Loss of adaptability rather than loss of sensitivity may be one of the initial signs of auditory impairment following exposure to noise. One way to examine the adaptability of hearing in experimental investigation is to measure the magnitude of the suppression, exerted by the medial olivocochlear efferent system, on the ipsilateral otoacoustic emissions in response to contralateral sound stimulation. Thus, in order to test the hypothesis it was decided to measure hearing thresholds (HT), the cubic DPOAE and suppression of cubic DPOAE by contralateral wide band noise in rats exposed to long-term, low level noise (90 days of 90 dBlin 4-20 kHz wide band noise 4 hours/day, 5 days/week). Measurements of HT were performed by assessment of the ABR, elicited by tone-pips from the same probe assembly used in the measurements of DPOAE. The suppression of the cubic distortion product (CDP) was determined in ketamine/xylazine anaesthesia, allowing a stable response for a minimum of 20 min. Of the frequencies tested, the rats exposed to noise had an increase in HT at 12.8 kHz only (6.8 dB, P<0.05), while a reduction on the CDP was evident with f2 going from 9.2 kHz to the upper limit at 17.4 kHz. Further, the rats exposed to noise had little suppression of the CDP at low levels of contralateral noise (CN), but no difference from the control animals was seen as the CN noise level was increased. The measurement of DPOAE suppression did not reveal any effects of the low level noise exposure that was not paralleled also by shifts in hearing thresholds. The most sensitive assessment of the auditory changes in the study was the measurements of DPOAE, and further elaboration on the bandwidth and frequency distribution of the CN is necessary, before auditory changes in the high frequency range can be probably assessed.
Keywords: Noise exposure, Rat, Hearing thresholds, Otoacoustic emissions, Distortion products, Contralateral suppression.
|How to cite this article:|
Lund SP, Jepsen GB, Simonsen L. Effect of long-term, low-level noise exposure on hearing thresholds, DPOAE and suppression of DPOAE in rats. Noise Health 2001;3:33-42
|How to cite this URL:|
Lund SP, Jepsen GB, Simonsen L. Effect of long-term, low-level noise exposure on hearing thresholds, DPOAE and suppression of DPOAE in rats. Noise Health [serial online] 2001 [cited 2020 Jun 4];3:33-42. Available from: http://www.noiseandhealth.org/text.asp?2001/3/12/33/31798
| Introduction|| |
Exposure to noise induce hearing loss, manifests as permanent shifts of the hearing thresholds of pure tones. The scientific literature and the general regulations of noise in the working environment are in fact dominated by the concept of noise induced hearing loss being a matter primarily of changes in hearing thresholds. This is in contrast to the difficulties of the hearing impaired, where changes in the adaptability of hearing, as loudness recruitment, hyperacusis, and loss of discrimination in background noise, may have considerable debilitating consequences.
One way to examine the adaptability of hearing in experimental investigation is to measure the magnitude of the suppression, exerted by the medial olivocochleare efferent system, on the ipsilateral otoacoustic emissions in response to contralateral sound stimulation (for a review cf. Guinan, 1996). Suppression of distortion product otoacoustic emissions (DPOAE) has been studied in humans. It has also been assessed in guinea pigs to reveal the effects of long-term, low-level noise exposure (Skellet, 1998) as well as the acute effects of gentamycin injection (Avan et al., 1996). To our knowledge DPOAE suppression has not previously been examined in rats.
The present study was initiated to evaluate the possibilities of measuring contralateral DPOAE suppression in rats, and to examine which parameters are of prime interest in the study of low-level exposure to noise and chemicals. Therefore rats were exposed to long-term, lowlevel noise and examined for changes in hearing thresholds, DPOAE, and suppression of DPOAE.
| Materials and methods|| |
24 Male Wistar rats (MOL:Wist Han) weighing 175-200 g were purchased from a local breeder (M & B, Ltd.). The rats were housed two by two in polypropylene cages (425 × 266 × 150 mm) with steam cleaned pinewood bedding (Lignocel S 8). Municipal water and rodent chow (Altromin 1324) was accessible ad libitum. In the animal quarters, the temperature was maintained at 21 ± 1 °C and humidity at 50 ± 5 %. Lights were on from 7.00 PM to 7.00 AM.
To be exposed to noise 12 rats were transferred daily from their home cages to closed climate chambers and kept two by two in wire mesh cages with free access to drinking water. The rats were exposed to 90 dB lin SPL rms wide band noise 4 hours/day, 5 days/week, for a period of 90 days. The noise was generated on a computer with a digital signal processing board (Ariel DSP16+), amplified by an audio amplifier (NAD 216 THX), and delivered by dome tweeters (Vifa D26TG-05-06) situated 56 cm above the floor of each cage. The sound field was measured at various points at the level of the floor of the cages with a ½ inch condenser microphone (B&K 4133) and a HP35670A spectrum analyser. Overall, the frequency distribution of the wide band noise was quite uniform between 4-20 kHz and the level varied less than ± 1 dB between measuring points see [Figure - 1].
General auditory test equipment
All auditory measurements were made with the same stimulus source, an Etymotic Research ER10B+ low noise microphone system coupled to ER-2 tubephones by standard front tubes. All measurements were performed only on the left ear with the animals laying on their right side on a heating plate in a closed sound booth (80x80x60 cm) lined with sound absorbing material (RockfonR).
The microphone probe were placed in the left external ear canal by a probe holder, a small brass funnel fixed to a micro manipulator. Before inserting the microphone, the position of the funnel were adjusted to allow a direct view of the ear drum with an otoscope. The primary tone f1 and f2 for the DPOAE were generated by a HP 8904 two-channel tone generator with phase control. The level of the stimulus from the individual ER-2 tubephones were adjusted in accordance with the frequency response curves, attained from the average response (84 ± 1.5 dB lin SPL rms ) from 1 to 17.5 kHz by an ER-7C probe tube microphone at the ear drum of 6 animals (12 ears). The output of the ER-10B and ER-7C microphones were adjusted in accordance with the frequency response curves supplied by the manufacturer.
The only DPOAE measured was the amplitude of cubic distortion product 2f1-f2 (CDP), and always with the level of f1 (L1) 10 dB higher than the level of f2 (L2 = L1 - 10 dB). The output of the ER-10B microphone was fed to a B&K (Nexus) amplifier and analysed on a HP 35670A spectrum analysator. All test procedures and equipment were controlled by a computer and were programmed in the visual programming language HP VEE (version 5.0). All auditory measurements were performed randomly cage by cage, and without any knowledge of the exposure status of the rats,
Determination of hearing thresholds was performed by analysis of the Auditory Brainstem Response (ABR) to pure tone stimulation (4096 Hz, 8192 Hz, 12800 Hz, and 16384 Hz) on anaesthetised rats (65 mg/kg pentobarbital sodium i.p.). The rectal temperature of the rats was throughout the ABR measurements kept at 38.0 ± 0.5oC. The pure tone stimuli were generated with a repetition rate of 19.9 per sec. by a programmable function generator (Hameg 8130) as symmetrical tapered 1.4 msec tonepips, and were after digital attenuation delivered by the ER-2 tubephones at levels ranging from 20 to 105 dB lin SPL rms in 5 dB steps.
The ABRs were recorded with a silver wire inserted subcutaneously at the back of the head as active electrode, a small roll of silver wire in the mouth as reference electrode and a stainless steel needle in the tail as ground electrode. The response was amplified 50,000 times, filtered through analogue bandpass filter (10 Hz to 10 kHz), and 15 msec were sampled at a rate of 51.2 kHz by a 16 bit data acquisition board. The ABR of each stimulus level consisted of 256 artefact free recordings that was averaged and stored on hard disk for later analysis. After further digitally filtering (FIR lowpass filter, 2.4 kHz cut of frequency and 5 kHz stopband) of the stored ABRs, the hearing thresholds were determined as the lowest stimulus level, where both the first wave and the first trough of the ABR could be clearly identified.
DP-gram was obtained by measuring the CDP with fixed ratio of f2/f1 = 16/13 = 1.23 and fixed level of the primary tones (L2 = 50 dB lin SPL rms ), but varying f2 from 4096 to 17408 Hz in steps of 512 Hz. Each spectrum was made on 64 time averaged recordings (time averaging). The DP-gram was made a long with the ABR measurements in pentobarbital anaesthesia (65 mg/kg pentobarbital sodium i.p.).
DPOAE Input/Output-curves( I/O-curves) were made with a fixed ratio of the primary tones (f2/f1 = 1.23) at 4 fixed frequencies of f2 (4096 Hz, 8192 Hz, 12800 Hz, and 16384 Hz). The I/O-curves were made by measuring the amplitude of the CDP to varying levels of the primary tones (L1= 20 - 90 dB lin SPL rms ) in 5 dB steps. While the level of the primary tones were decreased, the number of time averagings were increased.
The number of time averaged recordings for the I/O-curves were based on calculation of signal to noise ratio (S/N; N = mean of 8 bins, 4 on either side of the CDP), but was not allowed to exceed 512. The obtained amplitudes on the I/O-curves always had S/N better than 3 dB, and in general the noise floor at the low amplitudes was below -20 dB lin SPL rms. . The measurements of the I/O curves were in ketamine/xylazine anaesthesia (100 mg/kg ketamine hydrochloride i.p. / 10 mg/kg xylazine i.p.) along with the measurements of the suppression of the DPOAE.
Suppression of DPOAE
Suppression of DPOAE were made by feeding wide band noise (WBN) to the right ear (contralateral) while measuring the DPOAE on the left ear (ipsilateral). The noise was delivered from piezo-ceramic tweeter mounted on the backside of the heating plate. The plastic front of the tweeter was cut off to allow the rats to be placed on the side with the right ear above the centre of the speaker. The WBN was generated by the HP35670A spectrum analyse, and equalised by a 15 band graphic equaliser (KAM KEG15).
The frequency composition of the contralateral noise (CN) was adjusted by measurements of the soundfield above the tweeter with a condenser microphone (B&K 4133) and the HP35670A spectrum analyser. The noise was found to have a quite uniform frequency distribution ranging from 4 to 24 kHz. As the adjustment of the frequency distribution were made with a free field condenser microphone, the levels of the contralateral noise were also calibrated by measurements with the free field microphone and not at ear drum as the stimulus for the ABR and the primary tones for the DPOAE.
The determination of the DPOAE suppression were made at three primary tone frequencies (f2 = 8192 Hz, 12800 Hz, and 16384 Hz) in two ways : 1) Determination of the I/O-curves at a constant CN level (85 dB lin SPL rms ), and 2) by varying the CN level from 60 to 85 dB lin SPL rms at fixed levels of f1 and f2 (L1 = 60 dB lin SPL rms ).
The measurements of the DPOAE-suppression were made along the determinations of DPOAE I/O-curves in ketamine/xylazine anaesthesia (100 mg/kg ketamine hydrochloride i.p. and 10 mg/kg xylazine i.p.). A very strict time schedule for both induction of the anaesthesia and all the measurements was followed, and no supplementation of the anaesthetic agents was allowed. The ketamine was given 12 min. after the injection of xylazine, and measurements was initiated exactly 15 min. after the ketamine injection. The ketamine/xylazine anaesthesia allows measurements of a stable CN suppression for a minimum of 20 min., but all measurements were performed within 20 min from onset.
Comparison of the groups was performed using analysis of variance (ANOVA). In the following, the term significant is used unless stated otherwise when tests are rejected at a 5 % level.
| Results|| |
The hearing thresholds of both the noise exposed and the control rats are depicted in [Figure - 2]. There were no differences in the hearing thresholds at 4096 Hz and 8192 Hz. At 12800 Hz and 16384 Hz the hearing thresholds were elevated by 6.7 and 3.8 dB respectively, though the difference was significant only at 12800 Hz.
The DP-gram depicted in [Figure - 3] shows a significant reduction in the amplitude of the CDP with f2 ranging from 9.2 kHz all the way up to 17.4 kHz. From 12 to 16 kHz the reductions in CDP amplitude of the noise exposed rats were 10 dB or more.
DPOAE I/O-curves made at the four different frequencies of f2 are displayed in [Figure - 4].The I/O-curves made at high primary tone frequencies (f2 at 12800 Hz and 16384 Hz) reveal a reduction in the CDP amplitude in the noise exposed rats, the reduction being comparable to what was found in the DP-gram at the same primary tone levels. The only changes found in the I/O-curves of the two lower primary frequencies (f2 at 4096 Hz and 8192 Hz) is a highly significant reduction in the CDP amplitude at the highest stimulus levels (at L2 = 75 and 80 dB) at f2= 8192 Hz.
The DPOAE I/O curves at f2 = 12800 Hz an without CN suppression are displayed in [Figure - 5]. The size of DPOAE suppression with constant levels of CN (85 dB) are remarkably uniform even as the levels of the primary tones varied up to 60 dB. Although the magnitude of the CDP amplitude are approximately 10 dB lower in the noise exposed rats, the suppression of the CDP amplitude are of comparable magnitude in both the noise exposed and the control group. The corresponding DPOAE suppression at f2 = 8192 Hz and 16384 Hz are not shown, but the DPOAE suppression at these frequencies exhibit the same pattern as shown in [Figure - 5].
The suppression of DPOAE with varying levels of CN levels at the three frequencies of f2 are shown in [Figure - 6]. The DPOAE suppression are of equal magnitude at f2 = 8192 Hz at all levels of CN in both groups of rats. At f2 = 12800 Hz and 16384 Hz the magnitude of the DPOAE suppression differs at the low CN levels, while the suppression are of equal magnitude at the highest noise levels (80 and 85 dB). There is nearly no suppression at the lowest CN (50 dB) in the noise exposed group, which differs significantly from the control group.
No other significant differences where found in the magnitude of the DPOAE suppression between the to groups of rats. Altogether, the size of the DPOAE suppression are lower as the frequencies of the primary tones is increased, but especially the size of DPOAE suppression at f2= 16384 Hz seems rather low.
| Discussion|| |
The level of the noise used in the present study was expected to give a small, but significant loss of auditory sensitivity, as the 4 hours of daily exposure to noise at 90 dB SPL corresponds to L eq8hours = 87 dB SPL. This is confirmed by the results of the different auditory measurements, that altogether corresponds rather closely. The noise exposure has primarily caused a reduction in hearing thresholds and CDP amplitude due to impairment in the 12 - 16 kHz part of the cochlea, which is clearly seen in the hearing thresholds shown in [Figure - 2], and DP-gram shown in [Figure - 3], and the DPOAE I/O-curves in [Figure - 4].
The WBN applied in the noise exposure had a very uniform frequency distribution in the 4-20 kHz range. However, the frequency distribution of noise measured at the ear drum of a anaesthetised rat during noise exposure reveals a maximum at 6-12 kHz, where the sound pressure level is up to 15 dB higher than the level measured in a free field. This is a result of the resonance of the external ear canal (Henderson & Hamernik, 1995). Accordingly, the actual cochlear impairment is shifted approximately one octave upwards in relation the frequencies of maximum sound pressure level at the ear drum. This is quite similar to the findings of Borg & Engstrom (1989) in the chinchilla, where the effect of long-term, low-level noise exposure induced a hearing loss with a marked upward frequency shift in relation to the frequency composition of the exposure noise .
The studies of the suppression of DPOAE were performed in ketamine/xylazine anaesthesia. The CN suppression of the CDP is believed to be mediated by medial olivocochlear neurones, projecting efferent nervefibres through the crossed olivocochlear bundle to make synapses directly with the contralateral outer hair cells (Siegel & Kim, 1982; Puel & Rebillard, 1990; Guinan, 1996; Maison et al., 1999; Scates et. al., 1999). The anaesthetic agent may interact with the response under study and should be avoided (Avan et al., 1996) but, the microphone probe assembly must be kept strictly in same position to allow collection high quality measurements at the relevant frequencies. This is virtually impossible to achieve by keeping rats under rigorous restraint for longer periods of time without anaesthetic.
Several anaesthetics have been tested, but only ketamine/xylazine combined a reasonable preservation of the DPOAE suppression with an acceptable stability for more than 20 minutes. To secure that the rats had equal depth of the anaesthesia, several precautions were taken: The rats had to be fully anaesthetised from the start, and no supplementation of anaesthetic agents was given. Further, strict timetable was followed to secure that the measurements were made in the same order and at comparable points in time.
The assessments of the DPOAE suppression was performed with 4-24 kHz WBN in two ways, i.e. measuring the DPOAE I/O-curve at a constant high CN level (75 dB SPL, see [Figure - 5]) and varying the CN level (50 - 75 dB SPL) with fixed levels of the primary tones see [Figure - 6]. In neither of these assessments was there any difference between the two groups of rats at the high levels of CN at any of the frequencies tested. In contrast to this a reduction was seen in the suppression at the low level of CN at 12.8 kHz and to some extent also at 16.4 kHz. Consequently, the initial effect of noise exposure on CDP suppression should be assessed at low CN levels. This seems to explain why Skellett et al. (1996) found no suppression on CDP to a CN level of 70 dB SPL WBN in guinea pigs after chronic exposure to low-level noise. It also emphasises testing should include measurements in the high frequency range.
The maximum level of suppression at the three frequencies tested seems to follow rather closely the frequency distribution of the noise measured at the ear drum see [Figure - 1]. This seems to imply that same WBN cannot be applied for DPOAE suppression over a broad frequency range. In the high frequency area, where the noise induced impairment was quite evident, the level of CDP suppression were unsatisfactory. Thus, even if WBN may produce the best overall suppression of OAE (Maison et al., 2000), the CN has to be equalised to evaluate the functional changes at the different frequencies equally well.
The suppression DPOAE I/O-curves were made with a high level of CN, where the difference in DPOAE was at their maximum (f2= 12.8 kHz), there was no difference in the level of CDP suppression between the two groups. However, the I/O-curves with and without suppression runs remarkably parallel in both groups, and even more remarkably, the I/O-curve of the noise exposed group without suppression is nearly identical to the I/O-curve of the control group with suppression see [Figure - 5]. It is quite tempting to see the identity of these two I/Ocurves as a sign of an equal functional capacity of this part of the cochlea under the measurements, i.e. that the noise exposed rats without CN had a loss of the adaptive capacity equal to the induced reduction in the control group of CN level of 75 dB.
The present study has shown that measurements of suppression of otoacoustic emissions may be performed on anaesthetised rats with quite acceptable results. However, the measurement of DPOAE suppression did not reveal effects of the low level noise exposure that was not paralleled also by shifts hearing thresholds. Altogether, the most sensitive assessment of the auditory changes in the study was the measurements of DPOAE, and further elaboration on the bandwidth and frequency distribution of the CN is necessary, before auditory changes in the high frequency range can be probably assessed. We will continue to work within this field, as we believe that measurements of the suppression of otoacoustic emissions may reveal changes in the functional adaptability, which is an important part of the changes that follows impairment of hearing due to exposure to noise and chemical substances.
| Acknowledgements|| |
The authors would like to thank Lotte S. Drifte (NIOH) for her efforts throughout the study.
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Soren P Lund
National Institute of Occupational Health (NIOH), Lersų Parkallé 105, 2100 CopenhagenŲ
Source of Support: None, Conflict of Interest: None
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]