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Effects of industrial noise exposure on distortion product otoacoustic emissions (DPOAEs) and hair cell loss of the cochlea – long term experiments in awake guinea pigs
Hearing Research 148 (2000) 9^17 www.elsevier.com/locate/heares
E¡ects of industrial noise exposure on distortion product otoacoustic emissions (DPOAEs) and hair cell loss of the cochlea ^ long term experiments in awake guinea pigs E. Emmerich a
a;
*, F. Richter a , U. Reinhold a , V. Linss b , W. Linss
b
Institute of Physiology I, Department of Neurophysiology, Friedrich Schiller University, Teichgraben 8, D-07740 Jena, Germany b Institute of Anatomy I, Friedrich Schiller University, Jena, Germany Received 13 January 2000; accepted 21 April 2000
Abstract Distortion product otoacoustic emissions (DPOAEs), a sensitive detector of outer hair cell (OHC) function, cochlear microphonics (CM), and hair cell loss have been monitored in 12 awake guinea pigs before and after 2 h exposure to specific, playedback industrial noise (105 dB SPL maximal intensity). All animals had stable DPOAE levels before noise exposure. In the first hours after noise exposure DPOAE levels were reduced significantly. In about 70% a partial recovery of the DPOAEs was found within 4 months after noise exposure. In 16% of the investigated ears no recovery of DPOAEs was observed. However, in a few ears increased DPOAEs were observed after noise exposure. Exposure to industrial noise caused both morphological changes in the middle turns of the cochlea and electrophysiological changes in the middle frequency range. A close correlation existed between reduced DPOAE levels, loss in CM potentials, and area of damaged or lost OHCs, but not with the numbers of damaged or lost OHCs in the cochlea. It can be concluded that continuous industrial noise causes a damage to OHCs which differs form the damage caused by impulse noise. ß 2000 Elsevier Science B.V. All rights reserved. Key words: Distortion product otoacoustic emission; Outer hair cell; Continuous noise exposure; Cochlear microphonics ; Scanning electron microscopy; Hearing loss; Guinea pig
1. Introduction Numerous studies have examined the e¡ects of highly intense noise produced in industrial plants on the inner ear of man (Henderson and Salvi, 1998; Diero¡, 1994 ; Borg et al., 1983 ; Dobie, 1983). In many branches of light industry, the noise emitted consists of both impulse noise and noise with a broad frequency spectrum and a peak intensity below 100 dB SPL (Henderson and Hamernik, 1986). A typical example is noise from the washing and ¢lling machines in a brewery. Whether or not such continuous but non-painful noise would damage hearing function can be investigated in long term experiments on awake animals. In order to assess alterations of hearing function, in the present study we re-
* Corresponding author. Tel.: +49 (3641) 938811; Fax: +49 (3641) 938812; E-mail: [email protected]
corded in guinea pigs distortion product otoacoustic emissions (DPOAEs) that are evoked by bitonal stimulation and are routinely used for the clinical diagnostic screening of frequency-dependent cochlear function (Wagner and Plinkert, 1999 ; Lichtenstein and Stapells, 1996 ; Vinck et al., 1996 ; Zenner, 1994; Hauser et al., 1991). In humans, measurements of DPOAE are used for the early and di¡erential diagnosis of damage to the outer hair cells (OHCs) (Henley et al., 1996 ; LonsburyMartin et al., 1993). Experiments in animals have shown that DPOAE as well as transiently evoked otoacoustic emissions (TEOAE) are altered by ototoxic drugs, noise exposure and hypoxia (Zheng et al., 1997b ; Hofstetter et al., 1997 ; Hamernik et al., 1996; Henley et al., 1996; Brown and Gaskill, 1990 ; Cody and Russell, 1988, 1987 ; Kemp and Brown, 1983). We have previously shown that cochlear hair cells of the guinea pig can be damaged by a single noise impulse of 164 dB peak equivalent (pe) SPL (rise
0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 1 0 1 - 5
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time 6 0.1 ms) that was re£ected by reduced amplitudes of cochlear microphonics (CMs) (Emmerich and Biedermann, 1993; Emmerich et al., 1990; Linss et al., 1991 ; Richter and Biedermann, 1987; Meyer et al., 1985 ; Geyer et al., 1978). In impulse noise-exposed animals, DPOAE stimulated with higher frequencies were diminished and cochlear damage, i.e., hair cell loss, was shown morphologically in the ¢rst turn of the cochlea, which is sensitive to high frequencies (Emmerich et al., 2000). Prolonged exposure of awake chinchillas to continuous noise (Eddins et al., 1999; Hamernik et al., 1998) led to reduced DPOAE levels, but, in a few cases, DPOAEs were increased in the post-exposure period. The aim of our present study was to test the levels of DPOAEs after exposure to speci¢c industrial noise in awake guinea pigs, and to relate the changes in DPOAEs to CM and also to morphological changes in the cochlea. We wanted to test whether DPOAE is a reliable parameter for the prediction of hair cell loss, and whether DPOAEs could be used for monitoring the changes in inner ear function caused by noise exposure. The data should indicate the potential danger of continuous noise to employees in light industry, and they should be relevant for noise protection in these branches of industry. 2. Materials and methods 2.1. DPOAE recording Twelve female guinea pigs at an age of 3^4 months, weighing about 400 g, were used for these experiments. For the recording and subsequent analysis of DPOAE we used the ILO92 (Otodynamics Ltd., Herts, UK). The acoustic probe was held by hand to the opening of the external ear channel (meatus acusticus externus) with gentle pressure. The levels of DPOAE were measured in both ears at f2 stimulation frequencies of 1.5^ 6.0 kHz (frequency ratio: f1 /f2 = 1.22). Both tones had 60 dB SPL. DPOAEs were included in the analysis if they were at least 3 dB above background noise. During the measurements, both the experimenter and the experimental animal were placed in an sound-proof chamber. All measurements of the DPOAEs were performed in awake animals which were kept in a cage to restrict movements and were carried out by the same experimenter to whom the animals were habituated. This allowed the DPOAE to be recorded with a minimum of contaminating movements from the animals. A detailed description of these methods is given by Emmerich et al. (2000). Before noise exposure (see below), at least 25 recordings from both ears were performed in each guinea pig
within 8 weeks to obtain a stable pre-exposure baseline. Immediately after the ¢rst noise exposure DPOAEs were recorded at 5 min intervals for a total period of 2 h. The recording was repeated daily during the ¢rst 3 days after noise exposure and then at regular intervals of 2 days until DPOAE amplitudes had stabilized. Four months after noise exposure, the experimental protocol was terminated. Only when DPOAEs had recovered within 3 weeks to 65% or greater of pre-exposure values, a second exposure to the same noise was performed. Six animals without recovery of DPOAE therefore received only one noise exposure. For analysis, the DPOAEs before noise exposure were averaged and normalized in each animal. In addition, a grand mean over all animals before noise exposure was obtained. DPOAE data obtained after noise exposure were expressed as absolute levels and in percent of the pre-exposure values. Di¡erences between the pre-exposure values and the values after impulse noise exposure were compared for each test frequency with Student's t-test. Statistical signi¢cance was set at P 6 0.05. 2.2. Noise exposure Speci¢c industrial noise was recorded with a DATrecorder in the bottle washing and ¢lling department of Braugold GmbH Brewery at Erfurt. The frequency spectrum and intensities of that kind of noise are shown in Fig. 1. Noise exposure was performed by playing-back the recorded noise at an intensity of 105 dB SPL for 2 h to awake guinea pigs that were kept in the cage used for DPOAE recording. Movement of the head was restricted by using a small box which enclosed the whole body of the animal except the head. Noise presentation
Fig. 1. Frequency spectrum of the realistic industrial noise used in this study. Data are given as mean values (bars) with minimum (a) and maximum (#) values. The columns A and L show recordings with the ¢lters `A' and `L' without frequency distributions. Standard deviations were very low and are indicated (X).
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was performed by a high ¢delity sound system, consisting of a compact disc player (Pioneer, PD-306), an ampli¢er (Yamaha, P4500) and a loudspeaker. The distance between the loudspeaker and the ears of the animal was about 10 cm. The loudspeaker was placed in front of the head, so both ears were a¡ected by the noise with the same intensity. The frequency spectrum and the amplitudes were determined before the experiments using the Krones^Block con¢rming the identity of the original and the played-back noises. 2.3. CM potential recording and morphological analysis After the last DPOAE recording, animals were deeply anesthetized with sodium thiopental (Trapanal Byk Gulden, 100 mg/kg, i.p.) and placed in a head holder. The animals were breathing spontaneously room air. Body temperature was maintained by a heating pad at 37³. After administration of local anesthetics, skin and muscles were incised behind the right and left outer ear channels and a burr hole was made at each side that exposed the round window of the cochlea. An Ag^ AgCl-electrode was placed at the round window membranes of both ears for CM recordings. Test stimuli for CMs were sine waves using the frequencies of 0.5^ 10.0 kHz. The intensity of the sound was 60 dB SPL. Monopolar CMs were recorded, ¢ltered and then evaluated in dB by an audiofrequency spectrometer 2109 (Bru«el and Kjaer, Denmark). Amplitudes of CMs were compared to those obtained in non-exposed animals (Biedermann et al., 1977). After killing the anesthetized animals by decapitation, the cochleae were perfused with glutaraldehyde and the upper turn was opened. The cochleae were dehydrated with ethanol, critical point dried and sputtered with gold for scanning electron microscopy. The exposed apical turn of the organ of Corti was documented with the scanning electron microscope (Stereoscan 260, Cambridge Instruments, UK), then the fol-
Fig. 2. Mean values of pre-exposure DPOAE levels (12 guinea pigs and 25 individual tests in each animal, mean value þ S.E.M.). A slight drop at the stimulus frequency f2 = 4.0 kHz can be noted. Repeated pre-exposure tests con¢rmed that replicable results could be obtained with the hand-held probe.
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lowing turn was opened and similarly prepared. Using this method, the whole cochlea was investigated turn by turn. For evaluation, each cochlear turn was subdivided into four sectors (that is A1^A4, B1^B4, C1^C4, and D1^D3, see Fig. 6). In all sectors OHCs were counted and loss of OHCs compared to unexposed animals (Meyer et al., 1985) was documented. The care and use of the animals included in this study was approved by the Thu«ringer regional government (Landesverwaltungsamt), reg. no. 02-27/97). 3. Results 3.1. Noise e¡ects on DPOAE and CM Prior to noise exposure all animals showed reproducible DPOAEs. A pre-exposure mean value for all animals is given in Fig. 2. The levels of DPOAE exhibited a maximum at 5 and 6 kHz. At 4 kHz a relative reduction of the DPOAE was often noted. No di¡erences were found comparing right and left ears of the animals. After noise exposure, three di¡erent types of recovery of DPOAE were found. 3.1.1. Partial recovery In most of the animals (about 70% of the ears investigated), only a partial recovery was seen. An example from one animal with partial recovery restricted to the higher frequency range is shown in Fig. 3A that was characterized by a progressive decline and loss of DPOAEs at test frequencies lower than 3.0 kHz, and weakly diminished DPOAEs at frequencies of 4.0^ 6.0 kHz up to 1 day after noise exposure. Ten days after noise exposure, DPOAEs could be observed using test frequencies of 2.5^6.0 kHz. However, 7 weeks after noise exposure, only DPOAEs with lower levels were observed at 6.0 kHz, whereas no DPOAEs were recorded in the lower frequencies. In six animals a substantial recovery of DPOAEs to 65% or more of pre-exposure values was observed in the whole frequency range. Within 10 min to 1 h and also 2 h after noise exposure none of these guinea pigs showed any DPOAE at all. One to two days after noise exposure, recovery was seen at frequencies higher than 2.5 kHz, reaching nearly pre-exposure values. Recovery of DPOAEs at frequencies of 1.5 and 2.0 kHz started 3 days after noise exposure and reached levels of 60% or more of pre-exposure values 9 days after exposure. In these six animals a second exposure to the same noise after 3 weeks replicated the above mentioned changes. Again, only DPOAEs evoked by test frequencies higher than 5.0 kHz recovered very quickly, reaching initial values by 10 min post-exposure. DPOAEs obtained by test frequencies of 1.5^4.0 kHz remained
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Fig. 3. A,B: Representative example of partial recovery of DPOAE after exposure to industrial noise. A: Diagram showing partial recovery of DPOAE at high frequencies after one single exposure to industrial noise (2 h, 105 dB pe SPL). DPOAEs `before' are the mean values for this individual animal taken from 25 pre-exposure tests. At the end of the observation period of 7 weeks, DPOAEs could be recorded only at a frequency f2 6.0 kHz, no responses could be obtained at lower f2 frequencies. B: Scanning electron microscopic micrographs (1600:1) of damaged OHCs from sectors C3/C4 in this animal: the IHCs (top row) were not damaged by the noise. OHCs (three rows at bottom) are severely damaged, hairs are destructed and whole cells are lost. This damage a¡ected all three rows of OHC, preferentially the second and third rows (OHC 2 and OHC 3 in Fig. 6). In adjacent sectors, a similar pattern of destruction was seen.
diminished or were lost. Only little signs of restitution were found in DPOAE at test frequencies of 2.0 and 3.0 kHz. Fig. 4A shows a representative example for this subset of recovery from one animal of this group. The pre-exposure values given in the left part of the diagram resemble those from the other animals. It can be seen that recovery reached pre-exposure values in the whole frequency range 9 days after the ¢rst noise exposure, but was restricted to frequencies of 2.0 kHz and higher after the second noise exposure. In this particular animal, only little signs of recovery were seen at a test frequency of 4.0 kHz.
3.1.2. Overshooting recovery In two out of 12 animals (three ears), the initial loss of DPOAEs was very short-lived and complete recovery occurred within 9 days after noise exposure. A temporary increase in DPOAEs up to 170% of pre-exposure was registered especially at high frequencies. Between day 9 and 12 post-exposure, this increase, however, recovered to normal values. Then the two animals received a second noise exposure at the same time point as the above mentioned animals (Fig. 5). As after the ¢rst exposure, an initial drop in DPOAE was followed by increased DPOAEs that again exceeded pre-expo-
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sure values up to 170% in all frequencies tested. This increase could be observed at 2 weeks post-exposure and remained at that level up to 3 weeks, when this experiment was terminated. 3.1.3. No recovery of DPOAE In one guinea pig a complete and persistent loss of DPOAEs was observed already after the ¢rst noise exposure. As in the other animals, immediately after noise exposure DPOAE at frequencies of 1.5^3.5 kHz were severely diminished or even reduced to zero. DPOAE
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that could be recorded at frequencies of 4.0^5.0 kHz within 10 days after noise exposure were diminished in the second week after noise exposure and did not recover within 7 weeks. A complete survey of the observed e¡ects in all guinea pigs is given in Table 1. As representative examples, normalized data for two DPOAE stimulation frequencies and for a 6.0 kHz sine wave to elicit CM are shown. Data are depicted only from right ears, since no di¡erences existed to the left ears of the animals. In the ¢rst hour after noise exposure, DPOAEs were sig-
Fig. 4. A,B: Representative example of complete recovery of DPOAE after exposure to industrial noise. A: DPOAE recordings, values `before' give mean values for this individual animal taken from 25 pre-exposure tests. Recovery of DPOAE started after a short period following the ¢rst exposure to noise (indicated by arrow I). The animal was exposed for a second time after the complete recovery after 9 days to the same noise (indicated by arrow II). Again, after an initial drop in DPOAE a recovery was observed that was nearly complete in frequencies f2 higher than 2.5 kHz. A partial recovery was seen at lower f2 frequencies. B: Scanning electron microscopic micrographs (1600:1) of damaged OHCs in sector B4 in this animal: IHCs (top row) were not a¡ected by the noise. A small area of lost OHCs in the third row was found. Adjacent cells in the third row were damaged and had clotted or destructed hairs.
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Fig. 5. Typical example of DPOAEs with transient overshooting recovery. Values `before' give mean values for this individual animal taken from 25 pre-exposure tests. In a range of 1 h to 9 days after the ¢rst noise exposure (arrow I) DPOAEs exceeded pre-exposure values in most f2 frequencies tested. Between days 9 and 12 the DPOAE tended to lower (normal) values. A second exposure to the same noise (arrow II) replicated this result. In the whole frequency range tested 2 weeks after a second noise exposure, DPOAEs exceeded the pre-exposure values.
ni¢cantly diminished or lost in most of the animals. The animals in which the DPOAE levels recovered to 65% or more of pre-exposure values, received a second noise exposure following the 2 weeks data recording. As can be seen in Table 1, after the second noise exposure DPOAE were less diminished than after the ¢rst exposure. The reason for this phenomenon is unclear. CM recorded at the last day of the experimental protocol were declined in most of the noise exposed animals compared to normal hearing ones. This was in agreement with the DPOAE recordings. A positive and frequency-speci¢c correlation (slope 0.64 þ 0.22,
r = 0.45) was found. The decline in CM, however, did not parallel the decline in DPOAE level. 3.2. Results of scanning electron microscopy The cochlea of the guinea pig consists of four turns (turns A^D, Fig. 6) with three rows of OHCs and one row of inner hair cells (IHCs). In normal guinea pig cochleae usually very few OHCs are missing, and the typical formation of the three rows exists from the Ato the C-turn. In the D-turn the typical pattern of rows is absent, up to the helicotrema usually single OHCs were found.
Table 1 Percentage changes in the levels of DPOAE recorded from right ears after exposure to industrial noise in 12 guinea pigs (gp1^gp12) tested gp1
gp2
gp3
gp4
(a) DPOAE f2 : 3 kHz, data from right ears, *P 6 0.05 Control 100 100 100 100 1h 0 0 0 0 2 weeks 163.2* 0 0 79.3 1h 0 8.42* 3 weeks 44.44* 79.65* (b) DPOAE f2 : 6 kHz, data from right ears *P 6 0.05 Control 100 100 100 100 1h 0 0 0 0 2 weeks 15.42* 88.36 37.97* 97.07 1h 59.93* 72.9* 3 weeks 93.15 65.81* (c) CMs recorded at termination of experimental series, 6.0 kHz 60 100 0 90
gp5
gp6
gp7
gp8
gp9
gp10
gp11
gp12
100 49.01* 66.45 45.39* 10.86*
100 0 106.4
100 9.58* 79.04
100 10.77* 11.54* 38.46* 149.2*
100 61.02* 68.21 43.08* 68.2*
100 70.95 24.76*
100 22.22* 0
100 2.22* 54.07* 107.4 28.89*
100 75.59 86.29
100 2.29* 61.07*
100 33.23* 72.58* 71.29 90.97
95
87
56
100 100 0 44.63 92.91 85.54 50.39* 74.02 stimulus pure tone, 80 70
100 79.88* 96.28
100 100 98.51 82.86 62.83 77.14* 0 87.86 114.4 18.57* 60 dB SPL, data from right ears 100 98 40
The tables give values for the f2 frequencies 3.0 (a) and 6.0 kHz (b). The mean values of DPOAE levels in each animal before noise exposure were taken as 100% (control). After 2 weeks in six animals a second exposure to noise was performed, indicated by further data in the columns. In animals without a second noise exposure these columns are empty after the 2 week line, respectively. The 3 week line gives data that were obtained 3 weeks after the second noise exposure. Heavy numbers indicate an overshooting recovery. Asterisks indicate statistically signi¢cant di¡erences to pre-exposure (P 6 0.05, Student's t-test). CM (c) stimulated with a 6.0 kHz sine wave were recorded at the end of the experimental series. The table gives percentage levels related to normal hearing animals taken from Biedermann et al., 1977.
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Fig. 6. OHC loss in the di¡erent parts of cochlear turns. The diagram on the left shows percentage OHC loss, and the diagram on the right represents the four turns of a cochlea (not to scale) with the subsections indicated. Data on the left were taken from the guinea pig with DPOAE decline and OHC loss shown in Fig. 4A. Right panel: The three rows of OHCs are indicated by the di¡erent shades of the bars. Sector D3 was excluded, since the regular formation of the three rows of OHC started only at the basal part of the D-turn.
After exposure to industrial noise, the morphological observations showed di¡erent patterns of damage or loss of OHCs only. In none of the animals IHCs were damaged or lost. The cochleogram in Fig. 6 represents a typical pattern of OHC loss in an animal with reduced DPOAE and incomplete recovery. Little damage was seen in sectors A1 to B2, whereas large numbers of lost OHC were found in sectors B3 to D2, preferentially in the third row of the OHCs, but to a lesser extent also in the ¢rst and second rows of OHCs. This damaged area corresponded well to the loss of DPOAEs at lower test frequencies. As shown in Fig. 3B, damage to the second and third rows of OHC found in sectors C3 and C4 after single exposure to noise was re£ected by a complete loss of DPOAE evoked by 1.5^5.0 kHz. Fig. 4B shows an example of partial recovery of DPOAE that was re£ected by a pattern of lost and damaged OHC in sector B4, but almost restricted to the third row of OHC. However, no relation existed between numbers of lost OHC and percentage decline in DPOAE. 4. Discussion In the present study we tested e¡ects of a speci¢c continuous industrial noise on otoacoustic emissions (DPOAEs), on CMs, and on the morphology of OHCs. In previous experiments we found that DPOAE in guinea pigs under control conditions were similar to those in man if a frequency ratio f1 /f2 = 1.22 (Brown, 1987) was used. However, our level of stimulation was about 10 dB SPL higher for stimulus frequencies f2 s 2 kHz than that used in man. (Emmerich et al.,
2000 ; Brown and Gaskill, 1990; Kemp and Brown, 1983). It is known that rodent ears are most sensitive at higher frequencies (Hamernik et al., 1993). However, our equipment was designed for human investigations. The f2 stimulus frequencies used here were in the range of 1.5^6.0 kHz. In this range DPOAE levels rose with increased stimulus frequency. This slope was similar to the data in anesthetized guinea pigs (Brown and Gaskill, 1990), but had a slight drop at 4.0 kHz and levels in our guinea pigs were in mean 5^10 dB SPL higher. We relate these di¡erences to the use of awake animals that have a functional intact e¡erent innervation of the cochlea. The ear-damaging e¡ect of industrial noise can only be assessed by carrying out experiments in animals. Studies for the evaluation of noise e¡ects and investigations of DPOAEs or CMs have mostly been performed in anesthetized guinea pigs (Brown, 1987). It has been established that anesthesia exerts in£uences on inner ear's susceptibility to noise, probably due to an altered cochlear blood £ow (Angelborg et al., 1984; Angelborg et al., 1979 ; Hultcrantz et al., 1979). Therefore, our study used awake animals, which allowed repeated measurements of DPOAEs over long time periods with minimal strain for the animals. Stable DPOAEs before noise exposure showed that guinea pigs were well adapted to the experimental arrangement. Shaddock et al. (1985) reported on experiments on chinchillas that highly intense impulse noise a¡ected not only the morphology of OHC and IHC but also the vascular system of the inner ear. In previous experiments we con¢rmed these ¢ndings by using non-invasively recorded DPOAE. We could show that exposure
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of awake guinea pigs to impulse noise of 153 dB pe SPL caused OHC loss and degeneration of OHC between the ¢rst and second turns of the cochlea, and a reduction or even a loss of DPOAEs and CMs mainly with f2 stimulus frequencies greater than 4.0 kHz (Emmerich et al., 2000 ; Linss et al., 1991; Richter and Biedermann, 1987 ; Meyer et al., 1985). In the past, changes in DPOAE or in CM after continuous noise exposure were reported in chinchillas (Hamernik et al., 1998; Hamernik et al., 1996; Subramaniam et al., 1995; Hamernik et al., 1993) and in rabbits (Mensh et al., 1993a,b). In the present study we used real, played-back industrial noise with frequencies up to 21 kHz and peak intensities up to 105 dB SPL instead of a physically de¢ned octave band noise. We applied this noise for only 2 h. Interestingly, this short exposure already produced DPOAE shifts in the awake guinea pigs tested. The chosen application time agreed with Popelar and Syka (1993) and Popelar et al. (1987) who found damaged hearing function in guinea pigs after comparable short exposure times. After exposure to broadband industrial noise, hearing loss occurred at lower frequencies than after impulse noise. After industrial noise, responses to stimulus frequencies lower than 3.0 kHz were signi¢cantly more reduced, and recovered less, than responses to higher frequencies. This pattern of DPOAE changes corresponded to the morphological changes that were mostly found in turns B to D of the cochlea. In fact, our experiments showed a close correlation between frequency-speci¢c changes of DPOAE or of CM and the loss in OHC of a speci¢c cochlear turn. We could con¢rm that there is not always a close correlation between the number of damaged or lost hair cells in the whole cochlea and the extent of changes in DPOAE or in CM, and agree with Ahroon et al. (1993); Hamernik et al. (1993) and Lonsbury-Martin et al. (1993), who showed a discrepancy between audiometric data and numbers of lost or damaged OHCs after exposure to continuous or to impulse noise. What could be the reason for the increase in DPOAE after noise exposure? Zheng et al. (1997a) investigated noise e¡ects in chinchillas and found marked di¡erences between normally innervated and dee¡erented inner ears. Distortion product-grams obtained in awake and normal hearing guinea pigs or gerbils obviously di¡ered from those in anesthetized animals (Zheng et al., 1997b ; Brown, 1987). A temporary dee¡erentation by anesthesia could be an explanation for this ¢nding. There is some evidence that e¡erent projections to the OHC would be changed by noise and could increase hair cell motility (Zheng et al., 1997a). This hypothesis could explain a changed vulnerability of the cochlea (Henderson and Salvi, 1998). The particular reason for this increase, however, is unclear, especially, because only
a minority of animals produced such increased DPOAE without any predictability. It was speculated that tinnitus could be related to the increased DPOAE caused by a changed e¡erent cochlear innervation after acoustic trauma (Emmerich et al., 2000 ; Frolenkov et al., 1998 ; Kakigi et al., 1998). The experimental design used here could not solve this question. Our experiments showed that DPOAE can be recorded in awake animals in a reproducible manner over several months. Since morphological observations and recording of CM were only performed at the end of the experiment, no CM could be obtained during the follow-up of DPOAE changes after noise. We only checked whether or not a CM response existed after noise exposure. Earlier studies by Kirk et al. (1997) proposed a mechano-electric transduction transfer that should relate CM and the micromechanic phenomenon of DPOAE that could be a cause for the loss in CM in animals without DPOAE after noise exposure. In summary, the present study showed that continuous industrial noise produced in most of the animals a reduction in the levels of DPOAE that was probably caused by damage of the OHC. DPOAE seem to be a reliable parameter for the assessment of hearing loss and its recovery that can be used in non-invasive investigations. We assume that the increase in DPOAE after noise exposure could be due to changes in e¡erent innervation also caused by the damage. Since a noise exposure for only 2 h already resulted in severe and non-predictable damages, ear protection should be used in all noisy areas of light industry independently of the time of noise exposure. Acknowledgements The authors thank Professor Hans-Georg Schaible for comments and help during writing the manuscript and Ms. Gundula Kruse for her excellent assistance in the experiments with awake animals. The work was supported by the Deutsche Forschungsgemeinschaft (Em 57/1-1), and by Berufsgenossenschaft Nahrungsmittel und Gaststa«tten, Gescha«ftsbereich Pra«vention.
References Ahroon, W.A., Hamernik, R.P., Davis, R.I., 1993. Complex noise exposures: an energy analysis. J. Acoust. Soc. Am. 93, 997^ 1006. Angelborg, C., Axelsson, A., Larsen, H.C., 1984. Regional blood £ow in the rabbit cochlea. Arch. Otolaryngol. 110, 297^300. Angelborg, C., Hultcrantz, E., Beausang-Linder, M., 1979. The cochlear blood £ow in relation to noise and cervical sympathectomy. Adv. Otorhinolaryngol. 25, 41^48. Biedermann, M., Bu«ttner, G., Kaschowitz, H., Pingel, I., 1977. E¡ect
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E. Emmerich et al. / Hearing Research 148 (2000) 9^17 of single impulses on cochlear microphone potentials of the guinea-pig cochlea. Acta Biol. Med. Ger. 36, 1097^1105. Borg, E., Nilsson, R., Engstrom, B., 1983. E¡ect of the acoustic re£ex on inner ear damage induced by industrial noise. Acta Otolaryngol. Stockh. 96, 361^369. Brown, A.M., Gaskill, S.A., 1990. Measurement of acoustic distortion reveals underlying similarities between human and rodent mechanical responses. J. Acoust. Soc. Am. 88, 840^849. Brown, A.M., 1987. Acoustic distortion from rodent ears: A comparison of responses from rats, guinea pigs and gerbils. Hear. Res. 31, 25^39. Cody, A.R., Russell, I.J., 1988. Acoustically induced hearing loss: intracellular studies in the guinea pig cochlea. Hear. Res. 35, 59^ 70. Cody, A.R., Russell, I.J., 1987. The response of hair cells in the basal turn of the guinea-pig cochlea to tones. J. Physiol. Lond. 383, 551^ 569. Diero¡, H.G., 1994. La«rmschwerho«rigkeit. Gustav Fischer, Jena. Dobie, R.A., 1983. Reliability and validity of industrial audiometry: implications for hearing conservation program design. Laryngoscope 93, 906^927. Eddins, A.C., Zuskov, M., Salvi, R.J., 1999. Changes in distortion product otoacoustic emissions during prolonged noise exposure. Hear. Res. 127, 119^128. Emmerich, E., Richter, F., MeiMner, W., Diero¡, H.-G., 2000. The e¡ect of impulse noise exposure on distortion product otoacoustic emissions in the awake guinea pig. Eur. Arch. Otorhinolaryngol. 257, 128^132. Emmerich, E., Biedermann, M., 1993. Impulse noise e¡ects on evoked responses from hippocampus and auditory cortex. In: Heinze, H.-J., Mu«nte, T.F. (Eds.), Developments in Event-Related Potentials, Birkha«user, Boston, pp. 35^40. Emmerich, E., Biedermann, M., Richter, F., 1990. Auditory evoked responses in awake rabbits after exposure to high intensity noise impulses. Act. Nerv. Super. 32, 119^126. Frolenkov, G.I., Belyantseva, I.A., Kurc, M., Mastroianni, M.A., Kachar, B., 1998. Cochlear outer hair cell electromotility can provide force for both low and high intensity distortion product otoacoustic emissions. Hear. Res. 126, 67^74. Geyer, G., Biedermann, M., Schmidt, H., 1978. Endolymphatic leakage in case of acute loss of cochlear microphonics. Experientia 34, 363^364. Hamernik, R.P., Ahroon, W.A., Jock, B.M., Bennett, J.A., 1998. Noise-induced threshold shift dynamics measured with distortion-product otoacoustic emissions and auditory evoked potentials in chinchillas with inner hair cell de¢cient cochleas. Hear. Res. 118, 73^82. Hamernik, R.P., Ahroon, W.A., Lei, S.F., 1996. The cubic distortion product otoacoustic emissions from the normal and noise-damaged chinchilla cochlea. J. Acoust. Soc. Am. 100, 1003^1012. Hamernik, R.P., Ahroon, W.A., Hsueh, K.D., Lei, S.F., Davis, R.I., 1993. Audiometric and histological di¡erences between the e¡ects of continuous and impulsive noise exposures. J. Acoust. Soc. Am. 93, 2088^2095. Hauser, R., Probst, R., Harris, F., 1991. Die klinische Anwendung otoakustischer Emissionen kochlea«rer Distortionsprodukte. Laryngol. Rhinol. Otol. 70, 123^131. Henderson, D., Salvi, R.J., 1998. E¡ects on noise exposure on the auditory functions. Scand. Audiol. 48 (Suppl.), 63^73. Henderson, D., Hamernik, R.P., 1986. Impulse noise: critical review. J. Acoust. Soc. Am. 80, 569^584. Henley, C.M., Weatherly, R.A., Martin, G.K., Lonsbury-Martin, B.L., 1996. Sensitive developmental periods for kanamycin ototoxic e¡ects on distortion-product otoacoustic emissions. Hear. Res. 98, 93^103. Hofstetter, P., Ding, D.L., Powers, L., Salvi, R.J., 1997. Quantitative
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relationship of carboplatin dose to magnitude of inner and outer hair cell loss and the reduction in distortions product otoacoustic emissions amplitude in chinchillas. Hear. Res. 112, 199^215. Hultcrantz, E., Angelborg, C., Beausang-Linder, M., 1979. Noise and cochlear blood £ow. Arch. Otorhinolaryngol. 224, 103^106. Kakigi, A., Hirakawa, H., Harel, N., Mount, R.J., Harrison, R.V., 1998. Basal cochlear lesions result in increased amplitude of otoacoustic emissions. Audiol. Neurootol. 3, 361^372. Kemp, D.T., Brown, A.M., 1983. A comparison of mechanical nonlinearities in the cochlea of man and gerbil from ear canal measurements. In: Klinke, R., Hartmann, R. (Eds.), Hearing ^ Physiological Bases and Psychophysics. Springer, Berlin, pp. 104^132. Kirk, D.L., Molairinho, A., Patuzzi, R.B., 1997. Microphonic and DPOAE measurements suggest a micromechanical mechanism for the bounce phenomenon on following low-frequency tones. Hear. Res. 112, 69^86. Lichtenstein, V., Stapells, D.R., 1996. Frequency-speci¢c identi¢cation of hearing loss using transient-evoked otoacoustic emissions to clicks and tones. Hear. Res. 98, 125^136. Linss, W., Christner, A., Biedermann, M., 1991. Struktur und Funktion der Meerschweinchencochlea ein Jahr nach Impulsschallbelastung. Anat. Anz. 186 (Suppl.), 451^452. Lonsbury-Martin, B.L., Whitehead, M.L., Martin, G.K., 1993. Distortion-product otoacoustic emissions in normal and impaired ears: insight into generation processes. Progr. Brain Res. 97, 77^ 90. Mensh, B.D., Patterson, M.C., Whitehead, M.L., Lonsbury-Martin, B.L., Martin, G.K., 1993a. Distortion-product emissions in rabbit: I. Altered susceptibility to repeated pure-tone exposures. Hear. Res. 70, 50^65. Mensh, B.D., Lonsbury-Martin, B.L., Martin, G.K., 1993b. Distortion-product emissions in rabbit: II. Prediction of chronic-noise e¡ects by brief pure-tone exposures. Hear. Res. 70, 65^72. Meyer, C., Biedermann, M., Christner, A., 1985. Fru«h- und Spa«treaktionen der Meer-schweinchencochlea auf Impulsschall. Anat. Anz. 158, 5^12. Popelar, J., Syka, J., 1993. Middle latency responses to electrical stimulation of the auditory nerve in unanaesthetized guinea pigs. Hear. Res. 67, 69^74. Popelar, J., Syka, J., Berndt, H., 1987. E¡ect of noise on auditory evoked responses in awake guinea pigs. Hear. Res. 26, 239^247. Richter, F., Biedermann, M., 1987. Time lapse e¡ects of impulse noise on cochlear microphonics in rabbits observed in chronic experiments. Arch. Otorhinolaryngol. 244, 269^272. Shaddock, L.C., Hamernik, R.P., Axelsson, A., 1985. E¡ect of high intensity impulse noise on the vascular system of the chinchilla cochlea. Ann. Otol. Rhinol. Laryngol. 94, 87^92. Subramaniam, M., Henselman, L.W., Spongr, V., Henderson, D., Powers, N.L., 1995. E¡ect of high-frequency interrupted noise exposures on evoked-potential thresholds, distortion-product otoacoustic emissions, and outer hair cell loss. Ear Hear. 16, 372^ 381. Vinck, B.M., de Vel, E., Xu, Z.M., Van Cauwenberge, P.B., 1996. Distortion product otoacoustic emissions: A normative study. Audiology 35, 231^245. Wagner, W., Plinkert, P.K., 1999. The relationship between auditory threshold and evoked otoacoustic emissions. Eur. Arch. Otorhinolaryngol. 256, 177^188. Zenner, H.P., 1994. Ho«ren. Thieme, Stuttgart. Zheng, X.Y., Henderson, D., Hu, B.H., Ding, D.L., McFadden, S.L., 1997a. The in£uence of the cochlear e¡erent system on chronic acoustic trauma. Hear. Res. 107, 147^159. Zheng, Y., Ohyama, K., Hozawa, K., Wada, H., Takasaka, T., 1997b. E¡ect of anesthetic agents and middle ear pressure application on distortion product otoacoustic emissions in the gerbil. Hear. Res. 112, 167^174.
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E¡ects of industrial noise exposure on distortion product otoacoustic emissions (DPOAEs) and hair cell loss of the cochlea ^ long term experiments in awake guinea pigs E. Emmerich a
a;
*, F. Richter a , U. Reinhold a , V. Linss b , W. Linss
b
Institute of Physiology I, Department of Neurophysiology, Friedrich Schiller University, Teichgraben 8, D-07740 Jena, Germany b Institute of Anatomy I, Friedrich Schiller University, Jena, Germany Received 13 January 2000; accepted 21 April 2000
Abstract Distortion product otoacoustic emissions (DPOAEs), a sensitive detector of outer hair cell (OHC) function, cochlear microphonics (CM), and hair cell loss have been monitored in 12 awake guinea pigs before and after 2 h exposure to specific, playedback industrial noise (105 dB SPL maximal intensity). All animals had stable DPOAE levels before noise exposure. In the first hours after noise exposure DPOAE levels were reduced significantly. In about 70% a partial recovery of the DPOAEs was found within 4 months after noise exposure. In 16% of the investigated ears no recovery of DPOAEs was observed. However, in a few ears increased DPOAEs were observed after noise exposure. Exposure to industrial noise caused both morphological changes in the middle turns of the cochlea and electrophysiological changes in the middle frequency range. A close correlation existed between reduced DPOAE levels, loss in CM potentials, and area of damaged or lost OHCs, but not with the numbers of damaged or lost OHCs in the cochlea. It can be concluded that continuous industrial noise causes a damage to OHCs which differs form the damage caused by impulse noise. ß 2000 Elsevier Science B.V. All rights reserved. Key words: Distortion product otoacoustic emission; Outer hair cell; Continuous noise exposure; Cochlear microphonics ; Scanning electron microscopy; Hearing loss; Guinea pig
1. Introduction Numerous studies have examined the e¡ects of highly intense noise produced in industrial plants on the inner ear of man (Henderson and Salvi, 1998; Diero¡, 1994 ; Borg et al., 1983 ; Dobie, 1983). In many branches of light industry, the noise emitted consists of both impulse noise and noise with a broad frequency spectrum and a peak intensity below 100 dB SPL (Henderson and Hamernik, 1986). A typical example is noise from the washing and ¢lling machines in a brewery. Whether or not such continuous but non-painful noise would damage hearing function can be investigated in long term experiments on awake animals. In order to assess alterations of hearing function, in the present study we re-
* Corresponding author. Tel.: +49 (3641) 938811; Fax: +49 (3641) 938812; E-mail: [email protected]
corded in guinea pigs distortion product otoacoustic emissions (DPOAEs) that are evoked by bitonal stimulation and are routinely used for the clinical diagnostic screening of frequency-dependent cochlear function (Wagner and Plinkert, 1999 ; Lichtenstein and Stapells, 1996 ; Vinck et al., 1996 ; Zenner, 1994; Hauser et al., 1991). In humans, measurements of DPOAE are used for the early and di¡erential diagnosis of damage to the outer hair cells (OHCs) (Henley et al., 1996 ; LonsburyMartin et al., 1993). Experiments in animals have shown that DPOAE as well as transiently evoked otoacoustic emissions (TEOAE) are altered by ototoxic drugs, noise exposure and hypoxia (Zheng et al., 1997b ; Hofstetter et al., 1997 ; Hamernik et al., 1996; Henley et al., 1996; Brown and Gaskill, 1990 ; Cody and Russell, 1988, 1987 ; Kemp and Brown, 1983). We have previously shown that cochlear hair cells of the guinea pig can be damaged by a single noise impulse of 164 dB peak equivalent (pe) SPL (rise
0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 1 0 1 - 5
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time 6 0.1 ms) that was re£ected by reduced amplitudes of cochlear microphonics (CMs) (Emmerich and Biedermann, 1993; Emmerich et al., 1990; Linss et al., 1991 ; Richter and Biedermann, 1987; Meyer et al., 1985 ; Geyer et al., 1978). In impulse noise-exposed animals, DPOAE stimulated with higher frequencies were diminished and cochlear damage, i.e., hair cell loss, was shown morphologically in the ¢rst turn of the cochlea, which is sensitive to high frequencies (Emmerich et al., 2000). Prolonged exposure of awake chinchillas to continuous noise (Eddins et al., 1999; Hamernik et al., 1998) led to reduced DPOAE levels, but, in a few cases, DPOAEs were increased in the post-exposure period. The aim of our present study was to test the levels of DPOAEs after exposure to speci¢c industrial noise in awake guinea pigs, and to relate the changes in DPOAEs to CM and also to morphological changes in the cochlea. We wanted to test whether DPOAE is a reliable parameter for the prediction of hair cell loss, and whether DPOAEs could be used for monitoring the changes in inner ear function caused by noise exposure. The data should indicate the potential danger of continuous noise to employees in light industry, and they should be relevant for noise protection in these branches of industry. 2. Materials and methods 2.1. DPOAE recording Twelve female guinea pigs at an age of 3^4 months, weighing about 400 g, were used for these experiments. For the recording and subsequent analysis of DPOAE we used the ILO92 (Otodynamics Ltd., Herts, UK). The acoustic probe was held by hand to the opening of the external ear channel (meatus acusticus externus) with gentle pressure. The levels of DPOAE were measured in both ears at f2 stimulation frequencies of 1.5^ 6.0 kHz (frequency ratio: f1 /f2 = 1.22). Both tones had 60 dB SPL. DPOAEs were included in the analysis if they were at least 3 dB above background noise. During the measurements, both the experimenter and the experimental animal were placed in an sound-proof chamber. All measurements of the DPOAEs were performed in awake animals which were kept in a cage to restrict movements and were carried out by the same experimenter to whom the animals were habituated. This allowed the DPOAE to be recorded with a minimum of contaminating movements from the animals. A detailed description of these methods is given by Emmerich et al. (2000). Before noise exposure (see below), at least 25 recordings from both ears were performed in each guinea pig
within 8 weeks to obtain a stable pre-exposure baseline. Immediately after the ¢rst noise exposure DPOAEs were recorded at 5 min intervals for a total period of 2 h. The recording was repeated daily during the ¢rst 3 days after noise exposure and then at regular intervals of 2 days until DPOAE amplitudes had stabilized. Four months after noise exposure, the experimental protocol was terminated. Only when DPOAEs had recovered within 3 weeks to 65% or greater of pre-exposure values, a second exposure to the same noise was performed. Six animals without recovery of DPOAE therefore received only one noise exposure. For analysis, the DPOAEs before noise exposure were averaged and normalized in each animal. In addition, a grand mean over all animals before noise exposure was obtained. DPOAE data obtained after noise exposure were expressed as absolute levels and in percent of the pre-exposure values. Di¡erences between the pre-exposure values and the values after impulse noise exposure were compared for each test frequency with Student's t-test. Statistical signi¢cance was set at P 6 0.05. 2.2. Noise exposure Speci¢c industrial noise was recorded with a DATrecorder in the bottle washing and ¢lling department of Braugold GmbH Brewery at Erfurt. The frequency spectrum and intensities of that kind of noise are shown in Fig. 1. Noise exposure was performed by playing-back the recorded noise at an intensity of 105 dB SPL for 2 h to awake guinea pigs that were kept in the cage used for DPOAE recording. Movement of the head was restricted by using a small box which enclosed the whole body of the animal except the head. Noise presentation
Fig. 1. Frequency spectrum of the realistic industrial noise used in this study. Data are given as mean values (bars) with minimum (a) and maximum (#) values. The columns A and L show recordings with the ¢lters `A' and `L' without frequency distributions. Standard deviations were very low and are indicated (X).
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was performed by a high ¢delity sound system, consisting of a compact disc player (Pioneer, PD-306), an ampli¢er (Yamaha, P4500) and a loudspeaker. The distance between the loudspeaker and the ears of the animal was about 10 cm. The loudspeaker was placed in front of the head, so both ears were a¡ected by the noise with the same intensity. The frequency spectrum and the amplitudes were determined before the experiments using the Krones^Block con¢rming the identity of the original and the played-back noises. 2.3. CM potential recording and morphological analysis After the last DPOAE recording, animals were deeply anesthetized with sodium thiopental (Trapanal Byk Gulden, 100 mg/kg, i.p.) and placed in a head holder. The animals were breathing spontaneously room air. Body temperature was maintained by a heating pad at 37³. After administration of local anesthetics, skin and muscles were incised behind the right and left outer ear channels and a burr hole was made at each side that exposed the round window of the cochlea. An Ag^ AgCl-electrode was placed at the round window membranes of both ears for CM recordings. Test stimuli for CMs were sine waves using the frequencies of 0.5^ 10.0 kHz. The intensity of the sound was 60 dB SPL. Monopolar CMs were recorded, ¢ltered and then evaluated in dB by an audiofrequency spectrometer 2109 (Bru«el and Kjaer, Denmark). Amplitudes of CMs were compared to those obtained in non-exposed animals (Biedermann et al., 1977). After killing the anesthetized animals by decapitation, the cochleae were perfused with glutaraldehyde and the upper turn was opened. The cochleae were dehydrated with ethanol, critical point dried and sputtered with gold for scanning electron microscopy. The exposed apical turn of the organ of Corti was documented with the scanning electron microscope (Stereoscan 260, Cambridge Instruments, UK), then the fol-
Fig. 2. Mean values of pre-exposure DPOAE levels (12 guinea pigs and 25 individual tests in each animal, mean value þ S.E.M.). A slight drop at the stimulus frequency f2 = 4.0 kHz can be noted. Repeated pre-exposure tests con¢rmed that replicable results could be obtained with the hand-held probe.
11
lowing turn was opened and similarly prepared. Using this method, the whole cochlea was investigated turn by turn. For evaluation, each cochlear turn was subdivided into four sectors (that is A1^A4, B1^B4, C1^C4, and D1^D3, see Fig. 6). In all sectors OHCs were counted and loss of OHCs compared to unexposed animals (Meyer et al., 1985) was documented. The care and use of the animals included in this study was approved by the Thu«ringer regional government (Landesverwaltungsamt), reg. no. 02-27/97). 3. Results 3.1. Noise e¡ects on DPOAE and CM Prior to noise exposure all animals showed reproducible DPOAEs. A pre-exposure mean value for all animals is given in Fig. 2. The levels of DPOAE exhibited a maximum at 5 and 6 kHz. At 4 kHz a relative reduction of the DPOAE was often noted. No di¡erences were found comparing right and left ears of the animals. After noise exposure, three di¡erent types of recovery of DPOAE were found. 3.1.1. Partial recovery In most of the animals (about 70% of the ears investigated), only a partial recovery was seen. An example from one animal with partial recovery restricted to the higher frequency range is shown in Fig. 3A that was characterized by a progressive decline and loss of DPOAEs at test frequencies lower than 3.0 kHz, and weakly diminished DPOAEs at frequencies of 4.0^ 6.0 kHz up to 1 day after noise exposure. Ten days after noise exposure, DPOAEs could be observed using test frequencies of 2.5^6.0 kHz. However, 7 weeks after noise exposure, only DPOAEs with lower levels were observed at 6.0 kHz, whereas no DPOAEs were recorded in the lower frequencies. In six animals a substantial recovery of DPOAEs to 65% or more of pre-exposure values was observed in the whole frequency range. Within 10 min to 1 h and also 2 h after noise exposure none of these guinea pigs showed any DPOAE at all. One to two days after noise exposure, recovery was seen at frequencies higher than 2.5 kHz, reaching nearly pre-exposure values. Recovery of DPOAEs at frequencies of 1.5 and 2.0 kHz started 3 days after noise exposure and reached levels of 60% or more of pre-exposure values 9 days after exposure. In these six animals a second exposure to the same noise after 3 weeks replicated the above mentioned changes. Again, only DPOAEs evoked by test frequencies higher than 5.0 kHz recovered very quickly, reaching initial values by 10 min post-exposure. DPOAEs obtained by test frequencies of 1.5^4.0 kHz remained
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Fig. 3. A,B: Representative example of partial recovery of DPOAE after exposure to industrial noise. A: Diagram showing partial recovery of DPOAE at high frequencies after one single exposure to industrial noise (2 h, 105 dB pe SPL). DPOAEs `before' are the mean values for this individual animal taken from 25 pre-exposure tests. At the end of the observation period of 7 weeks, DPOAEs could be recorded only at a frequency f2 6.0 kHz, no responses could be obtained at lower f2 frequencies. B: Scanning electron microscopic micrographs (1600:1) of damaged OHCs from sectors C3/C4 in this animal: the IHCs (top row) were not damaged by the noise. OHCs (three rows at bottom) are severely damaged, hairs are destructed and whole cells are lost. This damage a¡ected all three rows of OHC, preferentially the second and third rows (OHC 2 and OHC 3 in Fig. 6). In adjacent sectors, a similar pattern of destruction was seen.
diminished or were lost. Only little signs of restitution were found in DPOAE at test frequencies of 2.0 and 3.0 kHz. Fig. 4A shows a representative example for this subset of recovery from one animal of this group. The pre-exposure values given in the left part of the diagram resemble those from the other animals. It can be seen that recovery reached pre-exposure values in the whole frequency range 9 days after the ¢rst noise exposure, but was restricted to frequencies of 2.0 kHz and higher after the second noise exposure. In this particular animal, only little signs of recovery were seen at a test frequency of 4.0 kHz.
3.1.2. Overshooting recovery In two out of 12 animals (three ears), the initial loss of DPOAEs was very short-lived and complete recovery occurred within 9 days after noise exposure. A temporary increase in DPOAEs up to 170% of pre-exposure was registered especially at high frequencies. Between day 9 and 12 post-exposure, this increase, however, recovered to normal values. Then the two animals received a second noise exposure at the same time point as the above mentioned animals (Fig. 5). As after the ¢rst exposure, an initial drop in DPOAE was followed by increased DPOAEs that again exceeded pre-expo-
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sure values up to 170% in all frequencies tested. This increase could be observed at 2 weeks post-exposure and remained at that level up to 3 weeks, when this experiment was terminated. 3.1.3. No recovery of DPOAE In one guinea pig a complete and persistent loss of DPOAEs was observed already after the ¢rst noise exposure. As in the other animals, immediately after noise exposure DPOAE at frequencies of 1.5^3.5 kHz were severely diminished or even reduced to zero. DPOAE
13
that could be recorded at frequencies of 4.0^5.0 kHz within 10 days after noise exposure were diminished in the second week after noise exposure and did not recover within 7 weeks. A complete survey of the observed e¡ects in all guinea pigs is given in Table 1. As representative examples, normalized data for two DPOAE stimulation frequencies and for a 6.0 kHz sine wave to elicit CM are shown. Data are depicted only from right ears, since no di¡erences existed to the left ears of the animals. In the ¢rst hour after noise exposure, DPOAEs were sig-
Fig. 4. A,B: Representative example of complete recovery of DPOAE after exposure to industrial noise. A: DPOAE recordings, values `before' give mean values for this individual animal taken from 25 pre-exposure tests. Recovery of DPOAE started after a short period following the ¢rst exposure to noise (indicated by arrow I). The animal was exposed for a second time after the complete recovery after 9 days to the same noise (indicated by arrow II). Again, after an initial drop in DPOAE a recovery was observed that was nearly complete in frequencies f2 higher than 2.5 kHz. A partial recovery was seen at lower f2 frequencies. B: Scanning electron microscopic micrographs (1600:1) of damaged OHCs in sector B4 in this animal: IHCs (top row) were not a¡ected by the noise. A small area of lost OHCs in the third row was found. Adjacent cells in the third row were damaged and had clotted or destructed hairs.
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Fig. 5. Typical example of DPOAEs with transient overshooting recovery. Values `before' give mean values for this individual animal taken from 25 pre-exposure tests. In a range of 1 h to 9 days after the ¢rst noise exposure (arrow I) DPOAEs exceeded pre-exposure values in most f2 frequencies tested. Between days 9 and 12 the DPOAE tended to lower (normal) values. A second exposure to the same noise (arrow II) replicated this result. In the whole frequency range tested 2 weeks after a second noise exposure, DPOAEs exceeded the pre-exposure values.
ni¢cantly diminished or lost in most of the animals. The animals in which the DPOAE levels recovered to 65% or more of pre-exposure values, received a second noise exposure following the 2 weeks data recording. As can be seen in Table 1, after the second noise exposure DPOAE were less diminished than after the ¢rst exposure. The reason for this phenomenon is unclear. CM recorded at the last day of the experimental protocol were declined in most of the noise exposed animals compared to normal hearing ones. This was in agreement with the DPOAE recordings. A positive and frequency-speci¢c correlation (slope 0.64 þ 0.22,
r = 0.45) was found. The decline in CM, however, did not parallel the decline in DPOAE level. 3.2. Results of scanning electron microscopy The cochlea of the guinea pig consists of four turns (turns A^D, Fig. 6) with three rows of OHCs and one row of inner hair cells (IHCs). In normal guinea pig cochleae usually very few OHCs are missing, and the typical formation of the three rows exists from the Ato the C-turn. In the D-turn the typical pattern of rows is absent, up to the helicotrema usually single OHCs were found.
Table 1 Percentage changes in the levels of DPOAE recorded from right ears after exposure to industrial noise in 12 guinea pigs (gp1^gp12) tested gp1
gp2
gp3
gp4
(a) DPOAE f2 : 3 kHz, data from right ears, *P 6 0.05 Control 100 100 100 100 1h 0 0 0 0 2 weeks 163.2* 0 0 79.3 1h 0 8.42* 3 weeks 44.44* 79.65* (b) DPOAE f2 : 6 kHz, data from right ears *P 6 0.05 Control 100 100 100 100 1h 0 0 0 0 2 weeks 15.42* 88.36 37.97* 97.07 1h 59.93* 72.9* 3 weeks 93.15 65.81* (c) CMs recorded at termination of experimental series, 6.0 kHz 60 100 0 90
gp5
gp6
gp7
gp8
gp9
gp10
gp11
gp12
100 49.01* 66.45 45.39* 10.86*
100 0 106.4
100 9.58* 79.04
100 10.77* 11.54* 38.46* 149.2*
100 61.02* 68.21 43.08* 68.2*
100 70.95 24.76*
100 22.22* 0
100 2.22* 54.07* 107.4 28.89*
100 75.59 86.29
100 2.29* 61.07*
100 33.23* 72.58* 71.29 90.97
95
87
56
100 100 0 44.63 92.91 85.54 50.39* 74.02 stimulus pure tone, 80 70
100 79.88* 96.28
100 100 98.51 82.86 62.83 77.14* 0 87.86 114.4 18.57* 60 dB SPL, data from right ears 100 98 40
The tables give values for the f2 frequencies 3.0 (a) and 6.0 kHz (b). The mean values of DPOAE levels in each animal before noise exposure were taken as 100% (control). After 2 weeks in six animals a second exposure to noise was performed, indicated by further data in the columns. In animals without a second noise exposure these columns are empty after the 2 week line, respectively. The 3 week line gives data that were obtained 3 weeks after the second noise exposure. Heavy numbers indicate an overshooting recovery. Asterisks indicate statistically signi¢cant di¡erences to pre-exposure (P 6 0.05, Student's t-test). CM (c) stimulated with a 6.0 kHz sine wave were recorded at the end of the experimental series. The table gives percentage levels related to normal hearing animals taken from Biedermann et al., 1977.
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Fig. 6. OHC loss in the di¡erent parts of cochlear turns. The diagram on the left shows percentage OHC loss, and the diagram on the right represents the four turns of a cochlea (not to scale) with the subsections indicated. Data on the left were taken from the guinea pig with DPOAE decline and OHC loss shown in Fig. 4A. Right panel: The three rows of OHCs are indicated by the di¡erent shades of the bars. Sector D3 was excluded, since the regular formation of the three rows of OHC started only at the basal part of the D-turn.
After exposure to industrial noise, the morphological observations showed di¡erent patterns of damage or loss of OHCs only. In none of the animals IHCs were damaged or lost. The cochleogram in Fig. 6 represents a typical pattern of OHC loss in an animal with reduced DPOAE and incomplete recovery. Little damage was seen in sectors A1 to B2, whereas large numbers of lost OHC were found in sectors B3 to D2, preferentially in the third row of the OHCs, but to a lesser extent also in the ¢rst and second rows of OHCs. This damaged area corresponded well to the loss of DPOAEs at lower test frequencies. As shown in Fig. 3B, damage to the second and third rows of OHC found in sectors C3 and C4 after single exposure to noise was re£ected by a complete loss of DPOAE evoked by 1.5^5.0 kHz. Fig. 4B shows an example of partial recovery of DPOAE that was re£ected by a pattern of lost and damaged OHC in sector B4, but almost restricted to the third row of OHC. However, no relation existed between numbers of lost OHC and percentage decline in DPOAE. 4. Discussion In the present study we tested e¡ects of a speci¢c continuous industrial noise on otoacoustic emissions (DPOAEs), on CMs, and on the morphology of OHCs. In previous experiments we found that DPOAE in guinea pigs under control conditions were similar to those in man if a frequency ratio f1 /f2 = 1.22 (Brown, 1987) was used. However, our level of stimulation was about 10 dB SPL higher for stimulus frequencies f2 s 2 kHz than that used in man. (Emmerich et al.,
2000 ; Brown and Gaskill, 1990; Kemp and Brown, 1983). It is known that rodent ears are most sensitive at higher frequencies (Hamernik et al., 1993). However, our equipment was designed for human investigations. The f2 stimulus frequencies used here were in the range of 1.5^6.0 kHz. In this range DPOAE levels rose with increased stimulus frequency. This slope was similar to the data in anesthetized guinea pigs (Brown and Gaskill, 1990), but had a slight drop at 4.0 kHz and levels in our guinea pigs were in mean 5^10 dB SPL higher. We relate these di¡erences to the use of awake animals that have a functional intact e¡erent innervation of the cochlea. The ear-damaging e¡ect of industrial noise can only be assessed by carrying out experiments in animals. Studies for the evaluation of noise e¡ects and investigations of DPOAEs or CMs have mostly been performed in anesthetized guinea pigs (Brown, 1987). It has been established that anesthesia exerts in£uences on inner ear's susceptibility to noise, probably due to an altered cochlear blood £ow (Angelborg et al., 1984; Angelborg et al., 1979 ; Hultcrantz et al., 1979). Therefore, our study used awake animals, which allowed repeated measurements of DPOAEs over long time periods with minimal strain for the animals. Stable DPOAEs before noise exposure showed that guinea pigs were well adapted to the experimental arrangement. Shaddock et al. (1985) reported on experiments on chinchillas that highly intense impulse noise a¡ected not only the morphology of OHC and IHC but also the vascular system of the inner ear. In previous experiments we con¢rmed these ¢ndings by using non-invasively recorded DPOAE. We could show that exposure
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E. Emmerich et al. / Hearing Research 148 (2000) 9^17
of awake guinea pigs to impulse noise of 153 dB pe SPL caused OHC loss and degeneration of OHC between the ¢rst and second turns of the cochlea, and a reduction or even a loss of DPOAEs and CMs mainly with f2 stimulus frequencies greater than 4.0 kHz (Emmerich et al., 2000 ; Linss et al., 1991; Richter and Biedermann, 1987 ; Meyer et al., 1985). In the past, changes in DPOAE or in CM after continuous noise exposure were reported in chinchillas (Hamernik et al., 1998; Hamernik et al., 1996; Subramaniam et al., 1995; Hamernik et al., 1993) and in rabbits (Mensh et al., 1993a,b). In the present study we used real, played-back industrial noise with frequencies up to 21 kHz and peak intensities up to 105 dB SPL instead of a physically de¢ned octave band noise. We applied this noise for only 2 h. Interestingly, this short exposure already produced DPOAE shifts in the awake guinea pigs tested. The chosen application time agreed with Popelar and Syka (1993) and Popelar et al. (1987) who found damaged hearing function in guinea pigs after comparable short exposure times. After exposure to broadband industrial noise, hearing loss occurred at lower frequencies than after impulse noise. After industrial noise, responses to stimulus frequencies lower than 3.0 kHz were signi¢cantly more reduced, and recovered less, than responses to higher frequencies. This pattern of DPOAE changes corresponded to the morphological changes that were mostly found in turns B to D of the cochlea. In fact, our experiments showed a close correlation between frequency-speci¢c changes of DPOAE or of CM and the loss in OHC of a speci¢c cochlear turn. We could con¢rm that there is not always a close correlation between the number of damaged or lost hair cells in the whole cochlea and the extent of changes in DPOAE or in CM, and agree with Ahroon et al. (1993); Hamernik et al. (1993) and Lonsbury-Martin et al. (1993), who showed a discrepancy between audiometric data and numbers of lost or damaged OHCs after exposure to continuous or to impulse noise. What could be the reason for the increase in DPOAE after noise exposure? Zheng et al. (1997a) investigated noise e¡ects in chinchillas and found marked di¡erences between normally innervated and dee¡erented inner ears. Distortion product-grams obtained in awake and normal hearing guinea pigs or gerbils obviously di¡ered from those in anesthetized animals (Zheng et al., 1997b ; Brown, 1987). A temporary dee¡erentation by anesthesia could be an explanation for this ¢nding. There is some evidence that e¡erent projections to the OHC would be changed by noise and could increase hair cell motility (Zheng et al., 1997a). This hypothesis could explain a changed vulnerability of the cochlea (Henderson and Salvi, 1998). The particular reason for this increase, however, is unclear, especially, because only
a minority of animals produced such increased DPOAE without any predictability. It was speculated that tinnitus could be related to the increased DPOAE caused by a changed e¡erent cochlear innervation after acoustic trauma (Emmerich et al., 2000 ; Frolenkov et al., 1998 ; Kakigi et al., 1998). The experimental design used here could not solve this question. Our experiments showed that DPOAE can be recorded in awake animals in a reproducible manner over several months. Since morphological observations and recording of CM were only performed at the end of the experiment, no CM could be obtained during the follow-up of DPOAE changes after noise. We only checked whether or not a CM response existed after noise exposure. Earlier studies by Kirk et al. (1997) proposed a mechano-electric transduction transfer that should relate CM and the micromechanic phenomenon of DPOAE that could be a cause for the loss in CM in animals without DPOAE after noise exposure. In summary, the present study showed that continuous industrial noise produced in most of the animals a reduction in the levels of DPOAE that was probably caused by damage of the OHC. DPOAE seem to be a reliable parameter for the assessment of hearing loss and its recovery that can be used in non-invasive investigations. We assume that the increase in DPOAE after noise exposure could be due to changes in e¡erent innervation also caused by the damage. Since a noise exposure for only 2 h already resulted in severe and non-predictable damages, ear protection should be used in all noisy areas of light industry independently of the time of noise exposure. Acknowledgements The authors thank Professor Hans-Georg Schaible for comments and help during writing the manuscript and Ms. Gundula Kruse for her excellent assistance in the experiments with awake animals. The work was supported by the Deutsche Forschungsgemeinschaft (Em 57/1-1), and by Berufsgenossenschaft Nahrungsmittel und Gaststa«tten, Gescha«ftsbereich Pra«vention.
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