25339
2014
AORXXX10.1177/0003489414525339Annals of Otology, Rhinology & LaryngologyLim et al
Article
Vulnerability to Acoustic Trauma in the Normal Hearing Ear With Contralateral Hearing Loss
Annals of Otology, Rhinology & Laryngology 2014, Vol. 123(4) 286–292 © The Author(s) 2014 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003489414525339 aor.sagepub.com
Hyun Woo Lim, MD1, Ji Won Lee, BA2, and Jong Woo Chung, MD2
Abstract Objectives: We undertook an animal study to investigate the functional and histological changes that occur in the normal hearing ear of following acoustic trauma. Methods: As an animal model of unilateral hearing loss, the right ears of CBA mice were deafened by cochlear destruction at 6 weeks of age (SSD group). The control groups included mice that underwent a sham surgery, and mice that were exposed to noise binaurally and monaurally (by plugging the right ear completely). At 10 weeks of age, all mice were exposed to acoustic trauma (110 dB sound pressure level for 1 hour) that induced a transient threshold shift (TTS). Changes in the hearing thresholds of the left ear were assessed over the next 4 weeks by measuring the auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). Results: Following the noise exposure, the SSD group showed a permanent threshold shift (PTS) of about 10 dB, whereas the other groups showed full recovery from the TTS. The threshold of the DPOAEs of the left ears were increased after noise exposure but returned to normal in all groups, with no significant differences among the groups. Histological evaluation showed no apparent cellular loss or apoptosis in the left ears of all groups, including the SSD group. Conclusions: These results suggest that normal hearing ears are more vulnerable to acoustic trauma following contralateral unilateral cochlear ablation. This increased vulnerability may be due to damaged neural structures. Keywords unilateral hearing loss, noise-induced hearing loss, temporary threshold shift, feedback, interaural coupling Neurons of the cochlear nucleus have been shown to switch from contralateral inhibition to contralateral excitation after ipsilateral unilateral conductive hearing loss.1 Similarly, compensatory enhancement of the cochlear nuclei of the normal hearing side has been shown to occur after contralateral unilateral cochlear ablation.2 These results suggest changes to the efferent regulation of the inner ear. Changes have been reported in vulnerability to noise exposure following acoustic injury. Unilateral cochlear destruction in guinea pigs renders the contralateral ear less vulnerable to acoustic injury immediately or a few days later.3,4 These results indicated that the loss of contralateral afferent input induces hyperactivation of ipsilateral olivocochlear efferent inhibition, resulting in protection from acoustic trauma. However, unlike acute unilateral cochlear destruction, acoustic trauma was found previously to be exacerbated in adult cats with chronic unilateral hearing loss.5 The discrepancy between these 2 results may be due to changes in the net olivocochlear effect from inhibition to excitation under conditions such as chronic unilateral hearing loss. To date, little is known about cochlear vulnerability to acoustic trauma in the absence of contralateral hearing.
Furthermore, changes in the cochlear response to acoustic trauma have not been correlated with structural changes. Hence, we here used a mouse model of contralateral unilateral hearing loss to investigate the functional and histological changes in the cochleae of normal hearing ears following acoustic trauma
Methods Experimental Animals CBA mice (Central Laboratory Animal, Seoul, Korea) were housed in cages in environmentally controlled rooms with a 1
Department of Otolaryngology, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Korea 2 Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Corresponding Author: Jong Woo Chung, MD, PhD, Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap2-dong, Songpa-gu, Seoul 138-736, Korea. Email: [email protected]
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Lim et al 12 h light/dark cycle and with food and water available ad libitum. All experimental procedures were approved by the Committee on Use and Care of Animals of the University of Ulsan. Animal care was performed under the supervision of the Laboratory Animal Unit of the Asan Institute for Life Sciences, Seoul, Korea.
Study Groups The right ears of 11 mice underwent a unilateral deafening procedure by cochlear destruction at 6 weeks of age (singleside deafened [SSD] group); the right ears of an additional 5 mice underwent the same surgical procedure except that cochlear destruction was not performed (sham surgery [SHAM] group). At 10 weeks of age, all mice were exposed to acoustic trauma by inducing a transient threshold shift (TTS). For the control groups, we used 12 mice without right ear surgery; 6 were binaurally exposed to noise (binaural exposure [BIN] group), whereas the other 6 were monaurally exposed to noise by plugging the right ear completely (monaural exposure [MON] group). All mice were euthanized at 14 weeks of age for histological evaluation.
Surgical Procedures Following anesthesia with zolazepam/tiletamine (25 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), the middle and inner ear were destroyed through the posterior approach to develop SSD mice. The penetrated oval window was covered with a collagen/fibrinogen fleece sealing material (TachoComb; Nycomed, Linz, Austria). Postauricular incisions were sutured with nylon suture materials. After surgery, the mice were injected intramuscularly once with an antibiotic (cefazolin, 50 mg/kg, i.m.). The SHAM group underwent the same procedure but the middle and inner ear was not destroyed.
Noise Exposure All mice were exposed to continuous white noise (300-10 000 Hz) at a 110 dB peak equivalent sound pressure level (SPL) for 1 hour to induce a TTS. White noise was generated with a personal computer and amplifier (R-399; Interm, Seoul, Korea) and delivered through a speaker (290-8L; Altec Lansing, Oklahoma City, Oklahoma, USA) in a noise booth. In the MON group, the right ear canal was plugged with earmold impression material (Precise II; Starkey Laboratories, South Eden Prairie, Minnesota, USA) during the noise exposure.
Measurements of the Auditory Brainstem Response and Distortion Product Otoacoustic Emission Thresholds Hearing thresholds were determined by measuring the auditory brainstem responses (ABRs) and distortion
product otoacoustic emissions (DPOAEs) as reported previously.6 Tone pips were generated with an auditory evoked potential workstation (Tucker-Davis Technologies, Alachua, Florida, USA) at frequencies of 4 kHz, 8 kHz, 16 kHz, and 32 kHz. Amplified ABR waveforms were recorded for 10 ms at a sampling rate of 11.1 kHz with bandpass filter settings of 0.5 to 3 kHz. ABR waveforms were recorded in 5-dB SPL steps by decreasing the stimulus intensity from the maximum tone-pip level of 90 dB SPL. The ABR threshold at each frequency was determined by visual inspection at the lowest input level at which wave V was identified for the consistency of the measurement. To evaluate outer hair cell (OHC) function, the DPOAE was measured. All stimuli were digitally synthesized with SigGen software applications, with a constant ratio of frequency 2 (f2) to frequency 1 (f1) of 1.2. The tones were simultaneously presented as continuous tones, with the f2 level varying from 10 to 80 dB SPL in 10 dB steps and f1 10 dB SPL greater than f2. The magnitude of the 2f1-f2 inputoutput functions was obtained by fast Fourier transformation of the resulting output with BioSig software (Tucker-Davis Technologies, Alachua, Florida, USA). Responses were recorded at 4 kHz, 8 kHz, 11.3 kHz, and 16 kHz. The DPOAE threshold at each frequency was defined as the primary dB SPL level of f2 required to produce a DPOAE above the noise wave. The ABR and DPOAE threshold shifts were defined relative to the initial thresholds.
Cochlear Tissue Processing For the histological cross-sections of the mouse cochlea, fixative (4% formalin and 1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4) was infused through the oval window and drained through the round window. Each cochlea was immersed in fixative for 48 h at 4°C and decalcified in 5.5% EDTA (Microdec; Microm, France) in phosphate-buffered saline (PBS) for 5 to 7 days. After dehydration and embedding in paraffin, serial 5-µm sections of cochleae were mounted onto gelatin-coated slides. After deparaffinization, the cells on the slides were permeabilized with a proteinase K working solution (DAKO Korea, Seoul, Korea). Expression of reactive oxygen species (ROS) was assessed by staining with a monoclonal antibody to 7,8-dihydro-8-oxoguanine (anti-8-oxoG; Chemicon International, Temecula, California, USA), a biomarker for the oxidative DNA damage involved in acoustic trauma. The 8-oxoG primary antibody was incubated (1:400) for 30 min, washed with PBS, and then incubated with biotinylated anti-mouse-IgG secondary antibody (Vector Laboratories, Burlingame, California, USA) for 30 min in room temperature. The immunoreaction was visualized using diaminobenzidine tetrahydrochloride (DAB; Zymed,
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San Francisco, California, USA) for few minutes at room temperature. As s negative control, the primary antibody was replaced with PBS. As a positive control, we used cross-section slides of the mice that were exposed to continuous noise at a 110 dB SPL for 3 hours per day for 3 consecutive days. The presence of apoptotic lesions in the cochleae was assessed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) staining (Fluorescein In Situ Cell Death Detection kit; Boehringer Mannheim, Mannheim, Germany). The slides were subsequently mounted in Vectashield containing 4,9,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining material (Vector Laboratories, Burlingame, California, USA), and viewed under a confocal microscope in epifluorescence mode.
Hair Cell Survival Count For the assessment of hair cell survival, the organ of Corti was harvested with a fine forceps. Fluorescein isothiocyanate (FITC)-phalloidin (1 μg/mL; Sigma, St Louis, Missouri, USA) was used for F-actin staining. Specimens were reacted with FITC-phalloidin for 1 hour at 4°C in a darkroom and rinsed with PBS. The tissue was counterstained with DAPI and observed under a confocal microscope. Hair cells were examined in 6 high-power fields of the cochlea (the upper and lower parts of each turn). In 1 high-power field (×500), 30 to 35 hair cells were found in each row. Hair cell survival was defined as a condition of intact F-actin appearance and normal cell nucleus morphology. Hair cells not stained with FITC and DAPI were designated as missing cells. The proportion of surviving hair cells was calculated as (the number of surviving hair cells / the number of total hair cells × 100) in 1 inner hair cell (IHC) row and 3 OHC rows, respectively.
Statistical Analysis Serial changes in the ABR and DPOAE threshold shifts of the left ear were statistically analyzed. To estimate the effect of both time and group on the outcomes, data were analyzed with repeated measures analysis of variance (ANOVA) that accounted for the effects of the mice. If the group-by-time interaction effect was significant, the outcomes were compared for multiple comparisons of the group effects within time points. If the group-by-time interaction effect was insignificant, the outcomes were compared for the overall group effects. The numbers of surviving hair cells of each group were compared with the Kruskal–Wallis test. Statistical analyses were conducted with SAS version 9.2 (SAS Institute, Cary, North Carolina, USA). Values of P < .05 were considered to indicate statistical significance.
Results In the right ears of SSD mice, there was no response in ABR and DPOAE at any times or at any test frequencies until the end of the experiment, confirming that they were unilaterally and permanently deafened with manipulation. In the right ears of the SHAM mice, the ABR and DPOAE thresholds increased after surgical manipulation and returned to resting levels 3 weeks later. Threshold shifts in the right ears of BIN and MON mice after noise exposure showed a TTS pattern recovery. The ABR thresholds of the left ears of all groups sharply increased immediately after noise exposure but gradually recovered to the resting level in the SHAM, BIN, and MON groups at 10 days after noise exposure, showing a TTS from noise exposure (Figure 1A). Threshold shifts in the left ears of the SSD group, however, did not recover to the resting level and showed a permanent threshold shift (PTS) of about 10 dB SPL until 28 days after noise exposure. The DPOAE thresholds of the left ears in all groups were also increased following noise exposure, but gradually recovered to the resting level (Figure 1B). Histological analysis showed no staining of 8-OxoG (Figure 2A) and no evidence of apoptosis (Figure 2B) in the inner ears of all groups after noise exposure. In terms of hair cell survival, the IHCs and OHCs of the SSD group were grossly intact and similar to those of other groups (Figure 2C). No statistically significant differences were found by comparing the hair cell survival proportions in all groups in the 6 distinct cochlea regions (Table 1).
Discussion In our present study, we found an increased vulnerability of normal hearing ears in mice with permanent contralateral hearing loss. Whereas an acoustic trauma resulted in TTS in other groups, the same acoustic stimulation induced PTS in the SSD group. Changes in the vulnerability of peripheral cochleae can be mediated by the olivocochlear efferent system that contacts hair cells in the organ of Corti and elicits bilateral feedback control.7,8 The medial olivocochlear (MOC) system projects axon fibers to OHCs and inhibits cochlear amplification by OHCs. The lateral olivocochlear (LOC) system contacts cochlear afferent fibers under IHCs and exerts both excitatory and inhibitory effects on auditory nerve response amplitudes. The MOC has been proven to protect against acoustic trauma, and transection of MOC bundles blocks its protective effect.4,9 It is unclear whether the LOC system can, like the MOC, be activated by acoustic stimulation. Although the exact function of the LOC is largely unknown, the net effect appears to be protective.10 Moreover, selective removal of LOC fibers also increases vulnerability to acoustic injury.11
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Figure 1. Changes in auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) threshold shifts of left ears after acoustic trauma. (A) ABR threshold shifts over time differed significantly among groups at 4k (P = .003), 16k (P < .001), and 32k Hz (P < .001). Multiple comparisons revealed that the threshold shifts in the single-side deafened (SSD) group at these frequencies differed significantly from those of all control groups at most time points (asterisks indicate P value < .05). In terms of ABR 8k Hz, overall threshold shift of the SSD group was different from those of monaural exposure (MON) (P = .002) and binaural exposure (BIN) (P = .001) groups. (B) DPOAE threshold shifts of all frequencies did not differ significantly among study groups over time or in overall threshold shifts.
Our results can be explained by the attenuated contralateral MOC reflex. The MOC reflex activated by contralateral sound suppresses the cochlear response to sound and protects the ear from acoustic injury.9,12 However, in the SSD group, the normal hearing ears of the mice cannot receive inhibitory efferent signals initiating from contralateral deaf ears. Despite a significant ABR threshold shift, there was no change in DPOAEs in the SSD group. This ABR-centered threshold shift suggests that the PTS seen after noise exposure in the SSD group was caused primarily by damage to cochlear neural components rather than the OHCs.
Likewise, deficits in ABR thresholds without DPOAE changes were found in a study of cochlear afferent damage by excitotoxicity.13 Glutamate excitotoxicity can damage synaptic terminals under the IHCs and can be clearly found during acoustic trauma, even transiently in TTS injury.14 It has also been reported that afferent terminal degeneration induces ABR threshold deficits without hair cell loss.15 In that study, TTS noise exposure has been shown to lead to permanent degeneration of cochlear neuronal components leaving cochlear hair cells intact. Similarly, histological examination of the current study demonstrated no apparent evidence of cellular loss despite PTS in the left ears of SSD
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Figure 2. Histologic findings of single-side deafened (SSD) group mice. No apparent oxidative DNA damage and apoptosis was noted in the 8-oxoG (A, ×200) and the TUNEL staining (B, ×400). A confocal image of lower middle turn hair cells of an SSD group mouse (C, ×500) shows outer hair cells and inner hair cells are grossly intact without missing cells. Closed arrow heads and open arrow heads indicate outer hair cells and inner hair cells, respectively. Abbreviations: L, limbus; OC, organ or Corti; SG, spiral ganglion; SV, stria vascularis. Table 1. Hair Cell Survival Proportions of 6 Distinct Cochlea Regions.a Hair Cell IHC OHC
Study Group
Lower Basal Turn
Upper Basal Turn
Lower Middle Turn
Upper Middle Turn
Lower Apical Turn
Upper Apical Turn
SSD SHAM MON BIN P value SSD SHAM MON BIN P value
98.6 ± 1.6 98.6 ± 1.6 98.6 ± 2.9 99.3 ± 1.4 .910 99.3 ± 0.5 99.3 ± 0.9 99.3 ± 0.9 98.8 ± 0.9 .722
99.3 ± 1.4 98.6 ± 2.8 97.9 ± 2.7 100 .447 99.3 ± 0.5 99.3 ± 1.4 98.8 ± 0.9 98.8 ± 1.2 .722
98.6 ± 1.7 99.3 ± 1.4 99.3 ± 1.4 98.4 ± 3.1 .788 98.8 ± 0.5 99.5 ± 0.6 99.3 ± 0.5 99.5 ± 0.6 .378
98.5 ± 1.7 98.6 ± 1.6 98.5 ± 1.8 100 .359 100 98.4 ± 1.0 99.5 ± 0.6 98.5 ± 1.9 .177
100 99.3 ± 1.5 98.5 ± 1.7 96.6 ± 2.8 .080 97.9 ± 0.03 96.8 ± 1.5 97.0 ± 1.0 96.6 ± 1.8 .192
93.8 ± 1.7 98.3 ± 1.9 97.2 ± 3.6 95.8 ± 1.7 .140 96.1 ± 1.1 95.1 ± 2.1 94.9 ± 1.1 97.1 ± 2.0 .376
Abbreviations: BIN, binaural exposure; IHC, inner hair cell; MON, monaural exposure; OHC, outer hair cell; SHAM, sham surgery; SSD, single-side deafened. a The mean percentages of remaining hair cells (± standard deviation) are demonstrated in each cochlear region.
mice. TUNEL and 8-oxoG staining also showed no apoptotic nuclei or oxidative DNA damage in the inner ear. A comparison of hair cell survival proportions among all study groups also indicated no statistically significant differences in the IHC and OHC survivals. However, presence of neural component damages in SSD mice cannot be concluded from our results, although no distinct sensory cellular loss in the current study suggests cochlear neural damage. In the MON group, only 1 ear was exposed to traumatic noise, which temporarily prevented the right ear from being stimulated by noise, similar to the deafened ear in the SSD group. The left-side MOC efferent inhibition from right ear stimulation was, therefore, also considered to be disturbed. We expected that the attenuated protective influence of MOC efferent inhibition would be demonstrated by the
observation of an increased vulnerability, as found in the SSD group. Our results, however, showed a TTS pattern of hearing recovery in the MON group. Thus, it was suggested that attenuation of the concurrent contralateral sound stimulation through ear plugging was unlikely to contribute to the increased vulnerability of the normal side. Although complete ear plugging provides sound attenuation of around 40 dB SPL from 0.5 to 16 kHz,16 the SPL directed at the plugged ear might be sufficient to induce a contralateral MOC reflex because a click sound of 50 to 60 dB SPL is reported to be sufficient to elicit the inhibitory MOC reflex.17 Because stress conditions such as surgery can influence vulnerability to acoustic noise,18 we included a SHAM group. However, given that the ABR threshold shift differed significantly between the SHAM and SSD groups, it can be stated that there was no influence of surgical stress.
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Lim et al Our results showing cellular loss-free PTS caused by TTS-inducing noise injury indicate permanent neural damage and exacerbated excitotoxicity. A drawback of current study is lack of further information to directly reveal fundamental mechanisms confirming the supposition, central auditory pathway-mediated cochlear neural damage. Although possible regulatory mechanisms that increase afferent neural damage are regarded as the attenuated protective effects of the olivocochlear system from current knowledge, it should be directly demonstrated from further investigations. For this purpose, it is necessary to measure the change of the strength of the MOC reflex according to the contralateral hearing loss but direct measurement of MOC activity is not possible so far. Researchers have used otoacoustic emissions produced by contralateral acoustic stimulation as an indicator of the MOC strength.19 A study tried to measure compensatory changes of MOC activity in SSD animals by DPOAE without contralateral acoustic stimulation and they found no significant changes in the response.20 To investigate the attenuated MOC reflex in SSD mice, a direct and sensitive tool not using contralateral stimulation should be developed. Since no gross cellular loss was found in this study, further examination of the neural structures is needed. It has been reported afferent terminal degeneration can be related with deficits in ABR thresholds without DPOAE changes.13-15 Degeneration of presynaptic ribbon and postsynaptic dendrites can be responsible for the decrease of neuronal responses. Quantification of presynaptic ribbons by immunostaining and examination of postsynaptic endings of the auditory nerve with transmission electron microscopy have been described in previous studies.13-15,21-23 It was reported that normal thresholds can be maintained even when ribbon count is reduced to 7 synapses per IHC, however, PTS can be associated with ribbon count at under 5 synapses per IHC.15 Repeated TTS noise exposure also can induce significant loss of ribbon synapses and subsequent permanent threshold change.23 Future studies on direct demonstration of neural response change and subsequent structural damage will support the current study as a fundamental mechanism of increased vulnerability in the situation of chronic lack of contralateral afferent input. Taken together, our present histological findings and audiological results (ie, PTS in ABR and no change in DPAOEs) suggest damage to neural components of the inner ear. The effects of the potential neural damage can be explained by a loss of the contralateral protective reflex and exacerbated excitotoxicity. Authors’ Note This work was partially presented at the 35th Annual Midwinter Research Meeting of the Association for Research in Otolaryngology, San Diego, California, USA, February 25-29, 2012.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2011-0026811).
References 1. Sumner CJ, Tucci DL, Shore SE. Responses of ventral cochlear nucleus neurons to contralateral sound after conductive hearing loss. J Neurophysiol. 2005;94:4234-4243. 2. Bledsoe SC Jr, Koehler S, Tucci DL, Zhou J, Le Prell C, Shore SE. Ventral cochlear nucleus responses to contralateral sound are mediated by commissural and olivocochlear pathways. J Neurophysiol. 2009;102:886-900. 3. Rajan R. Tonic activity of the crossed olivocochlear bundle in guinea pigs with idiopathic losses in auditory sensitivity. Hear Res. 1989;39:299-308. 4. Robertson D, Anderson CJ. Acute and chronic effects of unilateral elimination of auditory nerve activity on susceptibility to temporary deafness induced by loud sound in the guinea pig. Brain Res. 1994;646:37-43. 5. Rajan R. Unilateral hearing losses alter loud sound-induced temporary threshold shifts and efferent effects in the normalhearing ear. J Neurophysiol. 2001;85:1257-1269. 6. Kang WS, Lim HW, Suh JK, Chung JW. Effects of a zinc-deficient diet on hearing in CBA mice. Neuroreport. 2012;23:201-205. 7. Liberman MC, Puria S, Guinan JJ Jr.. The ipsilaterally evoked olivocochlear reflex causes rapid adaptation of the 2f1-f2 distortion product otoacoustic emission. J Acoust Soc Am. 1996;99:3572-3584. 8. Guinan JJ Jr.. Olivocochlear efferents: anatomy, physiology, function, and the measurement of efferent effects in humans. Ear Hear. 2006;27:589-607. 9. Maison SF, Luebke AE, Liberman MC, Zuo J. Efferent protection from acoustic injury is mediated via alpha9 nicotinic acetylcholine receptors on outer hair cells. J Neurosci. 2002;22:10838-10846. 10. Lendvai B, Halmos GB, Polony G, et al. Chemical neuroprotection in the cochlea: The modulation of dopamine release from lateral olivocochlear efferents. Neurochem Int. 2011;59:150-158. 11. Le Prell CG. Role for the lateral olivocochlear neurons in auditory function. Focus on “Selective removal of lateral olivocochlear efferents increases vulnerability to acute acoustic injury.” J Neurophysiol. 2007;97:963-965. 12. Kujawa SG, Liberman MC. Conditioning-related protection from acoustic injury: effects of chronic deefferentation and sham surgery. J Neurophysiol. 1997;78:3095-3106. 13. Le Prell CG, Yagi M, Kawamoto K, et al. Chronic excitotoxicity in the guinea pig cochlea induces temporary functional
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deficits without disrupting otoacoustic emissions. J Acoust Soc Am. 2004;116:1044-1056. 14. Puel JL, Saffiedine S, Gervais d’Aldin C, Eybalin M, Pujol R. Synaptic regeneration and functional recovery after excitotoxic injury in the guinea pig cochlea. Comptes rendus de l’Academie des sciences Serie III, Sciences de la vie. 1995;318:67-75. 15. Kujawa SG, Liberman MC. Adding insult to injury: cochlear nerve degeneration after “temporary” noise-induced hearing loss. J Neurosci. 2009;29:14077-14085. 16. Irving S, Moore DR, Liberman MC, Sumner CJ. Olivocochlear efferent control in sound localization and experience-dependent learning. J Neurosci. 2011;31: 2493-2501. 17. Guinan JJ, Backus BC, Lilaonitkul W, Aharonson V. Medial olivocochlear efferent reflex in humans: otoacoustic emission (OAE) measurement issues and the advantages of stimulus frequency OAEs. J Assoc Res Otolaryngol. 2003;4:521-540.
18. Wang Y, Hirose K, Liberman MC. Dynamics of noise induced cellular injury and repair in the mouse cochlea. J Assoc Res Otolaryngol. 2002;3:248-268. 19. Abdala C, Mishra SK, Williams TL. Considering distor tion product otoacoustic emission fine structure in measurements of the medial olivocochlear reflex. J Acoust Soc Am. 2009;125:1584-1594. 20. Larsen E, Liberman MC. Contralateral cochlear effects of ipsilateral damage: no evidence for interaural coupling. Hear Res. 2010;260:70-80. 21. Halmos G, Doleviczenyi Z, Repassy G, et al. D2 autoreceptor inhibition reveals oxygen-glucose deprivation-induced release of dopamine in guinea-pig cochlea. Neuroscience. 2005;132:801-809. 22. Khimich D, Nouvian R, Pujol R, et al. Hair cell synaptic ribbons are essential for synchronous auditory signalling. Nature. 2005;434:889-894. 23. Wang Y, Ren C. Effects of repeated “benign” noise exposures in young CBA mice: shedding light on age-related hearing loss. J Assoc Res Otolaryngol. 2012;13:505-515.
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2014
AORXXX10.1177/0003489414525339Annals of Otology, Rhinology & LaryngologyLim et al
Article
Vulnerability to Acoustic Trauma in the Normal Hearing Ear With Contralateral Hearing Loss
Annals of Otology, Rhinology & Laryngology 2014, Vol. 123(4) 286–292 © The Author(s) 2014 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003489414525339 aor.sagepub.com
Hyun Woo Lim, MD1, Ji Won Lee, BA2, and Jong Woo Chung, MD2
Abstract Objectives: We undertook an animal study to investigate the functional and histological changes that occur in the normal hearing ear of following acoustic trauma. Methods: As an animal model of unilateral hearing loss, the right ears of CBA mice were deafened by cochlear destruction at 6 weeks of age (SSD group). The control groups included mice that underwent a sham surgery, and mice that were exposed to noise binaurally and monaurally (by plugging the right ear completely). At 10 weeks of age, all mice were exposed to acoustic trauma (110 dB sound pressure level for 1 hour) that induced a transient threshold shift (TTS). Changes in the hearing thresholds of the left ear were assessed over the next 4 weeks by measuring the auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). Results: Following the noise exposure, the SSD group showed a permanent threshold shift (PTS) of about 10 dB, whereas the other groups showed full recovery from the TTS. The threshold of the DPOAEs of the left ears were increased after noise exposure but returned to normal in all groups, with no significant differences among the groups. Histological evaluation showed no apparent cellular loss or apoptosis in the left ears of all groups, including the SSD group. Conclusions: These results suggest that normal hearing ears are more vulnerable to acoustic trauma following contralateral unilateral cochlear ablation. This increased vulnerability may be due to damaged neural structures. Keywords unilateral hearing loss, noise-induced hearing loss, temporary threshold shift, feedback, interaural coupling Neurons of the cochlear nucleus have been shown to switch from contralateral inhibition to contralateral excitation after ipsilateral unilateral conductive hearing loss.1 Similarly, compensatory enhancement of the cochlear nuclei of the normal hearing side has been shown to occur after contralateral unilateral cochlear ablation.2 These results suggest changes to the efferent regulation of the inner ear. Changes have been reported in vulnerability to noise exposure following acoustic injury. Unilateral cochlear destruction in guinea pigs renders the contralateral ear less vulnerable to acoustic injury immediately or a few days later.3,4 These results indicated that the loss of contralateral afferent input induces hyperactivation of ipsilateral olivocochlear efferent inhibition, resulting in protection from acoustic trauma. However, unlike acute unilateral cochlear destruction, acoustic trauma was found previously to be exacerbated in adult cats with chronic unilateral hearing loss.5 The discrepancy between these 2 results may be due to changes in the net olivocochlear effect from inhibition to excitation under conditions such as chronic unilateral hearing loss. To date, little is known about cochlear vulnerability to acoustic trauma in the absence of contralateral hearing.
Furthermore, changes in the cochlear response to acoustic trauma have not been correlated with structural changes. Hence, we here used a mouse model of contralateral unilateral hearing loss to investigate the functional and histological changes in the cochleae of normal hearing ears following acoustic trauma
Methods Experimental Animals CBA mice (Central Laboratory Animal, Seoul, Korea) were housed in cages in environmentally controlled rooms with a 1
Department of Otolaryngology, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Korea 2 Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Corresponding Author: Jong Woo Chung, MD, PhD, Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap2-dong, Songpa-gu, Seoul 138-736, Korea. Email: [email protected]
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Lim et al 12 h light/dark cycle and with food and water available ad libitum. All experimental procedures were approved by the Committee on Use and Care of Animals of the University of Ulsan. Animal care was performed under the supervision of the Laboratory Animal Unit of the Asan Institute for Life Sciences, Seoul, Korea.
Study Groups The right ears of 11 mice underwent a unilateral deafening procedure by cochlear destruction at 6 weeks of age (singleside deafened [SSD] group); the right ears of an additional 5 mice underwent the same surgical procedure except that cochlear destruction was not performed (sham surgery [SHAM] group). At 10 weeks of age, all mice were exposed to acoustic trauma by inducing a transient threshold shift (TTS). For the control groups, we used 12 mice without right ear surgery; 6 were binaurally exposed to noise (binaural exposure [BIN] group), whereas the other 6 were monaurally exposed to noise by plugging the right ear completely (monaural exposure [MON] group). All mice were euthanized at 14 weeks of age for histological evaluation.
Surgical Procedures Following anesthesia with zolazepam/tiletamine (25 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), the middle and inner ear were destroyed through the posterior approach to develop SSD mice. The penetrated oval window was covered with a collagen/fibrinogen fleece sealing material (TachoComb; Nycomed, Linz, Austria). Postauricular incisions were sutured with nylon suture materials. After surgery, the mice were injected intramuscularly once with an antibiotic (cefazolin, 50 mg/kg, i.m.). The SHAM group underwent the same procedure but the middle and inner ear was not destroyed.
Noise Exposure All mice were exposed to continuous white noise (300-10 000 Hz) at a 110 dB peak equivalent sound pressure level (SPL) for 1 hour to induce a TTS. White noise was generated with a personal computer and amplifier (R-399; Interm, Seoul, Korea) and delivered through a speaker (290-8L; Altec Lansing, Oklahoma City, Oklahoma, USA) in a noise booth. In the MON group, the right ear canal was plugged with earmold impression material (Precise II; Starkey Laboratories, South Eden Prairie, Minnesota, USA) during the noise exposure.
Measurements of the Auditory Brainstem Response and Distortion Product Otoacoustic Emission Thresholds Hearing thresholds were determined by measuring the auditory brainstem responses (ABRs) and distortion
product otoacoustic emissions (DPOAEs) as reported previously.6 Tone pips were generated with an auditory evoked potential workstation (Tucker-Davis Technologies, Alachua, Florida, USA) at frequencies of 4 kHz, 8 kHz, 16 kHz, and 32 kHz. Amplified ABR waveforms were recorded for 10 ms at a sampling rate of 11.1 kHz with bandpass filter settings of 0.5 to 3 kHz. ABR waveforms were recorded in 5-dB SPL steps by decreasing the stimulus intensity from the maximum tone-pip level of 90 dB SPL. The ABR threshold at each frequency was determined by visual inspection at the lowest input level at which wave V was identified for the consistency of the measurement. To evaluate outer hair cell (OHC) function, the DPOAE was measured. All stimuli were digitally synthesized with SigGen software applications, with a constant ratio of frequency 2 (f2) to frequency 1 (f1) of 1.2. The tones were simultaneously presented as continuous tones, with the f2 level varying from 10 to 80 dB SPL in 10 dB steps and f1 10 dB SPL greater than f2. The magnitude of the 2f1-f2 inputoutput functions was obtained by fast Fourier transformation of the resulting output with BioSig software (Tucker-Davis Technologies, Alachua, Florida, USA). Responses were recorded at 4 kHz, 8 kHz, 11.3 kHz, and 16 kHz. The DPOAE threshold at each frequency was defined as the primary dB SPL level of f2 required to produce a DPOAE above the noise wave. The ABR and DPOAE threshold shifts were defined relative to the initial thresholds.
Cochlear Tissue Processing For the histological cross-sections of the mouse cochlea, fixative (4% formalin and 1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4) was infused through the oval window and drained through the round window. Each cochlea was immersed in fixative for 48 h at 4°C and decalcified in 5.5% EDTA (Microdec; Microm, France) in phosphate-buffered saline (PBS) for 5 to 7 days. After dehydration and embedding in paraffin, serial 5-µm sections of cochleae were mounted onto gelatin-coated slides. After deparaffinization, the cells on the slides were permeabilized with a proteinase K working solution (DAKO Korea, Seoul, Korea). Expression of reactive oxygen species (ROS) was assessed by staining with a monoclonal antibody to 7,8-dihydro-8-oxoguanine (anti-8-oxoG; Chemicon International, Temecula, California, USA), a biomarker for the oxidative DNA damage involved in acoustic trauma. The 8-oxoG primary antibody was incubated (1:400) for 30 min, washed with PBS, and then incubated with biotinylated anti-mouse-IgG secondary antibody (Vector Laboratories, Burlingame, California, USA) for 30 min in room temperature. The immunoreaction was visualized using diaminobenzidine tetrahydrochloride (DAB; Zymed,
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San Francisco, California, USA) for few minutes at room temperature. As s negative control, the primary antibody was replaced with PBS. As a positive control, we used cross-section slides of the mice that were exposed to continuous noise at a 110 dB SPL for 3 hours per day for 3 consecutive days. The presence of apoptotic lesions in the cochleae was assessed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) staining (Fluorescein In Situ Cell Death Detection kit; Boehringer Mannheim, Mannheim, Germany). The slides were subsequently mounted in Vectashield containing 4,9,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining material (Vector Laboratories, Burlingame, California, USA), and viewed under a confocal microscope in epifluorescence mode.
Hair Cell Survival Count For the assessment of hair cell survival, the organ of Corti was harvested with a fine forceps. Fluorescein isothiocyanate (FITC)-phalloidin (1 μg/mL; Sigma, St Louis, Missouri, USA) was used for F-actin staining. Specimens were reacted with FITC-phalloidin for 1 hour at 4°C in a darkroom and rinsed with PBS. The tissue was counterstained with DAPI and observed under a confocal microscope. Hair cells were examined in 6 high-power fields of the cochlea (the upper and lower parts of each turn). In 1 high-power field (×500), 30 to 35 hair cells were found in each row. Hair cell survival was defined as a condition of intact F-actin appearance and normal cell nucleus morphology. Hair cells not stained with FITC and DAPI were designated as missing cells. The proportion of surviving hair cells was calculated as (the number of surviving hair cells / the number of total hair cells × 100) in 1 inner hair cell (IHC) row and 3 OHC rows, respectively.
Statistical Analysis Serial changes in the ABR and DPOAE threshold shifts of the left ear were statistically analyzed. To estimate the effect of both time and group on the outcomes, data were analyzed with repeated measures analysis of variance (ANOVA) that accounted for the effects of the mice. If the group-by-time interaction effect was significant, the outcomes were compared for multiple comparisons of the group effects within time points. If the group-by-time interaction effect was insignificant, the outcomes were compared for the overall group effects. The numbers of surviving hair cells of each group were compared with the Kruskal–Wallis test. Statistical analyses were conducted with SAS version 9.2 (SAS Institute, Cary, North Carolina, USA). Values of P < .05 were considered to indicate statistical significance.
Results In the right ears of SSD mice, there was no response in ABR and DPOAE at any times or at any test frequencies until the end of the experiment, confirming that they were unilaterally and permanently deafened with manipulation. In the right ears of the SHAM mice, the ABR and DPOAE thresholds increased after surgical manipulation and returned to resting levels 3 weeks later. Threshold shifts in the right ears of BIN and MON mice after noise exposure showed a TTS pattern recovery. The ABR thresholds of the left ears of all groups sharply increased immediately after noise exposure but gradually recovered to the resting level in the SHAM, BIN, and MON groups at 10 days after noise exposure, showing a TTS from noise exposure (Figure 1A). Threshold shifts in the left ears of the SSD group, however, did not recover to the resting level and showed a permanent threshold shift (PTS) of about 10 dB SPL until 28 days after noise exposure. The DPOAE thresholds of the left ears in all groups were also increased following noise exposure, but gradually recovered to the resting level (Figure 1B). Histological analysis showed no staining of 8-OxoG (Figure 2A) and no evidence of apoptosis (Figure 2B) in the inner ears of all groups after noise exposure. In terms of hair cell survival, the IHCs and OHCs of the SSD group were grossly intact and similar to those of other groups (Figure 2C). No statistically significant differences were found by comparing the hair cell survival proportions in all groups in the 6 distinct cochlea regions (Table 1).
Discussion In our present study, we found an increased vulnerability of normal hearing ears in mice with permanent contralateral hearing loss. Whereas an acoustic trauma resulted in TTS in other groups, the same acoustic stimulation induced PTS in the SSD group. Changes in the vulnerability of peripheral cochleae can be mediated by the olivocochlear efferent system that contacts hair cells in the organ of Corti and elicits bilateral feedback control.7,8 The medial olivocochlear (MOC) system projects axon fibers to OHCs and inhibits cochlear amplification by OHCs. The lateral olivocochlear (LOC) system contacts cochlear afferent fibers under IHCs and exerts both excitatory and inhibitory effects on auditory nerve response amplitudes. The MOC has been proven to protect against acoustic trauma, and transection of MOC bundles blocks its protective effect.4,9 It is unclear whether the LOC system can, like the MOC, be activated by acoustic stimulation. Although the exact function of the LOC is largely unknown, the net effect appears to be protective.10 Moreover, selective removal of LOC fibers also increases vulnerability to acoustic injury.11
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Figure 1. Changes in auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) threshold shifts of left ears after acoustic trauma. (A) ABR threshold shifts over time differed significantly among groups at 4k (P = .003), 16k (P < .001), and 32k Hz (P < .001). Multiple comparisons revealed that the threshold shifts in the single-side deafened (SSD) group at these frequencies differed significantly from those of all control groups at most time points (asterisks indicate P value < .05). In terms of ABR 8k Hz, overall threshold shift of the SSD group was different from those of monaural exposure (MON) (P = .002) and binaural exposure (BIN) (P = .001) groups. (B) DPOAE threshold shifts of all frequencies did not differ significantly among study groups over time or in overall threshold shifts.
Our results can be explained by the attenuated contralateral MOC reflex. The MOC reflex activated by contralateral sound suppresses the cochlear response to sound and protects the ear from acoustic injury.9,12 However, in the SSD group, the normal hearing ears of the mice cannot receive inhibitory efferent signals initiating from contralateral deaf ears. Despite a significant ABR threshold shift, there was no change in DPOAEs in the SSD group. This ABR-centered threshold shift suggests that the PTS seen after noise exposure in the SSD group was caused primarily by damage to cochlear neural components rather than the OHCs.
Likewise, deficits in ABR thresholds without DPOAE changes were found in a study of cochlear afferent damage by excitotoxicity.13 Glutamate excitotoxicity can damage synaptic terminals under the IHCs and can be clearly found during acoustic trauma, even transiently in TTS injury.14 It has also been reported that afferent terminal degeneration induces ABR threshold deficits without hair cell loss.15 In that study, TTS noise exposure has been shown to lead to permanent degeneration of cochlear neuronal components leaving cochlear hair cells intact. Similarly, histological examination of the current study demonstrated no apparent evidence of cellular loss despite PTS in the left ears of SSD
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Figure 2. Histologic findings of single-side deafened (SSD) group mice. No apparent oxidative DNA damage and apoptosis was noted in the 8-oxoG (A, ×200) and the TUNEL staining (B, ×400). A confocal image of lower middle turn hair cells of an SSD group mouse (C, ×500) shows outer hair cells and inner hair cells are grossly intact without missing cells. Closed arrow heads and open arrow heads indicate outer hair cells and inner hair cells, respectively. Abbreviations: L, limbus; OC, organ or Corti; SG, spiral ganglion; SV, stria vascularis. Table 1. Hair Cell Survival Proportions of 6 Distinct Cochlea Regions.a Hair Cell IHC OHC
Study Group
Lower Basal Turn
Upper Basal Turn
Lower Middle Turn
Upper Middle Turn
Lower Apical Turn
Upper Apical Turn
SSD SHAM MON BIN P value SSD SHAM MON BIN P value
98.6 ± 1.6 98.6 ± 1.6 98.6 ± 2.9 99.3 ± 1.4 .910 99.3 ± 0.5 99.3 ± 0.9 99.3 ± 0.9 98.8 ± 0.9 .722
99.3 ± 1.4 98.6 ± 2.8 97.9 ± 2.7 100 .447 99.3 ± 0.5 99.3 ± 1.4 98.8 ± 0.9 98.8 ± 1.2 .722
98.6 ± 1.7 99.3 ± 1.4 99.3 ± 1.4 98.4 ± 3.1 .788 98.8 ± 0.5 99.5 ± 0.6 99.3 ± 0.5 99.5 ± 0.6 .378
98.5 ± 1.7 98.6 ± 1.6 98.5 ± 1.8 100 .359 100 98.4 ± 1.0 99.5 ± 0.6 98.5 ± 1.9 .177
100 99.3 ± 1.5 98.5 ± 1.7 96.6 ± 2.8 .080 97.9 ± 0.03 96.8 ± 1.5 97.0 ± 1.0 96.6 ± 1.8 .192
93.8 ± 1.7 98.3 ± 1.9 97.2 ± 3.6 95.8 ± 1.7 .140 96.1 ± 1.1 95.1 ± 2.1 94.9 ± 1.1 97.1 ± 2.0 .376
Abbreviations: BIN, binaural exposure; IHC, inner hair cell; MON, monaural exposure; OHC, outer hair cell; SHAM, sham surgery; SSD, single-side deafened. a The mean percentages of remaining hair cells (± standard deviation) are demonstrated in each cochlear region.
mice. TUNEL and 8-oxoG staining also showed no apoptotic nuclei or oxidative DNA damage in the inner ear. A comparison of hair cell survival proportions among all study groups also indicated no statistically significant differences in the IHC and OHC survivals. However, presence of neural component damages in SSD mice cannot be concluded from our results, although no distinct sensory cellular loss in the current study suggests cochlear neural damage. In the MON group, only 1 ear was exposed to traumatic noise, which temporarily prevented the right ear from being stimulated by noise, similar to the deafened ear in the SSD group. The left-side MOC efferent inhibition from right ear stimulation was, therefore, also considered to be disturbed. We expected that the attenuated protective influence of MOC efferent inhibition would be demonstrated by the
observation of an increased vulnerability, as found in the SSD group. Our results, however, showed a TTS pattern of hearing recovery in the MON group. Thus, it was suggested that attenuation of the concurrent contralateral sound stimulation through ear plugging was unlikely to contribute to the increased vulnerability of the normal side. Although complete ear plugging provides sound attenuation of around 40 dB SPL from 0.5 to 16 kHz,16 the SPL directed at the plugged ear might be sufficient to induce a contralateral MOC reflex because a click sound of 50 to 60 dB SPL is reported to be sufficient to elicit the inhibitory MOC reflex.17 Because stress conditions such as surgery can influence vulnerability to acoustic noise,18 we included a SHAM group. However, given that the ABR threshold shift differed significantly between the SHAM and SSD groups, it can be stated that there was no influence of surgical stress.
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Lim et al Our results showing cellular loss-free PTS caused by TTS-inducing noise injury indicate permanent neural damage and exacerbated excitotoxicity. A drawback of current study is lack of further information to directly reveal fundamental mechanisms confirming the supposition, central auditory pathway-mediated cochlear neural damage. Although possible regulatory mechanisms that increase afferent neural damage are regarded as the attenuated protective effects of the olivocochlear system from current knowledge, it should be directly demonstrated from further investigations. For this purpose, it is necessary to measure the change of the strength of the MOC reflex according to the contralateral hearing loss but direct measurement of MOC activity is not possible so far. Researchers have used otoacoustic emissions produced by contralateral acoustic stimulation as an indicator of the MOC strength.19 A study tried to measure compensatory changes of MOC activity in SSD animals by DPOAE without contralateral acoustic stimulation and they found no significant changes in the response.20 To investigate the attenuated MOC reflex in SSD mice, a direct and sensitive tool not using contralateral stimulation should be developed. Since no gross cellular loss was found in this study, further examination of the neural structures is needed. It has been reported afferent terminal degeneration can be related with deficits in ABR thresholds without DPOAE changes.13-15 Degeneration of presynaptic ribbon and postsynaptic dendrites can be responsible for the decrease of neuronal responses. Quantification of presynaptic ribbons by immunostaining and examination of postsynaptic endings of the auditory nerve with transmission electron microscopy have been described in previous studies.13-15,21-23 It was reported that normal thresholds can be maintained even when ribbon count is reduced to 7 synapses per IHC, however, PTS can be associated with ribbon count at under 5 synapses per IHC.15 Repeated TTS noise exposure also can induce significant loss of ribbon synapses and subsequent permanent threshold change.23 Future studies on direct demonstration of neural response change and subsequent structural damage will support the current study as a fundamental mechanism of increased vulnerability in the situation of chronic lack of contralateral afferent input. Taken together, our present histological findings and audiological results (ie, PTS in ABR and no change in DPAOEs) suggest damage to neural components of the inner ear. The effects of the potential neural damage can be explained by a loss of the contralateral protective reflex and exacerbated excitotoxicity. Authors’ Note This work was partially presented at the 35th Annual Midwinter Research Meeting of the Association for Research in Otolaryngology, San Diego, California, USA, February 25-29, 2012.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2011-0026811).
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