The aim of the following study is to evaluate immediate protective effect of ear plug from noise morphologically and functionally. An 1-month aged 29 male C57BL/6 mice. Subjects were divided into four groups as normal control(G1), bilaterally plugged group (G2), unilaterally plugged group (G3) and noise control group (G4) and later 3 groups were exposed to 110 sound pressure level white noise for 60 min. Immediately after noise exposure, audiologic tests were performed and cochlear morphology and expression levels of a-synuclein in the cochlea were investigated. There were no functional changes in G2 and plugged ears of G3 after noise exposure, whereas unplugged ears of G3 and G4 showed significant hearing loss. In morphological study, there were a significant degeneration of the organ of Corti and mean number and diameter of efferent buttons, in unplugged ears of G3 and G4. Plugged ears of G3 also showed mild changes in morphological study. Reduction of a-synuclein was observed at the efferent terminals or cochlear extracts after noise exposure. The protective effect of ear plug on noise exposure was proven morphologically and functionally in the animal model of noise-induced hearing loss. Further study on cellular or ultrastructural level with ear plug will be needed to reveal more precise mechanism.
Keywords: Ear plug, hearing loss, noise, protective effect
|How to cite this article:|
Kim DK, Park Y, Back SA, Kim HL, Park HE, Park KH, Yeo SW, Park SN. Protective effect of unilateral and bilateral ear plugs on noise-induced hearing loss: Functional and morphological evaluation in animal model. Noise Health 2014;16:149-56
|How to cite this URL:|
Kim DK, Park Y, Back SA, Kim HL, Park HE, Park KH, Yeo SW, Park SN. Protective effect of unilateral and bilateral ear plugs on noise-induced hearing loss: Functional and morphological evaluation in animal model. Noise Health [serial online] 2014 [cited 2019 Jul 20];16:149-56. Available from: http://www.noiseandhealth.org/text.asp?2014/16/70/149/134915
| Introduction|| |
Noise-induced hearing loss (NIHL) is the second most common self-reported occupational illness or injury, despite decades of study, regulation and work place interventions. World-wide, 16% of disabling hearing loss in adults is attributed to occupational noise.  In addition, exposure to combinations of continuous and impulse noise or the interactions of noise with chemicals in actual work place, can exacerbate NIHL of workers. 
It is important to avoid the noise environment or shield inner ear from the noise for NIHL prevention, because permanent and irreversible hearing loss is the natural course of NIHL and there have been many studies in the literature, which show and compare immediate and long-term effectiveness of various hearing protective devices (HPDs). ,, It is already known that various inner ear structures, especially outer hair cell (OHC), can be damaged by loud sound, but we still don't know how structures are affected with increasing noise level during symmetrical and asymmetrical exposures. We performed the present experiment to prove immediate effect of unilateral or bilateral ear plugs from noise by both audiological and histological methods.
In this study, we investigated efferent nerve endings of medial olivocochlear system (MOC) as well as the organ of Corti, after noise exposure. MOC fibers, which are thick and myelinated, originated from medial part of superior olivary complex and terminate on OHCs of both ipsilateral and contralateral side cochlea.  The main effect of MOC efferents is to inhibit cochlear responses by decreasing the gain of the cochlear amplifier. This inhibition can protect cochlea from noise and in noisy background, can actually enhance auditory nerve responses to transient sounds.  There have been a lot of research about the relation of MOC efferents to NIHL and recently interesting results of several papers, focused on the contralateral effects of MOC in unilateral hearing loss, are reported, ,,,,,,, but there are some differences between results. Some authors reported functional changes in contralateral ear, like tonic hyperactivity of MOC system,  or changes of suprathreshold cochlear neural responses,  but another said no functional changes of that. 
We conducted the present noise exposure study in two conditions; unilateral or bilateral ear plug insertion, since, we thought investigations of audiologic and histologic changes of the cochlea with ear-plugged side of the unilaterally plugged group (G3) could reveal contralateral effect of MOC in unilateral NIHL. We observed MOC efferent nerve endings by two proteins; synaptophysin and α-synuclein. Synaptophysin, an intrinsic membrane protein of synaptic vesicles, is present in the efferent synaptic terminals of neurons innervating both the inner and OHCs in a variety of species and have been used to show damage of efferent nerve from noise in several papers. , α-synuclein, synaptic proteins widely expressed within central nervous system, is known to be localized to the efferent auditory synapses below OHCs and we recently demonstrated reduction of α-synuclein in old C57 mice and suggested its role as a possible cause of early-onset presbycusis. , In this study, we investigated its change in NIHL and inferred its role from results.
| Methods|| |
An 1-month aged 58 ears of 29 male C57BL/6 mice (Jung-Ang Experimental Animal Center, Seoul, Korea) were used in this study, since we set up appropriate model for NIHL with C57 strain in the previous study.  They were housed in experimental animal raising room of the Catholic University of Korea prior to the initiation of experimental procedures. All procedures were performed in accordance with the national ethics guidelines and this study was approved by the institutional review board of our Hospital (CUMC-2011-0076-02).
Spongy type custom-made hearing protective earplugs (Ohropax, Germany, single number rating = 32 dB) were selected. We used punch to get regular-size (5 mm × 3 mm [diameter × height]) columnar shape earplugs that fit ear openings of C57BL/6s [Figure 1]. Experimental mice were divided into four groups and the description and the included number of ears of each group were as in the following. The first group (normal control [G1], N = 24) was normal-control group and was not exposed to noise, the second group (bilaterally plugged group [G2], N = 8) had earplug inserted in both ears and the third group was inserted earplugs in a single ear (left side). Each ear of G3 was subdivided as plugged ear subgroup and unplugged ear subgroup and evaluated separately (G3a, N = 16; G3b, N = 16). The last group (G4, N = 10) was designed for noise-control group, which had no earplugs.
|Figure 1: Specially designed ear plugs for mouse using punch (a, arrows) and a C57BL6 mouse with an ear plug on the right external auditory canal (b)|
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Three groups except normal-control were exposed to 110 dB sound pressure level (SPL) white noise (the band width from 2 kHz to 14 kHz) centered at 10 kHz for 60 min. We used a specially designed, separated, pie-shaped wire cage to avoid inappropriate exposure to noise caused by the mice gathering together and hiding their heads during noise stimulation and the detailed explanation of the apparatus is described in our previous paper.  During the procedure, the mice were exposed while awake to well induce NIHL depending on the recent study since general anesthesia could protect mice against developing NIHL during noise exposure procedure.
To investigate the hearing levels of C57 strain, auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs) were measured before and 12-14 h after noise exposure. Prior to the test, mice were anesthetized by intraperitoneal injection of a mixture of Xylazine (Rompun ® , Bayer, Germany; 0.4 mL/kg) and Tiletamine hydrochloride (Zoletil ® , Virbac, France; 0.6 mL/kg). Mouse body temperature was maintained with a heating pad. Hearing tests were conducted in all subjects.
The evoked acoustic brainstem response thresholds were differentially recorded from the scalps of the mice. Responses were recorded using subdermal needle electrodes at the vertex, below the pinna of tested ear (reference) and below the contralateral ear (ground) and the responses was recorded in both ear one by one. ABR thresholds were obtained for click (100-μs duration; 31 Hz). Evoked potentials were amplified (×200,000), bandpass filtered (100-3000 Hz) and averaged over 1024 sweeps. The responses are recorded during 12 ms after stimulus onset and thresholds were determined for a broadband click stimuli by decreasing the SPL in 10-dB decrements until the lowest level at which a distinct ABR wave pattern recognizable by two of the investigators was reached. ABR measurements were taken with an Intelligent Hearing System Smart EP System, running IHS high-frequency software (ver. 2.33) and using IHS high-frequency transducers (HFT9911-20-0035; IHS, Miami, FL).
DPOAE was also recorded by using the above-mentioned system. DPOAE measurements were conducted for pure tones from 6 to 32 kHz. An etymotic 10B+ probe was inserted into the external ear canal and used in conjunction with two different types of transducers depending on the range of the stimulation frequency. Two etymotic ER2 speakers were used for frequencies ranging from 6 to 16 kHz. For frequencies ranging from 16 to 32 kHz, two IHS high-frequency transducers were used. Stimulus response signals were sampled at a rate of 128 kHz using a 16-bit A/D converter. L1 amplitude was set to 65 dB SPL and L2 amplitude was set to 55 dB SPL. Frequencies were acquired with an F2-F1 ratio of 1.22.
At 1 day after noise exposure, 4 cochleae of each group were harvested for light microscopic examination. The mice were anesthetized and their cochleae were isolated and dissected. The cochleae were perfused through the round and oval windows with both 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and incubated in the same fixative overnight at 4°C. The cochleae were then rinsed with 0.1 M phosphate buffered saline (PBS) and incubated in 1% osmium tetroxide overnight, followed by immersion in 5% ethylenediaminetetraacetic acid (EDTA) for 2-4 days. The decalcified cochleae were then dehydrated in ethanol and propylene oxide and embedded in Araldite 502 resin (Electron Microscopy Sciences, Fort Washington, PA, USA). The cochleae were sectioned at 5 μm, stained with toluidine blue and mounted in permount on microscope slides.
Three different regions of the cochlea from mid-modiolar sections with clearly visible whole organs of Corti of basal turn, were selected for image magnification (×400 objective). A modified rank-order grading method , was used to rate the general shape of the organ of Corti. Briefly, numbers were assigned to indicate the following conditions: (5) Normal cytoarchitecture of the organ of Corti with intact hair cells; (4) maintenance of normal cytoarchitecture of the organ of Corti with all supporting cells intact, but loss of hair cells; (3) partial collapse of the organ of Corti, but with subtypes of supporting cells still recognizable; (2) a low cuboidal cell layer without recognizable supporting cells; and (1) complete degeneration of the organ of Corti into a single flattened and undifferentiated cell layer. The averaged regional values for the organ of Corti in the base turns were obtained from each group.
Scanning electron microscopic (SEM) study
Three cochleae of each group were subjected to SEM. The cochleae were perfused with 2% glutaraldehyde in 0.1 M PBS (pH 7.4) through the round and oval windows and then incubated in the same fixative overnight at 4°C. Tissues were then post-fixed in 1% osmium tetroxide for 2 h. After microdissection, cochleae were submerged in PBS and chemically dehydrated in a series of acetone solutions from 50% to 100% respectively (12 h at each concentration). The specimens were then transferred to hexamethyldisilazane and allowed to air-dry. All samples were gold coated using a sputter coater and examined under a JSM-5410 LV SEM (JEOL, Tokyo, Japan) operated with an accelerating voltage of 15 kV. Schematic drawings of mice cochleae from a previous study  showing the percent distance plotted were adopted for the observation of specific sites (8-, 16- and 32-kHz areas) in dissected cochleae and morphologic differences between exposed-to-noise group and protected-by-earplug group of P1mo C57 mice were compared.
Whole mount cochlear immunohistochemistry
Four cochleae of each group were tested for whole mount immunohistochemistry, 1 day after noise exposure. To evaluate MOC efferent innervation patterns in the cochlea, tissues excised from P1mo C57 mice were subjected to whole mount immunohistochemical staining for synaptophysin and α-synuclein. The cochleae of each group of P1mo C57 mice were perfused with 2% paraformaldehyde and maintained in fixative overnight at 4°C. After rinsing with PBS, the cochleae were decalcified with 5% EDTA for 2-4 days. The otic capsule was then removed, followed by the removal of the lateral wall, Reissner's membrane and the tectorial membrane. Polyclonal antibodies against synaptophysin (Zymed, South San Francisco, CA, USA) and α-synuclein (BD Bioscience, San Jose, CA) were used to visualize efferent nerve endings. Tissues were examined under a Zeiss confocal microscope with LSM imaging software (version 3.5, Zeiss, Germany). Considering the multiplicity of efferent terminals located at the different levels under OHCs, we took the Z-stacked pictures of efferent terminals with same parameters of depth, exposure time, step and magnification and these settings were maintained throughout image capture.
The images (×1000 objective) were imported into Image J (version 1.47, NIH, Bethesda, MD, USA) and quantitative analysis was performed by counting numbers of efferent buttons and measuring the diameter of them at each OHCs of the picture. Mean values of the size and numbers of efferent buttons were compared between groups.
Western blot assay
Total protein extracts were prepared from 4 cochleae of group G2, 3a and 3b and lysed with 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM Na 2 P 2 O 7 , 100 mM NaF, 2 mM Na 3 VO 4 , 1% NP-40, 1 mM PMSF, aprotinin and leupeptin. The cochlea homogenate was centrifuged (6500 rpm, 4°C, 20 s) and protein concentration in the supernatant was determined using a BCA protein assay. Equal amounts of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were probed with an anti-α-synuclein (BD Bioscience, San Jose, CA) primary antibody and appropriate horseradish peroxidase-conjugated secondary antibodies as per the manufacturer's recommendations. All western blots were performed in duplicate and visualized using an enhanced chemiluminescence system (Las3000, Fujifilm, Tokyo, Japan).
Statistical analysis was performed using statistical package for the social sciences (SPSS) software (ver. 16.0; SPSS Inc., Chicago, IL, USA) and P < 0.05 was considered to be significant.
| Results|| |
ABR and DPOAE recordings
In ABR tests, noise control group (G4) and unplugged ear subgroup of unilateral earplug group (G3b), which were exposed to noise without protection, showed significant increase of threshold in comparison with control group (G1). There was no significant difference between G4 and G3b. G4 and G3b also demonstrated significantly higher threshold than bilateral earplug group (G2) and plugged ear subgroup of unilateral ear plug group (G3a), which were protected from noise. G2 and G3a showed a little threshold increase compared with G1, but result was not statistically significant [Figure 2]a.
|Figure 2: Mean thresholds of auditory brain stem response (ABR, a) and mean responses of distortion product otoacoustic emission (DPOAE, b) in each group, immediately after noise exposure. Unplugged ear of G3 (group of unilateral ear plug) and noise control group (G4) show signifi cantly higher mean ABR thresholds and lower responses at DPOAE|
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G4 and G3b also showed significantly lower response than G1 at 6, 7, 10, 20 KHz in DPOAE tests. There was no significant difference between G3b and G4. G2 and G3a, which were protected from noise by earplugs showed no functional defect of OHCs when compared with G1 [Figure 2]b.
Light microscopic study and SEM study
In serial light microscopic images of the Organ of Corti, mean morphological grades of each group showed significant difference (P < 0.05, Kruskal-Wallis test). Mean grades of G1 (4.5 ± 0.5) and G2 (4.8 ± 0.4) were higher than those of G3b (2.8 ± 0.8) and G4 (3.0 ± 0.3) and G3a (3.8 ± 1.0) showed value between them [Figure 3]. Degenerations of the OHCs were observed at G4 and G3b, especially severe at the base region. Unlike them, G2 and G3a did not show severe degeneration of OHCs and especially G2, showed pretty much the same base organ of Corti grade with G1. SEM also revealed major morphological abnormality in G3b and G4, especially in base region, while not shown in G3a, G2 and G1 [Figure 4]. The morphological abnormality includes damage to stereocilia, which consists of tip fusion and scarring and depletion of OHC as well as inner hair cells.
|Figure 3: Light microscopy of the organ of Corti from each group at the basal turn of the cochleae. Mean morphologic grades of organ of Corti in normal control group (G1) and bilateral ear plug group (G2) were higher than those of unplugged ear of G3 and noise control (G4). Unilaterally plugged ear of G3 shows intermediate value between bilaterally plugged ear and unplugged ear of G3 in morphologic evaluation of organ of Corti|
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|Figure 4: Scanning electron micrographic study of each group in the same frequency area (at 32 KHz) demonstrates damage and missing (asterisks) of outer hair cells in the cochlea of unplugged ear of G3 and noise control (G4). Bilaterally plugged ears (G2) and unilaterally plugged ear of G3 did not show ultrastructural damage of stereocilia or missing of outer hair cells at 32 kHz area of the cochlea|
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Whole mount cochlear immunohistochemistry and western blot assay
Immunohistochemistry of synaptophysin showed much more intense and distinct efferent nerve terminals of G1, G2 and G3a than those of G3b and G4 [Figure 5]. In quantitative analysis of mean diameters and numbers of efferent buttons between G2, G3a and G3b, G2 showed significantly more numbers and larger mean diameter of efferent buttons than that of G3b and interestingly, G3a showed intermediate value between them [Figure 5]; P < 0.05, one way ANOVA test]. α-synuclein was predominantly localized in efferent nerve terminals as previously known and showed very similar expression pattern with synaptophysin, i.e., reduction of its expression in G3b and G4. Results of western blotting showed that similar expression level of α-synuclein in cochlea of G2 and G3a, but G3b showed much weaker expression [Figure 6].
|Figure 5: Immunohistochemical staining of efferent nerve endings by synaptophysin and α-synuclein. Much more intense and distinct staining of efferent nerve buttons with by synaptophysin and α synuclein were observed in control (G1), bilaterally plugged ears (G2) and plugged ear of G3 than those of unplugged ear of G3 and noise control (G4) (a). Signifi cant differences were observed in mean diameters (b) and numbers (c) of efferent buttons between bilaterally plugged ears and unilaterally plugged ears and between plugged and unplugged ears of unilaterally plugged group (G3). *P < 0.05 versus bilateral, †P < 0.05 versus unilateral|
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|Figure 6: Western blotting of α-synuclein in total homogenates of cochleae in the ear plug groups shows similar expression level of α-synuclein in bilateral ear plugged group (G2) and plugged ears of unilateral ear plug group (G3). Unplugged ear of G3 showed much weaker expression α-synuclein compared to plugged ears|
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| Discussion|| |
In this study, we investigated immediate protective effect of the ear plug from noise, by audiological and morphological study. In audiological study, we observed the difference of changes in hearing thresholds of ABR and responses of DPOAE tests after noise exposure between groups, there were not any functional damage of both bilaterally and unilaterally protected ear from noise. It has been know that wearing HPD, regardless of its kind, can attenuate noise around 20 dB at frequencies 0.5-8 kHz under field conditions. Ear plug as a HPD, also can provide protection equivalent to ear muffs, if properly inserted. , Until now, there have been few animal studies which show the real protective effect of ear plug on noise exposure.  In this study, we investigated the role of ear plug functionally and morphologically in the animal model of NIHL.
In morphological study, we observed protective effect of ear plug in protected ear, although there were some mild changes of the organ of Corti and efferent nerve endings of unilaterally plugged ear compared to bilaterally plugged ear. Especially, bilaterally plugged ear showed nearly normal appearance on all exams which was quite comparable to that of non-exposed control group. Hearing threshold and OHC function were perfectly preserved in the plugged ears after such intense noise exposure and the morphology of organ of Corti and OHCs were not changed at all in the group with plugged ear.
Interesting results of our study are audiologic and histologic results of G3, because comparing the results between plugged and unplugged side in this group could provide some information about MOC system. The main function of MOC efferents has been known to inhibit cochlear responses by decreasing the gain of cochlear amplifier and its effects tend to be largest on low-level responses, where the contributions of the cochlear amplifier are greatest.  MOC descends fibers to both sides of cochlea and mainly to contralateral side. However, this midline-crossed MOC fibers (to contralateral side) consists the arch of ipsilateral MOC acoustic reflex, because axons of cochlear nucleus cross the brainstem to innervate contralateral side MOC neurons. 
Because MOC efferent innervate bilaterally, contralateral effects of ipsilateral cochlear manipulation have been studied in several papers, to understand MOC systems. For example, unilateral cochlear destruction in guinea pig renders the opposite ear less vulnerable to acoustic injury for days to weeks after destruction because of tonic hyperactivity in the MOC system.  Moreover in other study, suprathreshold cochlear neural responses can be altered by hearing loss in the other ear.  In this study, we could not find any functional changes of unilaterally plugged ear compared to bilaterally plugged ear and non-exposed ear in ABR and DPOAE tests. There is a well-designed long-term study suggesting no functional change of contralateral ear by MOC activity change following ipsilateral ear manipulation. Larsen and Liberman  have reported similar results with ours that ipsilateral manipulation including tympanic membrane removal, or an acoustic overstimulation designed to produce a reversible or irreversible threshold shift, did not produced any systemic changes in contralateral cochlear responses, either in ABRs or DPOAEs. However, further study is needed to confirm whether there might be functional changes in MOC system and contralateral hearing after ipsilateral insult by noise, i.e., unilaterally plugged ear in our study, in not only short term but also longer term study
In contrast, we observed some mild changes of the organ of Corti and efferent nerve endings of unilaterally plugged ear compared to bilaterally plugged ear. Unilaterally plugged ear showed the reduction of the mean number and diameter of efferent buttons in immunohistochemistry of synaptophysin. This phenomenon seems to be related with the decrease of synaptic vesicles within the efferent nerve endings due to the contralateral reflex of MOC efferent nerves. In noisy environment, the efferent nerve endings of unilaterally plugged ear, would release synaptic vesicles due to the contralateral reflex of MOC system caused by the strong afferent stimuli of the contralateral unplugged ear. Furthermore, if the noise become intense and harmful, the efferent nerve endings of plugged side also could be changed or damaged with the reduction of storage of synaptic vesicles and transmitter substances due to fatigue effect. Damage of ipsilateral side efferent nerve endings from noise was proven by a study using electron microscopy. , The damage consisted of several ultrastructural alterations including a reduction in the number of synaptic vesicles at the presynaptic membrane, as well as swollen mitochondria in the efferent terminals. These morphological changes of ipsilateral efferent nerve degeneration after noise exposure also could be applied to contralateral side efferent nerve endings even in plugged condition in our study. The effects of asymmetrical noise exposure and the different morphological changes in unilaterally plugged cochleae and MOC efferent nerve endings shown in our study were interesting. There was a tendency of reduction of the organ of Corti grade as well as more degeneration of MOC efferent nerve endings of unilaterally plugged ear compared to those of bilaterally plugged ears. Further study which will show the real effect of MOC efferent nerves and the mechanism of contralateral effect of unilateral ear plugging will be necessary. Taken together, we could emphasize the importance of wearing bilateral ear plugs in noisy environment with the morphological evidence.
Immunostaining of α-synuclein in present study showed similar pattern with synaptophysin. Its co-localization with synaptophysin in the MOC efferent nerves has been already known. , α-synuclein is associated with endoplasmic reticulum-to-Golgi vesicle trafficking and have a role in neurotransmitter release and in the maintenance of the reserve pool of synaptic vesicle. In the present study, we could observe the reduction of α-synuclein in efferent nerve terminals and cochlear homogenates of noise exposed mice, which might be due to degeneration of efferent nerve terminals or reduction of synaptic vesicles within nerve terminals. This is a novel finding of morphological and biochemical proof of α-synuclein reduction within the efferent nerve endings after noise exposure. Since, we previously observed the rapid reduction of α-synuclein in the cochleae of C57 mice which might be related to their early onset hearing loss,  we assume that reduction of α-synuclein within efferent nerve terminals after intense noise exposure may be another causative factor for noise-induced damage of OHCs. Its causal relationship needs to be investigated in the future study. Another interesting topic on damage of OHCs and reduction of α-synuclein within efferent nerve endings after noise exposure is that these subjects with intense noise exposure may have greater vulnerability to additional noise or aging process of hearing.
In our study, we could demonstrate the protective role of ear plugs on OHCs as well as efferent nerve endings after noise exposure for the first time. Our morphologically and functionally proven role of ear plug can be used as educational purpose to the workers in noisy environment to emphasize the preventable NIHL.
| Conclusion|| |
In the present study, we revealed the protective effect of ear plug from noise in our functional and morphologic studies. Although, there were not any functional changes of both bilaterally and unilaterally protected ear after noise exposure, there were some mild changes in the grade of the organ of Corti and efferent nerve endings of unilaterally plugged ear in morphologic study. Contralateral MOC reflex is theorized to be a cause of this difference. Reduction of α-synuclein was also observed at the efferent terminal of noise damaged cochlea. Further long-term and ultrastructural study investigating the protective effect of ear plug on OHC damage as well as efferent nerve endings will be necessary.
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Department of Otolaryngology-Head & Neck Surgery, Seoul St. Mary's Hospital, 505 Banpo-dong, Seocho-gu, Seoul 137-701
Source of Support: This research was funded by grants from Seoul St. Mary’s Clinical Medicine Research Program year of 2011 through the Catholic University of Korea and from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0004744),, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]