# Selective Vulnerability of the Cochlear Basal Turn to Acrylonitrile and Noise

Authors: B. Pouyatos; C. A. Gearhart; A. Nelson-Miller; S. Fulton; L. D. Fechter
Journal: Journal of Toxicology
 (2009)

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## Abstract

Exposure to acrylonitrile, a high-production industrial chemical, can promote noise-induced hearing loss (NIHL) in the rat even though this agent does not itself produce permanent hearing loss. The mechanism by which acrylonitrile promotes NIHL includes oxidative stress as antioxidant drugs can partially protect the cochlea fromacrylonitrile+noise. Acrylonitrile depletes glutathione levels while noise can increase the formation of reactive oxygen species. It was previously noted that the high-frequency or basal turn of the cochlea was particularly vulnerable to the combined effects of acrylonitrile and noise when the octave band noise (OBN) was centered at 8 kHz. Normally, such a noise would be expected to yield damage at a more apical region of the cochlea. The present study was designed to determine whether the basal cochlea is selectively sensitive to acrylonitrile or whether, by adjusting the frequency of the noise band, it would be possible to control the region of the auditory impairment. Rats were exposed to one of three different OBNs centered at different frequencies (4 kHz, 110 dB and 8 or 16 kHz at 97 dB) for 5 days, with and without administration of acrylonitrile (50 mg/kg/day). The noise was set to cause limited NIHL by itself. Auditory function was monitored by recording distortion products, by compound action potentials, and by performing cochlear histology. While the ACN-only and noise-only exposures induced no or little permanent auditory loss, the three exposures to acrylonitrile+noise produced similar auditory and cochlear impairments above 16 kHz, despite the fact that the noise exposures covered 2 octaves. These observations show that the basal cochlea is much more sensitive to acrylonitrile+noise than the apical partition. They provide an initial basis for distinguishing the pattern of cochlear injury that results from noise exposure from that which occurs due to the combined effects of noise and a chemical contaminant.


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## Body

## 1. Introduction

Acrylonitrile (ACN; vinyl cyanide)
is an industrial chemical used extensively in the plastic, butyl rubber, and
textile industries (SRI 1984). Approximately 125 000
workers are exposed to ACN daily in the US [1]. While the maximum
permissible exposure level to ACN is quite low (1 ppm), exposure can quickly
reach levels that exceed 1 ppm via skin contact in case of accidental exposure [2].In laboratory animal models, high
level exposure to acrylonitrile does not cause permanent auditory damage by
itself [3–5],
but can increase the vulnerability of the cochlea to moderate noise. These
studies provide a means for testing the mechanisms responsible for potentiation
of NIHL by chemicals rather than a direct model for occupational exposure. Pouyatos et al. [4] demonstrated that, when
subjected to a combined exposure to ACN and to a 97 dB octave band noise
centered at 8 kHz (OBN-8 kHz), rats displayed large permanent distortion product
otoacoustic emissions (DPOAEs) deficits and compound action potential (CAP)
threshold shifts along with marked outer hair cell (OHC) loss. The same
exposure to noise alone did not yield any damage. Functional impairment was
characterized by large high-frequency hearing loss (30–35 dB above 16 kHz based
upon DPOAE and between 20–25 dB based upon CAP threshold), accompanied by a
near total disappearance of the cochlear basal turn’s OHCs. Because the cochlea
is organized in a tonotopic manner with high frequencies encoded at the base
and lower frequencies encoded at more apical locations in addition to the base,
this type of noise by itself should have given a maximum hearing loss and
cochlear damage centered on the middle turn at a location where tones of 12 kHz
are encoded according to the half octave shift rule postulated by McFadden [6]. This study was designed to
characterize further the role of noise and ACN exposure in auditory impairment
that results from the combined exposure to these agents. This study also aimed
to identify disparities between noise energy bands in the environment and
physiological impairments that may be useful in estimating the role that
chemical contaminants might play in hearing loss when noise is also present,
for example, in occupational settings.


## 2. Materials and Methods

### 2.1. Subjects

A
total of 57 male Long-Evans rats (225–249 g, 7–8 weeks old) obtained from
Harlan (Indianapolis, Ind, USA) were employed in these experiments. The subjects were
housed with free access to food and water in their home cages. Temperature was
maintained at21±1°C and lights were on from 6:30 am to 6:30 pm. The Loma Linda
Veteran Medical Center Institutional Animal Care and Use Committee (IACUC) approved
all the experimental protocols. All exposures and testing were performed during
the daytime.


### 2.2. Procedures

Groups of Long-Evans rats(n=4–9)
were exposed for 5 days (4 hours/day) to moderately intense octave band noise
(OBN) centered at 4 kHz (110 dB SPL), 8 kHz (97 dB SPL), or 16 kHz (97 dB SPL),
with or without co-exposure to ACN.  The
resultant permanent auditory impairments were assessed by evaluating loss of
DPOAE amplitude within subjects between a pre-exposure measurement and
assessment 4 weeks after exposure ended. Additionally, auditory thresholds were
measured 4 weeks postexposure using the CAP. Cochlear damage was quantified in
the same animals by systematic hair cell counts.Experimental groups and exposure schedule are
detailed in Table1.Table 1
Experimental groups and treatment schedules.Control(n=15) and ACN alone(n=3) animals were maintained for 4 hours daily in the exposure chambers with the
noise generator turned off. Due to technical reasons, DPOAEs were not recorded
in 3 rats that received OBN-16 kHz alone and in 2 rats that received ACN +
OBN-16 kHz. Control and noise-alone animals received saline injections in place
of ACN.


### 2.3. Acrylonitrile Exposure

Stabilized ACN(99%) was purchased
from Sigma-Aldrich (St. Louis, Mo, USA). ACN (50 mg/kg) injections were made daily
for 5 days, 30 minutes prior to the noise. Fechter et al. [3] showed that ACN caused slight
transient hearing impairment that reach a maximum at 10–20 minutes and resolve by
about 75–100 minutes. The interval between ACN injection and onset of noise was
selected based upon this known ACN-induced temporary threshold deficits.


### 2.4. Noise Exposure

Exposures were conducted in a
ventilated reverberant 40 L Plexiglas cylinder. The subjects were placed within
small wire-cloth enclosures (20×9×15 cm) within the chamber. They were
conscious and free to move within the enclosures. Broadband noises were
generated by a function-generator (Stanford Research System, Model DS335, Menlo
Park, Calif, USA) and bandpass filtered (Frequency Devices, 9002, Haverhill, Mass, USA) to
provide OBN with center frequencies of 4, 8 or 16 kHz. The roll-off for the
filter system was 48 dB/octave. This signal was amplified by a SAE 2200 Power
Amplifier (Scientific Audio Electronics Inc., Los Angeles, Calif, USA) and fed to
speakers (Vifa D25AG-05, Videbaek, Denmark) located approximately 5 cm above
the subjects’ wire-cloth enclosure. Sound intensities measured at the level of
the rats’ pinnae by a Quest Type 1 sound pressure meter with 1/1 octave filter
set (models 1700 and OB300, Oconomowoc, Wisc, USA) were 110 dB SPL for the OBN-4 kHz,
and 97 dB SPL for the OBN-16 kHz and the OBN-8 kHz (Figure 1). The noise level
chosen for the 4 kHz-OBN was 13 dB higher than the two other OBNs in order to
compensate for the rat’s higher auditory thresholds at low frequencies.
Empirically, the rat’s CAP threshold as determined in this laboratory is around
35 dB SPL in the octave band centered at 4 kHz, relative to 20 dB SPL for the
8-kHz and the 16-kHz OBNs. The noise spectra are displayed in Figure 1. Noise
exposures lasted 4 hours for 5 successive days. The noise varied less than 2 dB
within the exposure chamber.Figure 1
Noise spectra of the
110-dB-SPL octave-band centered at 4 kHz, and the 97-dB-SPL octave-bands
centered at 8 kHz and 16 kHz.


### 2.5. Distortion Product Otoacoustic Emissions (DPOAEs) Testing

The rats were lightly anesthetized
by injection of xylazine (7 mg/kgim)
and ketamine (44 mg/kg im), and
placed on a heating table in order to maintain the body temperature at 38°C. An
Etymotics Research probe (ER10) was inserted in the right auditory canal in
order to deliver the primary tones to the ear canal and record the DPOAE
response. The same ear was subsequently used for CAP determination.The primary tones,F1 and F2, were
generated by a dual-channel synthesizer (Hewlett Packard Model 3326A) and
attenuated, under computer control, using customized software. The F1 and F2
primaries were then presented through two separate earphones (Radio Shack,
Realistic Dual Radial Horn Tweeters, Tandy Corp., Ft. Worth, Tex, USA) and delivered
to the outer-ear canal through a probe, where they acoustically mixed to avoid
artifactual distortion. Ear-canal sound pressure levels, measured by an
emissions microphone assembly (Etymotic Research, ER-10B+, Elk Grove Village,
Ill, USA) embedded in the probe, were sampled, synchronously averaged, and Fourier
analyzed for geometric mean (GM) frequencies ((F1×F2)0.5) ranging
from 5.6 to 19.7 kHz (i.e., F2=6.3–22.5 kHz) by a
computer-based DSP board. Corresponding noise floors were computed by averaging
the levels of the ear-canal sound pressure for five frequency bins above and
below the DPOAE frequency bin (±54 Hz).For
test frequencies above 20.1 kHz, a computer-controlled dynamic-signal analyzer
(Hewlett Packard Model 3561A) was used. The related noise floors were estimated
by averaging the levels of the ear-canal sound pressure for the two FFT
frequency bins below the DPOAE frequency (i.e., for 3.75 Hz below the DPOAE).
No artifactual DPOAEs were ever measured in a hard-walled cavity that
approximated the size of the rat outer-ear canal, which was used to calibrate
the tonal stimuli. DPOAEs were considered to be present when they were at least
3 dB above the noise floor. DPOAEs were
measured as DP-grams. Specifically, DP-grams described emission levels in
response to primary tones atL1=L2=75 dB SPL as a function of the GM
frequencies, which ranged from 2.9 to 56.3 kHz (F2=3.2 to 63 kHz), in
0.1-octave increments. The ratio F2/F1 was 1.25.Between 20 and 25 kHz (F2=20.8,22.2,23.8, and 25.6 kHz), the DP-grams display an artifactual notch due to the resonance
of the rat’s outer auditory meatus. Therefore, these frequencies were excluded
from statistical analysis. Candreia et al. [7] previously observed a similar
notch in the mouse. This phenomenon was recently described in detail by Martin et al. [8].


### 2.6. Compound Action Potentials (CAPs)

CAP threshold assessment was
performed 4 weeks postexposure, the day after the last DPOAE measure. CAPs
recorded from the right round window were elicited with pure tones bursts at 2,
4, 6, 8, 12, 16, 20, 24, 30, 35, and 40 kHz. The test tones were presented using
a 1/2” ACO Pacific
(Belmont, Calif, USA) model 7013 condenser
microphone that was driven by a high impedence amplifier. The microphone was
held in a plastic speculum within the ear canal. The stimuli were 10 msec in
duration with a 1 msec onset and offset ramp. Stimuli were presented at a
frequency of 9.7 Hz. Auditory thresholds were assessed in a double walled sound
booth. The subjects were anaesthetized with xylazine (13 mg/kgim) and ketamine (87 mg/kg im) and normal body temperature was
maintained using a heating table. The temperature of the cochlea was maintained
using a low voltage high-intensity lamp. The auditory bulla was opened via a
ventrolateral approach to allow the placement of a fine (od 0.1 mm)
Teflon-coated silver wire electrode (A-M system, Inc., Carlsborg, Wash, USA) onto the
round window. A silver chloride reference electrode was inserted into neck
musculature. The CAP signals evoked by pure tones were amplified 1000x between
0.1–1.0 kHz with a Grass A.C. preamplifier (Model P15, W. Warwick, RI, USA). The
sound level necessary to generate a visually detectable CAP response averaged
over 4 sweeps on a digital oscilloscope (approximate response amplitude of 1 mV
at the output of the preamplifier) was identified.


### 2.7. Hair Cell Counts

Immediately after CAP measurements,
rats were decapitated and the cochleae harvested. Within 2 minutes, the cochleae
were fixed by perilymphatic perfusion with 1 mL of a trialdehyde fixative (3%
glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% DMSO in phosphate
buffered saline pH7.4). Following the primary 24-hour fixation, the tissue was
first washed with 0.1 M phosphate buffered saline, post-fixed with 2% osmium
tetroxide in water for 2 hours, and finally washed again with 0.1 M phosphate
buffered saline. The organ of Corti was dissected in 70% ethanol and mounted in
glycerin to allow counting of the hair cells using a surface preparation. Cells
were counted as present either when the stereocilia, the cuticular plate or the
cell nucleus could be visualized. No attempt was made to assess the degree of
possible cellular damage to surviving cells. The frequency-place map
established by Muller [9] was used to superimpose the
frequency coordinates on the length coordinates of the organ of Corti. This
“map” reflects the fact that the cochlea is organized in a tonotopic fashion
with high frequency sound producing maximum stimulation of cells in the base,
and low frequency sound in the apex. A cochleogram showing the percentage of
hair cell loss as a function of distance from the apex of the cochlea was
plotted for each animal. The results were averaged within each group of
subjects for comparison between groups. The custom programs used for counting
cochlear hair cells, plotting, and averaging cochleograms, were developed by R.
Lataye and Dr. P. Campo from the “Institut National de Recherche et Sécurité,”
Nancy, France. In some instances, the most basal region of the cochlea was
damaged or not recovered during dissection owing to the fragility of the organ
of Corti and difficulties extracting the hook at the extreme base from the
surrounding bone. The degree of loss was estimated as 10 hair cells in length.
In such cases all cells were considered to be present. Because such
difficulties do not affect the apical region of the cochlea, cell counts were
made from the apex of the cochlea to the base. Thus is was possible to be
confident of the relationship between damage along the organ of Corti and the
corresponding tone frequencies encoded at that locus.


### 2.8. Statistical Analysis

All statistical analyses were
conducted using Prism 4.0c (GraphPad Software, San Diego, Calif, USA).DPOAE amplitudes were analyzed with
a global 2-way ANOVA regrouping all experimental groups using “exposure” as a
between-subject factor and “F2 frequency” as within-subject factors. The 4
frequencies corresponding to the notch in the DPgram, (F2=20.8,22.2,23.8,
and 25.6 kHz) were excluded from the analyses. Planned post hoc comparisons
were performed between treatment groups using the Bonferroni’s test. P=.05
was considered as the significance threshold.Similarly, CAP thresholds were
analyzed with a global two-way ANOVA to evaluate the effects of “exposure”
(between-subject factor) at the different “frequencies” (within-subject).OHC loss obtained in all
experimental groups was analyzed conjointly with a global two-way ANOVA with
the “exposure” as a between-subject factor and “cochlear location” of the hair
cell loss as a within-subject factor. Bonferroni’s post-hoc tests were
performed between experimental groups.P=.05 was considered as the
significance threshold.


## 2.1. Subjects

A
total of 57 male Long-Evans rats (225–249 g, 7–8 weeks old) obtained from
Harlan (Indianapolis, Ind, USA) were employed in these experiments. The subjects were
housed with free access to food and water in their home cages. Temperature was
maintained at21±1°C and lights were on from 6:30 am to 6:30 pm. The Loma Linda
Veteran Medical Center Institutional Animal Care and Use Committee (IACUC) approved
all the experimental protocols. All exposures and testing were performed during
the daytime.


## 2.2. Procedures

Groups of Long-Evans rats(n=4–9)
were exposed for 5 days (4 hours/day) to moderately intense octave band noise
(OBN) centered at 4 kHz (110 dB SPL), 8 kHz (97 dB SPL), or 16 kHz (97 dB SPL),
with or without co-exposure to ACN.  The
resultant permanent auditory impairments were assessed by evaluating loss of
DPOAE amplitude within subjects between a pre-exposure measurement and
assessment 4 weeks after exposure ended. Additionally, auditory thresholds were
measured 4 weeks postexposure using the CAP. Cochlear damage was quantified in
the same animals by systematic hair cell counts.Experimental groups and exposure schedule are
detailed in Table1.Table 1
Experimental groups and treatment schedules.Control(n=15) and ACN alone(n=3) animals were maintained for 4 hours daily in the exposure chambers with the
noise generator turned off. Due to technical reasons, DPOAEs were not recorded
in 3 rats that received OBN-16 kHz alone and in 2 rats that received ACN +
OBN-16 kHz. Control and noise-alone animals received saline injections in place
of ACN.


## 2.3. Acrylonitrile Exposure

Stabilized ACN(99%) was purchased
from Sigma-Aldrich (St. Louis, Mo, USA). ACN (50 mg/kg) injections were made daily
for 5 days, 30 minutes prior to the noise. Fechter et al. [3] showed that ACN caused slight
transient hearing impairment that reach a maximum at 10–20 minutes and resolve by
about 75–100 minutes. The interval between ACN injection and onset of noise was
selected based upon this known ACN-induced temporary threshold deficits.


## 2.4. Noise Exposure

Exposures were conducted in a
ventilated reverberant 40 L Plexiglas cylinder. The subjects were placed within
small wire-cloth enclosures (20×9×15 cm) within the chamber. They were
conscious and free to move within the enclosures. Broadband noises were
generated by a function-generator (Stanford Research System, Model DS335, Menlo
Park, Calif, USA) and bandpass filtered (Frequency Devices, 9002, Haverhill, Mass, USA) to
provide OBN with center frequencies of 4, 8 or 16 kHz. The roll-off for the
filter system was 48 dB/octave. This signal was amplified by a SAE 2200 Power
Amplifier (Scientific Audio Electronics Inc., Los Angeles, Calif, USA) and fed to
speakers (Vifa D25AG-05, Videbaek, Denmark) located approximately 5 cm above
the subjects’ wire-cloth enclosure. Sound intensities measured at the level of
the rats’ pinnae by a Quest Type 1 sound pressure meter with 1/1 octave filter
set (models 1700 and OB300, Oconomowoc, Wisc, USA) were 110 dB SPL for the OBN-4 kHz,
and 97 dB SPL for the OBN-16 kHz and the OBN-8 kHz (Figure 1). The noise level
chosen for the 4 kHz-OBN was 13 dB higher than the two other OBNs in order to
compensate for the rat’s higher auditory thresholds at low frequencies.
Empirically, the rat’s CAP threshold as determined in this laboratory is around
35 dB SPL in the octave band centered at 4 kHz, relative to 20 dB SPL for the
8-kHz and the 16-kHz OBNs. The noise spectra are displayed in Figure 1. Noise
exposures lasted 4 hours for 5 successive days. The noise varied less than 2 dB
within the exposure chamber.Figure 1
Noise spectra of the
110-dB-SPL octave-band centered at 4 kHz, and the 97-dB-SPL octave-bands
centered at 8 kHz and 16 kHz.


## 2.5. Distortion Product Otoacoustic Emissions (DPOAEs) Testing

The rats were lightly anesthetized
by injection of xylazine (7 mg/kgim)
and ketamine (44 mg/kg im), and
placed on a heating table in order to maintain the body temperature at 38°C. An
Etymotics Research probe (ER10) was inserted in the right auditory canal in
order to deliver the primary tones to the ear canal and record the DPOAE
response. The same ear was subsequently used for CAP determination.The primary tones,F1 and F2, were
generated by a dual-channel synthesizer (Hewlett Packard Model 3326A) and
attenuated, under computer control, using customized software. The F1 and F2
primaries were then presented through two separate earphones (Radio Shack,
Realistic Dual Radial Horn Tweeters, Tandy Corp., Ft. Worth, Tex, USA) and delivered
to the outer-ear canal through a probe, where they acoustically mixed to avoid
artifactual distortion. Ear-canal sound pressure levels, measured by an
emissions microphone assembly (Etymotic Research, ER-10B+, Elk Grove Village,
Ill, USA) embedded in the probe, were sampled, synchronously averaged, and Fourier
analyzed for geometric mean (GM) frequencies ((F1×F2)0.5) ranging
from 5.6 to 19.7 kHz (i.e., F2=6.3–22.5 kHz) by a
computer-based DSP board. Corresponding noise floors were computed by averaging
the levels of the ear-canal sound pressure for five frequency bins above and
below the DPOAE frequency bin (±54 Hz).For
test frequencies above 20.1 kHz, a computer-controlled dynamic-signal analyzer
(Hewlett Packard Model 3561A) was used. The related noise floors were estimated
by averaging the levels of the ear-canal sound pressure for the two FFT
frequency bins below the DPOAE frequency (i.e., for 3.75 Hz below the DPOAE).
No artifactual DPOAEs were ever measured in a hard-walled cavity that
approximated the size of the rat outer-ear canal, which was used to calibrate
the tonal stimuli. DPOAEs were considered to be present when they were at least
3 dB above the noise floor. DPOAEs were
measured as DP-grams. Specifically, DP-grams described emission levels in
response to primary tones atL1=L2=75 dB SPL as a function of the GM
frequencies, which ranged from 2.9 to 56.3 kHz (F2=3.2 to 63 kHz), in
0.1-octave increments. The ratio F2/F1 was 1.25.Between 20 and 25 kHz (F2=20.8,22.2,23.8, and 25.6 kHz), the DP-grams display an artifactual notch due to the resonance
of the rat’s outer auditory meatus. Therefore, these frequencies were excluded
from statistical analysis. Candreia et al. [7] previously observed a similar
notch in the mouse. This phenomenon was recently described in detail by Martin et al. [8].


## 2.6. Compound Action Potentials (CAPs)

CAP threshold assessment was
performed 4 weeks postexposure, the day after the last DPOAE measure. CAPs
recorded from the right round window were elicited with pure tones bursts at 2,
4, 6, 8, 12, 16, 20, 24, 30, 35, and 40 kHz. The test tones were presented using
a 1/2” ACO Pacific
(Belmont, Calif, USA) model 7013 condenser
microphone that was driven by a high impedence amplifier. The microphone was
held in a plastic speculum within the ear canal. The stimuli were 10 msec in
duration with a 1 msec onset and offset ramp. Stimuli were presented at a
frequency of 9.7 Hz. Auditory thresholds were assessed in a double walled sound
booth. The subjects were anaesthetized with xylazine (13 mg/kgim) and ketamine (87 mg/kg im) and normal body temperature was
maintained using a heating table. The temperature of the cochlea was maintained
using a low voltage high-intensity lamp. The auditory bulla was opened via a
ventrolateral approach to allow the placement of a fine (od 0.1 mm)
Teflon-coated silver wire electrode (A-M system, Inc., Carlsborg, Wash, USA) onto the
round window. A silver chloride reference electrode was inserted into neck
musculature. The CAP signals evoked by pure tones were amplified 1000x between
0.1–1.0 kHz with a Grass A.C. preamplifier (Model P15, W. Warwick, RI, USA). The
sound level necessary to generate a visually detectable CAP response averaged
over 4 sweeps on a digital oscilloscope (approximate response amplitude of 1 mV
at the output of the preamplifier) was identified.


## 2.7. Hair Cell Counts

Immediately after CAP measurements,
rats were decapitated and the cochleae harvested. Within 2 minutes, the cochleae
were fixed by perilymphatic perfusion with 1 mL of a trialdehyde fixative (3%
glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% DMSO in phosphate
buffered saline pH7.4). Following the primary 24-hour fixation, the tissue was
first washed with 0.1 M phosphate buffered saline, post-fixed with 2% osmium
tetroxide in water for 2 hours, and finally washed again with 0.1 M phosphate
buffered saline. The organ of Corti was dissected in 70% ethanol and mounted in
glycerin to allow counting of the hair cells using a surface preparation. Cells
were counted as present either when the stereocilia, the cuticular plate or the
cell nucleus could be visualized. No attempt was made to assess the degree of
possible cellular damage to surviving cells. The frequency-place map
established by Muller [9] was used to superimpose the
frequency coordinates on the length coordinates of the organ of Corti. This
“map” reflects the fact that the cochlea is organized in a tonotopic fashion
with high frequency sound producing maximum stimulation of cells in the base,
and low frequency sound in the apex. A cochleogram showing the percentage of
hair cell loss as a function of distance from the apex of the cochlea was
plotted for each animal. The results were averaged within each group of
subjects for comparison between groups. The custom programs used for counting
cochlear hair cells, plotting, and averaging cochleograms, were developed by R.
Lataye and Dr. P. Campo from the “Institut National de Recherche et Sécurité,”
Nancy, France. In some instances, the most basal region of the cochlea was
damaged or not recovered during dissection owing to the fragility of the organ
of Corti and difficulties extracting the hook at the extreme base from the
surrounding bone. The degree of loss was estimated as 10 hair cells in length.
In such cases all cells were considered to be present. Because such
difficulties do not affect the apical region of the cochlea, cell counts were
made from the apex of the cochlea to the base. Thus is was possible to be
confident of the relationship between damage along the organ of Corti and the
corresponding tone frequencies encoded at that locus.


## 2.8. Statistical Analysis

All statistical analyses were
conducted using Prism 4.0c (GraphPad Software, San Diego, Calif, USA).DPOAE amplitudes were analyzed with
a global 2-way ANOVA regrouping all experimental groups using “exposure” as a
between-subject factor and “F2 frequency” as within-subject factors. The 4
frequencies corresponding to the notch in the DPgram, (F2=20.8,22.2,23.8,
and 25.6 kHz) were excluded from the analyses. Planned post hoc comparisons
were performed between treatment groups using the Bonferroni’s test. P=.05
was considered as the significance threshold.Similarly, CAP thresholds were
analyzed with a global two-way ANOVA to evaluate the effects of “exposure”
(between-subject factor) at the different “frequencies” (within-subject).OHC loss obtained in all
experimental groups was analyzed conjointly with a global two-way ANOVA with
the “exposure” as a between-subject factor and “cochlear location” of the hair
cell loss as a within-subject factor. Bonferroni’s post-hoc tests were
performed between experimental groups.P=.05 was considered as the
significance threshold.


## 3. Results

### 3.1. Functional Data

#### 3.1.1. Distortion Product Otoacoustic Emissions

Figure2 shows DP-grams obtained 4 weeks post-exposure in rats subjected to (a) 4, (b) 8, or (c) 16-kHz OBNs with or
without co-exposure to ACN. For reference, DP-grams obtained in controls and
ACN alone are included in each of the graphs.Four-week postexposure
DPOAE amplitudes obtained from rats exposed to (a) the 110 dB OBN-4 kHz with or
without ACN, (b) to the 97 dB OBN-8 kHz with or without ACN, or (c) to the 97 dB
OBN-16 kHz with or without ACN (see Table1 for details). DPOAE amplitude
measured in control and ACN alone animals were included in each of the graphs
for reference. DPgrams were obtained with the levels of the primaries F1 and F2
set at 75, and with F2/F1=1.25. The tested F2 frequencies ranged from 3.2 to
63 kHz (geometric mean frequencies: 2.9 to 56.3 kHz), in 0.1 octave increments.
The gray areas represent the theoretical octave-band noise frequency ranges.
Error bars: ±SEM. * for
technical reasons only 4 subjects could be measured with DPOAEs in the
OBN-16 kHz and in the ACN + OBN-16 kHz groups.
(a)(b)(c)Equivalent baseline DP grams were
obtained in the different groups prior to exposure (not shown).Four weeks post-exposure, the
global 2-way ANOVA reveals significant effects of “exposure” on the DPOAE
amplitudes (F(7,44)=5.955;P<.0001). The results of the post-hoc
Bonferroni’s tests are detailed in what follows.Figure2(a) displays the DPgrams
obtained 4 weeks after exposure to the 110 dB OBN-4 kHz noise with or without
ACN. Exposure to noise alone only induced a slight DPOAE decrease from about 6 kHz to the lower limit of the “notch.” Above this notch the decrease appears
more profound.  The difference from
controls is only significant from 36 to 55 kHz
by Bonferroni’s test (Figure 5(a)). The animals that were exposed to ACN and
noise show lower DPOAEs than the noise-alone exposed rats over a wide range of
frequencies. However, this difference is only significant from 16 to 28 kHz
(P<.05), while it approaches significance from 9 to 15 kHz. As noted
earlier, 4 frequencies (20.8, 22.2, 23.8, and 25.6 kHz were excluded from this
analysis.  ACN-alone animals do not show
any significant decrease of their DPOAE amplitudes when compared to control
animals.Figure2(b) displays the DPgrams
obtained 4 weeks after exposure to the 97-dB OBN-8 kHz noise with or without
ACN. The exposure to ACN + OBN-8 kHz (Figure 2(b)) induced a large potentiation of
NIHL. The region in which the
mean differences were greatest cannot be precisely determined due to the notch
in the responses, but it can be estimated that the difference between ACN +
OBN-8 kHz and the noise-alone animals is about 18–25 dB between 17 and 36 kHz. Bonferroni’s
post-test (Figure 5(a)) reveals that the ACN + noise animal’s DPOAE amplitudes differ
from the ones obtained in noise-alone animals from 15 to 55 kHz; while the
animals exposed to noise alone are not different from controls.In a similar fashion, the 97-dB
OBN-16 kHz exposure (Figure2(c)) did not induce any permanent decrease of DPOAE
amplitudes, while the ACN + OBN-16 kHz exposure caused a reliable DPOAE deficit
above 12 kHz. The mean difference was the greatest between 27 and 37 kHz where
it reached more than 30 dB. The only significant difference shown by
Bonferroni’s post-tests is between noise-alone and ACN + noise DPgrams between 12
and 45 kHz (P<.05).


#### 3.1.2. Compound Action Potentials

Figure3 presents the disruption of
CAP thresholds measured 4 week post-exposure in rats subjected to (a) 4, (b) 8,
or (c) 16-kHz OBN with or without co-exposure to ACN. For reference, thresholds
obtained in controls and ACN alone are included in each of the graphs.Effects of the different
experimental treatments on compound action potential (CAP) thresholds measured
4 weeks post-exposure for frequencies ranging from 2 to 40 kHz. CAP thresholds
obtained in rats exposed to (a) the 110 dB OBN-4 kHz with or without ACN, (b) to
the 97 dB OBN-8 kHz with or without ACN, or (c) to the 97 dB OBN-16 kHz with or
without ACN (see Table1 for details). Auditory thresholds measured in control
and ACN alone animals were included in each of the graphs for reference. The
colored areas represent the octave band-noise frequency ranges. Error bars: ±SEM.
(a)(b)(c)The two-way ANOVA carried out on
the CAP thresholds revealed a significant effect of “exposure” (F(7,49)=13.16;P<.0001). ACN alone animals
did not show any significant threshold difference compared to the control
thresholds (Bonferroni, P>.05; see Figure 5(b)). While the OBN-4 kHz animals
show slightly elevated thresholds between 6 and 16 kHz compared to the control
data, this increase is not statistically significant (P>.05). By
contrast, the animals that received combined exposures to ACN + OBN-4 kHz display
significantly elevated thresholds between 20 and 40 kHz when compared to the
noise-alone thresholds (P<.05; see Figure 5(b)). The difference between these
two groups reaches up to 41 dB at both 30 and 35 kHz.Neither the 8-kHz nor the 16-kHz
noise-alone groups show significantly elevated thresholds compared to controls
(Bonferroni,P>.05). By contrast, the ACN + OBN-8 kHz and the ACN +
OBN-16 kHz thresholds were significantly different from their noise-alone
counterparts, respectively, between 12 and 40 kHz and between 20 and 40 kHz
(Figure 5(b)).


### 3.2. Hair Cell Counts

To assess the magnitude of cochlear
damage, hair cells were counted from cochleae harvested from the same animals
used for physiological studies. The hair cell loss is presented as cochleograms
that display the percentage of hair cell loss as a function of distance from
the apex of the cochlea.Figure4 shows the mean
cochleograms obtained in the animals exposed to (a) ACN alone, (b) OBN-4 kHz
alone, (c) ACN + OBN-4 kHz, (d) OBN-8 kHz alone, (e) ACN + OBN-8 kHz, (f)
OBN-16 kHz alone, (g) ACN + OBN-16 kHz. The cochleae from controls (not shown)
and ACN alone subjects (Figure 4(a)) displayed no hair cell loss, while those
exposed to any of the OBNs without ACN (Figures 4(b), 4(d) and 4(f)) displayed limited
damage in the basal half of the organ of Corti, less than 10% OHC loss at any
tonotopic location. None of the exposures yielded any inner hair cell (IHC)
loss. Therefore, IHCs are not discussed here, and are not included in the
statistical analysis (see Section 2.8).Average cochleograms
showing hair cell loss in rats exposed to (a) ACN alone or to the 110 dB
OBN-4 kHz with (c) or without (b) ACN, in rats exposed to the 97 dB OBN-8 kHz with
(e) or without (d) ACN, and in rats exposed to the 97 dB OBN-16 kHz with (g) or
without (f) ACN. Abscissa (i) upper trace: length (mm) of the entire spiral
course of the organ of Corti from the bottom of the hook, (ii) lower trace:
frequency-map according to Muller [9]. Ordinate: hair cell loss in
percent. IHC: inner hair cells; OHC1: first row of outer hair cells; OHC2:
second row; OHC3: third row. Error bars represent the standard error.
(a)(b)(c)(d)(e)(f)(g)Statistical comparisons
between groups for (a) DPOAE amplitudes, (b) CAP thresholds, and (c) hair cell
loss at 4 weeks postexposure: Bonferroni’s post-hoc comparisons withP=.05
as the significant limit. The parent analyses were repeated measure ANOVAs with
“exposure” as between factor and “frequency” as a within
factor. The colored bands illustrate the frequency range for which Bonferroni’s
comparisons between groups yielded P<.05. ns: not significantly
different. Note: for the CAP
technique, the significance range is presented as “above x kHz” because it does
not allow measurements above 40 kHz, whereas the significance is likely more extended
toward high frequencies.
(a)
Post-hoc Bonferronil’s comparisons—DPOAEs(b)
Post-hoc Bonferronil’s comparisons —CAPs(c)
Post-hoc Bonferronil’s comparisons—HistologyConsistent with physiological
results, the cochleae from rats exposed to both ACN and noise (Figures4(c), 4(e), and
4(g)) exhibited substantial damage in the basal (or high frequency) third of the
organ of Corti. The pattern of OHC loss is actually surprisingly similar among
the different ACN + noise exposures. Despite the fact that the OHC loss
obtained in the rats exposed to ACN + OBN-8 kHz is scattered over a wider range
than the other two combined exposure groups, the hair cell loss averaged about
45% in the three OHC rows in the region corresponding to frequencies above 15 kHz for the three different combined exposures.The OHC loss obtained in all
experimental groups was analyzed with a global two-way ANOVA. The overall
effect of “exposure” was significant (F(7,47)=5.60;P=.0003). Bonferroni’s
post-tests (see graphic representation in Figure 5(c)) confirmed that
cochleograms obtained with either one of the OBNs were not significantly
different from the control cochleogram (not shown). By contrast, the ACN +
OBN-4 kHz, the ACN + OBN-8 kHz, and the ACN + OBN-16 kHz cochleograms are
significantly different from their noise-alone counterparts, respectively, from
22 to 64 kHz, from 23 to 64 and from 26 to 52 kHz (P<.05). Hair cell loss
in the ACN + OBN-8 kHz was visibly wider in terms of frequency than the OHC loss
obtained in the other two combined exposure groups. However, the difference
barely reached statistical significance at a few frequencies: OHC loss in the
ACN + OBN-8 kHz cochleae was different (P<.05) from the loss in the ACN +
OBN-4 kHz at 27, 28, 37–39, and 46–50 kHz, and from the ACN + OBN-16 kHz cochleae
at 21, 22–25, 27-28, and 38–42 kHz.


## 3.1. Functional Data

### 3.1.1. Distortion Product Otoacoustic Emissions

Figure2 shows DP-grams obtained 4 weeks post-exposure in rats subjected to (a) 4, (b) 8, or (c) 16-kHz OBNs with or
without co-exposure to ACN. For reference, DP-grams obtained in controls and
ACN alone are included in each of the graphs.Four-week postexposure
DPOAE amplitudes obtained from rats exposed to (a) the 110 dB OBN-4 kHz with or
without ACN, (b) to the 97 dB OBN-8 kHz with or without ACN, or (c) to the 97 dB
OBN-16 kHz with or without ACN (see Table1 for details). DPOAE amplitude
measured in control and ACN alone animals were included in each of the graphs
for reference. DPgrams were obtained with the levels of the primaries F1 and F2
set at 75, and with F2/F1=1.25. The tested F2 frequencies ranged from 3.2 to
63 kHz (geometric mean frequencies: 2.9 to 56.3 kHz), in 0.1 octave increments.
The gray areas represent the theoretical octave-band noise frequency ranges.
Error bars: ±SEM. * for
technical reasons only 4 subjects could be measured with DPOAEs in the
OBN-16 kHz and in the ACN + OBN-16 kHz groups.
(a)(b)(c)Equivalent baseline DP grams were
obtained in the different groups prior to exposure (not shown).Four weeks post-exposure, the
global 2-way ANOVA reveals significant effects of “exposure” on the DPOAE
amplitudes (F(7,44)=5.955;P<.0001). The results of the post-hoc
Bonferroni’s tests are detailed in what follows.Figure2(a) displays the DPgrams
obtained 4 weeks after exposure to the 110 dB OBN-4 kHz noise with or without
ACN. Exposure to noise alone only induced a slight DPOAE decrease from about 6 kHz to the lower limit of the “notch.” Above this notch the decrease appears
more profound.  The difference from
controls is only significant from 36 to 55 kHz
by Bonferroni’s test (Figure 5(a)). The animals that were exposed to ACN and
noise show lower DPOAEs than the noise-alone exposed rats over a wide range of
frequencies. However, this difference is only significant from 16 to 28 kHz
(P<.05), while it approaches significance from 9 to 15 kHz. As noted
earlier, 4 frequencies (20.8, 22.2, 23.8, and 25.6 kHz were excluded from this
analysis.  ACN-alone animals do not show
any significant decrease of their DPOAE amplitudes when compared to control
animals.Figure2(b) displays the DPgrams
obtained 4 weeks after exposure to the 97-dB OBN-8 kHz noise with or without
ACN. The exposure to ACN + OBN-8 kHz (Figure 2(b)) induced a large potentiation of
NIHL. The region in which the
mean differences were greatest cannot be precisely determined due to the notch
in the responses, but it can be estimated that the difference between ACN +
OBN-8 kHz and the noise-alone animals is about 18–25 dB between 17 and 36 kHz. Bonferroni’s
post-test (Figure 5(a)) reveals that the ACN + noise animal’s DPOAE amplitudes differ
from the ones obtained in noise-alone animals from 15 to 55 kHz; while the
animals exposed to noise alone are not different from controls.In a similar fashion, the 97-dB
OBN-16 kHz exposure (Figure2(c)) did not induce any permanent decrease of DPOAE
amplitudes, while the ACN + OBN-16 kHz exposure caused a reliable DPOAE deficit
above 12 kHz. The mean difference was the greatest between 27 and 37 kHz where
it reached more than 30 dB. The only significant difference shown by
Bonferroni’s post-tests is between noise-alone and ACN + noise DPgrams between 12
and 45 kHz (P<.05).


### 3.1.2. Compound Action Potentials

Figure3 presents the disruption of
CAP thresholds measured 4 week post-exposure in rats subjected to (a) 4, (b) 8,
or (c) 16-kHz OBN with or without co-exposure to ACN. For reference, thresholds
obtained in controls and ACN alone are included in each of the graphs.Effects of the different
experimental treatments on compound action potential (CAP) thresholds measured
4 weeks post-exposure for frequencies ranging from 2 to 40 kHz. CAP thresholds
obtained in rats exposed to (a) the 110 dB OBN-4 kHz with or without ACN, (b) to
the 97 dB OBN-8 kHz with or without ACN, or (c) to the 97 dB OBN-16 kHz with or
without ACN (see Table1 for details). Auditory thresholds measured in control
and ACN alone animals were included in each of the graphs for reference. The
colored areas represent the octave band-noise frequency ranges. Error bars: ±SEM.
(a)(b)(c)The two-way ANOVA carried out on
the CAP thresholds revealed a significant effect of “exposure” (F(7,49)=13.16;P<.0001). ACN alone animals
did not show any significant threshold difference compared to the control
thresholds (Bonferroni, P>.05; see Figure 5(b)). While the OBN-4 kHz animals
show slightly elevated thresholds between 6 and 16 kHz compared to the control
data, this increase is not statistically significant (P>.05). By
contrast, the animals that received combined exposures to ACN + OBN-4 kHz display
significantly elevated thresholds between 20 and 40 kHz when compared to the
noise-alone thresholds (P<.05; see Figure 5(b)). The difference between these
two groups reaches up to 41 dB at both 30 and 35 kHz.Neither the 8-kHz nor the 16-kHz
noise-alone groups show significantly elevated thresholds compared to controls
(Bonferroni,P>.05). By contrast, the ACN + OBN-8 kHz and the ACN +
OBN-16 kHz thresholds were significantly different from their noise-alone
counterparts, respectively, between 12 and 40 kHz and between 20 and 40 kHz
(Figure 5(b)).


## 3.1.1. Distortion Product Otoacoustic Emissions

Figure2 shows DP-grams obtained 4 weeks post-exposure in rats subjected to (a) 4, (b) 8, or (c) 16-kHz OBNs with or
without co-exposure to ACN. For reference, DP-grams obtained in controls and
ACN alone are included in each of the graphs.Four-week postexposure
DPOAE amplitudes obtained from rats exposed to (a) the 110 dB OBN-4 kHz with or
without ACN, (b) to the 97 dB OBN-8 kHz with or without ACN, or (c) to the 97 dB
OBN-16 kHz with or without ACN (see Table1 for details). DPOAE amplitude
measured in control and ACN alone animals were included in each of the graphs
for reference. DPgrams were obtained with the levels of the primaries F1 and F2
set at 75, and with F2/F1=1.25. The tested F2 frequencies ranged from 3.2 to
63 kHz (geometric mean frequencies: 2.9 to 56.3 kHz), in 0.1 octave increments.
The gray areas represent the theoretical octave-band noise frequency ranges.
Error bars: ±SEM. * for
technical reasons only 4 subjects could be measured with DPOAEs in the
OBN-16 kHz and in the ACN + OBN-16 kHz groups.
(a)(b)(c)Equivalent baseline DP grams were
obtained in the different groups prior to exposure (not shown).Four weeks post-exposure, the
global 2-way ANOVA reveals significant effects of “exposure” on the DPOAE
amplitudes (F(7,44)=5.955;P<.0001). The results of the post-hoc
Bonferroni’s tests are detailed in what follows.Figure2(a) displays the DPgrams
obtained 4 weeks after exposure to the 110 dB OBN-4 kHz noise with or without
ACN. Exposure to noise alone only induced a slight DPOAE decrease from about 6 kHz to the lower limit of the “notch.” Above this notch the decrease appears
more profound.  The difference from
controls is only significant from 36 to 55 kHz
by Bonferroni’s test (Figure 5(a)). The animals that were exposed to ACN and
noise show lower DPOAEs than the noise-alone exposed rats over a wide range of
frequencies. However, this difference is only significant from 16 to 28 kHz
(P<.05), while it approaches significance from 9 to 15 kHz. As noted
earlier, 4 frequencies (20.8, 22.2, 23.8, and 25.6 kHz were excluded from this
analysis.  ACN-alone animals do not show
any significant decrease of their DPOAE amplitudes when compared to control
animals.Figure2(b) displays the DPgrams
obtained 4 weeks after exposure to the 97-dB OBN-8 kHz noise with or without
ACN. The exposure to ACN + OBN-8 kHz (Figure 2(b)) induced a large potentiation of
NIHL. The region in which the
mean differences were greatest cannot be precisely determined due to the notch
in the responses, but it can be estimated that the difference between ACN +
OBN-8 kHz and the noise-alone animals is about 18–25 dB between 17 and 36 kHz. Bonferroni’s
post-test (Figure 5(a)) reveals that the ACN + noise animal’s DPOAE amplitudes differ
from the ones obtained in noise-alone animals from 15 to 55 kHz; while the
animals exposed to noise alone are not different from controls.In a similar fashion, the 97-dB
OBN-16 kHz exposure (Figure2(c)) did not induce any permanent decrease of DPOAE
amplitudes, while the ACN + OBN-16 kHz exposure caused a reliable DPOAE deficit
above 12 kHz. The mean difference was the greatest between 27 and 37 kHz where
it reached more than 30 dB. The only significant difference shown by
Bonferroni’s post-tests is between noise-alone and ACN + noise DPgrams between 12
and 45 kHz (P<.05).


## 3.1.2. Compound Action Potentials

Figure3 presents the disruption of
CAP thresholds measured 4 week post-exposure in rats subjected to (a) 4, (b) 8,
or (c) 16-kHz OBN with or without co-exposure to ACN. For reference, thresholds
obtained in controls and ACN alone are included in each of the graphs.Effects of the different
experimental treatments on compound action potential (CAP) thresholds measured
4 weeks post-exposure for frequencies ranging from 2 to 40 kHz. CAP thresholds
obtained in rats exposed to (a) the 110 dB OBN-4 kHz with or without ACN, (b) to
the 97 dB OBN-8 kHz with or without ACN, or (c) to the 97 dB OBN-16 kHz with or
without ACN (see Table1 for details). Auditory thresholds measured in control
and ACN alone animals were included in each of the graphs for reference. The
colored areas represent the octave band-noise frequency ranges. Error bars: ±SEM.
(a)(b)(c)The two-way ANOVA carried out on
the CAP thresholds revealed a significant effect of “exposure” (F(7,49)=13.16;P<.0001). ACN alone animals
did not show any significant threshold difference compared to the control
thresholds (Bonferroni, P>.05; see Figure 5(b)). While the OBN-4 kHz animals
show slightly elevated thresholds between 6 and 16 kHz compared to the control
data, this increase is not statistically significant (P>.05). By
contrast, the animals that received combined exposures to ACN + OBN-4 kHz display
significantly elevated thresholds between 20 and 40 kHz when compared to the
noise-alone thresholds (P<.05; see Figure 5(b)). The difference between these
two groups reaches up to 41 dB at both 30 and 35 kHz.Neither the 8-kHz nor the 16-kHz
noise-alone groups show significantly elevated thresholds compared to controls
(Bonferroni,P>.05). By contrast, the ACN + OBN-8 kHz and the ACN +
OBN-16 kHz thresholds were significantly different from their noise-alone
counterparts, respectively, between 12 and 40 kHz and between 20 and 40 kHz
(Figure 5(b)).


## 3.2. Hair Cell Counts

To assess the magnitude of cochlear
damage, hair cells were counted from cochleae harvested from the same animals
used for physiological studies. The hair cell loss is presented as cochleograms
that display the percentage of hair cell loss as a function of distance from
the apex of the cochlea.Figure4 shows the mean
cochleograms obtained in the animals exposed to (a) ACN alone, (b) OBN-4 kHz
alone, (c) ACN + OBN-4 kHz, (d) OBN-8 kHz alone, (e) ACN + OBN-8 kHz, (f)
OBN-16 kHz alone, (g) ACN + OBN-16 kHz. The cochleae from controls (not shown)
and ACN alone subjects (Figure 4(a)) displayed no hair cell loss, while those
exposed to any of the OBNs without ACN (Figures 4(b), 4(d) and 4(f)) displayed limited
damage in the basal half of the organ of Corti, less than 10% OHC loss at any
tonotopic location. None of the exposures yielded any inner hair cell (IHC)
loss. Therefore, IHCs are not discussed here, and are not included in the
statistical analysis (see Section 2.8).Average cochleograms
showing hair cell loss in rats exposed to (a) ACN alone or to the 110 dB
OBN-4 kHz with (c) or without (b) ACN, in rats exposed to the 97 dB OBN-8 kHz with
(e) or without (d) ACN, and in rats exposed to the 97 dB OBN-16 kHz with (g) or
without (f) ACN. Abscissa (i) upper trace: length (mm) of the entire spiral
course of the organ of Corti from the bottom of the hook, (ii) lower trace:
frequency-map according to Muller [9]. Ordinate: hair cell loss in
percent. IHC: inner hair cells; OHC1: first row of outer hair cells; OHC2:
second row; OHC3: third row. Error bars represent the standard error.
(a)(b)(c)(d)(e)(f)(g)Statistical comparisons
between groups for (a) DPOAE amplitudes, (b) CAP thresholds, and (c) hair cell
loss at 4 weeks postexposure: Bonferroni’s post-hoc comparisons withP=.05
as the significant limit. The parent analyses were repeated measure ANOVAs with
“exposure” as between factor and “frequency” as a within
factor. The colored bands illustrate the frequency range for which Bonferroni’s
comparisons between groups yielded P<.05. ns: not significantly
different. Note: for the CAP
technique, the significance range is presented as “above x kHz” because it does
not allow measurements above 40 kHz, whereas the significance is likely more extended
toward high frequencies.
(a)
Post-hoc Bonferronil’s comparisons—DPOAEs(b)
Post-hoc Bonferronil’s comparisons —CAPs(c)
Post-hoc Bonferronil’s comparisons—HistologyConsistent with physiological
results, the cochleae from rats exposed to both ACN and noise (Figures4(c), 4(e), and
4(g)) exhibited substantial damage in the basal (or high frequency) third of the
organ of Corti. The pattern of OHC loss is actually surprisingly similar among
the different ACN + noise exposures. Despite the fact that the OHC loss
obtained in the rats exposed to ACN + OBN-8 kHz is scattered over a wider range
than the other two combined exposure groups, the hair cell loss averaged about
45% in the three OHC rows in the region corresponding to frequencies above 15 kHz for the three different combined exposures.The OHC loss obtained in all
experimental groups was analyzed with a global two-way ANOVA. The overall
effect of “exposure” was significant (F(7,47)=5.60;P=.0003). Bonferroni’s
post-tests (see graphic representation in Figure 5(c)) confirmed that
cochleograms obtained with either one of the OBNs were not significantly
different from the control cochleogram (not shown). By contrast, the ACN +
OBN-4 kHz, the ACN + OBN-8 kHz, and the ACN + OBN-16 kHz cochleograms are
significantly different from their noise-alone counterparts, respectively, from
22 to 64 kHz, from 23 to 64 and from 26 to 52 kHz (P<.05). Hair cell loss
in the ACN + OBN-8 kHz was visibly wider in terms of frequency than the OHC loss
obtained in the other two combined exposure groups. However, the difference
barely reached statistical significance at a few frequencies: OHC loss in the
ACN + OBN-8 kHz cochleae was different (P<.05) from the loss in the ACN +
OBN-4 kHz at 27, 28, 37–39, and 46–50 kHz, and from the ACN + OBN-16 kHz cochleae
at 21, 22–25, 27-28, and 38–42 kHz.


## 4. Discussion

This study was carried out to
investigate the relationship between the frequency range of continuous OBN
exposures and the tonotopic location of the cochlear damage with and without
co-exposure to ACN, a compound that decreases cellular antioxidant defenses.
The results show that, when ACN is present, moderate noise exposures centered
at 4, 8, and 16 kHz all yield high frequency hearing impairment and basal OHC
damage. In fact, basal hair cell loss seems nearly independent of the noise
frequency band used in exposures. By contrast, without ACN treatment, the
damage induced by the different noise exposures were very limited. Also,
exposure to ACN alone did not cause permanent DPOAE decrease, CAP threshold
shift, or loss of hair cells.Despite some differences in the
pattern of the OHC loss, the maximal damage caused by ACN + noise exposures is
always located around 30–40 kHz, regardless of the center frequency of the OBN.
This observation underscores the fact that the noise exposures used in this
investigation include cochlear effects that are not limited to the frequency
ranges of the OBNs, but extend to the basal end of the cochlea.Histological observations confirm
that up to a 3-octave shift exists between the OBN center frequency and the
corresponding frequency sensitivity of the area of maximal cochlear damage.
Exposure to noise-alone caused minimal cochlear damage located within one
octave above the OBN (Figures4(b), 4(d), and 4(f)), which confirms that the losses at high
frequencies are a consequence of the combined ACN + noise effect.Our results are consistent with
those obtained by Chen and Fechter [10] who showed by exposing rats
to carbon monoxide and different OBNs that the potentiation was much bigger for
the high frequency exposures. Also, Rao and Fechter [11] demonstrated that
phenyl-N-tert-butylnitrone (PBN), a spin-trap agent which neutralizes ROS,
significantly protected the cochlea against the interactive effect of CO +
noise. This protection was, again, only significant at high frequencies. The
fact that we observed similar high frequency potentiation might suggest that
ACN and CO share common interactions with the cochlear defenses against NIHL.While ACN does not impair oxygen  delivery, it directly decreases tissue antioxidant defenses [12–15]. Further, injection of 50 mg/kg ACN in the rat
depletes liver and cochlear GSH by 60–80% within 30 minutes [5]. The potentiation of NIHL
(OBN-8 kHz, 97 dB SPL, 5×4 hours) by this ACN injection could be reduced by a
pre-treatment with L-N-acetylcysteine, a compound that increases cellular GSH
levels. This treatment was especially effective for the frequencies above 30 kHz.Several other studies offer a solid
basis to hypothesize that the shift of the impairment towards high frequencies
may be due to the fact that the cochlea has different susceptibilities to ROS
depending on the tonotopic location. In
vitro , Sha et al. [16] observed that cochlear
organotypic cultures of basal OHCs were more vulnerable to free-radical damage
than apical OHCs, and that basal OHC survival was improved by the addition of
L-NAC or GSH. In accordance with this result, Clerici and Yang [17]
showed in vivo that direct
perilymphatic generation of ROS (by instillation of artificial supplemented
perilymph) induced a rapid degradation in high-frequency CAP threshold
sensitivity, the pattern of which being surprisingly similar to the threshold
shift obtained in our ACN + noise exposed animals (Figure 3). The literature thus
suggests that the decrease of antioxidant defenses caused by ACN renders the
basal OHCs more susceptible to noise than the apical OHCs. This differential
vulnerability gives a possible explanation for the high-frequency shift
phenomenon observed in our experiments.In
the present study, two different audiometric techniques were used in order to
estimate both the global auditory sensitivity (CAPs) and the physiological
state of the OHC stereociliae (DPOAEs). As a general rule, data obtained with
these two techniques were in excellent agreement with the histology. However,
in terms of frequency range, CAPs threshold shifts were more closely related to
the OHC loss than to the DPOAEs (Figure5). The DPOAEs were especially inconsistent
with the histology for the ACN + OBN-4 kHz exposure (Figure 2(a) versus Figure 4(c) and
Figure 5(a) versus. Figure 5(c)). The potentiation appeared significant between 16 and 29 kHz with DPOAEs and between 22 and 64 kHz for the OHC loss. We suggest that
this discrepancy is likely due to the
fact that DPOAE decrements are better explained in terms of OHC dysfunction, rather
than actual OHC death. The DPOAE patterns obtained in the animals exposed to
the lower noise band suggest that this exposure was not traumatic enough to kill
the hair cells although it did alter OHC function.



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*Source: 908596-2009-05-06.xml*