This work was supported by the National Institute of Deafness and Other Communication Disorders grants R01DC015824 (K.M.S.) and T32DC00038 (supporting S.D.), the Department of Defense grant W81XWH-15-1-0472 (K.M.S.), the Bertarelli Foundation (K.M.S.), the Nancy Sayles Day Foundation (K.M.S.), and the Lauer Tinnitus Research Center (K.M.S.). We thank Jessica E. Sagers, B.A. for insightful comments on the manuscript.

The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Massachusetts Eye and Ear. Experiments were carried out according to the Code of Ethics of the World Medical Association.
1. Preparing the Dissection
<ol>
	<li>Preparing the surgical table
	<ol>
		<li>Use 70% ethanol to disinfect the surgical table.</li>
		<li>Place two nonsterile prep pads next to the microscope.</li>
		<li>Prepare the instrument tray, including heat-sterilized operating scissors, a scalpel handle, a micro knife, and forceps (two each of #4 &#38; #55). Place the instrument tray and a 50 mm, clear-walled, glass-bottomed petri dish onto one non-sterile pad.</li>
		<li>Obtain a sterile #15 scalpel blade; a sealable plastic bag or container (to dispose of the carcasses in line with institutional guidelines); ice in a bucket; and cold, sterile Hank&#39;s Balanced Salt Solution (HBSS). Place these items onto the other nonsterile pad.</li>
		<li>Transfer the HBSS into a 50 mL plastic laboratory tube and put it on ice.</li>
	</ol>
	</li>
	<li>Preparing the culture
	<ol>
		<li>Prepare all materials in a laminar flow hood. For every four specimens, put four autoclaved 10 mm round glass cover slips in the four inlays of the 35 mm 4-well petri dish.</li>
		<li>For every 4 specimens, mix a total volume of 300 &#181;L consisting of the following in a microcentrifuge tube: 10 &#181;L of tissue adhesive, 285 &#181;L of NaHCO3, and 5 &#181;L of NaOH.</li>
		<li>Put 45 &#181;L of the resulting solution on every cover slip and leave it there for at least 20 min at RT; this solution can be left on the coverslip for several hours but cannot be left O/N.</li>
		<li>Place one or two 35 mm 4-well petri dish(es) inside a 10 cm Petri dish in order to create two barriers against contamination.</li>
		<li>Prepare culture medium containing 98% Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), 1% N-2, and 1% ampicillin (65 &#181;L per well in a (micro)centrifuge tube). Warm the solution to 25 &#176;C prior to use.</li>
	</ol>
	</li>
</ol>
2. Murine Cochlear Explant Dissection
<ol>
	<li>Decapitation of the neonatal mouse
	<ol>
		<li>Obtain a 60 mm plastic petri dish and spray 70% ethanol in its lid so that the surface is wet.</li>
		<li>Pour cold HBSS from the plastic laboratory tube into the bottom part of the 60 mm plastic and the 50 mm clear-walled, glass-bottomed petri dish. Store the plastic laboratory tube and both petri dishes on ice to keep the tissue cold whenever it is not being dissected.</li>
		<li>Place a P3 -&#160;P5-aged neonatal mouse on ice in a cutoff finger of a latex glove and wait until the hypothermia induces loss of consciousness (approximately 1-3 min).</li>
		<li>Decapitate the mouse quickly with operating scissors and dispose of the body in the sealable plastic bag. Put the severed neonate head in the 70% ethanol in the lid of the 60 mm plastic petri dish.</li>
	</ol>
	</li>
	<li>Macroscopic dissection of the temporal bone
	<ol>
		<li>Remove the skin of the skull by making a superficial cut from the anterior to the posterior end (directly on top of the sagittal suture) using the scalpel blade.</li>
		<li>Cut both external auditory canals with the scalpel blade and fold the skin anteriorly to expose the whole cranium. 
		&#8203;NOTE: Although no dissection microscope is usually needed for this step, it can be used for improved visualization.</li>
		<li>Open the cranium along the sagittal suture using the #15 scalpel blade. Make sure to cut the cranium by gently moving the blade up and down from anterior to posterior to avoid compression of the cochleae. 
		&#8203;NOTE: The cranium should not be ossified at this age and should cut easily.</li>
		<li>Remove and discard the snout by making a vertical cut directly posterior to the orbits using the #15 scalpel blade. 
		&#8203;NOTE: The posterior part of the skull should still contain the brain and cochleae.</li>
		<li>Dispose of the forebrain, cerebellum, and brainstem through blunt dissection with #4 forceps.</li>
	</ol>
	</li>
	<li>Microscopic extraction of the cochleae
	<ol>
		<li>Adjust the microscope (6.5X&#160;magnification) and light source by placing forceps under the scope and optimizing the illumination and focus.</li>
		<li>Place the two halves of the cranium into the 60 mm plastic petri dish filled with cold HBSS.</li>
		<li>Completely expose the cochlea/e, identifiable as being adjacent to the surrounding stapedial artery (a tortuous artery within the temporal bone), and bluntly separate the organ from the temporal bone using #4 forceps.</li>
		<li>Transfer the cochlea into the 50 mm, clear-walled, glass-bottomed petri dish and position the dish under the microscope.</li>
		<li>Place the 60 mm plastic petri dish with the other half of the cranium on ice.</li>
		<li>Remove the cochlea from the vestibular system and carefully dissect the cochlear otic capsule (typically cartilaginous at this age) with #4 forceps. Use #55 forceps for all further steps.</li>
	</ol>
	</li>
	<li>Final steps of tissue processing
	<ol>
		<li>Continue with the processing of the inner ear tissue by carefully separating the spiral ligament adherent to the organ of Corti from the rest of the cochlea and the modiolus using the micro knife or two forceps.
		<ol>
			<li>Cut into the darker, translucent area at the crevice between the spiral ligament and the hair cell region and proceed to the subsequent removal of the spiral ligament by grasping it at the most basal aspect and gently unwinding it while moving apically.</li>
		</ol>
		</li>
		<li>Position the specimen to obtain a longitudinal, sagittal view of the cochlea and cut the cochlea into two to three sections using the micro knife, classifying these sections as the apical, (middle,)&#160;and basal turns. Remove the tissue superior and inferior to the actual layer of hair cells and neurites in order to achieve a cochlear explant piece that can lay horizontally on the petri dish. 
		&#8203;NOTE: An appropriate size for stable adhesion to the culture dish is approximately half of one cochlear turn.</li>
		<li>Remove the tectorial membrane, a very thin translucent layer immediately superior to the organ of Corti, by gently peeling it away with forceps.</li>
		<li>Remove Reissner&#39;s membrane, immediately superior to the SGNs, by touching it with forceps on both sides and peeling it away. 
		NOTE:&#160;Remove any laterally-located damaged hair cell areas by cutting them away with the micro knife.</li>
	</ol>
	</li>
</ol>
3. Transfer of Specimens to the Culture Plates
<ol>
	<li>Remove the 45 &#181;L of tissue adhesive solution from the four cover slips. Wash each cover slip with 50 &#181;L of sterile H2O. Aspirate the water.</li>
	<li>Pick up the 1 mL pipette. Wet the pipette tip with approximately 120 &#181;L of DMEM to prevent the specimen from adhering to the inside of the tip.</li>
	<li>Transfer the specimens individually using the 1 mL pipette. Pipette a maximum of 70 &#181;L from the HBSS-filled, small, clear-walled, glass-bottomed petri dish and place the specimen along with the liquid on one of the 4 inlays (prepared with cover slips) in the 4-well petri dish.</li>
	<li>Check under the microscope (50X magnification) that the specimen is in the correct orientation. Ensure that the area of hair cells from which the tectorial membrane was removed faces upwards. Ensure that the basilar membrane, together with the trimmed, basal part of the modiolus, faces downwards and adheres to the coverslip.
	<ol>
		<li>If the specimen is oriented upside-down upon inspection, deposit the HBSS that remains in the pipette tip into the inlay and reposition the piece until it is correctly oriented. 
		NOTE: This will prevent hair cells from floating in culture medium without structural support from the coverslip, which could lead to degeneration.</li>
	</ol>
	</li>
	<li>Aspirate excess HBSS using the 1 mL pipette. Wait for 15 s.</li>
	<li>Pipette 60 &#181;L of the prepared warm culture medium (98% DMEM, 1% N-2, and 1% ampicillin) directly onto the specimen. Take care not to touch the specimen with the pipette. Replace the inner and outer petri dish covers and incubate O/N at 37 &#176;C.</li>
	<li>Put all of the stock solutions back in their proper places, dispose of carcasses and residual waste, decontaminate the surgical table according to institutional guidelines (e.g., using ethanol or hypochlorite), clean and autoclave the instruments, and discard the sharps in the appropriate container.</li>
</ol>
4. Adding Final Culture Media and Substances of Interest
NOTE: Cochlear explants will be ready for use 10-16 h later.
<ol>
	<li>Prepare a mixture of 97% DMEM, 1% fetal bovine serum (FBS), 1% N-2, and 1% ampicillin (65 &#181;L per well in a microcentrifuge tube; warm the solution to 25 &#176;C prior to use).</li>
	<li>Add other substances of interest to the solutions for the respective treatment groups. If fluorescent substances are added (e.g., green fluorescent protein (GFP)-expressing viruses; in a recent publication, a concentration of 1010 GC of Adeno-Associated Viruses (AAVs) was used for 48 h in 50 &#181;L)18, protect from light.</li>
	<li>Transfer the cochlear explant-containing petri dishes from the incubator and place them under the laminar flow hood.</li>
	<li>Aspirate the old culture medium (w/o FBS) with the pipette from the inlays.</li>
	<li>Add 60 &#181;L per well of the prepared warm culture (treatment) medium (97% DMEM, 1% FBS, 1% N-2, 1% ampicillin, and the substances of interest).</li>
	<li>Place the petri dish back into the incubator (37 &#176;C).</li>
	<li>View regularly under the microscope to identify signs of contamination, cellular migration, or detachment of the cochlear explant and perform staining after a maximum of 7 days.</li>
</ol>
5. Immunofluorescence - Day 1
<ol>
	<li>Prepare blocking solution (94% Phosphate-Buffered Saline (PBS), 5% normal goat or horse serum&#160;(NGS/NHS, depending on the secondary antibodies), and 1% non-ionic surfactant (see the Table of Materials)) and stock antibody solution (98.6% PBS, 1% NHS, and 0.4% non-ionic surfactant with the respective antibodies). 
	NOTE: Antibodies include rabbit anti-Myo7A at a concentration of 1:400+ (myosin 7A, for inner and outer hair cells) and mouse anti-TuJ1 1:200+ (&#946;-tubulin, for SGNs) OR mouse(IgG1) anti-CtBP2 1:200+ (c-terminal binding protein, for presynapse), mouse(IgG2a) anti-PSD95 1:50+ (postsynaptic density, for postsynapse), and chicken anti-NF-H 1:1000+ (neurofilament, for SGNs and nerve fibers). &#34;+&#34; means &#34;or higher.&#34;</li>
	<li>Aspirate the culture medium using a pipette. Take care not to touch the specimen.</li>
	<li>Under the laminar flow hood, rinse the cochlear explants twice with sterile PBS, placing the specimens on a shaker for 5 min after each wash.</li>
	<li>Fix the tissue in 4% paraformaldehyde (PFA - CAUTION) for 20 min on the shaker (under the hood). Aspirate the PFA.</li>
	<li>Apply 60 &#181;L of PBS to the cochlear explants and place the petri dish on a shaker for 5 min. Aspirate the PBS. Repeat 3x.</li>
	<li>Place the cochlear explants in prepared blocking solution (94% PBS, 5% NHS, and 1% non-ionic surfactant) for 30 min on shaker.</li>
	<li>Place the cochlear explants in a prepared stock antibody solution (98.6% PBS, 1% NHS, and 0.4% non-ionic surfactant) with the respective primary antibodies (vortex before use) O/N on a counter at RT. 
	NOTE:&#160;Wrap the petri dishes with damp paper towels enclosed in plastic wrap (or aluminum foil if the added solution contains fluorescent materials such as GFP-expressing viruses) to keep the dishes humid and to prevent evaporation of the antibody solution O/N.</li>
</ol>
6. Immunofluorescence - Day 2
<ol>
	<li>Prepare the 4&#39;,6-diamidino-2-phenylindole (DAPI) stain (300 nM) and use the stock antibody solution to obtain the correct secondary antibody mixtures, complementary to the primary antibodies in step 5.1. (e.g., goat anti-rabbit 488 1:200+ and goat anti-mouse 568 1:200+ OR goat anti-mouse (IgG1) 568 1:1000+, goat anti-mouse (IgG2a) 488 1:500+, and goat anti-chicken 647 1:200+ OR Phalloidin 568 1:200+ (for inner and outer hair cells)).</li>
	<li>Aspirate the primary antibody solution.</li>
	<li>Apply 60 &#181;L of PBS to the cochlear explants and place the petri dish on a shaker for 5 min. Aspirate the PBS. Repeat 3x.</li>
	<li>Place the cochlear explants in prepared stock antibody solution (98.6% PBS, 1% NHS, and 0.4% non-ionic surfactant) with the respective secondary antibodies (vortex before use) for 1.5 h on a counter and protect from light.</li>
	<li>Aspirate the secondary antibody solution and rinse the cochlear explants with a DAPI stain (placing on a shaker for 1 min and then aspirate).</li>
	<li>Apply 60 &#181;L of PBS to the cochlear explants and place the petri dish on a shaker for 5 min. Aspirate the PBS. Repeat 3x.</li>
	<li>Use forceps to remove coverslips with specimens from the 4-well plate, dip them into a recipient filled with distilled H2O, and dry the coverslips by vertically bringing them into contact with paper towels.</li>
	<li>Place a drop of antifade mounting medium onto the microscope slide and invert the coverslips to immerse the downward-facing tissue in the medium.</li>
	<li>Seal the coverslip using clear nail polish circumferentially covering the edge (without crushing the specimen under the glass). Leave the slides lying flat in a covered area away from light for 15 - 20 min until dry and then place them in a box or examine under the confocal microscope. 
	NOTE: Fluorescent staining is best preserved by storing the slides at 4 or -20 &#176;C.</li>
</ol>
7. Confocal Imaging
<ol>
	<li>Obtain an overview and zoomed-in pictures for the organ of Corti region, including neurites and SGNs. 
	NOTE: Standard settings for the imaging process: Format of 1,024 x 1,024 pixels, speed of 400 Hz, frame average of 3, 20% overall laser intensity, and usually 20X and 63X&#160;lens with oil immersion (potentially combined with 2X digital zoom). Channel settings for the DAPI, Myo7A, and TuJ1 staining protocol are outlined above: 405 5% DAPI, &#34;Smart Gain&#34; 766.8V, &#34;Smart Offset&#34; 0.0% - 488 15% fluorescein isothiocyanate (FITC), &#34;Smart Gain&#34; 622.2V, &#34;Smart Offset&#34; 0.0% - 561 13% tetramethylrhomadine isothiocyanate (TRITC), &#34;Smart Gain&#34; 562.4V, &#34;Smart Offset&#34; 0.0%.</li>
	<li>Merge the images in a z-stack using the software to obtain a z-axis projection.</li>
</ol>

<ol>
	<li>Geleoc, G. S., Holt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sound+strategies+for+hearing+restoration).">Sound strategies for hearing restoration).</a> Science. 344 (6184), 1241062 (2014).</li><li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+age-related+hearing+loss+by+early+noise+exposure:+evidence+of+a+misspent+youth.">Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth.</a> J Neurosci. 26 (7), 2115-2123 (2006).</li><li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+insult+to+injury:+cochlear+nerve+degeneration+after+&amp;#34;temporary&amp;#34;+noise-induced+hearing+loss.">Adding insult to injury: cochlear nerve degeneration after &amp;#34;temporary&amp;#34; noise-induced hearing loss.</a> J Neurosci. 29 (45), 14077-14085 (2009).</li><li>Makary, C. A., Shin, J., Kujawa, S. G., Liberman, M. C., Merchant, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+primary+cochlear+neuronal+degeneration+in+human+temporal+bones.">Age-related primary cochlear neuronal degeneration in human temporal bones.</a> J Assoc Res Otolaryngol. 12 (6), 711-717 (2011).</li><li>Jensen, J. B., Lysaght, A. C., Liberman, M. C., Qvortrup, K., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+and+delayed+cochlear+neuropathy+after+noise+exposure+in+pubescent+mice.">Immediate and delayed cochlear neuropathy after noise exposure in pubescent mice.</a> PLoS One. 10 (5), (2015).</li><li>Landegger, L. D., Psaltis, D., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+audiometric+thresholds+do+not+predict+specific+cellular+damage+in+the+inner+ear.">Human audiometric thresholds do not predict specific cellular damage in the inner ear.</a> Hear Res. , 83-93 (2016).</li><li>Wang, H. C., 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=Spontaneous+Activity+of+Cochlear+Hair+Cells+Triggered+by+Fluid+Secretion+Mechanism+in+Adjacent+Support+Cells.">Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells.</a> Cell. 163 (6), 1348-1359 (2015).</li><li>Barclay, M., Ryan, A. F., Housley, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+vs+type+II+spiral+ganglion+neurons+exhibit+differential+survival+and+neuritogenesis+during+cochlear+development.">Type I vs type II spiral ganglion neurons exhibit differential survival and neuritogenesis during cochlear development.</a> Neural Dev. 6, (2011).</li><li>Dinh, C., 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=Short+interfering+RNA+against+Bax+attenuates+TNFalpha-induced+ototoxicity+in+rat+organ+of+Corti+explants.">Short interfering RNA against Bax attenuates TNFalpha-induced ototoxicity in rat organ of Corti explants.</a> Otolaryngol Head Neck Surg. 148 (5), 834-840 (2013).</li><li>Mazurek, B., Yu, Y., Haupt, H., Szczepek, A. J., Olze, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salicylate+modulates+Hsp70+expression+in+the+explanted+organ+of+Corti.">Salicylate modulates Hsp70 expression in the explanted organ of Corti.</a> Neurosci Lett. 501 (2), 67-71 (2011).</li><li>Kao, S. 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=Loss+of+osteoprotegerin+expression+in+the+inner+ear+causes+degeneration+of+the+cochlear+nerve+and+sensorineural+hearing+loss.">Loss of osteoprotegerin expression in the inner ear causes degeneration of the cochlear nerve and sensorineural hearing loss.</a> Neurobiol Dis. 56, 25-33 (2013).</li><li>Jan, T. A., Chai, R., Sayyid, Z. N., Cheng, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+LacZ-expressing+cells+from+mouse+inner+ear+tissues+using+flow+cytometry.">Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry.</a> J Vis Exp. (58), e3432 (2011).</li><li>Haque, K. D., Pandey, A. K., Kelley, M. W., Puligilla, C. <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+embryonic+mouse+cochlear+explants+and+gene+transfer+by+electroporation.">Culture of embryonic mouse cochlear explants and gene transfer by electroporation.</a> J Vis Exp. (95), e52260 (2015).</li><li>Parker, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+Corti.">Primary culture and plasmid electroporation of the murine organ of Corti.</a> J Vis Exp. (36), (2010).</li><li>Mulvaney, J. F., Dabdoub, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+time+lapse+imaging+of+mouse+cochlear+explants.">Long-term time lapse imaging of mouse cochlear explants.</a> J Vis Exp. (93), e52101 (2014).</li><li>Wang, Q., Green, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+neurotrophin-3+in+synapse+regeneration+by+spiral+ganglion+neurons+on+inner+hair+cells+after+excitotoxic+trauma+in+vitro.">Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro.</a> J Neurosci. 31 (21), 7938-7949 (2011).</li><li>Landegger, L. D., 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+synthetic+AAV+vector+enables+safe+and+efficient+gene+transfer+to+the+mammalian+inner+ear.">A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear.</a> Nat Biotechnol. 35 (3), 280-284 (2017).</li><li>Dilwali, S., Landegger, L. D., Soares, V. Y., Deschler, D. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Factors+from+Human+Vestibular+Schwannomas+Can+Cause+Cochlear+Damage.">Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage.</a> Sci Rep. 5, 18599 (2015).</li><li>Soares, V. 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=Extracellular+vesicles+derived+from+human+vestibular+schwannomas+associated+with+poor+hearing+damage+cochlear+cells.">Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.</a> Neuro Oncol. , (2016).</li><li>Tong, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerated+synapses+between+postnatal+hair+cells+and+auditory+neurons.">Regenerated synapses between postnatal hair cells and auditory neurons.</a> J Assoc Res Otolaryngol. 14 (3), 321-329 (2013).</li><li>Yuan, 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=Ouabain-induced+cochlear+nerve+degeneration:+synaptic+loss+and+plasticity+in+a+mouse+model+of+auditory+neuropathy.">Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.</a> J Assoc Res Otolaryngol. 15 (1), 31-43 (2014).</li><li>Fernandez, K. A., Jeffers, P. W., Lall, K., Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+after+noise+exposure:+acceleration+of+cochlear+synaptopathy+in+&amp;#34;recovered&amp;#34;+ears.">Aging after noise exposure: acceleration of cochlear synaptopathy in &amp;#34;recovered&amp;#34; ears.</a> J Neurosci. 35 (19), 7509-7520 (2015).</li><li>Barclay, M., Constable, R., James, N. R., Thorne, P. R., Montgomery, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+sensory+stimulation+alters+the+molecular+make-up+of+glutamatergic+hair+cell+synapses+in+the+developing+cochlea.">Reduced sensory stimulation alters the molecular make-up of glutamatergic hair cell synapses in the developing cochlea.</a> Neuroscience. , 50-62 (2016).</li><li>Zinn, 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=In+Silico+Reconstruction+of+the+Viral+Evolutionary+Lineage+Yields+a+Potent+Gene+Therapy+Vector.">In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.</a> Cell Rep. 12 (6), 1056-1068 (2015).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Mol Ther. 18 (1), 80-86 (2010).</li><li>Shu, 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=Identification+of+Adeno-Associated+Viral+Vectors+That+Target+Neonatal+and+Adult+Mammalian+Inner+Ear+Cell+Subtypes.">Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes.</a> Hum Gene Ther. , (2016).</li><li>Kao, S. Y., Soares, V. Y., Kristiansen, A. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRAIL-DR5+pathway+promotes+sensorineural+degeneration+in+the+inner+ear.">Activation of TRAIL-DR5 pathway promotes sensorineural degeneration in the inner ear.</a> Aging Cell. 15 (2), 301-308 (2016).</li></ol>

The authors have nothing to disclose.

Researchers must perfect the dissection technique before carrying out experiments involving cochlear explants. Hair cells are commonly damaged during dissections performed early on in the learning curve, and a particularly problematic moment for their integrity is the removal of the tectorial membrane, which requires steady hands, proper tools, and experience. To save time and resources, a visual control should be performed under the dissection microscope and potentially damaged areas should be noted. Instead of using expensive primary and secondary antibodies, cheaper reagents like phalloidin are more appropriate for preliminary experiments. Slightly damaged explants can potentially still be used as controls in certain experiments (analysis of sections that have not been affected) or for the testing of new combinations of antibodies.
Transfer of the specimens is also particularly important, because one must guarantee that the pieces are not oriented upside-down (&#34;floating&#34; hair cells without the guidance of the coated coverslip often degenerate). A central position on the coverslip facilitates all further pipetting steps.
For negative control experiments involving novel compounds (that obviously must be soluble in the culture medium), it is important to apply the appropriate vehicle to the explants and to not focus exclusively on untreated explants. For example, if a commercial drug includes sodium citrate in addition to the active ingredient, then sodium citrate should be added to the negative control cochlear explants because sodium citrate might itself have an effect.
Limitations to the use of cochlear explants include the fact that these organotypic cultures are typically derived from young pups that are still undergoing postnatal developmental changes and that there is a lack of an ionic gradient across the explant, which is essential for normal hearing in vivo. However, the utility of the protocol is compellingly shown in a recent publication regarding AAVs18. While this group of vectors is limited to about 4.7 kb of packaging capacity, it has become an important resource for the therapy of several human syndromes26. Using the cochlear explant model, the above-mentioned publication demonstrated that the Anc80 virus outperformed the traditional AAV serotypes in the transduction of a variety of cochlear cells, including hair cells and SGNs. These in vitro findings were subsequently confirmed by choosing the most promising candidates for injection through the round window in mouse pups in vivo. The ex vivo model provides a way to successfully recapitulate the anatomy of the cochlea, decreasing the time and resources needed for conducting in vivo work27.
Another advantage of the ex vivo model is its potential for application to the study of specific cellular mechanisms that contribute to SNHL. By applying compounds found in the tumor microenvironment (e.g., human tumor secretions) to an explant, the effect of these compounds on the cochlea can be directly visualized and the efficacy or toxicity of potential drug therapies assessed. This is important because the human cochlea cannot be biopsied, and the cells inside it cannot be visualized in vivo. For example, a recent publication examined the ototoxic potential of proteins secreted from vestibular schwannomas, which are intracranial tumors that arise from the vestibular nerves and cause hearing loss in 95% of patients. Applying human tumor secretions to cochlear explants, compared to secretions from control healthy human nerves, confirmed the toxic effects of these secretions on the cochlea19. The same experiment would be difficult to replicate in an in vivo model due to the immunogenic response against human proteins that would arise in an immunocompetent mouse.
In summary, this manuscript presents a technique that enables the simultaneous study of cochlear hair cells, synapses, neurites, and neurons in an ex vivo model. This model can help to provide insight into the mechanisms of action of molecules new to the cochlea28, including factors in human secretions19,20, while potentially accelerating the screening of candidate therapeutic compounds18 prior to their testing in vivo.

Sensorineural Hearing Loss (SNHL) reflects damage to the inner ear or ascending auditory pathway. While hearing loss is the most common sensory deficit in humans1, curative therapies do not yet exist2. Although cochlear or auditory brainstem implants can restore some degree of hearing to patients with severe to profound SNHL, the hearing provided by these devices is still very different from &#34;natural&#34; hearing, especially during attempts to understand speech in noise or to listen to music.
While hair cell degeneration has long been considered the primary consequence of traumatic auditory events (e.g., exposure to loud noise), there is growing evidence that the synapses transmitting information from hair cells to the auditory nerve are at least as vulnerable to acoustic trauma3,4,5,6. Since human audiometric thresholds, the current gold standard for the evaluation of hearing function, do not predict specific cellular damage in the inner ear, more refined tools are needed to detect cellular degeneration as soon as possible and to initiate adequate treatment7.
Promising pharmaceutical treatments for hearing loss are often tested on homogenous cell cultures in vitro, but such systems do not accurately model the cochlear microenvironment. Cochlear cells are known to secrete trophic factors that influence other cell types within the cochlea8,9, a crucial in vivo process that is lost when the organ of Corti10,11 or Spiral Ganglion Neurons (SGNs)12 are cultured in isolation or when molecular markers are analyzed13. However, in vivo studies that may be necessary for the validation of in vitro data to establish new, personalized treatments for hearing loss in the pursuit of &#34;precision medicine&#34; require significant resources and time. This is especially relevant when considering how much effort is required to perfect and perform middle ear or round window membrane injections with hearing tests and the subsequent dissection of cochlear whole-mounts. The efficient screening of promising compounds in organotypic ex vivo cultures known as cochlear explants provides an economic and reliable alternative14,15,16,17.
This article details a protocol by which to generate, maintain, and evaluate treated cochlear explants. Specific applications for this model are emphasized, including its use in the screening of potentially therapeutic compounds and the comparative evaluation of viral vectors for gene therapy. An ex vivo explant approach allows researchers to visualize the effects of a given treatment on different cell populations in situ, facilitating the identification of cell type-specific mechanisms and the subsequent refinement of targeted therapeutics.
Overall, this technique provides a model to study the cochlea ex vivo while preserving vital cross-talk between the vastly different cell types that coexist within the cochlea.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ampicillin, Sodium Salt</td>
			<td>Invitrogen</td>
			<td>11593-027</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CtBP2 antibody, mouse(IgG1)</td>
			<td>BD Transduction Laboratories</td>
			<td>612044</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Myo7A antibody, rabbit</td>
			<td>Proteus Biosciences</td>
			<td>25-6790</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-NF-H antibody, chicken</td>
			<td>EMD Millipore</td>
			<td>AB5539</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PSD95 antibody, mouse(IgG2a)</td>
			<td>Antibodies Inc.</td>
			<td>75-028</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-TuJ1 antibody, mouse</td>
			<td>BioLegend</td>
			<td>801202</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Tak Cell and Tissue Adhesive, 5 mg</td>
			<td>Corning</td>
			<td>354241</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR 15 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td>Greiner Bio-One</td>
			<td>188161</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Cell Culture Dish, 100x20 mm</td>
			<td>Greiner Bio-One</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Cell Culture Dish, 35x10 mm, four inner rings</td>
			<td>Greiner Bio-One</td>
			<td>627170</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Cell Culture Dish, 60x15 mm</td>
			<td>Greiner Bio-One</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR 50 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Nail Polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO&#174;, Sleeve/20, 50x7 mm</td>
			<td>Ted Pella Inc.</td>
			<td>14027-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13-0.16mm</td>
			<td>Ted Pella Inc.</td>
			<td>260368</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>15230-162&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate, no glutamine, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>10313-039</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 #55 Forceps (Dumostar)</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure, 200 proof, anhydrous, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>459836-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, USDA-approved regions, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>10437-028&#160;</td>
			<td>Aliquot in 50 ml tubes and store in -20&#176;C freezer</td>
		</tr>
		<tr>
			<td>Glutamate - GlutaMAX supplement, 100 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-chicken-647 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21469</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse(IgG)-568 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11004</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse(IgG1)-568 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21124</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse(IgG2a)-488 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21131</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-rabbit-488 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37116</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O, sterile, EmbryoMax Ultra Pure Water, 500ml</td>
			<td>EMD Millipore</td>
			<td>TMS-006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, calcium, magnesium, no phenol red, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument Tray with Lid Stainless Steel</td>
			<td>Mountainside Medical</td>
			<td>TechMed4255</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro (dissecting) knife &#8211; angled 30&#176;</td>
			<td>Fine Science Tools</td>
			<td>10056-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1&#34;), 1 mm thick, white, pack of 72</td>
			<td>VWR</td>
			<td>16004-382</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X), 5 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3, Sodium Bicarbonate 7.5% solution, 100 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>25080-094</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH, sodium hydroxide solution, 1 l</td>
			<td>Thermo Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Horse Serum (NHS)</td>
			<td>Invitrogen</td>
			<td>16050130</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-6806</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, Reagent Grade, Crystalline</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4, 500 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-023&#160;</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr>
			<td>Phalloidin, Alexa Fluor 568&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>Prep Pad, Non Sterile&#160;</td>
			<td>Medline</td>
			<td>05136CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 ml</td>
			<td>Eppendorf</td>
			<td>022363719</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr>
			<td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 ml</td>
			<td>Eppendorf</td>
			<td>022363204</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr>
			<td>Scalpel Blades - #15</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #4</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 2000-C Stereo Microscope</td>
			<td>Zeiss&#160;</td>
			<td>000000-1106-133</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP5 confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X (non-ionic surfactant)</td>
			<td>Integra</td>
			<td>T756.30.30</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield antifade mounting medium for fluorescence</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Zipper Bag, Reclosable, 4&#39;&#39; x 6&#39;&#39; - 2 mil. thick</td>
			<td>Zipline</td>
			<td>0609132541599</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

While many protocols focus on organ of Corti explants, this technique attempts to preserve the anatomy of the entire cochlear turn, including the SGNs. This gives researchers the opportunity to analyze the effects of a given treatment on neurites and somata of SGNs in addition to the organ of Corti. Performing a dissection that preserves part of the modiolus, as described here, is more technically challenging than explanting the organ of Corti alone. However, the neurite and SGN area is important, because significant pathological effects were observed in this region after applying vestibular schwannoma secretions (Figure 1) and extracellular vesicles in two recent publications using the same methodology19,20. Such changes could not have been revealed by using isolated organ of Corti explants.
The quantification of damaged or GFP-expressing cells for inner and outer hair cells, supporting cells, and SGNs can be performed by manual count or with an automated process, such as that built into the ImageJ plugin NeuronJ. Representative areas for these counts can be established after confirming their correlation with total counts. 3D reconstructions are particularly useful to exclude overlay effects in the z-axis projections of SGNs. Fiber organization, number of SGNs per given area, and SGN soma area can be evaluated. The staining and counting of synapses, hair cells, and neurites is depicted in Figure 2; the same protocol can also be used for cochlear whole mounts and has been shown to be a reliable model for these purposes17,21,22. Other researchers have established elegant alternative methods that can also be incorporated into experimental paradigms23,24.
The maximum length of culture is usually limited by the start of cellular migration and depends upon the concentration of FBS in the culture medium. When the proportion of FBS was reduced from five percent to three percent or one percent, the time period for maintaining an anatomically intact cochlear explant was extended to approximately one week (Figure 3).
Figure 4 demonstrates GFP-positive cochlear explant cells after transduction with the synthetic adeno-associated virus vector Anc80 for 48 h25. Inner hair cells, outer hair cells, and supporting cells can be quantified in this view, while a 3D reconstruction can help to quantify GFP-positive cells in the SGN area. Different viral serotypes can be compared in terms of their transduction efficiency in certain cell types using the outlined methodology18.
<img alt="Figure 1" src="/files/ftp_upload/55704/55704fig1.jpg" /> 
Figure 1: Representative Confocal Microscopy Images of Cochlear Explants from the Apical and Basal Regions after Incubation with Secretions from Great Auricular Nerves or Vestibular Schwannomas for 48 h. (A) Overview image of an explant. The rectangle marks the area in close-up images. Scale bar = 200 &#181;m. (B-E) Zoomed-in views of hair cells and neurites. Scale bar = 50 &#181;m. Green = Myosin 7A (Myo7A), stains hair cells; red = class III beta-tubulin (Tuj1), stains neuronal structures. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55704/55704fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55704/55704fig2.jpg" /> 
Figure 2: Confocal Microscopy Image of the Cochlear Explant Hair Cell Region. (A) Intact synapses can be assessed by labeling presynaptic (C-terminal binding protein, red) and postsynaptic (postsynaptic density-95, green) proteins together with the staining of neuronal structures (neurofilament, blue). OHC, outer hair cells; IHC, inner hair cells. Scale bar = 10 &#181;m. (B) Focus on the inner hair cell region with the count of inner hair cells (red arrowheads) and neurites (blue arrowheads). Due to the ramification of the neuronal structures, it is important to standardize the distance from the inner hair cells during counting (highlighted with the white frame, directly connected to the synapses). Scale bar = 5 &#181;m. (C) Close-up of separate synapses (2 pre- and postsynaptic pairs marked with white arrowheads). Scale bar = 1 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55704/55704fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55704/55704fig3.jpg" /> 
Figure 3: Representative Confocal Microscopy Images of Apical and Basal Cochlear Explants after 7 d in Culture with either 3% or 1% FBS. (A and B) Cells incubated in 3% FBS start to migrate between&#160;4 and 5 days (light gray arrows for hair cells, red arrows for neurites), (C and D) Explants in 1% FBS maintain organizational integrity until day 7. Light gray: Myosin 7A (Myo7A), stains all hair cells; red: class III beta-tubulin (Tuj1), stains neuronal structures. OHC, outer hair cells; IHC, inner hair cells. Scale bar = 100 &#181;m, applied to all panels. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55704/55704fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55704/55704fig4.jpg" /> 
Figure 4: Confocal Microscopy Image of a Cochlear Explant after Exposure to the Synthetic Adeno-associated Virus Vector Anc80 for 48 h. Transduced cells express GFP (green). Myosin 7A (Myo7A, blue) stains Outer Hair Cells (OHC) and Inner Hair Cells (IHC); class III beta-tubulin (Tuj1, red) stains neuronal structures. Supp.: area of Sox2-positive supporting cells; SGN, spiral ganglion neurons. Scale bar: 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55704/55704fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

Technika przeszczepu ślimaka u noworodków u myszy jako narzędzie badań przesiewowych in vitro w badaniach słuchu | Medycyna | Wideo

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.

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 ...

neonatal-murine-cochlear-explant-technique-as-an-vitro-screening-tool

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Medicine

Human Biology

Sensory Systems

SCIENTISTS IN ACTION

17.4K Views.  Massachusetts Eye and Ear. 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.

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

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Performing Intracochlear Electrocochleography During Cochlear Implantation

Electrocochleography (ECochG) measures inner ear potentials generated in response to acoustic stimulation of the ear. These potentials reflect the residual function of the cochlea. In cochlear implant candidates with residual hearing, the implant electrode can directly measure ECochG responses during the implantation process. Various authors have described the ability to monitor the inner ear function by continuous ECochG measurements during the surgery. The measurement of ECochG signals during surgery is not trivial. There are no interpretable signals in up to 20% of cases. For a successful recording, a standardized procedure is recommended to achieve the highest measurement reliability and avoid possible pitfalls. Therefore, seamless collaboration between the CI surgeon and CI technician is key. This video consists of an overview of the system setup and a stepwise procedure of performing intracochlear ECochG measurements during CI surgery. It shows the surgeon's and the CI technician's roles in the process, and how a smooth collaboration between the two is made possible.

Electrocochleography (ECochG) measures inner ear potentials generated in response to acoustic stimulation. In cochlear implant (CI) candidates, such inner ear potentials can be measured directly with the implant electrodes. In this video, we systematically explain how to perform ECochG recordings during CI surgery.

Electrocochleography (ECochG) measures inner ear potentials generated in response to acoustic stimulation of the ear. These potentials reflect the ...

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar ...

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...