194
Hearing Research, 62 (1992) 194-198 © 1992Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/~05.00
HEARES 01800
Hair cell damage after continuous and interrupted pure tone overstimulation: A scanning electron microscopic study in the guinea pig L. F r e d e l i u s a n d J. Wers~ill Department of Otolaryngology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden
(Received 14 November 1991; Revision received 15 May 1992;Accepted 29 May 1992)
In our earlier investigations[Fredelius et al., Hear. Res. 30, 157-167 (1987)] acoustic trauma was studied after continuous 3.85-kHz pure tone exposures of different intensities and durations. In the present investigation, the importance of the introduction of a break during longer 3.85-kHz pure tone exposures was studied. Female pigmented guinea pigs were exposed to 108, 114, or 120 dB SPL for 6 h with or without a 1-h break after the first 3 h. Four weeks after exposure the cochleas were prepared for scanning electron microscopy and the resulting hair cell damage was evaluated according to a 4-grade damage scale. Significantdifferences could be demonstrated in the hair cell damage in the animals exposed to continuous acoustic overstimulation and those exposed to intermittent overstimulation. The importance of rest periods to decrease hair cell damage during long periods of acoustic overstimulation was clearly demonstrated. Acoustic trauma; Cochlear damage; Scanning electron microscopy
Introduction
Information on the damaging effects of noise has been obtained by studying the relationship between exposure to noise and the effects on hearing by means of functional measurements in different animal models (Miller, 1963; Blakeslee, 1978) as well as morphological studies of the inner ear after exposue to different acoustic conditions (Nilsson et al., 1982; Stopp, 1983; Bohne et al., 1987; Fredelius et al., 1987). The damaging effects of noise on man have also been demonstrated (Ward 1976) by studying the relationship b6tween exposure to noise and deterioration in hearir, g using audiometric measurements. Systematic studies are difficult to make, however, since exposure to noise varies for different working periods and conditicris, Ward and Nelson (1971) pointed out that risk crit;:fia for damage which ignore the temporal pattern of the noise are erroneous. In our earlier investigations (Fredelius, 1988), re~ suits have been presented regarding electrophysiological and morphological changes occurring after continuous pure-tone exposures of varying intensities and duration. A close relationship was demonstrated between measured CAPN1 threshold shift and the morphological changes evaluated using the S E M and LM techniques. The SEM method for grading of hair cell
Correspondence to: L. Fredelius, Department of Otolaryngology, Karolinska Hospital, S-10401 Stockholm, Sweden.
damage made possible a statistical evaluation and comparison between the damaging effects of different sound energies and intensities. The aim of the present study was to investigate the effect on inner ear damage after the introduction of a break dividing continuous noise into two periods, using the above mentioned qualitative method to evaluate the damage.
M a t e r i a l and M e t h o d s
Twent3~-eight female pigmented guinea pigs with an approx, weight of 250 g were exposed to a 3.85-kHz pure tone. The animals were exposed to sound intensities of 108, 114, and 120 dB SPL for either 6 h or 3 + 3 h with a 1-h break (Table I). The exposures occurred in an anechoic chamber. The animals were able to move about ~reely during exposure in a wire-mesh cage mounted below a horn-speaker (James B. Lansing, TABLE I Experimental design Animals
Intensity (dB SPL)
Duration (h)
4 4 4 4 4 4 4
120 120 114 114 108 108 controls
6 3 + 3 (1-h paus) 6 3 + 3 (1-h paus) 6 3 + 3 (1-h paus)
195
Fig. 1. a) Grade-l-4 damage. Grade-1 damage represents 10-50% damage to or loss to individual hair cell stereocilia and grade 2 represents 50-100% damage to or loss of individual hair cell stereocilia. Gra~e 3 represents loss of individual hair cells and (b) grade 4 a total loss and collapse of the organ of Corti~
Mod. 2552 driver and a 2360 TH horn). The exposures were monitored with a calibrated microphone and the sound level within the cage was constant within + 1 dB. Four weeks after exposure the animals were sacrificed and temporal bones were resected before immediate fixation in 3% glutaraldehyde in sodiumphosphate buffer. After prefixation, one cochlea from each animal was microdissected for SEM evaluation according to standard procedures (Bredberg et al., 1970; Sodijn, 1976; Fredelius, 1988) and further processed using the tannin-fixation method (Murakami, 1974; Malik and Wilson, 1975). Earlier investigations have shown that no significant differences are found when comparing the number of lost HCs in the right and left cochlea (Fredelius, 1988). After dehydration the specimens were critcal-point dried (Balzerz CPD 010) and before examination .in the Philips 505 SEM microscope, the cochleas were mounted and gold-spluttered using a gold-spluttering unit (Polaron, E5000). Individual hair cell (HC) damage along the organ of Corti was evaluated and graded according to a 4-grade scale (Fredelius et al, 1987). Grade 1 represents 10-50% damage or loss of individual hair cell stereocilia and grade 2 a 50-100% loss or damage. Grade 3 represents a total
loss of surface structure or the hair cell, and grade 4 total destruction and collapse of the organ of Corti (Fig. 1). Using these criteria, the hair cell damage was estimated along the organ of Corti from a distance of 3-14 mm from the round window membrane. The total hair cell damage, including all grades of damage (grades 1-4) was statistically evaluated. A similar evaluation of hair cells (grades 3 + 4) was made separately. The evaluations were made in order to be able to compare the effect of introducting rest periods between exposures with the effect of constant noise exposure.
Results
A numerical reduction was noted in the mean values of damage to all hair cells, outer and inner hair cells, when a 1-h interval was introduced after 3 h compared to continuous exposure to noise. There was a considerable variation in values within each group. Grade-l-3 hair cell damage was found in all groups, but grade 4 only in the 114 dB and the 120 dB groups. The typical damage pattern in the areas of maximal damage is demonstrated in Figs. 2a-f. A statistical analysis was
TABLE II Mean number of damaged or lost hair cells Intensity Duration
120 dB 6h
120 dB 3+3h
114 dB 6h
114 dB 3+3h
108 dB 6h
108 dB 3+3h
OHC damage * IHC damage * Total HC damage * HC loss *
994+ 156 522 + 200 1516+ 318 513+ 115
744+ 130+ 875+ 375+
542+ 168 120+ 71 662+ 239 220+ 128
361+84 57+32 425+82 102+38
309+ 71 52+ 4 313+ 141 87+ 59
91~ 15+ 106+ 53+
* Grade-I + 2 + 3 + 4 damage; t Grade-3 + 4 damage.
193 50 188 162
7 8 11 14
196
made using the Mann-Whitney U test and demonstrated a significant reduction of total hair cell damage, including all grades of damage, in the 120 and 108 dB group, of the outer hair cell damage in the 108 dB group and of the inner hair cell damage in the 120 dB
group. The hair cell loss was not significantly reduced in any group.(Table ll.XFig 3) The extent of hair cell damage along the organ of Corti was reduced from 7 mm (6-12) in the 120 dB group to 3 mm (8-10) in the 108 dB group.
Fig. 2. Scanning electron micrographs from the area of maximal damage from the basal turn of the organ of Corti after different exposures. (a) Grade-4 damage in a 0.4-mm long section of the organ of Corti after exposure to 120 dB SPL for 6 h.(b) A collapse of the organ of Corti 9 nun from the round window in a specimen exposed to 120 dB SPL for 3 + 3 h. (c) A scanning micrograph demonstrating grade 4 damage with a gradual normalization of the epithelium basally and apically, after a 114 dB SPL exposure for 6 h. (d) After exposure to 114 dB SPL for 3 + 3 h a short grade-4 lesion can be seen. In the basal end of the lesion some of the supporting cells a~e present while the sensory epithelium, has degenerated. (e) Micrograph demonstrating damage an~l loss of both IHC and OHC after exposui'e to 108 dB for 6 h. (19 Damage to the first OHC row and light damage to the IHCs can be seen after exposure to 108 dB for 3 + 3 h.
Mean number damaged or lost HC 2000
I | 3
|
! m [] []
looo
e
OHCdamage =HCdamage HCdae~e HC~s
c) 0 120C
"~20P
114C 114P
108C
108P
Fig 3. ~'Hi¢ mean number of damaged hair cells for each exposure group. OHC, IHC, and HC damage represent the accumulated number of grade 1 + 2 + 3 + 4 damage. HC loss represents grade-3 + 4 damafe. "Ihe decrease in HC damage is evident on comparing groups of animals exposed to continuous sound (C) with those where a rest 9eriod (P) WdS introduced as well as exposure to lower sound intensities.
A significant difference could also l~e demonstrated ber~'een the 120-114 and the 114-108 dB groups with the same expost~re mode, on comparing total IHCs, OHCs, HC damage and HC loss. In the control animals, only occasional losses of HCs were found scattered over the investigated part of the organ of Corti. Damage of grades 1 and 2 did not occur in the control animals.
Discuss:m The results of the present investigation emphasize the importance of introducting rest periods between long-term acoustic overstimulations. By interrupting exposl~re, the amount of HC damage was significantly decreased in both the 120 and 108 dB groups. In our investigation, a significant decrease in IHC damage was demonstrated after the introduction of a break in the 120 dB SPL group, indicating a level of exposure where IHC damage dramatically decreases as a result of an introduction of a rest period. It is possible that this finding indicates a critical exposure level where the mechanical damage to the inner ear increases drastically compared to lower exposure levels. It has been demonstrated that 85-90% of the afferent innervation arises from the IHCs and one would expect that extensive IHC loss would cause a corresponding loss of auditory function (Morriaon et a~., 1975). A close correlation between IHC stereociliar changes and neural threshold values was also demonstrated by Stopp (1983) after exposures to 96 dB(A) broadband noise. Robertson and Johnstone (1980) discussed the correlation betwen N1 threshold shift and OHC and IHC damage. A different susceptibility was
suggested, where IHC damage would occur at higher exposure levels than OHCs. In the present material the 108 dB SPL exposure for 6 h was found to be a level where grade 4 damage did not develop, and even though not significant, a marked numerical decrease in IHC loss could be seen after introducing the rest period even at this exposure level. Grade-4 damage and subsequent loss of pillar cells was shown in earlier investigations (Fredelius, 1988) demonstrated to develop parallel with degeneration of radial nerve fibers in the same area. The noise-induced lesion in the present material was located at the site maximally stimulated by the 3.85 kHz tone. Lieberman (I984) reported a similar finding after narrow-band exposures in the mid-frequencies, whereas after high-frequency exposure, the maximal damage shifted toward higher-frequency regions and after exposure to low frequencies, the lesion was shifted to lower frequency regions. Both temporary and permanent threshold shift have been studied in the chinchilla by Blakeslee et al. (1978) and by Henderson et al. (1979) after exposure to noise for 10 continuous days or interrupted by night-time rest. In these investigations it was demonstrated that the amount of asymptotic TTS (ATS) recorded after the interrupted and uninterrupted exposures were about the same. However, when the cochleograms for the two groups were compared the uninterrupted exposure was much more traumatic. Ward (1976) observed that intermittent noise does not produce as much "ITS as the same total energy of continous noise. To evaluate the cochlear changes caused by acoustic trauma a method for statistical evaluation of both HC loss and slighter damage to the cochlea based on a 4-graded scale has been used (Fredelius et al., 1988). This type of evaluation makes it possible to demonstrate the total amount of damage to the organ rff Corti caused by the acoustic overstimulation so as to be able to compare and statistically evaluate the changes between different specimens and ~roups of specimens. In unpublished observations by Wersiill and colleagues a signific,:nt decrease in hair cell loss could be demonstrated after the introduction of a break, compared to continuous high-intensity exposure. It also seems plausible, based on ~ e information provided by earlier investigators (Miller et al., 1963; Bohne et al., !987), to draw the conclusion that this could also be applied to longer exposures to noise of low intensity such as under noisy working conditions. In the present investigation, we evaluated the total amount of HC damage, also including minor changes such as stereocilar disturbance, and performed a statistical analysis on the basis of this information. It can be concluded from the results that the introduction of a rest period during exposures to high sound energy produces a significant reduction of inner ear damage.
198 Acknowledgment This study was s u p p o r t e d by g r a n t s f r o m t h e Tysta Skolan F o u n d a t i o n a n d t h e Swedish M e d i c a l Associa~ tion.
References Blakeslee E.A., H~/nson K., Hamernick R.P. and Henderson D. (1978) Asymptotic threshold shift in chinchilla exposed to impulse noise. J. Acoust. Soc. Am. 63, 876-882. Bohne, B.A.,Yohman, L. and Gruner, M.M. (1987) Cochlear damage following interrupted exposure to high frequency noise. Hear. Res. 29, 251-264. Bredberg, G., Lindeman, H.H, Ades, H.W. and West, R. (1970) Scanning electron microscopy of the organ of Corti. Science 170, 861-863. Fredelius, L., Johansson B., Bagger-Sj/~blick, D. and Wers~iil, J. (1987) Quantitative and qualitative changes in the guinea pig organ of Corti after pure tone overstimulation. Hear. Res. 30, 157-167. Fredelius, L.(1988) Degeneration pattern in the guinea pig organ of Corti after pure tone overstimulation. A morphological and electrophysiological investigation. Thesis, Stockholm. Henderson, D., Hamernik, R.P. and Hynson, K. (1979) Hearing loss from simulated work-week exposure to impulse noise. J. Acoust. Soc. Am. 65, 1231-1237. Liberman, M.C. (1984) Single-neuron labeling and chronic cochlear pathology. 1. Threshold shift and characteristic-frequency shift. Hear. Res. 16, 33-41.
Malick, L.E. and Wilson, R.B. (1975) Evaluation of a modified technique for SEM examination of vertebrate specimens without evaporated metal layers. Scan. Electron. Micr. I, 269-274. Miller, J.D., Watson, C.S. and Covell, W.P. (1963) Deafening effects of noise on the cat. Acta Otolaryngol. Suppl. 176, 1-91. Morrison, D., Schindler, R.A. and Wersiill, J. (1975) A quantitative analysis of the afferent innervation of the organ of Corti in the guinea pig. Acta Otolaryngol. 79, 11-23. Murakami, T. (1974) A revised tannin-osmium method for non-coated scanning electron microscope specimens. Arch. Histoi. Jap 36, 189-193. Nilsson, P., Erlandsson, B., H~kansson, H., Ivarsson, A. and Wers:,ill, J. (1982) Anatomical changes in the cochlea of the guinea pig following industrial noise exposure. In: R.P. H a m e r n ~ , D. Henderson and R. Saivi (Eds.), New Perspectives on Noise-Induced Hearing Loss. Raven Press, New York, pp. 69-86. Robertson, D. and Johnstone, B.M. (1980) Acoustic trauma in the guinea pig cochlea: eacly changes in ultrastructure and neural threshold. Hear. Res. 3, 167-179. Soudijn, E.R. (1976) Scanning electron microscopic study of the organ of Corti in normal and sound-damaged guinea pigs. Ann. Otol. Rhinol. Laryngol. (suppl) 29, 1-58. Stopp, P.E. (1983) Effects on guinea pig cochlea from exposure to moderately intense broad-band noise. Hear. Res. 11, 55-72. Ward, W.D. and Nelson, D.A. (1971) Reduction of permanent threshold shifts through intermittency (chinchilla). J. Acoust. Soc. Am. 49, 91. Ward, W.D. (1976) A comparison of the effects of continuous, intermittent, and impulse noise. In: D. Henderson, R.P. Hamernik, D.S. Dosanjh and J-H. Mills (Eds.), Effects of Noise on Hearing, Raven Press, New York, pp. 407-419.
Hearing Research, 62 (1992) 194-198 © 1992Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/~05.00
HEARES 01800
Hair cell damage after continuous and interrupted pure tone overstimulation: A scanning electron microscopic study in the guinea pig L. F r e d e l i u s a n d J. Wers~ill Department of Otolaryngology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden
(Received 14 November 1991; Revision received 15 May 1992;Accepted 29 May 1992)
In our earlier investigations[Fredelius et al., Hear. Res. 30, 157-167 (1987)] acoustic trauma was studied after continuous 3.85-kHz pure tone exposures of different intensities and durations. In the present investigation, the importance of the introduction of a break during longer 3.85-kHz pure tone exposures was studied. Female pigmented guinea pigs were exposed to 108, 114, or 120 dB SPL for 6 h with or without a 1-h break after the first 3 h. Four weeks after exposure the cochleas were prepared for scanning electron microscopy and the resulting hair cell damage was evaluated according to a 4-grade damage scale. Significantdifferences could be demonstrated in the hair cell damage in the animals exposed to continuous acoustic overstimulation and those exposed to intermittent overstimulation. The importance of rest periods to decrease hair cell damage during long periods of acoustic overstimulation was clearly demonstrated. Acoustic trauma; Cochlear damage; Scanning electron microscopy
Introduction
Information on the damaging effects of noise has been obtained by studying the relationship between exposure to noise and the effects on hearing by means of functional measurements in different animal models (Miller, 1963; Blakeslee, 1978) as well as morphological studies of the inner ear after exposue to different acoustic conditions (Nilsson et al., 1982; Stopp, 1983; Bohne et al., 1987; Fredelius et al., 1987). The damaging effects of noise on man have also been demonstrated (Ward 1976) by studying the relationship b6tween exposure to noise and deterioration in hearir, g using audiometric measurements. Systematic studies are difficult to make, however, since exposure to noise varies for different working periods and conditicris, Ward and Nelson (1971) pointed out that risk crit;:fia for damage which ignore the temporal pattern of the noise are erroneous. In our earlier investigations (Fredelius, 1988), re~ suits have been presented regarding electrophysiological and morphological changes occurring after continuous pure-tone exposures of varying intensities and duration. A close relationship was demonstrated between measured CAPN1 threshold shift and the morphological changes evaluated using the S E M and LM techniques. The SEM method for grading of hair cell
Correspondence to: L. Fredelius, Department of Otolaryngology, Karolinska Hospital, S-10401 Stockholm, Sweden.
damage made possible a statistical evaluation and comparison between the damaging effects of different sound energies and intensities. The aim of the present study was to investigate the effect on inner ear damage after the introduction of a break dividing continuous noise into two periods, using the above mentioned qualitative method to evaluate the damage.
M a t e r i a l and M e t h o d s
Twent3~-eight female pigmented guinea pigs with an approx, weight of 250 g were exposed to a 3.85-kHz pure tone. The animals were exposed to sound intensities of 108, 114, and 120 dB SPL for either 6 h or 3 + 3 h with a 1-h break (Table I). The exposures occurred in an anechoic chamber. The animals were able to move about ~reely during exposure in a wire-mesh cage mounted below a horn-speaker (James B. Lansing, TABLE I Experimental design Animals
Intensity (dB SPL)
Duration (h)
4 4 4 4 4 4 4
120 120 114 114 108 108 controls
6 3 + 3 (1-h paus) 6 3 + 3 (1-h paus) 6 3 + 3 (1-h paus)
195
Fig. 1. a) Grade-l-4 damage. Grade-1 damage represents 10-50% damage to or loss to individual hair cell stereocilia and grade 2 represents 50-100% damage to or loss of individual hair cell stereocilia. Gra~e 3 represents loss of individual hair cells and (b) grade 4 a total loss and collapse of the organ of Corti~
Mod. 2552 driver and a 2360 TH horn). The exposures were monitored with a calibrated microphone and the sound level within the cage was constant within + 1 dB. Four weeks after exposure the animals were sacrificed and temporal bones were resected before immediate fixation in 3% glutaraldehyde in sodiumphosphate buffer. After prefixation, one cochlea from each animal was microdissected for SEM evaluation according to standard procedures (Bredberg et al., 1970; Sodijn, 1976; Fredelius, 1988) and further processed using the tannin-fixation method (Murakami, 1974; Malik and Wilson, 1975). Earlier investigations have shown that no significant differences are found when comparing the number of lost HCs in the right and left cochlea (Fredelius, 1988). After dehydration the specimens were critcal-point dried (Balzerz CPD 010) and before examination .in the Philips 505 SEM microscope, the cochleas were mounted and gold-spluttered using a gold-spluttering unit (Polaron, E5000). Individual hair cell (HC) damage along the organ of Corti was evaluated and graded according to a 4-grade scale (Fredelius et al, 1987). Grade 1 represents 10-50% damage or loss of individual hair cell stereocilia and grade 2 a 50-100% loss or damage. Grade 3 represents a total
loss of surface structure or the hair cell, and grade 4 total destruction and collapse of the organ of Corti (Fig. 1). Using these criteria, the hair cell damage was estimated along the organ of Corti from a distance of 3-14 mm from the round window membrane. The total hair cell damage, including all grades of damage (grades 1-4) was statistically evaluated. A similar evaluation of hair cells (grades 3 + 4) was made separately. The evaluations were made in order to be able to compare the effect of introducting rest periods between exposures with the effect of constant noise exposure.
Results
A numerical reduction was noted in the mean values of damage to all hair cells, outer and inner hair cells, when a 1-h interval was introduced after 3 h compared to continuous exposure to noise. There was a considerable variation in values within each group. Grade-l-3 hair cell damage was found in all groups, but grade 4 only in the 114 dB and the 120 dB groups. The typical damage pattern in the areas of maximal damage is demonstrated in Figs. 2a-f. A statistical analysis was
TABLE II Mean number of damaged or lost hair cells Intensity Duration
120 dB 6h
120 dB 3+3h
114 dB 6h
114 dB 3+3h
108 dB 6h
108 dB 3+3h
OHC damage * IHC damage * Total HC damage * HC loss *
994+ 156 522 + 200 1516+ 318 513+ 115
744+ 130+ 875+ 375+
542+ 168 120+ 71 662+ 239 220+ 128
361+84 57+32 425+82 102+38
309+ 71 52+ 4 313+ 141 87+ 59
91~ 15+ 106+ 53+
* Grade-I + 2 + 3 + 4 damage; t Grade-3 + 4 damage.
193 50 188 162
7 8 11 14
196
made using the Mann-Whitney U test and demonstrated a significant reduction of total hair cell damage, including all grades of damage, in the 120 and 108 dB group, of the outer hair cell damage in the 108 dB group and of the inner hair cell damage in the 120 dB
group. The hair cell loss was not significantly reduced in any group.(Table ll.XFig 3) The extent of hair cell damage along the organ of Corti was reduced from 7 mm (6-12) in the 120 dB group to 3 mm (8-10) in the 108 dB group.
Fig. 2. Scanning electron micrographs from the area of maximal damage from the basal turn of the organ of Corti after different exposures. (a) Grade-4 damage in a 0.4-mm long section of the organ of Corti after exposure to 120 dB SPL for 6 h.(b) A collapse of the organ of Corti 9 nun from the round window in a specimen exposed to 120 dB SPL for 3 + 3 h. (c) A scanning micrograph demonstrating grade 4 damage with a gradual normalization of the epithelium basally and apically, after a 114 dB SPL exposure for 6 h. (d) After exposure to 114 dB SPL for 3 + 3 h a short grade-4 lesion can be seen. In the basal end of the lesion some of the supporting cells a~e present while the sensory epithelium, has degenerated. (e) Micrograph demonstrating damage an~l loss of both IHC and OHC after exposui'e to 108 dB for 6 h. (19 Damage to the first OHC row and light damage to the IHCs can be seen after exposure to 108 dB for 3 + 3 h.
Mean number damaged or lost HC 2000
I | 3
|
! m [] []
looo
e
OHCdamage =HCdamage HCdae~e HC~s
c) 0 120C
"~20P
114C 114P
108C
108P
Fig 3. ~'Hi¢ mean number of damaged hair cells for each exposure group. OHC, IHC, and HC damage represent the accumulated number of grade 1 + 2 + 3 + 4 damage. HC loss represents grade-3 + 4 damafe. "Ihe decrease in HC damage is evident on comparing groups of animals exposed to continuous sound (C) with those where a rest 9eriod (P) WdS introduced as well as exposure to lower sound intensities.
A significant difference could also l~e demonstrated ber~'een the 120-114 and the 114-108 dB groups with the same expost~re mode, on comparing total IHCs, OHCs, HC damage and HC loss. In the control animals, only occasional losses of HCs were found scattered over the investigated part of the organ of Corti. Damage of grades 1 and 2 did not occur in the control animals.
Discuss:m The results of the present investigation emphasize the importance of introducting rest periods between long-term acoustic overstimulations. By interrupting exposl~re, the amount of HC damage was significantly decreased in both the 120 and 108 dB groups. In our investigation, a significant decrease in IHC damage was demonstrated after the introduction of a break in the 120 dB SPL group, indicating a level of exposure where IHC damage dramatically decreases as a result of an introduction of a rest period. It is possible that this finding indicates a critical exposure level where the mechanical damage to the inner ear increases drastically compared to lower exposure levels. It has been demonstrated that 85-90% of the afferent innervation arises from the IHCs and one would expect that extensive IHC loss would cause a corresponding loss of auditory function (Morriaon et a~., 1975). A close correlation between IHC stereociliar changes and neural threshold values was also demonstrated by Stopp (1983) after exposures to 96 dB(A) broadband noise. Robertson and Johnstone (1980) discussed the correlation betwen N1 threshold shift and OHC and IHC damage. A different susceptibility was
suggested, where IHC damage would occur at higher exposure levels than OHCs. In the present material the 108 dB SPL exposure for 6 h was found to be a level where grade 4 damage did not develop, and even though not significant, a marked numerical decrease in IHC loss could be seen after introducing the rest period even at this exposure level. Grade-4 damage and subsequent loss of pillar cells was shown in earlier investigations (Fredelius, 1988) demonstrated to develop parallel with degeneration of radial nerve fibers in the same area. The noise-induced lesion in the present material was located at the site maximally stimulated by the 3.85 kHz tone. Lieberman (I984) reported a similar finding after narrow-band exposures in the mid-frequencies, whereas after high-frequency exposure, the maximal damage shifted toward higher-frequency regions and after exposure to low frequencies, the lesion was shifted to lower frequency regions. Both temporary and permanent threshold shift have been studied in the chinchilla by Blakeslee et al. (1978) and by Henderson et al. (1979) after exposure to noise for 10 continuous days or interrupted by night-time rest. In these investigations it was demonstrated that the amount of asymptotic TTS (ATS) recorded after the interrupted and uninterrupted exposures were about the same. However, when the cochleograms for the two groups were compared the uninterrupted exposure was much more traumatic. Ward (1976) observed that intermittent noise does not produce as much "ITS as the same total energy of continous noise. To evaluate the cochlear changes caused by acoustic trauma a method for statistical evaluation of both HC loss and slighter damage to the cochlea based on a 4-graded scale has been used (Fredelius et al., 1988). This type of evaluation makes it possible to demonstrate the total amount of damage to the organ rff Corti caused by the acoustic overstimulation so as to be able to compare and statistically evaluate the changes between different specimens and ~roups of specimens. In unpublished observations by Wersiill and colleagues a signific,:nt decrease in hair cell loss could be demonstrated after the introduction of a break, compared to continuous high-intensity exposure. It also seems plausible, based on ~ e information provided by earlier investigators (Miller et al., 1963; Bohne et al., !987), to draw the conclusion that this could also be applied to longer exposures to noise of low intensity such as under noisy working conditions. In the present investigation, we evaluated the total amount of HC damage, also including minor changes such as stereocilar disturbance, and performed a statistical analysis on the basis of this information. It can be concluded from the results that the introduction of a rest period during exposures to high sound energy produces a significant reduction of inner ear damage.
198 Acknowledgment This study was s u p p o r t e d by g r a n t s f r o m t h e Tysta Skolan F o u n d a t i o n a n d t h e Swedish M e d i c a l Associa~ tion.
References Blakeslee E.A., H~/nson K., Hamernick R.P. and Henderson D. (1978) Asymptotic threshold shift in chinchilla exposed to impulse noise. J. Acoust. Soc. Am. 63, 876-882. Bohne, B.A.,Yohman, L. and Gruner, M.M. (1987) Cochlear damage following interrupted exposure to high frequency noise. Hear. Res. 29, 251-264. Bredberg, G., Lindeman, H.H, Ades, H.W. and West, R. (1970) Scanning electron microscopy of the organ of Corti. Science 170, 861-863. Fredelius, L., Johansson B., Bagger-Sj/~blick, D. and Wers~iil, J. (1987) Quantitative and qualitative changes in the guinea pig organ of Corti after pure tone overstimulation. Hear. Res. 30, 157-167. Fredelius, L.(1988) Degeneration pattern in the guinea pig organ of Corti after pure tone overstimulation. A morphological and electrophysiological investigation. Thesis, Stockholm. Henderson, D., Hamernik, R.P. and Hynson, K. (1979) Hearing loss from simulated work-week exposure to impulse noise. J. Acoust. Soc. Am. 65, 1231-1237. Liberman, M.C. (1984) Single-neuron labeling and chronic cochlear pathology. 1. Threshold shift and characteristic-frequency shift. Hear. Res. 16, 33-41.
Malick, L.E. and Wilson, R.B. (1975) Evaluation of a modified technique for SEM examination of vertebrate specimens without evaporated metal layers. Scan. Electron. Micr. I, 269-274. Miller, J.D., Watson, C.S. and Covell, W.P. (1963) Deafening effects of noise on the cat. Acta Otolaryngol. Suppl. 176, 1-91. Morrison, D., Schindler, R.A. and Wersiill, J. (1975) A quantitative analysis of the afferent innervation of the organ of Corti in the guinea pig. Acta Otolaryngol. 79, 11-23. Murakami, T. (1974) A revised tannin-osmium method for non-coated scanning electron microscope specimens. Arch. Histoi. Jap 36, 189-193. Nilsson, P., Erlandsson, B., H~kansson, H., Ivarsson, A. and Wers:,ill, J. (1982) Anatomical changes in the cochlea of the guinea pig following industrial noise exposure. In: R.P. H a m e r n ~ , D. Henderson and R. Saivi (Eds.), New Perspectives on Noise-Induced Hearing Loss. Raven Press, New York, pp. 69-86. Robertson, D. and Johnstone, B.M. (1980) Acoustic trauma in the guinea pig cochlea: eacly changes in ultrastructure and neural threshold. Hear. Res. 3, 167-179. Soudijn, E.R. (1976) Scanning electron microscopic study of the organ of Corti in normal and sound-damaged guinea pigs. Ann. Otol. Rhinol. Laryngol. (suppl) 29, 1-58. Stopp, P.E. (1983) Effects on guinea pig cochlea from exposure to moderately intense broad-band noise. Hear. Res. 11, 55-72. Ward, W.D. and Nelson, D.A. (1971) Reduction of permanent threshold shifts through intermittency (chinchilla). J. Acoust. Soc. Am. 49, 91. Ward, W.D. (1976) A comparison of the effects of continuous, intermittent, and impulse noise. In: D. Henderson, R.P. Hamernik, D.S. Dosanjh and J-H. Mills (Eds.), Effects of Noise on Hearing, Raven Press, New York, pp. 407-419.
