Auditory sensori-neural alterations induced by salicylate

Progress in Neurobiology 62 (2000) 583±631

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Auditory sensori-neural alterations induced by salicylate Yves Cazals* Inserm EPI 9902 Pathologies de l'oreille interne et reÂhabilitation, Laboratoire Otologie NeuroOtologie, Faculte de MeÂdecine Nord, Univ. MeÂditerraneÂe Aix-Marseille II, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France Received 6 March 2000

Abstract Early after the development of aspirin, almost 150 years ago, its auditory toxicity has been associated with high doses employed in the treatment of chronic in¯ammatory diseases. Tinnitus, loss of absolute acoustic sensitivity and alterations of perceived sounds are the three auditory alterations described by human subjects after ingestion of large doses of salicylate. They develop over the initials days of treatment but may then level o€, ¯uctuate or decrease, and are reversible within a few days of cessation of treatment. They may also occur within hours of ingestion of an extremely large dose. Individual subjects vary notably as to their susceptibility to salicylate-induced auditory toxicity. Tinnitus may be the ®rst subjective symptom, and is often described as a continuous high pitch sound of mild loudness. The hearing loss is slight to moderate, bilaterally symmetrical and a€ects all frequencies with often a predominance at the high frequencies. Alterations of perceived sounds include broadening of frequency ®ltering, alterations in temporal detection, deterioration of speech understanding and hypersensitivity to noise. Behavioral conditioning of animals provides evidence for mild and reversible hearing loss and tinnitus, similar to those observed in humans. Anatomical examinations revealed signi®cant alterations only at outer hair cell lateral membrane. Electrophysiological investigations showed no change in endocochlear resting potential, and small changes in the compound sensory potentials, cochlear microphonic and summating potential, at low acoustic levels. Measures of cochlear mechanical responses to sounds indicated a clear loss of absolute sensitivity and an associated broadening of frequency ®ltering, both of a magnitude similar to audiometric alterations in humans, but at extremely high salicylate levels. Otoacoustic emissions demonstrated changes in the mechano-sensory functioning of the cochlea in the form of decrease of spontaneous emissions and reduced nonlinearities. In vitro measures of isolated outer hair cells showed reduction of their fast motile responses which are thought to be at the origin of cochlear absolute sensitivity and associated ®ne ®ltering. Acoustically evoked neural responses from the eighth nerve to the auditory cortex showed reversible and mild losses of absolute sensitivity and associated broadening of frequency ®ltering. There is no evidence of a direct alteration of cochlear e€erent innervation. Evidence was obtained for decreases in cochlear blood supply under control of autonomous innervation. Spontaneous neural activity of the auditory nerve revealed increases in ®rings and/or in underlying temporal synchronies. Similar e€ects were found at the inferior colliculus, mostly at the external nucleus, and at the cortex, mostly at the anterior and less at the secondary auditory cortex but not at the primary auditory cortex. These changes in spontaneous activity might underlie tinnitus as they a€ect mostly neural elements coding high frequencies, can occur without a loss of sensitivity, are dose dependent, develop progressively, and are reversible. Biochemical cochlear alterations are poorly known. Modi®cations of oxydative phosphorylation does not seem to occur, involvement of inhibition of prostaglandin synthesis appears controversial but could underlie changes in blood supply. Other biochemical alterations certainly also occur at outer hair cells and at a€erent nerve ®bers but remain unknown. 7 2000 Elsevier Science Ltd. All rights reserved.

Abbreviations: NSAIDs, nonsteroidal antiin¯ammatory drugs; EP, endocochlear potential; CM, cochlear microphonic potential; SP, summating potential; OAE, otoacoustic emissions; DPOAE, distortion product otoacoustic emissions; CMDP, cochlear microphonic distortion products; CAP, compound action potential; ATP, adenosine triphosphate; PG, prostaglandin. * Tel.: +33-4-91-69-89-46; fax: +33-4-91-69-87-31. E-mail address: [email protected] (Y. Cazals). 0301-0082/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 1 - 0 0 8 2 ( 0 0 ) 0 0 0 2 7 - 7

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Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

2.

Auditory perceptive alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Hearing alterations measured in humans . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Loss of absolute hearing sensitivity. . . . . . . . . . . . . . . . . . . . 2.1.2. Supra-threshold e€ects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Tinnitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Loss in absolute hearing sensitivity and tinnitus inferred from animals' 2.2.1. Hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Tinnitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Physiological alterations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Anatomical observations . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Cochlear sensory processes. . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Endocochlear potential . . . . . . . . . . . . . . . . . . . . . 3.2.2. Cochlear microphonic and summating potentials . . . 3.2.3. Cochlear mechanics . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Otoacoustic emissions . . . . . . . . . . . . . . . . . . . . . . 3.2.5. Isolated outer hair cells . . . . . . . . . . . . . . . . . . . . . 3.3. Eighth nerve and higher auditory structures . . . . . . . . . . . . 3.3.1. Acoustically-evoked activities . . . . . . . . . . . . . . . . . 3.3.2. Cochlear e€erents . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Cochlear autonomous innervation and blood supply 3.3.4. Spontaneous activity . . . . . . . . . . . . . . . . . . . . . . . 3.4. Biochemical processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Pharmacokinetics in serum and cochlea . . . . . . . . . 3.4.2. Biochemical alterations in the cochlea by salicylate .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

1. Introduction Therapeutic compounds containing salicylate have been used for thousands of years to treat various pains. Indeed they are constituents of several plants where they seem to work in defense against infection (Thomma et al., 1998; Hunt et al., 1996; Romeis et al., 1999). Prescriptions of medicaments from plants containing salicylate have been found in ancient Egyptian documents, dated around 1500 BC, and Hippocrates, around 400 BC, used a powder of willow's bark to treat pain. In the second century Galen's pharmacopeia included remedial powers of willow derivatives. It was in the 19th century that salicylic acid and salicylate were identi®ed as active ingredients of these medications. The active ingredient of the willow bark was identi®ed by Leroux in 1829 which he called salicine, salicylic acid was synthetized in 1860, and several years later acetylsalicylic acid was synthetized by Ho€man and soon after given the brand name aspirin. At the beginning of the twentieth century aspirin was widely recognized for its antipyretic, analgesic and anti-in¯ammatory properties. Various other drugs were later discovered which present the same therapeutic actions, such as for instance, acetaminophen,

and ibuprofen. These drugs, which have very diverse chemical compositions, are grouped under the name nonsteroidal anti-in¯ammatory drugs (NSAIDS) in distinction from the other major group of anti-in¯ammatory drugs, the glucocorticosteroids. Today, aspirin remains one of the most consumed drug in the world. Various estimates of aspirin consumption vary considerably but all indicate very high and increasing values: for 1970 its consumption in the USA had been estimated at 19  109 tablets (Lucente, 1971); in France estimated in 1979 as 3  109 aspirin tablets consumed annually (Oudot et al., 1979); in 1994 in the USA aspirin consumption had been estimated at 80  109 tablets a year (PharminfoNet, 1994, 1999), and according to Vane et al. (1998) 15  1012 aspirin tablets are consumed annually worldwide. Aspirin, and NSAIDS have several side e€ects: they can induce gastrointestinal irritation, ringing in the ears and hearing loss, renal dysfunction, hepatic and allergic reactions. The physiological mechanisms underlying these side e€ects are not clearly identi®ed. The observation of prostaglandin synthesis inhibition by NSAIDS (Vane, 1971) has considerably developed biochemical research in this area. The more recent discovery of two isoforms of the inhibited enzyme (Xie et al., 1991;

Y. Cazals / Progress in Neurobiology 62 (2000) 583±631

Fletcher et al., 1992) has led to development of a new group of NSAIDS speci®cally inhibiting one isoform, and presently there are several indications that these drugs show a lesser degree of side e€ects. Several of these new NSAIDS are now available on the market and large scale veri®cation of their side e€ects will probably take several years. In auditory science the toxicity of aspirin is of special concern as it presents a unique pattern of auditory dysfunctions which are described hereafter. These features were earlier observed in response to aspirin and sodium salicylate; because salicylate was soon thought to be the active compound and because early observations localized the alterations in the inner ear, this area of research has been labeled salicylate ototoxicity. To remain in line, the present review has kept this naming, however these auditory alterations are certainly also relevant to possible similar side e€ects of some other nonsteroidal anti in¯ammatory drugs, and although the inner ear seems to be the origin of auditory toxicity, the central nervous system may also be sensitive to salicylate and central auditory structures are necessarily involved in perceptual alterations. The pattern of salicylate-induced auditory sensori-neural alterations is di€erent from that found for other ototoxic drugs, so salicylate constitutes a tool of special interest for auditory research. Three previous articles reviewed the ototoxicity of salicylates. One review (Boettcher and Salvi, 1991) developed the functional audiologic alterations more whereas another review (Jung et al., 1993) elaborated more on biochemical aspects, and the third review (Brien, 1993) made a brief presentation of some essential aspects. The present review aims at extending to recent data and emphasizing new perspectives which they o€er. 2. Auditory perceptive alterations Tinnitus, loss of absolute acoustic sensitivity and alterations of perceived sounds are the three auditory alterations described by human subjects after ingestion of large doses of salicylate. Very early reports of salicylate auditory toxicity were given by MuÈller (1877) and Schwabach (1984). SeÂe (1877) indicated that salicylates relieve chronic rheumatic arthritis, and various early measures of auditory toxicity came from observations of arthritic patients who received large doses of aspirin. It soon appeared that there was a considerable individual variability as to the degree of these auditory e€ects. Since all auditory side e€ects appeared reversible, increasing the dosage until the start of tinnitus or hearing loss has been used as a titration of dosage for individuals. Many observations of the auditory alterations

585

induced by aspirin and associated drugs were made. We present here below results of studies which speci®cally examined and quanti®ed with audiometric tests the various auditory e€ects. First are presented data on loss of absolute sensitivity in hearing, i.e. deafness to the faintest sounds representing a decrease of the auditory ®eld. Then are presented more elaborate audiometric measures which emphasize alterations of auditory perceptions within the auditory ®eld. Finally are presented data on tinnitus. The speci®c features of the various auditory alterations induced by salicylate are underlined and the variable e€ects are related with available data on plasma salicylate levels. 2.1. Hearing alterations measured in humans 2.1.1. Loss of absolute hearing sensitivity 2.1.1.1. E€ects of moderate to large doses. Early observations of deafness induced by salicylate came from subjective impressions and were approximately quanti®ed with rudimentary instruments such as tuning forks and watch ticks. The advent of electroacoustics, i.e. the recording and the production of acoustic signals with electric devices, provided a considerable improvement in audiometry through highly reproducible and precisely calibrated acoustic stimuli. Macht et al. (1920) (as reported by Pohlman and Kranz in 1922) assessed the e€ects of various antipyretics, including sodium salicylate and acetylsalicylic acid, upon the hearing sensitivity of normal individuals. Using the distance at which a watch tick could be heard before and one hour after the administration of the drug, these authors reported mild alterations of hearing. Pohlman and Krantz (1922) re-examined these e€ects using an early electroacoustic device, a thermophone. Their results con®rmed a loss of hearing sensitivity after sodium salicylate and acetylsalicylic acid, but in®rmed results on other antipyretics. In later studies electroacoustic audiometers essentially similar to the audiometers of nowadays started to be employed. Falbe-Hansen (1941) reported about several experiments on humans in which some measures were performed with an audiometer although many were still performed with tuning forks. Over all their experiments, 51 subjects were tested. Results indicated that a single intake of sodium salicylate at the dose of 2 g did not induce any detectable change, whereas at doses of 3 or 5 g a hearing loss could be detected which was not subjectively clearly felt, which at audiometry a€ected mostly the high frequencies and could reach about 20 dB. In additional experiments, doses of 3±7 g/day fractionated in 1 g/h produced similar results. E€ects of successive daily doses were tested on 21 normally hearing subjects who took from 2 to 6 g daily over 3±18 days. Among them six subjects received 3 g

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or less daily and presented no deafness, the other 15 subjects received 4 g or more daily and 13 of them suffered some hearing loss. In most cases deafness disappeared within 3±5 days after treatment cessation. Subjects with a preexisting hearing loss were also tested. 13 cases had a middle ear conductive deafness and ®ve cases had a sensori-neural deafness. In two subjects who received a single dose of 3 g of sodium salicylate, no hearing deterioration was observed. In the 16 other subjects who received daily doses of 3.8±7 g from 3 to 39 days a further hearing loss was detected which was similar to that observed for subjects with normal ears. Jager and Alway (1946) observed 38 patients, 27 with rheumatic fever and 11 with other diseases. They were treated with daily intravenous injections of sodium salicylate, for 4±16 weeks in 30 patients, and less for the other eight patients. Plasma salicylate levels were determined two to three times a week, or at least once a week. Subjective impression of deafness ``to a noticeable degree'' was present in only ®ve patients. Subjective symptoms appeared more pronounced with higher plasma salicylate level. Within 2±3 days following cessation of medication, deafness disappeared. Two other subjects received oral salicylate medication and their hearing was measured audiometrically every week. Their plasma salicylate levels ranged from 220 to 550 mg/l and their hearing losses ranged from 20 to 35 dB. At cessation of treatment hearing went back to normal within 3±4 days. Graham and Parker (1948) studied salicylate therapy in 40 rheumatic subjects and 30 nonrheumatic patients serving as controls. Blood samples were taken whenever the patient reported a toxic e€ect in the ®rst 7 days of treatment. 58 patients made such complaints. Three cases complained of deafness and presented plasma salicylate levels of 72 mg/l. In nine other patients deafness appeared for plasma salicylate levels of 100±480 mg/l, the average value being about 250 mg/l. Among 33 patients whose plasma salicylate level rose above 350 mg/l, all complained of deafness. Therapy was maintained in spite of these e€ects which disappeared in a few days. When they reappeared they were controlled by omission of the treatment for 1±2 days and did not recur with subsequent attainment of much higher plasma salicylate levels. Deafness was always reversible after cessation of treatment. Waltner (1955) reported on a young woman who ingested approximately 11 g of aspirin per day for 6 days and su€ered tinnitus, deafness and vertigo. Her audiogram showed a loss of approximately 50 dB at all frequencies. Two days after cessation of the treatment the hearing loss was reduced to about 20 dB and after a week, thresholds were normal. Weiss and Lever (1964) presented three cases of adults treated for psoriasis with skin ointment contain-

ing salicylic acid. After several days of treatment, all three su€ered tinnitus and hearing loss, and presented plasma salicylate levels of 460±640 mg/l. A few days after treatment cessation all symptoms disappeared. Myers et al. (1965) undertook to further quantify the audiometric loss together with associated blood salicylate levels. Twenty-one patients with rheumatoid arthritis were studied, for these patients salicylate treatment was withdrawn and replaced with propoxyphene hydrochloride so that at the beginning of the study the salicylate plasma level was negligible. The subjects were then given increasing doses of acetylsalicylic acid and magnesium aluminum hydroxide until high-pitched tinnitus and subjective hearing loss were noted by the patients. This required 6±8 g of the drug per day. Audiometric assessment indicated that subjects with a normal audiogram before the start of the study, showed, in both ears, threshold elevations at all frequencies ranging from 20 to 40 dB. In subjects with a preexisting hearing loss, threshold elevation a€ected more the frequencies with best sensitivity and resulted in a ¯at audiometric loss at about 40 dB for all frequencies. After cessation of treatment, in all cases, salicylate-induced hearing losses were completely reversible within approximately 1 week, improvement started within 24±72 h. Blood salicylate levels varied threefold between subjects, approximately from 200 to 600 mg/l, and exhibited a weak nonsigni®cant correlation with the audiometric loss. Myers and Bernstein (1965) additionally studied four patients who had been taking large doses of salicylate for several months or years and who su€ered from tinnitus and hearing loss. Audiometric testings were performed, and a value of 20 dB of hearing loss is indicated as being typical. Then the salicylate was withdrawn from treatment and replaced by propoxyphene hydrochloride. Within three days tinnitus disappeared and hearing improved. McCabe and Dey (1965) experimented on ®ve normally-hearing women about the e€ects of aspirin tablets ingested at a dose of approximately 5 g of salicylate per day for 4 days. Audiometric evaluation was based upon six audiometric tests including the audiogram. Threshold shifts were progressive over the 4 days of treatment, a€ected all frequencies but were more pronounced at high frequencies where they reached 30 dB on average. Measured blood salicylate levels varied less than twofold between subjects, from 215 to 348 mg/l. Unfortunately, no indication is available as to their relation with auditory alterations. The authors note an inverse relation between body weight and salicylate plasma concentration. All subjects returned to their normal thresholds within 3 days of the last dose. Perlman (1966) reports on two women having generalized psoriasis who applied on their skin, three times

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a day, an ointment containing 5% salicylic acid and who complained of hearing loss. Audiometry indicated symmetrical bilateral hearing losses of 60 and 50 dB while their plasma salicylate levels were measured at 350 and 240 mg/l, respectively. A week after cessation of treatment hearing had returned to normal. Bernstein and Weiss (1967) studied twelve patients with rheumatoid arthritis who received increasing doses of acetylsalicylic acid and magnesium aluminum hydroxide until subjective hearing loss was noted. This occurred generally for a dose of 6±8 g per day. The hearing loss was bilaterally symmetrical with nearly equal threshold elevation at all frequencies ranging from 25 to 45 dB. Plasma salicylate levels varied approximately twofold, from 225 to 475 mg/l, and appeared correlated to the hearing loss. All patients had recovered by 4 days after the end of the treatment. Perez de Moura and Hayden (1968) reported on a 76-year-old woman with rheumatoid arthritis taking up to 5 g of acetylsalicylic acid per day. She presented an audiogram with a symmetrical bilateral loss at all frequencies ranging from 30 to 70 dB. Three days after discontinuing drug ingestion her audiogram had improved by about 25 dB at all frequencies. Mongan et al. (1973) performed a study speci®cally assessing tinnitus induced by salicylate in a group of 67 patients with rheumatoid arthritis and seven healthy volunteers. Hearing loss data for individuals is unfortunately not available. An example case is given presenting a loss of hearing sensitivity of approximately 30 dB at all frequencies and presenting a plasma salicylate level of 258 mg/l. Pedersen (1974) tested 21 subjects with normal hearing, seven untreated served as controls and 14 were treated with aspirin at 4 g/day, this dosage being reduced for some subjects in whom tinnitus or dizziness appeared. After 3 or 4 days audiometric testing was performed and blood samples drawn. Treatment was stopped and a week later audiometric testing was performed again. Seven persons without treatment served as a control group. Various degrees of hearing loss of up to 40 dB were observed for all treated subjects. Audiometric measures were back to normal after a one week recovery. A good correlation was found between plasma salicylate levels, ranging from 60 to 300 mg/l, and audiometric losses. An unpublished study of May (1976), as indicated by McFadden and Plattsmier (1983), reported on a series of 21 normally-hearing subjects who took a single ingestion of aspirin at the dose of 13.2 mg per kg of body weight, doses ranged between subjects from 0.67 to 1.25 g. No loss of hearing sensitivity was detected in them. Jardini et al. (1978) studied thirty patients with rheumatoid arthritis. They were divided in two groups,

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one receiving a lactose placebo and the other receiving acetyl salicylic acid capsules for 7 days. At days 0, 3 and 7 blood salicylate levels were taken and audiological tests were performed. Over the 7 days, blood salicylate levels underwent a sixfold diminution on average for the placebo receiving group, whereas it remained approximately stable for the aspirin group. For subjects of the placebo group blood salicylate level declined systematically and was associated with a signi®cant improvement in audiometry. Individual audiograms are not given. For subjects of the aspirin group blood salicylate levels varied over the three test days, highest or lowest blood salicylate levels being recorded on the third day. Greater threshold shifts in audiometry occurred on the day with higher blood salicylate level. Low or moderate plasma salicylate levels were measured, and presented, respectively, average values of 16 and 114 mg/l. Bonding (1979) tested 16 normally hearing subjects treated with acetylisalicylic acid at 4 g/day during 4 days. Subjects who developed tinnitus and/or hearing loss during treatment were tested audiometrically. A group of ®ve normal untreated subjects was used for control. All treated subjects showed a ¯at hearing loss of 10±40 dB. A good correlation was found between salicylate plasma levels, ranging approximately from 150 to 400 mg/l, and hearing loss. A control audiometry performed 2±3 weeks later indicated normal results. Johnsen and Elberling (1982) report on a normallyhearing woman who was given 10 g of acetylsalicylate in a day. Her audiogram showed a relatively ¯at loss at all frequencies of 25±30 dB, and her plasma salicylate level was about 400 mg/l. Two days later the audiogram was back to normal. Young and Wilson (1982) studied ®ve subjects (two with rheumatoid arthritis, one with tendinitis and two volunteers). The two volunteers were treated for 1 day with doses of 1.5 g of aspirin every 4 h. If tinnitus occurred the subjects decreased the dose so that tinnitus no longer continued. The three patients were treated with doses of 6±8 g/day during up to a week. Audiometric testing was performed before treatment and at the end of day 1 to 1 week treatment. Small hearing losses of up to 20 dB maximum were observed which could a€ect all frequencies with a dominance at the highest frequencies. McFadden and Plattsmier (1983) studied four normally-hearing subjects ingesting either 1.95 or 3.9 g of aspirin per day for 2.75 days. While no signi®cant hearing loss was detected for the lower dose, hearing losses of a few dB to more than 15 dB were observed for the higher dose. Blood samples were taken at the end of the treatment and ranged from 71 to 271 mg/l. The two authors also studied on themselves progressive doses of aspirin ranging from 4.4 to 5.8 and 3.9 to

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4.9 g per day for 4 days. Their hearing loss amounted to 10±25 dB. In an additional study McFadden et al. (1984a) report about eight subjects with normal hearing who ingested 3.9 g of aspirin per day for 4 days. The subjects presented hearing losses of 5±15 dB. A similar e€ect was observed for treatment with identical doses of sodium salicylate. McFadden et al. (1984b) investigated on ®ve normally-hearing persons the e€ects of a dose of 3.9 g of aspirin per day for 5 days. Hearing losses were measured every day at 3500 Hz and on the third day at 500 Hz. A blood sample was taken on the fourth day to measure salicylate levels which ranged from 150 to 407 mg/l. Two subjects with light eye-color showed less than 6 dB of hearing loss and low serum levels. The three other subjects showed an approximately constant threshold elevation around 10±20 dB at 3500 Hz over the 5 days and a threshold elevation of 6±15 dB at 500 Hz on the third day. McFadden and Plattsmier (1984) measured hearing losses of up to 20 dB in ®ve young subjects with normal hearing after 3.75 days of aspirin ingestion at a rate of 975 mg every 6 h. Detailed values of hearing losses are given for two subjects and show considerable variations as a function of both sound frequency and time during treatment. Values of salicylate plasma levels are not available. Koegel (1985) reports on a 52-year-old woman complaining of bilateral ¯uctuant hearing loss, aural fullness, unsteadiness and tinnitus, who had taken approximately 2 g of aspirin per day for many months. Audiometric testing indicated a ¯at loss of approximately 45 dB. The patient was instructed to discontinue the drug and when she came back to the clinic 24 days later her hearing had returned to normal. Lindgren and Axelsson (1986) studied 10 male volunteers with normal hearing who received 1 g of acetyl salicylic acid per day during 2±5 days. No signi®cant alteration of hearing was observed. Wier et al. (1988) studied four adult male subjects ingesting 3.9 g of aspirin per day for 1±4 days. Variable threshold shifts were observed at di€erent frequencies and for the di€erent subjects. Hearing losses of up to about 10 dB were measured. Long and Tubis (1988a, 1988b) examined the absolute hearing threshold microstructure, i.e. the threshold variations for sound frequencies very close to each other. Four normally-hearing subjects ingested 3.9 g of aspirin per day for 3±4 days. Threshold microstructure was measured before, during and after aspirin consumption. At the ®rst day of aspirin consumption subjects often presented improved thresholds (better absolute hearing sensitivity) specially at maxima of their threshold microstructure curve. In later days a threshold deterioration was observed. Threshold changes were variable for di€erent frequencies and

between subjects, changes of up to 15 dB were measured. Day et al. (1989) experimented on eight normal volunteers the e€ects of 1-week treatments with aspirin in slow release tablets at four dosages ranging from approximately 2 to 6 g/day, the 1-week treatments being administered in random order, double blind and 2 weeks apart. At the end of each treatment week, blood samples were taken and hearing loss was measured with a pure tone audiogram. No e€ect was observed for the lowest dose of 1.95 g/day. Quasilinear relationships were observed between higher aspirin doses and hearing loss. The hearing loss a€ected all frequencies and was small, less than 10 dB, but for the higher dose of 5.85 g/day it reached about 20 dB. All measures of total and unbound plasma salicylate concentrations plotted as a function of all corresponding hearing losses showed a rather linear relation. Two studies by Halla and Hardin (1988) and Halla et al. (1991) examined the presence of tinnitus and subjective hearing loss together with blood salicylate levels in large populations of arthritic patients. Halla and Hardin (1988) examined 134 patients with rheumatoid arthritis and treated with salicylate in comparison with 182 healthy untreated subjects. In a complementary study Halla et al. (1991) examined 260 patients with rheumatoid arthirtis and 112 patients with osteoarthrirtis, both groups being not treated with salicylate. Their results indicate that often the subjective symptoms of tinnitus and hearing loss were equally present frequently in these populations, and that signi®cant salicylate side e€ects are recognized as an aggravation of these symptoms and not simply as the presence of these symptoms. Overall these data indicate that subjective symptoms correlate poorly with a single random salicylate serum measurement. Additional results reinforce this point since they observed that audiometric measures correlated poorly with subjective impressions. McFadden and Champlin (1990) studied ®ve subjects who took 3.9 g of aspirin per day for 4 days. Audiometric testing indicated a hearing loss of 5±16 dB. Jordan (1991) reports on a 76-year-old man who complained of a bilateral hearing loss and roaring tinnitus. His audiogram revealed a typical presbyacusic loss of 20±60 dB increasing progressively at high frequencies. On enquiry the subject indicated taking 6±7 g of aspirin per day for vertebral pain relief. A month after cessation of aspirin consumption the subject presented threshold improvements of approximately 20 dB but for the high frequencies a€ected by presbyacusis. Brown et al. (1993) tested four normally-hearing subjects who took 3.8 g of aspirin per day for 2 days. Hearing losses were variable between subjects,

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threshold elevation a€ected all frequencies and could reach 25 dB. Carlyon and Butt (1993) examined eight normally hearing volunteers who took 3.8 g of aspirin per day for 2 days. The same subjects were used as controls by taking a placebo for 2 days. Hearing losses were small, up to 15 dB, and when clearly present a€ected all frequencies. Beveridge and Carlyon (1996) studied on nine normally hearing subjects the e€ects of ingestion of 3.8 g of aspirin per day for 3 days. The same subjects were used as controls by taking placebo for 3 days. Six subjects presented a hearing loss of 3±12 dB while the other three showed a di€erence of one dB or less. Hicks and Bacon (1999) observed six normally-hearing subjects who received a dose of 3.9 g of aspirin per day for 4 days, hearing losses a€ecting all tested frequencies. The hearing loss for brief signals averaged over four frequencies, from 375 to 3000 Hz, ranged from 3.7 to 19 dB for the di€erent subjects at the fourth day. Plasma salicylate levels taken at the fourth day ranged from 138 to 365 mg/l and were well correlated with the hearing loss. The hearing losses measured at the second and third days also correlated well with plasma salicylate levels measured at the fourth day. However, hearing losses measured for longer duration signals, lasting 300 ms rather similar to signals usually employed in audiometry, and at di€erent days for di€erent subjects, do not show such a correlation. Quaranta et al. (1999) studied eight young normallyhearing subjects who ingested 3.84 g of aspirin per day (doses of 960 mg of aspirin every sixth hour) for 2 days (eight doses). Subjects were tested before the ®rst aspirin ingestion, after the fourth and the eighth ingestion, and ®nally 7 days after the last aspirin ingestion. The mean blood salicylate levels were 102.5 and 132.5 mg/l after the fourth and the eighth ingestions. Losses in absolute hearing sensitivity were on average at about 6 dB for frequencies above 2 kHz at the fourth ingestion and up to 9 dB at the eighth ingestion. No individual data is available and no correlation is indicated. 2.1.1.2. E€ects of extreme doses. Salicylate poisoning contributes a signi®cant proportion of hospital admissions for poisoning. Tinnitus and deafness are major symptoms in cases of ingestion of very high doses of salicylate and plasma salicylate levels of up to 1000 mg/l have been recorded as indicated by Ghose and Joekes (1964) and Mantz et al. (1978). Gignoux et al. (1966) reported on a 16-year-old woman who attempted to commit suicide by ingesting 22 g of aspirin. By the third hour she noticed a subjectively `total' bilateral deafness. By the seventh hour

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she was hospitalized and had a plasma salicylate level of 830 mg/l. After two days, deafness regressed. Oudot et al. (1979) reported aspirin to have been used in approximately 4% of suicide attempts and in ten such patients they could document induced hearing alterations. The ingested doses could reach 50 g of aspirin sometimes accompanied by another drug. The reported symptoms included tinnitus, hearing loss, a sensation of fullness of the ear and less often vertigo. Audiometric losses and plasma salicylate levels were measured at the earliest 6 h after the aspirin ingestion, and then at various times for the di€erent subjects. Plasma salicylate concentrations ranged from 100 to 300 mg/l. Hearing loss a€ected all frequencies and amounted to a maximum of approximately 40 dB. For all subjects, within 2 days of hospitalization, subjective auditory symptoms had disappeared and the audiogram showed recovery. Ramsden et al. (1985) reported on two cases of attempts to commit suicide. One case had consumed 100 aspirin tablets, presumably about 30 g of aspirin, and was examined four 4 h later. He had a plasma salicylate level of 606 mg/l and a bilateral symmetrical hearing loss at all frequencies of approximately 30 dB. The second subject had taken about 30 tablets, presumably about 10 g of aspirin. Two hours later, he had a plasma salicylate level of 448 mg/l, and showed a bilateral symmetrical hearing loss of approximately 40 dB. After a few days of hospitalisation all symptoms disappeared for both subjects. Janssen et al. (2000) examined a 22-year-old woman who ingested a dose of presumably 10 g of aspirin with suicidal intent. At 22 h post-aspirin ingestion, they measured a hearing loss of 15±25 dB at low to mid-high frequencies with a loss of 40±50 dB at the highest frequencies. One day later hearing was back to normal. 2.1.1.3. Features of salicylate-induced loss of absolute acoustic sensitivity. From the data reviewed above several characteristic features can be drawn. The hearing loss induced by salicylate may not be the ®rst auditory e€ect. When present it is slight to moderate. It can be so mild as to remain unnoticed or hardly detectable with audiometry. There is evidence that in an early stage an opposite e€ect, i.e. a sporadic improvement of hearing sensitivity may occur. For subjects with a normal hearing, the salicylate-induced hearing loss is bilateral, symmetrical and it a€ects all frequencies with often a predominance at the high frequencies. For the same ear the hearing loss can vary considerably from one frequency to the next. The loss did not exceed about 40±50 dB even for the highest doses. In cases of a preexisting hearing loss the salicylate-induced loss is not simply additive as the total loss appears limited to about 50 dB. The hearing loss develops over the initial

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days of treatment but may then level o€ and ¯uctuate or even show marked decreases in the longer term. Individual subjects appear to vary considerably as to their susceptibility to salicylate-induced hearing loss. The hearing loss is reversible within a few days of cessation of treatment. An aspect of major concern in the clinical studies is the considerable inter-individual variability observed in the results. It should be noted that in various studies a similarly considerable day-to-day intra-individual variability was observed. In addition, but to a lesser degree, there can be a notable variability in threshold shifts at di€erent frequencies for a given ear especially when the hearing losses are mild. Because aspirin and other related compounds such as sodium salicylate are known to quickly degrade to salicylate which is thought to be the active compound, dosages were made of plasma salicylate levels in various studies presented above. The raw results of these studies appear contradictory, as some experimenters observed a correlation between individual plasma salicylate levels and hearing loss, while others did not. Many of these studies concerned only a few individuals and their results are therefore very sensitive to one or two marginal data. In order to further assess a relation between hearing loss and plasma salicylate levels we collected and plotted together the available data from these studies. It must be emphasized that although these studies used conventional audiometry, data given in written reports are not exhaustive and did not provide identical measures. Indeed, studies di€er considerably in audiometric procedures used, in particular in their precision in dB, in frequencies tested, and in signal durations used. Other very signi®cant factors such as drug composition, administration schedule, and delay between plasma extraction and audiometric measures also di€ered notably. In the collection and plotting of data, we selected, as much as possible, data corresponding to middle frequencies, or average over low to high frequencies, and long duration signals. Fig. 1 presents the data for more than a hundred subjects extracted from 16 studies. It can be seen that overall, together with a signi®cant scatter of data, there seems to be an approximately linear relation between plasma salicylate level and the amount of hearing loss. Statistical analysis of the data indicate a very signi®cant correlation coecient value of 0.7 which approximately accounts for half of the total variance of the data. Little data is available as to factors associated with this individual variability. The plasma salicylate levels measured for individuals receiving the same drug dose show several-fold variations. Various studies which measured plasma salicylate levels at di€erent times during salicylate treatment indicate large variations in time. Jager and Alway (1946) documented these aspects very demonstratively. They observed that

during the ®rst two weeks of oral administration of constant doses of sodium salicylate to patients with rheumatic fever, plasma salicylate level initially increased but later tended to drop. In spite of careful regulation of dosage, plasma salicylate level for a given individual often varied as much as 100 mg/l from day to day or from week to week. Throughout the day variations of 50±90 mg/l could be observed. In two cases sudden changes of 200±300 mg/kg were accompanied by toxic manifestations. Some studies mention the body weight of the subjects to be of signi®cant in¯uence. The most demonstrative data are from the study of McCabe and Dey (1965) which clearly indicate an inverse relation between body weight and plasma salicylate level. In one of their studies McFadden et al. (1984b) remarked that two subjects with light-eye color presented the lowest plasma salicylate levels associated with the lowest hearing losses. The reversibility of hearing loss after cessation of treatment is a constant observation in the above-mentioned studies and appears to be a characteristic feature of salicylate ototoxicity. Only two articles report

Fig. 1. Loss on absolute sensitivity as reported from the 16 references indicated in the insert at upright. To obtain more comparable data losses were taken as much as possible for middle frequencies. Continuous line: linear regression line.

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irreversible hearing losses after salicylate ingestion which, however, cannot be attributed with certainty to salicylate. Kapur (1965) reports on a 13-year-old boy with a normal audiogram who developed a mild fever. On the second day he took two tablets of aspirin, presumably 600 mg, and another tablet on the morning of the third day. He noticed a hearing loss soon after taking the two tablets. Fever came down on the third day but the child was dizzy for 2±3 days afterwards. He did not experience tinnitus. A month later he was still su€ering from a hearing loss, was examined and his audiogram showed a severe bilateral hearing loss of approximately 80 dB. After two months his audiograms still showed a bilateral ¯at loss of around 60 dB. It is quite surprising that a dose of a little more than a gram could have been by itself the cause of the observed severe hearing loss. It is also surprising that no tinnitus had been experienced since it is a common complaint in other studies. Jarvis (1966) reports on a young female who, after having had a tooth extraction, ingested 2±3 aspirin tablets every 2 h, presumably amounting to above 9 g/day, for 3 days and then noticed vertigo, with unilateral tinnitus and hearing loss. The hearing loss was still present 4 months later. The unilaterality of hearing loss and tinnitus are in opposition with all other reports on salicylate otoxicity and cast doubt as to the aspirin being the only cause of these symptoms. It seems likely that in these two studies aspirin ingestion may have triggered or potentialized more permanent causes of hearing impairments. 2.1.2. Supra-threshold e€ects Together with deterioration of absolute hearing sensitivity many reports indicate alterations of perceived sounds as induced by salicylate toxicity. At very high doses salicylate can a€ect the central nervous system with drowsiness or euphoria to delirium and coma, and thus might indirectly a€ect auditory perceptions. Jager and Alway (1946) indicate drowsiness and euphoria as common manifestations at high plasma salicylate levels; for several subjects with plasma salicylate levels over 500 mg/l they observed hallucinations, mental confusion and coma. Graham and Parker (1948) report drowsiness and mental upsets as uncommon e€ects of salicylate. Oudot et al. (1979) indicate in cases of attempts to commit suicide with salicylate, a hypervigilance followed, if no treatment is applied, by coma. There are auditory-speci®c e€ects of salicylate which occur at much lower doses. These e€ects were earlier noticed as subjective diculties in identi®cation of sounds and have been a growing research area over the last decades. Hereafter, are presented audiometric measures of these alterations in the perception of sounds above threshold.

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2.1.2.1. Audiometric observations. Myers and Bernstein (1965) observed that 40% of their 25 patients presented a deterioration of speech audiometry. For normal hearing, as acoustic level increases above hearing threshold, the recognition of words improves and reaches 100% recognition score. Patients with deteriorated speech perception presented a loss of at least 10% in maximum recognition score. A third of the 25 patients presented a better than normal sensitivity to brief and small amplitude modulations as measured with the short increment sensitivity index (sisi) test. Recruitment, i.e. a steeper than normal growth of loudness as a function of acoustic level, was observed in one patient with a preexisting sensori-neural deafness. These results are consistent with an impairment at the level of the cochlea. McCabe and Dey (1965) made a selection of six di€erent audiometric tests aiming at discriminating between cochlear (sensory), retrocochlear (cochlear nerve) and central auditory processes. Hearing thresholds for tones were found more deteriorated when presented as a series of brief pulses than when presented as a steady signal. Average threshold elevations for pulsed tones were more elevated than for steady tones at high frequencies, a di€erence of up to 10 dB was observed. Threshold adaptation, i.e. the decay in time of the perception of a tone which decreases progressively in amplitude, was found unchanged. A test of temporal integration at threshold of hearing was used which indicates a slope of 10 dB of threshold elevation for reduction of tone duration by a factor of ten for normal hearing. On average the slope of the temporal integration function was found reduced from 10 to 5 dB per decimation of signal duration during salicylate treatment. These results appear contradictory with the deterioration of pulsed versus steady tones. Two speech tests were used, one consisting of sentences and the other of word pairs di€ering in the last syllable (rhyme test) presented in background noise. Average results for these speech tests were found una€ected. Measures of temporary threshold shifts induced by exposure to a loud sound of 112 dB SPL for 5 min were performed. The threshold shifts were tracked over 5 min after the loud sound exposure. Results showed less threshold shift during salicylate treatment. However, when the exposure level was adjusted to account for the loss in absolute hearing sensitivity, the di€erence disappeared but for a small di€erence which remained at 3±5 min post-exposure. The majority of subjects reported some diculty with the normal perception of sounds in their home environment, with more concentration being required in most everyday listening situations. Bernstein and Weiss (1967) studied twelve patients. Five patients had speech intelligibility scores reduced by 20% or more. For three patients who did not show

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such reduced intelligibility scores a more re®ned test of ®ltered speech audiometry was performed. In this test speech was ®ltered in two bands, one containing high frequencies and the other low frequencies, and the respective contribution of each and both bands to speech recognition was found unchanged. Pedersen (1974) measured temporal integration at frequencies of 500, 1000, 4000 and 8000 Hz in 14 subjects treated with salicylate. Measures were taken during salicylate treatment and in the same subjects a week after cessation of treatment. After 3±4 days of salicylate treatment there was, at all frequencies, a decrease in temporal integration of approximately 5 dB per decimation of signal duration. The amount of temporal integration decrease varied among the di€erent subjects and presented a signi®cant correlation with plasma salicylate levels and with loss of hearing sensitivity at the same frequency. In previous studies the same authors observed similar audiometric alterations after acoustic trauma and in subjects with presbyacusis. Unpublished data by May (1976) as reported by McFadden and Plattsmier (1983), indicate an aspirininduced potentiation of sensitivity to acoustic trauma even in the absence of a loss of hearing sensitivity induced by aspirin. A single dose of about 1 g of aspirin was ingested by subjects an hour and a half before exposure to a 103 dB broad band noise for 20 min. Several days apart a similar noise exposure without previous aspirin injection was performed. After aspirin ingestion an increase of about 7 dB was observed in the temporary noise-induced hearing loss. Jardini et al. (1978) studied speech audiometry in 18 patients with rheumatoid arthritis already under treatment with salicylate. They were divided in two groups, one receiving a lactose placebo and the other receiving acetylsalicylic acid capsules for 7 days. At days 0, 3 and 7, three measures of speech intelligibility were performed: threshold for 50% speech recognition (SRT), percentage of speech recognition at 40 dB above SRT in quiet and in a background noise with a signal to noise ratio of ÿ4 dB. For subjects of the placebo group blood salicylate level declined systematically and was associated with a signi®cant improvement in both pure tone audiometry and speech threshold scores. Individual data for audiogram and speech scores are not given. Overall at days 3 and 7 the aspirin group presented poorer results at all three tests of speech audiometry than the placebo group. Bonding (1979) investigated values of critical bandwidths in 16 normally hearing subjects treated with acetylsalicylic acid. Several auditory perceptions remain unchanged when the frequency bandwidth of acoustic stimuli is progressively increased until an abrupt change occurs when the bandwidth is increased beyond a critical value. The study investigated the

loudness matching of noise bands of various bandwidths to the loudness of a broadband noise. In 14 of the 16 cases critical band estimates were wider during than after salicylate treatment. A good correlation was found between the amount of widening and the loss of hearing sensitivity. A widening appeared when the loss exceeded about 20 dB. Loudness matches indicated maximal widening usually at mid-intensities around 50 dB whereas at high acoustic levels no di€erence was observed indicating loudness recruitment. Young and Wilson (1982) studied speech audiometry using words presented at 70 dB SPL either in the quiet or in the presence of a background noise. Three of the ®ve subjects showed a signi®cant loss of speech recognition in noise specially at the poorest signal to noise ratio of ÿ8 dB. These three subjects were retested several days after salicylate treatment and presented performances back to normal. There was no correlation between de®cits in speech perception in noise and losses of hearing sensitivity. McFadden and Plattsmier (1983) investigated the potentiation by aspirin of temporary hearing loss induced by a loud sound in six subjects. The exposure was a 2500 Hz tone of 10 min duration, the level of which was adjusted for each individual to induce a temporary hearing loss of about 15 dB. A temporary hearing loss of 18±27 dB was observed if subjects had taken about 4 g, or more, of aspirin per day for the previous 2 days, or more. Lesser doses or shorter durations of aspirin consumption produced smaller or no such exacerbation of noise-induced hearing loss. A slower recovery was also apparent in the data. In an additional study, McFadden et al. (1984a) report about 11 subjects with normal hearing who ingested 3.9 g of aspirin per day for 4 days. The subjects presented hearing losses of 5±15 dB. A potentiation of acoustic trauma by 10±15 dB was again observed. A similar e€ect was observed for treatment with identical doses of sodium salicylate. Two other NSAIDS, sulindac and di¯unisal, administered at approximately equivalent therapeutic dosages, induced no hearing loss and no potentiation of acoustic trauma. McFadden et al. (1984b) investigated the e€ects of aspirin upon three audiometric tests of temporal auditory perceptions: forward masking, temporal integration and gap detection. Measures were taken from ®ve normally-hearing persons ingesting a dose of 3.9 g of aspirin per day for 5 days. Among the ®ve subjects, three presented a signi®cant loss of hearing sensitivity of 10±20 dB. A loud sound may render a fainter sound inaudible. This phenomenon is called masking, and forward masking refers to the masking of a sound by another sound which occurred a brief time earlier. Experiments were performed using tones at 3500 Hz at the three time intervals of 0, 15 and 30 ms. In control tests the subjects showed a forward masking slope of

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about ÿ0.5 dB/ms. Measures at the second day of aspirin treatment indicated that two of the three subjects with a hearing loss showed a slight reduction in the slope of forward masking. Temporal integration was measured at the two frequencies of 500 and 3500 Hz. Slopes of temporal integration at control sessions were about 9 dB per decimation in signal duration. Measures taken on the third day of treatment showed slopes reduced to about 3 dB per decimation of signal duration for the high frequency. For the lower frequency, at which subjects presented less hearing losses, results were more irregular and did not show clear changes. The test of gap detection consisted in measuring the minimum duration of a silent interval placed within a noise, to be heard. Two noise bands were used and centered around 600 and 3500 Hz and tested at ®ve levels between 25 and 75 dB SPL. For four of the ®ve subjects aspirin diminished the temporal sensitivity, i.e. longer silent gap durations were needed for detection. Changes were observed for both frequency bands and appeared clear for the two lowest acoustic levels used. Koegel (1985) observed a subject who had taken approximately 2 g of aspirin per day for many months. Audiometric testing indicated a ¯at loss of approximately 45 dB and speech intelligibility reduced to 40 and 28% in the right and left ear, respectively. Several days after drug cessation hearing returned to normal. Lindgren and Axelsson (1986) studied 10 male volunteers with normal hearing who were exposed from 2 to 5 days to a third octave ®ltered noise centered around 2000 Hz with an acoustic level of 105 dB SPL for 20 min. The same subjects, on other sessions ingested 1 g of acetylsalicylic acid per day 1 h before being exposed to the same loud noise. Each subject underwent in a randomized order the noise alone or acetylsalicylic acid ingestion plus noise exposure sessions. No change in absolute sensitivity of hearing was detected. No evidence of exacerbation of noise induced loss by aspirin was observed either. McFadden and Champlin (1990) investigated the e€ects of aspirin upon the perception of a phenomenon called masking onset overshoot. When a brief tone is presented at the onset of a long duration sound it is more masked than if presented several hundreds of milliseconds after the onset of the long sound. Di€erence in masking between onset and later occurrence, expressed in dB, is used to measure masking overshoot. Five subjects took 3.9 g of aspirin per day for 4 days and presented hearing losses of 5±16 dB at the tested frequency of 3550 Hz. A mid-intensity noise level was used which produced a threshold detection of the brief signal at approximately 50± 60 dB SPL. Under aspirin treatment four of the ®ve subjects presented reduced masking overshoot, this being due to a better than normal detectability of the brief sound at

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onset. The reduction in masking overshoot did not appear related to the loss of absolute hearing sensitivity. Such e€ects were also observed after exposure to an intense sound, although then, only when the loss of absolute sensitivity was 10 dB or more. A constant background noise aimed at mimicking a threshold elevation induced by aspirin did not result in similar reduction of overshoot. Jordan (1991) studied the case of a 76-year-old man who took 6±7 g of aspirin per day for vertebral pain relief, presented with a loss of hearing sensitivity of about 20 dB accompanied with a loss of 20% in speech recognition score. Carlyon and Butt (1993) performed measures of the sharpness of auditory ®lters under aspirin treatment. Auditory perception of a sound at a given frequency is more perturbed by sounds of nearby than of remote frequencies. This represents a frequency ®ltering of sounds. The authors used a 4000-Hz brief tone signal and estimated auditory ®lter width by measuring the masking e€ects of a broad-band noise with a notch in frequency. Three widths of notch and three positions of notch in reference to signal frequency were tested. At the widest notch-width for most subjects, a broadening of ®lter shape was measured under aspirin treatment. There was no correlation with loss of absolute sensitivity of hearing for the di€erent subjects. Altogether the data also permitted to observe that there was no reduction of temporal integration, a fact that the authors suggest could be due to the limited aspirin treatment used. In a further study on sharpness of auditory ®lters Beveridge and Carlyon (1996) measured the masking of narrow-band noises on a brief tone, using both a forward and a simultaneous masking paradigm. Forward and simultaneous masking produced similar results. A signi®cant broadening of auditory ®lter was detected with no correlation to the loss of absolute sensitivity. Long (1998) studied the phenomenon of monaural diplacusis, i.e. the perception of multiple tones when only one tone is presented to an ear. One of the three subjects who presented monaural diplacusis ingested 2.9 g of aspirin over a day. Psychoacoustic measures were taken before, during and 2 days after aspirin consumption. At the end of aspirin consumption improved thresholds and disappearance of monaural diplacusis were measured. The authors also indicate that other subjects reported disappearance of monaural diplacusis after aspirin ingestion. Hicks and Bacon (1999) investigated three tests of auditory nonlinearity at two frequencies: 750 and 3000 Hz, their tests measured sharpness of auditory ®ltering, two-tone suppression and growth of masking with acoustic level. Measures of loss of absolute sensitivity performed with tones of two di€erent durations showed that temporal integration was reduced by

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about 2 dB only on average for all subjects and was similar at low, middle and high frequencies. These reductions presented notable variations between subjects and for ®ve of the six subjects measures revealed no reduction at, at least, one frequency. Data indicate no correlation with loss of absolute sensitivity. Sharpness of auditory ®ltering measured with notched noise and at two acoustic levels, was found broadened under aspirin. The broadening was slight and mostly at low acoustic level for 750 Hz. It was more pronounced and independent of acoustic level for 3000 Hz. At 750 Hz results were not correlated with loss of absolute hearing sensitivity whereas at 3000 Hz they were. The twotone suppression test refers to the decrease, or suppression, of masking of a sound onto a signal by the presence of a third sound. For a 750-Hz signal, addition of a third suppressor tone did not induce masking suppression and no change was observed under aspirin. At 3000 Hz clear suppression was observed which, under aspirin, was notably reduced for all subjects. There was no signi®cant correlation with losses of absolute sensitivity. A sound may mask less a signal when both acoustic levels are increased in parallel. Under aspirin, masking growth as a function of acoustic level was found slightly decreased at 750 Hz and more clearly at 3000 Hz. Results were signi®cantly correlated with loss of absolute hearing sensitivity. Quaranta et al. (1999) investigated remote masking, i.e. the masking of a low frequency signal by a high frequency sound, which can be related to nonlinear distortion e€ects arising at the auditory periphery. In eight normally-hearing subjects who ingested 3.84 g of aspirin per day for 2 days, they measured a signi®cant average reduction of remote masking from 22 to about 16 dB at the end of the second day. The subjects simultaneously had a slight, 9 dB, loss of hearing sensitivity on average. No individual data and no correlation is available. 2.1.2.2. Features of supra-threshold alterations of hearing. As from the above-mentioned reports it is clear that aspirin can induce notable changes in perceptions within the auditory ®eld. The e€ects studied can be ranged schematically in four categories: deterioration of speech understanding either in quiet or in noise, broadening of frequency ®ltering, alterations in temporal detection, hypersensitivity to noise-induced temporary elevation of absolute thresholds of hearing. Taken all together the data do not give consistent evidence for a clear correlation with the loss of absolute hearing sensitivity. There appears to be a trend, however, for supra-threshold auditory alterations to be more salient in subjects with greatest losses of absolute hearing sensitivity. Such a correlation may be real but obscured in the data by several factors. Indeed, as was indicated above, these threshold losses were estimated

with very di€erent procedures and at di€erent times often not well synchronous with the time of obtainment of the various test results. In addition it must be recalled that these threshold losses appear to ¯uctuate in time and/or at di€erent frequencies for the same individual under aspirin in¯uence. All results of suprathreshold alterations show a considerable inter-individual variability and average results are often presented. Very few data provide estimates of the repeatability in time of results to a given test for individuals. In these studies supra-threshold alterations followed over days of treatment did not always develop evenly but could show notable ¯uctuations. It is possible that the variability in the results re¯ects time-to-time intra-individual variability as well as pervasive inter-individual variability. Deterioration in speech understanding either in quiet or in noise was one of the most early observed suprathreshold alteration, probably because it was originally subjectively felt and reported by the patients. Measures of speech audiometry established that considerable deteriorations can be induced by aspirin. Results vary greatly from one study to another, may be in part, because of di€erent test procedures used. Subjectively reported diculties in following speech conversation may not necessarily show in tests of speech intelligibility in quiet in which a listening concentration e€ort may overcome the diculties. Indeed, it is well known that speech understanding in quiet is very resistant to strong distortions of speech signals. In contrast within a noisy background even moderate distortions of speech signals can have a strong e€ect on intelligibility. Correspondingly under aspirin, speech intelligibility was found more a€ected in a noisy background than in the quiet. Speech is a complex acoustic signal comprising many spectral and temporal features whose roles in intelligibility are still not very well understood. This is probably one reason why more recent studies investigated alterations in the perception of more elementary acoustic features. Various measures on auditory processing of sound frequency have shown aspirin-induced alterations as indicated by measures of sharpness of ®ltering, twotone suppression and monaural diplacusis. Broadening of the sharpness of auditory ®lters has been found repeatedly. The amount of broadening appears correlated to the loss of absolute hearing sensitivity when this loss is at least 20 dB. However, a signi®cant broadening was also measured in subjects who showed very little loss of absolute hearing sensitivity. The broadening of auditory ®lters and reduction of twotone suppression appear to occur consistently more at high than at low frequencies, in line with the trend observed for the loss of absolute hearing sensitivity. Aspirin-induced de®cits in auditory temporal processing were observed from tests of temporal integration,

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forward masking, gap detection and onset overmasking. As for other tests, large inter-individual and interstudy variabilities can be observed, and correlation with loss of absolute hearing sensitivity is not clear although largest e€ects tend to be associated with greatest losses. These alterations in temporal processing seem to a€ect all tested frequencies. A remarkable aspect is the aspirin-induced improvement in the perception of brief sounds. As indicated by temporal integration measures, losses of absolute hearing sensitivity are less for brief than for long tones, as brief tones become relatively better detected. In onset overmasking a brief tone at masker onset which is inaudible for a normal ear can become perceptible under aspirin treatment. This represents an absolute improvement. These improvements in detectability could be linked to the changes in auditory frequency processing. The broadening of frequency ®ltering, or the reduction in two-tone suppression may produce an unusual spread of tonotopic cochlear excitation, a spread which could be so much wider as briefer sounds have a wider physical spectrum. Instances of potentiation of noise-induced temporary hearing loss by aspirin consumption have been demonstrated repeatedly. This potentiation seems to occur only when high doses of aspirin or salicylate are ingested over a period of several days. The consistency of this e€ect however is unclear as studies are few, and results present notable inter-study and inter-individual variations, as do other results on supra-threshold e€ects of aspirin consumption. All aspirin-induced supra-threshold e€ects can be observed in other pathological conditions of hearing resulting from a di€erent cause. All the e€ects can be found in sensori-neural cochlear hearing impairments, a fact arguing in favor of a mostly cochlear origin of the e€ects of aspirin. However, functional impairments of auditory structures of the central nervous system could also intervene specially at very high doses of aspirin ingestion. An interesting aspect from a functional point of view is the possible decoupling observed on various instances between the loss of absolute hearing sensitivity and the occurrence of supra-threshold alterations of hearing. Indeed, several studies indicated subjects with clear e€ects on one aspect without obvious manifestation on the other or conversely. 2.1.3. Tinnitus Tinnitus is a common feature of salicylate-induced auditory impairment. Tinnitus is a subjective and often labile symptom on which, unfortunately, until now objective audiometric studies provide only approximate assessments. Audiometric procedures of matching loudness and pitch or other psychoacoustic aspects of tinnitus to an external sound are the best procedures

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available. However, such procedures are not used commonly and many studies provide mostly frequency of occurrence, delay and duration of tinnitus together with brief subjective descriptions. 2.1.3.1. Audiometric observations. SeÂe (1877) indicates tinntius to be the most constant and the ®rst appearing auditory e€ect of salicylate. In his observations tinnitus appeared in patients for doses of 5±6 g or more of salicylic acid or for doses of 10 g or more of sodium salicylate. Subjectively, tinnitus was described as roaring or hissing noises which disappeared quickly on cessation of medication. Falbe-Hansen (1941) reports on four young healthy subjects who received a single dose of 5 g of sodium salicylate, after 3 h all subjects noticed tinnitus which was still present after 24 h and disappeared only after 48 h. Another group of four subjects received a single dose of 3 g, three subjects experienced tinnitus after two or three hours. In another group of four subjects receiving a single dose of 2 g no tinnitus was noticed. Doses of 3, 5 and 7 g/day, fractionated in 1 g/h, also produced tinnitus in two tested subjects. Long-term daily doses were studied on 21 normally-hearing subjects who received from 2 to 6 g daily on average, over 3±18 days. Six subjects receiving 3 g or less daily did not experience tinnitus. Among the other 15 subjects, who received 4 g or more daily, nine subjects noticed tinnitus. In all but one case tinnitus disappeared within 3±5 days after treatment cessation. Several experiments were also conducted on subjects with abnormal ears, 13 cases had a middle ear conductive deafness and ®ve cases had a sensori-neural deafness. For two subjects a single dose of 3 g induced tinnitus after 2±3 h. Seven subjects received doses of 5±6 g in a day fractionated in 1 g/h, six subjects experienced tinnitus which disappeared after 24 or 48 h but for one case for whom tinnitus was still present after 48 h. A last group of nine subjects received an average daily dose of 3.8±7 g for 3±39 days. Contrary to previous studies subjects of this group were not tested in a silent cabin but in a somewhat noisy environment. In these conditions only three subjects experienced tinnitus (not those who received the higher doses). Reported tinnitus in this last group of nine subjects was more pronounced than in other studies, and lasted several days after treatment arrest. In the study of Jager and Alway (1946), tinnitus was experienced by 34 of the 38 patients treated with daily intravenous injections of sodium salicylate for several weeks. Tinnitus appeared with blood salicylate levels as low as 200 mg/l whereas some patients with levels as high as 500 mg/l did not mention tinnitus. Within 2±3 days following cessation of medication, deafness disappeared while tinnitus persisted for several days more.

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Graham and Parker (1948) studied 40 rheumatic subjects and 30 nonrheumatic patients. Blood samples were taken whenever the patient reported a toxic e€ect in the ®rst 7 days of treatment. 58 patients made such complaints. Three cases experienced tinnitus, deafness and vomited with plasma salicylate levels of 72 mg/l. In nine other patients tinnitus, deafness and nausea appeared for plasma salicylate levels of 100±480 mg/l, the average value being about 250 mg/l. Among 33 patients whose plasma salicylate level rose above 350 mg/l, all complained of tinnitus and deafness. Therapy was maintained in spite of these e€ects which disappeared in a few days. When they reappeared they were controlled by omission of the treatment for 1±2 days and did not recur with subsequent attainment of much higher plasma salicylate levels. Tinnitus and deafness were always reversible upon cessation of treatment. Waltner (1955) reports on a young woman who ingested approximately 11 g of aspirin per day for 6 days and su€ered from tinnitus, deafness and vertigo. Myers et al. (1965) indicate that among the 21 patients treated with salicylate they studied, most had severe high-pitched tinnitus. Withdrawal of salicylate in four patients resulted in rapid disappearance of tinnitus. McCabe and Dey (1965) experimented the e€ects of aspirin tablets ingested at a dose of approximately 5 g of salicylate per day for 4 days on ®ve normally-hearing women. Tinnitus of a high frequency character was the most common subjective symptom. Tinnitus was reported to be as buzzing bees, crickets, humming, birds and musical tones. Pitch matching to an external tone was made to frequencies ranging from 7000 to 9000 Hz. Mongan et al. (1973) studied 67 patients with rheumatoid arthritis and seven healthy volunteers. Subjects were given bu€ered aspirin in gradually increasing doses by steps of 0.6 g/day every 2±3 days, until a tinnitus of at least ®ve consecutive min was noted. Blood sample was drawn while the subject had tinnitus. Salicylate was discontinued for at least 2 days, then audiometric tests were performed, and serum salicylate level was measured again to ascertain there was no detectable level. Audiometric tests included pure tone audiometry and speech reception threshold. Audiometric data for individuals are unfortunately not available. Fifty-nine subjects experienced tinnitus, while 15 did not. All seven volunteers and 52 patients experienced tinnitus. All subjects who had normal hearing at the beginning of the study noted tinnitus before they appreciated any hearing loss. Among the subjects who experienced tinnitus, there were large individual variations, a threefold variation in aspirin dosage and more than twofold in plasma salicylate levels. There was no correlation between their aspirin dosage and plasma levels. The 15 patients who did not experience

tinnitus were among the 22 patients with a pre-existing hearing loss. All 15 patients had a high frequency hearing loss, and nine of them also had an additional low frequency hearing loss. The 15 patients who did not note tinnitus had an average serum salicylate level in the highest range. In Oudot et al. (1979) 10 patients were studied who took very large doses of aspirin in attempts to commit suicide. Subjective reports from eight patients indicate tinnitus occurring in a few hours on aspirin ingestion. They sounded as whistles in six cases and as humming noises in the remaining two cases. McFadden and Plattsmier (1983) studied on themselves progressive doses of aspirin ranging from 4.4 to 5.8 and 3.9 to 4.9 g of aspirin per day for 4 days. Soon after beginning the aspirin treatment both noted a high-pitch tinnitus that sounded as a tone or sometimes as the chirping of crickets. Ramsden et al. (1985) reported on two cases of attempts to commit suicide. One had taken probably about 30 g of aspirin, and the other probably about 10 g. They were each admitted to hospital 4 and 2 h after aspirin ingestion. Both had tinnitus at the time of hospital examination. After a few days the tinnitus disappeared. Wier et al. (1988) studied four adult male subjects ingesting 3.9 g of aspirin per day for 1±4 days. One subject ceased aspirin consumption at the end of the ®rst day because of perception of a strong tinnitus. Two studies by Halla and Hardin (1988) and Halla et al. (1991) examined the presence of tinnitus and subjective hearing loss together with blood salicylate levels in large populations of arthritic patients. Halla and Hardin (1988) examined 134 patients with rheumatoid arthritis and treated with salicylate in comparison with 182 healthy untreated subjects. In a complementary study Halla et al. (1991) examined 260 patients with rheumatoid arthritis and 112 patients with osteoarthritis, neither group being treated with salicylate. Their results indicate that often the subjective symptoms of tinnitus and hearing loss were present equally frequently in these populations, and that signi®cant salicylate side e€ects are recognized as an aggravation of these symptoms. Thus of the 56 subjects who were taking salicylate and who were complaining about tinnitus few tolerated an upward salicylate dose adjustment although about half of them had plasma salicylate levels of less than 200 mg/l. Retrospective enquiry was performed in a group of 94 patients who were found to have had, on one or more occasions, plasma salicylate levels above 350 mg/l. These patients were then asked whether they remembered having had tinnitus at the time of blood test; about half of them indicated they did not notice tinnitus, while about a third did notice tinnitus and the rest indicated they already had tinnitus.

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Day et al. (1989) experimented the e€ects of 1-week treatments with aspirin in slow release tablets at four dosages ranging from approximately 2 to 6 g/day on eight normal volunteers. The one-week treatments were administered in random order, double blind and 2 weeks apart. At the end of each treatment week, blood samples were taken, hearing loss measured with a pure tone audiogram, and tinnitus pitch and loudness were assessed objectively by matching to an external tone, and subjectively by pointing onto a ®xedintervals scale. No e€ect was observed for the lowest dose of 1.95 g/day. Quasi-linear relationships were observed between higher aspirin doses and both hearing loss and tinnitus. The hearing loss a€ected all frequencies and was small, being less than 10 dB, but for the higher dose of 5.85 g/day it reached about 20 dB. Tinnitus loudness levels appeared more prominent since on average they were matched to an external tone level of 25, 36, 51 and 62 dB SPL, respectively for the aspirin doses of 1.95, 3.25, 4.55 and 5.85 g/day. All measures of total and unbound plasma salicylate concentrations plotted as a function of all corresponding hearing losses showed a rather linear relation, while the relation with tinnitus loudness levels presented a reduced increase at higher levels for some subjects. Tinnitus pitch is only very brie¯y presented. It was matched to tones of frequencies ranging from 900 to 14,500 Hz and did not present a correlation with the salicylate ingested dose or plasma concentration. Penner (1989) reported on a woman who was su€ering from tinnitus described as a steady tone and a hum. The patient underwent a 3-day treatment with aspirin at 975 mg every 6 h. This resulted in tinnitus being changed to a noise in the head. Penner and Coles (1992) reported on another case of a woman who complained of tinnitus described as ringing. She took 600 mg of aspirin every 6 h, after the ®rst day of treatment tinnitus had disappeared and remained inaudible for the 3 days of aspirin ingestion. Within a day of treatment cessation tinnitus returned and was felt worse, but subsequently in the next 2 days tinnitus remained audible. Hicks and Bacon (1999) experimented on six normally-hearing subjects who received 3.9 g/day of aspirin for 4 days. All subjects reported tinnitus by the second day of aspirin use. Janssen et al. (2000) reported on a 22-years-old woman seen 22 h after having ingested presumbly 10 g of aspirin, she indicated a tinnitus sounding like a high frequency hissing noise in both ears, one day later tinnitus had disappeared. 2.1.3.2. Features of salicylate-induced tinnitus. Tinnitus is a subjective perception of a sound in the absence of a corresponding external source. For the vast

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majority of persons su€ering from tinnitus, whatever its origin, its loudness is matched to only 5±15 dB above their absolute sensitivity of hearing. This corresponds to a faint sound which indeed is clearly perceived in a quiet surrounding and can very often become unnoticeable in a somewhat noisy environment. Tinnitus induced by salicylate presents these same features. Under salicylate treatment tinnitus can be unnoticed unless attention is drawn to it and quiet surroundings, as best obtained in a silent cabin, are provided. This indeed explains many of the apparent discrepancies between or even within studies. In everyday life incipient tinnitus may go unnoticed or considered negligible and not reported unless special attention and/or appropriate conditions for detecting the presence of tinnitus are provided. It is striking that all studies which were dedicated to the study of tinnitus report a high incidence of salicylate-induced tinnitus whereas in many other studies it may not be reported at all or very brie¯y indicated. Many subjects indicated that tinnitus often preceded hearing loss. However, in various studies losses of absolute hearing sensitivity when not specially under study may have been small and neglected. As for other alterations of hearing induced by salicylate, there seems to be a delay of a few hours after ingestion of salicylate until tinnitus becomes perceptible. A large individual variability is observed concerning susceptibility to tinnitus since some subjects did not experience tinnitus in spite of ingestion of high doses of salicylate and high plasma levels of salicylate. One consistently reported factor associated with the absence of tinnitus induction by salicylate is a preexisting loss of absolute sensitivity of hearing, particularly a loss at the high frequencies. In all cases where tinnitus was induced by salicylate there appears, however, to be a clear correlation with plasma salicylate level. In Fig. 2, we have gathered data from di€erent studies to illustrate this relation. Unconnected symbols refer to individual measures of minimum plasma salicylate levels associated with tinnitus. Data from the two studies of Jager and Alway (1946) and Graham and Parker (1948) were arbitrarily plotted at a loudness level of 10 dB. It can be seen that minimum plasma salicylate levels associated with occurrence of tinnitus were almost always below 100 mg/l. At such values the losses of absolute sensitivity of hearing are minimal or inexistent. The increase of tinnitus loudness as a function of plasma salicylate level appears to follow an approximately linear rise which, at about 300 mg/l, associates a tinnitus loudness of about 60 dB. This appears to be a steep rise compared with the slope for loss of absolute hearing sensitivity.

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The pitch of salicylate-induced tinnitus appears to be for almost all cases in the high frequency range. Often matching to external tones was made to the highest frequency range. More detailed descriptions of tinnitus most often indicated tone-like sounds or a variety of high frequency noises. In all studies tinnitus disappeared within a few days of cessation of drug ingestion. Such a de®nite reversibility of tinnitus, and hearing loss, is also observable after exposure to a loud sound, but is in contrast with many other cases of tinnitus which are recurrent or continuous. 2.2. Loss in absolute hearing sensitivity and tinnitus inferred from animals' behavior 2.2.1. Hearing loss Myers and Bernstein (1965) performed a study on ten squirrel monkeys. The animals were trained to avoid an electric shock delivered through the training cage ¯oor by jumping over a barrier to the other side of the cage when they heard a tone which preceded the electric shock. Absolute hearing sensitivity so measured was found 90% reliable from day to day within 5 dB and the threshold values ®tted well the

Fig. 2. Loudness matches of tinnitus in relation to plasma salicylate level for the six studies indicated in the insert at upright. Individual points represents lower individual values at tinnitus appearance. For two studies, incipient tinnitus points were placed at 10 dB loudness level. Connected points represent average values of loudness growth (match to an external sound: ®lled symbols, subjective scale: open symbols) as a function of increase in salicylate plasma level.

otherwise known acoustic sensitivity of these animals (Fay, 1988). Each animal was given a subcutaneous injection of sodium salicylate at an extremely large dose of 500±600 mg/kg of body weight which is slightly below the dose producing death in 50% of such small animals. Thresholds of hearing were determined 24 h later. Behavioral measures indicated hearing losses in all animals ranging from 17 to 36 dB. After behavioral testing a blood sample was taken, and plasma salicylate levels found ranged from 190 to 580 mg/l. Surprisingly, the individual data show a clear inverse correlation between the injected dose and the plasma concentration. The data do not show any correlation between either dose or plasma salicylate level and the amount of hearing loss. The authors' statement that salicylate plasma levels below 400 mg/l correlate with the degree of hearing loss does not appear statistically signi®cant from their raw data. Stebbins et al. (1973) tested one monkey with a positive reinforcement (food pellet delivery) conditioning procedure. Such a procedure avoids the emotional perturbations often produced by the electric shock in avoidance conditioning. One intramuscular injection of sodium salicylate was given at the dose of 250 mg/kg, and threshold variations in response to a high frequency tone of 4 kHz were monitored over several hours. The initial threshold value was at 13 dB SPL (Sound Pressure Level) which is in agreement with the otherwise known acoustic sensitivity of these animals (Fay, 1988). After an hour the threshold showed a loss of about 22 dB, after 3 h about 20 dB and after 6 h threshold was back to normal. A second experiment on the same animal was performed using a single dose of 500 mg/kg. The threshold rose to 23 dB after 1 h, and to 33 dB after 3 h, 35 dB after 6 h and was back to normal after 24 h. As for the study of Myers and Bernstein (1965) mentioned above, the doses employed are extremely high, and the authors indicate problems associated with administration and the presence of other toxic e€ects interfering with behavioral testing. Crifo (1975) tested ten albino guinea pigs using the technique of shiver audiometry which consists of making the animal shiver by a cold air draught and detecting the stoppage of shivering when a tone is presented. To obtain shivering the animals seem to have been placed in a refrigerator at 0 to ÿ28C. The animals were conditioned to respond to sound by associating an electric shock. In addition the authors tested the acoustic pinna re¯ex, i.e. the re¯ex of ear pinna de¯ection which can be detected in response to loud sounds at approximately 60 dB above hearing threshold. Unfortunately, no data is available concerning the initial values of thresholds in terms of absolute sensitivity or dB SPL, and results were expressed in reference to the pre-treatment values adjusted to zero. In all experiments inter- individual variability was ap-

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proximately 20 dB for all measures and the average results are indicated hereafter. Animals received intraperitoneal injections of lysine acetyl salicylate. In one series of experiments an injection of 350 mg/kg was given and shiver audiometry and pinna re¯ex were monitored after 2, 7, 24 and 72 h. The authors speculate that salicylate levels could have been about 500 mg/l after 2 h and about 23 mg/l after 7 h. After 2 h no shiver-audiometry threshold change was observed whereas after 7 h a threshold shift of approximately 20 dB was observed at all frequencies. After 24 h threshold shifts were almost unchanged and after 72 h thresholds had returned to their initial value. In a second series of experiments six animals daily received an injection of lysine acetyl salicylate corresponding to 50 mg/kg of aspirin for 20 days. After ®ve and until 10 days shiver-audiometry thresholds indicated a shift of approximately 10±20 dB, being more pronounced at high frequencies. After 15 days thresholds shifts ranged approximately from 20 to 35 dB, and after 20 days shifts ranged between 25 and 40 dB. Threshold values of pinna re¯ex, ranging between 80 and 100 dB, are shown for the 5th and the 20th days and present some improvement at the high frequencies on the 20th day. Eddy et al. (1975, 1976) experimented on seven adult chinchillas using an electric shock avoidance procedure. Chinchillas were considered well conditioned when showing an 80% correct response rate at 30 dB SPL. The animals received a subcutaneous injection of sodium salicylate at time zero, then after 3 h and every 6 h for 72 h. Sodium salicylate was diluted at the concentration of 200 mg/cm3. Blood samples were taken at 4, 6, 7, 12, 36, 60 and 78 h. The animals received various doses adjusted to obtain a serum salicylate level of 200±300 mg/l. A reference threshold value for the 1 kHz frequency indicates an absolute sensitivity at ÿ5 dB (presumably SPL) which ®ts the known acoustic sensitivity of these animals (Fay, 1988.). Thresholds shifts were about 30 dB on average, and at the mid frequency of 1 kHz they reached about 25 dB at the end of the ®rst day and on the second day. The combined e€ects of noise and salicylate were investigated. The animals were exposed to a broad band noise at a level of 85 dB for 2 days, temporary threshold shifts were measured and the animals recovered for 45 days. Then they were treated with salicylate at doses adjusted to obtain plasma levels of 200±300 mg/l. After a day and a half of salicylate treatment the animals were exposed to the same broad band noise for 2 days while salicylate treatment was continued. Noise alone induced threshold shifts of about 35 dB while the combination of noise and salicylate resulted in shifts of approximately 55 dB. Chen and Aberdeen (1980) showed that salicylate can potentiate the induction of audiogenic seizure in

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mice. Some inbred strains of mice can present epileptic reactions in response to a loud sound. In another breed which is genetically resistant to audiogenic seizures, the authors showed that prime exposure to a 125±127 dB bell sound for 10 s could induce audiogenic seizure at exposure to a similar sound a week later, and the percentage of reacting mice was increased if 500 mg/kg salicylate was given orally to mice 24 or 6 h before the second sound exposure. Deer and Hunter-Duvar (1982) tested ®ve chinchillas which received an intramuscular injection of sodium salicylate at the dose of 300 mg/kg followed 4 h later by a subcutaneous injection at the dose of 200 mg/kg. This procedure was previously determined as reliably giving high but sublethal salicylate serum levels, and it was found not to have an adverse e€ect on the behavior of the animal. Behavioral conditioning used the shock avoidance procedure. A reliability of 5 dB or less over three consecutive tests was obtained. Reference thresholds in absolute values are not available, and changes are expressed as threshold shifts in dB. One and a half hour after the second injection, animals were behaviorally tested and then blood samples were taken. Serum salicylate levels of 370±670 mg/l were measured. Average threshold shifts were 31, 19, 27 and 30 dB, respectively, for the four tested frequencies of 1, 2, 4 and 8 kHz. Data from individual animals show that higher salicylate levels appear associated with higher threshold shifts at the two frequencies of 1 and 4 kHz but not at the two other frequencies. Kurata et al. (1997) used a combination of negative and positive reinforcement conditioning procedures to test hearing of rats. The animals were deprived of water to a 10% loss in body weight. In the positive reinforcement procedure at the end of a tone the animal had to press a lever to obtain a drop of water. In the negative reinforcement procedure the press of the lever at the end of the tone delivered an electric shock. A ratio of lever pressings in both conditions was used as a measure of response to sound. Best hearing thresholds obtained from control animals were measured at 45 dB SPL at 8 kHz, a value grossly above threshold determined in experiments by other authors. The animals were intravenously injected with a dose of 225 mg/kg of aspirin DL-lysine. Behavioral measures indicated a threshold elevation of 15 dB at 2 kHz but, quite surprisingly, none at 4, 8 and 10 kHz. This threshold elevation was observed 1, 24 and 48 h after the aspirin injection, and at 72 h post-injection the threshold shift was about 5 dB. Overall these animal experiments demonstrated that losses of absolute hearing sensitivity can be induced by salicylate in di€erent animal species. In agreement with typical features observed in human data, the e€ects appear to develop within a few hours of a salicylate administration, to increase with the dose of salicylate,

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to impair both ears, to a€ect various frequencies by some tens of dB only, and to be reversible within days after cessation of treatment. As for humans, data also support a potentiation of noise trauma by salicylate. 2.2.2. Tinnitus Validating a behavioral procedure to assess the presence of tinnitus in animals is an unusual and dicult task to which a research group devoted many years. Jastrebo€ et al. (1988a, 1988b) and Jastrebo€ and Sasaki (1994) reviewed their own work over years concerning the development of an animal model of tinnitus. They experimented on the behavior of rats to ®nd evidence for the presence of tinnitus after administration of salicylate. To reach this aim they reversed the usual conditioning paradigm by associating a behavioral reaction to silence. Their peculiar procedure, elaborated from a classical procedure to measure fear behavior by Estes and Skinner (1941), consists in exposing animals to a mild sound and to associate cessation of this background sound with an electric foot shock. Animals are rendered thirsty by depriving water producing a constant percentage of weight loss. Then, in the testing cage animals are given water and their lickings are counted. For a constant level of thirst a stable licking behavior is observed. The occurrence of a silence produces a diminution of licks. Extinction of this reaction to silence can be obtained by suppressing the electric foot shock associated with silence occurrence. As described in related literature on behavior, the ratio of licks occurring during 1 min of silence divided by the sum of licks occurring during 1 min before the silence and during the silent minute, is taken as an index of behavioral change in response to the silence. Measures of licks changes indicate that training animals to associate silence with electric foot shock takes 4±10 associations corresponding to 1±2 days of training. Extinction of this behavior takes 4±5 days. The rationale then is that if tinnitus, i.e. a subjective sensation of sound, is induced by salicylate, it will interfere with the perception of silence occurrence and will modify the extinction behavior. If tinnitus is induced after the animal has been trained, the extinction would be accelerated. If tinnitus is induced while training, the animal would associate tinnitus with electric shock and extinction would be slowed. Behavioral measures indeed ®tted with these predictions. As an additional control, a continuous sound, aimed at mimicking tinnitus, was introduced in nonsalicylatetreated animals at the same times as tinnitus would start in treated animals. As presumed, animals' behavior was similar whether the continuous sound or the salicylate treatment were applied. Consistent data were obtained from several hundreds of rats treated with subcutaneous injections of sodium salicylate at the dose of 300 mg/kg per day. Various ratios of licking

activities including measures after the silence were assessed and reinforced the value of the ratio used. Results from experiments using light instead of sound as the conditioning stimulus demonstrated the auditory speci®city of the salicylate induced behavioral response. A hearing loss induced by salicylate together with tinnitus does not seem to interfere. Indeed, it would disturb the sound-to-silence contrast, however, animals trained under the in¯uence of salicylate showed normal licking suppression during training, associated with slowed extinction. In subsequent studies the same research group used the method of auditory similarity to assess the pitch of salicylate-induced tinnitus in rats. The basic idea is that a continuous sound close to tinnitus in pitch would alter extinction more than a sound with a pitch quite di€erent from that of the tinnitus. Results indeed showed marked di€erences between tones of di€erent frequencies, with continuous tones of 10 and 11 kHz showing the strongest e€ect. This is consistent with human reports of high-pitch tinnitus induced by salicylate. Because human reports indicate an increase in tinnitus loudness with increasing doses of salicylate administration, di€erences in animal behavior were examined as a function of the dose of salicyate injected. Doses of 50±300 mg/kg were tested on animals treated before or after licking suppression training. For the low dose of 50 mg/kg no di€erence was observed with saline injection. For larger doses animals treated after training showed faster extinction than animals treated before training. A strong correlation was found between these behavioral di€erences and the injected salicylate dose. Similar experiments were performed using a continuous sound of 10 kHz and of variable sound pressure level to mimic tinnitus loudness. Similar behavioral di€erences were obtained and when plotted on the same graph as for salicylate doses they show approximately parallel growths and allow an estimation of tinnitus loudness in salicylate treated animals. Following average data tinnitus was matched to a 10 kHz external tone of 35±72 dB SPL for salicylate doses of 100± 300 mg/kg. In another series of experiments (Jastrebo€ et al., 1988b), the same group of authors measured salicylate blood levels in conditioned animals as a function of salicylate dose. Taken together the animal data relating tinnitus loudness match to an external tone as a function of blood salicylate level ®t remarkably well with the human data of tinnitus loudness match to an external tone as a function of blood salicylate level (Day et al., 1989). This matching is illustrated in Fig. 3. A recent study by the same group (Brennan et al., 1996) used two variants of a classical avoidance conditioning and assessed the hearing loss induced by salicylate on postweanling and on adult rats. Sodium salicylate was given as a subcutaneous injection at the

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doses of 300 and 200 mg/kg, 2 h before behavioral testing. Hearing thresholds were measured at 29 frequencies from 1 to 35 kHz. Testing lasted for several days to ensure repeated measures at each frequency. A total of 132 rats were tested. In control animals threshold values reached 5±10 dB SPL which ®ts the otherwise known hearing sensitivity of the rat (Fay, 1988). Treated animals presented a hearing loss increasing progressively for frequencies above about 2 kHz and reaching at most 17 and 10 dB, respectively, for the doses of 300 and 200 mg/kg. Two other series of experiments validate these behavioral measures as manifestations of tinnitus. Quinine, which is also known to often induce tinnitus in humans, was tested and the animals' behavior exhibited responses similar to those observed after salicylate infusion (Jastrebo€ et al., 1991). Another well-known means of inducing tinnitus is exposure to very loud sounds. Very recently, Jastrebo€ et al. (1999) and also another research group, (Kaltenbach et al., 1999), reported behavioral manifestations of tinnitus using the same behavioral procedures. These studies fully support the validity of these behavioral measures for studying tinnitus. A very recent report by Bauer et al. (1999) used a slightly di€erent training procedure to test for tinnitus in salicylate-treated rats. The authors fed rats with sodium salicylate in the drinking water of the animals. This produced a serum salicylate level of about 150±

Fig. 3. Tinnitus loudness matches to an external tone from human data (®lled symbols, same data as in Fig. 2) and animal experiments (open symbols).

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400 mg/l in the various animals. The rats were trained to press a lever to obtain food pellets in the presence of a white noise and to cease during periods of silence which were associated with a noxious stimulus. Then pure tones were presented in place of the white noise. In this condition the salicylate-treated animals showed less lever pressing suppression than the control animals and a maximal e€ect was obtained for a tone at 15 kHz. Such a di€erence was not observed for white noise or silent periods. This is coherent with salicylatetreated rats experiencing tonal high frequency tinnitus. All the behavioral studies investigating the presence of tinnitus in animals treated with salicylate provided demonstrative evidence and the associated control studies provided validating results. The results indicate tinnitus developing progressively over days of treatment, having a high frequency content with a mild to moderate loudness level, and disappearing within a few days of cessation of treatment. These features are quite similar to those observed in humans. These results are of particular signi®cance for validating the relevance of the many physiological studies which were performed, most often on rodents. 3. Physiological alterations 3.1. Anatomical observations Early anatomical observations of the inner ear of animals treated with salicylate reported rather contradictory results. An overview can be found in the monograph of Falbe-Hansen (1941). Kirchner's (1883) studies indicated hemorrhages in the cochlea and the labyrinth. After Wittmaack (1903), Bech (1913) reported atrophy of ganglion cells and nerve ®bers with displacements of Reissner and tectorial membranes. However, Lindt (1913) and Schroder and Hinsberg (1915) did not ®nd similar morphopathologies, or to a small extent, and also in untreated animals. Lurie (1935) reported degeneration of outer hair cells and hemorrhages in perilymphatic spaces, Covell (1936, 1938) indicated vacuolisations of outer hair cells and distension of the stria vascularis. Mosher (1938) reported hemorrhages in scala tympani and around cranial nerves. Falbe-Hansen (1941) indicated alterations in cells of the spiral ganglion and in positions of the Reissner and tectorial membranes. Gotlib (1957) noted changes in spiral ganglion cells, acoustic and vestibular nuclei and cortex. In later studies most of these above-mentioned alterations were not con®rmed to be associated with salicylate treatment. Myers and Bernstein (1965), using both optic and transmission electron microscopy did not ®nd any signi®cant change in the cochleas of the twelve monkeys for which they had behaviorally measured salicylate-

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induced loss of absolute hearing sensitivity. An additional study was performed on two temporal bones of humans who su€ered from rheumatoid arthritis and had undergone treatments with salicylate doses of 5± 10 g daily. Morphologic observations of these temporal bones were restricted to optic microscopy because of delayed post-mortem ®xation and showed no signi®cant alteration in the appearance of sensory cells or stria vascularis. Perez de Moura and Hayden (1968) examined the temporal bones of a 76-year-old woman who died a year after discontinuing salicylate treatment. Light microscopy observations revealed several alterations which could all be due to aging such as shrinking and loss of neural cells in the spiral ganglion, and atrophy of the stria vascularis. Falk (1974) injected daily subcutaneous doses of 200 mg/kg of sodium salicylate for 5 days to young albino guinea pigs. He used transmission electron microscopic observations of the spiral ganglion and auditory nerve and did not ®nd any sign of pathology. Crifo (1975) mentions brie¯y that the cochlear duct of a guinea pig sacri®ced after 10 days of salicylate treatment appeared normal. Hawkins (1976) brie¯y mentioned light microscopic examinations of cochleas from guinea pigs treated with salicylate within more documented data on quinine treatment. He reported constriction of capillaries in both the stria vascularis and in the spinal lamina. Oliveira and Marseillan (1976) found on examination with light microscopy no loss of hair cells in guinea pigs treated with various doses of salicylate, including lethal doses of up to 3 g/kg given intraperitoneally. The animals were sacri®ced between 2 and 48 h post-injection. No hair cell loss was observed in guinea pigs treated at 100 or 200 mg/kg/day for 10±300 days. Woodford et al. (1978) injected ®ve chinchillas with an intramuscular injection of salicylate at a dose of 400 mg/kg. Electrophysiological measures ascertained a loss of absolute hearing sensitivity of 10±20 dB within a few hours post-injection. After several days hearing was back to normal and the animals were sacri®ced. On examination with light microscopy, the authors report, in one animal, a small loss of slightly more than 10% of outer hair cells in a restricted area of the upper cochlea, having observed smaller losses of outer hair cells in control animals. The authors indicate that this loss could be attributed to salicylate. The authors examined combination of salicylate with three di€erent loud sound exposures, and concluded that additional hair cell loss was observed by the combination. Deer and Hunter-Duvar (1982) performed examinations, by light microscopy and by scanning and transmission electron microscopy, of the cochleas of chinchillas on which behavioral measures of loss of ab-

solute hearing sensitivity had been obtained. No clearly signi®cant morphological alterations were observed in treated animals compared with control animals. However, the authors reported their impression of increased lysosomes and vacuolisation in the subcuticular zone of outer hair cells in the salicylate-treated animals. Douek et al. (1983) performed studies with light microscopy and with transmission and scanning electron microscopy on albino guinea pigs treated with salicylates. The ®rst group of animals received a subcutaneous injection of 375 mg/kg daily for 5±7 days and were sacri®ced from day 1 to 6 weeks after the end of the treatment. The other animals received a single injection of 500 mg/kg and were sacri®ced 4 and 24 h later. For both groups control animals were similarly treated with saline. Light microscopy examination of hair cells did not indicate any anomaly. On animals sacri®ced at day 1 after ®ve daily injections, scanning electron microscopy revealed bent and ¯accid stereocilia of outer hair cells of the outermost row with protrusion of phalangeal processes above the cuticular plate and distorted and reduced microvilli. Transmission electron microscopy on the same group of animals con®rmed the bending of stereocilia of outer hair cells and revealed vacuolisations of the smooth endoplasmic reticulum. Inner hair cells also showed similar signs but to a smaller extent. At 4 h after a single dose of salicylate, the outer hair cells showed vacuolisation of the lateral smooth endoplasmic reticulum. At 24 h after salicylate injection the same aspects were observed but to a lesser degree, similar much less marked aspects were seen in inner hair cells. No alteration were observed in the stria vascularis. Lambert et al. (1986) using light microscopy observed no hair cell loss in chicks fed aspirin for 5 days at doses producing serum salicylate levels of about 250 mg/l. The animals were killed ten days after the end of treatment. In parallel, the group of chicks was exposed to a 1.5 kHz tone at 115 dB SPL for 8 h. This group showed no di€erence in hair cell loss. Cazals et al. (1988) examined with light microscopy and morphometry the widths of vessels in the stria vascularis and spinal lamina of guinea pigs which received a single intramuscular injection of lysine acetyl salicylate at the dose of 400 mg/kg. Animals were sacri®ced 3 h after salicylate injection, a time at which loss of absolute hearing sensitivity was ascertained by electrophysiological measures. No signi®cant change in vessel widths was detected after infusion of salicylate whereas, after injection of other drugs, modi®cations could be observed. Carson et al. (1989) fed rats with aspirin for 18 days at doses of up to 200 mg/kg twice daily, thus producing blood salicylate levels to above 350 mg/l. All animals were killed 3 weeks after aspirin treatment. Light

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microscopy examination indicated no loss of hair cells. Combination of daily loud noise exposure with salicylate treatment for 8 or 12 days could result in increased hair cell loss compared to noise exposure alone. This was true only for the group treated and exposed for 12 days. However, evoked potential recordings from the same animals revealed no functional loss due to the combination and in this group six out of 15 animals died during the experiment. Boettcher and Salvi (1991) indicated no loss of hair cells in chinchillas treated with 300 or 450 mg/kg of salicylate for 15 days, or treated with 200 mg/kg twice daily for 15 days. Dieler et al. (1991) examined with transmission electron microscopy ultrastructural changes produced by exposing isolated cochlear hair cells of guinea pig to various doses of sodium salicylate, In the same study functional alterations of electromotility of these cells were also demonstrated. The authors observed dilation, vesiculation and modi®cations of the parallel arrangement of lateral membrane subsurface cisternae. No additional alteration was observed in other organelles. These alterations occurred in a time and dosedependent manner and were reversible in close parallel to the recorded alterations in electromotility. Spongr et al. (1992) examined with light microscopy the cochleas of chinchillas which received daily intraperitoneal injection of salicylate at 300 mg/kg for 15 days. The animals were sacri®ced approximately 40 days after salicylate treatment and showed little or no hair cell loss. The authors also studied the combination of such a salicylate treatment with exposure during the 15 days to a continuous octave band noise at 85 or 105 dB SPL. The hair cell losses were not greater than those observed for salicylate or noise alone treatments. Rybak et al. (1992a, 1992b) examined with electron transmission microscopy the stria vascularis of chinchillas which received an intravenous injection of quite a low dose of sodium salicylate (50 mg/kg). They observed no ultrastructural anomaly. Pollice and Brownell (1993) used two di€erent ¯uorescent membrane probes and performed optical examination of outer hair cells exposed to salicylate. They used a probe known to label Golgi apparatus and another probe preferentially labeling endoplasmic reticulum. In salicylate exposed outer hair cells the ®rst probe showed a patchy, instead of a normal continuous, labeling along the subsurface cisternae. The width of this probe ¯uorescence presented an increase of about 50% compatible, although less extensive, with the widening observed with transmission electron microscopy. Dieler et al. (1994) performed further experiments on isolated outer hair cells but this time they examined pigeon auditory hair cells that, contrary to mammalian

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hair cells, do not present subsurface cisternae. They observed that salicylate produced a dilation of endoplasmic reticulum, which, like subsurface cisternae, has layered membranes, and an increased number of membrane-bound vesicles ®lled with electron-dense bodies. Zheng and Gao (1996) examined the ototoxicity of salicylate on organotypic cultures of postnatal cochlear explants of rats. They exposed explants to sodium salicylate at various concentrations from 1 to 10 mM for 2 or more days. Explants were then ®xed and histological staining of surviving cells was performed using a neuro®lament antibody for neural cells and phalloidin for hair cells. The results showed a neural degeneration starting at 3 mM and being almost complete at 10 mM, whereas there was no hair cell loss even at the highest dose. In summary the anatomical observations agree in indicating no loss of hair cells, neural cells or vascular and supporting tissues in the cochlea, whereas in organotypic culture losses of neural cells clearly occurred. The data provide very limited evidence for an increase by salicylate of hair cell loss induced by a noise trauma. These observations on sensory structures are quite in line with the mildness and reversibility of the perceptive alterations described above. This led to searching for ultrastructural signs of dysfunction. The alterations found from examinations with transmission electron microscopy point to cochlear hair cells and most particularly outer hair cells. Although the data are few they are in accordance with a series of physiological observations as indicated hereafter. 3.2. Cochlear sensory processes 3.2.1. Endocochlear potential The cochlear endolymph bathes the upper part of the organ of Corti, and so the top of the sensory hair cells. It presents the speci®c pattern of a high concentration of potassium, and low concentrations of sodium and calcium, it is the only extra cellular body ¯uid with such a composition. Homeostasis of these speci®c concentrations is necessary for a normal functioning of the cochlea, in particular because transduction current through hair cells is extremely dependent upon potassium concentration. This ionic composition is believed to originate from the stria vascularis. This composition produces a positive endocochlear potential (EP) of approximately 80 mV which corresponds to the resting potential of the cochlear endolymph. The potassium channels in hair cells generate a negative membrane potential of about ÿ60 mV. The resulting potential di€erence of about 140 mV is the driving force of transduction currents through hair cells. Experimental alterations of EP results in clear dysfunctions of the cochlea. A recent overview can be consulted in Wangemann and Schacht (1996).

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Tanaka and Brown (1970) injected acetyl salicylate into scala media at an undocumented dose and observed no signi®cant change in EP. Thalmann et al. (1973) experimented on guinea pigs given an intraperitoneal injection of salicylate at doses of 350 and 500 mg/kg. Very little EP change was observed for 3 h post-injection, while simultaneously neural responses showed a strong decrease. Rybak and Whitworth (1987) and Rybak et al. (1986, 1990, 1992a, 1992b) continuously monitored for an hour the EP of chinchillas who received a single intravenous injection of sodium salicylate at the low dose of 50 mg/kg. Measures of EP did not show signi®cant changes. Tran Ba Huy et al. (1987) did not ®nd a change in EP after an intraperitoneal injection of 350 mg/kg lysine salicylate in rats. Puel et al. (1990) injected salicylate in the perilymph of guinea pigs through scala vestibuli at doses of up to 10 mmol/l. These injections did not a€ect EP but for a small decrease of 6 mV at a high dose of salicylate. In the same experiments clear changes were observed on other sensori-neural responses. Stypulkowski (1990) injected sodium salicylate intravenously in cats at doses of 200 or 400 mg/kg. Blood levels so obtained reached about 600±700 mg/l. The EP remained unchanged while other sensori-neural cochlear potentials were a€ected. Fitzgerald et al. (1993) did not ®nd any signi®cant change in EP of guinea pigs administered salicylate in scala vestibuli at di€erent concentrations of up to 10 mM. Simultaneously signi®cant changes were recorded in other sensori and neural responses of the animals. ShehataDieler et al. (1994) administered salicylate either directly to the endolymph or to the perilymph of pigeons at doses of up to about 18 mmol/l. No signi®cant change in EP was measured, while changes were observed for other sensori-neural responses. It appears clearly from all these data that salicylate did not change EP in a variety of experimental conditions, in many of which other sensori and/or neural cochlear responses were clearly modi®ed. 3.2.2. Cochlear microphonic and summating potentials Under the vibration of sounds the ciliary tuft of sensory cells is de¯ected and transduction channels situated in the cilia are opened. Through the electromotive force of EP and intracellular hair cell potential, these channel openings result in a current of ions, mostly potassium ions, which are the basis of the cochlear microphonic (CM) potential. Because these transduction currents follow the rhythm and polarity of the acoustic vibration, as a microphone does, the resulting potential has been called microphonic potential. A variety of experiments have established that in normal cochleas CM originates mostly from outer hair cells. During sound stimulation a dc component can also be recorded which is called summating potential (SP). It

is believed to represent the dc receptor potential from both inner and outer hair cells. A detailed presentation of CM and SP is available in Dallos (1973). In animal experiments an easily accessible electrode place for recording CM and SP is the base of the cochlea and appears to be a relevant choice for salicylates which seem to a€ect the cochlear base more. Silverstein et al. (1967) studied CM with an electrode near the round window of cats which received an intraperitoneal injection of salicylate at a dose of 350 mg/kg. A measure of serum salicylate level of 510 mg/l is indicated at the time of electrophysiological recordings. A nonfrequency-speci®c sound stimulus was used, a brief click having a broad frequency spectrum. About 30 min after salicylate injection CM started to decrease and stabilized within 3±4 h. Data for four cats showed CM amplitude decreases of 28± 66%. Click stimuli were presented at moderate sound pressure levels where CM amplitude is a linear function of sound pressure level. Thus the observed decreases correspond to a drop of about 6±10 dB. Simultaneous recordings of neural responses indicated a larger loss of about 20±25 dB. Tanaka and Brown (1970) measured CM in guinea pigs from an electrode introduced into the scala media. They used tone bursts at a sound pressure level within the linear range of CM and injected via a micropipette into scala media acetyl salicylate at a dose calculated to give a ®nal concentration of 0.7 mM in endolymph. CM measures showed no signi®cant change. Matsuura et al. (1971) recorded no change in microphonic potential from the sacculus of the gold®sh after injection via a micropipette directly into the sacculus of aspirin or sodium salicylate at concentrations of 3 mg/ml, whereas other drugs did produce modi®cations. Thalmann et al. (1973) detected very small changes in CM in response to a 6-kHz tone recorded at the cochlear base in guinea pigs given an intraperitoneal injection of salicylate at doses of 350 and 500 mg/kg. Simultaneously, neural responses showed a strong decrease. Mitchell et al. (1973) studied CM in guinea pigs after a subcutaneous injection of 268 and 545 mg/kg of sodium salicylate. At the higher dose, within 2 h post-injection blood salicylate levels rose quickly to about 400 mg/l and then continued to increase over the next 4 h to about 450 mg/l. CM measures taken from 100 to 20,000 Hz and at low acoustic levels did not show any signi®cant change over 6 h post-injection. Simultaneous recordings of eighth nerve evoked potentials indicated clear losses. McPherson and Miller (1974) studied CM from a round window electrode in guinea pigs given an oral dose of 300 mg/kg of choline salicylate. They used pure tones of variable frequencies from 200 to 20,000 Hz. Their results indicate very strong decreases in CM

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amplitude at all frequencies amounting to an equivalent loss of about 50±60 dB at 4±7 h after choline salicylate administration. Oliveira and Marseillan (1976) treated guinea pigs with intraperitoneal injections of salicylate at doses of 500, 750 and 1000 mg/kg. They measured CM from the round window and observed in response to 1 and 4 kHz tones. They observed no change or a decrease of a few dB for the dose of 500 mg/kg and of up to 15 dB for the dose of 1000 mg/kg. Simultaneous recordings of neural responses showed greater losses. Ramsden et al. (1985) recorded from an electrode on the bony promontory at the base of the cochlea in two patients who had taken overdoses of salicylate. Their measures of CM remained approximately constant during hearing recovery while the eighth nerve responses did show clear changes. Cazals et al. (1988) did not ®nd change in CM and SP recorded from a round window electrode in awake guinea pigs injected an intramuscular dose of 500 mg/ kg lysine salicylate. CM was recorded every hour for 3 h in response to various pure tones from 250 to 32,000 Hz presented at 80 dB sound pressure level. In the same animals simultaneously recorded neural responses were considerably modi®ed. Puel et al. (1989, 1990) recorded CM and SP from scala vestibuli at the basal cochlear turn in guinea pigs. Through a hole in scala tympani salicylic acid sodium salt or acetyl salicilic acid were perfused into the cochlea at concentrations of 0.04±20 mmol/l. CM was recorded in response to a 10-kHz tone. Little or no change in CM were observed. CM evoked at 68 dB SPL was unchanged, and a slight decrease was measured for CM evoked at 98 dB SPL for salicylate concentrations above 1 mmol/l. No change in SP was observed. Neural responses from the same animals showed considerable alterations. Stypulkowski (1990) experimented on cats with an electrode on the round window edge. Sodium salicylate was administered intravenously at doses of 200 or 400 mg/kg over 10±30 min and salicylate blood levels of 600±700 mg/l were measured for several hours postinjection. CM was recorded in response to clicks and 8-kHz tones. Slight increases in CM were measured corresponding to an elevation of 3±4 dB at all tested levels from 50 to 90 dB SPL. SP was found to decrease at low sound intensities. Simultaneous recordings from the same animals exhibited much more pronounced decreases in neural responses. Kujawa et al. (1992) measured CM in response to an 8-kHz tone in guinea pigs with an electrode in the basal turn of scala vestibuli and perfused sodium salicylate in scala tympani at concentrations of 0.6±5 mM. The CM remained unchanged in this case. Fitzgerald et al. (1993) experimented on guinea pigs perfused into scala media with salicylate and recorded

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with an electrode on the round window. They measured CM in response to a 1-kHz tone and observed some increase in CM amplitude more pronounced at low sound pressure levels. This e€ect was dose dependent and, for a 10 mM perfusate, it could reach a three-fold increase which is equivalent to a 10dB increment in sensitivity. On the same animals large decreases in sensitivity of neural responses were measured. Didier et al. (1993) measured CM with a round window electrode in guinea pigs given sodium salicylate either intramuscularly at doses up to 300 mg/kg, or on the round window at doses up to 300 mM. CM measures in response to tones of 2±32 kHz were taken 2 h post-systemic and 15 min post-topical administration of salicylate. No clear changes were seen, mostly decreases in sensitivity of a few dB were observed, a few being statistically signi®cant. Simultaneous measures of neural responses exhibited very clear losses of sensitivity. Murugasu and Russell (1995) recorded CM in response to tones of 15 and 1 kHz from an electrode on the round window of guinea pigs and perfused salicylate at various concentrations up to 5 mM in scala tympani. Their recordings showed quite variable results, namely slight increases or clear decreases for the 15 kHz tone, and no change or a slight decrease for the 1-kHz tone. These changes were not correlated with simultaneous measures of basilar membrane mechanical movement and neural responses which both showed strong decreases in sensitivity. Bian and Cherto€ (1998) undertook a detailed analysis of CM changes induced by salicylate in gerbils. Two drops of 100 mM salicylate were deposited onto the round window and left for 10 min. The round window was then dried. CM was recorded from the round window in response to a broad-band noise presented at sound pressure levels of 68 and 88 dB SPL. CM analysis was performed using spectral data and a nonlinear systems identi®cation technique based on a third order polynomial model of the transduction function. The data obtained after infusion of salicylate were compared with those obtained after acoustic trauma by an 8-kHz tone which resulted in similar hearing loss as determined with eighth nerve responses. Salicylate was found to induce complex changes in CM as a function of frequency and sound pressure levels. The results indicated mostly increases in CM amplitudes, together with an increased range, a steeper slope and an increased asymmetry of the derived transduction function. The results permit to ®t and explain some apparent contradictions in the results of several previous experiments from other authors. Overall the results provide evidence for very limited changes in CM or SP after salicylate administration. The variability in the results even within one study can

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originate in the complex nature of these ®eld potentials which are a composite sum of phases and amplitudes of responses originating from at least several hundreds of hair cells. Therefore, the end result in amplitude of CM and SP can bear a very nonlinear relationship with changes occurring at a cellular level. In addition, CM and SP are the result not only of one simple resistive component of transducer channels but also of other active processes such as hair cells membrane components and the compound impedance of the cochlear partition. Therefore, CM and SP are too complex to allow for simple interpretations. Detailed recordings in association with elaborate mathematical analysis might prove useful in this perspective. It remains that the bulk of experimental data indicate little change, if any, in the compound potentials CM or SP after salicylate administration whereas in many studies simultaneous recordings of neural evoked responses were found more strongly a€ected. 3.2.3. Cochlear mechanics The measurements of cochlear movements in response to sounds have much progressed over the last tens of years thanks to availability of ®ner physical tools, (see Wilson, 1991 for an overview). The movement of the various cochlear parts in response to sounds at threshold of hearing is smaller than the dimension of an atom, being less than 10ÿ10 m. Two studies reported mechanical measures of cochlear motion at threshold, one by Khanna and Leonard (1982) used laser interferometry and the other by Sellick et al. (1982) which used the MoÈssbauer technique, i.e. detection of a Doppler shift in the radiation of a radioactive source in movement. Since then several studies have been performed using mostly laser interferometry. These measures together with knowledge on otoacoustic emissions, have re-emphasized the necessity of excellent physiological conditions, and thereby the cochlear fragility, for the obtainment of a normal functioning of outer hair cells at hearing threshold. Two studies dealt with alterations of cochlear mechanics by salicylate intoxication. Mammano and Ashmore (1993) performed laser interferometry measurements from isolated cochleas. Movements were taken at the apical part of the organ of Corti and at the basilar membrane in response to electrical stimulation applied across the whole organ of Corti. The perfusion of 10 mM salicylate for 5 min in the bathing medium resulted in a decrease of approximately 50% of the amplitude of movements. This is equivalent to a loss of 6 dB. This e€ect occurred after about 5 min and was reversible within about 15 min. Murugasu and Russell (1995) used laser interferometry to measure movements of the basilar membrane in guinea pigs whose cochlear base was perfused with salicylate concentrations of 2.5, 5 or 10 mM. All

measurements were made at the basal end of the cochlea in a tonotopic region of approximately 15 kHz. Sound stimuli used were centered around this frequency. The normal mechanical response at the recorded point of the basilar membrane has the form of a tuning curve made of a narrow band-pass ®ltering at low sound pressure levels, de®ning the tip of the tuning curve, and a low-pass ®lter at high levels above about 50 dB SPL de®ning the tail of the tuning curve. Measures were taken of sound pressure levels necessary to produce basilar membrane motions of 10ÿ9 m, that is within about 10 dB of normal threshold values. After salicylate administration several clear and reproducible changes were seen. The tip of the tuning curve was desensitized by 20±30 dB after 2.5 or 5 mM salicylate infusion and up to 40 dB after 10 mM salicylate infusion. Its most sensitive frequency was shifted by about 2 kHz, its width at 10 dB above best threshold was broadened by a factor of about 3, while the high and low frequency slopes of the tuning curve were shallower, and in several instances the tail of the tuning curve could present a better sensitivity. The time course of these e€ects were measured using a 2-min perfusion of salicylate followed by a washout. The alterations in basilar membrane mechanics started within about 5 min, reached a maximum after about 30 min and then recovered within the next 10 min. Neural responses showed a time course of deteriorations parallel to that of mechanical tuning but evidenced little or no recovery suggesting an additional action of salicylate on the auditory nerve. The data from measurements of cochlear mechanics provide evidence that salicylate can induce losses in the basic mechanical properties of the cochlea in response to sound vibrations. However, these studies appear to have used mostly unphysiological levels of salicylate concentration. As indicated in a subsequent section on salicylate pharmacokinetics, various experiments have shown that perilymph salicylate concentration is roughly 30% plasma salicylate concentration. Measures in humans and in various animal experiments, as indicated above, showed that plasma concentrations near or above approximately 4 mM or 640.44 mg/l can be lethal. Associated perilymph concentration would be about 1.3 mM. So, in these studies on cochlear mechanics many e€ects observed may not re¯ect processes reported from all other in vivo or in toto experiments. The dose/e€ect relation for mechanical e€ects is unknown but seems to be nonlinear. If it can be assummed that at physiological concentrations qualitatively similar mechanical e€ects occur, the physiological signi®cance of the data can be considered. Then in the perspective of the active mechanical feedback provided by outer hair cells in vivo in the organ of Corti, it can be thought that a similar alteration of the normal functioning of these hair cells

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is produced by salicylate. Supraliminal e€ects such as changes in the shape of the mechanical tuning could likely be linked to similar anomalies in supraliminal auditory perceptions. 3.2.4. Otoacoustic emissions Otoacoustic emissions (OAE) are sounds emitted by the cochlea and recordable with a microphone tightly sealed into the external auditory canal. They were ®rst recorded by Kemp (1978). Their characteristics provide evidence for an active mechanical ampli®cation of sounds reaching the cochlea, the gain of this ampli®cation being greater as the acoustic pressure of the incoming sound is lower. A series of experimental data indicate that this mechanical ampli®cation originates from outer hair cells. There are several types of recordable OAE. The ear can produce OAE without being excited by an external sound. These are called spontaneous OAEs. All other are evoked OAEs and can either show emission of acoustic energy in a frequency area contained in the acoustic stimulus, or they can show energy at di€erent frequencies and re¯ect mechanical distortion originating within the cochlea. Evoked OAE can be recorded in response to sounds presented at very low sound pressure levels which is a relevant aspect for salicylate investigations. Two reviews can be consulted in Patuzzi (1996) and Whitehead et al. (1996). Johnsen and Elberling (1982) recorded OAE evoked by a click from the ear of a normally hearing woman given 10 g of acetylsalicylate. Her hearing threshold for the click rose by about 15 dB and her OAE simultaneously showed a sensitivity loss of approximately 15 dB. McFadden and Plattsmier (1984) studied spontaneous otoacoustic emissions from the ears of ®ve young normally-hearing subjects who took 975 mg of aspirin every 6 h for 3.75 days. All spontaneous OAE progressively decreased and disappeared during drug treatment and progressively returned to normal within several days of cessation of treatment. In some ears where multiple spontaneous OAE were present, they showed notable variations in their relative amplitudes in both disappearance and recovery processes. In the available data, changes in OAEs do not seem simply correlated with losses of hearing sensitivity. Wier et al. (1988) compared spontaneous OAEs and distortion product otoacoustic emissions (DPOAEs) from four normally-hearing subjects ingesting 975 mg of aspirin every 6 h for 1±4 days. The disappearance of spontaneous OAE during the course of aspirin treatment was con®rmed, whereas distortion product DPOAEs were only reduced. DPOAEs were more reduced when the two pure tone acoustic stimuli were of low sound pressure level. Audiometric hearing losses for the same subjects were very small.

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Long and Tubis (1988a) explored spontaneous and evoked OAEs in four subjects who took 975 mg of aspirin every 6 h for 3 or 4 days. All spontaneous OAEs disappeared during aspirin treatment but for one OAE from one ear of a subject which was only reduced. In the early phase of aspirin treatment evoked OAEs were reduced but remained present while spontaneous OAEs disappeared, but later in the course of aspirin treatment evoked OAEs also disappeared. The time course of reduction di€ered between subjects and even between the two ears of the same subject. Audiometric hearing thresholds before aspirin treatment were measured for frequencies very close to each other so as to reveal small dips and troughs of a few dB de®ning a threshold microstructure. During aspirin consumption, small changes in threshold microstructure were observed which followed in time changes in OAEs. Changes in evoked OAEs and threshold microstructure lasted longer and recovered sooner than spontaneous OAEs. Long and Tubis (1988b) performed several additional experiments to investigate the association between threshold microstructure and OAEs. In one experiment they present more detailed data on one subject of the above-mentioned study. They indicate that at the time of disappearance of spontaneous OEAs, audiometric sensitivity was improved while later when all OEAs were suppressed there was a deterioration of audiometric sensitivity. For several OAEs a slight shift in frequency was observed. Martin et al. (1988) measured spontaneous and evoked OAEs in one rhesus monkey after intramuscular injection of acetyl salicylic acid at 50 and 100 mg/ kg. No clear e€ect was observed at the dose of 50 mg/ kg. Within 70 min post-injection at 100 mg/kg spontaneous OAEs had disappeared. Distortion product OAEs remained unchanged even after 3 h post-salicylate injection. Two days later all OAEs were back to pre-treatment values. Penner (1989) reported on a woman who was su€ering of tinnitus in her right ear only, described as a steady tone and a hum. She was a very exceptional case of spontaneous OAEs linked with tinnitus. Her right ear showed ®ve spontaneous OAEs in the range of 1±2 kHz. In a series of subjective assessments she matched the tonal aspect of her tinnitus to tones comprised between about 1 and 2 kHz. The patient underwent a 3-day treatment with aspirin at 975 mg every 6 h. After the third day all spontaneous OAEs had disappeared and then she su€ered a tinnitus sounding like a noise in the head. Stypulkowski (1990) recorded distortion products OAEs in cats with a microphone in the external ear canal and also from CM responses with an electrode on the edge of the round window. An hour after sali-

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cylate administration DPOAEs were notably reduced and after about 3 h, one component was reduced by about 5 dB while another component had disappeared. In contrast CM measures of distortion products simultaneously increased by about 3 dB. Discrepancies between decreases measured in OAEs and increases observed in CM were tentatively related by the authors to a propagated mechanical activity for OEAs, and for CM to a local source of transduction currents at the site of primary tones excitation at the base of the cochlea. Long et al. (1991) explored the e€ects of aspirin on the suppression and synchronization of spontaneous OAEs by external tones in four young subjects with normal hearing. Subjects ingested 975 mg of aspirin every 6 h for 18 h. Their spontaneous OAEs were progressively reduced during aspirin treatment. Tones of frequency close to that of an OAE are more ecient in suppressing the OAE. The acoustic levels of di€erent tones necessary to reduce an OAE by a given amount de®ne a frequency selectivity curve. During reduction of OAE after aspirin ingestion, these frequency selectivity curves kept their sharpness but showed an elevation along the sound pressure level axis in proportion to the reduction of the OAE amplitude. A spontaneous OAE of a given frequency can be shifted to become synchronized to an external tone of a nearby frequency. The width of frequency span synchronizing an OAE was found broadened after aspirin consumption. The physiological signi®cance of these observations is unclear and the authors' ®t of the data to a physical model is only partly successful. Penner and Coles (1992) reported on another extremely rare case of tinnitus possibly linked with spontaneous OAEs. The subject was a woman who complained of binaural tinnitus, louder in the left ear, and described as ringing. A spontaneous OAE recorded in her right ear was unstable from day to day whereas an OAE in her left ear at a frequency of about 0.5 kHz was found stable and was used as a monitoring measure. The patient undertook a treatment of 600 mg of aspirin every 6 h for 3 days. After the ®rst day of treatment the spontaneous OAE had disappeared, and it reappeared a day after treatment arrest with a slightly greater level and was back to its original level in the next 2 days. The patient reported a parallel disappearance of her tinnitus followed by a rebounce. Kujawa et al. (1992) investigated distortion products in both OAEs and CM in guinea pigs. In order to avoid possible nonspeci®c e€ects of systemic administration sodium salicylate was perfused in scala tympani at concentrations of 0.6±5 mM. Distortion products were measured in response to two tones of 10 and 12 kHz providing a distortion product at 8 kHz. Measures were taken at various sound pressure levels

of the two primary tones. After salicylate CM in response to an 8-kHz tone alone was unchanged at all sound pressure levels. DPOAEs were reduced after salicylate perfusion, the reduction being greater at low sound pressure levels. CMDP were strongly reduced at low sound pressure levels but unchanged at high sound pressure levels. These data clearly indicate greatest changes occurring at low sound pressure levels for both OAEs and CMDP. They also demonstrate changes occurring in the nonlinear two-tone distortion of CM responses while response to a single tone remained unaltered. Kumagai (1992) studied electrically evoked otoacoustic emissions in guinea pigs after an intravenous injection of sodium salicylate at doses from 100 to 400 mg/kg. After salicylate injection electrically evoked otoacoustic emissions disappeared reversibly or irreversibly. Brown et al. (1993) investigated DPOAEs in eight normally hearing subjects who ingested 3.84 g of aspirin per day for 2 days. The e€ects of aspirin varied greatly between subjects. Fine variations of DPOAEs were investigated in response to frequency sweeps of one primary tone while the other primary frequency was held constant. These variations de®ne a band-pass ®lter shape of DPOAEs. After aspirin infusion three subjects showed a downward shift in the resonance frequency of this band-pass ®lter of DPOAE but no change in its width of tuning. In four subjects there was a reduction in the phase slope of DPOAEs. Fitzgerald et al. (1993) measured DPOAEs at 8 kHz produced by two tones of 10 and 12 kHz presented at 20±90 dB of sound pressure level. After perfusion of scala tympani with concentrations of 2 and 10 mM salicylate very strong reductions of DPOAEs were observed for low sound pressure levels whereas little reductions were observed at high sound pressure levels. Murphy et al. (1995) recorded spontaneous OAEs in one young normally-hearing subject. In previous experiments the authors had shown that the time course of suppression of spontaneous OAEs by an external tone can present undershoot at onset of suppression and overshoot at o€set. These e€ects were explained by the variations in masking by a neighboring OAE responding more slowly to suppression. The e€ect of aspirin was found to be a reduction in the magnitude of the overshoot by reduction of the neighboring OAE. Frank and Kossl (1996) measured DPOAEs in gerbils after iontophoretic injection of sodium salicylate into scala media. The rationale of their study was that two di€erent distortion products, the inferior cubic di€erence tone and the di€erence tone, represent respectively asymmetric and symmetric components in the nonlinearity of transduction transfer function. During the initial phase of salicylate e€ects, they

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observed a decrease in the inferior cubic distortion tone OAE with a simultaneous increase in di€erence tone OAE, consistent with the interpretation of a shift of the resting position of the transduction transfer function. Ueda et al. (1996) examined click-evoked OAEs in guinea pigs after intravenous injection of aspirin at the dose of 100 and 400 mg/kg. OAEs decreased on average by about 3 dB during the ®rst 20 min post-injection of a dose of 100 mg/kg, at the dose of 400 mg/kg OAEs decreased by about 10 dB during the ®rst 40 min post-injection. Recovery was observed after several hours. Decreases were more pronounced at lower sound pressure levels. The time course of OAEs decreases was parallel to losses observed in neural responses simultaneously measured in the same animals. Arruda et al. (1996) studied click-evoked OAEs in chinchillas after application onto the round window of a gelfoam soaked with 150 mg of sodium salicylate left for 2 h. After salicylate application there was a clear decrease of more than 10 dB in the amplitude of the higher spectral peak of the OAE whereas no signi®cant change was observed when the overall spectral power was taken as a measure. Quaranta et al. (1999) recorded click-evoked OAEs in eight normally-hearing subjects who ingested 3.84 g of aspirin per day for 2 days. OAEs were reduced on average by about 4 dB after the ®rst day of aspirin treatment and by about 6 dB after the second day. Takahashi et al. (1999) measured DPOAEs in guinea pigs after cochlear perfusion of 5 mM salicylate. DPOAEs were reversibly reduced by 5±10 dB after salicylate administration, the reduction being more pronounced as the stimuli were of lower sound pressure level. Stewart and Hudspeth (2000) administered a single intraperitoneal dose of 62 mg/kg sodium salicylate to Tokay gecko lizards. For ears with higher power SOAEs, these SOAEs diminished by about 30% in the ®rst hour post-injection and were back to normal after 3 h. For ears with a lower SOAEs, an increase of SOAE was observed in the ®rst hour with a return to normal at the third hour. The authors also gave 35 mg/kg of acetylsalicylic acid orally twice daily for 2±7 days. This resulted in all SOAEs being diminished by about 40% and a return to original values was observed 1 week after the end of treatment. Janssen et al. (2000) measured DPOAEs and clickevoked OAEs in a 22-year-old woman at 22 h after ingestion with suicidal intent of a dose of presumably 10 g of aspirin and a day later when both hearing loss and tinnitus had disappeared. At time of aspirin poisoning click-evoked OAEs had disappeared wheras DPOAEs were still recordable even at high frequencies associated with a hearing loss of 40±50 dB and corresponding to the frequency region of tinnitus. The vari-

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ation of DPOAEs amplitude as a function of sound level was more linear than normal, in agreement with loss of outer hair cell compression. A day later hearing loss and tinnitus had disappeared and both clickevoked OAEs and DPOAEs were within normal limits. Overall the data indicate that clear alterations are produced on OAE by salicylate. The most conspicuous e€ect is the disappearance of spontaneous OAEs. The functional signi®cance of spontaneous OAEs is unknown. Spontaneous OAEs are recordable in a majority of ears from subjects with a normal hearing sensitivity. Although there is no direct demonstration it is generally accepted that they are the result of a local high gain in the electro-mechanical feed-back loop on the mechano-electrical transduction performed by outer hair cells. Too much feed back gain would represent some sort of self-triggered ringing in the organ of Corti. Contrary to ®rst intuition these spontaneous OAEs are inaudible, but in extremely rare cases, and do not seem related to subjective hearing sensations as tinnitus. The disappearance of spontaneous OAEs appears contingent with the eventual appearance of salicylate-induced tinnitus. A case-report of salicylate e€ects on OAEs apparently linked with tinnitus, provides a demonstrative example. The data do not indicate either for amount or time course, clear correlations between the reduction and/or disappearance of spontaneous OAEs and the losses of absolute hearing sensitivity. Studies on click-evoked OAEs indicate reductions in response amplitude and associated threshold elevation, in good correspondence with loss of absolute hearing sensitivity. DPOAEs indicate reductions more pronounced as the level of the stimuli are of lower sound pressure level. Detailed measures of various DPOAEs components and/or characteristics further revealed more subtle changes induced by salicylate on di€erent aspects of nonlinear phenomena of cochlear functioning. The interpretation of the results of all these studies is dicult because underlying physiological processes are poorly known as are their possible correspondences with auditory perceptions. However, the data clearly indicate salicylate-induced changes in the mechano-sensory functioning of the cochlea in the form of an overall decrease in spontaneous otoacoustic emissions in response to low sound pressure levels and also in the form of alterations in various nonlinear processes. A disappearance of spontaneous OAEs can be thought of as altering the spontaneous activity of the auditory nerve and subsequently of higher auditory structures associated with the subjective presence of an extraneous, tinnitus sound. A decrease in response sensitivity is in line with a loss of absolute hearing sensitivity. Alterations of various aspects of sound-

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evoked cochlear nonlinearities might be related to various aspects of suprathreshold hearing alterations. 3.2.5. Isolated outer hair cells Cochlear outer hair cells can be isolated and kept in good condition for several hours in vitro. This approach allowed Brownell (1983) and Brownell et al. (1985) to discover that these cells present motile responses to electrical stimulation. In various subsequent studies it was shown that astonishingly this motility covers the whole range of hearing frequencies which includes several tens of kilohertz. These observations together with the ®ndings of otoacoustic emissions and the sharp tuning of cochlear mechanics at low acoustic levels led to the notion that the motile properties of outer hair cells make them act in the cochlea as mechanical ampli®ers with a nonlinear gain inversely proportional to the acoustic level of incoming sounds. Many studies are still devoted to the investigation of the unique motile properties of outer hair cells. Dallos (1991) provides a concise review on essentials aspects, and several chapters of a book edited by Dallos et al. (1996) give abundant and detailed informations. On the basis of converging indirect evidences of the action of salicylate upon outer hair cells as indicated above, various studies investigated the action of salicylate upon isolated outer hair cells. Shehata et al. (1991) observed a reversible loss of turgidity, seen as a de¯ated appearance, and a diminution of electromotility in outer hair cells exposed to perfusion in the bathing medium of salicylate at concentrations of 0.1±10 mM. These e€ects and their occurrence times were dose dependent and both turgor and electromotility were restored by rinsing the bathing medium. They also observed a change in membrane conductance occurring later in time, independent of salicylate dose, and not reversible after rinsing. A further study by the same group (Dieler et al., 1991) performed transmission electron microscopy on isolated outer hair cells exposed to salicylate as reported above. The observed changes in subsurface cisternae were dose-dependent and reversible and they followed the same time-course as alterations in electromotility. Tunstall et al. (1995) used the patch clamp whole cell recording technique to examine e€ects of salicylate upon isolated outer hair cells. Salicylate (1±10 mM) reduced the fast charge movements, the membrane capacitance and the electrically-induced length changes of outer hair cells. Additional experiments indicated that the reduction in pH induced by salicylate played a minor role in decreases of electromotility. The alterations in capacitance were found independent of voltage holdings of membrane potentials over a very large range suggesting that an uncharged form of salicylate is involved. The data are interpreted as the result of salicylate partitioning into the cell membrane.

Russell and Schauz (1995) used a glass ®ber of calibrated sti€ness to measure the axial movement, sti€ness and forces generated by isolated outer hair cells in response to electrical stimulation. Electromotile forces and axial sti€ness of outer hair cells were reduced by about two-thirds when bathed in 5 mM salicylate. A closely linear relation appeared for di€erent cells between sti€ness in control bathing solution and in salicylate, whereas a large scatter appeared for force measurements between control bathing solution and salicylate. So that force generation is in¯uenced by at least another factor, such as for instance the previously reported conductance changes induced by salicylate, the amount of observed decrease in sti€ness seems to be directly correlated with the mechanical alterations reported by Murugasu and Russell (1995). Kakehata and Santos-Sacchi (1996) had previously shown that whole cell patch clamp recording can modify intracellular pressure and thereby alter membrane capacitance and motility of outer hair cells. So they examined the e€ects of salicylate while minimizing or controling variations of intracellular pressure. Mammalian outer hair cells present a bell-shaped voltagedependent capacitance function. Salicylate applied either extracellularly or intracellularly reduced the amplitude of the nonlinear peak capacitance. It also reduced the voltage-induced length changes, which became more linear as a function of applied voltage. E€ects started to be detected at concentration of 0.3 mM and increased sigmoidally to 10 mM. Intracellular application of trypsin destroys the cortical skeleton of outer hair cells and deteriorates subsurface cisternae. In spite of intracellular trypsin, the application of salicylate still reversibly reduced the nonlinear peak capacitance. Hallworth (1997) used a glass ®ber applied on the cuticular plate of isolated outer hair cells to measure e€ects of salicylate upon compliance and force. Compliance, the inverse of sti€ness, was measured in response to motion applied to the ®ber glass and force was measured in response to electrical stimulation. Exposure to salicylate concentrations of 0.01±10 mM resulted in reduction of force and no signi®cant change in compliance. The dose-e€ect relation was sigmoidal from 0.01 to 10 mM. Jung et al. (1997) examined optically morphologic changes of isolated outer hair cells exposed to 10 mM salicylate. Previous studies by these authors provided arguments for the ototoxicity of salicylate to be linked to increased leukotrienes in the cochlea so they also exposed outer hair cells to leukotrienes and to a leukotriene inhibitor. Salicylate induced a shortening and swelling of outer hair cells after 20 min and the leukotrienes induced similar e€ects but the leukotriene inhibitor prevented these alterations. Nenov et al. (1997) observed an almost complete

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suppression of the early capacitive transient in response to voltage steps, when outer hair cells were exposed to 10 mM salicylate, these e€ects were reversible. Wu and Santos-Sacchi (1998) observed that two lipophilic ions, a negative and a positive, respectively, shifted and/or increased the voltage dependent capacitance peak of outer hair cells. After exposure to the negative lipophilic ion solution, exposure of outer hair cells to salicylate (10 mM) still induced a decrease of the peak capacitance and did not alter the voltage driven motility. The results suggest that salicylate acts rather directly on the outer hair cell motor and not through nonselective charge changes. Chan and Ulfendahl (1999) investigated movement and axial sti€ness of outer hair cells under mechanical vibration at very low infra sound frequencies. Clear responses were observed for apical cells only. Salicylate perfusion at 5 mM concentration induced a decrease in response threshold, and showed either a decrease or an increase in axial sti€ness. Lue (1999) and Brownell (1983) investigated outer hair cells lateral wall sti€ness by measuring the length of membrane aspirated in a pipette when an aspiration pressure of 10 cm of water was applied. After 10 mM salicylate perfusion a reversible decrease of lateral wall membrane sti€ness was observed. Overall, the studies on isolated outer hair cells de®nitely demonstrated that salicylate does interfere with the special mechanical and motile properties of these cells. However, as indicated in the section on cochlear mechanics, it should be recalled that perilymph salicylate concentrations near 1.3 mM or above may be considered outside the physiological range. In line with characteristic features of salicylate ototoxicity the alterations in electromotility and membrane capacitance appear dose dependent and reversible. The studies of Russell and Schauz (1995) and Murugasu and Russell (1995) o€er a link between isolated hair cell changes and alterations in mechanical responses of the whole cochlea in vivo. Thus salicylate-induced alterations of outer hair cells appear reasonably linked with other human and animal data on losses in absolute hearing sensitivity and associated broadening in frequency selectivity induced by salicylate. 3.3. Eighth nerve and higher auditory structures 3.3.1. Acoustically-evoked activities The activity of the auditory nerve conveys to the brain all auditory information originating from the sensory organ and therefore must contain alterations of cochlear origin leading to perceptive alterations induced by salicylate. Recording from higher auditory structures was used to investigate the impact of salicylate on the central nervous system. Recordings of com-

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pound potentials from the whole nerve are easily performed. They can provide accurate threshold measurements within a few dB and have often been used to assess salicylate ototoxicity. Using scalp electrodes is technically easy and allows recording of compound brainstem auditory evoked potentials, and potentials taken from a gross electrode in an auditory structure are also easily obtained. Recordings from single cells can bring much more detailed information but is much more time consuming and few studies have used this approach to evaluate salicylate auditory toxicity. Gold and Wilpizeski (1966) recorded the eighth nerve compound action potential (CAP) with an electrode stereotaxically descended onto the nerve trunk. They used a click which is a very broad frequency band stimulus. Various measurements were made. Absolute threshold changes and amplitude variations as a function of stimulus level were taken. Threshold adaptation was measured as the amount of threshold drift over 1 min of stimulation by a continuous train of clicks at 200 clicks/s. Suprathreshold adaptation was measured as the amplitude decreases observed in response to a brief 1 kHz tone presented during one min at 100 dB SPL with various interstimulus intervals from 0 to 500 ms. Experiments were performed on cats and guinea pigs. Four cats received a single intraperitoneal injection of sodium salicylate at the dose of 300 mg/kg and four other cats received 125 mg/kg/day fractionated in two doses during 2-weeks. In both groups absolute threshold elevations of approximately 10 dB were observed, associated with amplitudes at high sound levels similar to normal, resulting in a steeper than normal growth similar to loudness recruitment. Measures of threshold and suprathreshold adaptation were found unchanged. Serum salicylate levels were about 400 mg/l after the single injection, and only 300 mg/l after the 2-week treatment. Other experiments were performed on six guinea pigs, which received a single intraperitoneal injection of 300 mg/kg or 10 daily doses of 150, 200 or 300 mg/kg/day, in three divided doses per day, for three and a half days. Only absolute threshold changes were measured and the single injection of 300 mg/kg resulted in a threshold shift of about 10dB whereas the treatment at 300 mg/ kg/day produced a threshold shift of 3±4 dB only. The treatments at 225 and 150 mg/kg/day produced no signi®cant change. Silverstein et al. (1967) recorded from the round window of cats CAP evoked by clicks at 40±50 dB above threshold. Cats were given one intraperitoneal injection of 350 mg/kg of sodium salicylate and electrophysiological recordings were followed for 5±7 h. Within 3 h post-injection CAP amplitude dropped by 50±80% which is equivalent to an attenuation of ap-

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proximately 20±30 dB. In accordance thresholds were found elevated by 20±24 dB. Thalmann et al. (1973) measured CAP in response to a 6-kHz tone burst from guinea pigs after an intraperitoneal injection of sodium salicylate at the dose of 350 mg/kg, They observed a decrease of CAP amplitude at low acoustic levels while no change occurred for high acoustic levels. A threshold elevation of approximately 40 dB was measured. Simultaneous measures of cochlear microphonic indicated little or no change. Higher doses of salicylate up to 500 mg/kg, or perilymphatic perfusion at a concentration of 300 mg/l provided similar e€ects. Mitchell et al. (1973) studied CAP in guinea pigs after a subcutaneous injection of 545 mg/kg sodium salicylate. Within 2 h post-injection blood salicylate levels rose quickly to about 400 mg/l and then continued to increase over the next 4 h to about 450 mg/l. CAP exhibited a loss of sensitivity of up to 20 dB after approximately 5 h. A day later a return to normal was observed. McPherson and Miller (1974) gave guinea pigs an oral dose of 300 mg/kg of choline salicylate. They measured click-evoked CAP and reported CAP amplitude reduction of about 60% at 3±5 h after drug administration. Such a decrease likely corresponds to an attenuation of about 20±30 dB in acoustic level and a similar threshold elevation can be assumed. Oliveira and Marseillan (1976) observed CAP amplitude, decreases expressed in dB, of 6±12 dB in guinea pigs given intraperitoneal injections of sodium salicylate at doses of 500 and 1000 mg/kg. CAP responses were probably taken in response to a click well above threshold, and amplitude decreases seem to correspond to a drop of about 50% and 75% equivalent to an attenuation of about 20±30 dB. Woodford et al. (1978) experimented on chinchillas implanted with a screw electrode through the skull to record auditory brainstem responses evoked by 0.5±8 kHz stimuli. Chinchillas were injected intramuscularly with 400 mg/kg sodium salicylate which produced a plasma salicylate level of 650 mg/l. This resulted in a threshold elevation of about 10 dB maximum at the middle frequencies and up to 20 dB at the highest frequency. Within a few days all thresholds went back to pretreatment values. In the same study the authors investigated the combination of salicylate injection with exposure to three di€erent noise traumas, and found no e€ect of these combinations over noise alone. Evans et al. (1981) and Evans and Borerwe (1982) intravenously injected sodium salicylate at the dose of 400 mg/kg in three divided doses over a period of 10± 20 min in cats. They measured the eighth nerve CAP and single ®ber responses over up to 18 h post-injection. Serum salicylate concentrations were about 600

mg/l at 10±25 min post-injection and 340 mg/l or greater 16 h post-injection. CAP thresholds were found elevated by 10 dB at the low frequencies and up to 30 dB at the high frequencies 40 min post-injection. Surprisingly, 15 h later threshold elevations were further increased to 30 and 46 dB, respectively, for the low and high frequencies. Minimum thresholds of single ®bers followed a very similar time course and showed similar threshold elevations. Within hours post-salicylate injection, the tuning curves of single ®bers presented a progressive elevation of their tip of about 40 dB and up to 60 dB for ®bers tuned to the highest frequencies whereas the tail of the tuning curve did not seem altered. The dynamic range, or sound pressure range over which a ®ber increases its mean discharge rate, decreased from about 40±50 dB before salicylate to 25±35 dB several hours after salicylate. Ramsden et al. (1985) measured CAP evoked by a broad-band click from an electrode on the bony promontory at the base of the cochlea in two human subjects who had taken overdoses of salicylate. They observed CAP-threshold elevations of 30 and 40 dB, respectively, for the two subjects, in accordance with subjective audiometric losses. The amplitudes of CAP were reduced at low acoustic levels and remained unchanged at high acoustic levels, similar to subjective loudness recruitment. Both CAP and subjective audiometry thresholds followed a closely parallel recovery for each of the two subjects. Schreiner and Snyder (1987) recorded eighth nerve CAP in cats after intravenous injection of sodium salicylate at the dose of 200 mg/kg followed 3 h later by a dose of lidocaine, a drug known to temporarily suppress tinnitus in some patients, followed an hour later by 125 mg/kg of sodium salicylate. CAP responses to 5 and 20 kHz tone bursts indicated a threshold elevation of 20±30 dB 5 h after the ®rst dose of salicylate. Twenty hours later, the thresholds had improved only by 5±10 dB. Cazals et al. (1988) experimented on guinea pigs given an intramuscular injection of lysine salicylate at the dose of 500 mg/kg. CAPs recorded from the round window presented threshold elevations reaching about 10±20 dB for low to high frequencies 2 h post-injection that remained stable to the third hour and recovered after 24 h. Gunther et al. (1988, 1989a, 1989b) used rats and recorded auditory brainstem responses from subcutaneous electrodes in response to 10 and 20 kHz stimuli. One day after giving an oral dose of 700 mg/kg of salicylic acid, they found a threshold elevation of 15± 20 dB. One week later the thresholds were back to normal. Boettcher et al. (1989) implanted electrodes into the inferior colliculus of chinchillas. They gave intraperitoneal injections of sodium salicylate at 450 mg/kg/day

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for 5±15 days and they observed associated serum salicylate levels of 250±500 mg/l. Thresholds measured from 250 to 16,000 Hz in octave steps indicated losses of a few dB on average with large interindividual variability and maximum losses of about 30 dB. Frequency selectivity curves were obtained at 1 and 4 kHz and showed some broadening at 4 kHz. Temporal measures of forward masking did not show any change. Carson et al. (1989) used subcutaneous electrodes and recorded brainstem auditory potentials in rats in response to a tone pip at 8 kHz. They gave the rats an oral administration of aspirin for 18 days at the dose of 200 mg/kg/day. Serum salicylate levels were checked to be above 350 mg/l at the tenth day, being signi®cantly more elevated in female rats. No change in threshold was observed. Combination of daily loud noise exposure with salicylate treatment during 8 or 12 days did not result in increased thresholds compared with noise exposure alone. Puel et al., (1989, 1990) recorded from the auditory nerve of xenopus and guinea pig. In the xenopus salicylate was injected in the lateral line organ at concentrations of 0.3±2.5 mM, which resulted in reduction and suppression of spontaneous activity. In guinea pig cochleas they perfused 0.6±10 mM sodium salicylate. They measured CAP evoked by a 10-kHz tone burst and the CAP threshold was found elevated by approximately 20 dB with a decreased amplitude in response to low but not to high acoustic stimulus levels. Stypulkowski (1990) studied the e€ects of intravenous injection of sodium salicylate at doses of 200 and 400 mg/kg on cats with an electrode on the cochlear round window. Plasma salicylate levels reached 600± 700 mg/l for the highest dose. Within 1±2 h post-injection CAP evoked by a broad band click or a tone burst at 8 kHz showed a threshold elevation of up to 20 dB and a decreased amplitude in response to low but not high acoustic levels. CAP evoked by cochlear electrical stimulation were much reduced after salicylate for low but not for high level stimuli. Auditory nerve single ®ber recordings with a microelectrode revealed threshold elevations of about 20 dB. Tuning curves of auditory nerve ®bers showed a threshold elevation and, consequently, a broadening of their sharp tip. Discharge rate and phase as a function of stimulus acoustic level presented a loss at low levels and no change at high levels. Rybak et al. (1990) administered an intravenous injection of a low dose, 50 mg/kg of sodium salicylate to chinchillas and measured auditory nerve CAP evoked by a broad band click. They observed no change in CAP after salicylate. Bancroft et al. (1991) implanted chinchillas with an indwelling electrode into the inferior colliculus. Evoked response thresholds were taken for stimuli ranging

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from 0.25 to 16 kHz. They administered one daily intraperitoneal injection of sodium salicylate at the dose of 300 mg/kg for 15 days. Testing at 2±4 h postinjection, at the time when serum salicylate levels were measured around 300 mg/l, did not indicate any threshold change. The combination of salicylate treatment with exposure to an octave band noise at 0.5 kHz at 85 or 105 dB SPL did not result in threshold elevation greater than exposure to the noise alone. Johnson (1992) fed rats with aspirin at 100 mg/kg twice daily for 10 days. Auditory brainstem responses were recorded from subcutaneous needles. Threshold were measured in response to tone bursts of 1±20 kHz. No threshold change was observed. Jung et al. (1992a, 1992b) applied 50±200 mg of sodium salicylate onto the round window of chinchillas. Subcutaneous needles were used to record auditory brainstem responses evoked by a broad band click. They observed threshold elevations of 10±30 dB as the salicylate dose increased. Kumagai (1992) examined CAP responses evoked acoustically or electrically in guinea pigs after an intravenous injection of sodium salicylate at doses from 100 to 400 mg/kg. After salicylate acoustically evoked CAP responses were diminished specially at low sound pressure levels and electrically evoked responses were also diminished but less. Fitzgerald et al. (1993) perfused salicylate, bromosalicylate and acetylsalicylate into the cochlea of guinea pigs and monitored the eighth nerve CAP in response to tone bursts of 2±24 kHz. After perfusion of 5 and 10 mM salicylate they observed a threshold elevation of about 40 dB at all frequencies. For a concentration of 2 mM threshold loss was only about 15 dB. For bromosalicylate similar threshold changes were observed for concentrations of 1±0.2 mM only. For acetylsalicylate threshold elevation of 6 and 40 dB were observed for concentrations of 1 and 10 mM. Kay and Davies (1993) studied the e€ects of intraperitoneal injection of sodium salicylate at the dose of 300 mg/kg to rats. Plasma salicylate levels reached a maximum of 360 mg/l, 4 h post-injection. At this time threshold elevation of 30 dB was observed in response to a broad band click. Didier et al. (1993) recorded auditory nerve CAP in guinea pigs given an intravenous injection of sodium salicylate at doses of 100, 200 and 300 mg/kg. They observed no change in threshold for the lower dose and threshold elevations of about 25 dB at high frequencies for the higher dose. Martin et al. (1993) recorded auditory brainstem responses from scalp electrodes as well as CAP from the round window and the nerve trunk in cats given an intravenous injection of 150 mg/kg of sodium salicylate. Preoperative thresholds were high (44 dB SPL) and as the nerve trunk was pulled to record from it,

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after salicylate injection thresholds rose to very high levels of up to 100 dB SPL. Shehata-Dieler et al. (1994) recorded eighth nerve CAP and single ®bers from pigeons after scala media perfusion of sodium salicylate at doses of up to about 18 mM. CAP-threshold elevation of 5±35 dB were observed for the mid and high frequencies. These were dose-dependent and reversible. Similar threshold elevation were observed for single ®bers. The tuning curves of ®bers presented an elevated and broadened tip with the high frequency side tending to be more a€ected. Park et al. (1994) experimented the e€ects of intraperitoneal injection of sodium salicylate on chinchillas at the dose of 300 mg/kg followed 2 h later by a dose of 200 mg/kg. They measured auditory brainstem responses evoked by a broad band click and found a threshold elevation of 30 dB within 6 h post-injection. The latencies of the brainstem responses were found increased at all levels of acoustic stimulation. This could simply correspond to the absence of the earliest contribution of neural elements normally responding to the high frequencies. Murugasu and Russell (1995) measured eighth nerve CAP from an electrode onto the cochlear round window in guinea pigs. They perfused 2.5±10 mM salicylate into the cochlea and observed a CAP-threshold elevation of about 20 dB in response to a 20-kHz tone burst. Aoyagi et al. (1996) injected guinea pigs with an intravenous dose of 500 mg/kg of sodium salicylate. They measured eighth nerve CAP thresholds in response to 2±12 kHz tone bursts and observed threshold elevations of about 10 dB at the lowest frequency and of about 35 dB at high frequencies. Interaction between salicylate and noise was investigated but the data do not provide clear evidence for a potentiation of noise-induced threshold shifts by salicylate. Ueda et al. (1996) measured auditory brainstem response in guinea pigs given an intravenous injection of aspirin at 100 and 400 mg/kg. Using click stimuli they found a threshold elevation of about 12 dB at 1 and 2 h post-injection. Duan and Canlon (1996) performed intracochlear perfusion of 1 mM salicylate in guinea pigs by using an osmotic pump connected to the cochlea by a catheter. Using brainstem auditory responses they noticed one day after implantation of the osmotic pump a threshold elevation of about 45 dB at 1, 4 and 8 kHz. Temporal course of forward masking was found unchanged. Kurata et al. (1997) implanted screws through the skull as permanent electrodes to record auditory brainstem responses evoked by tone bursts of 2, 4 and 8 kHz. Rats were given an intravenous injection of 225 mg/kg of sodium salicylate. Surprisingly, the authors

detected losses at 2 kHz but smaller and not signi®cant losses at 4 and 8 kHz, respectively. Very small increases in latencies, up to about 0.3 ms, were observed at some sound pressure levels for the ®rst peak corresponding to the auditory nerve response which can certainly be attributable to small amplitude and threshold changes. Very small increases in interpeak latencies were also observed which may simply correspond to some desynchronization of these compound evoked potentials due to decreased responsiveness of some neural elements as observed at the ®rst peak. Ochi and Eggermont (1996) administered a dose of 200 mg/kg sodium salicylate to cats and measured at the primary auditory cortex the acoustically-evoked local ®eld potential and responses from single cells. They succeeded in recording from 21 single units starting before and over 6 h post-salicylate injection. The local ®eld potential presented a frequency of best sensitivity similar to that of tuning curves from single cells. Both local ®eld potential and single cells showed a 20± 30 dB of threshold elevation 2 h post-injection and no recovery during the next 4 h. Wallhausser-Franke et al. (1996) investigated activation of brain structures induced by salicylate using radioactive deoxyglucose which is a marker of energetic consumption. They gave gerbils for four consecutive days an intraperitoneal injection of 200, 300 or 350 mg/kg sodium salicylate, 2 h after the last salicylate injection they injected of radioactive deoxyglucose. Compared to control animals they observed desactivation of the cochlear nucleus, the inferior colliculus and the lateral lemniscus together with activation of the medial geniculate body and auditory cortex. At the inferior colliculus desactivation was observed mostly in the high frequency tonotopic area. At the auditory cortex activation was observed in tonotopic areas of low frequencies while desactivation was observed at high frequency areas. The correspondence between radioactive dexoyglucose labelling and electrophysiological spontaneous activity is not clear so that the results may indeed be compatible with electrophysiological ®ndings from other authors. Wallhausser-Franke et al. (1997) performed additional experiments using c-fos immunochemistry labeling early gene activation. After salicylate treatment they found that the cochlear nucleus and medial geniculate body were devoid of labelled neurons and there was much individual and dose-independent variation in labeling at the inferior colliculus. The primary and anterior auditory cortices were clearly labelled in a dose-dependent manner. Besides, labelings were observed in prefrontal, parietal, perirhinal, pririform and enthorhinal cortices and in many nuclei of the limbic system especially the amygdala pointing to involvement of associated emotional aspects. Correspondence

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between c-fos immunolabeling and electrophysiological activity is unclear and no inference can be made. Kenmochi and Eggermont (1997) recorded from the primary auditory cortex in cats given one intraperitoneal injection of sodium salicylate at 200 mg/kg. From single cell recordings they observed that the best modulation frequency in response to periodic click trains decreased from 10 to 9 Hz after salicylate infusion. Eggermont and Kenmochi (1998) recorded in cats from three cortical areas simultaneously, the primary and secondary auditory cortex and the anterior auditory ®eld. They measured the local ®eld potentials and single cell responses. One intraperitoneal injection of sodium salicylate was performed which resulted in an approximately 15±20 dB threshold elevation mostly at frequencies above 2 kHz in all three cortical areas. The (onset) responses to sounds were found unaltered. Cazals et al. (1998) implanted indwelling electrodes on the round window of guinea pigs and monitored eighth nerve responses evoked by tone bursts of 0.5±32 kHz. Guinea pigs were treated with intramuscular injections of sodium salicylate at the dose of 200 mg/ kg delivered twice a day for 3 or 2 weeks. Very small threshold alterations of less than 10 dB, were observed for some animals in response to the highest frequencies at the end of the 3-week treatment while no threshold change was observed for the 2-week treatment. Sha and Schacht (1999) gave subcutaneous injections of sodium salicylate twice daily at 100 mg/kg for 19 days to guinea pigs. At the end of treatment plasma salicylate level was 142 mg/l. Auditory brainstem responses evoked by 3, 9 and 18 kHz tone bursts showed no threshold change. It appears clearly that recordings from the eighth nerve to the auditory cortex in these studies showed clear threshold elevations induced by salicylate. These changes were dose dependent and reached several tens of dB for the highest doses used. As indicated earlier these values are notably higher than those observed on the sensory potentials, CM and SP, and on otoacoustic emissions, they are closer to those observed in humans' or in animals' behavior. The data provide little evidence for a potentiation by salicylate of noise-induced threshold elevations. Most studies which compared acoustically-evoked eighth nerve responses to these other measures did notice a clear di€erence and point to an additional action of salicylate at the cochlear neural level. There is no discrepancy between eighth nerve and brainstem or auditory cortex as to threshold elevations after salicylate infusion. This suggests that these changes in evoked activity originate in the cochlea. Overall, the changes in eighth nerve, i.e. threshold elevation with broadening of tuning curve tip, appear to convey alterations leading to similar auditory perceptive alterations.

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3.3.2. Cochlear e€erents The cochlea receives nerve ®bers which constitute the cochlear e€erents from the olivary nuclei in the brainstem. These e€erents innervate mostly the basal part of the cochlea but their role in auditory physiology is still poorly understood. Three studies dealt with alterations of e€erents activity by salicylate. Stypulkowski (1990) used electrical stimulation of the e€erent bundle at the level of the ¯oor of the fourth ventricle. For control cats they observed, as shown previously by several authors, that stimulation of e€erents produced a reduction of amplitude of the eighth nerve CAP more pronounced at low sound levels. This reduction is accompanied by a threshold shift of about 20 dB. Electrical stimulation of e€erents also results in an increase in CM amplitude of about 3 dB independent of sound level. Electrical stimulation of e€erents also produces a decrease in EP amplitude. All three e€ects, on CAP, CM and EP, were diminished after intravenous administration of sodium salicylate at 400 mg/kg. These diminutions appeared more pronounced as the e€ects of salicylate increased over 2 h post-injection. Fitzgerald et al. (1993) also used electrical stimulation of the e€erents at the ¯oor of the fourth ventricle in guinea pigs. They measured e€erents' e€ects on CM and observed that after intracochlear perfusion of 5 and 10 mM sodium salicylate, e€erents stimulation produced smaller increases in CM amplitude. In agreement with previous studies perfusion of scala tympani with 100 mg of d-tubocurarine completely suppressed the e€ects of electrical stimulation of e€erents on CM. Perfusion of a mixture of d-tubocurarine and 5 mM salicylate still resulted in an increase of CM suggesting that the e€ects of salicylate on CM are independent from that of the e€erents. Cazals et al. (1998) used acoustic stimulation of the contralateral ear as one means of assessing cochlear e€erent functioning. They had previously shown that the spontaneous neurophysiological activity at the round window in silent conditions can be decreased when a contralateral broad band noise is presented (Cazals and Huang, 1996). Over 3 weeks of sodium salicylate treatment at the dose of 200 mg/kg twice a day on the guinea pigs did not show CAP-threshold changes and no change was observed in the e€ects of a contralateral noise. The e€ects of cochlear e€erents can be a€ected by salicylate treatment, but this seems to be a possible secondary e€ect of alterations in the CM and/or eighth nerve responses induced by salicylate and from which measures of e€erents activity were inferred. 3.3.3. Cochlear autonomous innervation and blood supply The cochlear blood supply has two anatomical com-

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ponents, one comprises the stria vascularis along the external wall of the scala media and the other is constituted of modiolar vessels which run in the central axis of the cochlea, the modiolus, and reach the hair cells area. As on other organs alterations of blood supply can have drastically deleterious e€ects on the cochlear functioning. The cochlea has an autonomous innervation which is restricted to the modiolus and which appears to be noradrenergic. The possible involvement of discrete alterations in cochlear blood supply by salicylate has been examined in a few studies. Cazals et al. (1988) tested the e€ects of combining salicylate and vasoactive drugs on guinea pigs. They used three vasodilators Ð a sympathetic adrenolytic, a sympathetic antagonist of serotonin and a dopaminergic agent, and a strong sympathomimetic vasoconstrictor. Guinea pigs were injected lysine salicylate intramuscularly at the dose of 500 mg/kg. One and a half hour later di€erent experimental groups received an intraperitoneal injection of the di€erent vasoactive agents. Measures of eighth nerve CAP indicated that the threshold elevation induced by salicylate was further increased by the vasoconstrictor and decreased by the adrenenolytic vasodilator, whereas the other drugs had no e€ect. These e€ects of noradrenergic-related drugs is in line with an action upon the modiolar vessels' tone control. Morphometric analysis of diameters of the strial and modiolar vessels were performed on animals so treated and the only signi®cant result was a constriction of modiolar vessels under hair cells in guinea pigs treated with lysine salicylate and the vasoconstrictor. Jung et al. (1990) administered an intraperitoneal injection of sodium salicylate at 300 mg/kg followed 2 h later by an injection at 200 mg/kg to chinchillas. They measured auditory brainstem responses and perilymph levels of various catecholamines. Auditory brainstem responses revealed a hearing loss and perilymph analyses with high performance liquid chromatography indicated increased levels of norepinephrine, epinephrine, dopamine, and the two metabolites 5hydroxyindole-3-acetic acid and homovanilic acid. An additional study from the same group (Fratianni et al., 1990) used the same techniques to examine the e€ects of two adrenergic blockers (an alpha antagonist Ð phenoxybenzamine, and a beta antagonist Ð propranolol) on salicylate ototoxicity. They reported a partial prevention of the salicylate-induced hearing loss with both adrenergic blockers, while perilymph levels of catecholamines were normal. Didier et al. (1993) measured cochlear blood ¯ow using the laser Doppler ¯owmetry technique together with eighth nerve CAP in guinea pigs given one intramuscular injection of sodium salicylate at doses of 100±300 mg/kg. At 100 mg/kg they observed a decrease in cochlear blood ¯ow of about 10% starting

within 30 min post-injection. No CAP-threshold elevation was observed. For the two higher doses cochlear blood ¯ow was reduced by about 20% and CAP thresholds were elevated at high frequencies, above 4 kHz, by approximately up to 10 and 20 dB, respectively, or 200 and 300 mg/kg. Application of sodium salicylate on the round window at concentrations of 100 mM produced no change in cochlear blood ¯ow or CAP. Concentrations of 200 and 300 mM induced CAP-threshold elevation of about 15 dB restricted to the highest frequencies whereas blood ¯ow was reduced by about 15%. Application of the vasodilating drug hydralazine onto the round window produced a 100% increase in cochlear blood ¯ow with no change in CAP threshold. When hydralazine was combined with salicylate cochlear blood ¯ow was increased by about 20%. However, CAP thresholds were overall approximately as elevated as when salicylate was given alone. Thresholds were measured in response to 2±32 kHz tone bursts and a slight reduction in threshold elevation was observed only for the two frequencies of 8 and 16 kHz. Jung et al. (1995) measured cochlear blood ¯ow on chinchillas after systemic or topic administration of sodium salicylate. They observed a decrease in cochlear blood ¯ow of 20±35%, respectively, 1 and 2 h after application onto the round window of 150 mg of sodium salicylate. After one intraperitoneal injection of sodium salicylate at the dose of 300 mg/kg they observed decreases in cochlear blood ¯ow of about 10%, 30% and 40% at 2, 4 and 6 h post-injection. From some of their previous studies the authors had observed an increased level of vasoconstricting leukotrienes in the cochlea after salicylate. Pretreatment with a leukotriene inhibitor completely suppressed the decreases in cochlear blood ¯ow induced by salicylate. Didier et al. (1996) tested the e€ects of EGb761, a complex drug with various e€ects including vasodilation through a noradrenergic antagonist property. Morphometric studies of cochlear vessels of guinea pigs showed that EGb761 produced a vasodilation of vessels of the modiolar component but not of the strial component of cochlear vasculature. Treatments for several weeks with EGb761 were applied to guinea pigs before receiving an intramuscular injection of 200 mg/kg sodium salicylate and cochlear blood ¯ow was monitored. EGb761 treated animals showed less cochlear blood ¯ow reduction induced by salicylate during the ®rst hour post-salicylate injection but no di€erence at the next half hour. These data clearly establish that cochlear blood supply is a€ected by administration of sodium salicylate. From the limited available data it is not possible to argue whether this e€ect could be at the origin of other aspects of salicylate auditory toxicity. The studies which associated CAP recordings with alterations

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in cochlear vasculature suggest partly dissociated e€ects. 3.3.4. Spontaneous activity Spontaneous activity in auditory neural structures is a primary concern for understanding the physiological processes underlying salicylate-induced tinnitus. Various electrophysiological techniques were used in studies of salicylate toxicity. Studies have used intracellular recordings with a microelectrode, extracellular near ®eld recordings as in multi-unit activity, or extracellular far ®eld recordings using a gross electrode. In contrast to central auditory structures, the auditory nerve activity is known to be a€ected very little by anesthesia. Recent developments in recordings from the auditory nerve with a gross electrode have provided a novel approach. Evans et al. (1981) and Evans and Borerwe (1982) intravenously injected sodium salicylate at the dose of 400 mg/kg in three divided doses over a period of 10± 20 min in cats. Serum salicylate concentrations were about 600 mg/l 10±25 min post-injection and 340 mg/l or more 16 h post-injection. Experiments were performed on six cats but data from two animals were discarded because their body temperature exceeded normal limits. Spontaneous activity in ®bers from control cats showed a bimodal distribution with about 36% of ®bers having spontaneous discharge rates below 20 spikes/s, and 64% having a spontaneous discharge above that rate. The authors reported on a signi®cant increase of about 1±20 spikes/s on average for ®bers with a high spontaneous rate. These estimates were based on recordings from approximately 200 ®bers. Their data show a tendency for ®bers with the highest characteristic frequency to display the greater increase in spontaneous activity. In control cats, ®bers responses show a tendency for ®bers with a higher spontaneous discharge rate to have more sensitive absolute thresholds. After salicylate, this relationship appears inverted as ®bers with higher characteristic frequencies which showed the most increased spontaneous activity also presented the larger threshold elevation. The interspike interval histograms of spontaneous activity showed an unusual peak at very brief interspike intervals. This histogram in control cats showed a unimodal distribution with a peak at 4±7 ms. After salicylate infusion 19 of 30 ®bers so examined presented an early peak at 0.6±1 ms interspike interval. This had apparently the form of double spikings most of the time and not of many spikes trains. There was no signi®cant di€erence in the absolute refractory period of the ®bers after salicylate infusion. It was found to be 0.67 ms in controls and 0.68 ms in salicyate-treated animals. Only ®bers with a spontaneous rate above 20 spikes/s displayed these very brief spontaneous interspike intervals. Intravenous

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injection of 1.5 mg/kg of lignocaine, a drug which temporarily reduces tinnitus in many patients, was measured on one ®ber. It induced a small nonsigni®cant reduction of spontaneous activity. It has been well established that tinnitus can be suppressed in humans by electrical stimulation of the cochlear round window by positive currents whereas negative currents induce auditory sensations (Cazals et al., 1978). Similarly, the authors found that positive electrical stimulation at the round window could reduce or suppress the spontaneous activity in salicylate treated animals. Jastrebo€ and Sasaki (1986) measured spontaneous activity from single cells of the inferior colliculus in albino guinea pigs before and after one intraperitoneal injection of sodium salicylate at the dose of 450 mg/kg which induced a serum salicylate level of 300±400 mg/ l. Results are based upon recordings from 169 cells before and 89 cells after salicylate prefusion. Before salicylate prefusion the mean spontaneous rate was around 25 spikes/s but after salicylate prefusion it rose to 65 spikes/s. This increase reached a maximum after about 6±8 h post-salicylate injection and then started to drop. In the inferior colliculus cells most responsive to high frequencies are located in the upper part while cells most sensitive to the low frequencies are deeper, so de®ning a tonotopy. After salicylate, cells at all depths had increases of spontaneous activity. Control recordings were taken from the lobulus V of the cerebellum because it is an adjacent structure similarly a€ected by the surgery, and because the cerebellum is known to be very sensitive to anesthesia, and lobulus V does not receive auditory innervation. Spontaneous activity from cells of this structure remained unchanged after salicylate. Schreiner and Snyder (1987) pioneered in analyzing the spontaneous activity of the eighth nerve from a gross electrode on the nerve trunk. They showed that spectral averaging of spontaneous activity revealed the presence of underlying periodic activities. They observed that after cats were given one intravenous injection of sodium salicylate at the dose of 200 mg/kg the average spectrum of the nerve spontaneous activity showed an increase in spectral peak at 200 Hz often accompanied by a decrease of a broader peak at 1 kHz. The salicylate-increased amplitude of the 200 Hz peak was reduced after injection of 6 mg/kg lidocaine, a drug known to temporarily reduce tinnitus in many patients. A further dose of 125 mg/kg of sodium salicylate increased again the 200 Hz peak which returned to normal within approximately the next 10 h. Acoustically evoked compound action potentials simultaneously by recorded showed an increase after salicylate prefusion with little recovery within the last ten hours, thus indicating partly dissociated underlying processes for spontaneous and evoked eighth nerve activity. In an additional experiment the authors indicate

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that positive electrical stimulation at the cochlear round window did suppress the 200 Hz peak increased by salicylate infusion. Stypulkowski (1990) performed single ®ber recordings from the auditory nerve of cats after one intravenous injection of sodium salicylate at the dose of 200 mg/kg. Recordings of spontaneous activity from 98 ®bers before salicylate injection gave an average of about 6 and 53 spikes/s for the two populations of ®bers with a spontaneous discharge rate below and above 18 spikes/s. After salicylate infusion there was no signi®cant increase in mean spontaneous rates, the average values of about 6 and 52 spikes/s being observed for the two populations of ®bers. However, the author observed a few examples of ®bers which displayed increased spontaneous activity by about 20 spikes/s after salicylate infusion. The 200 mg/kg dose employed in this study is notably lower that that used in the above-mentioned experiments from other authors. Kumagai (1992) recorded from single ®bers from the auditory nerve of guinea pigs given an intravenous injection of 200 or 400 mg/kg of sodium salicylate. At 200 mg/kg, the 102 ®bers recorded post-salicylate infusion did not show any change in spontaneous activity, while after the 400 mg/kg dose the rate of the 112 recorded ®bers increased signi®cantly. Martin et al. (1993) used the technique of spectral averaging of the eighth nerve spontaneous activity to study in cats the e€ects of an intravenous injection of 150 mg/kg sodium salicylate. After salicylate infusion an increase in the spectral peak at about 200 Hz was observed which was reduced by a subsequent lidocaine injection at 6 mg/kg. The physiological condition of the animals had certainly deteriorated since acoustically evoked brainstem responses simultaneously recorded showed extremely elevated thresholds up to 100 dB SPL. A large part of this elevation was observed to be the result of surgical cerebellar retraction and nerve stretching. Martin (1994) studied spectral averaging of spontaneous eighth nerve activity from 14 human subjects in the course of cerebello-pontine angle surgery. Seven of the patients had a pre- and post-operative history of continuous tinnitus. A spectral peak at about 200 Hz was found for these seven subjects. This spectral peak was also observed for three other subjects who had tinnitus before but not after surgery and for one subject who had tinnitus only after the surgery. One subject without complaint of tinnitus also showed a 200 Hz peak. Two subjects without tinnitus complaint did not show a 200 Hz peak. Shehata-Dieler et al. (1994) recorded spontaneous activity of eighth nerve ®bers from pigeons after scala media perfusion of sodium salicylate at doses of up to about 18 mM. Data obtained for a few, approximately

14 ®bers, indicated that after salicylate infusion the mean spontaneous discharge rate slightly increased or remained unchanged. Chen and Jastrebo€ (1995) investigated the spontaneous activity of 471 cells of the external nucleus of the inferior colliculus in rats after one intraperitoneal injection of sodium salicylate at 233 mg/kg which induced serum salicylate levels of about 700 mg/l at 4± 6 h post-injection. In these experiments the rat was chosen as the same authors had obtained behavioral evidence of tinnitus in rats. The external nucleus of the inferior colliculus was chosen because the authors had previously shown increased activity in the main nucleus and wanted to test the hypothesis of involvement of extra-lemniscal auditory pathways. The average spontaneous rate before salicylate infusion was found to be about 5±6 spikes/s. At one and half hour post-salicylate injection this value remained unchanged, then it rose to about 10±11 spikes/s at 2.5 and 3.5 h post-injection and it went back to 5 spikes/s at 4.5 h post-injection. The interval histograms of di€erent units were further analyzed and related to whether the units had an acoustic receptor ®eld, or tuning, with a sharp frequency response or not. Units which had a sharp acoustic tuning revealed after salicylate infusion an increase of around 5% in the proportion of intervals below 2 ms and an increase of around 14% in the range 30±50 ms. Units which did not respond to contralateral stimulation up to 60 dB SPL showed very large increases of around 15±20% in the proportion of interspike intervals below 10 ms and maximal increases of about 36% at around 31 ms. Units responding with inhibition to contralateral sound stimulation presented after salicylate an increase of around 5% in the proportion of interspike intervals below 10 ms and a decrease in the proportion of intervals above about 100 ms. After salicylate infusion units most sensitive to 12±16 kHz tones presented the greatest increases in spontaneous activity, while units most sensitive to frequencies above 16 kHz presented a decrease of spontaneous activity. This is in line with evidence from animal behavior, given by the same research group, for tinnitus having a pitch assessed around 10±11 kHz. Further analysis of data revealed that increased activity took the form of bursts of 4±20 spikes with interspike intervals of less than 3 ms most of the time. The same research group had shown salicylate-induced calcium changes in the perilymph and cerebro-spinal ¯uid and also evidence from behavioral animal testing that such a calcium treatment could attenuate manifestations of tinnitus (see Penner and Jastrebo€, 1996). Consequently calcium supplement was given to rats by adding 50 mM CaCl2 in the drinking water for three days before salicylate injection. This suppressed all alterations in spontaneous activity induced by salicylate. Examination of correlation

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between pairs of simultaneous recordings was also undertaken. The proportion of units presenting a correlated activity was about 50% both before and after salicylate. Martin et al. (1996) recorded spectral averaging of spontaneous eighth nerve activity from the round window of 10 patients. Three subjects su€ered tinnitus at least part of the time, whereas in two of these subjects a peak at 200 Hz was observed. For the remaining subjects abnormal peaks at di€erent frequencies were observed. Four subjects with tinnitus did not present the spectral peak. Three subjects had no tinnitus and showed no spectral peak about 200 Hz. Ochi and Eggermont (1996) measured extracellular multi units recordings using two microelectrodes simultaneously exploring the primary auditory cortex of cats before, during and up to 6 h after one intraperitoneal injection of sodium salicylate at the dose of 200 mg/kg. Because of the large range of spontaneous activity of cells at the auditory cortex the authors set to record from the same cells before and after salicylate administration. The authors succeeded in recording continuously from 21 cells before and up to 6 h post-salicylate injection. At about 2±3 h post-salicylate injection, 9 out of 12 cells with a high ®ring rate greater than 1 spike/s presented a decrease of spontaneous activity, whereas seven of the nine units with a low ®ring rate, lesser than 1 spike/s presented with an increase in spontaneous activity. Interspike interval histograms did not indicate signi®cant changes. The ®rst autocorrelation peak of each unit spontaneous activity was the most salient periodicity in spike intervals. A small but signi®cant shift towards lower periodicities was observed after salicylate infusion. Correlations between simultaneous recordings from one or both electrodes were analyzed. The obtained correlograms revealed a strong peak centered around 0 ms with a width of approximately 200 ms, indicating a very high probability of observing a ®ring of a second unit at 2100 ms of the ®ring of a ®rst unit. After salicylate infusion the width of these peaks narrowed signi®cantly indicating a briefer time-dependence, or increased synchrony between spontaneous ®rings from two units. The number of spikes in burst discharges and the number of isolated ®rings were found unchanged. Acoustically evoked ®rings of units in response to click trains of varying periodicities were used to assess capacities to synchronize the various sound rhythms; analyses revealed after salicylate infusion a decrease in the best and maximum modulation frequencies indicating a reduced ability to synchronize at rapid rhythms. Kenmochi and Eggermont (1997) provided additional data on spontaneous and sound evoked

619

rhythms from the local ®eld potential and for 251 single units at the primary auditory cortex of cats before and after one intraperitoneal injection of sodium salicylate at the dose of 200 mg/kg. Periodicities in the spontaneous activity measured with the autocorrelation of the local ®eld potential indicated an average value of 8.7 Hz which after salicylate infusion decreased to 7.6 Hz. Recordings from 251 units responses to various click trains indicated a decrease from 10.3 to 8.9 Hz in best modulation frequency, or acoustic frequency producing the highest ®ring synchrony. Manabe et al. (1998) performed extracellular recordings in the inferior colliculus of guinea pigs before and after an intravenous injection of 200 mg/kg sodium salicylate. Spontaneous activity was on average around 5 spikes/s before salicylate. It reached a maximum of about 20 spikes/s at two 2 h post-injection and then progressively decreased to pre-salicylate value within the next 8 h. Serum salicylate levels were found to decrease exponentially from about 450 mg/l a few minutes after injection to 100 mg/l at 10 h post-injection. This salicylate-induced increase in spontaneous activity could be inhibited by an intravenous injection of lidocaine, a drug known to temporarily relieve tinnitus in many patients. Some neurons showed a limited inhibition whereas others showed a complete inhibition for more than 30 min. Eggermont and Kenmochi (1998) analyzed local ®eld potentials and single unit activity at the primary and secondary auditory cortices and at the anterior auditory ®eld in cats before and after an intraperitoneal injection of sodium salicylate at 200 mg/kg. Spontaneous ®ring rate from multi units recordings revealed no signi®cant change at the primary auditory cortex, a very slight increase at the secondary auditory cortex and, a strong decrease from 4.5 to 1.5 Hz at the anterior auditory ®eld. Responses of a unit to di€erent sounds de®ne a best sensitivity to an acoustic frequency called its characteristic frequency. More detailed analysis indicated that at the primary auditory cortex units with characteristic frequency above 10 kHz showed a decrease in spontaneous rates whereas no signi®cant change was observed for units with a characteristic frequency below 10 kHz. At the secondary and anterior auditory areas units with a high characteristic frequency showed increased spontaneous ®ring rates, whereas other units showed a decrease of spontaneous rates. There was no signi®cant correlation between the amount of change in spontaneous rate and the loss of absolute sensitivity. Cazals et al. (1998) recorded the average spectrum of spontaneous eighth nerve activity from the round window of guinea pigs. Because tinnitus is known to develop over days of salicylate treatment in humans and animals, a salicylate treatment of several weeks

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was performed. Recordings were taken from chronically implanted animals thus permitting easy monitoring and avoiding interfering e€ects of anesthesia on average spectrum shown by Cazals and Huang (1996). Intramuscular doses of 200 mg/kg sodium salicylate were delivered twice a day during 2 or 3 weeks. No CAP-threshold changes were observed. In guinea pigs, as shown previously, the average spectrum of spontaneous activity of the eighth nerve does not show a peak at 200 Hz but only the broad peak at about 1 kHz. This spectral peak was found to be acutely decreased during a few hours post-salicylate injection which could show habituation in some animals. In contrast over days of treatment the 1-kHz peak increased progressively and returned to normal within one to several weeks after cessation of treatment. Similar increases in the 1-kHz peak amplitude could be produced by delivering a white noise at about 55 dB SPL. This noise induced a CAP-threshold elevation of about 15 dB mostly at high frequencies, 8±20 kHz, in control animals. In salicylate-treated animals a similar noise produced a greater CAP-threshold increase of up to 25 dB as if it added to a preexisting internal noise. The reduction of the 1-kHz peak by ipsilateral pure tones of 16 and 20 kHz was found diminished by salicylate. In contrast, the reduction of the 1-kHz peak by a contralateral noise, mediated by cochlear e€erents, remained unchanged over the whole experiment. All these data provide clear evidences for salicylateinduced alterations of the spontaneous neural activity in auditory structures. At the auditory nerve, recordings from single ®bers showed increased spontaneous activity which was dose dependent, a€ected most the ®bers with high characteristic frequencies, and was reversible within several hours. An increase of eighth nerve spontaneous activity is a feature unique to salicylate, and somewhat also to quinine, another tinnitusinducing drug, whereas other ototoxic drugs, acoustic trauma or aging produce decreases of spontaneous actitvity of the auditory nerve. Recordings from the auditory nerve with a gross electrode in animals showed simultaneous increases and decreases of di€erent spectral peaks after salicylate infusion. The spectral peaks represent underlying periodicities in the auditory nerve spontaneous activity. In cats and rabbits one injection of salicylate produced within hours the emergence of a sometimes preexisting peak at about 200 Hz which was most often accompanied by a decrease of the broad spectral peak around 1-kHz. In guinea pigs the 200 Hz peak was never observed but the decrease of the 1-kHz peak also occurs within hours after one injection of salicylate. In guinea pigs, long-term salicylate treatment produced a progressive increase of the 1 kHz peak. At the brainstem level recordings from the central nucleus of the inferior colliculus showed an increase in

spontaneous activity during hours of one injection of salicylate. This increase a€ected all cells regardless of their high or low frequency tonotopy. Recordings from the external nucleus of the inferior colliculus also showed an overall increase in spontaneous activity but associated mostly with cells having a characteristic frequency in the 12±16 kHz range whereas cells with higher characteristic frequencies showed a decrease of spontaneous activity. At the primary auditory cortex cells presented an increase or a decrease of spontaneous activity during hours after an injection of salicylate. Spontaneous ®rings between pairs of cells showed an increased synchrony associated with a signi®cant decrease of their own spontaneous periodicities. These cells were also less able to synchronously follow high acoustic rates. At the secondary auditory cortex a small increase of spontaneous activity was observed while at the anterior auditory ®eld a clear increase was observed. Increase in spontaneous activity was associated to ®bers with a high characteristic frequency. The data provide evidence of changes from the auditory nerve to the auditory cortices. Involvement of auditory cortices is essential if data are to be considered tinnitus-linked since tinnitus is a conscious auditory sensation. There is no clear evidence of salicylate acting speci®cally on central auditory structures. It appears that the e€ects observed at the auditory nerve could be at the base of alterations observed at the higher auditory centers. The changes at all levels seem to be somewhat dissociated from threshold elevations of acoustically evoked responses. Whether the observed changes in spontaneous activity are indeed related to a tinnitus perception cannot be de®nitely stated. However, all arguments seem to be converging in support of this hypothesis as they all essentially re¯ect the characteristic features of salicylate- induced tinnitus. The changes can occur before alterations in threshold elevations are observed in a dose-dependent manner. It is well documented, as indicated above from human data, that a single injection of a very high dose of salicylate can induce tinnitus so that the majority of studies which used only one salicylate injection at a high dose may be representative of a tinnitus condition. Data from studies on long-term salicylate treatment present remarkable similarities in delay of occurrence, development and reversibility with the salicylate-induced tinnitus reported by humans or inferred from animal behavior. The high frequency pitch most often reported by humans seem to be re¯ected in the speci®cally greatest changes of spontaneous activity observed for units preferentially responding to high frequency sounds. The only two reliable ways of suppressing tinnitus in many patients are electrical stimulation of the cochlea and administration of lidocaine. Experimental data provide evi-

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dence that both indeed could suppress the salicylateinduced alterations of spontaneous activity. Quinine at high dose can induce tinnitus in humans. Jastrebo€ et al. (1991) provided behavioral evidence of quinineinduced tinnitus in rats. Mulheran (1999) showed an increased proportion of ®bers with high spontaneous activity together with a decrease of absolute refractory period, after an intravenous injection of quinine in guinea pigs. At the auditory cortex, Kenmochi and Eggermont (1997), Ochi and Eggermont (1998) and Eggermont and Kenmochi (1998) observed quite similar alterations induced by salicylate and quinine. All these similar observations on quinine and salicylate support the idea of such alterations being related to tinnitus. 3.4. Biochemical processes 3.4.1. Pharmacokinetics in serum and cochlea Needs and Brooks (1985) and Miners (1989) present reviews of general pharmacokinetics of the salicylates. After oral ingestion, aspirin is rapidly absorbed in the stomach and its concentration rises quickly in the plasma to reach a peak within about 30 min. Aspirin is rapidly hydrolyzed into salicylic acid by nonspeci®c esterases found in many body tissues. Thus aspirin concentration decreases rapidly as salicylic acid concentration increases. Salicylic acid binds extensively to plasma proteins primarily albumin, at therapeutic concentrations of 180±350 mg/l salicylic acid is in molar excess to albumin and 80±90% of salicylic acid is bound to plasma proteins. As plasma concentration increases, such as in many instances of salicylate ototoxicity, the unbound fraction increases. Salicylates are distributed extensively through body ¯uids including the cerebrospinal ¯uid. Salicylic acid is removed from the body by renal elimination and in parallel by four metabolic pathways leading to the four metabolites Ð salicyluric acid, salicyl phenolic glucuronide, salicyl acid glucuronide and gentisic acid. Day et al. (1989) undertook a study on the relation between ototoxicity and plasma concentration of salicylate on humans (eight male normal volunteers). Total and unbound plasma levels were measured for aspirin treatments of 1.95±5.85 g/day for a week. Hearing loss was measured with classical subjective audiometry from 250 to 8000 Hz, and tinnitus subjective features were assessed by pitch and loudness matching to an external sound and pointing to a ®xed interval scale. Plasma samples were taken on the last day of treatment. Two plasma samples were taken per subject the results of which did not di€er by more than about 10%. Unbound salicylate increased from 4 to 10% of total plasma salicylate from the weakest to the strongest dose treatment. There was a linear relation between unbound salicylate and hearing loss.

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With total salicylate a less linear relation was found as very similar results were observed for concentrations below 150 mg/l. Tinnitus loudness showed a rather linear relation with total salicylate whereas relation with some leveling for concentrations above about 30 mg/l. These data clearly established a relation between ototoxic symptoms and total salicylate plasma concentrations below 250 mg/l. Salicylate concentration in the ear has been measured in a few studies. Silverstein et al. (1967) collected cerebrospinal ¯uid and perilymph from cats approximately 6 h after they had been given an intraperitoneal injection of 350 mg/kg of sodium salicylate. The authors found an average salicylate concentration of 690 mg/l in the serum, 310 mg/l in the cerebrospinal ¯uid and 250 mg/l in the perilymph. Juhn et al. (1985) measured serum, cerebrospinal ¯uid, and perilymph salicylate concentrations in chinchillas after an intraperitoneal injection of sodium salicylate at the dose of 300 mg/kg. They found that serum salicylate level peaked at about 650 mg/l after 0.5±1 h post-injection, whereas the cerebrospinal ¯uid concentration peaked at 150 mg/l at 1 h post-injection and perilymph peaked at about 100 mg/l at 2 h postinjection. All levels decreased moderately to the fourth hour post-injection when the last measure was taken. Jastrebo€ et al. (1986) investigated the time course of salicylate concentrations in serum, cerebrospinal ¯uid and perilymph in albino rats and albino guinea pigs over eight hours after an intraperitoneal injection of sodium salicylate at 460 mg/kg. Serum levels reached a peak of about 700 mg/l within one and half hour and slowly decreased to 500 mg/l in the next 6 h. Cerebrospinal ¯uid and perilymph concentrations showed very similar values and time courses. They rose to approximately 200±300 mg/l within 1 h and remained at this level for the next 3 h, and then decreased slowly to 150±200 mg/l in the next 4 h. The perilymph salicylate concentration appears to be about 36% of that in serum. In guinea pigs serum levels rose to about 600 mg/l within 2 h and further to 700 mg/l in the next 2 h, then slowly decreased to 600 mg/l in the next 4 h. The cerebrospinal ¯uid levels rose regularly to about 200 mg/l (33% of that in serum) in 5 h and declined slightly in the next 2 hours. For both rats and guinea pigs concentration values varied between animals but all three were strongly correlated together. In an additional study, Jastrebo€ et al. (1988a, 1988b) investigated the e€ects of pigmentation, anesthesia, daily treatment and behavioral activity upon salicylate concentrations in serum, cerebrospinal ¯uid and perilymph in rats. They measured salicylate concentrations for 6 h following an intraperitoneal injection of sodium salicylate at doses of 350 and 460 mg/kg. The pigmented animals showed signi®cantly

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higher levels of serum salicylate whereas perilymph salicylate concentration was not signi®cantly higher. Salicylate concentration in perilymph was about 35% of that in serum. Compared to anesthetized animals, the awake animals showed a steeper rise of serum salicylate concentration and a steeper decrease. Daily injections of 350 mg/kg for 5 days resulted in a moderate increase in serum salicylate levels from the ®rst to the second day from about 600 to 640 mg/l followed by a small decrease over the next three days with concentration being about 600 mg/l at the ®fth day. Behavioral conditioning of the animals as used for tinnitus assessments did not signi®cantly a€ect the salicylate concentrations. Boettcher et al. (1990) measured salicylate concentrations in serum and perilymph of chinchillas after an intraperitoneal injection of sodium salicylate at the dose of 150, 300 and 450 mg/kg. After a dose of 450 mg/kg serum salicylate concentrations reached a peak of about 520 mg/l at 2±4 h post-injection, then decreased linearly and clearance appeared complete at 16 h post-injection. Perilymph salicylate concentration was measured together with serum salicylate concentration at 4 h after an injection at doses of 150, 300 and 450 mg/kg. A close correlation was found, at high levels perilymph concentration was about 33% of serum concentration whereas at low levels the ratio was about 25%. Only one study by Ishii et al. (1967) investigated the distribution of tritium labeled salicylic acid in the cochlea at di€erent times (0.25, 1, 6 and 13 h) after an intravenous or intraperitoneal injection in guinea pigs. Fifteen minutes after intravenous injection radioactivity was found in the vessels of the stria vascularis and spiral ligament. After 1 h radioactivity was detected in the capillaries of the stria and spiral ligament, in cells of the stria, over the whole organ of Corti and in the spiral ganglion. After 6 h little radioactivity was found and none was detected after 13 h. Overall these studies, together with more partial results from other studies, indicate that the time courses of salicylate concentration are roughly similar in the serum, cerebrospinal ¯uid, and cochlear perilymph. Serum concentrations as a function of administered dose appear rather similar in man and other animals although species di€erences can be observed. The salicylate concentration in cochlear perilymph can be estimated to be approximately 30% of that measured in the serum. In long-term daily administration, salicylate levels appear to stabilize without showing continuous increase. Within the cochlea salicylate seems to enter quickly through blood supply, then to be distributed homogeneously to all cochlear parts and not to be accumulated or retained in any particular cochlear area.

3.4.2. Biochemical alterations in the cochlea by salicylate The e€ects of aspirin and other salicylates cover a wide range such as Ð analgesic antipyretic and anti-in¯ammatory properties Ð prevention of heart attacks and cerebral thrombosis Ð prevention of colorectal cancer. They also produce undesirable side-e€ects such as Ð gastrointestinal ulceration, inhibition of platelet aggregation, inhibition of uterine motility, ototoxicity, inhibition of neutrophils, and allergic reactions. Biochemical processes underlying these e€ects are still matters of research and numerous metabolic interferences by salicylate have been identi®ed (Abramson and Weissmann, 1989; Levy, 1997; Vane and Botting, 1998) such as: inhibition of prostaglandins synthesis, inhibition of NADPH oxydase, of phospholipase C, of cholesterol ester synthase, of ATPase, of antigen±antibody interactions, insertion into membranes and interference with ion transport, uncoupling of oxydative phosphorylation, hyperglycemia, activation of heat shock transcription factor and, inhibition of free radicals. Concerning salicylate ototoxicity very few of these processes have been a subject of investigation and the presentation given here below is limited to studies which provided experimental data while various purely speculative considerations were discarded. 3.4.2.1. Interference with oxydative phosphorylation. Silverstein et al. (1967) analyzed cerebrospinal ¯uid, perilymph and endolymph from cats after an intraperitoneal injection of 350 mg/kg sodium salicylate. Malic dehydrogenase, an enzyme known to be inhibited by salicylate in liver tissue and participating in oxydative phosphorylation, was found signi®cantly decreased in endolymph and perilymph. Glucose levels were signi®cantly elevated in serum, perilymph and endolymph. Simultaneous measures of sodium potassium and total protein concentration appeared una€ected by salicylate. These results suggest that energetic metabolism was a€ected by salicylate. Upon the hypothesis that acetyl salicylate inhibits ATP production, Tanaka and Brown (1970) perfused the cochlea of guinea pigs with acetyl salicylate at a concentration of 0.7 mM. They observed no signi®cant e€ect upon the cochlear microphonic potential, whereas other drugs inhibiting ATP production did have an e€ect. Krzanowski and Matschinsky (1971) examined ATP and P-creatine levels in the cochlea of control guinea pigs and after an intraperitoneal injection of 400 mg/ kg salicylate. They took measures from the organ of Corti, the stria vascularis, Reisssner's membrane, the cochlear nerve, the spiral ganglion and cochlear blood vessels. Their results in control guinea pigs indicated that ATP had the highest concentration in the cochlea and was evenly distributed from base to apex, whereas P-creatine showed a fourfold decreasing gradient from

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apex to base. Salicylate-treated animals showed ATP levels in Reissner's membrane lowered (by 40%) and both ATP and P-creatine levels slightly increased (by 10%) in stria vascularis and in the cochlear nerve. Thalmann et al. (1973) examined ATP and P-creatine levels in the same cochlear areas after an injection at a slightly lower dosage (350 mg/kg), but did not ®nd any signi®cant change. Krzanowski and Matschinsky (1975) further investigated ATP and P-creatine in di€erent cochlear areas of guinea pigs after an intraperitoneal injection of 100 or 600 mg/kg sodium salicylate or after ®ve consecutive doses at 12 h intervals of 400 mg/kg sodium salicylate. Doses of 100 mg/kg, 600 mg/kg and repetitive 400 mg/kg resulted in average serum salicylate levels, respectively, of 110, 675 and 502 mg/l. After the dose of 100 mg/kg ATP was found increased in the stria vascularis, the cochlear nerve, the spiral ganglion, the modiolar blood vessels and in the organ of Corti mostly towards the apex of the cochlea. P-creatine levels were found increased in the cochlear nerve and in modiolar blood vessels. After the high dose of 600 mg/kg ATP levels were slightly elevated in the cochlear nerve, and in the organ of Corti mostly at the cochlear apex, P-creatine levels showed increases in the stria vacularis and in the Reissner's membrane. The authors acknowledge that these results contradict those of their previous study in which they assume sample contaminations might have occurred. After repetitive salicylate doses, ATP and P-creatine levels were found decreased in all cochlear areas except the modiolar blood vessels; however, the signi®cance of these results is questioned by the authors since a third of the treated animals did not survive the treatment. So while their results do not support the view that inhibition of oxydative phosphorylation could be the mechanism of salicylate ototoxicity, the authors point to the possible role of prostaglandins and to a speci®c action at the neural level. Overall these studies do not provide clear evidence for an interference with oxidative phosphorylation as being a possible mechanism of salicylate ototoxicity. 3.4.2.2. Inhibition of prostaglandins synthesis. After the demonstration by Vane (1971) that aspirin, sodium salicylate and indomethacin inhibit prostaglandin synthesis, this mechanism of action has been considered as a possible basis of salicylate ototoxicity (Krzanowski and Matschinsky, 1975; Hawkins, 1976). Jung and Juhn (1984) measured levels of two prostaglandins, PGE2 and 6-Keto-PGF1a in the perilymph and cerebrospinal ¯uid of chinchillas by radioimmunoassay. In control animals they observed much higher levels of these prostaglandins in the perilymph than in the cerebrospinal ¯uid. After administration of aspirin or indomethacin, prostaglandin levels were found

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decreased by two to three times in the perilymph but unchanged in the cerebrospinal ¯uid. Escoubet et al. (1985) using radiolabeled arachidonic acid, the membrane constituent from which prostaglandins are synthetized, found that the stria vascularis and the spiral ligament of guinea pigs produced PGI2, PGF2a and PGE2. PGI2, PGF2a were also found in the perilymph. Prostaglandin levels were measured at 30 min, 3 h and 3 days after an intraperitoneal injection of 350 mg/kg aspirin. At 30 min and 3 h post-aspirin injection 6KPGF1a, PGF2a and PGE2 levels were found decreased whereas at 3 days post-aspirin injection levels were back to normal. Similar e€ects were observed after treatment with the ototoxic antibiotic gentamicin. The action of these prostaglandins in the cochlea is not known but it can be assumed that they may participate in the control of blood supply since it is known from studies on other organs that PGI1 and PGE2 have vasodilating properties whereas PGF2a is vasoconstrictor. Puel et al. (1990) showed, as described above, that salicylate perfusion in the cochlea of guinea pigs reduced CM and CAP evoked at low sound intensities. In the same experiments they performed cochlear perfusion of two prostaglandin synthesis inhibitors, mefenamate and meclofenamate, which had no e€ect on cochlear potentials. Jung et al. (1992a) applied to the round window of the cochlea of chinchillas sodium salicylate or indomethacin. By measuring brainstem evoked responses they observed a hearing loss of 20±50 dB after 1±2 h and complete recovery after 8 h. In the same experiments they measured in the perilymph the two arachidonic acid metabolites prostaglandins and leukotrienes by high performance liquid chromatography and radioimmunoassay. Arachidonic acid is competitively metabolized by cyclooxygenase to prostaglandins and tromboxanes and by lipooxygenase to leukotrienes. After sodium salicylate application, over the same time course as the reversible hearing loss they found a reversible decrease of prostaglandin 6KPGF1a and of tromboxane B2, with an increase of leukotrienes B4 and C4. Indomethacin resulted in lower levels of prostaglandin 6KPGF1a but with an increase of tromboxane B2, PGF2a, PGD2 and PGA2. In additional experiments, Jung et al. (1992b) applied various cyclooxygenase products or lipooxygenase products onto the round window of chinchillas and tested hearing with auditory brainstem responses and collected perilymph for analysis. They observed that PG application produced little or no hearing loss, application of tromboxane B2 produced up to 18 dB losses, application of leukotriene B4, C4 and of 15HETE, another lipooxygenase product, resulted in hearing losses of 20±40 dB at 1±2 h post-application.

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Perilymph variations in levels of prostaglandins, tromboxane and leukotriene were quite variable as a function of drug applied and time elapsed since application. There was a trend for prostaglandins or tromboxane application to produce some increase in prostaglandins or tromboxane levels and a clearer increase in leukotriene levels. Application of leukotrienes resulted in some decrease of perilymph prostaglandin levels and increase in leukotriene levels together with a decrease in 15-HETE levels. 15-HETE application produced little or no change in prostagladin or thromboxane levels and increases in leukotriene and 15-HETE levels. Fitzgerald et al. (1993) showed, as described above, that intracochlear perfusion of salicylate and bromosalicylate resulted in CAP-threshold losses, increase in CM amplitude and reduction in acoustic otoemissions. Perfusion with indomethacin did not produce similar e€ects unless at doses about 50 times greater than that required to inhibit prostaglandin synthesis in cochlear tissues. Perfusion with acetyl salicylate, a more potent prostaglandin synthesis inhibitor than salicylate, did not a€ect cochlear responses more than salicylate. Park et al. (1994) reasoning that corticosteroids inhibit the production of arachidonic acid by phospholipase A2 from cells membrane phospholipids, and thus block prostaglandins, thromboxanes and leukotrienes synthesis, investigated the e€ect of a corticosteroid treatment upon salicylate ototoxicity. Dexamethasone was administered either intraperitoneally or locally at the round window and two intraperitoneal injections of sodium salicylate, one at 300 mg/ kg and 2 h later, another at 200 mg/kg were given to chinchillas. Auditory brainstem responses and perilymph analysis with high performance liquid chromatography were performed. Corticosteroid treatment was found to reduce salicylate-induced hearing losses from 30 dB to 20±15 dB. Dexamethasone applied locally reduced prostaglandin 6KPGF1a level and applied systematically, it resulted in a small reduction of reduced prostaglandin 6KPGF1a. Levels of leukotrienes B4 and C4 were small in control animals and were found undetectable after systemic or local dexamethasone administration. Sodium salicylate treatment resulted in a strong decrease of prostaglandin 6KPGF1a level, a small increase in leukotriene B4 and a large increase in leukotriene C4 levels. Combinations of dexamethasone and sodium salicylate treatments produced a decrease in prostaglandin 6KPGF1a level and a decrease in B4 but not C4 leukotrienes. Overall the changes in perilymph concentrations of 6KPGF1a prostaglandin or B4 and C4 leukotrienes do not show a clear relation with auditory threshold elevations. Jung et al. (1995) measured the e€ect of a leukotriene inhibitor upon salicylate-induced changes in cochlear blood ¯ow in chinchillas. The leukotriene in-

hibitor was given orally and sodium salicylate was administered either intraperitoneally or locally at the round window. Sodium salicylate alone produced a reduction of cochlear blood ¯ow of 30±40% and the leukotriene inhibitor pretreatment prevented the salicylate-induced changes in cochlear blood ¯ow. Arruda et al. (1996) measured otoacoustic emissions after round window application of sodium salicylate or leukotriene C4 combined with pre-treatment with a leukotriene inhibitor. Both salicylate and leukotriene C4 application resulted in decreases of click-evoked otoacoustic emissions, which were prevented by the leukotriene inhibitor pre-treatment. The data from all these experiments are too partial and contradictory to allow any clear conclusion as to the involvement of prostaglandin inhibition in the ototoxicity of salicylate. It is clear that various NSAIDs inhibit prostaglandin synthesis; however, for each drug the dose required may vary greatly as a function of in vitro or in vivo experimental conditions and also according to various tissues or cells (Vane et al., 1998). Information available concerning cochlear tissues is too meagre to permit any ®rm conclusion. 3.4.2.3. Catecholamines and blood ¯ow. The sympathetic innervation in the cochlea was shown to be adrenergic (Spoendlin and Lichtensteiger, 1966; Eybalin et al., 1983) and to in¯uence cochlear blood ¯ow (Ohlsen et al., 1991; Laurikainen et al., 1994). The studies mentioned above in the section on cochlear autonomous innervation and blood supply provide demonstration of a vascular component in salicylate otoxicity certainly in¯uenced by catecholamines. However, data do not permit estimation whether it could be an essential or a subsidiary mechanism in salicylate otoxicity. 3.4.2.4. Increased salicylate ototoxicity by zinc and magnesium de®ciencies. Gunther et al. (1988) reported that rats de®cient in zinc showed a slightly greater increase in salicylate-induced hearing loss compared to control rats. Rats were fed with a zinc de®cient diet for 1 month and then they were administered an oral dose of 700 mg/kg of sodium salicylate. One day later, auditory brainstem responses were measured from scalp electrodes in response to 10 and 20 kHz. The animals were fed on a normal diet and a week later similar measures were taken again. The zinc-de®cient diet was found to have no e€ect on hearing thresholds. Salicylate alone produced a threshold elevation of 50± 55 dB on average, while its combination with the zincde®cient diet resulted in a threshold elevation of 60±62 dB on average. A week later thresholds were back to normal for all animals. Gunther et al. (1989a) observed similar results on rats fed for one month with a magnesium de®cient diet. For normally fed rats salicylate increased thresholds by 10±20 dB, while for rats fed

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with the magnesium de®cient diet salicylate administration produced threshold increases of about 30±40 dB. The magnesium de®cient diet itself produced a threshold elevation limited to 3±6 dB. After a week of normal feeding thresholds were back to normal. Gunther et al. (1989b) showed that threshold elevation induced by an oral dose of 700 mg/kg sodium salicylate could be completely prevented by a simultaneous subcutaneous injection of zinc at the dose of 6 mg/kg. Salicylate induced threshold elevations of about 10±20 dB on average. Simultaneous subcutaneous injection of MgCl2 at 1.5 mM/kg had no e€ect. Simultaneous subcutaneous injection of desferioxamine, an iron chelator, at 100 mg/kg reduced threshold loss by about 5 dB, suggesting no involvement of iron accumulation in salicylate ototoxicity. Various biochemical processes can be a€ected by zinc and magnesium and the data do not provide indication as to the involvment of any particular one. 3.4.2.5. Calcium involvement. In a series of experiments Jastrebo€ and colleagues investigated the involvement of calcium changes in salicylate ototoxicity. An overall view is given in Jastrebo€ and Sasaki (1994) and in Penner and Jastrebo€ (1996). A series of behavioral experiments were performed measuring the e€ects of nimodipine, a calcium channel antagonist, or of a calcium supplementing diet by giving 50 mM CaCl2 in drinking water. These were combined with daily salicylate administration. Both the calcium supplementing diet and nimodipine attenuated the behavioral signs of salicylate-induced tinnitus. Similar experiments were also conducted using, instead of salicylate, quinine, another drug known to induce tinnitus, and similar attenuation e€ects were observed. In addition a series of experiments was conducted using the same drug treatments as in behavioral experiments but measuring intracochlear free perilymphatic calcium with a calcium sensitive microelectrode introduced in the round window. Data showed that salicylate treatment caused a dose-dependent decrease in free perilymphatic calcium. The calcium supplement did not change the calcium level before salicylate treatment but attenuated its salicylate-induced decrease. In their study on spontaneous activity of single cells in the inferior colliculus (Chen and Jastrebo€, 1995), a calcium supplemented diet suppressed the salicylate-induced increase in spontaneous activity. Kay and Davies (1993) reported that an intraperitoneal injection of sodium salicylate at the dose of 300 mg/kg to rats induced a threshold elevation of 30 dB measured from scalp electrodes and brainstem responses evoked by a broad band click. A subcutaneous injection of nimodipine, a calcium channel antagonist, simultaneous with that of salicylate did not modify the threshold elevation but was

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found to very slightly reduce salycilate-induced latency shifts in brainstem waves. These reports suggest an involvement of calcium in salicylate ototoxicity and rather speci®cally an involvement in tinnitus related processes. Calcium movements and concentration changes are associated with many cellular activities whether in normal or pathological conditions and present data do not indicate in which cochlear tissue or cells calcium alterations occur and which metabolism is a€ected. 3.4.2.6. Interactions with other ototoxic drugs. Rybak and colleagues performed a series of studies on the combined e€ects of salicylate and furosemide. Furosemide is a diuretic drug known to be ototoxic a€ecting specially the stria vascularis where it induces shrinkage and extracellular edema associated with a drop in endocochlear resting potential (EP). Rybak et al. (1986) and Rybak and Whitworth (1987) showed that an intravenous injection of 25 mg/kg furosemide in chinchillas resulted in a drop of about 70% in EP whereas pretreatment with an intravenous injection of 50 mg/kg sodium salicylate attenuated this drop to about 20%. Rybak et al. (1990) reported that salicylate had no such protective e€ect upon the ototoxicity of another diuretic, ethacrynic acid, which very similarly a€ects the stria vascularis and the EP. Rybak et al. (1992a, 1992b) examined with transmission electron microscopy the stria vascularis after treatment with furosemide, salicylate and the combination of both. They observed that salicylate alone did not induce any pathology and that it did reduce the furosemideinduced edema at 10 min post-injection whereas it increased it at 20 min and had no e€ect at 30 min. The authors experimented on other organic acids than salicylate and found large di€erences in e€ects between di€erent organic acids so that the data give little insight into processes of salicylate ototoxicity. Johnson (1992) showed that acetyl salicylic acid potentiated the hearing loss induced by exposure to toluene in rats. She exposed rats to toluene at 1000 ppm 16 h/day for 10 days. This resulted in threshold elevations of 10±15 dB at high frequencies. Rats which were simultaneously given acetyl salicylic acid orally at 100 mg/kg/day for the 10 days, showed additional 10± 30 dB losses at all frequencies. The mechanism of auditory toxicity of toluene is unknown and no experimental data is available to argue upon how salicylate and toluene ototoxicities might interfere. Sha and Schacht (1999) worked on the hypothesis that the ototoxicity of aminoglycoside antibiotics could be due to iron chelation followed by production of free radicals. Salicylic acid can be partly converted, under oxidative stress, to 2,3-dihydroxybenzoic acid which is an iron chelator. Thus protective e€ects against iron dependent aminoglycoside ototoxicity

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could be expected. The aminoglycosidic antibiotic gentamicin was administered via a subcutaneous injection at the dose of 120 mg/kg/day for 6.5 weeks and sodium salicylate was given subcutaneously at the dose of 100 mg/kg twice daily. Auditory brainstem responses indicated hearing losses of 40±60 dB on average at high frequencies for gentamicin alone, and losses of 10±20 dB when combined with salicylate, and the associated hair cell loss was also much reduced. Serum salicylate level at the end of the treatment was 142 mg/l and did not a€ect the serum gentamicin level. The biochemical processes associated with salicylate ototoxicity are at present poorly known. Experimental data are few and do not ®rmly establish involvement of any metabolic pathway. One of the diculties has been the smallness of the inner ear which makes collection of biochemical samples limited to small quantities and very sensitive to contaminations. Quantitative biochemical methods have recently greatly improved and molecular genetic techniques provide new promising tools. Physiological data conclusively demonstrate that alterations of cochlear blood ¯ow, of outer hair cells and of a€erent cochlear nerve ®bers activity occur in salicylate ototoxicity. Vascular modi®cations seem compatible with salicylate interfering with sympathetic control possibly through inhibition of prostaglandin synthesis. Outer hair cells alterations are often referred to salicylate interference with cell membrane motor elements and/or permeability. Changes in a€erent nerve ®bers certainly involve inner hair cell synaptic transmission and interference of salicylate with neurotransmitters including catecolamines can be implicated possibly involving calcium. Whether the alterations at these three levels are more or less independent seems doubtful on the basis of present experimental evidence or theoretical knowledge. In the ®eld of salicylate auditory toxicity, as in many scienti®c areas, unifying apparently disparate facts into a coherent explanation, or de®nitely demonstrating the existence of separate processes, is one of the challenges of future research. The potential socioeconomic implications of understanding and controling salicylate, and other NSAIDs, auditory side e€ects and tinnitus generating processes, are strong incentives for developing research on salicylate ototoxicity.

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