Name: cochlear surface preparation in the adult mouse
Uploaded: 2019-11-06T16:00:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about Cochlear Surface Preparation in the Adult Mouse at JoVE.com

The research project described was supported by grant R01 DC009222 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. This work was conducted in the WR Building at MUSC in renovated space supported by grant C06 RR014516. Animals were housed in MUSC CRI animal facilities supported by grant C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources. The authors thank Dr. Jochen Schacht for his valuable comments and Andra Talaska for proofreading of the manuscript.

All research protocols involving male adult CBA/J mice at the ages of 10&#8211;12 weeks and C57BL/6J mice at the ages of 6&#8211;8 weeks were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical University of South Carolina (MUSC). Animal care was under the supervision of the Division of Laboratory Animal Resources at MUSC.
NOTE: For the procedures presented below, mice are anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injections. Mice are decapitated after the animal no longer responds to painful stimuli, such as a toe pinch.
1. Extraction of Temporal Bones
<ol>
	<li>Decapitate the mouse immediately postmortem with surgical scissors (17 cm long) and cut the skull bone with scissors from the posterior aspect forward along the center line of the skull after exposing the skull bone by pulling the skin anteriorly.</li>
	<li>Remove the brain tissue using forceps and manually remove the temporal bones with the thumb and index finger for mice three months of age or older. Use small surgical scissors (11 cm long) to cut the temporal bone of mice younger than three months old.</li>
	<li>Put the temporal bone into a Petri dish (30 mm in diameter) containing ice-cold fresh 4% paraformaldehyde (PFA) solution dissolved in phosphate-buffered saline (PBS), pH 7.4. 
	NOTE: Prepare fresh 4% PFA solution and adjust the pH to 7.4 just before fixing the temporal bones, because improper pH balancing of the PFA solution will decrease the quality of the immunolabeling.</li>
</ol>
2. Fixation and Perfusion
<ol>
	<li>Remove the stapes from the oval window and puncture the round window membrane with size #5 forceps. Under a stereo dissection microscope make a small hole into the apex of the cochlea using a 27 G needle connected to a 1 mL syringe.</li>
	<li>Gently and slowly perfuse the cochlea with 4% PFA solution via the round and oval windows until the solution washes out of the small hole at the apex. Transfer the cochlea (one or two cochleae per vial) to 20 mL volume scintillation vials containing 10 mL of 4% PFA solution.</li>
	<li>Gently agitate the scintillation vials at room temperature (RT) for 2 h and leave overnight in a refrigerator (4 &#176;C) on a rotator. 
	NOTE: The time of fixation can be adjusted depending on the antibody used. For example, limiting the fixation to 1.5 h will allow for successful immunolabeling of post synaptic terminals when using the GluA 2 antibody.</li>
</ol>
3. Decalcification of the Temporal Bone
<ol>
	<li>Remove the PFA solution and wash the cochleae with fresh PBS 3x for 5 min each.</li>
	<li>Add 20 mL of 4% ethylenediaminetetraacetic acid disodium salt (EDTA) solution (pH 7.4) to the scintillation vials and keep in a refrigerator for 48&#8211;72 h on a rotator with gentle agitation. Change the EDTA solution daily until the cochleae are decalcified. Check if the cochleae are decalcified by touching the bony vestibular portion with forceps to assess for elasticity or simply cut a small piece from the edge of the vestibular portion. If such cut results in a crushed piece, the cochlea is not decalcified. 
	NOTE: The EDTA solution may interfere with the immunoreaction of primary antibodies depending on the solution concentration. Prepare 4% EDTA in PBS and adjust the pH to 7.4. For consistent decalcification of temporal bones, fresh EDTA solution is used.</li>
	<li>Change the solution to PBS for microdissection after decalcification is complete. 
	NOTE: If the cochleae are not sufficiently decalcified, dissection of the cochleae cannot be performed.</li>
</ol>
4. Microdissection of the Cochlear Epithelium
NOTE: Once decalcification is complete, microdissection of cochlear epithelium for immunolabeling needs to be performed as soon as possible. In a clean 30 mm Petri dish containing PBS, the inner ear is oriented in the following manner: in reference to the Petri dish's top (lid) and bottom, the cochlear round and oval windows face the top. In reference to dissector, the cochlear portion is oriented toward the front (away from dissector) and vestibular portion toward the back (near dissector). The following describes the steps in detail.
<ol>
	<li>Hold the vestibular portion of the temporal bone with forceps and cut the apical turn with the scalpel at a 45&#176; angle as indicated by the red line (Figure 1a).</li>
	<li>Cut vertically along the faint line between the round window and the oval window, as indicated by the red line (Figure 1b) to separate the cochlea from the vestibular portion (Figure 1c). 
	NOTE: This cochlear portion contains the middle, basal, and hook portions of the epithelium.</li>
	<li>Place the cochlear portion with the basal turn toward the bottom and the middle turn toward the top of the Petri dish and cut the bony capsule and lateral wall of the middle turn toward the end from which the apical section was removed, as indicated by the red line (Figure 1d,e).</li>
	<li>Continue cutting to completely separate the middle portion from the basal and hook regions (Figure 1f,g). 
	NOTE: The hook region of the cochlea is the end of the cochlear epithelium corresponding to sensitivity to tones 48 kHz and higher in mice.</li>
	<li>Put the basal and hook portion with the basilar membrane site oriented toward the bottom of the dish and vertically cut the modiolus off the hook region to remove the modiolus as indicated by the vertical red line (Figure 1g). 
	NOTE: The modiolus is the conical-shaped central axis of the cochlea that consists of spongy bone and cochlear nerve as well as the spiral ganglion. It may be preferable to perform this cut with scissors.</li>
	<li>Cut the basal and hook regions as the other red line indicates in Figure 1g to separate the hook portion (Figure 1h). 
	NOTE: The cochlea has now been separated into apical, middle, basal, and hook portions. The next steps will be the final dissection of each of the individual turns, using the middle turn as an example.</li>
	<li>Cut away the relatively large portions of bony capsule and lateral wall tissue of the middle turn as indicated by the red line (Figure 1i). 
	NOTE: The lateral wall of the cochlear duct is formed by the spiral ligament and the stria vascularis.</li>
	<li>Hold the lateral wall with forceps to align the bony capsule and lateral wall with the bottom of the Petri dish and cut these tissues from the basilar membrane side. Then flatten the specimen to orient the sensory hair cell surface side up. Trim away the rest of the bony capsule and lateral wall (Figure 1j).</li>
	<li>Remove the tectorial membrane using forceps to completely separate the middle region (Figure 1k).</li>
	<li>Repeat steps 4.7&#8722;4.9 for the final dissections of the remaining turns or regions as shown in Figure 1l.</li>
	<li>Prepare a 10 mm round coverslip for adhesion of the sensory epithelium. Use a pipette to hand-spread 0.5 &#181;L of cell and tissue adhesive on the center of the round coverslip. After drying (3&#8211;5 min at RT), put the coverslips in PBS in Petri dishes.</li>
	<li>Stick all four pieces of the sensory epithelium on the 10 mm round coverslip in the Petri dish. Then hold one edge of the coverslip to transfer it to a four-well dish for immunolabeling or immunohistochemistry (Figure 1m). 
	NOTE: Figure 2 illustrates the location of the major cuts.</li>
</ol>
5. Immunolabeling for Cochlear Synapses
NOTE: The protocol for immunolabeling for cochlear synapses followed in this study has been previously described13. Presynaptic ribbons were labeled with a CtBP2 antibody (mouse anti-carboxyl-terminal binding protein 2 IgG1, labeling the B domain of the RIBEYE scaffolding protein). Post synaptic terminals were labeled with GluA2 antibody (mouse anti-glutamate receptor 2 IgG2a, labeling subunits of the AMPA receptor).
<ol>
	<li>Wash the sensory epithelium with PBS 3x for 5 min each wash in a four-well dish and then add 2 mL of 2% nonionic surfactant (i.e., Triton-X 100) into the dish for 30 min at RT on a rotator.</li>
	<li>Remove the nonionic surfactant solution from the dish using a pipette and add 100 &#181;L of blocking solution containing 10% normal goat serum to each well for 1 h on a rotator with gentle agitation at RT.</li>
	<li>Remove the blocking solution from each well, wash with PBS 3x for 5 min each wash under gentle agitation at RT.</li>
	<li>Add 100 &#181;L of the primary antibodies diluted with PBS to each well: mouse anti-GluA2 IgG2a (1:2,000), mouse anti-CtBP2 IgG1 (1:400). Cover each four-well dish with its lid and place into a large humidified container that protects from light. Incubate at 37 &#176;C for 24 h. 
	NOTE: As an option, rabbit anti-myosin VIIa (1:400) for immunolabeling sensory hair cells can be added.</li>
	<li>Wash 3x with PBS for 5 min each wash with gentle agitation at RT.</li>
	<li>Add 100 &#181;L of the secondary antibodies Alexa 594 goat anti-mouse IgG1a (1:1,000), Alexa 488 goat anti-mouse IgG2a (1:1,000) diluted with PBS to each well. Cover each four-well dish with its lid and place into a large humidified container that protects from light. Incubate at 37 &#176;C for 2 h. 
	NOTE: If myosin VIIa is used, add Alexa 350 goat anti-rabbit (1:200).</li>
	<li>Wash 3x with PBS for 5 min. Then, transfer the 10 mm round coverslip onto a slide with the samples on top.</li>
	<li>Carefully add 8 &#181;L of Fluoro-Gel with Tris buffer into the center of the coverslip. Then hold the edge of another 10 mm round coverslip with forceps to mount on top, sandwiching the two coverslips together.</li>
	<li>Use nail polish to seal the slides, put them in a cardboard slide folder, and store in the refrigerator. 
	NOTE: If some Fluoro-Gel leaks out between the coverslips during mounting, clean the edges of the coverslips before sealing the slide with nail polish. Confocal images need to be taken within 7 days.</li>
</ol>

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The authors have nothing to disclose.

Cochlear microdissection of whole-mount surface preparations in combination with immunolabeling provides a basic tool for investigation of inner ear pathologies and molecular mechanisms. This modified adult mouse cochlear dissection method using the cell and tissue adhesive simplifies this difficult procedure.
Although this modified cochlear surface preparation method is relatively easy and accessible, it still requires practice in order to achieve proficiency. To make the correct cuts, the dissector needs careful concentration. Because the cochlear sensory hair cells in the basal turn are so close to the spiral limbus, it is difficult to fully remove the limbus tissue to allow the surface preparation to lie completely flat, but confocal Z-projections can compensate for this issue. Additionally, the hook region of the cochlea is still the most difficult portion to dissect. Separations between OHCs and IHCs of the hook region may occur. The hook region displays large anatomic variations in humans and is important for bone-conduction hearing22,23. Based on the cochlear frequency mapping reported by Muller1 and Viberg and Canlon2, the hook region, beginning around 4.7 mm from the apex, corresponds to sensitivity to 48 kHz tones and higher (Table 1), whereas auditory functional assessments of acquired hearing loss in mice, including noise-induced, aminoglycoside-induced hearing loss, and age-related hearing loss, are generally measured at 8, 16, and 32 kHz with auditory brainstem response (ABR)5,6,7,9,24 and from 6&#8211;45 kHz with distortion product otoacoustic emission (DPOAE)25. In agreement with Montgomery et al., distortion of the epithelium is not commonly seen when the spiral is divided into 3&#8211;4 pieces19.
The modified method presented here involves dissecting the cochlear spiral into only four pieces (apex, middle, basal turns, and the hook region), whereas the Eaton-Peabody technique produces six pieces18. The Eaton-Peabody technique starts with a cochlear bisection, which avoids making tangential cuts through the epithelium to first separate the apical turn from the rest of the spiral, as described in this modified technique. By producing smaller pieces, the Eaton-Peabody technique is designed to minimize the flattening required to view a large piece with an immersion objective. In fact, the larger portions of tissue facilitate measurement of frequency mapping from the apical to the basal turn, in line with Montgomery et al.19. Additionally, a difference between this method and Montgomery et al.19 is that the modified cochlear dissection method described here employs a scalpel for most cuts and only one step is done with scissors (i.e., the separation of basal and hook regions as illustrated in the third cut in Figure 2), whereas Montgomery et al.19 used scissors with a silicone elastomer-coated dissection dish for surface preparations. To avoid distortion of the tissue, disconnection of the basilar membrane to remove the modiolus is critical.
This protocol uses a cell and tissue adhesive (Table of Materials) for adhesion of the pieces of cochlear epithelium to the 10 mm round coverslip for immunolabeling or immunohistochemistry, which makes the processes more convenient and avoids loss of epithelium tissues during the multiple washes of immunolabeling procedures. Cell and tissue adhesive is a formulation of polyphenolic proteins that adheres to cells and tissues and has been widely used in many common in-vitro techniques, including immunohistochemistry, in-situ hybridization, and immunoassays20. Consistent with the notion that the cell and tissue adhesive does not interfere with immunoreactions, parallel immunolabeling of myosin VIIa with surface preparations shows no difference of immunoreactions or uniformity with and without the cell and tissue adhesive. Comparing these three dissection methods (Eaton-Peabody Laboratories, Montgomery et al.19, and the modified method described), the graduate students in the lab agree that this modified method using the cell and tissue adhesive is easier to learn and master.
In summary, producing whole-mount adult mouse surface preparations is a basic skill for evaluation of cochlear pathologies. The described modified protocol for adult mice surface preparations simplifies this difficult procedure.

The cochlea is dedicated to the detection of sound and responsible for hearing. The cochlear duct is coiled in a spiral shape in the bony labyrinth and holds the auditory sensory end organ, the organ of Corti (OC). The OC rests on the basilar membrane, making up the cochlear epithelium, with a length of about 5.7 mm when uncoiled in adult CBA/CaJ mice1,2. Because the OC is tonotopically organized with high frequencies detected in the base and low frequencies in the apex, the cochlear epithelium is often divided into three parts for analytical comparisons: the apical, middle, and basal turns corresponding to low, middle, and high frequency detection, respectively. In addition to an array of supporting cells, the OC is composed of one row of inner hair cells (IHCs) located medially and three rows of outer hair cells (OHCs) located laterally with respect to the cochlear spiral.
Correct auditory processing depends on the integrity of the sensory hair cells in the cochlea. Damage to or loss of sensory hair cells is a common pathological feature of acquired hearing loss, caused by numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process3. Additionally, the integrity and function of the inner hair cell/auditory nerve synapses can be impaired by mild insults4. With the availability of molecular and genetic information and manipulation of genes by knockout and knock-in techniques, mice have been widely used in hearing science. Although the adult mouse cochlea is minuscule and the cochlear epithelium is surrounded by a bony capsule resulting in technically difficult microdissections, surface preparations of the epithelium in combination with immunolabeling or immunohistochemistry and confocal imagery have been broadly used for investigation of cochlear pathologies, including losses of ribbon synapses and hair cells, changes in levels of proteins in sensory hair cells and supporting cells, and hair cell regeneration. Cochlear surface preparations have also been used to determine the pattern of expression of reporter genes (i.e., GFP) and confirm successful transduction and identify transduced cell types. These techniques have been previously used for the study of molecular mechanisms underlying noise-induced hearing loss using adult CBA/J mice5,6,7,8,9.
Unlike immunohistochemistry using paraffin sections or cryosections to obtain small cross-sectional portions of the cochlea that contain three outer hair cells (OHCs) and one inner hair cell (IHC) on each section, cochlear surface preparations allow visualization of the entire length of the OC for counting sensory hair cells and ribbon synapses and immunolabeling of sensory hair cells corresponding to specific functional frequencies. Table 1 shows the mapping of hearing frequencies as a function of distance along the length of the cochlear spiral in adult CBA/J mouse according to studies from Muller1 and Viberg and Canlon1,2. Cochlear surface preparations have been widely used for investigation of cochlear pathologies4,5,6,7,8,9,10,11,12,13,14,15. The whole-mount cochlear dissection method was originally described in a book edited by Hans Engstrom in 196616. This technique was subsequently refined and adapted to a variety of species as described in the literature by a number of scientists in hearing science10,11,12,13,15,17 and by the Eaton-Peabody Laboratories at Massachusetts Eye and Ear18. Recently, another cochlear dissection method was reported by Montgomery et al.19. Microdissection of the cochlea is an essential and critical step for cochlear surface preparations. However, dissecting mouse cochleae is a technical challenge and requires considerable practice. Here, a modified cochlear surface preparation method is presented for use in adult mouse cochleae. This method requires decalcification and use of cell and tissue adhesive (i.e., Cell-Tak) to adhere the pieces of cochlear epithelium to 10 mm round coverslips for immunolabeling. Cell and tissue adhesive has been widely used for immunohistochemistry20. This modified cochlear microdissection method is relatively simple compared to those previously reported18,19.

<table><tbody><tr><td>10 mm Rund Coverslips</td><td>Microscopy products for science and industry</td><td>260367</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-mouse IgG2</td><td>Thermo Fisher Scientific</td><td>A-21131</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td></td></tr><tr><td>Alexa Fluor 594 Goat Anti-mouse IgG1</td><td>Thermo Fisher Scientific</td><td>A-21125</td><td></td></tr><tr><td>Alexa Fluor 594 Goat Anti-rabbit IgG (H+L)</td><td>Thermo Fisher Scientific</td><td>A11012</td><td></td></tr><tr><td>Carboard Micro Slide Trays</td><td>Fisher Scientific</td><td>12-587-10</td><td></td></tr><tr><td>Cell-Tak</td><td>BD Biosciences</td><td>354240</td><td></td></tr><tr><td>Corning Petri Dishes</td><td>Fisher Scientific</td><td>353004</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher Scientific</td><td>62247</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>FST fine science tools</td><td>11251-20</td><td></td></tr><tr><td>EDTA Disodium Salt</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Fluoro-gel with Tris Buffer</td><td>Electron Microscopy Sciences</td><td>17985-10</td><td></td></tr><tr><td>Four-well Cell Culture Dishes</td><td>Greiner Bio-One</td><td>627170</td><td></td></tr><tr><td>Goat Anti-myosin VIIa Antibody</td><td>Proteus Biosciences</td><td>25-6790</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-544-7</td><td></td></tr><tr><td>Mouse Anti-CtBP2 Antibody</td><td>BD Biosciences</td><td>#612044</td><td></td></tr><tr><td>Mouse Anti-Glu2R Antibody</td><td>Millipore</td><td>MAB397</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Thermo Fisher Scientific</td><td>31872</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>441244</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Fisher Scientific</td><td>BP665-1</td><td></td></tr><tr><td>Scalpel</td><td>VWR</td><td>100491-038</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15001-08</td><td></td></tr></tbody></table>

cochlear surface preparation in the adult mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

Surface preparations of the cochlear epithelium, in combination with immunolabeling and confocal imaging, have been used broadly in hearing science for the investigation of cochlear pathologies, such as quantification of ribbon synapses, sensory hair cells, and protein expression in sensory hair cells5,6,7,8. Although the dissection of adult mouse cochleae for surface preparations is not simple, new graduate students are able to learn this modified method after practicing with 10&#8211;15 ears (Figure 1 and Figure 2). With this technique, in combination with immunolabeling for CtBP2 (a marker for presynaptic ribbons) and GluA2 (a marker for post synaptic terminals), counting IHC/auditory nerve synapses using confocal images under Z projections with 0.25 &#181;m intervals, based on the size of mouse ribbon synapses, is possible21. This is consistent with published results4,6,13. Surface preparations of CBA/J mice (without treatment) at the age of 10&#8211;12 weeks immunolabeled with CtBP2 (red) and GluA2 (green) showed that both presynaptic ribbons and post synaptic terminals are located below the IHC nuclei and are juxtaposed, indicating functional synapses (Figure 3)6,13.
By immunolabeling surface preparations with different antibodies, assessment of molecular signaling and structure in sensory hair cells is possible. For example, Figure 4 shows the results for immunolabeling for myosin VIIa and counterstaining with phalloidin and 4&#8217;,6-diamidino-2-phenylindole (DAPI). Parallel immunolabeling experiments with and without the cell and tissue adhesive using identical solutions have been conducted to assess if the adhesive used interferes with the immunoreactions. Cochlear surface preparations were immunolabeled with myosin VIIa and counterstained with phalloidin. Confocal images were taken with a 63x magnification lens under identical conditions and equal parameter settings for laser gains and photomultiplier tube (PMT) gains. There was no difference in immunoreactions or uniformity with and without the cell and tissue adhesive (Figure 5). Using different fixatives, surface preparations have provided the basis for scanning electron microscopy (SEM) images for visualization of cochlear stereocilia9. C57BL/6J mice (without treatment) at the age of 6&#8211;8 weeks show well-organized V-shaped stereocilia of OHCs in three rows (Figure 6). Additionally, cochlear surface preparations have been used to determine the pattern of expression of a report gene (i.e., GFP) and confirm successful transduction and identify transduced cell types.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60299/60299fig01.jpg" /> 
Figure 1: Depiction of the steps for adult mouse surface preparations.&#160;(a) The cochlear bony capsule of the temporal bone faces up. The red line indicates the first cut to separate the apical turn. RW = round window, OW = oval window. (b) The apex is separated from the cochlea as indicated by the arrow. The red line between round window and oval window indicates the second cut made in the cochlea. (c) The cochlear portion that contains the middle, basal, and hook regions is separated from the vestibular portion. (d) The cochlear portion is situated facing up. The red line indicates the third cut, directed towards the end from which the apical section was removed. (e) The arrow indicates the gap left after the completion of the third cut. (f) This image shows the middle region. (g) This image shows the combined basal and hook regions. The vertical red line indicates the cut between the modiolus and hook region. The cut between the basal and hook regions is indicated by the other red line. (h) Basal and hook regions are separated as indicated by the arrows. (i) The middle region of the bony capsule is cut as indicated by the red line. (j) The image shows the middle region after one side of the bony capsule is removed. (k) This image shows the middle region after completion of dissection. (l) All four regions are completely dissected. (m) Cochlear turns affixed to a 10 mm round coverslip transferred to a four-well Petri dish with the four regions as indicated. All images were taken under a stereo-dissection microscope at 1.2x, 2.5x, and 0.6x magnifications. Scale bars are indicated in each image. <a href="https://www.jove.com/files/ftp_upload/60299/60299fig01large.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/60299/60299fig02.jpg" /> 
Figure 2: Location of the major cuts.&#160;The entire cochlear bony capsule was removed. The locations of the major cuts are indicated. 1) The location where the apical turn is cut. 2) The site where the middle turn is separated. 3) The critical cut to remove the modiolus. 4) The line to divide the basal and hook regions. Scale bar = 0.5 mm. <a href="https://www.jove.com/files/ftp_upload/60299/60299fig02large.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/60299/60299fig03.jpg" /> 
Figure 3: Confocal images reveal immunolabeling for CtBP2 and GluA2 on adult mouse surface preparations.&#160;The apical, middle, and basal regions are labelled. The lower magnification confocal images were taken with a 10x lens. Red = CtBP2, green = GluA2. Scale bar = 100 &#181;m. Confocal images of 5-, 16-, and 32-kHz regions were taken with a 63x lens. Enlarged views of the areas indicated by the white rectangles in the apex, middle, and base portions. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60299/60299fig03large.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/60299/60299fig04.jpg" /> 
Figure 4: Confocal images reveal immunolabeling of sensory hair cells of a surface preparation from the 32-kHz region.&#160;All images were merged Z-stack projections. Phalloidin (green) stained the structure of the three rows of OHCs and one row of IHCs. Myosin VIIa (red) immunolabeled three rows of OHCs and one row of IHCs. DAPI-stained hair cell nuclei. Merged confocal images were reconstructed for side views of sensory hair cells. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60299/60299fig04large.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/60299/60299fig05.jpg" /> 
Figure 5: Cochlear surface preparations with and without the cell and tissue adhesive were processed in parallel, immunolabeled for myosin VII (red), and counterstained with phalloidin (green) using identical solutions. The cell and tissue adhesive used does not interfere with immunoreactions.&#160;Confocal images were taken with a 63x magnification lens under identical conditions and equal parameter settings. Images were taken from the 32 kHz region. Immunolabeling for myosin VIIa and staining for phalloidin in OHCs was similar with and without the cell and tissue adhesive. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60299/60299fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60299/60299fig06.jpg" /> 
Figure 6: Scanning electron microscopy shows three rows of OHC stereocilia from C57BL/6J mice.&#160;Images were taken from the middle region. (a) A low-magnification view of three rows of OHCs. (b) Enlarged images show three rows of well-organized OHC stereocilia that appear in &#8220;V&#8221; shapes. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60299/60299fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Name</td>
			<td colspan="7"></td>
		</tr>
		<tr>
			<td colspan="2">Distance from the apex (mm)</td>
			<td>0.4</td>
			<td>1</td>
			<td>2.4</td>
			<td>3.3</td>
			<td>3.9</td>
			<td>4.7</td>
			<td>5.7</td>
		</tr>
		<tr>
			<td colspan="2">Distance from the apex (%)</td>
			<td>7.7</td>
			<td>18</td>
			<td>43</td>
			<td>54</td>
			<td>68</td>
			<td>82</td>
			<td>100</td>
		</tr>
		<tr>
			<td colspan="2">Frequencies (kHz)</td>
			<td>6</td>
			<td>8</td>
			<td>16</td>
			<td>22</td>
			<td>32</td>
			<td>48</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Mapping of the CBA/J mouse cochlear frequency sensitivity as a function of distance from the apex according to Muller1 and Viberg and Canlon2.

Watch this Scientific Journal Video about Cochlear Surface Preparation in the Adult Mouse at JoVE.com

Cochlear Surface Preparation in the Adult Mouse

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

cochlear-surface-preparation-in-the-adult-mouse

Korea Advanced Institute of Science and Technology (KAIST)

Research

JoVE Journal

Neuroscience

15.6K Views.  Medical University of South Carolina. This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Video: Cochlear Surface Preparation in the Adult Mouse

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

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

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

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

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

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 ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

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A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection