Morphometric Evaluation of Facial and Vestibulocochlear Nerves Using Magnetic Resonance Imaging in Patients with Menière’s Disease

Several studies proposed a loss of neural structures (such as hair cells or neurons within the spiral ganglion) in Menière’s disease (MD). It has been shown that VII th and VIII th cranial nerves are enlarged within MD patients compared to normal controls. We now aimed to investigate potential differences in these two nerves in patients with MD. Etiology of endolymphatic hydrops, central pathological hallmark of MD, includes genetic predisposition, autoimmune processes, viral infections, cellular apoptosis and oxidative stress. We evaluated morphometric properties (long and short diameter, cross-sectional area) of the VII th and VIII th cranial nerves passing from the cerebellopontine angle to the inner ear modiolus, acquired on a clinical 3T magnetic resonance imaging scanner. 71 patients with MD were included, 53 of whom clinically showed a unilateral affection. Our data showed no differences in nerve morphometry between the clinically non-affected and the clinically affected side in patients with clinically unilateral MD. There was also no correlation to duration of symptoms, in contrast to previously demonstrated correlations between clinical features and the extent of endolymphatic hydrops. A disease process starting before the onset of clinical symptoms could be a potential explanation.

The course of cranial nerves VII and VIII from the pons through the cerebellopontine angle and the internal auditory canal (IAC) make these nerves amenable to morphometric evaluation in anatomical and MRI studies. The VIII th cranial nerve divides within the IAC into its three branches: cochlear nerve (CN), superior vestibular nerve (SVN) and inferior vestibular nerve (IVN). In the anterosuperior portion of the IAC the facial nerve is found, and inferior to it the cochlear nerve (18)(19)(20)(21)(22). To our knowledge, no morphometric analyses of these nerves have been performed using different MRI sequences with different spatial resolution in patients with clinically unilateral MD in correlation to symptom duration.
We previously demonstrated a swelling of VII th and VIII th cranial nerves on MR imaging in patients with MD (17). The aim of this study was to investigate morphometric differences of the VII th and VIII th cranial nerve within MD patients using different MR imaging techniques.

Study group
Ethical review board approval was provided by the institutional review board of University of Munich / LMU Munich, Protocol-Nr. 093 − 09. All examinations were performed in concordance with the Helsinki Declaration revised 2013.
Patients with certain MD according to the AAO-HNS classi cation (1995) (23) and with MR morphologic con rmation of endolymphatic hydrops (ELH) as an equivalent to the required histopathological ndings were included in the study (12,(24)(25)(26). All of these patients underwent MRI on a clinical 3 Tesla scanner investigation with a sequence protocol comprising several different MR sequences described below. All patients gave their informed consent for all the methods including them in the study, also for the locally enhanced inner ear MRI (LEIM) and the electrophysiological examinations. Morphometric analysis of the VII th and VIII th cranial nerves was performed retrospectively. 24 hours prior to the MRI scan a gadoliniumbased contrast agent diluted 8-fold in saline solution, was intratympanically injected (27,28). After administration, the patient remained in a supine position for another 30 minutes with the head turned approximately 45 degrees toward the contralateral side.
Patient data consisting of CISS-0.6-sequence measurements was compared in our previous study to clinical healthy controls (17). In the current study we compared the affected side with the non-affected side of unilaterally affected MD patients as well as the bilaterally affected MD patients vs. unilateral affected MD patients using the mentioned different MRI sequences.

MR Imaging
All MR imaging examinations were performed on a 3T MR unit (Magnetom Verio, Siemens Healthcare, Erlangen, Germany) using a commercially available 4-channel exible surface coil combined with an 8channel head coil.
The following MR-sequences were acquired of the temporal bone: CISS-0.6: A strongly T2-weighted constructive interference in steady state sequence (CISS) with the following parameters was used: TR 7. On each transverse section the long diameter (LD), short diameter (SD) perpendicular to LD and crosssectional area (CSA) were measured. Several dot-markers were positioned on the outline of the examined nerves. These markers were linked and the CSA was calculated. All measurements were performed by the same two readers based on consensus readings. Both readers were blinded for the diagnosis of the patients. All these measurements were made for the CISS-0.4, the CISS-0.6, the IR-0.3 and the IR-0.5 sequences to investigate different nerve sizes depending on the used MRI sequence.
All patients received locally enhanced inner ear MRI (LEIM Clinical patient`s data including age and sex, the investigated LEIM-side, symptom duration, four-tone average calculated from the hearing at 0.5, 1, 2 and 3 kHz, cervical vestibular evoked myogenic potentials (cVEMP´s), electrocochleography data and caloric canal paresis including the measurements of the slow phase speed (SPS, °/s) is shown in Table 1 for the unilaterally affected and in Table 2 for bilaterally affected MD patients.
No signi cant differences were observed when comparing the affected side to the non-affected side of the VII th and VIII th cranial nerves in the cohort of clinically unilaterally affected MD patients (Table 3) when adjusting for multiple testing. Without adjustment for multiple testing, in order for catering for an explorative approach, signi cant differences were found following no certain pattern, e.g. p < 0.05 could be found for SD at the level of CPA in the VIII th nerve and LD at level of CPA and fundus of the VII th nerve. These results were found to be independent of the used MRI sequence (CISS-0.4, CISS-0.6, IR-0.5 or IR-0.3) when adjusting for multiple testing (Tables 4 and 5) at the level of the CPA. Without Bonferroni correction signi cant differences were found in the VIII th nerve at this level only for SD in CISS-0.4 and CSA in IR-0.3 at the level of CPA (Table 3), obviously also not following a speci c pattern.
Also, no signi cant differences between the morphometric measurements of the VII th and VIII th cranial nerves of the different sides were observed after Bonferroni correction in the cohort of clinically bilaterally affected patients.
When comparing the affected side of the unilaterally affected cohort with the LEIM-assessed side of the bilaterally affected cohort, no signi cant difference was shown along our measuring points of VII th and VIII th cranial nerves (Table 6, cross sectional areas within CISS-0.4, CISS-0.6, IR-0.3 and IR-0.5). Similar results were found investigating the clinically non-affected side of the rst cohort and the contralateral ear of the bilaterally affected cohort. These results were again independent of the used MRI sequence.
An overview of the signi cant p-values without adjustment for multiple testing is summarized in Table 7, no speci c pattern is recognizable.
We furthermore evaluated the morphometric properties of the VII th and VIII th cranial nerves of the rst cohort in dependence of symptom duration. We initially divided the cohort of unilaterally affected patients in a subgroup with a symptom duration of a maximum of 12 months (n = 9) and compared these to the rest of the group (n = 26); we observed no signi cant differences between the groups neither when comparing the affected sides nor when comparing the non-affected sides. Subsequently, we compared patients with a symptom duration of at least 120 months (n = 14) to the patients with a symptom duration of a maximum of 12 months (n = 9). Again, there were no signi cant differences for the morphometric parameters (Table 8).
Scatterplots (diagrams 1-4), Pearson Correlation Coe cients (tables 9 and 10) and Bland-Altman plots (diagrams 5-8) were used for visualization of the degree of correlation as well as differences of measurements between each two of the 4 employed sequences.

Discussion
In our patient cohorts we observed no signi cant differences between the clinically non-affected side and the clinically affected side of unilaterally affected patients with MD independently of the used MRI sequence when using the Bonferroni correction for multiple testing.
The pathogenesis of MD is still incompletely understood and the disease can be di cult to diagnose in early stages (29). Autoimmune processes and viral infections, such as latent herpes simplex virus type-1 that may cause vestibular neuritis (30), may play a role in the induction of MD. The involved immunological mechanisms are still not clear, but approximately one-third of the MD cases may have an autoimmune origin (31)(32)(33)(34)(35). Further pathophysiological aspects have been discussed, such as genetic predisposition (36-38), excitotoxicity, chronic otitis media (33), cellular apoptosis and oxidative stress. Reactive oxygen species, speci cally nitric oxide generated by nitric oxide synthase, regulate the cochlear blood ow and lead to the release of mitochondrial cytochrome c, which is an important mediator of the intrinsic pathway of apoptosis (39,40). The presence of hydrops may thus cause neuronal damage in the inner ear via a process of excitotoxicity (2). This neuronal damage could possibly affect the VIII th and potentially also the VII th cranial nerve.
Our data, however, demonstrated no differences in nerve diameters when comparing the clinically nonaffected to the clinically affected side of unilaterally affected MD patients, possibly pointing toward a more systemic process that leads to subclinical reaction of the contralateral ear in clinically unilaterally affected MD patients. Kariya et al found equal results when comparing the mean number of spiral ganglion cells, the mean loss of inner and outer hair cells in patients and the damage of the stria vascularis with unilateral MD (6). This may support autoimmune processes or genetic predispositions, whereas local processes such as excitotoxicity and reactive oxygen species seem less likely. Moreover, our data did not show a correlation to clinical symptom duration, which again may point toward a very longstanding underlying process prior to the onset of clinical symptoms. On the other hand, it could also indicate that there is no change in the 7th and 8th cranial nerves in MD. However, this contradicts our previous study, which showed thicker nerves in patients with MD compared to a healthy control group (17).
Moreover our data showed different means for the same patients at the same measuring levels in dependence of the employed MRI sequence. These differences occur due to different variable sequence parameters such as slice thickness, different partial volume effects and the relatively small sample size.
Where measurements varied largely between VII th and VIII th cranial nerve, differences between the 4 employed sequences were small. However, due to the limited sample size, a signi cant positive correlation (Tables 7 and 8) could only be shown for the IR-0.3 and IR-0.5 for VII th and VIII th cranial nerve. This re ects the di culty of comparing absolute morphometric parameters in different MRI-studies performed with different sequences on different scanners.
In an explorative approach, without catering for multiple testing, scattered single parameters were found to be signi cant, these ndings showed no speci c pattern. Therefore Bonferroni correction was applied.

Study Limitations
Our study has several limitations that need to be taken into account when interpreting the data. First, our sample size was limited, especially when performing the subgroup analyses. Larger studies are warranted to con rm our results. Second, we did not compare our measurements to an age-and sexmatched healthy control cohort to analyze differences to a clinically unaffected control population. As shown in our previous study (17) only few surveys (14)(15)(16)22) with measurements of the VII th and VIII th cranial nerves had been published, mostly in normal hearing patients. The comparison of these published data also showed differences of nerve diameters according to the measuring point and the used MRI sequence.

Conclusion
Our data showed no signi cant differences in diameters and cross-sectional areas of the VII th and VIII th cranial nerves between the affected and the clinically non-affected side of unilaterally affected patients with MD after Bonferroni correction for multiple testing. There was also no correlation to duration of clinical symptoms. This may potentially point toward a systemic process starting long before the onset of rst clinical symptoms. On the other hand, it could also indicate that there is no change in the 7th and 8th cranial nerves in MD. However, this contradicts our previous study, which showed thicker nerves in patients with MD compared to a healthy control group (17).
Normative nerve diameters should only be used if comparable MRI sequence parameters are used as our data showed different means for the same patients at the same measuring points depending on the MRI sequences used.  Degree of correlation (x-axis) and differences of measurements (y-axis) between sequence CISS-0.6 and IR-0.5 for cranial nerves VII and VIII (upper right diagram). 7. Degree of correlation (x-axis) and differences of measurements (y-axis) between sequence CISS-0.6 and IR-0.3 for cranial nerves VII and VIII (lower left diagram). 8. Degree of correlation (x-axis) and differences of measurements (y-axis) between sequence IR-0.3 and IR-0.5 for cranial nerves VII and VIII (lower right diagram).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.