Degeneration of the vestibular nerve in unilateral Meniere’s disease evaluated by galvanic vestibular-evoked myogenic potentials

Accepted Manuscript Degeneration of the vestibular nerve in unilateral Meniere’s disease evaluated by galvanic vestibular-evoked myogenic potentials Chih-Ming Chang, Yi-Ho Young, Fu-Shan Jaw, Chi-Te Wang, Po-Wen Cheng PII: DOI: Reference:

S1388-2457(17)30220-1 http://dx.doi.org/10.1016/j.clinph.2017.06.004 CLINPH 2008170

To appear in:

Clinical Neurophysiology

Received Date: Revised Date: Accepted Date:

10 September 2016 2 June 2017 9 June 2017

Please cite this article as: Chang, C-M., Young, Y-H., Jaw, F-S., Wang, C-T., Cheng, P-W., Degeneration of the vestibular nerve in unilateral Meniere’s disease evaluated by galvanic vestibular-evoked myogenic potentials, Clinical Neurophysiology (2017), doi: http://dx.doi.org/10.1016/j.clinph.2017.06.004

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Degeneration of the vestibular nerve in unilateral Meniere’s disease evaluated by galvanic vestibular-evoked myogenic potentials

Chih-Ming Chang, MD1,2, Yi-Ho Young, MD 3, Fu-Shan Jaw, PhD1, Chi-Te Wang, MD 2, Po-Wen Cheng, MD 2,4

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Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan

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Department of Otolaryngology, Far Eastern Memorial Hospital, Taipei, Taiwan

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Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan

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Oriental Institute of Technology, Taipei, Taiwan

Correspondence and reprints request to: Po-Wen Cheng, MD Department of Otolaryngology, Far Eastern Memorial Hospital, Taipei, Taiwan 21, Sec.2, Nanya S. Rd, Banqiao Dist., New Taipei City, Taiwan Tel.: + 886-2-89667000 ext 2833 Fax: + 886-2-77282149 E-mail: [email protected]

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Abstract Objective: The staging system of Meniere’s disease utilizes audiograms to probe cochlear dysfunction. We explored the addition of galvanic vestibular-evoked myogenic potentials (VEMP) to further explore vestibular function. Methods: Seventy patients with unilateral Meniere’s disease were enrolled in this study. Within 2 weeks of diagnosis, all subjects underwent pure tone audiometry, cervical and ocular VEMP, and caloric test. The prevalence of abnormal tests and the VEMP characteristic parameters such as latencies and amplitudes were analyzed. Results: In affected ears, the abnormal rate of acoustic cVEMPs, galvanic cVEMPs, vibratory oVEMPs and galvanic oVEMPs was 37%, 17%, 20% and 9%, respectively. No significant differences existed in VEMP latencies and amplitudes between affected ears and unaffected ears. Conclusions: The impairment of otolithic organs was found to be more than that of vestibular afferents. The deterioration of the saccule was more than that of the utricle, whereas retrolabyrinthine degeneration of sacculo-collic reflex and vestibulo-ocular reflex was similar. Significance: This study is the first to use an electrophysiological test to evaluate the retrolabyrinthine function of patients with unilateral Meniere’s disease.

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Highlights 1. Retrolabyrinthine degeneration in Meniere's disease was assessed with vestibular myogenic potentials evoked by Galvanic vestibular stimulation (GVS-VEMPs). 2. The impairment of otolithic organs was more than that of vestibular afferents in Meniere's disease. 3. Meniere's disease duration positively correlated with disease stage and abnormal GVS-VEMP responses.

Keywords: Galvanic vestibular stimulation, cervical vestibular-evoked myogenic potential, ocular vestibular-evoked myogenic potential, Meniere’s disease.

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1. Introduction Meniere’s disease is an inner ear disease characterized by relapsing-remitting aural symptoms and vertigo. The diagnosis is established on the most widely-used guidelines proposed by the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) and by clinical presentation (Committee on Hearing and Equilibrium, 1995). AAO-HNS defines definite Meniere’s disease as two or more spontaneous episodes of vertigo, tinnitus or aural fullness, documented hearing loss on at least one occasion, and other causes excluded. The pathogenesis for Meniere’s disease has been widely supported to be endolymphatic hydrops, which may result from over-production or inadequately resorption of endolymph in the inner ear (Paparella, 1985). Although the cochlea is the most involved area, vestibular endorgans, including the saccule, utricle and semicircular canals, may also be involved in the endolymphatic hydrops (Okuno and Sando, 1987). According to audiometric criteria with 4-frequency pure-tone averages at 0.5, 1, 2, and 3 kHz of the worst audiogram during the interval of 6 months before treatment, the AAO-HNS staging system defined Meniere’s disease with 4 stages of different disease severities. However, because pure tone audiogram only reflects the decline of cochlear function, additional information of vestibular function seems to be necessary for mapping the disease in detail.

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Newly-developed cervical vestibular-evoked myogenic potential (cVEMP) has been progressively utilized for assessing the descending sacculo-collic reflex (SCR) pathway and saccule function (Colebatch et al., 1994), whereas the novel ocular vestibular-evoked myogenic potential (oVEMP) has been successfully demonstrated for evaluating the ascending vestibulo-ocular reflex (VOR) pathway and utricle function (Iwasaki et al., 2007). Air conducted sound (ACS) was first proposed to be best for generating cVEMPs and bone conducted vibration (BCV) for eliciting oVEMPs by Welgampola and Carey (2010), in order to achieve the best otolith afferent selectivity. However, even though abnormal ACS-cVEMPs or BCV-oVEMPs may indicate impaired SCR or VOR pathway, it is still insufficient to discriminate between labyrinthine and retrolabyrinthine deficits. VEMPs via galvanic vestibular stimulation (GVS) are recognized as fundamental in the assessment of retrolabyrinthine function because GVS directly elicits vestibular afferents and bypasses otolithic organs. In other words, GVS-VEMPs combined with the results of ACS-cVEMP or BCV-oVEMP testing may differentiate a labyrinthine lesion from a retrolabyrinthine one (Murofushi et al., 2002; Iwasaki et al., 2005; Cheng et al., 2009). Most patients with Meniere’s disease who manage their illness by medication have an acceptable control of associated symptoms. Few patients will undergo

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vestibular destructive surgery, such as vestibular neurectomy and labyrinthectomy due to intractable vertigo. Clinically, even without neurectomy, vertigo will subside gradually after deterioration of the vestibular nerve and compensation of the central nervous system. Previously, various studies have investigated the correlation between Meniere’s disease and VEMPs via ACS or BCV modes for evaluating the otolithic function of the disease (Huang et al., 2011; Katayama et al., 2010; Manzari et al., 2010; Young et al. 2003). This study further investigates the physiological function of vestibular nerve and brainstem by adding GVS-VEMP tests into the inner ear test battery. Considering the retrolabyrinthine involvement of Meniere’s disease pathologically (Ritter et al., 1981a, 1981b; Tsuji et al., 2000), physicians may offer patients additional information regarding the likelihood of future vertigo attacks due to electrophysiological GVS-VEMP testing.

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2. Material and methods 2.1 Patients This prospective study enrolled seventy patients with unilateral Meniere’s disease. The diagnostic criteria of an ear with Meniere’s disease were based on the AAO-HNS guidelines proposed in 1995. If Meniere’s disease was established in one ear while the opposite ear had inner ear symptoms, such as documented hearing loss, tinnitus, or aural fullness, the case was considered as bilateral Meniere’s disease and excluded from this study. Within 2 weeks of the disease diagnosis, all subjects underwent pure tone audiometry, ACS-cVEMP, GVS-cVEMP, BCV-oVEMP, GVS-oVEMP and caloric test in a random order. The prevalence of all tests and the characteristic parameters of VEMP tests, such as wave latencies (p13, n23, nI, pI), intervals and amplitudes, were further compared and analyzed. The research ethics review committee approved this study. All subjects provided written informed consent.

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2.2 Pure tone audiometry A patient was considered to have hearing loss if their mean hearing level was >25 dBHL. According to the AAO-HNS staging system of Meniere’s disease, the four stages of different disease severities were defined by mean hearing level as follows—stage I: <26 dB, stage II: 26 to 40 dB, stage III: 41 to 70 dB, and stage IV: >70 dB. Each patient underwent pure tone audiometry to assess the mean hearing level, which was measured as the average of hearing thresholds at 4 frequencies (0.5, 1, 2, and 3 kHz). 2.3 cervical VEMP test via ACS mode The acquisition of VEMP data was performed as described previously (Huang et al., 2004). In brief, VEMP data were obtained by randomizing the stimulation modes to minimize order effect. Electromyography (EMG) activity was monitored by an evoked potential system (Medelec Synergy, Surrey, UK) and measured to correct and minimize the effects of muscle fatigue. There was a surface electrode placed on the upper half of each sternocleidomastoid (SCM) muscle. Ground and reference electrodes were placed on the forehead and the lateral end of the upper sternum, respectively. Impedance of electrodes was kept under 8 kΩ. Unilateral rarefaction clicks (stimulation rate 5/sec, 105 dB nHL, 0.5 ms) were sequentially delivered to each ear through a headphone. All subjects were supine but instructed to

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elevate their heads during the recording. Activation of their SCM muscles allowed EMG activities to be monitored during VEMP acquisition. The minimal background muscle activity of 50 µV was maintained when each ear was tested. EMG signals were band-pass filtered between 20 and 2k Hz and amplified. The analysis window for each test was 50 ms. The results were averaged for each run (128 click stimuli per run), and two reproducible runs were averaged to provide the final ACS-cVEMP response. At our laboratory, the mean and standard deviation (SD) of p13 latency of ACS-cVEMPs were 11.7 and 1.5 ms, respectively. Those with a p13 latency of greater than 14.7 (mean+2SD) were defined as delayed responses. For specifying the side difference, asymmetry ratio of cVEMPs was measured as the difference of the p13-n23 amplitude of both ears divided by the sum of p13-n23 amplitude of both ears (larger amplitude – smaller amplitude / sum of amplitudes). Those with asymmetry ratio greater than 0.36 (mean+2SD) were defined as asymmetric responses. 2.4 cervical VEMP test via GVS mode In GVS-cVEMP tests, cathode and anode galvanic stimuli electrodes were placed on the mastoid process of the test side and forehead, respectively. EMG recording conditions were as described above for ACS-cVEMP. Because electrical artifacts existed in the original GVS-cVEMP waveform, the patients were placed in

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a supine position and administered galvanic stimuli (5 mA, 1 msec pulses) with and without the SCM muscle contraction (Watson et al., 1998; Murofushi et al., 2002; Iwasaki et al., 2005). The original response recorded without muscle contraction was subtracted from that with SCM muscle contraction, and then the corrected GVS-cVEMP response was obtained (Watson and Colebatch, 1998; Watson et al., 1998). Galvanic stimuli (128 pulses) were averaged for each run with two reproducible runs. At our laboratory, the mean and standard deviation (SD) of p13 latency of GVS-cVEMPs were 10.8 and 0.8 ms, respectively. Those with a p13 latency of greater than 12.4 (mean+2SD) were defined as delayed responses. Those with asymmetry ratio greater than 0.36 were defined as asymmetric responses. 2.5 ocular VEMP test via BCV mode For BCV-oVEMP tests, all EMG activity was recorded from surface electrodes as described in Cheng et al., 2009. Briefly, the active electrode was positioned inferior to each eye (about 1 cm below the medial aspect of each lower eyelid) and the reference electrode was placed 1-2 cm below the respective active one. A ground electrode was positioned over the sternum with the electrode impedance maintained at a level under 8 kΩ. BCV mode utilized a hand-held electromechanical vibrator (V201 vibrator; Ling Dynamic Systems, Royston, UK) fitted with a short M4 bolt terminated in a bakelite cap. The input signal was a condensation square wave (0.5

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ms), which was driven by a custom amplifier combination. The initial BCV impulse was approximately a half-cycle of a 600 Hz sine wave. The drive voltage was fixed and adjusted to produce a 12-Newton peak force. As measured by an artificial mastoid, the force was about 142 dB force level from the vibrator. The operator held the vibrator and supported most of the weight by hand so that the axis of the connected bakelite cap was perpendicular and delivered a repeatable tap at the Fpz site of subject’s skull. The subject was instructed to look upward at the same fixed target during recording. The EMG signals were bandpass-filtered between 1 and 1k Hz and amplified. The stimulation rate was 5 Hz and analytical period was 50 ms. The 50 responses of each run were averaged, and two reproducible runs were averaged to produce the final BCV-oVEMP response. At our laboratory, the mean and standard deviation (SD) of nI latency of BCV-oVEMPs were 10.2 and 1.4 ms, respectively. Those with a nI latency of greater than 13.0 (mean+2SD) were defined as delayed responses. Asymmetry ratio of oVEMPs was measured as the difference of the nI-pI amplitude of both ears divided by the sum of nI-pI amplitude of both ears (larger amplitude – smaller amplitude / sum of amplitudes). Those with asymmetry ratio greater than 0.36 were defined as asymmetric responses. 2.6 ocular VEMP test via GVS mode In GVS-oVEMP tests, anode and cathode electrodes for producing galvanic

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stimuli were positioned on the forehead and mastoid process of the test side, respectively. The settings of the EMG recording were the same as described earlier. The subjects were in a sitting position and instructed to gaze up or down. The galvanic stimuli with 5-mA intensity and 1-ms duration were given (Watson et al., 1998; Sung et al., 2014). To avoid electric artifacts and calculate the corrected response of GVS-oVEMP, the average responses of 128 pulses obtained upon gazing down was subtracted from that obtained upon gazing up (Rosengren et al., 2009). At our laboratory, the mean and standard deviation (SD) of nI latency of GVS-oVEMPs were 8.5 and 0.8 ms, respectively. Those with a nI latency of greater than 10.1 (mean+2SD) were defined as delayed responses. Those with asymmetry ratio greater than 0.36 were defined as asymmetric responses. 2.7 Caloric test Electronystagmographic examination (Nagashima, OK-5, Tokyo, Japan) consisted of recording the spontaneous nystagmus, followed by eye tracking, optokinetic nystagmus and bithermal caloric tests. When compared to the sum of slow phase velocities between each ear, a difference greater than 25% between the maximum slow phase velocity measurements was defined as canal paresis. If the caloric test failed to elicit a response, the subject underwent ice water (0 °C, 10 ml) caloric testing to further confirm the caloric areflexia (Young et al., 2003).

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2.8 Statistical methods The response rate, which was defined as the percentage of ears in which clear responses were identified, was evaluated by McNemar test. VEMP parameters such as latencies and amplitudes were compared by paired t test. The regression method with stepwise regression selection method was used to examine which independent variables, including disease stage (1~4), results of ACS-cVEMP, BCV-oVEMP, GVS-cVEMP, GVS-oVEMP and caloric test (0=normal; 1=abnormal), have a statistically significant influence on the disease duration. A difference was regarded as significant if p < 0.05.

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3. Results 3.1 Pure tone audiometry There were 70 patients with unilateral Meniere’s disease enrolled in this study, including 35 men and 35 women. Their ages ranged from 22 to 77 years, with a mean of 58 years. The right ear was affected in 33 patients (47%), and the left in 37 patients (53%). There were 6 patients (9%) in stage I Meniere’s disease, 18 patients in stage II (26%), 27 patients in stage III (38%) and 19 patients in stage IV (27%). The abnormal rate of hearing with 4-tone average >26 dBHL was 91%. The mean duration of the disease was 8.5 years, and that of 4 stages was 3.2, 6.5, 8.5, 12 years, respectively. 3.2 cervical VEMP testing In affected ears, 24 absent responses and 2 delayed responses were found in ACS-cVEMPs, and 6 absent responses, 5 delayed responses and 1 asymmetric response were noted in GVS-cVEMPs. The abnormal rate of ACS-cVEMPs was significantly larger than that of GVS-cVEMP (37% v.s. 17%) (p=0.001, McNemar’s test; table 1) (Figure 1). However, there was no significant difference between the abnormal rates of ACS-cVEMPs and GVS-cVEMP (7% v.s. 1%) in unaffected ears (p=0.13, McNemar’s test; table 1). Furthermore, the abnormal rates of cVEMPs in affected ears were significantly higher than those in unaffected ears, regardless of whether ACS (p=0.0001, McNemar’s test) or GVS (p=0.0001, McNemar’s test)

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stimulation was used. All GVS-abnormal responses were always abnormal for ACS-cVEMPs (Table 1). In affected ears, the mean p13 latency, n23 latency and p13-n23 amplitude of ACS-cVEMPs were 12.1±1.6 ms, 18.5±2.4 ms and 84.7 ± 63.2μV, respectively; these values significantly larger than GVS-cVEMPs values of 11.3±1.5 ms, 17.3±2.1 ms and 60.9 ± 43.2μV (p=0.01, p=0.01, p=0.03; paired t test; table 2). Likewise, the mean p13 latency, n23 latency and p13-n23 amplitude of ACS-cVEMPs were 12.0±1.5 ms, 18.5±2.4 ms and 100.2 ± 79.6μV in unaffected ears, respectively; these values also significantly larger than GVS-cVEMPs values of 11.0±1.0 ms, 17.2±1.8 ms and 60.1 ± 44.6μV in unaffected ears (p<0.0001, p=0.0002, p=0.001; paired t test; table 2). However, no significant differences existed in these parameters (p13 latency, n23 latency, p13-n23 interval and p13-n23 amplitude) between affected ears and unaffected ears, regardless of whether ACS or GVS stimulation was used (p>0.05, paired t test; table 2). 3.3 ocular VEMP testing In affected ears, 12 absent responses and 2 delayed responses were found in BCV-oVEMPs, and 5 absent responses and 1 delayed response were noted in GVS-oVEMPs. Similar with the results of cVEMP testing, the abnormal rate of BCV-oVEMPs was significantly larger than that of GVS-oVEMP (20% v.s. 9%) in

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affected ears (p=0.01, McNemar’s test; table 1) (Figure 1). Also, there was no significant difference between the abnormal rates of BCV-oVEMPs and GVS-oVEMP (6% v.s. 1%) in unaffected ears (p=0.25, McNemar’s test; table 1). The abnormal rates of oVEMPs in affected ears were significantly higher than those in unaffected ears, regardless of whether BCV (p=0.01, McNemar’s test) or GVS (p=0.001, McNemar’s test) stimulation was used. All GVS-abnormal responses were always abnormal for BCV-oVEMPs (Table 1). In affected ears, the mean nI latency, pI latency and nI-pI amplitude of BCV-oVEMPs were 10.4±1.8 ms, 13.7±2.5 ms and 6.3 ± 5.0μV, respectively; these values were significantly larger than the GVS-oVEMPs values of 8.6±0.8 ms, 11.6±1.1 ms and 3.6 ± 5.0μV (p<0.0001, p<0.0001, p=0.004; paired t test; table 2). Likewise, the mean nI latency, pI latency and nI-pI amplitude of BCV-oVEMPs were 10.5±1.9 ms, 13.7±2.8 ms and 6.5 ± 5.2μV in unaffected ears, respectively; these values were also significantly larger than the GVS-oVEMPs values of 8.4±1.1 ms, 11.9±1.6 ms and 3.8 ± 3.7μV in unaffected ears (p<0.0001, p<0.0001, p=0.001; paired t test; table 2). No significant differences existed in these parameters (nI latency, pI latency, nI-pI interval and nI-pI amplitude) between affected ears and unaffected ears, regardless of whether BCV or GVS stimulation was used (p>0.05, paired t test; table 2).

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3.4 cervical VEMPs v.s. ocular VEMPs In affected ears, the abnormal rate of ACS-cVEMPs (37%) was significantly higher than that of BCV-oVEMPs (20%) (p=0.03, McNemar’s test). However, no significant difference of abnormal rate existed between GVS-cVEMPs and GVS-oVEMPs (17% v.s. 9%) (p=0.18, McNemar’s test). In non-disease ears, the abnormal rates of ACS-cVEMPs, BCV-oVEMPs, GVS-cVEMPs and GVS-oVEMPs were 7%, 6%, 1%, 1%, respectively. No significant differences were found between cVEMPs (p=1.00, McNemar’s test) and oVEMPs (p=0.48, McNemar’s test). 3.5 Caloric test 52 affected ears (74%) revealed abnormal caloric responses, including areflexia in 40 ears and canal paresis in 12 ears. 18 unaffected ear (26%) showed abnormal caloric responses, including areflexia in 1 ear and canal paresis in 17 ears. There was a significant difference between the abnormal rates of both ears (p=0.01, McNemar’s test). 3.6 Multivariate linear regression analysis According to the regression model, the stage of Meniere’s disease, abnormal GVS-cVEMP response and abnormal GVS-oVEMP response had significant positive correlations with the disease duration (p =0.001, R2 = 0.281; table 3). A scoring scale using these significant parameters and their regression coefficients in combination was proposed as: predicted disease years = 1.348 + 2.121 * (stage) + 5.249 *

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(GVS-cVEMP) + 7.412 * (GVS-oVEMP) (Figure 2).

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4. Discussion For further evaluation of Meniere’s disease localization and prevalence, the inner ear test battery, including audiometry, cVEMP, oVEMP and caloric tests, was utilized for exploration of inner ear function, namely, the cochlea, saccule, utricle and semicircular canals (Yang et al., 2010; Young, 2013). Involvement of labyrinth or retrolabyrinth was further defined by combined GVS-VEMP results with ACS-VEMP or BCV-VEMP results. In general, abnormal ACS-VEMPs plus normal GVS-VEMPs may imply a labyrinthine lesion, whereas abnormal ACS-VEMPs plus abnormal GVS-VEMPs may indicate a retrolabyrinthine lesion at least (Murofushi et al., 2002; Iwasaki et al., 2005; Cheng et al., 2009). 4.1 Audiometry for assessing cochlear function The clinical course of Meniere's disease varies considerably between patients, from intervals of uncompromising attacks to long periods of remission interposed by episodic attacks. With long-standing disease of more than 10 years, the hearing loss of most patients stabilizes (Friberg et al., 1984), and profound sensorineural hearing loss occurs in 1 to 2% of patients (Stahle, 1976). In addition, auditory function may decline over time (Minor et al., 2004). In this study, 91% affected ears had abnormal hearing with 4-tone average >26 dBHL, and the abnormal rate was higher than those of cVEMPs, oVEMPs and caloric tests. The result indicated that the prevalence of

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the cochlear dysfunction was at least 91%, which is compatible with the histopathological findings that the endolymphatic hydrops was all observed in the cochlea (22/22=100%) (Okuno and Sando, 1987). The multivariate linear regression analysis revealed that the disease stages according to audiometric criteria had a significant positive correlation with the disease duration. It also showed that the hearing status is closely related to the natural course of Meniere’s disease. 4.2 VEMPs for assessing SCR and VOR pathway ACS-cVEMPs examine the saccule function and the SCR pathway (Colebatch et al., 1994), whereas BCV-oVEMPs mainly evaluate the utricle function and the VOR pathway (Manzari L et al., 2010). In affected ears, ACS mode had a significant higher abnormal rate (37%) than GVS mode (17%) to elicit cVEMP responses. Similar with cVEMP results, BCV mode had a significant higher abnormal rate (20%) than GVS mode (9%) to generate oVEMP responses. According to the results, the rate of abnormalities of the otolith organs is greater than for otolith afferents in Meniere's disease. Previous studies had revealed that the number of vestibular afferents in Meniere patients was diminished by 13-37% in cases of 5-7 years of disease duration and by 73% in cases of 15-21 years of disease duration. At later stages of the disease, thinner fibers could be observed with progression (Ritter et al., 1981a). Degenerative

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changes in both vestibular nerve and Scarpa's ganglion were also found by microscopy at later stages of the disease (Ritter et al., 1981b). In this study, multiple linear regression method showed the positive correlation of the abnormal GVS-VEMP results with the disease duration, implying that prolonged diseases have a higher proportion of nerve dysfunction than new-onset diseases. Besides, abnormal GVS-VEMP responses were only present in patients with disease duration of 10 years or more (Figure 3). This reinforces the fact that nerve dysfunction is a late phenomenon. The retrograde neural degeneration of cochlear and vestibular innervations had been previously demonstrated using animal models after inner ear injection of aminoglycoside intoxication. Dynamic morphologic changes of afferent degeneration were noted after destruction of the inner ear neuroepithelia (Dupont et al., 1993). In our study, higher abnormal rates of ACS-cVEMPs and BCV-oVEMPs compared to GVS-cVEMPs and GVS-oVEMPs may imply the phenomenon of retrograde neural degeneration in Meniere’s disease. This brings into question why anterograde degeneration was ruled out in our hypothesis. Anterograde degeneration means that nerves are damaged earlier than inner ear endorgans. In such circumstances, the impaired reflex pathway will not only cause abnormal GVS-VEMPs, but also abnormal ACS-VEMPs and BCV-VEMPs. Consequently,

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abnormal rates of VEMPs via different stimulation modes would be similar. In addition, correlation of disease duration with only abnormal GVS-VEMPs in our results also supports the hypothesis of retrograde neural degeneration. In the condition of anterograde neural degeneration, disease duration would not only correlate with abnormal GVS-VEMPs, but also with abnormal ACS- and BCV-VEMPs. ACS-cVEMPs had a significantly higher abnormal rate than BCV-oVEMPs in affected ears (37% v.s. 20%), further supporting that the endolymphatic hydrops was more frequently observed in saccule (19/22=86%) than in utricle histopathologically (11/22=50%) (Okuno and Sando, 1987). There is no significant difference of abnormal rate between both galvanic cVEMPs and oVEMPs (17% v.s. 9%), indicating that the retrolabyrinthine degeneration of SCR and VOR pathways are similar. In addition, abnormal ACS-cVEMP or BCV-oVEMP response was found in those with abnormal GVS-VEMP response. It may imply that impaired retrolabyrinthine neural pathway not only affect GVS-VEMPs, but also ACS/BCV-VEMPs. In terms of characteristic parameters of VEMPs, the latencies and the amplitudes of ACS-cVEMPs or BCV-oVEMPs were significantly larger than those of GVS-cVEMPs or GVS-oVEMPs, regardless of affected ears or unaffected ears. It

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is likely because the galvanic stimulation directly activates the vestibular afferents and bypasses the otolithic organs, leading to earlier VEMP waveform generation. Besides, the galvanic stimulation may fire less vestibular neurons than acoustic or mechanical stimulation, leading to smaller amplitudes of GVS-VEMPs. In this study, no significant differences were noted between affected ears and unaffected ears in terms of latencies and amplitudes of VEMPs. As a result, it can be explained by the fact that most of the abnormal responses were absent responses (ACS-cVEMP: 24/26, GVS-cVEMP: 6/12, BCV-oVEMP: 12/14, GVS-oVEMP: 5/6), which were excluded from statistical analysis. Consequently, a small number of delayed and small VEMP responses could not influence the mean and variance of the affected ear group. However, exclusion of absent responses may artificially inflate the mean value of amplitude, leading to no difference in amplitude between affected and unaffected ears. Another presumption is based on the fact that endolymphatic hydrops were noted in 44-75% asymptomatic ears of unilateral Meniere’s disease by Magnetic Resonance Imaging (Pyykkö et al., 2013; Yoshida et al., 2013). Temporal bone histopathology also showed that 30% unaffected ears of unilateral Meniere’s disease had hydrops (Yazawa and Kitahara, 1990), and the contralateral inner ears had significantly more damage compared to the inner ears of normal subjects (Kariya et al., 2007). Because some unaffected ears may be not

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really healthy, it is not surprising that no significant differences existed between parameters of both groups. 4.3 Caloric test for assessing semicircular canal function A significant loss of type II hair cells for all 3 cristae and Scarpa’s ganglion cells was found in Meniere’s disease when compared to healthy subjects (Tsuji et al., 2000). In this study, abnormal caloric responses were found in 74 % affected ears, exhibiting some extents of semicircular canal or retrolabyrinthine deficits. No correlation was established between the caloric response and the disease duration by multiple linear regression method. These findings disclosed that the impaired caloric function was not related to disease duration, compatible with histopathological finding that ampullary membrane distortion and vestibular atelectasis may occur at any stage of the disease (Gürkov et al., 2012; Merchant and Schuknecht, 1988). 4.4 Strength of the study To our best knowledge, there have only been temporal bone studies concerning the retrolabyrinthine degeneration of Meniere’s disease (Ritter et al., 1981a, 1981b; Tsuji et al., 2000). The current study is the first one using an electrophysiological inner ear test to assess nerve function for Meniere’s disease, and the results were compatible with the histopathological finding that the vestibular afferents and ganglion cells deteriorate with time (Ritter et al., 1981a, 1981b). In addition,

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according to the result of multiple linear regressions, patients with longer duration of Meniere’s disease were significantly correlated to more auditory dysfunction and vestibular nerve deterioration, but not to more impairment of saccule and utricle function. In our clinical experience, patients with abnormal GVS-VEMPs have a reduction in vertigo attacks. However, lack of clinical data to support this finding is a drawback of our study. Further study is mandatory to determine the clinical implication of GVS-VEMPs. Overall, it maybe of clinical value that GVS-VEMP testing may aid clinicians to better localize the lesion site and predict vertigo attacks in the future. 4.5 Limitation Caloric test has been used for assessing the function of horizontal VOR in dizzy patients for decades. However, caloric test is and uncomfortable, time-consuming and non-physiological test in the low-frequency range of up to 0.003 Hz. Nowadays, video head impulse test (vHIT) with a physiological working range of 4-7 Hz offers an alternative choice to test horizontal VOR in an easy and comfortable way (Weber et al., 2009). Although both caloric and vHIT tests had higher abnormal rates in Meniere’s disease than in normal subjects, dissociation between these two tests was reported (McGarvie et al., 2015; McCaslin et al., 2015). Caloric test has been shown to be more sensitive than vHIT test in Meniere’s disease (Blödow et al., 2014;

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McGarvie et al., 2015), and this is the reason why we only chose caloric test for evaluation of horizontal VOR pathway in the current study. However, both tests can be used in a complementary way for the detection of frequency-dependent horizontal VOR impairment in future studies.

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5. Conclusions According to the functional evaluation of unilateral Meniere’s disease via the inner ear test battery, the impairment of otolithic organs was found to be more than that of vestibular afferents. Furthermore, the deterioration of the saccule was more than that of the utricle, whereas the degeneration of the retrolabyrinthine part of SCR and VOR was similar. No obvious differences in latency, interval or amplitude between affected and unaffected ears may imply some contralateral ears of unilateral Meniere’s disease have asymptomatic endolymphatic hydrops. As the duration of the disease increased, Meniere’s disease stage and abnormal GVS-VEMP responses also increased with a significant correlation.

Conflicts of interest statement None of the authors have potential conflicts of interest to be disclosed.

Acknowledgements This work was supported by Far Eastern Memorial Hospital: Grant no. FEMH – 2013 – C – 002. Special thanks to Hillary Chiao Lee for text editing.

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Figure legends Figure 1. Cervical and ocular vestibular-evoked myogenic potentials (cVEMP and oVEMP) elicited by air-conducted sound (ACS), bone-conducted vibration (BCV) and galvanic vestibular stimulation (GVS) modes on the right (R) and left (L) ears in a 50 year-old female patient who had left Meniere’s disease for 16 years. Figure 2. Correlation between actual disease years and predicted disease years using the scoring scale. Figure 3. Abnormal rate of cervical and ocular vestibular-evoked myogenic potentials elicited by galvanic vestibular stimulation versus disease duration in 70 patients with unilateral Meniere’s disease.

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Table 1. Relationships between VEMPs triggered by different stimuli in patients with unilateral Meniere’s disease

ACS-cVEMP

Unaffected ears

GVS-cVEMP

GVS-cVEMP

Normal

Abnormal

Total

Normal

Abnormal

Total

Normal

44

0

44 (63%)

65

0

65 (93%)

Abnormal

14

12

26 (37%)

4

1

5 (7%)

Total

58 (83%)

12 (17%)

69 (99%)

1 (1%)

p value

BCV-oVEMP

Affected ears

<0.01

0.13

GVS-oVEMP

GVS-oVEMP

Normal

Abnormal

Total

Normal

Abnormal

Total

Normal

56

0

56 (80%)

66

0

66 (94%)

Abnormal

8

6

14 (20%)

3

1

4 (6%)

Total

64 (91%)

6 (9%)

69 (99%)

1 (1%)

p value

0.01

0.25

ACS: air-conducted sound; BCV: bone-conducted vibration; GVS: galvanic vestibular stimulation

cVEMP: cervical vestibular-evoked myogenic potential; oVEMP: ocular vestibular-evoked myogenic

potential.

p value: calculated by McNemar’s test

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Table 2. Comparison of characteristic parameters of cVEMPs and oVEMPs in patients with unilateral Meniere’s disease

Affected ears

Unaffected ears

cVEMP

cVEMP

p13 latency

n23 latency

Amplitude

p13 latency

n23 latency

Amplitude

(ms)

(ms)

(μV)

(ms)

(ms)

(μV)

ACS

12.1±1.6

18.5±2.4

84.7 ± 63.2

12.0±1.5

18.5±2.4

100.2 ± 79.6

GVS

11.3±1.5

17.3±2.1

60.9 ± 43.2

11.0±1.0

17.2±1.8

60.1 ± 44.6

p value

0.01

0.01

0.03

<0.0001

0.0002

0.001

oVEMP

oVEMP

nI latency

pI latency

Amplitude

nI latency

pI latency

Amplitude

(ms)

(ms)

(μV)

(ms)

(ms)

(μV)

BCV

10.4±1.8

13.7±2.5

6.3 ± 5.0

10.5±1.9

13.7±2.8

6.5 ± 5.2

GVS

8.6±0.8

11.6±1.1

3.6 ± 5.0

8.4±1.1

11.9±1.6

3.8 ± 3.7

p value

<0.0001

<0.0001

0.004

<0.0001

<0.0001

0.001

Data are expressed as mean ± SD

ACS: air-conducted sound; BCV: bone-conducted vibration; GVS: galvanic vestibular stimulation

cVEMP: cervical vestibular-evoked myogenic potential; oVEMP: ocular vestibular-evoked myogenic

potential.

p value: calculated by paired t test

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Table 3. Multiple linear regression analysis

Unstandardized coefficients Model

B

Standard error

(constant)

1.348

2.128

Stage

2.121

0.697

GVS-cVEMP

5.249

GVS-oVEMP

7.412

Standardized coefficients

t

Significance

0.633

0.529

0.311

3.045

0.003

1.713

0.316

3.064

0.003

2.308

0.331

3.211

0.002

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