This research was supported in part by the Intramural Research Program of the NIH, NIDCD (NIH Intramural Research Funds Z01-DC000060 and ZIC DC000081 to the Advanced Imaging Core). We are grateful to Drs. Raoul D. Nelson and R. Lance Miller (University of Utah, Salt Lake City, UT) and Dr. Susan Wall (Emory University School of Medicine, Atlanta, GA) for sharing the Tg(ATP6V1B1-Cre) mice and to the NIDCD animal facility staff for caring for our animals. We express our gratitude to Dr. Philine Wangeman for the beautiful illustration of the inner ear. We thank Drs. Thomas B. Friedman and Robert J. Morell for critically reviewing this manuscript.

All animal experiments and procedures were performed according to protocols approved by the Animal Care and Use Committees of the National Institute of Neurological Diseases and Stroke/National Institute on Deafness and Other Communication Disorders (#1264) and the Institutional Animal Care and Use Committee at Tokyo Medical and Dental University (A2020-058A).
NOTE: C57BL/6J mice (stock number no. 000664), Pds-/-&#160;also known as Slc26a4-/-&#160;mice (129S-Slc26a4tm1Egr/AjgJ, no. 018424) 11, B6;CBA-Tg(ATP6V1B1-Cre)1Rnel/Mm mice12 and Ai9(LSL-RCL-tdT) (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze, no. 007909) mice13 are available from the Jackson Laboratory and upon request. All genotyping procedures have previously been described. B6;CBA-Tg(ATP6V1B1-Cre)1Rnel/Mm&#160;mice previously backcrossed to strain C57BL/6J for at least five generations, were bred for experiments. Animals of both sexes can be studied with this approach without an obvious difference. The first day after overnight mating is counted as embryonic day 0.5 (E0.5).
1. Harvest and fixation of the intact inner ear in the skull
<ol>
	<li>Euthanize experimental animal or time-pregnant dam using an institutionally approved procedure, for example, using CO2 inhalation. Work under a fume hood if possible, to avoid or minimize the spread of mouse allergens. After verifying the lack of response to painful stimuli such as paw or tail pinches, decapitate the animal. For a time-pregnant dam, cervical dislocation can be used as an alternative to decapitation if its inner ears are not used for further experiments.</li>
	<li>Harvest the embryos by laparotomy14 using artery scissors with a ball tip to protect the embryos during the process. Manipulate the embryos using ring forceps. Place the uterine horns in 4 &#176;C PBS on ice and isolate each embryo as previously described15 before decapitating them.</li>
	<li>Bisect the head from back to front: first incise the top of the head by first incising the skin, then the top of the skull, incising about one third of it with each cut so there is no bending of the skull. Then cut the bottom of the skull at once. 
	NOTE: For embryos, the use of a sharp blade, rather than scissors, is advised. It is critical that, at all ages, the skull is not deformed while bisecting it.</li>
	<li>Without touching or bending the skull, carefully remove the hemi-brain to reveal the bony labyrinth (Figure 1B, Supplementary Figure 1A-B).</li>
	<li>For mice postnatal day 5 or older, transect the external auditory canal and pull the skin forward towards the nose. Trim the skull around the inner ear (Supplementary Figure 1C).</li>
	<li>Transfer the two hemi-skulls of each animal with the inner ear, as intact as possible, to a glass vial or tube containing 10 mL of 4 &#176;C 4% paraformaldehyde (PFA) diluted in 1x PBS and keep on ice under a fume hood. 
	NOTE: Do not try to remove the inner ear from the skull at this step as this may damage the endolymphatic duct and sac while doing so.</li>
	<li>Incubate for 1 h at 4 &#176;C on a rocking shaker. If working with tissue from an animal with a fluorescent reporter, maintain the tube containing the specimen in aluminum foil or in a box to minimize exposure to light. 
	NOTE: If interested in studying microtubules and associated molecules, tissue fixation should be performed at room temperature (RT) with RT fixative.</li>
	<li>Discard the PFA in an appropriate container in a fume hood. Wash three times with 10 mL of 1x PBS for 15 min each time on a rocking shaker at RT to eliminate the fixative before dissection.</li>
</ol>
2. Endolymphatic sac microdissection
<ol>
	<li>Transfer each inner ear preparation to a 35 mm tissue culture dish containing 1x PBS.</li>
	<li>Place the preparation in the dish so that the root of the 8th cranial nerve is oriented up. Hold the tissue in this position by grasping the cochlea with a #4 forceps (Figure 1C).</li>
	<li>Identify important landmarks: the vestibular aqueduct, the anterior and posterior semicircular canals, common crus, and sigmoid sinus (vein) (Figure 1D). 
	NOTE: The endolymphatic sac in the wild type mouse is translucent and hard to visualize. For successful microdissection of the endolymphatic sac, it is important to understand its anatomy and localization (Figure 1E).</li>
	<li>Incise the dura mater and vestibular aqueduct, as well as the underlying connective tissues surrounding the endolymphatic sac, using a 27 G needle on a 1 mL syringe (Figure 1D,F). 
	NOTE: For an adult mouse, decalcification of the preparation using 10% EDTA (pH 7.4) before microdissection is useful, but not essential, to facilitate incision of the vestibular aqueduct. For dissection of opened endolymphatic sac older than postnatal day 5, it is recommended to position the incision line on the vestibular aqueduct slightly anterior to make an incision into the endolymphatic sac lumen (Figure 2A).</li>
	<li>Hold the connective tissue located lateral to the endolymphatic sac with forceps and pull up the tissue to peel it from the temporal bone (Figure 1G).</li>
	<li>Carefully remove any remaining debris. The preparation usually includes the endolymphatic sac epithelium, surrounding connective tissues, a portion of the vestibular aqueduct, and the sigmoid sinus (Figure 1H-I). If necessary, separate the endolymphatic sac epithelium from surrounding tissues (Figure 1J).</li>
	<li>(Optional) For dissection of the opened endolymphatic sac, hold the stem part of the preparation so that the cross-section of the lumen can be observed. Insert a 27 G needle into the lumen and move it to cut the endolymphatic sac into two sheets (Figure 2B-C). Hold the edge of each sheet-like tissue with forceps and separate them from each other (Figure 2D-F).</li>
</ol>
3. Immunohistochemistry
<ol>
	<li>After microdissection, use #5 forceps to transfer endolymphatic sacs into a 9-well spot glass plate.</li>
	<li>Using the microscope to make sure the endolymphatic sacs remain untouched, use a 200 &#181;L pipette to remove all 1x PBS except one drop containing the tissue. Add 200 &#181;L of permeabilizing and blocking solution (1x PBS with 0.15% Triton X-100 (PBS-TX) and 5% bovine serum albumin (BSA)) and incubate for 1 h at RT on an orbital shaker.</li>
	<li>After removing all blocking solution except one drop containing the tissue, add 200 &#181;L of primary antibodies and incubate overnight at 4 &#176;C. Anti-pendrin antibodies6 diluted 1:1000 in blocking solution can be used to identify the mitochondria-rich cells of the endolymphatic sac, although a faint expression is also seen in a subset of ribosome-rich cells16 (Figure 5).</li>
	<li>After three washes in 1x PBS for 15 min at RT, incubate with secondary antibodies. For the labeling presented here, incubate 200 &#181;L of fluorescent dye-conjugated secondary antibodies, diluted from the commercial stock at 1:500 in blocking solution, with the tissue on a rocking platform for 1 h at RT in the dark. Fluorescent dye-conjugated phalloidin, which recognizes &#946;-actin (ACTB), can be used to highlight the presence of the endolymphatic sac and duct as well as the conjunctive tissue around them (Figure 5A, C, F-G).</li>
	<li>Mount endolymphatic sacs between a glass slide and coverslip using an antifade mounting medium with DAPI. After letting the slides dry protected from light for 1 h at RT, apply transparent nail polish at the junction between coverslip and glass slide to seal them and limit air bubble formation. If trying to visualize the differential distribution of proteins at the apical versus the basolateral surface of the epithelial cells, it is recommended to open the sac during the mounting step by incising an edge and opening it.</li>
	<li>Image using a confocal microscope. First visualize endolymphatic sac preparations with a 10x objective to provide a global view of the sac labeling (Figure 5A-D). Then use a 63x objective to gain a detailed view of the distribution of the protein of interest in mitochondria-and ribosome-rich cells of the endolymphatic sac (Figure 5F-G).</li>
</ol>

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	<li>Dror, A. A., Brownstein, Z., Avraham, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+human+and+mouse+genetics+reveals+pendrin+function+in+hearing+and+deafness.">Integration of human and mouse genetics reveals pendrin function in hearing and deafness.</a> Cellular Physiology and Biochemistry. 28 (3), 535-544 (2011).</li><li>Reardon, W., CF, O. M., Trembath, R., Jan, H., Phelps, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+vestibular+aqueduct:+a+radiological+marker+of+pendred+syndrome,+and+mutation+of+the+PDS+gene.">Enlarged vestibular aqueduct: a radiological marker of pendred syndrome, and mutation of the PDS gene.</a> QJM. 93 (2), 99-104 (2000).</li><li>Griffith, A. J., Wangemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+loss+associated+with+enlargement+of+the+vestibular+aqueduct:+mechanistic+insights+from+clinical+phenotypes,+genotypes,+and+mouse+models.">Hearing loss associated with enlargement of the vestibular aqueduct: mechanistic insights from clinical phenotypes, genotypes, and mouse models.</a> Hearing Research. 281 (1-2), 11-17 (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> Journal of Neurology. 263, 71-81 (2016).</li><li>Eckhard, 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=Water+channel+proteins+in+the+inner+ear+and+their+link+to+hearing+impairment+and+deafness.">Water channel proteins in the inner ear and their link to hearing impairment and deafness.</a> Molecular Aspects of Medicine. 33 (5-6), 612-637 (2012).</li><li>Choi, B. Y., 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=Mouse+model+of+enlarged+vestibular+aqueducts+defines+temporal+requirement+of+Slc26a4+expression+for+hearing+acquisition.">Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition.</a> Journal of Clinical Investigation. 121 (11), 4516-4525 (2011).</li><li>Miyashita, T., 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=Presence+of+FXYD6+in+the+endolymphatic+sac+epithelia.">Presence of FXYD6 in the endolymphatic sac epithelia.</a> Neuroscience Letters. 513 (1), 47-50 (2012).</li><li>Royaux, I. 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=Localization+and+functional+studies+of+pendrin+in+the+mouse+inner+ear+provide+insight+about+the+etiology+of+deafness+in+pendred+syndrome.">Localization and functional studies of pendrin in the mouse inner ear provide insight about the etiology of deafness in pendred syndrome.</a> Journal of the Association for Research in Otolaryngology. 4 (3), 394-404 (2003).</li><li>Kim, H. M., Wangemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+stretching+and+luminal+acidification+lead+to+a+retarded+development+of+stria+vascularis+and+deafness+in+mice+lacking+pendrin.">Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin.</a> PLoS One. 6 (3), 17949 (2011).</li><li>Dahlmann, A., von During, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endolymphatic+duct+and+sac+of+the+rat:+a+histological,+ultrastructural,+and+immunocytochemical+investigation.">The endolymphatic duct and sac of the rat: a histological, ultrastructural, and immunocytochemical investigation.</a> Cell and Tissue Research. 282 (2), 277-289 (1995).</li><li>Everett, L. 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=Targeted+disruption+of+mouse+Pds+provides+insight+about+the+inner-ear+defects+encountered+in+Pendred+syndrome.">Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome.</a> Human Molecular Genetics. 10 (2), 153-161 (2001).</li><li>Miller, R. L., 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=The+V-ATPase+B1-subunit+promoter+drives+expression+of+Cre+recombinase+in+intercalated+cells+of+the+kidney.">The V-ATPase B1-subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney.</a> Kidney International. 75 (4), 435-439 (2009).</li><li>Madisen, L., 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=A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nature Neuroscience. 13 (1), 133-140 (2010).</li><li>Wang, L., Jiang, H., Brigande, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+the+developing+mouse+inner+ear+by+in+vivo+electroporation.">Gene transfer to the developing mouse inner ear by in vivo electroporation.</a> Journal of Visualized Experiments. (64), e3643 (2012).</li><li>Currle, D. S., Hu, J. S., Kolski-Andreaco, A., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+mouse+neural+stem+cell+precursors.">Culture of mouse neural stem cell precursors.</a> Journal of Visualized Experiments. (2), 152 (2007).</li><li>Honda, K., 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=Molecular+architecture+underlying+fluid+absorption+by+the+developing+inner+ear.">Molecular architecture underlying fluid absorption by the developing inner ear.</a> Elife. 6, (2017).</li></ol>

The authors have nothing to disclose.

Many researchers have used paraffin-embedded or frozen sections for morphological studies of the endolymphatic sac. The disadvantage of sectioned preparations is that it can be difficult to fully observe the complicated cell-to-cell contacts because the size and shape of each epithelial cell in the endolymphatic sac is extremely variable, forming a wrinkled and pseudostratified cell layer. Whole-mount immunostaining and z-stack imaging, described in this protocol, permit better visualization of the three-dimensional structure of the endolymphatic sac.
For successful microdissection, the critical point is to mentally visualize the entire outline of the endolymphatic sac, even if it is invisible in the wild type mouse. Observation of the fluorescent reporter shown in Figure 3 and in Supplementary Figure 1-2, can be helpful for recognizing localization and size of the endolymphatic sac in mice.
While this whole-mount dissection method was used for immunostaining in the above protocol, this technique can also be used to harvest endolymphatic sac tissue for gene expression analysis, such as RT-qPCR, microarray expression, and even single-cell RNA-seq. The results of single-cell RNA-seq analysis using tissue prepared with this protocol have been previously reported16. For gene expression analysis of endolymphatic sac epithelial cells, it is preferable to collect isolated epithelium so that there is no contamination with other adjacent tissues. Although the embryonic endolymphatic sac can be separated from the adjacent connective tissues, it is much harder to do at later ages. For specimens from older mice, incubation with collagenase/dispase for 5 min at 37 &#176;C is encouraged and facilitates the isolation of the endolymphatic sac epithelium.
One of the drawbacks of the whole-mount immunostaining described in this protocol is that the original three-dimensional structure may be affected by trauma during dissection or mounting of the preparation between glass slide and coverslip. Care is needed to avoid artefacts due to manipulation.

The mammalian inner ear consists of the cochlea, the saccule, the utricle, three semi-circular canals, and the endolymphatic sac (Figure 1A). These organs comprise a continuous, fluid-filled epithelium called the membranous labyrinth, with adjacent organs connecting directly or through small canal structures such as the ductus reuniens, saccular duct, utricular duct, or endolymphatic duct. Compared to other organs of the inner ear, the endolymphatic sac has unique characteristics. First, it lacks sensory epithelial cells. Instead, the endolymphatic sac has cells specialized for ion transport. Second, although the membranous labyrinth is enclosed in the bony labyrinth, the endolymphatic sac is an exception, protruding partially out of the petrous bone and into the intracranial cavity. This morphology appears to be highly conserved during evolution of the inner ear. Third, the endolymphatic sac is the first structure to develop from the otocyst at an early embryonic stage prior to formation of other organs. In addition several pathologies have been associated with an enlarged endolymphatic sac or an abnormal endolymphatic compartment. The presence of pathogenic variants in SLC26A4 (also known as the pendrin gene) leads to a comparatively common form of sensorineural hearing loss, caused by the enlargement of the endolymphatic sac associated with the presence of an enlarged vestibular aqueduct (EVA)1. When associated with goiter, it is refered to as Pendred syndrome2,3. Meniere&#39;s disease is also thought by some to be associated with abnormality of the endolymphatic compartment (hydrops)4. The unique features of the endolymphatic sac and pathologies associated with a change of its morphology are consistent with a critical role for development and maintenance of the inner ear.
Despite its importance, knowledge is still limited regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is at least partly due to the difficulty of visualizing and microdissecting this tissue. The endolymphatic sac is a pouch-shaped structure, composed of a single layer of epithelial cells, which is often translucent and very difficult to identify out of the conjunctive tissue which surrounds it by light stereomicroscopy. Although a small number of researchers have developed whole-mount dissection techniques and published their experimental findings5,6,7,8,9,10, there is no publication focusing on the technical details of this procedure.
In this study, the microdissection approaches developed to access and isolate the endolymphatic sac from the wild-type mouse inner ear at different ages, in whole-mount, is described. The results of a similar dissection is shown in a mouse model, lacking expression of SLC26A4, which has enlargement of the endolymphatic duct and sac. A mouse line with a transgene encoding a Cre recombinase expressed in the endolymphatic sac is reported. In presence of a fluorescent reporter of Cre expression, here tdTomato, the fluorescence can be used to readily visualize the endolymphatic sac with limited dissection and to obtain information about its size. This reporter mouse line can also be used as an educational tool to practice and perfect endolymphatic sac dissections. The ability to dissect endolymphatic sac tissue should facilitate further characterization of this understudied but essential component of the inner ear.

<table><tbody><tr><td>EDTA 0.5 M</td><td>Crystalgen</td><td>221-057</td><td></td></tr><tr><td>1 mL TUBERCULIN syringe with 27G x 13mm</td><td>BD</td><td>309623</td><td></td></tr><tr><td>9-well spot glass plate</td><td>Pyrex</td><td>13-748B</td><td></td></tr><tr><td>Alexa Fluor 555-conjugated phalloidin</td><td>ThermoFisher Scientific</td><td>A34055</td><td></td></tr><tr><td>Artery&amp;nbsp;Scissors&amp;nbsp;- Ball Tip</td><td>Fine Science Tools</td><td>14080-11</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A3059</td><td></td></tr><tr><td>Camera to capture images in incident light</td><td>Leica</td><td>DFC 495</td><td></td></tr><tr><td>Camera to capture fluorescent images</td><td>Leica</td><td>DFC 7000 GT</td><td></td></tr><tr><td>Centrifuge tubes 15 mL</td><td>Corning</td><td>430053</td><td></td></tr><tr><td>Centrifuge tubes 50 mL</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>Collagenase/Dispase</td><td>Roche</td><td>10269638001</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 880</td><td></td></tr><tr><td>Cover Glass</td><td>Corning</td><td>2940-223</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine Science Tools</td><td>11241-30</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Dumont #55 Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Alternative: Extra Fine Micro Dissecting Scissors; Straight; Sharp Points; 20mm Blade Length; 3 1/2&quot; Overall Length</td><td>Roboz</td><td>RS-5880</td><td></td></tr><tr><td>Goat anti-rabbit Alexa Fluor 488-conjugated secondary antibody</td><td>ThermoFisher Scientific</td><td>A11034</td><td></td></tr><tr><td>MicroSlides Superfrost</td><td>VWR</td><td>48311-702</td><td></td></tr><tr><td>Orbital shaker, for example Mini Shaker</td><td>Daigger</td><td>980275</td><td></td></tr><tr><td>Paraformaldehyde (PFA) 16% Aqueous Solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Phosphate-Buffered Saline (10X) pH 7.4</td><td>ThermoFisher Scientific</td><td>AM9624</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>ThermoFisher Scientific</td><td>P36931</td><td></td></tr><tr><td>Rabbit anti-Pendrin antibodies</td><td>In-house</td><td></td><td>RRID:AB_2713943, PB826 (Choi &lt;em&gt;et al.&lt;/em&gt;, 2011)</td></tr><tr><td>Ring Forceps</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>Rocking shaker, for example GyroMini</td><td>Labnet</td><td>S0500</td><td></td></tr><tr><td>Stereomicroscope equipped with a PlanApo 1.0x objective</td><td>Leica</td><td></td><td></td></tr><tr><td>Stainless Steel Single Edge Blades, .009&quot;</td><td>GEM Personna</td><td>62-0176</td><td></td></tr><tr><td>Tissue culture dish 60 mm</td><td>Falcon</td><td>353002</td><td></td></tr><tr><td>Transparent nail polish</td><td></td><td></td><td></td></tr><tr><td>Triton X-100</td><td>ACROS Organics</td><td>32737-1000</td><td></td></tr></tbody></table>

dissection of the endolymphatic sac from mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

Each step of this microdissection of a wild type endolymphatic sac from a postnatal day 5 (P5) mouse is detailed in the associated video and snapshots of the key steps of this dissection and opening of the endolymphatic sac are presented in Figure 1 and Figure 2. 
Representative results of the dissections of the endolymphatic sac with tdTomato fluorescence at embryonic day 16.5 (E16.5), P5 and P30, following this protocol are presented in Figure 3 and in Supplementary Figure 1 and Supplementary Figure 2. Using a dissection microscope with fluorescence, the endolymphatic sac can be readily visualized in the skull after removal of the brain in mice carrying the transgene Tg(ATP6V1B1-Cre) in the presence of the reporter of Cre expression: Ai9 (LSL-RCL-tdT) (see Figure 3A-B, and Supplementary Figure 1 for images captured in incident light and tdTomato fluorescence of the endolymphatic sac at different stages of the dissection). In these mice, Cre is expressed as a mosaic in cells all along the endolymphatic sac and duct (Figure 3, see also Supplementary Figure 2 for visualization of the endolymphatic sac with versus without fluorescent reporter). Although the expression of this reporter is not specific for the endogenous expression of Atp6v1b1, which is restricted to the mitochondria-rich cells of the endolymphatic sac16, one of the two cell types of this epithelium (Figure 5E), it is a helpful aid to identify and dissect the endolymphatic sac.
Inner ears dissected from a mouse model for enlargement of the vestibular aqueduct are presented in Figure 4. As compared to control littermates, the endolymphatic sacs and ducts of mice deficient for SLC26A4 (pendrin) are enlarged.
Representative images of immunohistochemistry of intact endolymphatic sac with and without conjunctive tissue are shown in Figure 5. Results of immunohistochemistry of opened endolymphatic sac of a P5 wild type mouse are&#160;also presented (Figure 5F-G). The endolymphatic sac is composed of a single layer of epithelium folded in a pouch-shaped structure, containing in its lumen endolymph. This epithelium consists of two cell types, mitochondria-rich cells and ribosome-rich cells (Figure 5E). A whole-mount&#160;endolymphatic sac will be flattened because endolymph is no longer present. The sac will appear as two intertwined layers of epithelium, making it difficult to determine subcellular localization of proteins expressed in this epithelium. By opening the endolymphatic sac, the single layer epithelium can be readily visualized and the relative distribution of the proteins of interest, with respect to the endolymphatic sac lumen, can be determined more definitively. A potential apical (luminal, where endolymph would be) versus basal enrichment of these proteins can be more accurately determined. As an example, SLC26A4 is enriched on the apical side of mitochondria-rich cells (Figure 5F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62375/62375fig01.jpg" /> 
Figure 1. Whole-mount dissection of the endolymphatic sac of a wild type mouse at post natal day 5 (P5). 
(A) Schematic of the membranous labyrinth of a developed mouse inner ear (right). The location of the endolymphatic sac, and the cochlea and vestibular structures are indicated. This figure has been modified from Honda et al.16. 
(B) Mid-sagittal section showing the left hemi-skull. The location of the endolymphatic sac is indicated (arrow). (O, occipital; F, frontal) 
(C) Otic capsule along with the squamous part of the temporal bone. 
(D) Important landmarks overlaid onto the dotted box in panel C. (ES, endolymphatic sac; ED, endolymphatic duct; VA, vestibular aqueduct; ASC, anterior semicircular canal; PSC, posterior semicircular canal; CC, common crus; SS, sigmoid sinus) 
(E) Schematic illustration of the cross-section along with the continuous white line on panel D through the endolymphatic sac. The proximal side of the endolymphatic sac is covered by a bony canal called the vestibular aqueduct, along with the endolymphatic duct. In the distal side, the endolymphatic sac, surrounded by conjonctive tissue (CT), extends and protrudes to the outside of the bony labyrinth and is sandwiched between the dura mater (DM) and sigmoid sinus with the squamous part of the temporal bone (TB). 
(F) A fine needle is used to cut around the endolymphatic sac (following the dotted yellow lines on panel D). 
(G) The preparation is carefully removed from the temporal bone by holding the tissue at the position shown by the asterisk (*, also shown in panel E) and peeling it up. 
(H) An isolated whole endolymphatic sac with surrounding tissues. 
(I) Schematic version of this isolated endolymphatic sac with surrounding tissues. 
(J) Isolated endolymphatic sac without surrounding tissues. 
Scale bars: 2 mm (B), 1 mm (J). <a href="https://www.jove.com/files/ftp_upload/62375/62375fig01large.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/62375/62375fig02.jpg" /> 
Figure 2. Dissection of the opened endolymphatic sac of a P5 wild type mouse. 
(A) On step 2.4, the incision line on the vestibular aqueduct is positioned slightly anterior (red dotted line) to make an incision into the endolymphatic sac lumen. 
(B) Hold the stem part of the preparation, insert a 27-gauge needle into the lumen, and move it to cut the endolymphatic sac into two sheets. 
(C) Schematic illustration of panel B. 
(D) Hold the edge of each sheet with forceps and separate them from each other. 
(E) Schematic illustration of panel D. 
(F) The endolymphatic sac is separated into two sheets, including the epithelium and the surrounding tissues. <a href="https://www.jove.com/files/ftp_upload/62375/62375fig02large.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/62375/62375fig3v3.jpg" /> 
Figure 3. Dissection of R26LSL-RCL-tdT/+;Tg(ATP6V1B1-Cre)1Rnel/Mn mouse endolymphatic sac at E16.5, P5, and P30.&#160;All images presented show tdTomato fluorescence obtained using a stereomicroscope with a 1 x objective equipped for detection of tdTomato fluorescence. 
(A, B) Mid-sagittal section of the skull of a E16.5 R26LSL-RCL-tdT/+;Tg(ATP6V1B1-Cre)1Rnel/Mn mouse before (A) and after (B) half-brain removal. tdTomato fluorescence outlines the position of the inner ear. The endolymphatic sac is readily visible even without dissection (arrowhead). 
(C, E, G) Isolated inner ears from E16.5, P5 and P30 mice, respectively. 
(D, F and H) Higher magnification images of the corresponding microdissected endolymphatic sacs and ducts. At P30, the endolymphatic duct is encapsulated in bone making it particularly difficult to isolate. Scale bars: 2 mm (A, B, C, E, G), 500 &#181;m (D, F, H). <a href="https://www.jove.com/files/ftp_upload/62375/62375fig3v2large.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/62375/62375fig04.jpg" /> 
Figure 4. Gross anatomy of the otic capsule with an enlarged endolymphatic sac.&#160;Inner ears from Slc26a4+/- (left) and Slc26a4-/- (right) mouse littermates at P105. The vestibular aqueduct and endolymphatic sac and duct (black dotted lines) are enlarged in the Slc26a4-/-&#160;mouse as compared to those in a Slc26a4+/- mouse. Scale bar: 2 mm. <a href="https://www.jove.com/files/ftp_upload/62375/62375fig04large.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/62375/62375fig5v2.jpg" /> 
Figure 5. SLC26A4 expression of mitochondria-rich cells in the endolymphatic sac epithelium at E16.5 and P5. 
(A, B) Isolated endolymphatic sac from E16.5 mouse labeled with anti-SLC26A4 antibody (green) and phalloidin which labels &#946;-actin (ACTB, red). 
(C) A low-magnification image of opened endolymphatic sac from P5 mouse labeled with an anti-SLC26A4 antibody (green). Phalloidin (ACTB, red) can be used to highlight the presence of the endolymphatic sac as well as the conjunctive tissue around it. 
(D) Isolated endolymphatic sac labeled with an anti-SLC26A4 antibody (green). 
(E) Schematic illustration of endolymphatic sac epithelium highlighting the presence of two cell types: the mitochondria-rich cells, which apical surface is covered with microvilli, and the ribosome-rich cells. 
(F-G) High-magnification&#160;images of endolymphatic sac epithelium at P5, after the endolymphatic sac was opened and labeled with an anti-SLC26A4 antibody (green) and phalloidin (ACTB, red). A representative image at the apical membrane level is shown in G, and a reconstructed cross-section from z-stack at the level of the punctate white line is shown in F. The nucleus of the cells is labeled with DAPI (blue). 
Scale bars: 100 &#956;m (A, B); 200 &#956;m (C, D) ; 20 &#956;m (F, G). <a href="https://www.jove.com/files/ftp_upload/62375/62375fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. Incident light and corresponding tdTomato fluorescence images of a P5 R26LSL-RCL-tdT/+;Tg(ATP6V1B1-Cre)1Rnel/Mn mouse inner ear at three steps of the dissection.&#160;The endolymphatic sac and duct can be readily visualized using tdTomato fluorescence. 
(A-B) Region of the inner ear in the posterior part of an intact half skull of a P5 R26LSL-RCL-tdT/+;Tg(ATP6V1B1-Cre)1Rnel/Mn&#160;mouse. 
(C-D) Removal of the skin and part of the skull allows better visualization of the region of the endolymphatic sac. 
(E, F) Isolated inner ear with the endolymphatic sac still associated with conjunctive tissue. 
(A, C, E) Images captured in incident light. (B, D, F) tdTomato fluorescence of the corresponding tissue. Scale bars: 2 mm (A-B), 1 mm (C-F). <a href="https://www.jove.com/files/ftp_upload/62375/JoVE_HondaLee_SupplementaryFigure1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2. Comparison of the fluorescence of isolated endolymphatic sacs of&#160;R26LSL-RCL-tdT/+;Tg(ATP6V1B1-Cre)1Rnel/Mn&#160;and R26LSL-RCL-tdT/+ littermate mice at E16.5 and P5.&#160;tdTomato fluorescence greatly facilitates the recognition of the endolymphatic sac and duct. 
(A-C) Microdissected endolymphatic sacs of E16.5 R26LSL-RCL-tdT/+;Tg(ATP6V1B1-Cre)1Rnel/Mn&#160;(A) and R26LSL-RCL-tdT/+&#160;littermate mice (B, C). 
(D-F) Microdissected endolymphatic sacs of P5 R26LSL-RCL-tdT/+;Tg(ATP6V1B1-Cre)1Rnel/Mn&#160;(D) and R26LSL-RCL-tdT/+ littermate mice (E, F). 
(A, B, D, E) tdTomato fluorescence. (C, F) Incident light images corresponding to B and E. Scale bars: 500 &#956;m. <a href="https://www.jove.com/files/ftp_upload/62375/JoVE_HondaLee_SupplementaryFigure2.pdf" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Dissection of the Endolymphatic Sac from Mice at JoVE.com

Dissection of the Endolymphatic Sac from Mice

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

dissection-of-the-endolymphatic-sac-from-mice

Research

JoVE Journal

Biology

2.1K Views.  National Institute on Deafness and Other Communication Disorders, National Institutes of Health. This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

Video: Dissection of the Endolymphatic Sac from Mice

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetics

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Neuroscience

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Bioengineering

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Medicine

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

An Integrated (Microscopic/Endoscopic) Dissection Ear Surgery Course

Traditionally, otologic surgical training consisted of microscopic cadaveric dissections. However, during the last decades the endoscope has significantly changed the surgical perspective in the otologic field. Thus, the modern ear and lateral skull base surgeon should master the entire spectrum of endoscopic and microscopic approaches, with the aim of tailoring the procedure and guaranteeing the best possible functional outcome. This work proposes a step-by-step guided and illustrated dissection course, including indications for the setup of the cadaver lab and the integration of the microscope and endoscope to enhance the use of both instruments. The alternation of the endoscope and microscope allows the novice to train the correct handling of the instruments in the surgical field under both optical views. This aspect is of utmost importance since it is not advisable to start off a technique without practicing the other one, as both are important and complementary in the modern otologic surgery setting.

An Integrated Microscopic/Endoscopic Dissection Ear Surgery Course

Traditionally, otologic surgical training consisted of microscopic cadaveric dissections. However, during the last decades the endoscope has significantly ...

Surgical Treatment of an Endolymphatic Sac Tumor

Endolymphatic sac tumors (ELST) are low-grade papillary adenocarcinoma originating from the endolymphatic sac. Usually slow-growing, with local aggressiveness and a low risk of distant metastases, ELST can be sporadic but also frequently associated with von Hippel Lindau disease. The current treatment of ELST is primarily surgical resection. A 55-year-old woman accessed our otologic tertiary level referral center for a sudden worsening of hearing loss in her left ear and vertigo. A magnetic resonance (MRI) and computer tomography scan study subsequently showed a mass in the petrous bone; hence, the presence of an ELST was hypothesized. After embolization of the mass, the patient underwent surgical removal of the lesion. The resection of the mass was done through a translabirinthine approach, with an uneventful procedure. No residual disease remained after surgery. After 24 months of radiologic follow up with MRI, there are no signs of recurrence disease. This paper reports the management of this sporadic ELST, as well as the follow up results, providing clinicians this protocol for the handling of such a challenging otologic skull base surgery and rare disease.

Here, we discuss clinical and radiological features of endolymphatic sac tumors and report the methodology and results of a surgical removal in a case in care at our otologic referral center.

Endolymphatic sac tumors (ELST) are low-grade papillary adenocarcinoma originating from the endolymphatic sac. Usually slow-growing, with local ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.