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 ORIGINAL RESEARCH ARTICLE
AGING NEUROSCIENCE
published: 16 February 2015
doi: 10.3389/fnagi.2015.00007

Swept-sine noise-induced damage as a hearing loss model

for preclinical assays
Lorena Sanz 1,2,3 † , Silvia Murillo-Cuesta 1,2,4 *† , Pedro Cobo 5 † , Rafael Cediel-Algovia 1,2,6 , Julio Contreras 1,2,6 ,
Teresa Rivera 1,2,3 , Isabel Varela-Nieto 1,2,4 † and Carlos Avendaño 4,7 †
1
Institute for Biomedical Research “Alberto Sols” (IIBM), Spanish National Research Council–Autonomous University of Madrid (CSIC-UAM), Madrid, Spain
2
Centre for Biomedical Network Research (CIBER), Institute of Health Carlos III (ISCIII), Madrid, Spain
3
Príncipe de Asturias University Hospital, University of Alcalá, Alcalá de Henares, Madrid, Spain
4
Hospital La Paz Institute for Health Research (IdiPAZ), Madrid, Spain
5
Institute for Physical and Information Technologies (ITEFI), Spanish National Research Council (CSIC), Madrid, Spain
6
Veterinary Faculty, Complutense University of Madrid, Madrid, Spain
7
Department of Anatomy, Histology and Neuroscience, Medical School, Autónoma University of Madrid, Madrid, Spain

Edited by: Mouse models are key tools for studying cochlear alterations in noise-induced hearing
Rodrigo Orlando Kuljiš, Zdrav Mozak
loss (NIHL) and for evaluating new therapies. Stimuli used to induce deafness in mice are
Limitada, Chile
Gemma Casadesus, Case Western usually white and octave band noises that include very low frequencies, considering the
Reserve University, USA large mouse auditory range. We designed different sound stimuli, enriched in frequencies
Reviewed by: up to 20 kHz (“violet” noises) to examine their impact on hearing thresholds and cochlear
Miguel A. Merchán, University of cytoarchitecture after short exposure. In addition, we developed a cytocochleogram to
Salamanca, Spain
quantitatively assess the ensuing structural degeneration and its functional correlation.
Jiri Popelar, Institute of
Experimental Medicine AS CR, Finally, we used this mouse model and cochleogram procedure to evaluate the potential
Czech Republic therapeutic effect of transforming growth factor β1 (TGF-β1) inhibitors P17 and P144 on
*Correspondence: NIHL. CBA mice were exposed to violet swept-sine noise (VS) with different frequency
Silvia Murillo-Cuesta, Institute for ranges (2–20 or 9–13 kHz) and levels (105 or 120 dB SPL) for 30 min. Mice were evaluated
Biomedical Research “Alberto Sols”
by auditory brainstem response (ABR) and otoacoustic emission tests prior to and 2, 14
(IIBM), Spanish National Research
Council–Autonomous University of and 28 days after noise exposure. Cochlear pathology was assessed with gross histology;
Madrid (CSIC-UAM), Arturo hair cell number was estimated by a stereological counting method. Our results indicate
Duperier 4, 28029 Madrid, Spain that functional and morphological changes induced by VS depend on the sound level
e-mail: [email protected]
and frequency composition. Partial hearing recovery followed the exposure to 105 dB
†
These authors have contributed
SPL, whereas permanent cochlear damage resulted from the exposure to 120 dB SPL.
equally to this work.
Exposure to 9–13 kHz noise caused an auditory threshold shift (TS) in those frequencies
that correlated with hair cell loss in the corresponding areas of the cochlea that were
spotted on the cytocochleogram. In summary, we present mouse models of NIHL, which
depending on the sound properties of the noise, cause different degrees of cochlear
damage, and could therefore be used to study molecules which are potential players in
hearing loss protection and repair.

Keywords: cytocochleogram, hair cells, hearing loss, transtympanic, TGF-β inhibition, violet noise

INTRODUCTION 2009; Le Prell, 2012). Inner and particularly outer hair cells (IHC
Noise-induced hearing loss (NIHL) is the most common form and OHC, respectively) especially those located in the basal turn
of acquired deafness in developed countries and therefore it is of the cochlea in mammals, are very sensitive to noise damage.
a public health priority (Sliwinska-Kowalska and Davis, 2012). Thus, the disruption of stereocilia leads to a severe alteration in
Hearing impairment may be induced by a single impulsive noise HC structural integrity that has been correlated to permanent
or after repetitive exposure to moderate or high intensity noise threshold shifts (TS) in many species (Hamernik and Qiu, 2000;
(Kirchner et al., 2012). The cumulative damaging effects on the Chen and Fechter, 2003; Chen et al., 2003; Hu et al., 2006; Bohne
inner ear depend on the noise characteristics (frequency, level), et al., 2007; Harding and Bohne, 2009).
chronicity and individual susceptibility to noise (Konings et al., Mice models are essential tools for studying the
pathophysiology of NIHL and for evaluating new potential
Abbreviations: ABR, auditory brainstem responses; BM, basilar membrane;
therapies (Ohlemiller, 2006; Park et al., 2013). As reported
DPOAE, distortion product otoacoustic emissions; IHC, inner hair cell; NIHL, in human cases, functional and structural alterations depend
noise-induced hearing loss; OC, organ of Corti; OHC, outer hair cell; TS, on noise level and duration of exposure, and also on strain
threshold shift; V, violet noise; VS, violet swept-sine noise. susceptibility. Thus, C57BL/6 and BALB/c mice are particularly

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

vulnerable to noise, whereas CBA/Ca mice appear to be more HEARING EVALUATION

resistant (Ohlemiller et al., 2000, 2011; Ou et al., 2000a; Gratton Auditory brainstem responses (ABR) and distortion product
et al., 2011). There are many reports on the functional and otoacoustic emissions (DPOAE) were obtained before and 2, 14
morphological characterization of different NIHL mouse models days and 28 days after noise exposure (Figure 1) with a System
in which mice are usually exposed for a few (1–4) hours to III Evoked Potential Workstation (Tucker-Davis Technologies,
high intensity (95–120 dB SPL) white or octave band noises, Alachua, FL, USA). Briefly, mice were anesthetized with ketamine
usually centered on low frequencies up to 10 kHz (Park et al., (Imalgene, Merial, 100 mg/kg) and xilacyne (Rompun, Bayer, 10
2013). These frequencies are in the lowest frequency range of mg/kg) and placed on a heating pad inside a soundproof acoustic
the mouse hearing spectrum (Greenwood, 1996). Exposures chamber.
to noise over 10 kHz are much less frequent (i.e., 8–16 kHz For the ABR test, click and tone burst stimuli (8, 16, 20,
in Kujawa and Liberman, 2009; 4–45 kHz in Ohlemiller et al., 28 and 40 kHz) were presented with an MF1 magnetic speaker
2011). In this work we generated NIHL by short exposure to (TDT) from 90 to 20 dB SPL in 5–10 dB SPL steps (Cediel
“violet” swept-sine noise (VS), a 10 s linear sweep in frequencies et al., 2006; Riquelme et al., 2010; Murillo-Cuesta et al., 2012).
from 2 kHz to 20 kHz designed with high-pass filtering and Click stimuli were 0.1 ms and tone burst stimuli were 5 ms
linear with frequency gain (Cobo et al., 2009). Additionally VS duration (2.5 ms each for rise and decay, without plateau).
noise with frequencies from just 9 to 13 kHz was used to injure Threshold of click-evoked and tone-evoked ABR, peak latencies
a specific stretch of the basilar membrane (BM). To illustrate and amplitudes were determined. For DPOAE, an ER10B+ probe
and quantify the distribution of HC along the length of the (Etymotic Research Inc., IL, USA) was inserted into the external
cochlea under the different conditions used, a cytocochleogram auditory canal and mice were stimulated with two synchronic
was performed, applying a stereological approach to previously tones, whose frequencies (f1, f2; relation f1/f2 = 1.2) were
published procedures (Ou et al., 2000b; Viberg and Canlon, calculated from a central frequency (F = 8, 10, 14, 18 and
2004; Müller and Smolders, 2005; Müller et al., 2005; Boyce et al., 22 kHz; f1 = F ∗ 0.909, f2 = F ∗ 1.09), and presented with decreasing
2010). intensity from 80 to 30 dB SPL (f1 level = f2 level). The distortion
Here we describe the functional and morphological product 2f1–f2 was determined for each sound level from the
consequences, and their correlation, of exposure to VS noises FFT waveforms. DPOAE thresholds were defined as the minimum
of different sound levels and frequency ranges. We found that level of the primary tones that elicit a 2f1–f2 response higher than
the characteristics of the VS noise determine the pattern of background noise.
hair cell density along the cochlea and, accordingly, the ABR
electrophysiological response. Therefore, the study of NIHL and NOISE EXPOSURE AND EXPERIMENTAL GROUPS
of potential prevention and repair therapies could be further Mice were exposed for 30 min to noise in a reverberant
improved by refining the design of noise stimuli in experimental chamber acoustically designed to reach maximum sound level
models. Furthermore, we tested the local actions of two peptides with minimum deviation in the central exposure area (Cobo
P17 or P144 inhibitors of transforming growth factor beta 1 et al., 2009). Two sound stimuli, named violet (V) and violet
(TGF-β1) to repair NIHL. TGF-β1 is a cytokine involved in swept-sine (VS), were designed with Wavelab Lite software
the cochlear inflammatory response that is released early after (Steinberg Media Technologies GmbH, Hamburg, Germany).
cochlear injury caused by aminoglycoside (Wissel et al., 2006), Both have a linear-with-frequency gain to compensate for the
antigens (Satoh et al., 2006), otitis media (Ghaheri et al., 2007) high frequency losses inside the chamber, and present a spectrum
and noise exposure (Murillo-Cuesta et al., submitted). Our
functional and cochleogram data indicated that a single local
administration of either peptide did not exert a therapeutic effect
on NIHL.

MATERIALS AND METHODS

MOUSE HOUSING AND HANDLING
The study was carried out on 2 month old male mice
from CBA/CaOlaHsd (CBA) and C57BL/6JOlaHsd (C57)
strains (Harlan Laboratories). Patterns of cochlear damage
after noise exposure in these strains have been previously
described; both strains show consistent lesions but different
susceptibilities to noise injury (Ohlemiller et al., 2000,
2011; Ou et al., 2000a; Wang et al., 2002; Ohlemiller and
FIGURE 1 | Chronogram of the experiment. Mice were exposed to Violet
Gagnon, 2007; Park et al., 2013). All procedures and animal Swept-sine (VS) noise with different intensities (105 or 120 dB SPL) and
handling were conducted in accordance with the European frequency ranges (2–20 or 9–13 kHz) for 30 min. Hearing was evaluated
(2010/63/EU) and Spanish (RD 1201/2005) legislation on with Auditory Brainstem Responses (ABR) and Distortion Products of
the experimental use of animals, and were approved by the Otoacoustic Emissions (DP) before (baseline) and 2, 14 and 28 days after
noise exposure. At the end of the study, cochleae were dissected and
Ethics Committee of the Spain National Research Council
processed for histology and cell counting.
(CSIC).

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

biased towards high frequencies, a kind of violet coloring. V bony and membranous labyrinths and the tectorial membrane
noise was synthesized from white noise, whereas VS noise was were carefully removed to expose the organ of Corti (OC).
a 10 s linear sweep in frequency from 2 kHz up to 20 kHz Total removal of the BM along the entire cochlea is technically
that was repeated during the 30 min of exposure. Specific difficult; the basal-most region or “hook” is more delicate and
characteristics of these stimuli are described in Cobo et al. it is easily injured during dissection. Therefore, the cochleogram
(2009). shown in this work represents the 80% (range across cases:
Preliminary experiments with V and VS noise were conducted 70–85%) of the cochlea that can be dissected while maintaining
in C57 and CBA mice in order to select the appropriate mouse cellular integrity. The 20% of the cochlea which is destroyed
strain and noise. For further studies, CBA mice were used and results in a loss of information regarding the 50–80 kHz
four experimental groups were established (Figure 1). Mice frequencies, which, by definition, should not be greatly affected
of the control group were not exposed (n = 12) whereas by noise containing frequencies in the range of 2–20 kHz
other mice were exposed to VS noise with the following (Figure 2).
level and frequency ranges: 105 dB SPL and 2–20 kHz (105 OC was sectioned into two portions, one containing the
VS2−20 , n = 12), 120 dB SPL and 2–20 kHz (120 VS2−20 , apex and middle turns and the other containing the basal
n = 8), and 105 dB SPL and 9–13 kHz (105 VS9−13 , turn of the cochlea, placed on plates (Nunclon Microwell
n = 12). Plates, Sigma Aldrich) filled with PBS, and stored at 4◦ C.
Finally, to evaluate the efficacy of P17 and P144 peptides, mice Samples were permeabilized with 0.5% Triton X-100 (Sigma-
were exposed to 105 VS2−20 for 30 min and operated on 48 h Aldrich) for 15 min, at room temperature on a shaker, and
after noise damage, once the increase in hearing thresholds was then incubated 1 h with 1:1000 phalloidin (R Alexa Fluor 488
confirmed. The effect of noise exposure on hearing function was phalloidin, Invitrogen) in PBS. After three washes in PBS, the
evaluated with the ABR test as described before, 2, 14 and 28 days samples were transferred to concave microscope slides (Menzel-
after noise exposure. Gläser) and mounted with Vectashield mounting medium
with DAPI (Vector Laboratories). A total of 43 cochleae were
DRUG ADMINISTRATION obtained from the 22 animals (105 VS2−20 , n = 6), 120
Chemically synthesized peptide inhibitors with high affinity VS2−20 , n = 6, 105 VS9−13 , n = 6, control non-exposed mice,
for TGF-β1 (Ezquerro et al., 2003) were gently defrosted, n = 4).
diluted and sonicated (only P144) to completely dissolve them.
Local administration of inhibitors into the inner ear was STEREOLOGY AND COCHLEOGRAM PLOTTING
performed 24 h after noise exposure, once the increase in In order to plot a standardized cochleogram and to correlate cell
hearing thresholds was confirmed. Briefly, the tympanic bulla damage with increased hearing thresholds at specific frequencies,
was exposed via ventral surgical approach, and a bullostomy a stereological approach was designed to determine the number
was performed at the posterolateral aspect using a small hook. and distribution of HCs along the length of the cochlea (Viberg
Once the round window and stapedial artery were clearly and Canlon, 2004; Müller et al., 2005; Boyce et al., 2010; Figure 2).
visible, we applied directly 10 µl of a concentrated (40 mg/ml) “Flattening” of the BM that resulted from cochlear extraction
P17 or P144 solution or saline (n = 6 each) to the round and mounting procedure helped to ensure an unbiased sampling
window using a gelatin sponge vehicle (Murillo-Cuesta et al., of cells. High resolution photomicrographs of the OC samples
2009). were taken through a BX51 microscope with a DP70 camera
(Olympus). These images were studied offline using CorelDRAW
COCHLEAR PROCESSING FOR HISTOLOGY AND HAIR CELL COUNTING X3 software (Corel Corp., Ottawa, Ontario, Canada) to define
At the end of the experiment (Figure 1), mice were anesthetized an area of interest along the BM that included the rows of
with pentobarbital (Dolethal, Bayer, 150 mg/kg) and cochleae HCs, This area was further divided into sectors, each covering
were extracted for light microscopy or HC counting. 5% of the total length, from apex to basal region. The OC
For histological evaluation, mice were perfused with 4% samples were then evaluated with the same microscope under
paraformaldehyde (in 0.1 M saline buffer, pH 7.4) and the ultraviolet epi-illumination (Olympus U-RFL-T) with 4X and
cochleae were removed, fixed overnight, decalcified with 10% 40X magnification to detect phalloidin immunofluorescence,
EDTA (0.3 M, pH 6.5) and embedded in paraffin as described and previously defined sectors were located in the sample
(Riquelme et al., 2010; Murillo-Cuesta et al., 2012). Mid- in order to perform cell counts. Interactive test grids and
modiolar 10 µm sections were Nissl-stained and evaluated control of the motorized stage (Prior Scientific, Rockland, MA,
with a Zeiss Diaplan microscope and a digital camera (Leitz USA) were provided by CAST stereological software package
DFC300 FXC). For HC counting, mice were sacrificed by cervical (v.2.3.2.0, Visiopharm, Hørsholm, Denmark). HC loss and
dislocation and the inner ear was carefully dissected. After stereocilia abnormalities are thought of as the principal correlate
removing the bony wall of the tympanic bulla and the stapes, in NIHL. Since each HC is unequivocally represented by its
the cochlea was exposed and two openings were performed, hair bundle, phalloidin-stained stereocilia were used as counting
one between the round and oval windows and one in the units and HC were counted as present if the hair bundle was
apex, to circulate 300 µl paraformaldehyde. The cochleae were intact.
immersed in paraformaldehyde for 24 h and decalcified with HC count was carried out by placing unbiased frames in a
EDTA for 4–6 days. Using an angled sharp micro scalpel, the uniformly random sampling strategy on the focus planes of the

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

FIGURE 2 | Cochleogram and stereology. (A) Photomontage of digital divided in 5% bins (top) and the corresponding frequency map (bottom).
images of the two blocks of a CBA mouse dissected cochlea. The region (D–F) Phalloidin-stained whole mounts of portions of the apical
containing the BM is roughly delineated in green (apical-middle block) or (D), middle (E) and basal (F) cochlear turns in a control mouse. The
blue (basal block); white traverses divide the whole length of the cochlea stereological method used to estimate HC densities is summarized in
into 5% intervals. (B) Diagram of the BM of the same cochlea, with 10% (E): A set of unbiased frames is randomly placed on the stereociliary
intervals marked with yellow dots. The blue line separates the portion fringe, the convex hull area bounding the hair tufts of the IHC and OHC;
reconstructed in A (roughly 80% from the apex) from the basal the HCs to be included in the count in this example are delineated by
quarter-turn, which was never included in the reconstruction. (C) Diagram white profiles; dots highlight the frame corners that hit the stereociliary
showing a place-frequency map according to Viberg and Canlon (2004) fringe, and serve to estimate the reference space sampled with the
and Müller et al. (2005), which combines a schematic drawing of the BM frames. Scale: 25 µm. IHC, inner hair cells; OHC, outer hair cells.

cilia. The reference space was considered the stereociliary fringe, sampled with the unbiased frames within the given sector.
that is, the convex hull area bounding the region containing the The precision of this method was approximated by computing
rows of hair tufts of the HCs. the coefficient of error (CE) of the estimates (NA ) obtained
The IHC and OHC cell densities were estimated for each sector on each sector, applying Cochran’s equation for ratio
as follows, P estimators (eq. 10.32 in Howard and Reed, 2005). Our strategy
Q(HC) · 1000 yielded CE values of ∼17% (0.17 ± 0.015) in the control
NA (HC) =
animals, and ∼21% (0.21 ± 0.020) in the noise-damaged
P
a(SF)
groups.
where NA is the P cell density expressed as number of Cytocochleograms were constructed by plotting the number
cells/1000 µm2 , PQ is the sum of HC counted within of present OHCs or IHCs, or the OHC and IHC densities, as a
each sector, and a(SF) is the area of interest, in µm2 , function of percent distance from the apex of the cochlea.

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

STATISTICAL ANALYSIS of threshold 2 weeks after damage, with significant differences

Data obtained from hearing test and hair cell counting were compared to exposure to 105 VS2−20 (F (3) = 202.64, p = 0.026)
analyzed using v.19 IBM SPSS Statistics software. Results were and 105 VS9−13 (p = 0.000) noises. No differences were found
expressed as mean ± SEM. Differences in ABR and DPOAE between 105 VS2−20 and 105 VS9−13 noises at any time after
thresholds were analyzed using analysis of variance (ANOVA) insult, although the latter showed a non-significant but consistent
with repeated measures. Bonferroni and T2 Tamhane post hoc small recovery of click thresholds. Similarly, baseline DPOAE
tests (for equal or unequal variances, respectively) were used thresholds were similar in all the experimental groups. Noise-
to make pairwise comparisons of the repeatedly measured data exposed mice showed marked threshold increases in all the tested
in different measurement times for each experimental group. frequencies, with statistically significant differences compared to
ANOVA tests were also performed for comparison of HC the control group, but without evident differences among them
numbers and densities among experimental groups. Results were (Figure 3B).
considered significant at p ≤ 0.05. In contrast, differences were found in the audiogram between
exposure to 105 VS2−20 and 105 VS9−13 noises. Thus, mice
exposed to VS9−13 noise showed higher TS 2-days after
RESULTS
noise exposure, especially when high frequencies were tested
LONGITUDINAL STUDY OF HEARING-LOSS AFTER EXPOSURE TO
(Figure 4A). Significant differences were found at stimuli
DIFFERENT NOISE TYPES
frequencies of 16 (F (3) = 32.7, p = 0.002), 20 (F (3) = 94.6,
Preliminary experiments were conducted in C57 and CBA
p = 0.000), 28 (F (3) = 86.7, p = 0.000) and 40 (F (3) = 177.1,
mice in order to select the appropriate noise for subsequent
p = 0.000) kHz.
cytocochleogram studies. Mice were exposed to noise for 30 min
To study potentially selective damage on specific cochlear
and threshold of click-evoked and tone-evoked ABR were
regions, the audiogram of mice exposed to VS9−13 noise was
determined before and at 1 h, and 3 and 7 days after exposure
extended to include frequencies of 4 and 10 kHz. As expected, the
to noise. Results are summarized in Table 1. C57 mice exposed
TS at 10 kHz was larger than TS found at the nearby frequencies
to noise at 120 dB SPL showed severe cochlear damage with
of 4, 8 and 16 kHz. This TS peak in the audiogram was maintained
irreversible TS, whereas following exposure at 100 dB SPL,
throughout the study (Figure 4B).
baseline thresholds were practically recovered in the first week
Statistical analysis of ABR latencies showed a significant
after insult, especially when V noise was used. CBA mice exposed
decrease of the peak and interpeak latencies, especially I–II,
to VS 105 dB SPL noise showed smaller TS than C57 mice, thus
in animals exposed to noise compared to baseline values. This
CBA was the strain chosen for further studies.
reduction was similar in the three exposed groups, without
In these advanced studies, CBA mice from the four
significant differences, and persisted throughout the times
experimental groups (Figure 1) showed baseline thresholds
examined (data not shown).
of click-evoked and tone-evoked ABR below 20 dB SPL,
without significant differences among them (Figure 3A). The
control group maintained normal hearing thresholds throughout COCHLEAR MORPHOLOGY AND HAIR CELL DENSITY AFTER NOISE
the experiment, with highly significant differences (Wald EXPOSURE
χ2(3) = 2282.9, p = 0.000) when compared to all groups of At the end of the experiment, CBA mice exposed for 30 min
noise-exposed mice. A click TS of around 50 dB occurred 2 to VS stimulus showed some of the typical chronic cochlear
days after noise exposure in all mice exposed to VS noise. alterations reported in this strain after noise damage (Wang
No significant differences were found among damaged groups, et al., 2002); no gross differences were detected among 105
although mice with the greatest TS were those exposed to VS2−20 , 120 VS2−20 and 105 VS9−13 groups. The main cellular
120 dB SPL noise. The evolution of click thresholds presented feature detected in Nissl-stained sections was fibrocyte loss
specific patterns for the three noise-exposed groups (Figure 3A). in the spiral ligament and spiral limbus (Figure 5), whereas
Thus, mice exposed to 120 VS2−20 noise showed a worsening no evident changes in the gross anatomy of the OC were

Table 1 | Evolution of ABR threshold in response to click stimulus.

ABR threshold (dB SPL)

Time after noise exposure

Strain Stimulus Level (dB SPL) N Baseline 1h 3 days 7 days

C57BL/6JOlaHsd V2–20 100 5 42 ± 3 55 ± 10 48 ± 12 41 ± 2

120 3 43 ± 8 90 ± 10 90 ± 17 87 ± 12
VS2–20 100 4 42 ± 6 63 ± 5 60 ± 8 53 ± 5
120 4 40 ± 8 88 ± 10 90 ± 8 83 ± 17
CBA/CaOlaHsd VS2–20 105 12 17 ± 5 n.d. 69 ± 4 70 ± 6

Exposure of mice to noxious noises at 100 dB SPL for 30 min induced moderate TS with almost complete recovery, especially when V 2-20 noise was used. In
contrast, 120 dB SPL noise exposure irreversibly damaged the cochlea. Exposure to VS2-20 noise in CBA mice was further selected to study functional effects and
concomitant hair cell loss. V, violet noise; VS, violet swept-sine noise. Superscripts indicate the noise frequency range (in kHz).

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

FIGURE 3 | Evaluation of hearing. (A) Evolution of threshold of click-evoked p < 0.01, ##). (B) DPOAE thresholds at individual frequencies at the end of
ABR. Control non-exposed mice (n = 12) maintained normal hearing the experiment. 28 days after noise exposure (105 VS2−20 , n = 5; 105 VS9−13 ,
thresholds along the study, with statistically significant differences (p < 0.001, n = 8; 120 VS2−20 , n = 4) mice showed significant (p < 0.001, ***) higher
***) compared to noise damaged mice. Two days after noise insult, a notable DPOAE thresholds for all the frequencies as compared to control mice
TS occurred in exposed animals; at longer post-exposure periods, mice (n = 6). No significant differences were found among exposed animals. VS,
exposed to 120 VS2−20 (n = 7) showed significantly higher values compared violet swept-sine noise; superscripts indicate the noise frequency range (in
to those exposed to 105 VS2−20 (n = 11, p < 0.05, &) and 105 VS9−13 (n = 12, kHz); the coefficient indicates the noise level in dB SPL.

observed. Stereocilia damage and HC loss were in fact evident density from apex to base, whereas in the group of mice exposed
in phalloidin-stained whole mount samples (Figures 5C,F,G,J). to 105 VS9−13 noise, OHC loss was evident in the basal region
These preparations were further used for stereological hair when compared to non-exposed mice (p = 0.000).
cell counting. Figure 2 shows that the whole length of the
dissected BM was divided into 5% sectors from apical to basilar EVALUATION OF TGF-β1 INHIBITORS IN THE TREATMENT OF NIHL
cochlear regions. Present IHC and OHC were counted and NIHL mouse models and the cochleogram procedures were
densities were estimated for each sector in control and noise validated in a preclinical assay with TGF-β1 inhibitors
exposed mice (Figure 6). Control non-exposed mice showed (Figure 7A). Mice were exposed to 105 VS2−20 noise and
similar hair cell density values in all sectors, both for OHC after 2 days they were operated on for local delivery of a single
(over 10 cell/1000 µm2 ) and IHC (over 4 cell/1000 µm2 ). dose of TGF-β1 inhibitor (P17, P144) or saline. Analysis of
Differences among noise-damaged mice were evident from thresholds of click-evoked and tone-evoked ABR showed notable
sector 20 onwards, especially for OHC. Mice exposed to permanent TS 1 day after noise exposure and then a minimal
120 VS2−20 noise showed a clear decrease in OHC density, recovery regardless of the treatment. No statistically significant
mainly at the basal region, where no OHCs were detected. differences were found in ABR thresholds in response to click or
A similar but smaller density decrease was observed in 105 tone burst stimuli among mice treated with P17, P144 or saline at
VS2−20 exposed mice when compared to those exposed to any of the times evaluated (1, 14 and 28 days after noise exposure)
120 VS2−20 . Whereas mice exposed to 105 VS9−13 noise (Figure 7B).
showed an acute loss of OHCs in sectors 25 to 30%, and At the end of the study, cochleae were collected to perform the
a progressive decrease of cellular density from sector 55% cochleogram as described. As mentioned before, mice exposed to
onwards (Figures 6A,B). Similar results were observed when 105 VS2−20 noise showed a statistically significant decrease in HC
IHC density was evaluated in all the experimental groups number and density, both for IHCs and OHCs (data not shown).
(Figures 6C,D). However, no significant differences were observed in cochleogram
Sectors were grouped into three cochlear regions (apical, data between mice treated with TGF-β1 inhibitors and those with
5–25%; middle, 30–55%; basal, 60–80%) for comparison saline (Figure 7C).
(Figure 6E). As expected, the control group presented higher HC
densities in the three regions compared to noise-exposed mice. A DISCUSSION
notable decrease in OHC density was observed in mice exposed NIHL is an important medical concern and reliable animal
to 120 VS noise, especially in the middle and basal regions. Mice models are key to understanding its pathophysiology and
exposed to 105 VS2−20 noise showed a gradual decrease of OHC developing new therapeutic strategies. In humans and mice,

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

FIGURE 4 | Evolution of thresholds of tone-evoked ABR. (A) Control p < 0.01, &&& p < 0.001). Mice exposed to 105 VS9−13 (N, n = 12,)
non-exposed mice (◦, n = 12) maintained baseline (B) hearing showed significant early damage followed by a partial, but quick,
thresholds and showed statistically significant differences (p < 0.001, recovery. (B) Mice exposed to VS noise showed an elevation in the
***) when compared to noise-damaged mice. Noise 120 VS2−20 (, audiogram, compared to control mice. A TS peak in the 105 VS9−13
n = 7) induced the most severe damage (compared to 105 VS noises; was evident in response to 8 and 10 kHz. VS, violet swept-sine noise;
# p < 0.05, ## p < 0.01, ### p < 0.001), whereas TSs in 105 VS2−20 superscripts indicate the noise frequency range (in kHz); the coefficient
(•, n = 11,) were significantly lower (compared to the other noises; && indicates the noise level in dB SPL.

noise exposure induces temporary or permanent hearing loss During the first 2 weeks after noise exposure, mice can show
depending on the noise intensity and the individual susceptibility temporary and reversible increased threshold (temporal TS),
(reviewed in Davis et al., 2003). Other physical parameters of but one month after injury, the auditory threshold elevation
noise as frequency spectra, duration and intermittency also is considered permanent (permanent TS) (Hamernik and Qiu,
contribute to injury, but they are less frequently examined in 2000; Harding et al., 2002; Wang et al., 2002; Chen et al., 2003).
NIHL studies (Mahendra Prashanth and Venugopalachar, 2011) Here we show that 30 min of exposure to VS noise of intensities
or animal models. of either 105 or 120 dB SPL could induce a notable increase in
In this work we demonstrate that by modulating noise thresholds of click-evoked and tone-evoked ABR; the magnitudes
characteristics, the hearing loss outcome in noise-exposed mice of temporal and permanent TSs are proportional to the intensity
can vary largely. Thus, by modifying the physical properties as reported (Ohlemiller et al., 2000, 2011; Park et al., 2013).
of the sound, we have generated and characterized a range of Interestingly, small changes in the noise frequency spectrum also
experimental NIHL mouse models with the aim of obtaining induced different ABR profiles. Two days after exposure to 105
experimental tools for studying human NIHL and for testing VS9−13 there was an acute TS greater than that observed after
potential therapeutic strategies. exposure to 105 VS2−20 noise, particularly at high frequencies.

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

FIGURE 5 | Comparative study of cochlear morphology after noise conditions show similar cytoarchitectural alterations with no evident
insult. Comparison of the spiral limbus (SL) (A,B), organ of Corti (D,E) differences among them. At the focal plane shown, phalloidin-stained
and spiral ligament (Spl) (H,I) of control (A,D,H) and VS noise-exposed whole mounts for actin exhibit the disrupted sequence (arrows in C) of
(B,E,I) mice at the cochlear middle level. Main features observed after otherwise well-defined outlines of OHC and a substantial OHC loss
short time noise exposure included fibrocyte loss at SL (B) and loss of 28 days after VS noise insult (C,F,G,J). Scale bars: 100 µm
type IV fibrocytes at Spl (white arrowheads at I). The organ of Corti (A,B,D,E,H,I), 25 µm (C,F,G,J). BM, basilar membrane; IHC, inner hair
reveals an essentially normal gross anatomy (no collapse of tunnel of cells; OHC, outer hair cells; TM, tectorial membrane; SL, spiral limbus;
Corti, Hensen cells or spaces of Nuel). The different noise exposure Spl, spiral ligament.

It is known that exposure to noise of a certain frequency swept-sine “intermittent” noises produce greater cochlear damage
does not induce a higher TS at that frequency, but rather at than “continuous” ones, including white, violet and broadband
0.5 to 2 octaves above that frequency, depending on the species noises, possibly because they expose HCs to higher activity and
(Ou et al., 2000a). Therefore, to assess functional impairment stress.
after noise exposure, it is necessary to measure thresholds of Concomitantly with noise-induced TS, we observed a decrease
tone-evoked ABR over a range of frequencies by stimulation in ABR peak latencies, which could be interpreted as acceleration
with multiple tone bursts. In this work we performed a large in neural transmission along the auditory pathway. This
audiogram from 8 to 40 kHz. A notch was evident in the observation is consistent with many studies both in human
ABR audiogram from 105 VS9−13 noise-exposed mice with and animal models (Strelcyk et al., 2009; Scheidt et al., 2010;
higher thresholds for pure tones at 8 and 10 kHz. Similar Henry et al., 2011), and diverse theories have been proposed,
frequency-related variations in hearing thresholds have been including recruitment phenomenon and hyperexcitability of
reported in human studies. Exposures to broadband noise induce central auditory fibers. Cochlear recruitment occurs when
maximum TS at frequencies between 3 and 6 kHz but, if the damaged HC call up those close to them to cover their
noise is a pure tone, the higher the frequency, the greater activity, creating a paradoxical situation of hyperexcitability in
the resulting TS (Mahendra Prashanth and Venugopalachar, the central auditory fibers, in which there is an abnormal
2011). In addition to level and frequency composition, other increase in the response to sound (Moore, 2004; Sherlock
noise characteristics could influence the hearing outcome. Thus, and Formby, 2005). It is has been proposed that this is

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

FIGURE 6 | Hair cell counting data. (A–D) Inner and outer hair cell regions (apex, 5–25; middle, 30–55; base, 60–80) and the mean hair cell
density (mean ± SEM, expressed as number of cells/1000 µm2 ) for each density was calculated. Statistically significant differences among groups
5% sector across the total length of the BM (A,C) and focused on were found, indicating that exposure to 120 VS2−20 noise induced the
sectors 25–30 (B,D). Exposure to 105 VS9−13 noise induced a significantly most severe damage, especially towards the basal region. * p < 0.05; **
higher loss of hair cells in the cochlear region corresponding to sectors p < 0.01; *** p < 0.001. VS, violet swept-sine noise; superscripts
25 and 30 (see Figure 2C) compared to exposure to 105 VS2−20 noise. indicate the noise frequency range (in kHz); the coefficient indicates the
(E) To better assess regional variations, sectors were grouped into three noise level in dB SPL.

a mechanism for balancing neuronal activity after cochlear 2009). Wave amplitudes also decreased after noise-exposure,
damage (Syka, 2002; Cai et al., 2009; Rybalko et al., 2011). a pattern that was maintained throughout the study and
However, the consequences of noise damage on ABR latencies which has been related to loss of afferent nerve terminals
are still unclear and other authors have observed increased (Popelar et al., 2008; Kujawa and Liberman, 2009; Henry et al.,
values after exposure (Chen et al., 2002; Gourévitch et al., 2011).

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

FIGURE 7 | Continued
expressed as %) in mice treated with TGF-β1 inhibitors compared to saline
treated mice, in the three standard regions (apex, 5–25; middle, 30–55;
base, 60–80) at the end of the experiment. No significant differences were
observed between mice treated with P17 (orange bars) or P144 (pink) and
those treated with saline (white) after exposure.

DPOAE is a method for early detection of NIHL in patients

as their alteration may reflect subclinical cochlear changes that do
not show up yet in the tone threshold audiometry (Attias et al.,
2001; Martin et al., 2006; Fetoni et al., 2009). In parallel with
changes in ABR parameters, we observed an increase in DPOAE
thresholds in the three noise-exposed mice groups, without
significant differences among them. DPOAE TS correlated with
that observed in the ABR and also with the extent of OHC loss
detected in the cochleogram, as described previously by other
authors (Vázquez et al., 2004; Jamesdaniel et al., 2011; Park et al.,
2013).
The CBA mouse strain has been reported to be more resistant
to the harmful effects of noise than the C57 strain (Ohlemiller
et al., 2011), a fact that was confirmed in our work by comparing
two noise types, V and VS, at two intensities. C57 mice exposed to
noise often showed an extreme response and became irreversibly
profoundly deaf. Therefore CBA mice were used for further
comparison of the lesions on different features caused by VS
noises. CBA mice showed a characteristic noise-induced pattern
of injury which includes changes in the spiral ligament, the
limbus, the stria vascularis and the HC (Ou et al., 2000a; Wang
et al., 2002; Ohlemiller et al., 2011; Park et al., 2013). In this study,
in conjunction with the functional alterations observed, CBA
mice exposed to VS noise showed cellular lesions in fibrocytes
and HC. To fully characterize the effects of noise on HCs it is
necessary to examine the entire OC from apex to base. However,
surgical dissection of the organ of Corti is difficult, especially in
the basal tip region or “hook”, which is usually lost or artificially
damaged. We estimated that this portion represents around 20%
of the total length of the OC, therefore the cochleogram includes
the 80% of the cochlea that maintained cellular integrity. Based
on the frequency location map of the mouse cochlea by Ou
et al. (2000b), this portion results in a loss of information on
the 50–80 kHz frequencies. As mentioned before, noise exposure
induces higher TS in a range of frequencies 0.5 to 2 octaves around
FIGURE 7 | TGF-β1 inhibitors in NIHL treatment. (A) Chronogram of the the noise frequency. Therefore this hook region should not be
experiment. Mice were exposed for 30 min to VS noise at 105 dB SPL and much affected by our VS. To further study HC loss resulting from
after 2 days they were operated on to deliver P17, P144 or saline (n = 6 mice different VS noise exposures, stereological methods were applied
per group) to the inner ear through a bullostomy. ABR tests were
performed before and 1, 14 and 28 days after noise exposure. (B) Evolution
to develop a cytocochleogram, a topographical map of the cochlea
of threshold of click-evoked ABR in mice exposed to 105 VS2−20 noise for which associates frequencies to defined HC positions along the
30 min and treated locally with a single dose of TGF-β1 inhibitors P17 length of the BM, offering key information on the function to
(orange line), P144 (pink) or saline (black), compared to non-exposed (green) structure relationship, under both physiological and pathological
and sham-operated (purple) mice . Noise induced notable TS in the first day
conditions (Viberg and Canlon, 2004). Due to intraspecies BM
after damage without noticeable recovery, compared to non-exposed
controls (***p ≤ 0.001). No significant differences were observed between
length variation, it is preferable to construct percentage, rather
mice treated with TGF-β1 inhibitors and those treated with saline. No than absolute length (in mm) cochleograms. When length values
statistically significant increase in click-evoked ABR threshold was observed are expressed as percentages, this results in a linear topographic
in sham-operated mice. (C) Outer hair cell density (mean ± SEM, map that can be safely used to correlate the location of the HC
expressed as %) in mice treated with TGF-β1 inhibitors compared to saline
(or a lesion thereof) to sound frequency by using appropriate
(Continued)
frequency-place equations (Viberg and Canlon, 2004). For this

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Sanz et al. Mouse NIHL and TGF-β1 inhibition

study with CBA mice, we chose to follow the Müller equation TGF-β1 inhibitors applied to the middle ear using a gelatin
(Müller et al., 2005) and to plot the cytocochleogram dividing the sponge, after noise exposure. The potential therapeutic effect
length of the BM in 5% equidistant sectors from the apex to the was evaluated with ABR and a cochleogram, as described. Mice
base of the cochlea. exposed to noise showed a similar pattern of damage, both
Cell counting procedures most often consist of direct counts functional and morphological, with no significant differences
of HC cell bodies or nuclei (or their absence from identified compared to saline treated mice. Therefore, our results suggest
spots) at fixed length intervals from the apex, which may then that a single dose of these peptides is not enough to prevent noise
be transformed into percentage distance from the cochlear apex damage.
(Nordmann et al., 2000; Minami et al., 2007; Harding and In summary, we show that CBA mice exposed for 30 min
Bohne, 2009; Choudhury et al., 2011). We opted instead for to noise enriched in frequencies up to 20 kHz and presented
estimating HC densities at regular 5% length intervals from in a swept-sine mode suffered a TS of around 50 dB.
the apex, an efficient and consistent novel procedure, which Depending on the intensity and frequency range, this noise
relied on applying a stereological method (Boyce et al., 2010) induced specific functional and morphological changes, with a
on phalloidin-stained stereocilia bundles as counting units. tonotopic correlation between the noise-frequency input and the
Single bundles are unambiguously identified on flat mounts injury output measured by means of functional data and cell
of the cochlea, and their disappearance marks an irreversibly density counting. The methodology described herein, combining
degenerating or already absent HC, since hair bundle loss standardized NIHL mouse models, friendly local delivery systems,
is not recoverable in mammalian cochlear HC (Jia et al., and precise evaluation techniques such ABR and cochleogram,
2009). should be useful in further understanding NIHL and for
Using this cytocochleogram, we evidenced different patterns evaluating novel therapeutic strategies.
of HC loss depending on the level and frequency range of the VS
noise, as well as a correlation with functional data. As reported AUTHORS AND CONTRIBUTORS
for other sound stimuli in mice, VS noise induced a greater loss LS: acquisition, analysis, and interpretation of cytocochleogram
of OHC than IHC (Wang et al., 2002; Ohlemiller and Gagnon, data. SM-C: acquisition, analysis, and interpretation of functional
2007; Ohlemiller, 2008; Park et al., 2013). After exposure to noise data; drafting and revision of the manuscript. PC: substantial
with a particular range of frequencies, HC loss fundamentally contribution to the conception and design of the noises
occurs within the cochlear region that codifies this signal, but and exposition chamber. RC: substantial contribution to the
also outside this zone (Harding and Bohne, 2009). Consequently, conception and design of the noises and exposition chamber.
we observed a notable HC loss, especially in OHC, in sectors JC: acquisition, analysis, and interpretation of morphological
5–55% after exposure to 105 VS2−20 and a focal lesion in sectors data. TR: design of the work; revision of the manuscript.
25–30% when 105 VS9−13 was used, but also we detected cell IVN: design of the work; analysis, and interpretation of data;
loss in the basal cochlear region. This is because the acoustic drafting and revision of the manuscript. CA: design of the
energy delivered by a noise is maximal in its frequency range work, analysis and interpretation of data; critical revision of the
and declines above and below it. As expected, after exposure to manuscript.
120 VS2−20 noise the cochleogram showed generalized cochlear
lesions with a marked decrease in HC density, with combined
ACKNOWLEDGMENTS
lesions in OHC and IHC and with large areas where no HC were
We warmly thank our colleagues from the Neurobiology
detected.
of Hearing group for helpful discussions and for sharing
The NIHL paradigm and cochleogram procedure shown here
unpublished data and procedures. We also thank the technical
constitute valuable tools in preclinical assays of new molecules
support of the Non-invasive Neurofunctional Evaluation
with potential therapeutic effects. Inhibitors are key regulators
facility (IIBm, CSIC-UAM and SEFALer, CIBERER). This
of the inflammatory and immune response in several tissues
work was supported by grants from MINECO (SAF2011-
including the inner ear. Upregulation of TGF-β1 has been
24391), Fundación de Investigación Médica Mutua Madrileña
confirmed in some animal models with otic damage, including
(FMM2012), European FP7-INNOVA2-AFHELO and FP7-
ototoxicity and antigen injection, chronic otitis media (Satoh
PEOPLE-IAPP-TARGEAR to IVN and FIS PI 10/00394 for TR.
et al., 2006; Wissel et al., 2006; Ghaheri et al., 2007) and NIHL
S.M.-C. holds a CIBERER (ISCIII) postdoctoral contract.
(Murillo-Cuesta et al., submitted). In this context, the use of
TGF-β1 inhibitors could be useful in ameliorating pathological
changes in the cochlea after noise insult. Systemic treatment REFERENCES
Attias, J., Horovitz, G., El-Hatib, N., and Nageris, B. (2001). Detection and clinical
with P17 and P144 before noise exposure as well after damage diagnosis of noise-induced hearing loss by otoacoustic emissions. Noise Health
has been shown to improve the TSs and the evolution of 3, 19–31.
thresholds of click-evoked and tone-evoked ABR, compared to Bohne, B. A., Harding, G. W., and Lee, S. C. (2007). Death pathways in noise-
saline treated mice (Murillo-Cuesta et al., submitted). In order damaged outer hair cells. Hear. Res. 223, 61–70. doi: 10.1016/j.heares.2006.10.
to deliver molecules to the inner ear with accuracy and to avoid 004
Boyce, R. W., Dorph-Petersen, K. A., Lyck, L., and Gundersen, H. J. (2010). Design-
adverse effects, new localized approaches to the cochlea are now based stereology: introduction to basic concepts and practical approaches
being tested (Rivera et al., 2012). In this work we showed the for estimation of cell number. Toxicol. Pathol. 38, 1011–1025. doi: 10.
functional and morphological results of a preclinical assay with 1177/0192623310385140

Frontiers in Aging Neuroscience www.frontiersin.org February 2015 | Volume 7 | Article 7 | 11

Sanz et al. Mouse NIHL and TGF-β1 inhibition

Cai, S., Ma, W. L., and Young, E. D. (2009). Encoding intensity in ventral Hu, B. H., Henderson, D., and Nicotera, T. M. (2006). Extremely rapid induction of
cochlear nucleus following acoustic trauma: implications for loudness outer hair cell apoptosis in the chinchilla cochlea following exposure to impulse
recruitment. J. Assoc. Res. Otolaryngol. 10, 5–22. doi: 10.1007/s10162-008- noise. Hear. Res. 211, 16–25. doi: 10.1016/j.heares.2005.08.006
0142-y Jamesdaniel, S., Hu, B., Kermany, M. H., Jiang, H., Ding, D., Coling, D., et al.
Cediel, R., Riquelme, R., Contreras, J., Díaz, A., and Varela-Nieto, I. (2006). (2011). Noise induced changes in the expression of p38/MAPK signaling
Sensorineural hearing loss in insulin-like growth factor I-null mice: a new model proteins in the sensory epithelium of the inner ear. J. Proteomics 75, 410–424.
of human deafness. Eur. J. Neurosci. 23, 587–590. doi: 10.1111/j.1460-9568.2005. doi: 10.1016/j.jprot.2011.08.007
04584.x Jia, S., Yang, S., Guo, W., and He, D. Z. (2009). Fate of mammalian cochlear hair
Chen, T. J., Chen, S. S., Wang, D. C., and Hsieh, Y. L. (2002). Increased vulnerability cells and stereocilia after loss of the stereocilia. J. Neurosci. 29, 15277–15285.
of auditory system to noise exposure in mdx mice. Laryngoscope 112, 520–525. doi: 10.1523/JNEUROSCI.3231-09.2009
doi: 10.1097/00005537-200203000-00021 Kirchner, D. B., Evenson, E., Dobie, R. A., Rabinowitz, P., Crawford, J., Kopke,
Chen, G. D., and Fechter, L. D. (2003). The relationship between noise-induced R., et al. (2012). Occupational noise-induced hearing loss: ACOEM task force
hearing loss and hair cell loss in rats. Hear. Res. 177, 81–90. doi: 10.1016/s0378- on occupational hearing loss. J. Occup. Environ. Med. 54, 106–108. doi: 10.
5955(02)00802-x 1097/JOM.0b013e318242677d
Chen, Y. S., Liu, T. C., Cheng, C. H., Yeh, T. H., Lee, S. Y., and Hsu, C. J. Konings, A., Van Laer, L., and Van Camp, G. (2009). Genetic studies on noise-
(2003). Changes of hair cell stereocilia and threshold shift after acoustic induced hearing loss: a review. Ear Hear. 30, 151–159. doi: 10.1097/AUD.
trauma in guinea pigs: comparison between inner and outer hair cells. ORL J. 0b013e3181987080
Otorhinolaryngol. Relat. Spec. 65, 266–274. doi: 10.1159/000075224 Kujawa, S. G., and Liberman, M. C. (2009). Adding insult to injury: cochlear
Choudhury, B., Adunka, O. F., Demason, C. E., Ahmad, F. I., Buchman, C. A., nerve degeneration after "temporary" noise-induced hearing loss. J. Neurosci.
and Fitzpatrick, D. C. (2011). Detection of intracochlear damage with cochlear 29, 14077–14085. doi: 10.1523/JNEUROSCI.2845-09.2009
implantation in a gerbil model of hearing loss. Otol. Neurotol. 32, 1370–1378. Le Prell, C. G. (2012). Noise-induced hearing loss: from animal models to
doi: 10.1097/MAO.0b013e31822f09f2 human trials. Adv. Exp. Med. Biol. 730, 191–195. doi: 10.1007/978-1-4419-
Cobo, P., Murillo-Cuesta, S., Cediel, R., Moreno, A., Lorenzo-García, P., and 7311-5_43
Varela-Nieto, I. (2009). Design of a reverberant chamber for noise exposure Mahendra Prashanth, K. V., and Venugopalachar, S. (2011). The possible influence
experiments with small animals. Appl. Acoust. 70, 1034–1040. doi: 10.1016/j. of noise frequency components on the health of exposed industrial workers–a
apacoust.2009.03.005 review. Noise Health 13, 16–25. doi: 10.4103/1463-1741.73996
Davis, R. R., Kozel, P., and Erway, L. C. (2003). Genetic influences in individual Martin, G. K., Stagner, B. B., and Lonsbury-Martin, B. L. (2006). Assessment
susceptibility to noise: a review. Noise Health 5, 19–28. of cochlear function in mice: distortion-product otoacoustic emissions.
Ezquerro, I. J., Lasarte, J. J., Dotor, J., Castilla-Cortázar, I., Bustos, M., Curr. Protoc. Neurosci. Chapter 8:Unit8.21C. doi: 10.1002/0471142301.ns0
Peñuelas, I., et al. (2003). A synthetic peptide from transforming growth 821cs34
factor beta type III receptor inhibits liver fibrogenesis in rats with carbon Minami, S. B., Yamashita, D., Ogawa, K., Schacht, J., and Miller, J. M. (2007).
tetrachloride liver injury. Cytokine 22, 12–20. doi: 10.1016/s1043-4666(03) Creatine and tempol attenuate noise-induced hearing loss. Brain Res. 1148, 83–
00101-7 89. doi: 10.1016/j.brainres.2007.02.021
Fetoni, A. R., Garzaro, M., Ralli, M., Landolfo, V., Sensini, M., Pecorari, G., Moore, B. C. (2004). Testing the concept of softness imperception: loudness near
et al. (2009). The monitoring role of otoacoustic emissions and oxidative stress threshold for hearing-impaired ears. J. Acoust. Soc. Am. 115, 3103–3111. doi: 10.
markers in the protective effects of antioxidant administration in noise-exposed 1121/1.1738839
subjects: a pilot study. Med. Sci. Monit. 15, PR1–PR8. Müller, M., and Smolders, J. W. (2005). Shift in the cochlear place-frequency
Ghaheri, B. A., Kempton, J. B., Pillers, D. A., and Trune, D. R. (2007). Cochlear map after noise damage in the mouse. Neuroreport 16, 1183–1187. doi: 10.
cytokine gene expression in murine chronic otitis media. Otolaryngol. Head Neck 1097/00001756-200508010-00010
Surg. 137, 332–337. doi: 10.1016/j.otohns.2007.03.020 Müller, M., von Hünerbein, K., Hoidis, S., and Smolders, J. W. (2005). A
Gourévitch, B., Doisy, T., Avillac, M., and Edeline, J. M. (2009). Follow-up of physiological place-frequency map of the cochlea in the CBA/J mouse. Hear.
latency and threshold shifts of auditory brainstem responses after single and Res. 202, 63–73. doi: 10.1016/j.heares.2004.08.011
interrupted acoustic trauma in guinea pig. Brain Res. 1304, 66–79. doi: 10. Murillo-Cuesta, S., Camarero, G., González-Rodríguez, A., De La Rosa, L. R., Burks,
1016/j.brainres.2009.09.041 D. J., Avendano, C., et al. (2012). Insulin receptor substrate 2 (IRS2)-deficient
Gratton, M. A., Eleftheriadou, A., Garcia, J., Verduzco, E., Martin, G. K., Lonsbury- mice show sensorineural hearing loss that is delayed by concomitant protein
Martin, B. L., et al. (2011). Noise-induced changes in gene expression in the tyrosine phosphatase 1B (PTP1B) loss of function. Mol. Med. 18, 260–269.
cochleae of mice differing in their susceptibility to noise damage. Hear. Res. 277, doi: 10.1016/s1096-6374(12)60045-8
211–226. doi: 10.1016/j.heares.2010.12.014 Murillo-Cuesta, S., García-Alcántara, F., Vacas, E., Sistiaga, J. A., Camarero, G.,
Greenwood, D. D. (1996). Comparing octaves, frequency ranges and cochlear- Varela-Nieto, I., et al. (2009). Direct drug application to the round window: a
map curvature across species. Hear. Res. 94, 157–162. doi: 10.1016/0378- comparative study of ototoxicity in rats. Otolaryngol. Head Neck Surg. 141, 584–
5955(95)00229-4 590. doi: 10.1016/j.otohns.2009.07.014
Hamernik, R. P., and Qiu, W. (2000). Correlations among evoked potential Nordmann, A. S., Bohne, B. A., and Harding, G. W. (2000). Histopathological
thresholds, distortion product otoacoustic emissions and hair cell loss following differences between temporary and permanent threshold shift. Hear. Res. 139,
various noise exposures in the chinchilla. Hear. Res. 150, 245–257. doi: 10. 13–30. doi: 10.1016/s0378-5955(99)00163-x
1016/s0378-5955(00)00204-5 Ohlemiller, K. K. (2006). Contributions of mouse models to understanding of age-
Harding, G. W., and Bohne, B. A. (2009). Relation of focal hair-cell lesions to noise- and noise-related hearing loss. Brain Res. 1091, 89–102. doi: 10.1016/j.brainres.
exposure parameters from a 4- or a 0.5-kHz octave band of noise. Hear. Res. 254, 2006.03.017
54–63. doi: 10.1016/j.heares.2009.04.011 Ohlemiller, K. K. (2008). Recent findings and emerging questions in cochlear noise
Harding, G. W., Bohne, B. A., and Ahmad, M. (2002). DPOAE level shifts injury. Hear. Res. 245, 5–17. doi: 10.1016/j.heares.2008.08.007
and ABR threshold shifts compared to detailed analysis of histopathological Ohlemiller, K. K., and Gagnon, P. M. (2007). Genetic dependence of cochlear cells
damage from noise. Hear. Res. 174, 158–171. doi: 10.1016/s0378-5955(02) and structures injured by noise. Hear. Res. 224, 34–50. doi: 10.1016/j.heares.
00653-6 2006.11.005
Henry, K. S., Kale, S., Scheidt, R. E., and Heinz, M. G. (2011). Auditory brainstem Ohlemiller, K. K., Rybak Rice, M. E., Rellinger, E. A., and Ortmann, A. J. (2011).
responses predict auditory nerve fiber thresholds and frequency selectivity in Divergence of noise vulnerability in cochleae of young CBA/J and CBA/CaJ
hearing impaired chinchillas. Hear. Res. 280, 236–244. doi: 10.1016/j.heares. mice. Hear. Res. 272, 13–20. doi: 10.1016/j.heares.2010.11.006
2011.06.002 Ohlemiller, K. K., Wright, J. S., and Heidbreder, A. F. (2000). Vulnerability to noise-
Howard, C. V., and Reed, M. G. (2005). Unbiased Stereology. Three Dimensional induced hearing loss in ‘middle-aged’ and young adult mice: a dose-response
Measurement in Microscopy. 2nd Edn. New York: Garland Science/BIOS approach in CBA, C57BL and BALB inbred strains. Hear. Res. 149, 239–247.
Scientific Publishers. doi: 10.1016/s0378-5955(00)00191-x

Frontiers in Aging Neuroscience www.frontiersin.org February 2015 | Volume 7 | Article 7 | 12

Sanz et al. Mouse NIHL and TGF-β1 inhibition

Ou, H. C., Bohne, B. A., and Harding, G. W. (2000a). Noise damage in the Strelcyk, O., Christoforidis, D., and Dau, T. (2009). Relation between derived-
C57BL/CBA mouse cochlea. Hear. Res. 145, 111–122. doi: 10.1016/s0378- band auditory brainstem response latencies and behavioral frequency selectivity.
5955(00)00081-2 J. Acoust. Soc. Am. 126, 1878–1888. doi: 10.1121/1.3203310
Ou, H. C., Harding, G. W., and Bohne, B. A. (2000b). An anatomically based Syka, J. (2002). Plastic changes in the central auditory system after hearing loss,
frequency-place map for the mouse cochlea. Hear. Res. 145, 123–129. doi: 10. restoration of function and during learning. Physiol. Rev. 82, 601–636. doi: 10.
1016/s0378-5955(00)00082-4 1152/physrev.00002.2002
Park, S. N., Back, S. A., Park, K. H., Seo, J. H., Noh, H. I., Akil, O., et al. (2013). Vázquez, A. E., Jimenez, A. M., Martin, G. K., Luebke, A. E., and Lonsbury-Martin,
Comparison of functional and morphologic characteristics of mice models of B. L. (2004). Evaluating cochlear function and the effects of noise exposure in
noise-induced hearing loss. Auris Nasus Larynx 40, 11–17. doi: 10.1016/j.anl. the B6.CAST+Ahl mouse with distortion product otoacoustic emissions. Hear.
2011.11.008 Res. 194, 87–96. doi: 10.1016/s0378-5955(04)00130-3
Popelar, J., Grecova, J., Rybalko, N., and Syka, J. (2008). Comparison of Viberg, A., and Canlon, B. (2004). The guide to plotting a cochleogram. Hear. Res.
noise-induced changes of auditory brainstem and middle latency response 197, 1–10. doi: 10.1016/j.heares.2004.04.016
amplitudes in rats. Hear. Res. 245, 82–91. doi: 10.1016/j.heares.2008. Wang, Y., Hirose, K., and Liberman, M. C. (2002). Dynamics of noise-induced
09.002 cellular injury and repair in the mouse cochlea. J. Assoc. Res. Otolaryngol. 3, 248–
Riquelme, R., Cediel, R., Contreras, J., la Rosa Lourdes, R. D., Murillo-Cuesta, 268. doi: 10.1007/s101620020028
S., Hernandez-Sanchez, C., et al. (2010). A comparative study of age-related Wissel, K., Wefstaedt, P., Miller, J. M., Lenarz, T., and Stöver, T. (2006).
hearing loss in wild type and insulin-like growth factor I deficient mice. Front. Differential brain-derived neurotrophic factor and transforming growth factor-
Neuroanat. 4:27. doi: 10.3389/fnana.2010.00027 beta expression in the rat cochlea following deafness. Neuroreport 17, 1297–
Rivera, T., Sanz, L., Camarero, G., and Varela-Nieto, I. (2012). Drug delivery to the 1301. doi: 10.1097/01.wnr.0000233088.92839.23
inner ear: strategies and their therapeutic implications for sensorineural hearing
loss. Curr. Drug Deliv. 9, 231–242. doi: 10.2174/156720112800389098 Conflict of Interest Statement: The authors declare that the research was conducted
Rybalko, N., Bureš, Z., Burianová, J., Popeláˇr, J., Grécová, J., and Syka, J. in the absence of any commercial or financial relationships that could be construed
(2011). Noise exposure during early development influences the acoustic startle as a potential conflict of interest.
reflex in adult rats. Physiol. Behav. 102, 453–458. doi: 10.1016/j.physbeh.2010.
12.010 Received: 18 December 2014; accepted: 19 January 2015; published online: 16 February
Satoh, H., Billings, P., Firestein, G. S., Harris, J. P., and Keithley, E. M. 2015.
(2006). Transforming growth factor beta expression during an inner ear Citation: Sanz L, Murillo-Cuesta S, Cobo P, Cediel-Algovia R, Contreras J, Rivera
immune response. Ann. Otol. Rhinol. Laryngol. 115, 81–88. doi: 10.1177/ T, Varela-Nieto I and Avendaño C (2015) Swept-sine noise-induced damage
000348940611500112 as a hearing loss model for preclinical assays. Front. Aging Neurosci. 7:7.
Scheidt, R. E., Kale, S., and Heinz, M. G. (2010). Noise-induced hearing loss alters doi: 10.3389/fnagi.2015.00007
the temporal dynamics of auditory-nerve responses. Hear. Res. 269, 23–33. This article was submitted to the journal Frontiers in Aging Neuroscience.
doi: 10.1016/j.heares.2010.07.009 Copyright © 2015 Sanz, Murillo-Cuesta, Cobo, Cediel-Algovia, Contreras, Rivera,
Sherlock, L. P., and Formby, C. (2005). Estimates of loudness, loudness Varela-Nieto and Avendaño. This is an open-access article distributed under the terms
discomfort and the auditory dynamic range: normative estimates, comparison of the Creative Commons Attribution License (CC BY). The use, distribution and
of procedures and test-retest reliability. J. Am. Acad. Audiol. 16, 85–100. doi: 10. reproduction in other forums is permitted, provided the original author(s) or licensor
3766/jaaa.16.2.4 are credited and that the original publication in this journal is cited, in accordance with
Sliwinska-Kowalska, M., and Davis, A. (2012). Noise-induced hearing loss. Noise accepted academic practice. No use, distribution or reproduction is permitted which
Health 14, 274–280. doi: 10.4103/1463-1741.104893 does not comply with these terms.

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