Robert G&#252;rkov received fundings from the German Ministry of Research and Education BMBF, Grant No. 01 EO 0901.

All the procedures were approved by the local ethical committee (Institutional review board of the University of Munich/LMU Munich Protocol No. 093-09). All patients gave their informed consent to the performed procedures.
1. Clinical Examination
<ol>
	<li>Identify patients suffering from suspected MD in cooperation with the Department for Ear, Nose and Throat (ENT).
	<ol>
		<li>Perform clinical evaluation; vertigo, tinnitus/ringing of the ear, and hearing loss (possibly fluctuating) need to be evaluated. Check for associated nausea and vomiting. Check for the duration of the symptoms.</li>
		<li>Take the clinical and functional test results into consideration for diagnosis of MD: check for the results of audiometry, caloric video-oculography, vestibular evoked myogenic potentials (VEMP), and electrocochleography (ECoG) in the hospital papers or electronic medical record system.</li>
		<li>Check for typical findings in MD: audiometry may show impaired hearing levels in pure tone averaging (PTA), caloric irrigation can reveal horizontal semicircular paresis, SP/AP ratio may be pathologically high in ECoG, and VEMP interaural amplitude ratio may be significantly lower in MD patients.</li>
	</ol>
	</li>
</ol>
2. MRI Image Acquisition in Patients Suffering from MD and Healthy Controls
<ol>
	<li>Apply an intratympanic gadolinium-injection 24 h prior to the MRI scan in the patient group. Inject 0.4 mL of a gadolinium-based contrast agent intratympanically, (e.g., Magnograf diluted 8-fold in saline), 24 h prior to the scheduled MRI-scan.</li>
	<li>Prepare the patient for MRI examination: check for metal implants (exams are feasible with a pacemaker if adequate precautions are taken; dental implants are usually feasible), claustrophobia, etc. Use noise reduction devices such as noise reduction headphones for protection of patient hearing.</li>
	<li>Position the patient adequately into the scanner. Position the patient&#39;s head straight, and fit and close the MR-head coil. Position the patient&#39;s head/temporal bone at the isocenter of the MR scanner.</li>
	<li>Perform the MRI-scan according to the study protocol, including 3D-FLAIR- and 3D-real IR-sequences for detection of endolymphatic hydrops in the patient group and strongly T2-weighted 3D-CISS for morphologic analysis of cranial nerves in the patient group and healthy controls.</li>
	<li>Set the sequence parameters for the morphometric scan 3D-CISS as follows: repetition time (TR) 5.79 ms, echo time (TE) 2.58 ms, flip angle 34&#176;, field of view (FoV) 160 x 160 mm2, matrix size 320 x 320, number of averages 1, slice thickness 0.5 mm (Table 1). Perform the scans of the 3D-FLAIR using a TR of 9,000 ms, TE 128 ms, inversion time 2,500 ms, flip angle of 180&#176;, matrix size 384 x 384, slice thickness 2 mm. Set the parameters for the 3D-real IR as follows: TR 6,000 ms, TE 155 ms, inversion time 1,500 ms, flip angle 180&#176;, matrix size 320 x 320, slice thickness 0.5 mm.</li>
</ol>
3. MRI Quality Check and Identification of Endolymphatic Hydrops in MRI
<ol>
	<li>Check the MRI image quality with regard to artifacts like fold-over artifacts, pulsation artifacts, metal artifacts, and take special account of the target of evaluation, in this case the cranial nerves VII and VIII throughout their course.</li>
	<li>Evaluate the endolymphatic hydrops in the MRI-scans of patient group. Check for degree of cochlear and labyrinthine endolymphatic hydrops made visible by examining the acquired 3D-FLAIR- and 3D-real IR-sequences (Figure 1).</li>
</ol>
4. Image Based Measurements of Cranial Nerves
<ol>
	<li>General preparations
	<ol>
		<li>Install a DICOM viewer of choice for image evaluation and measurements on the evaluation workstation, e.g., OsiriX or Horos.</li>
		<li>Run the DICOM viewer by double clicking the icon of the application; the database window will be displayed.</li>
		<li>Import the patient image data by left-mouse clicking on &#34;File&#34; in the upper dropdown menu, then select &#34;Import&#34; &#8594; &#34;Import file&#34;. In the file selector, select the patient image data; the patient name and data will be displayed in the database window after successful import. 
		NOTE: Importing of compressed files (e.g.,.zip) or uncompressed directories of DICOM files is feasible with the aforementioned DICOM viewers.</li>
		<li>In the database window expand the patient image folder by left-clicking onto the triangle symbol on the left side of the patient name. Select the sequence of choice from this folder (here CISS sequence) and double left click on it to open the corresponding image data. The patient image data will be displayed.</li>
	</ol>
	</li>
	<li>Reconstructions of cranial nerves 
	Note: Because of the long and not always in-plane course of the nerves from the brain stem through the cerebellopontine angle (CPA) into the acoustic meatus and further to the fundus of the internal auditory canal (IAC), the reconstruction and evaluation of the nerve&#39;s diameters and CSAs at different levels is necessary.
	<ol>
		<li>Prepare for reconstructing transverse sections at the following locations throughout the course of the cranial nerves to avoid measurement errors derived from oblique slices throughout the course of the nerve, by selecting &#34;3D MPR&#34; in the &#34;3D Viewer&#34; dropdown menu at the top of the screen; the MPR-window will be displayed.</li>
		<li>Adjust zoom levels to accommodate for the structures to be reconstructed (here cranial nerves VII and VIII) by selecting the zoom tool (magnifying glass) from the &#34;Change mouse button function&#34; area in the toolbar in the upper left part of the MPR-window. Then move the mouse cursor to each of the 3 planes in the MPR-window and adjust zoom levels by left click and dragging the mouse (the mouse cursor will transform into a magnifying glass).</li>
		<li>Reconstruct the central VIII nerve and set the reconstruction plane orthogonal to the nerves course in the middle of the CPA. Check and adapt the orientation of the reconstructed plane in all 3 planes/windows (it should be reconstructed orthogonal to the direction of the crossing of the nerve in order to avoid partial volume effects at the following measurements).
		<ol>
			<li>Check for out of plane traversing of the nerve and correct the plane orientation respectively.
			<ol>
				<li>To correct plane orientation, move the mouse cursor to the center of the axis crosshair of each plane in the MPR-window (when located correctly the mouse cursor will transform into a hand symbol).</li>
				<li>Grab the axis crosshair in each of the 3 planes/windows individually with the grab tool indicated by the hand-icon, and move the axis approximately to the entry to the internal acoustic meatus in each of the 3 planes.</li>
				<li>Adjust the orientation of the 3 axes to the nerve&#39;s course using the rotate-function available by moving the mouse to the lateral aspects of each axis (the correct rotate-function is depicted by a change of the mouse cursor to a curvilinear icon). Then hold the left mouse button pressed and drag the mouse to adjust the plane orientation.</li>
				<li>Adjust the plane orientation in all 3 windows of the MPR-window. In order to reconstruct a plane transverse to the VIII nerves course at the level of the middle of the CPA, go to the left lower window in the MPR-window, and move the mouse to the middle of the axis crosshair so the mouse cursor will transform again into a hand symbol. Then left click and drag the plane (orange line) to the desired location (here, to the middle of the CPA).</li>
				<li>Adjust the zoom levels if necessary with the zoom tool (the mouse will change its icon to a magnifying glass) by left clicking and dragging.</li>
				<li>Left click into the upper right window of the MPR-window to select this plane. Select &#34;File&#34; &#8594; &#34;Export&#34; &#8594; &#34;Export to DICOM file(s)&#34;. In the &#34;DICOM Export&#34;-window select &#34;Sequence:&#34; &#8594; &#34;Current image only&#34; by left clicking into adjacent circle selector.</li>
				<li>Rename the series appropriately, here &#34;VIII CPA&#34;. Then left click the &#34;OK&#34;-button on the right lower aspect of the DICOM export window. 
				&#8203;NOTE: This will close the DICOM export window, will save the reconstructed image into the patient database, and return to the MPR window.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Reconstruct the orthogonal views of the branches of VIII nerve: cochlear nerve (CN), superior vestibular nerve (SVN), and inferior vestibular nerve (IVN) at the level of the meatus of the IAC, where representative visualization is usually well feasible. Check for out of plane traversing of the nerve and correct the plane orientation respectively.
		<ol>
			<li>Grab the axis crosshair in each of the 3 planes/windows with the grab tool indicated by a hand-icon, move the axis toward the CN, SVN, and IVN, respectively at the level of the meatus of the IAC, and adjust their orientation to the nerve&#39;s course using the rotate-function available at the lateral aspects of each axis depicted by the curved icon, as described under step 4.2.3.1.1 - 4.2.3.1.5.</li>
			<li>Export and rename the reconstructed planes as under step 4.2.3.1.6 - 4.2.3.1.7.</li>
		</ol>
		</li>
		<li>Reconstruct the orthogonal views of the facial nerve (cranial nerve VII) at the level of the CPA, meatus of the IAC, and fundus of the IAC as described under step 4.2.3. Check for out of plane traversing of the nerve and correct the plane orientation respectively at each level of reconstruction.</li>
	</ol>
	</li>
	<li>Measurements 
	Note: Perform the following measurements: measure CSA, long diameter (LD) and perpendicular short diameter (SD) of the facial nerve (cranial nerve VII) and vestibulocochlear nerve (cranial nerve VIII) in the reconstructed transverse images (Figure 4 and Figure 5). Pay attention to consistent windowing levels between scans to avoid partial volume effects influencing the quantificational measurements in a non-systematic way.
	<ol>
		<li>Select the previously reconstructed image of cranial nerve VIII at the level of CPA by left clicking the corresponding image file (previously named &#34;VIII CPA&#34;) in the database window of the DICOM viewer. Open it by double left-clicking on the filename. The reconstructed image will open in a single window.</li>
		<li>Zoom in into the image structures if necessary as instructed at step 4.2.2. Select &#34;Length&#34; by left clicking with the mouse on the triangular symbol next to the &#34;Change the mouse button function&#34; in the toolbar on top of the screen. Left click and hold the left mouse button pressed to draw a line of measurement for the longest diameter of the cranial nerve VIII; this measurement is LD.</li>
		<li>Perform a measurement perpendicular to LD for the SD measurement. 
		&#8203;NOTE: The measurements will be automatically stored if using OsiriX or Horos as DCIOM viewer.
		<ol>
			<li>Repeat these measurements of LD and SD also in the reconstructions of the VIII nerve at the level of the meatus of the IAC by measuring in the image file from the image database named &#34;VIII meatus&#34; and at the level of the fundus of the IAC, file name &#34;VIII fundus&#34;.</li>
		</ol>
		</li>
		<li>Evaluate CSA preferably using the closed polygon region-of-interest (ROI) to account for possible inhomogeneities in the contour of the cranial nerve&#39;s cross section. In the toolbar on the upper part of the screen, press the triangular symbol on the right of the &#34;Mouse button function&#34; area and select &#34;Closed Polygon&#34; (the previously selected line symbol will change to a polygon).</li>
		<li>Outline the contour of the cranial nerve VIII by left clicking multiple times on the border of the nerve. To close the polygon double left click at the desired point; the complete contour will be displayed. 
		Note: If necessary correct the position of the polygon points by left clicking and moving.</li>
		<li>Open the previously performed reconstruction of the facial nerve at the level of the CPA (image file name &#34;VII CPA&#34;) and perform measurements of LD, SD, and CSA for cranial nerve VII following steps 4.3.1 - 4.3.5.</li>
		<li>Open the reconstructions at the level of the meatus of the IAC and perform measurements of LD, SD, and CSA for the CN, SVN, IVN, and cranial nerve VII following steps 4.3.1 - 4.3.5.</li>
		<li>Open the reconstructions at the level of the fundus of the IAC and perform measurements of LD, SD, and CSA for the CN, SVN, IVN, and cranial nerve VII following steps 4.3.1 - 4.3.5.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gurkov, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualized+endolymphatic+hydrops+and+inner+ear+functions+in+patients+with+electrocochleographically+confirmed+Meniere's+disease.">In vivo visualized endolymphatic hydrops and inner ear functions in patients with electrocochleographically confirmed Meniere's disease.</a> Otol Neurotol. 33 (6), 1040-1045 (2012).</li><li>Gurkov, R., Flatz, W., Louza, J., Strupp, M., Krause, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+endolyphatic+hydrops+in+patients+with+Meniere's+disease:+correlation+with+audiovestibular+function.">In vivo visualization of endolyphatic hydrops in patients with Meniere's disease: correlation with audiovestibular function.</a> Eur Arch Otorhinolaryngol. 268 (12), 1743-1748 (2011).</li><li>Gurkov, R., Pyyko, I., Zou, J., Kentala, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+Meniere's+disease?+A+contemporary+re-evaluation+of+endolymphatic+hydrops.">What is Meniere's disease? A contemporary re-evaluation of endolymphatic hydrops.</a> J Neurol. 263, 71-81 (2016).</li><li>Plontke, S. K., Gurkov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meniere's+Disease.">Meniere's Disease.</a> Laryngorhinootologie. 94 (8), 530-554 (2015).</li><li>Klockars, T., Kentala, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+Meniere's+disease+in+the+Finnish+population.">Inheritance of Meniere's disease in the Finnish population.</a> Arch Otolaryngol Head Neck Surg. 133 (1), 73-77 (2007).</li><li>Greco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meniere's+disease+might+be+an+autoimmune+condition.">Meniere's disease might be an autoimmune condition.</a> Autoimmun Rev. 11 (10), 731-738 (2012).</li><li>Ozdogmus, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connections+between+the+facial,+vestibular+and+cochlear+nerve+bundles+within+the+internal+auditory+canal.">Connections between the facial, vestibular and cochlear nerve bundles within the internal auditory canal.</a> J Anat. 205 (1), 65-75 (2004).</li><li>Nakamichi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+normal+diameter+range+of+the+cochlear+and+facial+nerves+with+3D-CISS+at+3T.">Establishing normal diameter range of the cochlear and facial nerves with 3D-CISS at 3T.</a> Magn Reson Med Sci. 12 (4), 241-247 (2013).</li><li>Kang, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normative+diameters+and+effects+of+aging+on+the+cochlear+and+facial+nerves+in+normal-hearing+Korean+ears+using+3.0-tesla+magnetic+resonance+imaging.">Normative diameters and effects of aging on the cochlear and facial nerves in normal-hearing Korean ears using 3.0-tesla magnetic resonance imaging.</a> Laryngoscope. 122 (5), 1109-1114 (2012).</li><li>Jaryszak, E. M., Patel, N. A., Camp, M., Mancuso, A. A., Antonelli, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+nerve+diameter+in+normal+hearing+ears+using+high-resolution+magnetic+resonance+imaging.">Cochlear nerve diameter in normal hearing ears using high-resolution magnetic resonance imaging.</a> Laryngoscope. 119 (10), 2042-2045 (2009).</li><li>Henneberger, A., Ertl-Wagner, B., Reiser, M., Gurkov, R., Flatz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+evaluation+of+facial+and+vestibulocochlear+nerves+using+magnetic+resonance+imaging:+comparison+of+Meniere's+disease+ears+with+normal+hearing+ears.">Morphometric evaluation of facial and vestibulocochlear nerves using magnetic resonance imaging: comparison of Meniere's disease ears with normal hearing ears.</a> Eur Arch Otorhinolaryngol. 274 (8), 3029-3039 (2017).</li><li>Zwergal, A., Brandt, T., Magnusson, M., Kennard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIZZYNET:+the+European+network+for+vertigo+and+balance+research.">DIZZYNET: the European network for vertigo and balance research.</a> J Neurol. 263, 1 (2016).</li><li>Zwergal, A., Brandt, T., Magnusson, M., Kennard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIZZYNET--a+European+network+initiative+for+vertigo+and+balance+research:+visions+and+aims.">DIZZYNET--a European network initiative for vertigo and balance research: visions and aims.</a> J Neurol. 263, 2-9 (2016).</li><li>Grill, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DizzyReg:+the+prospective+patient+registry+of+the+German+Center+for+Vertigo+and+Balance+Disorders.">DizzyReg: the prospective patient registry of the German Center for Vertigo and Balance Disorders.</a> J Neurol. , (2017).</li><li>Semaan, M. T., Alagramam, K. N., Megerian, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+science+of+Meniere's+disease+and+endolymphatic+hydrops.">The basic science of Meniere's disease and endolymphatic hydrops.</a> Curr Opin Otolaryngol Head Neck Surg. 13 (5), 301-307 (2005).</li><li>Hayashi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+the+advanced+neuroimaging+protocol+based+on+plain+and+gadolinium-enhanced+constructive+interference+in+steady+state+images+for+gamma+knife+radiosurgery+and+planning+microsurgical+procedures+for+skull+base+tumors.">Usefulness of the advanced neuroimaging protocol based on plain and gadolinium-enhanced constructive interference in steady state images for gamma knife radiosurgery and planning microsurgical procedures for skull base tumors.</a> Acta Neurochir Suppl. 116, 167-178 (2013).</li></ol>

The authors have nothing to disclose.

We have demonstrated a feasible and accessible method of evaluation of morphometric changes of cranial nerves, as they may occur in several pathophysiological situations, here in MD compared to normal hearing controls.
Modifications and Troubleshooting:
Similar measurements to the ones reported here for the 7th and 8th cranial nerves can be performed using the employed 3D-CISS-sequence scans for all other cranial nerves at different levels, as long as they are still surrounded by cerebral fluid, otherwise contrast issues may arise with the mentioned MR-scanning sequence technique. For morphometric analysis of cranial nerves at levels where they are not surrounded by liquid, changes of the MR scanning protocol become mandatory, e.g., application of intravenous Gadolinium or employment of MRI fat-suppression techniques. Measurements in orthogonal reconstructions throughout the nerves course remain mandatory.
When combining MR-based evaluation of endolymphatic hydrops with morphometric analysis, the MR-scan can not only be used for describing anatomical changes of cranial nerves but can also aid in the diagnosis of MD. In the future, automated morphometric analysis techniques including machine based learning and artificial intelligence may speed up the evaluation and improve consistency of measurements and evaluations.
In a general approach for morphometric analysis of cranial nerves, MRI examinations can be performed &#34;natively&#34; without the use of intravenous or intratympanic administration of MRI contrast agents. In the protocol diluted intratympanic contrast agent has been applied in order to quantify the severity of endolymphatic hydrops occurring in MD, relevant for the diagnosis of the disease. The small amount and small concentration of Gadolinium-based contrast agent via intratympanic application in this study does not show effects on quantitative measurements of signal intensities of cerebrospinal fluid or the nerves when comparing the diseased side and contralateral side in MD patients, a finding corroborated by other studies. Signal intensities, image quality, and contrast do not differ when comparing strongly T2-weighted images of Gadolinium-injected patients with images of the non-injected controls16. Therefore, the effects of the contrast agent on the morphometric measurements do not play a role. Until today no evidence was found that Gadolinium might play a role on brain or cranial nerves with regard to alterations of volume. However, the long-term effects of Gadolinium-based contrast agents on cranial nerves remains to be studied. Intratympanic application of Gadolinium in healthy controls remains unethical and therefore wasn&#39;t performed in the normal hearing patients in this study.
Future Applications:
The depicted method allows for comparison of morphometric changes of cranial nerves in a very large variety of diseases and in several neural structures. Future transfer of the method for evaluating morphometric changes of cranial nerves e.g., in chronic pain, Alzheimer&#39;s Disease, or multiple sclerosis (MS) and comparing those findings to healthy controls is feasible.
Critical Steps Within the Protocol and Limitations of the Technique:
When evaluating morphometric parameters using the described techniques, set consistent windowing levels throughout the scans and/or let the measurements be performed by two or more readers. To avoid inter-rater variability, let each reader evaluate all scans. Using thin slice thickness and slice orientation perpendicular to the course of the cranial nerves is mandatory, throughout the whole course of the nerves. When comparing different studies performed on different MR-scanners, it must be considered that differences in MR scan parameters can result in differences in partial volume effects, as well as differences with regard to image contrasts and image quality. The levels at which morphometric analysis throughout the course of the cranial nerves was performed in different studies need to be considered when comparing studies.

Magnetic resonance imaging (MRI) plays a major role in visualizing and analyzing anatomy as well as physiological and pathological processes in the human body. Since clinical and electrophysiological diagnosis of Meni&#232;re&#39;s Disease (MD) can be challenging, using additional information derived from MRI is more than helpful1,2,3,4. An in vivo method was developed to analyze endolymphatic hydrops in MD and morphometric changes of cranial nerves using MRI. With this combined approach, diagnosis of definite MD was confirmed, and morphometric changes of cranial nerves were studied at different levels throughout the course of the nerves. Etiology of MD is still unclear5,6,7. It was proposed that neural cell loss could be involved in MD, but this has yet to be confirmed.
Suitable cranial nerves for morphometric analysis in MD are the 7th and 8th nerve with its branches, which were analyzed in this study. Only a few studies can be found analyzing morphometric aspects of these nerves using MRI8,9,10. The study by Henneberger et al. analyzed morphometric changes of the 7th and 8th cranial nerve in MD ears compared to normal hearing ears11.
The method presented here enables in vivo visualization and morphometric analysis of the 7th and 8th cranial nerves throughout their course from the brain to the temporal bone. Using this method, we have shown that there are significant differences between the patient group of MD patients and healthy ears. We propose the described method for use in several situations/diseases whenever potential morphometric changes of cranial nerves are of interest. Whether this method will be established in clinical diagnostic workups remains to be evaluated by future studies. Real alternatives to the described method for in vivo evaluation of morphometric changes of cranial nerves are not available, and while computed tomography (CT) has its strengths such as wide availability, speed, and depiction of bony changes, it also exhibits too low tissue contrasts to visualize subtle changes in cranial nerves within the neurocranium and temporal bone. Post mortem analysis of cranial nerve changes in MD patients remains to be studied. With special imaging and evaluation techniques as described here, it is possible to analyze morphometric changes of cranial nerves in MD patients and healthy controls using MRI. Routine MRI workup of the brain often does not include high resolution, strongly T2-weighted imaging techniques, which are mandatory for the evaluation of morphometric changes of cranial nerves 7 and 8.
The developed method may have further diagnostic impact on evaluating different levels of severity in MD, as well as play a role in the evaluation of vertigo, hearing deficits, and tinnitus. Specialized centers for diagnostic and therapeutic workup of vertigo play a major role in today&#39;s health care systems and our method could provide specialists with a possible tool for their diagnostic workup12,13,14. Vertigo is a complex symptom occurring in several diseases, requiring a thorough interdisciplinary cooperation between different specialties, as demonstrated in specialized centers for diagnostic and therapeutic workup of vertigo12,13,14.
To our knowledge, there is no method available in the literature for in vivo morphometric analysis of cranial nerves in MD and healthy controls.

<table><tbody><tr><td>&lt;strong&gt;MR-scanner&lt;/strong&gt;, e.g. Siemens Magnetom Verio, or appropriate &lt;strong&gt;MR-scans in DICOM format&lt;/strong&gt;, e.g. 3D-CISS</td><td>Siemens Healthcare GmbH, Erlangen, Germany, or MR scans by any other vendor</td><td>1</td><td>Instead of the MR scanner, appropriately acquired MR-scans can be used for morphometric analysis</td></tr><tr><td>Osirix or any other &lt;strong&gt;DICOM-Viewer&lt;/strong&gt; with appropriate evaluation tools</td><td>Pixmeo SARL, Geneva, Switzerland</td><td>2</td><td>Software for viewing and evaluating DICOM images</td></tr><tr><td>MedCalc or any other &lt;strong&gt;statistical analysis software&lt;/strong&gt;, e.g. SPSS&amp;nbsp;</td><td>MedCalc Software bvba, Ostend, Belgium</td><td>3</td><td>Software for statistical analysis</td></tr><tr><td>&lt;strong&gt;Computer&lt;/strong&gt; running Windows or MacOSX/macOS</td><td>e.g. Lenovo, Apple or anything selfmade</td><td>4</td><td>Hardware on which the above software can be employed</td></tr></tbody></table>

in vivo morphometric analysis of human cranial nerves using magnetic resonance imaging in meni&#232;re's disease ears and normal hearing ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

Statistical analysis was performed using statistical analysis software, and two-sided independent samples t-test was applied. Image evaluation was performed by two readers. A significant difference between the mean values of the patient group (n = 21) and healthy control group (n = 39) can be found for measurements of the CSA of the facial nerve, CN, SVN, and IVN (Table 2). CSA measurements in the patient group showed significantly larger CSA values (Figure 2 and Figure 3). Evaluation of measurements of the LD and SD showed varying results, depending on the site of measurement, and differences in LD and SD between the two groups were found. For example, at the level of the meatus, SD of the SVN was significantly larger in the patient group compared to the healthy control group, whereas LD was found to be not significantly different (Table 3 and Table 4). Mediator-based theories of MD support these findings7,15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57091/57091fig1.jpg" /> 
Figure 1: Endolymphatic hydrops in MRI scans. High grade endolymphatic hydrops of the cochlea (straight arrows) and the vestibule (curved arrows) in 3D-FLAIR (A) and 3D-Real-IR (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57091/57091fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57091/57091fig2.jpg" /> 
Figure 2: Morphometric evaluation of cochlear nerve. Significant differences of mean values and interquartile ranges of the cross-sectional area (CSA) of the cochlear nerve were found in the patient group compared to healthy controls. The upper and lower green horizontal lines depict minimal and maximal values, connected by the whisker. The purple star shows the arithmetic mean. The green middle line represents the median. The blue error bars depict 1 SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57091/57091fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57091/57091fig3.jpg" /> 
Figure 3: Morphometric evaluation of cranial nerve VII. Significant differences of the cross-sectional area (CSA) of the facial nerve at the level of the cerebellopontine angle (CPA) were found in the patient group compared to healthy controls. The upper and lower green horizontal lines depict minimal and maximal values connected by the whisker. The purple star shows the arithmetic mean. The green middle line represents the median. The blue error bars depict 1 SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57091/57091fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57091/57091fig4.jpg" /> 
Figure 4: Measurement of the cross-sectional area (CSA) of the cochlear nerve. Measurement performed at the fundus of the internal meatus on a reconstructed slice perpendicular to the nerve&#39;s course. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57091/57091fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57091/57091fig5.jpg" /> 
Figure 5: Measurement of the long diameter (LD) and perpendicular short diameter (SD) of the cochlear nerve. Measurement performed at the fundus of the internal meatus on a reconstructed slice perpendicular to the nerve&#39;s course. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57091/57091fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Mr-sequence parameters</td>
			<td>3D-CISS</td>
		</tr>
		<tr>
			<td>TR</td>
			<td>5.79 ms</td>
		</tr>
		<tr>
			<td>TE</td>
			<td>2.58 ms</td>
		</tr>
		<tr>
			<td>Flip angle</td>
			<td>34&#176;</td>
		</tr>
		<tr>
			<td>Field of view</td>
			<td>160 x 160 mm2</td>
		</tr>
		<tr>
			<td>Matrix size</td>
			<td>320 x 320</td>
		</tr>
		<tr>
			<td>Averages</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Slice thickness</td>
			<td>0.5 mm</td>
		</tr>
	</tbody>
</table>
Table 1: MRI sequence parameters. Set MRI sequence parameters as described using Constructive Interference in Steady State (CISS)-sequence technique for achieving strongly T2-weighted image contrast for optimal depiction of nerves surrounded by cerebrospinal fluid.
<img alt="Table 2" src="/files/ftp_upload/57091/57091table2.jpg" /> 
Table 2: Morphometric analysis results of cross-sectional area measurements (CSA). Comparison of patients vs. healthy controls measuring CSA of the 7th and 8th cranial nerve at different levels through their course. Analysis of unilaterally affected patients, bilaterally affected patients, and healthy controls including mean value, standard deviation, and p-values (independent samples t-test, patient group n = 21, healthy controls n = 39); significant results with p &#60;0.000595 after Bonferroni correction are marked bold.
<img alt="Table 3" src="/files/ftp_upload/57091/57091table3.jpg" /> 
Table 3: Morphometric analysis results of long diameter (LD). Comparison of patients vs. healthy controls measuring LD of the 7th and 8th cranial nerve at different levels through their course. Analysis of unilaterally affected patients, bilaterally affected patients, and healthy controls including mean value, standard deviation, and p-values (independent samples t-test, patient group n = 21, healthy controls n = 39).
<img alt="Table 4" src="/files/ftp_upload/57091/57091table4.jpg" /> 
Table 4: Morphometric analysis results of short diameter (SD). Comparison of patients vs. healthy controls measuring SD of the 7th and 8th cranial nerve at different levels through their course. Analysis of unilaterally affected patients, bilaterally affected patients, and healthy controls including mean value, standard deviation, and p-values (independent samples t-test, patient group n = 21, healthy controls n = 39).

Watch this Scientific Journal Video about In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in MeniÃ¨re's Disease Ears and Normal Hearing Ears at JoVE.com

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

in-vivo-morphometric-analysis-human-cranial-nerves-using-magnetic

Nanyang Technological University

Research

JoVE Journal

Medicine

10.5K Views.  LMU Munich. Om te evalueren van de morfologische veranderingen van de hersenzenuwen zoals verlies van neurale is structuren of zwelling van de craniale zenuwen in de ziekte van Menière van (MD) of in gezonde personen in vivo, een protocol van evaluatie ontwikkeld met behulp van magnetische resonantie beeldvorming (MRI) . Extra MRI gebaseerde bevestiging van MD werd uitgevoerd.

Video: In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Menière's Disease Ears and Normal Hearing Ears

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Neuroscience

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

High-Speed Human Temporal Bone Sectioning for the Assessment of COVID-19-Associated Middle Ear Pathology

Histopathologic analysis of human temporal bone sections is a fundamental technique for studying inner and middle ear pathology. Temporal bone sections are prepared by postmortem temporal bone harvest, fixation, decalcification, embedding, and staining. Due to the density of the temporal bone, decalcification is a time-consuming and resource-intensive process; complete tissue preparation may take 9-10 months on average. This slows otopathology research and hinders time-sensitive studies, such as those relevant to the COVID-19 pandemic. This paper describes a technique for the rapid preparation and decalcification of temporal bone sections to speed tissue processing.
Temporal bones were harvested postmortem using standard techniques and fixed in 10% formalin. A precision microsaw with twin diamond blades was used to cut each section into three thick sections. Thick temporal bone sections were then decalcified in decalcifying solution for 7-10 days before being embedded in paraffin, sectioned into thin (10 &#181;m) sections using a cryotome, and mounted on uncharged slides. Tissue samples were then deparaffinized and rehydrated for antibody staining (ACE2, TMPRSS2, Furin) and imaged. This technique reduced the time from harvest to tissue analysis from 9-10 months to 10-14 days. High-speed temporal bone sectioning may increase the speed of otopathology research and reduce the resources necessary for tissue preparation, while also facilitating time-sensitive studies such as those related to COVID-19.

This article describes a technique for rapid human temporal bone sectioning that utilizes a microsaw with twin diamond blades to generate thin slices for rapid decalcification and analysis of temporal bone immunohistochemistry.

Histopathologic analysis of human temporal bone sections is a fundamental technique for studying inner and middle ear pathology. Temporal bone sections ...

Immunology and Infection

Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly understand the mechanism of NIHL, and subsequently optimize the corresponding treatment strategies. This study aims to create an improved protocol for developing a mouse model of NIHL. Male C57BL/6J mice were used in this study. Unanesthetized mice were exposed to loud noises (1 and 6 kHz, presented simultaneously at 115-125 dB SPL-A) continuously for 6 h per day for 5 consecutive days. Auditory function was assessed 1 day and 1 week after noise exposure, using auditory brainstem response (ABR). After the ABR measurement, the mice were sacrificed, and their organs of Corti were collected for immunofluorescence staining. From the auditory brainstem response (ABR) measurements, significant hearing loss was observed 1 day after noise exposure. After 1 week, the hearing thresholds of the experimental mice decreased to ~80 dB SPL, which was still a significantly higher level than the control mice (~40 dB SPL). From the results of immunofluorescence imaging, outer hair cells (OHCs) were shown to be damaged. In summary, we created a model of NIHL using male C57BL/6J mice. A new and simple device for generating and delivering pure-tone noise was developed and then employed. Quantitative measurements of hearing thresholds and morphological confirmation of OHC damage both demonstrated that the applied noise successfully induced an expected hearing loss.

Here, we present a protocol for a mouse model of noise-induced hearing loss (NIHL). To induce NIHL, we developed a new and simple device using corrugated plastic, a rat trap cage, and a speaker. Auditory brainstem response and immunofluorescence imaging were employed to assess the hearing function and outer hair cell damage, respectively.

An animal model of noise-induced hearing loss (NIHL) is useful for pathologists, therapists, pharmacologists, and hearing researchers to thoroughly ...

Measuring the Influence of Magnetic Vestibular Stimulation on Nystagmus, Self-Motion Perception, and Cognitive Performance in a 7T MRT

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called magnetic vestibular stimulation (MVS). In this article, we present an experimental setup in a 7T MRT scanner (MRI scanner) that allows the investigation of the influence of strong magnetic fields on nystagmus as well as perceptual and cognitive responses. The strength of MVS is manipulated by altering the head positions of the participants. The orientation of the participants&#39; semicircular canals with respect to the static magnetic field is assessed by combining a 3D magnetometer and 3D constructive interference in steady-state (3D-CISS) images. This approach allows to account for intra- and inter-individual differences in participants&#8217; responses to MVS. In the future, MVS can be useful for clinical research, for example, in the investigation of compensatory processes in vestibular disorders. Furthermore, it could foster insights into the interplay between vestibular information and cognitive processes in terms of spatial cognition and the emergence of self-motion percepts under conflicting sensory information. In fMRI studies, MVS can elicit a possible confounding effect, especially in tasks influenced by vestibular information or in studies comparing vestibular patients with healthy controls.

In this article, we describe the experimental setup, material, and procedures to assess reflexive eye movements, self-motion perception, and cognitive tasks under magnetic vestibular stimulation, as well as the anatomical orientation of the vestibular organs, in a 7 Tesla Magnetic resonance tomography (7T-MRT) scanner.

Strong magnetic fields induce dizziness, vertigo, and nystagmus due to Lorentz forces acting on the cupula in the semi-circular canals, an effect called ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Menière's Disease Ears and Normal Hearing Ears

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