Otology & Neurotology 36:755Y762 Ó 2015, Otology & Neurotology, Inc.

Therapeutic Effect of Dexamethasone for Noise-induced Hearing Loss: Systemic Versus Intratympanic Injection in Mice Min Ah Han, Sang A Back, Hong Lim Kim, So Young Park, Sang Won Yeo, and Shi Nae Park Department of OtorhinolaryngologyYHead and Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul, Korea

Objective: Dexamethasone is commonly used clinically to treat noise-induced hearing loss (NIHL) because the drug exerts multiple anti-inflammatory effects. In the present study, we investigated the post-noise therapeutic effects of dexamethasone given systemically or via intratympanic injection in the mouse. Animals: Twenty-four C57BL/6J mice were used. Eighteen experimental mice were exposed to 110 dB sound pressure level white noise and then divided into three groups: the noise, intraperitoneal dexamethasone injection (IP), and intratympanic dexamethasone injection (IT) groups. Methods: Dexamethasone (3 mg/kg/d) was injected intraperitoneally for five successive days in the IP group. Intratympanic injections were given on post-noise days 1 and 4 in the IT group. We compared hearing levels, the architecture of the organ of Corti (OC), and the microscopic appearance of the medial olivocochlear efferent terminals (MOC ETs) among the groups. Results: Both the IP and IT groups exhibited hearing recovery as revealed by auditory brainstem responses (ABRs), but recovery was not apparent in distortion product otoacoustic emissions

(DPOAEs). OC degeneration as revealed by light microscopy was most extensive in the noise group and least extensive in the IP group. Scanning electron microscopy showed that the OC ultrastructure was better preserved in the IP than the IT group. Confocal microscopy showed that the ETs were shrunken in all noise-exposed groups as compared to the control group, but more shrunken in the dexamethasone-treated groups. Transmission electron microscopy showed that the MOC ET-outer hair cell (OHC) synapses were damaged in all noise-exposed groups, but the extent of degeneration was less in the IT than in the noise group. Conclusion: Dexamethasone exerts reliable therapeutic effects when used to treat NIHL. It seems that the protective effects may differ by the routes of administration as the OCs were better preserved in the IP group and the ET-OHC synapses were more intact in the IT group. Key Words: GlucocorticoidV Intratympanic injectionVMedial olivocochlear efferentVOrgan of CortiVOuter hair cellVOxidative stressVSteroid. Otol Neurotol 36:755Y762, 2015.

Noise-induced hearing loss (NIHL) is a prevalent acquired condition that can be difficult to either prevent or cure. Many complex mechanisms trigger NIHL, and clinicians find it difficult to formulate a consensus definition. Nevertheless, some general mechanistic concepts have emerged over the past decades. NIHL features mechanical destruction followed by metabolic disturbance. Mechanical damage is initiated by excessive vibration of the basilar membrane, inducing detachment of the tectorial membrane, disconnection of interciliary bridges, and, finally, even rupture of the basilar membrane and hair cell loss (1,2). Such insults to cells and

the extracellular matrix often trigger apoptosis (3). Metabolic activity may be stimulated at the time of mechanical destruction in efforts to repair and minimize cell damage, but, paradoxically, it actually aggravates cochlear destruction. Briefly, rupture of inner ear structures, not only in gross terms but also at the cellular level, including destruction of gap junctions and loss of fibrocytes of types II and IV, compromise cochlear fluid homeostasis. Ionic imbalances (among Na+, Ca2+, K+, and Clj) influence cell-to-cell interactions and scala chamber activities, accelerating cell death and dysfunction. Also, inner ear trauma triggers the release of various cytokines by fibrocytes; such cytokines include tumor necrosis factor > (TNF->), interleukin-6 (IL-6), and interleukin-1b (IL-1b), which cause inner ear destruction (4,5). In turn, this stimulates mitochondrial activity, increasing free radical levels. Free radicals contain unpaired electrons, are short-lived (unstable), and break down lipid molecules, finally triggering cell death. Reactive

Address correspondence and reprint requests to Sang Won Yeo, M.D., Ph.D., and Shi Nae Park, M.D., Ph.D., Department of OtorhinolaryngologyY Head and Neck Surgery, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137Y701, Korea; E-mail: [email protected]; [email protected] The authors report no conflicts of interest.

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oxygen species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide, and reactive nitrogen species (RNS) are known to contribute to cochlear oxidative stress (5). The greater the effort made by mitochondria to meet the metabolic demands of repair, the more oxygen is used, and the higher the levels of superoxides generated as byproducts of an overactive mitochondrial electron transport chain. Also, excessive release of glutamate from inner hair cells (IHCs) to afferent terminals triggers neuronal swelling or even rupture, and causes a threshold shift. A reduction in blood flow to the stria vascularis accelerates cochlear destruction: ischemia/reperfusion injury is in play. Destruction of strial marginal cells and ROS formation triggered by noise stress render capillaries avascular, causing further cochlear damage (4,6). Also, blood flow is affected by activation of the sympathetic nervous system. An increase in the norepinephrine response to noise stress may constrict the capillary vessels (7). All of the events described above interact, as they occur contemporaneously, and finally trigger cell death via both necrosis and apoptosis (8,9). Therapeutic strategies should target one or more of the destructive pathways described above. As the damage mechanisms are both complex and intertwined, it is difficult to inhibit cochlear destruction by using one or a few agents. Glucocorticoids are most widely used to treat NIHL because the drugs exert multiple anti-inflammatory effects, thus affecting several destructive pathways simultaneously. Glucocorticoid receptors are present in the spiral ligament, spiral ganglion cells, spiral limbus, stria vascularis, and hair cells of rodents and humans (10). Dexamethasone is the most common clinically prescribed steroid and may be given either systemically or via intratympanic injection. Many studies have shown that the drug is well distributed to the organ of Corti (OC), crossing the round window membrane via diffusion and also via a systemic route (11,12). The purpose of the present study was to evaluate the therapeutic effect of dexamethasone in NIHL. We compared two different administration routes using practical, conventional clinical protocols. These were daily systemic administration for 5 days and intratympanic injection once every 3 days. The morphologies of both the OC and medial olivocochlear efferent terminals (MOC ETs) were examined by light, electron, and confocal microscopy. We focused particularly on the cochlear materials preserved according to the two different treatment routes. MATERIALS AND METHODS Experimental Design Twenty-four male C57BL/6J (B6) mice (4 wk of age; weight 13Y17 g) were purchased from the Central Experimental Animal Center (Orient Bio, Seoul, Korea) and kept in the animal colony of the Catholic University of Korea before initiation of experimental procedures. All mice received regular rodent chow and water ad libitum. All animal procedures were performed in accordance with national ethical guidelines and relevant laws, and were approved by and complied with the dictates of the Animal Care and Use Committee of the Catholic University of Korea (IACUC-CUMC-2012-0042-04). Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE)

tests were performed on all mice before noise exposure. The 18 experimental mice were exposed to 110-dB white noise for 60 min in a specially designed acrylic box (53 cm  35 cm  53 cm  2 cm) with eight compartments, placed in a pie-shaped wire-mesh cage; a single mouse was noise-exposed in each compartment. White noise centered at 10 kHz was generated by random noise generators (B&K Type 1027) placed on top of the box. The six controls were not exposed to noise. ABR and DPOAE tests were performed immediately after noise exposure and on post-noise day 7 to record threshold shifts. The experimental mice were randomly divided into three groups after noise exposure: a noise-only group, an intraperitoneal dexamethasonetreated group (IP), and an intratympanic dexamethasone-treated group (IT) (n = 6 in each group). Dexamethasone sodium phosphate (5 mg/ml; Huonz, Humedix, Korea) was used for treatment. For systemic administration, each mouse received 3 mg/kg/d of the drug in an average volume of 9 Kl. The drug was gently delivered into the intraperitoneal cavity through a 27-gauge needle without anesthesia on each of five consecutive days. Before intratympanic injection, mice were placed on a heating pad and anesthetized via intraperitoneal injection of a mixture of Rompune (0.4 ml/kg) and Zoletil (0.6 ml/kg). The tympanic membrane was exposed using a surgical stereomicroscope. Dexamethasone was injected gently through the superior part of the tympanic membrane using a 30-gauge needle until the middle ear cavity was nearly full. All mice remained in the anesthetic chamber for about 1 h, a time sufficient to allow recovery from anesthesia. No steroid leakage occurred. Injections were given on post-noise days 1 and 4, as commonly performed in clinics. On day 4, pinpoint holes were observed in the tympanic membranes, but no dexamethasone leakage was apparent. All mice underwent hearing tests once more on day 7 and were then killed. Cochleae were harvested for morphological study of the OCs and MOC ETs.

Hearing Tests All hearing tests were performed under anesthesia using a mixture of Rompune (0.4 ml/kg) and Zoletil (0.6 ml/kg). ABRs was recorded using an Intelligent Hearing system (IHS) Smart EP fitted with high-frequency transducers (HFT9911-20-0035) and running high-frequency software version 2.33 (IHS, Miami, FL). Subdermal needle electrodes placed in the vertex (active) and below the left pinna (reference) were connected to a preamplifier filtering from 0.1 to 3 kHz. The acoustic stimuli used included a click (100 Ks in duration; 31 Hz) and tone bursts of 8, 16, and 32 kHz (1,562 Ks in duration; cos2-shaped; 21 Hz) that were presented through an inserted earphone, attenuated in 5- to 10-dB steps, to determine the thresholds. Evoked potentials were amplified (200,000), band pass-filtered (100Y3,000 Hz), and averaged over 1,024 sweeps. Thresholds were determined for the broadband click and for the 8, 16, and 32 kHz tone bursts until the lowest level at which distinct ABR wave patterns were recognizable by two investigators were attained. DPOAEs were recorded over 6 to 32 kHz geometric-mean (GM) frequencies using an IHS Smart OAE 4.26 system. An etymotic 10B+ probe was inserted into the external ear canal in conjunction with two transducers: the Etymotic ER2 stimulator for frequencies 6 to 16 kHz and an IHS high-frequency transducer for frequencies 16 to 32 kHz. The response signals were sampled at a rate of 128 kHz using a 16-bit D/A converter. The L1 amplitude was set at 65 dB SPL and the L2 amplitude at 55 dB SPL. Frequencies were acquired with an f1/f2 ratio of 1.22. Five stimulation levels, ranging from 65 to 25 dB SPL in 10-dB steps, were used. Four blocks were acquired in total, and each block consisted of 32 sweeps. ABR thresholds and DPOAE levels were compared across the four groups. The ABR thresholds obtained on post-noise day 7 were

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THERAPEUTIC EFFECT OF DEXAMETHASONE FOR NIHL TABLE 1.

cochleae were submerged in PBS and next chemically dehydrated in a series of acetone solutions (from 50 to 100% v/v) for 12 h at each concentration. The specimens were transferred to hexamethyldisilazane and gold-coated before examination under scanning electron microscopy (SEM) (JSM-5410LV; JEOL, Japan). The mouse frequency-place map was adopted to localize frequencyspecific areas: 8 kHz (18% distant from the apex), 16 kHz (43%), and 32 kHz (68%) (14). The number of missing IHCs/OHCs and the pathologies of OHC stereocilia were assessed in a single typical section from each of the above three regions and categorized as none (0Y1), mild (2,3), moderate (4Y6), and severe (97).

ABR thresholds to click and tone bursts on post-noise day 7 (dB)

Group

Click

8 kHz

Control IP IT Noise

20.8 T 3.8 25.0 T 3.2 24.2 T 2.0 40.8 T 2.0

24.2 T 3.8 25.8 T 2.0 25.8 T 3.8 42.5 T 4.2

16 kHz 21.7 T 23.3 T 25.8 T 40.8 T

4.1 2.6 3.8 2.0

32 kHz 25.0 T 3.2 27.5 T 2.8 25.8 T 2.0 42.5 T 4.2

Data are presented as mean T SD. IP indicates intraperitoneal; IT, intratympanic.

compared using ANOVA and the post hoc Tukey test, and the DPOAE levels were compared using the Kruskal-Wallis test and the post hoc Mann-Whitney test with the Bonferroni correction. The two-tailed significance level was set for 0.05.

Confocal Microscopy of Immunofluorescent Cochlear ETs Labeled with Anti->-Synuclein Whole-mount processed cochleae (n = 4 for each group) were immunostained with a monoclonal antibody against >-synuclein (BD Bioscience, San Jose, CA), an efferent synaptic vesicular protein. Specimens were rinsed twice for 5 min each time in 0.1 M PBS (pH 7.4), pre-incubated for 1 h in 0.3% (v/v) Triton X100 and 5% (v/v) normal goat serum (NGS), and incubated in a humidified chamber overnight at 4-C with anti >-synuclein antibody diluted to 1:100 in 5% (v/v) NGS in PBS. The tissues were rinsed and incubated for 2 h in Alexa 488 goat-anti-mouse antibody diluted 1:40,000 in PBS. The secondary antibody was removed and the specimens were exposed to DAPI (Vectashield, Burlingame, CA) and cover-slipped. Both the immunofluorescent regions and the nuclei of the OHCs were visualized. Confocal laser-scanning microscopy (LSM 510 Meta; Zeiss, Germany) was used to examine the ETs at the OHC bases of whole-mount cochleae labeled with anti->-synuclein. The captured images were imported into ImageJ software to allow quantitative analysis. A single examiner measured the diameters of approximately 80 ETs at each cochlear turn of the four groups and compared the data using ANOVA with the post hoc Tukey test.

Morphology of the Organ of Corti Experimental and control mice were sacrificed under anesthesia after the second hearing test and the cochleae were harvested. No pathology was identified in either the outer or middle ear of any mouse. After cochlear isolation and dissection, the cochleae were perfused through both the round and oval windows with a cocktail of 2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), incubated in the same fixative overnight at 4-C, rinsed in 0.1 M PBS, incubated in 1% (w/v) osmium tetroxide overnight, and immersed in 5% (w/v) EDTA for 2 to 4 days. The decalcified cochleae were dehydrated in ethanol and propylene oxide and embedded in Araldite 502 resin (Electrode Microscopy Sciences, Fort Washington, PA). The embedded cochleae were sliced at 5 Km thickness, stained with toluidine blue, and mounted on microscope slides. NIH ImageJ software was used to quantify morphological observations. The morphologies of the basal, middle, and apical turns were evaluated by light microscopy of six randomly selected sections from the mid-modiolar plane of each turn. The extent of OC degeneration was graded from 1 to 5 in whole numbers using a modification of the rank order method of Leake (13). This method rated both the condition of supporting cells and general OC architecture: 5 = normal cytoarchitecture with intact hair cells; 4 = normal cytoarchitecture of the OC and all supporting cells, but loss of hair cells; 3 = partial OC collapse, but supporting cell subtypes still recognizable; 2 = presence of a low cuboidal cell layer without recognizable supporting cells; 1 = complete degeneration of the OC into a single flattened and undifferentiated cell layer. The area of the stria vascularis was measured in the same sections at 25 magnification using NIH ImageJ software (National Institutes of Health, USA). The average grades of the OC/stria vascularis areas at the base, middle, and apical turns were compared across the four groups using ANOVA with the post hoc Tukey test (n = 4 cochleae from each group). Fixed cochleae (n = 4 from each group) were placed in 25% (v/v) EM-grade glutaraldehyde overnight at 4-C and post-fixed in 1% (w/v) osmium tetroxide for 2 h. After microdissection, the TABLE 2. Group

6 kHz

7 kHz

Control IP IT Noise

j3.3 T 5.6 j6.7 T 4.8 j14.0 T 14.3 j12.7 T 3.3

10.7 T 5.2 0.3 T 2.9 j0.8 T 3.4 j0.3 T 1.0

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Transmission Electron Microscopy of MOC Efferent-OHC Synapses Cochleae (n = 4 for each group) were fixed in 4% (v/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M PBS overnight, and post-fixed with 1% (w/v) osmium tetroxide in the same buffer for 1 h. Specimens were dehydrated in a graded series of ethyl alcohol/acetone baths and embedded in Epon 812. After polymerization at 60-C for 3 days, ultrathin radial sections were obtained at 70 nm thickness. Ultrastructural features of the MOC ETs in the OHC region were examined via TEM (JEM1010; JEOL, Japan).

RESULTS Hearing The detailed data on ABR thresholds and DPOAE levels of each group are presented in Tables 1 and 2. ABR thresholds on post-noise day 7 for the click and tone

DPOAEs in geometric-mean frequencies on post-noise day 7 (dB) 10 kHz 11.0 T 10.1 j13.7 T 8.2 j13.7 T 2.7 j11.2 T 3.1

17 kHz j1.2 j4.7 j2.0 j2.5

T T T T

7.1 8.5 2.0 2.1

20 kHz j7.2 j3.7 j1.2 j0.7

T 6.7 T 3.9 T 5.5 T 1.4

24 kHz j13.5 T j8.7 T j11.5 T j15.3 T

4.2 4.3 4.4 4.6

28 kHz j1.5 1.0 j1.5 j1.0

T 6.6 T 5.8 T 6.1 T 3.8

32 kHz j0.3 j3.5 j0.3 j0.2

T 3.8 T 6.2 T 3.9 T 4.5

Data are presented as mean T SD. IP indicates intraperitoneal; IT, intratympanic. Otology & Neurotology, Vol. 36, No. 5, 2015

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M. A. HAN ET AL.

ABR thresholds on post-noise day 7. *p G 0.001 vs. the control/IP/IT group. Error bars indicate SEM.

bursts were significantly lower in the control, IP, and IT groups than in the noise group (Fig. 1). DPOAE levels differed significantly between the control and IP/IT/noise groups at the 7 and 10 kHz GM frequencies. At higher GM frequencies, all DPOAE responses were small, and thus intergroup differences were not apparent (Fig. 2).

OC reticular lamina revealed various extents of ultrastructural damage in the experimental groups. The IP group exhibited mild OHC stereociliary changes in the 32 kHz regions; the IT group severely disturbed stereocilia and some OHC loss; the noise group severe stereociliary damage and moderate OHC loss (Fig. 4).

Morphology of the Organ of Corti Light microscopic examination of the OC revealed less damage to basal turn cytoarchitecture in the IP, as compared to the IT and noise groups (Fig. 3A). Quantitative data supported these microscopic findings. The grades for basal OC degeneration were 4.2 T 0.4, 3.5 T 1.1, 3.0 T 0.9, and 2.9 T 0.5 (mean T SD) in the control, IP, IT, and noise groups, respectively. Significant differences were evident between the control and IP/IT/noise groups, and between the IP and noise groups upon post hoc testing (Fig. 3B). The base stria vascularis areas (in pixels) were 58,497 T 833, 52,017 T 2,842, 50,615 T 2,594, and 48,875 T 2,091 (mean T SD) in the control, IP, IT, and noise groups, respectively. Significant differences were evident between the control and IP/IT/noise groups (Fig. 3C). SEM of the

Morphology of MOC Efferent Terminals Whole-mount cochlear ETs labeled with anti->-synuclein were most prominent in the middle turn and least prominent in the basal turn in all groups (Fig. 5A). At the apex, the ET diameters of the control, IP, IT, and noise groups were 1.54 T 0.50, 1.20 T 0.40, 1.21 T 0.35, and 1.40 T 0.50 (mean T SD) Km, respectively. Significant differences were evident between the control and IP/IT/noise groups (p G 0.001/G0.001/=0.001), and between the noise and IP/IT groups (all p values G0.001). In the middle turn, the ET diameters of the control, IP, IT, and noise groups were 1.61 T 0.49, 1.29 T 0.40, 1.33 T 0.38, and 1.52 T 0.46 Km, respectively, with significant differences evident between the control and IP/IT/noise groups (p G 0.001/G0.001/=0.039), and between the noise and IP/IT groups (all p values G0.001).

FIG. 2.

Average DP-grams for a 65-dB SPL primary tone on post-noise day 7. *p G 0.01 vs. the IP/IT/noise group. Error bars indicate SEM.

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FIG. 3. A, Representative photomicrographs of the OC taken from the basal turn. Compared to the noise and IT groups, the cytoarchitecture of the IP group is preserved. B, The OC grade of the IP group is significantly higher than that of the noise group. C, The area of the stria vascularis (SV) shows a similar trend. *p G 0.05, **p G 0.001. Error bars indicate SEM.

In the basal turn, the ET diameters of the control, IP, IT, and noise groups were 1.19 T 0.42, 1.18 T 0.32, 1.32 T 0.40, and 1.33 T 0.38 Km, respectively, with significant differences evident between the control and IT/noise groups (all

p values G0.001), and between the IP and IT/noise groups (all p values G0.001) (Fig. 5B). Transmission electron microscopic findings of the MOC efferent-OHC synapses revealed frequent degenerative

FIG. 4. Representative scanning electron micrographs of the OC reticular lamina in the 32-kHz region. The IP group demonstrates subtle disarray of OHC stereocilia. Disturbed stereocilia and presumptive hair cell loss (asterisks) are evident in the noise and IT groups. The extents of ultrastructural changes in this region are shown in the Table. j, none (0Y1); +, mild (2,3); ++, moderate (4Y6); +++, severe (97). IHC, inner hair cell; OHC, outer hair cell. Otology & Neurotology, Vol. 36, No. 5, 2015

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FIG. 5. A, Confocal micrographs of fluorescent >-synuclein-labeled efferent terminals (ETs) at OHC base in whole-mount cochleae. ETs in contact with OHC nuclei stained with DAPI are shown. B, Diameters of the ETs are compared across the groups in the apical, middle, and basal turns. *p G 0.005, **p G 0.05.

changes in the noise-exposed experimental groups. Basal OHC apoptosis was observed in the noise group. In contrast, the OHC ultrastructures of the IP and IT groups were less affected. The details are shown in Figure 6.

DISCUSSION Both systemic and intratympanic steroid therapies are commonly prescribed to treat NIHL. In the present study, we examined the regions rescued by steroids given via two routes and the therapeutic effects of the agents per se. ABR

threshold shifts have recovered in the dexamethasonetreated groups, whereas reduced DPOAE levels have not. The innate low DPOAE levels in the middle to high frequency range of this mouse strain as demonstrated in the control group are thought to have resulted in no differences across all groups. With regard to low frequencies, the differential damage between the outer and inner hair cells (more vulnerable OHCs) may explain in part the mechanisms related with unrecoverable DPOAEs after noise exposure regardless of the treatment. It is postulated that the reduced DPOAEs mirrored OHC loss or dysfunction resulting from altered stereocilia.

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FIG. 6. TEM radial sections through the OHCs at the basal turn. Within the ETs, tiny electron-dense presynaptic vesicles are wrapped in thick walls (black arrowheads). The noise group displays apoptotic OHCs with dark cytoplasm, shrunken nuclei, and blebs in the plasma membrane (black arrow). Damaged OHCs and swollen ETs contain abnormal vacuoles (white arrows). The IP and IT groups exhibit less damage to the OHCs and ETs than did the noise group. A, afferent; E, efferent; N, nucleus; M, mitochondrion.

Microscopically, we found that noise destroyed OC architecture. Both light and electron microscopy showed that dexamethasone protected against OC damage, probably because of the anti-inflammatory and neuroprotective effects of the steroid. The cochlea was rescued from the effects of damaging cascades before destruction had become irreversible. In addition, the area of the stria vascularis (a structure critical to maintenance of endolymph ion homeostasis) was less in the noise than the control group, consistent with the data of other studies showing that the stria vascularis becomes acutely swollen after noise exposure, and is disrupted and thinned after 2 weeks of noise trauma (15). We suggest that the larger stria vascularis areas in steroid-treated groups reflect the fact that the steroid exerted anti-inflammatory effects. Notably, the IP group exhibited greater preservation of OC architecture (higher grading scores and a greater stria vascularis area) than did the IT group, probably attributable to the different entry routes of the steroid. Upon systemic administration, the agent is thought to spread through the stria vascularis and then to diffuse into the scala media exerting direct effects on the OC. In contrast, upon intratympanic injection, the agent can enter the scala tympani first, reaching the OC more slowly at lower levels as compared to systemic administration. Confocal microscopy showed that the ET diameter had shrunk after noise exposure. In general, MOC fibers are known to synapse to the OHCs, inhibiting basilar membrane responses to reduce acoustic trauma (16). >-Synuclein is a ‘‘natively unfolded’’ small protein of 140 amino acids, and helical monomers thereof interact with many cellular proteins and membranes (17). In the present study, reactions with anti->-synuclein antibodies may identify >-synuclein

proteins bound to neurotransmitters that are ready to be released from ETs into synapses with OHCs, reflecting the fact that the ETs are functional. ET diameter was smallest in the basal cochlear turn and largest in the middle turn (Fig. 5B). This feature may be innate to B6 mice. It is notable that the ETs shrunk somewhat after noise exposure, and shrunk even more in the apical and middle turns after steroid therapy. Such a pattern was not evident in the basal turn, probably because the ET diameter in this region was initially small. We speculate that the ETs shrink because of high-level neurotransmitter release in an effort to control the OHC responses to excessive noise. We tentatively suggest that steroids may trigger neurotransmitter release, thus protecting the OHCs. This means that neurotransmitter levels may be low in the steroid-treated groups. The precise mechanisms of steroids related with neurotransmitter release require further study.

CONCLUSION This study is significant in that we experimentally clarified the therapeutic effects of glucocorticoids used to treat NIHL. Dexamethasone protected noise-exposed cochleae from damage and preserved hearing. Notably, the mechanisms of drug action differed by the routes of administration. We speculate that dexamethasone enters the scala media through the lateral wall of the OC upon systemic administration, whereas, after intratympanic injection, the agent enters the scala tympani directly to act first on the ET-OHC synapses and then diffuses into the scala media at lower concentration. Further studies are needed to Otology & Neurotology, Vol. 36, No. 5, 2015

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explore the detailed anti-inflammatory actions of steroids in the inner ear. Acknowledgments: This work was supported in part by grants from the Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (2010-0004744 and 2013-011870).

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8. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Caner 1972;26:239Y57. 9. Hu BH, Henderson D, Nicotera TM. Involvement of apoptosis in progression of cochlear lesion following exposure to intense noise. Hear Res 2002;166:62Y71. 10. Xu RB. Function and clinical study on receptor. Med J China 1997; 77:311Y6. 11. Chandrasekhar SS, Rubinstein RY, Kwartler JA, et al. Dexamethasone pharmacokinetics in the inner ear: comparison of route of administration and use of facilitating agents. Otolaryngol Head Neck Surg 2000;122:521Y8. 12. Harqunani CA, Kempton JB, DeGaqne JM, et al. Intratympanic injection of dexamethasone: time course of inner ear distribution and conversion to its active form. Otol Neurotol 2006;27:564Y9. 13. Leake PA, Kuntz AL, Moore CM, et al. Cochlear pathology induced by aminoglycoside ototoxicity during postnatal maturation in cats. Hear Res 1997;113:117Y32. 14. Viberq A, Canlon B. The guide to plotting a cochleogram. Hear Res 2004;197:1Y10. 15. Hirose K, Liberman MC. Lateral wall histopathology and endocochlear potential in the noise-damaged mouse cochlea. J Assoc Res Otolaryngol 2003;4:339Y52. 16. Guinan JJ Jr. Olivocochlear efferents: anatomy, physiology, function, and the measurement of efferent effects in humans. Ear Hear 2006;27:589Y607. 17. Scarlata S, Golebiewska L. Linking alpha-synuclein properties with oxidation: a hypothesis on a mechanism underling cellular aggregation. J Bioenerg Biomembr 2014;46:93Y8.

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Therapeutic Effect of Dexamethasone for Noise-induced Hearing Loss: Systemic Versus Intratympanic Injection in Mice.

Dexamethasone is commonly used clinically to treat noise-induced hearing loss (NIHL) because the drug exerts multiple anti-inflammatory effects. In th...
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