone. The explanation of for this vary from one individual to the next. is that the exposed rats developed tinnitus that was around 10 kHz in pitch and it interacted with the 10- 8. The test cannot be used to follow an animal’s tinnitus kHz tone to make it “noiser”. over time as the foot shock is turned off during testing allowing the animals to learn that they do not This procedure does not meet the first two criteria. need to stop drinking when the noise is turned off. With regard to the sound used to induce tinnitus, it is known that exposing humans to that amplitude of octave noise will cause tinnitus with a 5-min. exposure 2. Interaction of tinnitus with external tones (Atherley et al., 1968; Loeb & Smith, 1967). Thus, the In one such test, the animals are trained to press a lever one hour exposure the rats received was probably to get food while a broadband noise is on and to stop sufficient to cause tinnitus. However, the onset of pressing when the noise is turned off to avoid foot tinnitus due to exposure to loud sound in humans is shock (Bauer & Brozoski, 2001; Turner et al., 2006). immediate and, at least at the shorter durations used in The animals may be exposed to a tinnitus inducing the human studies, is transient. Thus, it is surprising agent before training begins, in which any tinnitus that the animals did not immediately test positive for would then be associated with shock. Alternatively, the tinnitus. In addition, there is no convincing evidence of animals can be exposed after training, in which case humans developing tinnitus months after exposure to a tinnitus might serve as a signal that it is safe to press sound that initially did not cause tinnitus. Moreover, the lever. tinnitus in humans is not known to interact with The test for tinnitus involves presenting various tones physical sounds to produce a “noisier” sound, although and observing whether an animal’s response to a hyperacusis does. Finally, presenting a tone of the same particular tone differs from the responses of unexposed pitch as one’s tinnitus is known to suppress the tinnitus control animals, a result taken to indicate tinnitus with a (e.g., McFadden, 1982). Therefore, the results of this pitch similar to that of the tone. procedure do not match what we know about tinnitus in humans and it therefore seems unlikely that this is a Figure 2 presents the results of a test in which rats were valid test for tinnitus in animals. trained to lever press when background noise was on (safe signal) and to stop pressing when the noise was turned off (warning signal). The animals were then 3. Startle reflex inhibition gap procedure The startle reflex inhibition procedure involves reducing an animal’s startle response to a sudden, loud sound by presenting a weaker stimulus just before the startle sound is presented; a reduction in the amplitude of the startle response indicates that the animal perceived the preceding stimulus. A common use of this procedure has been to determine the audibility of sounds by observing whether they reduce the startle response. However, in the tinnitus test, the startle response is reduced not by another sound, but by preceding the startle stimulus with a brief gap in an on- Fig. 2. Rats trained to press a lever in the presence of going background sound. The hypothesis is that tinnitus background noise (safe signal) and to stop pressing during would interfere with detecting the gap if it were similar silence (warning signal) also responded to a 10-kHz tone in pitch to the background sound. as a safe signal. Rats exposed to loud sound pressed more In this procedure, an animal is placed in a test cage with than the unexposed animas 8-9 weeks post-exposure, a low-level background sound, such as 60-dB which was taken as the appearance of delayed tinnitus that made the 10-kHz tone sound noisier. (From Turner et al., narrowband noise. A startle sound (e.g., 115-dB, 20-ms 2006.) broadband noise burst) is presented at random intervals
5 and the animal’s startle response is measured by a strain human subjects show that it does not (e.g., Fournier & gauge attached to the test cage. The startle sound is Hebert, 2012; Shadwick & Sun, 2015). Finally, this either presented alone or preceded by a gap in the procedure overlooks the fact that sounds similar in pitch background sound, typically a 50-ms gap beginning to a subject’s tinnitus would be expected to suppress the 100 ms before the startle stimulus. A reduction in the tinnitus. average startle response that is caused by preceding the startle sound with a gap is used to indicate that the animal perceived the gap (e.g., Turner et al., 2006). 6. Two-choice sound localization procedure Thus, by testing animals with different background The sound localization procedure is based on the idea sounds, typically narrow band noise centered at that exposing one ear to a loud sound will cause tinnitus different frequencies, it is thought possible to determine in that ear and that an animal trained to report whether a the pitch of their tinnitus. sound came from its left or right side will, in the This is a popular procedure because it involves no absence of a physical sound, respond by going to the training of either the animal or the experimenter. One side of the ear that has tinnitus (Heffner & Koay, 2005; simply purchases the equipment, puts the animal in the Heffner, 2011). test box, and the computer does the testing In this test, an animal is trained to make a left or right automatically (Fig. 3). response to sounds coming from its left or right side, respectively. Correct responses are rewarded with water whereas incorrect responses are followed by a mild shock (Fig. 4). Silent trials, in which no sound is presented, are interspersed among the sound trials. The animal receives neither reward nor punishment for its responses on silent trials and its side preference on these trials is recorded. Importantly, feedback on the sound trials is changed so that randomly half of the sound trials are followed by reward or punishment to reduce the possibility that an animal will notice that responses to silent trials are never rewarded or punished. This permits long term testing of an animal’s tinnitus. Fig. 3. Startle Reflex Response System. The rat is placed in a cylinder sitting on sensors that detect its startle response. A computer presents the startle sound and records the animal’s reflexive response. The gap detection test suffers from some of the same problems as the previous procedure. First, there is no Fig. 4. Sound localization test cage with a hamster. An evidence that tinnitus interacts with external sounds, animal is rewarded for going left and right to left and right including affecting the detection of gaps in sound. sounds, respectively. Trials in which no sound is presented Hearing loss, on the other hand, is known to affect gap (silent trials) are given and tinnitus is induced in the ear detection thresholds. Second, it presumes that tinnitus opposite the animal’s preference on those trials. The animal would interfere with the perception of gaps in sounds is then tested to see if it shifts its responding to the side of similar in pitch to the tinnitus, but recent studies of the exposed ear.
6 The side preference of an animal on the silent trials is 2. Asking subjects to report in which ear they hear have determined and the animal is exposed to a loud sound tinnitus is a standard procedure with human patients. in the ear opposite its side preference; it is then tested to 3. Animals show a positive response when presented see if it shifts responding on silent trials to the side of with sounds that simulate tinnitus. the exposed ear. This is conceptually equivalent to 4. Hearing loss in one ear is unlikely to cause a positive human patients reporting the ear in which they hear response because, if anything, an animal would their tinnitus. Besides being able to indicate whether an respond to the unexposed (normal) ear in which they individual animal has lateralized tinnitus, the two- hear the incidental sounds of their movements. choice procedure would not be expected to be confounded by the hearing loss that accompanies 5. There is no reason to think that hyperacusis would exposure to loud sound; indeed, a hearing loss in the affect the results as the animals are responding when exposed ear would be expected to cause an animal to no external sound is presented. respond to the side of its unexposed ear as any 6. The procedure does not indicate the pitch of an incidental sounds made by the animal would be animal’s tinnitus. perceived in that ear. 7. The test uses each animal as its own control so that Because the animals are never given feedback on the individual results, as opposed to group averages, are silent trials, and their responses on sound trials are only analyzed, providing for greater statistical power. given feedback half of the time, responding to their 8. The test can follow an animal over time allowing tinnitus may not easily habituate, making it possible to recovery to be monitored. However, there is always follow the time course of the tinnitus. An example of the possibility that an animal will habituate to its the results of this test are shown in Figure 5. tinnitus. Side Note: After recovering from one exposure, the animals were tested by exposing them to another loud tone, with each exposure requiring the animal to be sedated. This revealed an unexpected effect of anesthesia (halothane/nitrous oxide) on tinnitus. That is, whereas anesthetizing unexposed rats did not cause them to test positive for tinnitus, anesthesia alone would sometimes reinstate tinnitus in animals that had previously been exposed. That is, a rat that was no longer testing positive for tinnitus would occasionally shift its responding on the silent trials to the side of its previous exposure after being anesthetized even though it was not exposed to any sound at that time (Heffner, 2011). Neural Mechanisms of Tinnitus Fig. 5. Rats (A-F) were trained to respond in the direction of There is an on-going debate as to the site of the left and right sounds. Trials were inserted in which no sound generation of tinnitus (e.g., Eggermont, 2013). One was presented and their side preference on those trials determined. The animals were exposed in the ear opposite obvious site is the Organ of Corti. Another is the dorsal their side preference on Day 0 with testing begun 10 minutes cochlear nucleus. And then there is the idea that afterwards. The exposure (4-kHz tone, 110 dB, 10 min) was auditory cortex may be necessary for the perception of sufficient to cause all six animals to shift their responding on tinnitus. silent trials to the side of their exposed ear, indicating The main problem is that electrophysiological studies tinnitus in that ear. (From Heffner, 2011.) look for an increase in neural activity following either administration of salicylate or exposure to loud sound. With regard to the criteria: However, both of these treatments will cause a hearing 1. The sounds used to induce tinnitus were within the loss. The assumption that a hearing loss will reduce intensity and duration of those that cause tinnitus in neural activity overlooks the fact that most neural humans (Davis et al., 1950). activity within the central nervous system is inhibitory, not excitatory. Thus, when auditory neurons cease
7 activity, they may release other neurons from Day, R. O., Graham, G. G., Bieri, D., Brown, M., inhibition, causing those neurons to increase their Cairns, D., Harris, G., Hounsell, J., Platt-Hepworth, neural activity. Thus, increased neural activity in the S., Reeve, R., Sambrook, P. N., & Smith, J. (1989). auditory system following administration of salicylate Concentration-response relationships for salicylate- or exposure to loud sound is likely a sign of hearing induced ototoxicity in normal volunteers. British loss, rather than tinnitus. Journal of Clinical Pharmacology, 28, 695-702. A case in point is the increase in activity in the dorsal Eggermont, J. J. (2013). Hearing loss, hyperacusis, or cochlear nucleus following exposing an ear to intense tinnitus: What is modeled in animal research? sound (e.g., 10 kHz, 120 dB, 4 hrs). Although it is Hearing Research, 295, 1404-149. argued that this increased activity is the basis for Fournier P. & Hebert, S. (2012). Gap detection deficits tinnitus (e.g., Kaltenbach & McCaslin, 1996), there is in humans with tinnitus as assessed with the acoustic good reason to think that it is not. Whereas the onset of startle paradigm: Does tinnitus fill in the gap? tinnitus to a loud sound is immediate, the increased Hearing Research, 295, 16-23. activity in the dorsal cochlear nucleus does not occur Heffner, H. E. (2011). Two-choice sound-localization until many days later. Second, a behavioral study that procedure for detecting lateralized tinnitus in animals. took hearing loss into account found that the increased Behavior Research Methods, 43, 577-589. activity in the dorsal cochlear nucleus was correlated with hearing loss, rather than tinnitus (Zhang et al., Heffner, H. E. & Harrington, I. A. (2002). Tinnitus in 2004). hamsters following exposure to loud sound. Hearing Research, 170, 83-95. Of all the possible sites for the generation of tinnitus, the one with the strongest evidence is the Organ of Heffner, H. E. & Heffner, R. S. (2012). Behavioral tests Corti. In giving high doses of aspirin as a medical for tinnitus in animals. In J. J. Eggermont, F.-G. treatment, physicians often use the rule of increasing Zheng, A. N. Popper & R. R. Fay (Eds.) Tinnitus. the dosage until the patient reports tinnitus and then Springer: NY, pp. 21-58. reducing it until the tinnitus disappears. However, this Heffner, H. E. & Koay, G. (2005). Tinnitus and hearing procedure does not work with elderly patients who have loss in hamsters exposed to loud sound. Behavioral a hearing loss resulting from loss of the hair cells in the Neuroscience, 119, 734-742. cochlea that encode high frequencies (presbycusis)— Jastreboff, P. J., Brennan, J. F., Coleman, J. K., & these patients do not develop tinnitus when given Sasaki, C. T. (1988). Phantom auditory sensation in salicylate (Schuknect & Gacek, 1993; Mongan et al., rats: an animal model for tinnitus. Behavioral 1973). This observation suggests that it is the effect of Neuroscience, 102, 811-822. salicylate on hair cells that causes tinnitus. Jastreboff, P. J. & Brennan, J. F. (1994). Evaluating the loudness of phantom auditory perception (tinnitus) in References rats. Audiology, 33, 202-217. Atherley, G. R. C., Hempstock, T. I., & Noble, W. G. Kaltenbach, J. A. & McCaslin, D. L. (1996). Increases (1968). Study of tinnitus induced temporarily by in spontaneous activity in the dorsal cochlear nucleus noise. Journal of the Acoustical Society of America, following exposure to high intensity sound: a 44, 1503-1506. possible neural correlate of tinnitus. Auditory Neuroscience, 3, 57-78. Bauer, C. A. & Brozoski, T. J. (2001). Assessing tinnitus and prospective tinnitus therapeutics using a Loeb, M., & Smith, R. P. (1967). Relation of inducted psychophysical animal model. Journal of the tinnitus to physical characteristics of the inducing Association for Research in Otolaryngology, 2, 54- stimuli. Journal of the Acoustical Society of America, 64. 42, 453-455. Cazals, Y. (2000). Auditory sensori-neural alterations McFadden, D. (1982). Tinnitus: facts, theories, and induced by salicylate. Progress in Neurobiology, 62, treatments. Washington, DC: National Academy 583-631. Press. Davis, H., Morgan, C. T., Hawkins, J. E., Jr., McCabe, P. A. & Dey, F. L. (1965). The effect of Galambos, R., & Smith, F. W. (1950). Temporary aspirin upon auditory sensitivity. Annals of Otology, deafness following exposure to loud tones and noise. Rhinology and Laryngology, 74, 312-325. Acta Oto-Laryngologica Supplement, 88, 1-57. Mongan, E., Kelly, P., Nies, K., Porter, W. W., & Pulus, H. E. (1973). Tinnitus as an indication of
8 therapeutic serum salicylate levels. Journal of the Turner, J. G., Brozoski, T. J., Bauer, C. A., Parrish, J. American Medical Association, 226, 142-145. L., Myers, K., Hughes, L. F., & Caspary, D. M. Myers, E. N. & Bernstein, J. M. (1965). Salicylate (2006). Gap detection deficits in rats with tinnitus: a ototoxicity. Archives of Otolaryngology, 82, 483-493. potential novel screening tool. Behavioral Neuroscience, 120, 188-195. Shadwick, K. & Sun, W. (2015). Acoustic startle reflex and pre-pulse inhibition in tinnitus patients. Journal Zhang, J., Heffner, H. E., Koay, G., & Kaltenbach, J. A. of Otology, 1-5, in press. (2004). Hyperactivity in the hamster dorsal cochlear nucleus: Its relationship to tinnitus. Abstracts of the Schuknecht, H. F. & Gacek, M. R. (1993). Cochlear Twenty Seventh Meeting of the Association for pathology in presbycusis. Annals of Otology, Research in Otolaryngology, 27, 302. Rhinology, and Laryngology, 102, 1–16.
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