The effect of ‘conditioning’ on hearing loss from a high frequency traumatic exposure

Hearing Research, 58 (1992) 57-62 © 1992 Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/$05.00

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The effect of 'conditioning' on hearing loss from a high frequency traumatic exposure M a l i n i S u b r a m a n i a m 1, D o n a l d H e n d e r s o n ~, P i e r r e C a m p o 2 a n d V l a s t a S p o n g r 1 i Hearing Research Laboratory, Department of Communicatire Disorders Sciences, State Unirersity of New York, Buffalo, NE, U.S.A., and 2 Institut National de Recherche et de Securite, Vandoeucre, France (Received 19 April 1991~ accepted 2 October 1991)

The role of high frequency, low level "conditioning" exposures as moderators of hearing loss from subsequent exposure to the same noise at a higher level was studied using monaural chinchillas. All the animals in the experimental groups were exposed to an octave band noise centered at 4 kHz at 85 dB SPL for 6 h a day for 10 days. One of the experimental groups was allowed to recover for 5 days and the other was allowed to recover for 18 h, prior to the higher level exposure at 100 dB for 48 h. A third group exposed only to the higher level constituted the control group. A comparison of threshold shifts and hair cell loss after 4 weeks of recovery across the three groups revealed: (a) the 5-day recovery group incurred greater threshold shifts than the other two groups; the hair cell loss in this group was greater than in the 18-h recovery group, but the same as in the control group and (b) the 18-h recovery group incurred considerably less threshold shift as well as hair cell loss than the other two groups. The results were also compared with the results from similar exposures using low frequency noise which indicated that the base vs. apex differences in the cochlea appear to extend to th¢ effects of "conditioning" exposures. Hearing loss; 'Conditioning'; High frequency traumatic exposure; Base vs. apex

Introduction

Since the pioneering work of Bekesy certain systematic differences between the base and the apex of the cochlea have been recognized. The obvious differences include a stiffness gradient between the base and the apex of the cochlea, the base being 100 to 200 times stiffer than the apex (Bekesy, 1947). In addition, outer hair cells (OHC) at the base are substantially smaller than those at the apex (Smith, 1968). Developmentally, the basal OHCs mature first and reach a higher degree of differentiation. On closer inspection, more subtle differences are evident. Large efferent nerve endings are more prevalent in the basal end of the cochlea than at the apex (Pujol and Lenoir, 1986). Conversely, OHCs at the apical end receive greater afferent input (Spoendlin, 1969). In addition, the infra-cuticular network of actin is more prevalent in the OHCs of the apical turns of the cochlea (Thorne et al., 1987). PhysiOlogically, frequency coding studied using tuning curves and distribution of VIII nerve characteristic frequencies appear to be different above and below 1 kHz, with the high characteristic frequency (CF) fibers being more sharply tuned than the low CF fibers (Liberman

Correspondence to: Malini Subramaniam, Ph.D., Hearing Research Laboratory, 215, Parker Hail, SUNY at Buffalo, NY 14214, U.S.A.

and Kiang, 1978). Furthermore, the base and the apex of the cochlea also respond differently to noise and ototoxic drugs with the basal region of the cochlea being more vulnerable t~ both the agents (Miller et al., 1963; Hawkins, 1976). Given these documented differences between the base and the apex of the cochlea, it would be interesting to see if the basal region of the cochlea can be made more resistant to noise induced hearing loss by prior exposures to high frequency low level noise, as seen in the case of low frequencies. There is evidence that low level "conditioning' exposures can actually provide protection against damage from further exposures at higher levels at low and mid frequencies (Canlon et al., 1988; Campo et al., 1991). Canlon et al. (1988) studied the protective effects of 'conditioning' on hearing loss from exposures to a 1 kHz tone. A 1 kHz tone presented continuously at 81 dB SPL for 21 days constituted the 'conditioning' exposure. This was followed by an exposure to the same tone at 105 dB for 72 h. Campo et al. (1991) reported a related experiment with 'conditioning' exposures of OBN centered at 0.5 kHz at 95 dB SPL (6 h / d a y for 10 days) followed by exposure to the same noise at 106 dB SPL for 48 h. Despite methodological differences both Canlon et al. and Campo et al. showed that prior exposures to a low level 'conditioning" sound resulted in 10 to 25 dB less permanent threshold shift (PTS) compared to control

58 subjects. The aim of this study was to determine if similar protective effects are also observed in response to high frequency exposures. The initial results from the experiment prompted a second question: what is the role of the recovery time in the protection from traumatic exposure?

Methods

Subjects Seventeen adult chinchillas (500 to 600 g) served as subjects. Each animal was anesthetized with a subcutaneous injection of acepromazine (0.56 mg/kg) and ketamine (36 mg/kg) and made monaural by surgical destruction of the left cochlea. A chronic recording electrode was then stereotaxically implanted in the left inferior colliculus and a ground electrode was implanted just below the dura mater (Henderson et al., 1973). Following surgery, the animals were given antibiotics (chloramphenicoi palmitate twice a day for 4 days) and allowed to recover for at least 2 weeks prior to testing.

Audiometry After the 2 week recovery period, hearing thresholds of all the animals were measured at least five times using evoked potential recording. The thresholds were observed to be stable across the five sessions. The average of these five measures constituted the pre-exposure threshold. Each animal was tested separately in a sound treated booth. A yoke-like harness kept the animal's head in a fixed position within the calibrated sound field. In addition to pre-exposure testing the animals were tested just before and immediately after each 'conditioning' (85 dB) exposure. Hearing thresholds were measured in ascending steps of frequency and intensity so that a given frequency was tested approximately at the same time over the days of exposure. The total duration of testing for each animal was about 20 min. Hearing thresholds were also measured immediately after, 24 h and 5 days after the higher level (100 dB) exposure. After 4 weeks of recovery, thresholds were determined at least five times to calculate the PTS. All threshold shifts were calculated with reference to the pre-exposure baseline. Test stimuli consisted of tone pips (5 ms Blackman rise/fall ramp, constant starting phase) at frequencies from 0.5 to 16 kHz at octave intervals including the mid-octave frequency of 5.6 kHz. Details of stimulus generation and response analysis have been described in detail elsewhere (Campo et al., 1991).

Noise exposure The noise (Gaussian) was generated by a D / A converter on a signal processing board (Loughborough

TMS 32020) in a personal computer (IBM compatible). It was routed through an attenuator (HP 350 D), a filter (Krohn-Hite 3550 R) and a power amplifier (NAD 2200) to an acoustic horn (JBL 2360). The first group of experimental animals (N = 6) was exposed to the noise - OBN centered at 4 kHz - at 85 dB for 6 h a day for 10 days. They were then allowed to recover for 5 days before the traumatic exposure. The same spectrum of noise presented at 100 dB for 48 h constituted the traumatic exposure. The exposure conditions for the second group of experimental animals (N = 5) were the same as the first group, except that subjects were allowed a recovery time of 18-h. The control group (N = 6) was exposed only to the 100 dB noise for 48 h. The animals were exposed in groups of two or three. They were introduced in to the exposure at intervals of 20 min to permit each subject to be tested just before and immediately after the exposure. Each animal was housed in a separate cage (8"× i-'2 ~±" X 8") and given free access to food and water. The cages were placed just below the loud speaker so that the difference in the sound pressure across the cages was less than 1 dB. The animals were rotated to different cages to minimize any effects of differences in sound pressure.

Histology After PTS measurements, each animal was anesthetized using 1 c.c. of pentobarbitol ( I / P ) and decapitated. The right cochlea was then removed and prepared for light microscopy. The cochlea was first perfused with 2.5% gluteraldehyde in veronal acetate buffer (pH 7.3-7.4), through the round window membrane. Twelve to 24 h later it was perfused with 1% osmium tetroxide buffer for one h. The cochlea was then rinsed in bt,.ffer, dissected in 70% ETOH and evaluated using phase-contrast microscopy. Inner and outer hair cell populations were assessed to determine if the hair cell was present or not for each subjec~ and the results were averaged for each condition. The status of the stereocilia or the hair cell were not included in the assessment.

Results

Pre-exposure thresholds Mean pre-exposure thresholds of the three groups of animals are presented in Fig. 1. There are no systematic differences across the three groups in terms of mean thresholds. Further, the thresholds estimated with the evoked potential technique are in good agreement with previously published norms using the conventional but more time consuming behavioral conditioning procedure (Miller, 1970).

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Threshold shift from 'conditioning' exposure

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The results of the 'conditioning' exposures are discussed in depth elsewhere (Subramaniam et ai., 1991). Briefly, the daily exposures at 85 dB did not cause any threshold shift (re: pre-exposure mean) at 0.5 and 1 kHz. At 2 kHz, a 10 dB threshold shift (TS) was seen on day 1 and 2 which decreased progressively to 5 dB or less by day 7. The threshold shifts at 4 kHz and above, recorded just after the 85 dB exposure over the 10 days of exposure are presented in Fig. 2a. It may be seen that the TS decreased dramatically over the 10 days of the 'conditioning' exposure. The thresholds decreased by 12 dB at 4 kHz and 24 dB at 5.6 and 8 kHz with reference to the shift recorded on the first day of exposure. The trend was consistent across all animals and the magnitude of 'toughening' (the difference between the peak shift and the shift on day 10) was about the same in each of the animals (Fig. 2b).

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The threshold shifts recorded at various recovery times after the traumatic exposure are presented in Fig. 4. As expected, animals in all three groups incurred the greatest shift soon after the traumatic exposure at all frequencies. The initial threshold shift as well as the recovery at 2 ld-lz were approximately the same across the three groups. At 8 and 16 kHz the three groups

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Residual hearing loss from 'conditioning' exposure The threshold shift (re: pre-exposure threshold) recorded just before the 100 dB exposure in the two experimental groups is presented in Fig. 3. It may be seen that the group allowed to recover for only 18 h had 10-15 dB residual hearing loss at 8 and 16 kHz. On the other hand, the group allowed a recovery time of 5 days had no residual hearing loss at any frequency.

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DAY OF EXPOSURE Fig. 2. (a) Post-exposure threshold shifts from the 85 dB SPL "conditioning" exposures across the 10 days of exposure. (b) Mean post-exposure threshold shift (dotted line) and the post-exposure threshold shift at 5.6 kHz among the individual animals of group ! (symbols)

incurred different amounts of threshold shift initially but showed little difference at recovery times of 24 h or more. The results at 4 and 5.6 kHz are strikingly different. Here the animals in the 5 day recovery group had the least threshold shift soon after the exposure. Thresholds of animals in the 18-h recovery group and in the control group were often above the limits of the test equipment. In such cases thresholds were assumed to be 5 dB above the upper intensity limit of the test equipment used in the study. Threshold shift in the three groups converged at 24 h. From then on, animals in the 18-h recovery group and the control group recovered substantially more than the animals in the 5 day recovery group. The PTS recorded at various test frequencies are plotted in the form of an audiogram in F~g. 5. It may be seen from Fig. 5 that the three groups had about

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the same amount of PTS at all frequencies except 4 and 5.6 k_Hz where the 5 day recovery group had the greatest PTS and the 18-h recovery group had the least PTS. An ANOVA carried out on the PTS data indicated that the effect of frequency as well as the interaction of frequency and experimental condition were significant (P < 0.01). Further analysis indicated that the effect of frequency was significant (P < 0.01) at 4 and 5.6 kHz, the two frequencies where the audiogram shows a significant difference. When between group differences were compared at these frequencies it was observed that (i) the two experimental ('conditioned') groups were significantly different (P < 0.01) at both frequencies; (ii) the difference between the 5 day recovery group and the control group was significant at both frequencies but only at P < 0.05; and (iii) the difference between the 18-h recovery group and the control group was significant only at 4 kHz (P < 0.1).

Histologicalfindings The average cochleograms obtained for the three groups are presented in Fig. 6. which indicates that all groups incurred a narrow lesion specific to the exposure frequency. The differences across the three groups analyzed separately for the outer and inner hair cell loss using ANOVA. The analysis showed a significant difference between the 18-h recovery group and the control group (P < 0.01) as well as between the two experimental ('conditioned') groups (0.05 > P > 0.01). No significant difference was found between the 5 day recovery group and the control group in terms of OHC loss. The three groups did not differ significantly in terms of IHC loss. However, further study is warranted to better understand the possible sub-cellular changes.

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creased threshold shift is a sign that the auditory system has become more resistant to damage from further exposures, then it is reasonable to expect that the 4 kHz daily exposures would render the subjects more resistant to higher level exposures. Unfortunately, the results are complicated and it is difficult to draw a definite conclusion about the relation between the 'conditioning' exposures and resistance to damage from future exposures.

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Discussion The primary goal of this experiment was to see whether an intermittent exposure to high frequency noise (4 kHz OBN) made that region of the cochlea more resistant to subsequent higher level exposures. In some respects the results of this experiment are like those of previous experiments with low frequency exposures (Canlon et al., 1988; Campo et al., 1991) and in other respects they are quite different.

Conditioning exposures The results of the 4 kHz OBN "conditioning' exposures have been reported in detail earlier (Subramaniam et al., 1991). Like the 0.5 kHz OBN exposure, the 4 kHz OBN exposure produced a reduction in threshold shift from day 1 to day 10. The initial threshold shift at one octave above the exposure frequency was about the same for the 0.5 and 4 kHz exposures. Over the 10 days of exposure, the magnitude of 'toughening' at one octave above the exposure frequency was 15 dB for the low frequency exposure and 24 dB for the high frequency exposure. If the de-

In the Campo et al. (1991) experiment using low frequency exposures, there was a 5 day waiting period between the last 'conditioning' exposure and the higher level exposure. This recovery period was chosen to ensure that the hearing thresholds had completely recovered and returned to baseline. In their experiment the 'conditioned' animals incurred less threshold shift than the control animals. Conversely, in the current experiment the animals allowed to recover for 5 days incurred greater PTS than the control animals. Certainly there is no hint of increased resistance, instead there is a suggestion that the 'conditioned' ears might have become more vulnerable. However, given that the size of the cochlear lesion in this group was not different from that in the control group, there is no supportive evidence for this suggestion. The results from the second experimental group are more puzzling. Eighteen h after the last exposure, there was a residual hearing loss of 10 to 15 dB at frequencies from 4 to 16 kHz. However, this group developed significantly less PTS and hair cell loss than the five day recovery group. In addition, the PTS at 4 kHz as well as the total hair cell loss were significantly less than in the control group. The smaller outer hair cell lesions lend additional credibility to the notion that the 18-h recovery group was more resistant to the noise than the other two groups. The increased resistance suggests two interesting possibilities. First, the 18-h recovery group might have become more resistant to the 100 dB exposure because they had some residual hearing loss at the onset of the higher level exposure. Alternatively, there is possibility that 'conditioning' exposures actually rendered the ears more resistant, but the 'protective' changes were transitory and dissipated by 5 days, thus the 5 day recovery group did not show any protective effect.

Base vs. apex differences These experiments were designed to evaluate base vs. apex differences in response to 'conditioning' exposures. The first difference seen was in terms of hair cell loss. The findings of the histological examination were consistent with those of Bohne et al. (1987) in that the damage from the 4 kHz OBN exposure was narrow and

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restricted to the frequency of exposure. A comparison with the results of the low frequency exposures (Campo et al., 1991) suggests that the cochlear damage from the two exposures is strikingly different i.e., the low frequency exposure results in a more diffuse damage, whereas the high frequency exposure causes a narrow frequency-specific lesion. The differences in PTS in the five day recovery group in this study and in the previous studies by Canlon et al. (1988) and Campo et al. (1991) with low frcquency exposures probably reflects either basic biological difference between the base and the apex of the cochlea or in the action of the acoustic reflex (AR). The role of the acoustic reflex in these 'conditioning' exposures is ambiguous. On one hand, change in the effectiveness of the AR is a possible cause for the protection provided for low frequency exposures (Canlon et al., 1988; Campo et al., 1991). However, because of its limited attenuation effect at frequencies above 2 kHz, the AR is less likely to afford protection from 4 kHz OBN exposures. Therefore, it is difficult to conceive of how the acoustic reflex would play a role in the consistent reductions in the threshold shift from daily 'conditioning' exposures both for low and high frequency exposures. Conversely, if the differences in the results are related to anatomical a n d / o r physiological differences, then there are several possible contenders such as the differences in the outer hair cells, distribution of infra-cuticular actin, afferent a n d / o r efferent innervation, and differences in neurotransmitters.

Conclusions It may be concluded from the findings of the present study that 'conditioning' exposures do protect the auditory system from subsequent higher level exposures. However, the time-course of recovery following 'conditioning' exposure appears to be an important factor in determining its ('conditioning') effect on furtheir exposures. The results also suggest that the mechanisms of 'toughening' may be differeat at low and high frequencies. Further anatomical studies are war~,,nted t~ answer the questions raised in this study.

Acknowledgements The authors thank Mr. Samuel S. Saunders for his assistance in statistical analysis. This research was supported by a grant from NIOSH (5ROIOHO115209).

References Bekesy, G. yon (1947) Variation of phase along the basilar membrane with sinusoidal vibrations. J. Acoust. Soc. Am. 452-460. Bohne, B.A., Yohman, L. and Gruner, M.M. (1987) Cochlear damage following interrupted exposures to high frequency noise. Hear. Res. 29. 251-264. Campo, P., Subramaniam, M. and Henderson, D. (1991) The effect of 'conditioning' exposures on hearing loss from traumatic exposure. Hear. Res. 55, 195-200. Canlon, B., Borg, E. and Flock, A. (1988) Protection against noise trauma by pre-exposure to a low level acoustic stimulus. Hear. Res. 34, !97-200. Henderson, D., Hamernik, R.P., Woodford, C., Sitler, R.W. and Salvi, R. (1973) Evoked-response audibility of the chinchilla. J. Acoust. Soc. Am. 54, 1099-1101. Hawkins, J.E. (1976) Drug Ototoxicity. In: W.D. Keidei and W.D. Neff (Eds.). Handbook of Sensory Physiology, Vol. V. Auditory System. Springer-Verlag, pp. 707-748. Liberman, M.C. and Kiang, N.Y. (1978) Acoustic trauma in cats. Cochlear pathology and auditory nerve activity. Acta. Otolaryng. 358, 1-63. Miller, J.D. (1970) Audibility curve of the chinchilla. J. Acoust. Soc. Am. 48, 513-523. Miller, J.D., Watson, C.W. and Coveli, W.P. (1963) Deafening effects of noise on the cat. Acta Otolaryng. Suppl. 176. Pujol, R. and Lenoir, M. (1986) The four types of synopsis in the organ of Corti. In: R.A. Altschuler, R.P. Bobbin and D.W. Hoffman (Eds.). Neurobiology of Hearing: the Cochlea. Raven Press, New York, pp. 161-172. Smith, C.A. (1968) Ultrastructure of the organ of corti. Adv. Sci. 24, 419-433. Spoendlin, H. (1969) Neural connections of the outer hair cell system. Acta Otolaryng. 87, 381-387. Subramaniam, M., Campo, P. and Henderson, D. (1991) Development of resistance to hearing loss from high frequency noise. Hear. Res., in press. Thorne, P.R., Carlisle, L., Zajic, G., Schacht, J. and Aitschul~r, R.A. (1987) Differences in distribution of F-actin in hair cells along the guinea pig organ of Corti. Hear. Res. 30, 253-266.