Basal Cochlear Lesions Result in Increased Amplitude of Otoacoustic Emissions

Transcription

1 Original Paper Audiol Neurootol 1998;3: Received: March 28, 1997 Accepted after revision: March 31, 1998 Akinobu Kakigi Haruo Hirakawa Noam Harel Richard J. Mount Robert V. Harrison Basal Cochlear Lesions Result in Increased Amplitude of Otoacoustic Emissions Department of Otolaryngology, Auditory Science Laboratory, University of Toronto and Hospital for Sick Children, Toronto, Canada OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Key Words Aminoglycoside Amikacin Ototoxicity Otoacoustic emission Monitoring Scanning electron microscopy Cochleogram Auditory brainstem evoked responses OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Abstract We have measured the changes in transient otoacoustic emissions (TEOAEs) and distortion product otoacoustic emissions (DPOAEs) during and after ototoxic amikacin treatment in an animal (chinchilla) model. TEOAE and DPOAE were recorded from 6 adult chinchillas over a 6-week time course starting just before a 5-day or 7-day treatment period with amikacin sulphate (400 mg/kg/day, i.m.). After final recordings, cochlear morphology was assessed by scanning electron microscopy. Generally, both DPOAE and TEOAE amplitudes change during and after treatment in a systematic fashion. High-frequency components change first, followed by lower-frequency components. We note that there is often a long latency to the onset of changes in otoacoustic emissions (OAE), and that these changes can continue for weeks after treatment. Most importantly we report that when the basal region of the cochlea is damaged in the frequency region above the OAE recording bandwidth (0.6 6 khz for TEOAE; khz for DPOAE), we often find an increase in OAE amplitudes. More specifically, we note that as a cochlear lesion progresses apically, there is often a transient increase in a frequency-specific OAE before it reduces or is lost. Our results suggest that the increase in OAE amplitudes precedes the expression of detectable cochlear pathology. OOOOOOOOOOOOOOOOO ABC Fax karger@karger.ch S. Karger AG, Basel /98/ $15.00/0 Accessible online at: Prof. Robert V. Harrison Department of Otolaryngology Hospital for Sick Children 555 University Avenue, Toronto, Ont. M5G 1X8 (Canada) Tel. +1 (416) , Fax +1 (416)

2 Introduction Ototoxicity and nephrotoxicity are known side effects of treatment with aminoglycosides. It is important to identify these effects at an early stage to prevent severe, long-term damage. Nephrotoxicity and ototoxicity are correlated with the increasing drug levels [1 3] and serum concentration of aminoglycoside [2, 4 6]. Ototoxicity is typically revealed by subjective symptoms such as dizziness, tinnitus, and hearing loss or by using objective measures of change to the auditory system such as electrocochleography and evoked response audiometry [7, 8]. Morphologically, aminoglycosides progressively destroy the sensory epithelium from basal to apical turns of the cochlea; especially susceptible are the outer hair cells (OHCs) [9 12]. It is well accepted that otoacoustic emissions (OAEs) reflect some aspect of the active cochlear mechanisms which are mainly attributed to the function of the OHCs [13 16]. Theoretically then, OAEs should be helpful in the detection of ototoxicity, especially since most ototoxins predominantly affect the OHCs of the inner ear. In clinical studies Zorowka et al. [17] and Hotz et al. [18] reported that OAEs are useful for monitoring aminoglycoside ototoxicity. Animal studies [14, 19, 20] have also shown that OAEs could be used to detect early stages of aminoglycoside-induced pathology. In the present study we administered the aminoglycoside amikacin to chinchillas and made frequent OAE recordings, over a long time period before, during, and after treatment. At the end of this experimental period we examined the pattern of cochlear damage using scanning electron microscopy. The study was designed to ask a number of questions concerning the sensitivity of OAE recordings to monitor cochlear changes which might be remote from the specific frequency regions of generation of the emissions. First, given that aminoglycoside poisoning of the cochlea starts out in the most basal, high-frequency areas, can OAE recording devices which generally monitor only up to 6 8 khz be used to detect such basal cochlear dysfunction? In preliminary (unpublished) experiments, we had noted that OAE amplitudes could, paradoxically, increase in animals with basal cochlear lesions. The present study was designed to explore more systematically this observation. Our main experimental hypothesis is that during the progression of a cochlear lesion from basal to more apical areas, OAEs generated from sites apical to the lesion have an increased amplitude as measured at the level of the external meatus. Secondly, we ask whether OAEs can provide useful information concerning the dynamic aspects of ototoxicity, for example, the rate of progression of cochlear damage. More practically, how long do we need to monitor OAEs after aminoglycoside treatment to ascertain the full and final extent of any possible ototoxic effect? Materials and Methods Subjects Six adult chinchillas ( g; purchased locally, all female, of same age) were treated with the ototoxic drug amikacin sulphate (400 mg/kg/day, via i.m. injection) to produce bilateral lesions to the cochleas. Three animals were treated for 5 days, the others for 7. Based on previous experience this dosage regime was known to produce, over time, extensive cochlear lesions. ABR Recording To determine auditory threshold levels, auditory brainstem evoked responses (ABRs) to tone-pip stimuli were used [21] under anaesthesia (atropine sulphate 0.04 mg/kg, i.m., xylazine hydrochloride 2.4 mg/kg, i.m. and ketamine hydrochloride 15 mg/kg, i.m.). These recordings were done on only two occasions, before amikacin injection and before sacrifice and histological evaluations (6 10 weeks after amikacin injec- 362 Audiol Neurootol 1998;3: Kakigi/Hirakawa/Harel/Mount/ Harrison

3 tion). Acoustic stimuli were delivered in a closed system earphone (ER-2, Etymotic Research). The rise and fall time of tone pips was 1 ms and plateau was 2 ms. All 12 ears of the 6 chinchillas had normal ABR response thresholds before amikacin administration (i.e. were not outside of the normal threshold range based on our laboratory standard). At each recording session, the middle ear condition was checked with the otoscope through the external auditory meatus. There was no evidence of otitis media or externa. OAEs Recording Transient evoked OAEs (TEOAEs) were recorded in a sound-attenuating room using the ILO88 system (Otodynamics Ltd., software version 3.92) [22]. The stimulus consisted of a non-filtered click of 80 Ìs duration, the level being adjusted to obtain a peak stimulus level of 81 db SPL B 3 db. Click rate was 50 per second and post-stimulus analysis time was ms after stimulation onset. The recording time window was set at 2.5 ms to eliminate the initial section of the transient response corresponding to linear resonance of the ear canal and the middle ear. The number of sweeps averaged was 260 and the system passband was approximately khz. Recordings were carried out using the non-linear mode as formulated by Kemp [23] allowing at least partial elimination of stimulus artifact. The overall TEOAE spectrum was determined by taking a 512-point FFT of the temporal waveforms. Components at 1, 2, 3, 4, and 5 khz were represented in terms of signal-to-noise ratio. The data from the 1-, 2-, and 4-kHz components were used for monitoring TEOAE change. Distortion product otoacoustic emissions (DPOAEs) were recorded using the ILO92 system (Otodynamics Ltd., software version 3.92). Each DPOAE recording was made immediately subsequent to a TEOAE recording in order to keep the same probe conditions. The f 1 /f 2 ratio was maintained at The frequency of f 2 was varied in 1/3-octave steps from 1 to 8 khz (9 points). The intensities of f 1 and f 2 were 50 db SPL re: 20 ÌPa. DPOAE (2f 1 f 2 ) results at f 2 values of 1, 2, 4, and 6.7 khz were used for monitoring DPOAE change. Both DPOAE and TEOAE were recorded from left and right ears alternately 3 times during each session. All OAE recordings were made in the awake animal, lightly held in a head holder. In our experience chinchillas remain very calm and apparently unstressed with this type of restraint. Recording sessions were conducted at various intervals before, during and after amikacin treatment until 6 or 10 weeks after amikacin injection in 5 or 7 times injected animal groups, respectively. There was no evidence of otitis media or externa. Morphological Study After the final recordings, the animals were euthanized by anaesthetic overdose (sodium pentobarbital) and temporal bones dissected out. The cochlea was exposed, the round and oval windows opened and the perilymphatic scalae perfused with fixative (1% glutaraldehyde/4% formalaldehyde in phosphate buffer). After fixation for a total of 2 h, the cochleas were washed overnight in 0.1 M phosphate buffer (ph 7.4), then postfixed in buffered 1% OsO 4, all at 4 C. Following dehydration to 70% ethanol, the cochleas were dissected to expose the organ of Corti. After complete dehydration through absolute ethanol, the specimen was critical-point-dried from CO 2, sputter-coated with gold and examined on a Hitachi scanning electron microscope at 15 kev. Morphologic observations of the cochlea were associated with functional properties (ABR threshold, OAE responses) using a cochlear place-frequency map derived for the chinchilla [24]. Further, cochleograms were made according to the following grading: (1) normal: no missing OHCs; (2) mild: less than 1/3 of OHCs missing; (3) moderate: from 1/3 to 2/3 of OHCs missing; (4) severe: more than 2/3 of OHCs missing and/or missing inner hair cells. These experiments were specifically approved by the Animal Care Committee of the Faculty of Medicine, University of Toronto. All procedures involving animals were carefully carried out and the level of care was within the guidelines of the Canadian Council on Animal Care. Results ABR thresholds of all ears were within normal range before amikacin treatment (the normal range is based on our laboratory experience with hundreds of subjects). Figure 1 shows ABR threshold shift of the 6 animals after amikacin treatment and just before morphological study. The upper three audiograms represent the threshold changes resulting from 5 amikacin injections, and the lower plots represent loss resulting from 7 injections. Each animal shows a different pattern of high-frequency hearing loss ranging from Amplitude Increase of OAEs Audiol Neurootol 1998;3:

4 Fig. 1. Change in ABR thresholds (to frequency-specific stimuli) resulting from treatment with amikacin (400 mg/kg/day, i.m.) for 5 days (upper series) and 7 days (lower series). The threshold shift represents the difference between the pre-treatment baseline and the final (presacrifice) ABR recording. little or no threshold elevation at 8 khz (e.g. No. 434) to significant threshold elevation across a range of frequencies (e.g. No. 439). Figures 2 5 show the time course of change of TEOAEs and DPOAEs during and after amikacin treatment together with the (final) cochleogram. Results from these 8 ears have been ordered to show a range of cochlear damage varying from a restricted basal lesion (fig. 2) to very extensive and severe hair cell damage throughout the cochlea (fig. 5). We note, as with the ABR audiograms, that similar dosage regimes can produce very different cochlear hair cell lesions. In figure 2 the cochleograms show 2 ears in which there is a severe to moderate lesion in the very high-frequency region (basal turn) but mild or no damage in lower-frequency regions (upper turns). In both cases DPOAEs show little change except 6.7-kHz components during the 42-day recording period. The 6.7-kHz components decreased approximately 10 db even though ABR threshold shift was up to 30 db. The TEOAEs show little or no change during the early post-treatment period, but at later stages (after approximately 10 days) show an increased amplitude. This is particularly the case for No. 434L at 1 and 2 khz. In figure 3 the cochleograms indicate basal turn lesions up to the 4-kHz region. In both ears, low-frequency (1- and 2-kHz) DPOAEs 364 Audiol Neurootol 1998;3: Kakigi/Hirakawa/Harel/Mount/ Harrison

5 Fig. 2. Left-hand panels show time courses of TEOAE and DPOAE changes during a 5-day amikacin treatment period (indicated by thick bar) and thereafter for a period of many weeks, for 2 ears, No. 434 R (right ear) and 434 L (left ear). OAE frequency components are as indicated by the key in each plot. In both ears there is ultimately a severe to moderate basal (high-frequency) lesion (right-hand cochleograms). Amplitude Increase of OAEs Audiol Neurootol 1998;3:

6 Fig. 3. Left-hand panels show time courses of TEOAE and DPOAE changes (OAE frequency components as indicated by key; NR = no response, i.e. signal was below recording system noise level) in 2 ears (No. 438R and 440R) with lesions extending apically up to the 4-kHz region as shown in the cochleograms (right). 366 Audiol Neurootol 1998;3: Kakigi/Hirakawa/Harel/Mount/ Harrison

7 Fig. 4. Left-hand panels show time courses of TEOAE and DPOAE changes (OAE frequency components as indicated by key; NR = no response, i.e. signal was below recording system noise level) in 2 ears (No. 431L and 430L) with extensive lesions as indicated by the cochleograms (right). Amplitude Increase of OAEs Audiol Neurootol 1998;3:

8 and TEOAEs change very little in amplitude during and after treatment. However, the 4-kHz component of both types of emission shows an increased amplitude for many weeks after treatment. This is particularly the case for the TEOAE responses. Concerning the final amplitudes of the 6.7-kHz component of DPOAEs, subject No. 438R showed a decrease and subject No. 440R an increase in the amplitude. These changes correlate with (final) ABR threshold shifts and cochleograms. During the initial treatment period, for both subjects No. 438R and No. 440R, there is some evidence that increase in DPOAE occurs earlier than the increase in TEOAEs. However, some caution in interpretation is required given the variation from subject to subject. Figure 4 shows examples of the 2 ears in which there is more severe damage of the cochlea. In subject No. 431L, both TEOAE and DPOAE amplitudes fall over time, with high-frequency components (4 khz) reducing before lower frequencies. However, the time course of these changes appears to be different in that DPOAEs appear to reduce in amplitude over a long time period (30 days) compared to TEOAEs. In both cases the amplitude reductions continue well after the amikacin treatment period. In subject No. 430L TEOAE and DPOAE changes do not correlate well. For the DPOAEs, 6.7-, 4-, and 2-kHz components reduce in amplitude over time with the 1-kHz emission remaining level. For the TEOAEs the 4-kHz component changes relatively little, whilst lower-frequency components (1- and 2-kHz) increase in amplitudes. The cochleograms of figure 5 show that there is severe damage to most of the cochlea in both ears. Both DPOAE and TEOAE amplitudes change in a systematic fashion. Highfrequency components change first, followed by lower-frequency components. An important observation can be noted in these ears: the amplitudes of the emissions increase before they start to reduce. This phenomenon can also be seen in, for example, figure 4 (No. 431L) where increases in both TEOAE and DPOAE precede the reduction in amplitudes. In other subjects, the increased OAE amplitudes observed (e.g. fig. 2, TEOAEs) may be a similar phenomenon, however in these cases the cochlear lesion is not ultimately extensive enough to cause OAE reductions. Figure 6 shows recordings of DPOAE and TEOAE over a period of 28 days in a nontreated control animal. This serves to indicate the relatively stable recordings (low variability) over time compared with the experimental subjects. Discussion Because OAEs can be measured quickly and non-invasively, requiring no voluntary response from the patient, it is a useful objective test for monitoring drug ototoxicity including that of aminoglycosides [clinical studies: 17, 18; animal studies: 14, 20; reviews: 19, 25]. These studies highlight a number of important aspects relating to monitoring of aminoglycoside ototoxic effects. For example, Hotz et al. [18] used click- and tone burstevoked TEOAEs for monitoring ototoxicity of human patients whose individual treatment durations ranged from 9 to 33 days. They found that TEOAEs decreased when a treatment period lasted longer than 16 days. This study illustrates the delay in detecting OAE changes, most likely because of a progression of basal cochlear dysfunction. In our present study we have explored this progressive effect more fully. In another clinical study, Zorowka et al. [17] used TEOAE recording to monitor the hearing of perinatally infected newborns 368 Audiol Neurootol 1998;3: Kakigi/Hirakawa/Harel/Mount/ Harrison

9 Fig. 5. Left-hand panels show time courses of TEOAE and DPOAE changes (OAE frequency components are as indicated by the key in each plot; NR = no response, i.e. signal was below recording system noise) in 2 ears (No. 439L and 439R) both of which ultimately developed a near-total lesion of the cochlea. Amplitude Increase of OAEs Audiol Neurootol 1998;3:

10 Fig. 6. No-treatment control subject in which TEOAE and DPOAE recordings were made over a period of 28 days (OAE frequency components are as indicated by the key in each plot). who received ampicillin plus either cefotaxime or aminoglycosides. Of particular interest was their report of increased TEOAE emissions in 10 of 21 ears of aminoglycoside treatment and 4 of 13 ears of cefotaxime treatment. We have made similar observations (see below). A general issue in these studies relates to the fact that many ototoxic drugs (including aminoglycosides) affect basal high-frequency regions above the frequency range of emission recording or conventional pure tone audiometry. Many authors [e.g. 26] suggest using highfrequency audiometric monitoring in order to detect an early aminoglycoside-induced hearing loss. However, such subjective audiometric tests are often not useful for infants or children. It is still a problem that commercially available OAE systems cannot monitor highfrequency regions, for example 8 khz and above. In animal studies, Brown et al. [14] used DPOAE for monitoring gentamicin ototoxicity in guinea pigs. They investigated DPOAE changes for 21 days and reported that the emissions change before there are obvious morphologic changes to hair cells. Because animal studies can allow ultimate morpholog- 370 Audiol Neurootol 1998;3: Kakigi/Hirakawa/Harel/Mount/ Harrison

11 ical assessment of the cochlea, they are useful to explore in detail various aspects of monitoring drug ototoxic effects. For this reason, in the present study, we investigated both TEOAEs and DPOAEs during amikacin treatment using adult chinchillas. An important observation in our own study, and which confirms previous reports, is that the duration of pathological change is often much longer than the treatment period. Changes to OAEs were sometimes first observed many days after the treatment had finished, for example, as shown in animals No. 434L and No. 430L of figures 2 and 4, respectively. These results are consistent with previous studies [27, 28] and suggest that clinically we need long monitoring periods both during and after aminoglycoside treatment in order to determine the full and final ototoxic effects of a drug. Amikacin induced various levels of high tone threshold shift of ABRs and correspondingly various degrees of cochlear hair cell loss. Some cochleograms showed severe damage restricted to the (highfrequency) basal turn. Such lesions were not reflected as an OAE amplitude reduction but rather, in some cases, OAE amplitude increase (as is clearly seen in fig. 2). Relating to this, we often observed, as in figure 5, a temporary increase in OAE amplitude occurring before reduction, as a lesion progressed through the frequency region being monitored. This phenomenon may be the basis of the increase in OAE amplitudes reported by Zorowka et al. [17] after aminoglycoside treatment in newborns with perinatal infection. They interpreted this as relating to an improvement of the general condition of the subject over time. Based on our results we suggest that this may relate to the presence of a lesion more basal to the frequency region being monitored. We speculate that this increased amplitude OAE could be the result of a more effective basal transmission of the emission signal across a less active, and therefore less interfering cochlear region. It might be useful clinically to look for increased OAEs to detect very early high-frequency cochlear damage. Acknowledgements This study was supported by Medical Research Council (Canada) and the Masonic Foundation of Ontario. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO References 1 Sarubbi FA Jr, Hull JH: Amikacin serum concentrations: Prediction of levels and dosage guidelines. Ann Intern Med. 1978;89: Meyer RD: Amikacin. Ann Intern Med. 1981;95: Gatell JM, San Miguel JG, Zamora L, Araujo V, Bonet M, Bohe M, Jimenez de Anta MT, Farre M, Elena M, Ballesta A, Marin JL: Comparison of the nephrotoxicity and auditory toxicity of tobramycin and amikacin. Antimicrob Agents Chemother 1983;23: Barza M, Scheife RT: Antimicrobial spectrum, pharmacology, and therapeutic use of antibiotics. IV. Aminoglycosides. J Maine Med Assoc 1977;68: Dahlgren JG, Anderson ET, Hewitt WL: Gentamicin blood levels: A guide to nephrotoxicity. Antimicrob Agents Chemother 1975;8: Plaut ME, Schentag JJ, Jusko WJ: Aminoglycoside nephrotoxicity: Comparative assessment in critically ill patients. J Med. 1979;10: Henley CH, Schacht J: Pharmacokinetics of aminoglycoside antibiotics in blood, inner-ear fluids and tissues and their relationship to ototoxicity. Audiology 1988;27: Hotz MA, Allum JH, Kaufmann G, Follath F, Pfaltz CA: Shifts in auditory brainstem response latencies following plasma-level-controlled aminoglycoside therapy. Eur Arch Otorhinolaryngol 1990;247: Hawkins JE, Lurie MH: The ototoxicity of dihydrostreptomycin and neomycin in cat. Ann Otol Rhinol Laryngol 1953;62: Amplitude Increase of OAEs Audiol Neurootol 1998;3:

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