Name: neonatal murine cochlear explant technique as an in vitro screening tool in hearing research
Uploaded: 2017-06-08T15:00:00
Description: Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

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 ope...

<ol>
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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 ex...

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">
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			<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>
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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 i...

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

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 ...

The overall goal of this procedure is the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants to be used as an in vitro screening tool in hearing research. This method can help answer key questions in the hearing research field, such as enabling the simultaneous study of cochlear hair cells, synapses, neurites, and neurons in an ex vivo model and providing insight into mechanisms of action of molecules new to the cochlea, including factors found in human secretions. The main advantage of this technique is that the screening process of candidate therapeutic compounds can be accelerated prior to their testing in vivo.Begin with a freshly collected neonate head contained in 70%ethanol in the lid of a Petri dish. Use a scalpel blade to make a superficial cut in the skin from the anterior to the posterior end of the skull. Then cut both external auditory canals and fold the skin anteriorly to expose the whole cranium.Next, open the cranium along the sagittal suture by gently moving the scalpel blade up and down from anterior to posterior to avoid compression of the cochlea. Remove and discard the snout by making a vertical cut directly posterior to the orbits. The posterior part of the skull should still contain the brain and cochlea.Then use 4 forceps to bluntly dissect and dispose of the forebrain, cerebellum, and brain stem. Place the two halves of the cranium into a 60 millimeter plastic Petri dish filled with cold HBSS. Then use 4 forceps to completely expose the cochlea which is adjacent to the surrounding stapedial artery and bluntly separate the organ from the temporal bone.Transfer the cochlea into a 50 millimeter clear walled glass bottom Petri dish containing HBSS and position the dish under the microscope. Place the 60 millimeter plastic Petri dish with the other half of the cranium on ice. Next, use 4 forceps to carefully dissect the cochlear otic capsule.Now, use a micro knife or two 55 forceps to carefully separate the spiral ligament that is adhered to the organ of Corti from the rest of the cochlea and the medialis. This can be done by grasping the spiral ligament by the most basal aspect and gently unwinding it while moving apically. After the spiral ligament is removed, position the specimen to obtain a longitudinal sagittal view of the cochlea.Then cut the cochlea into two or three sections using the micro knife, classifying these sections as the apical and basal turns. Remove the tissue superior for the more apical piece and inferior for the more basal piece, down to the actual layer of hair cells and neurites to allow the cochlear explant to lay horizontally on the Petri dish. Next, use forceps to gently grasp and peel away the tectorial membrane, a very thin translucent layer immediately superior to the organ of Corti.The most technically challenging step is likely the removal of the thin translucent tectorial membrane as one can easily damage the hair cells underneath. By starting from the edges of the tissue, hair cells along the length of the explant are preserved, except at the very edge where peeling of the tectorial membrane began. Lastly, remove Reissner's membrane immediately superior to the spiral ganglion neurons by touching it with forceps on both sides and peeling it away.The cochlear specimen is now ready for culturing after removing any damaged areas on the edges. Begin the culture procedure by wetting the tip of a one milliliter pipette with approximately 120 microliters of DMEM to prevent the explant from adhering to the inside of the tip. Then use the pre-wetted tip to pipette a maximum of 70 microliters from the glass bottom Petri dish and place the specimen along with the liquid on one of four inlays in the four-well Petri dish.Check under the microscope that the explant is in the correct orientation. Ensure that the area of hair cells from which the tectorial membrane was removed faces upwards and that the basilar membrane together with the trimmed basal part of the medialis faces downwards and adheres to the coverslip inlay. If the specimen is orientated upside down upon inspection, as shown here, add HBSS from the pipette tip and reposition the piece until it is correctly oriented.Once the explant is correctly oriented, aspirate excess HBSS then wait for 15 seconds. Now, pipette 60 microliters of warm culture medium directly unto the specimen, taking care not to touch the specimen with the pipette. Replace the inner and outer Petri dish covers and incubate overnight at 37 degrees Celsius.The next day, transfer the cochlear explant containing Petri dishes from the incubator to the laminar flow hood. Aspirate the old culture medium which does not contain FBS from the inlay. Add 60 microliters per well of the prepared warm treatment medium.Place the Petri dish back into the incubator until staining of the cultures after a maximum of seven days. As seen in this confocal image, this technique preserves the anatomy of the entire cochlear turn including the spiral ganglion neurons or SGNs, allowing analysis of treatment effects on neurites and somata of SGNs in addition to the organ of Corti. As seen here, these explants can be used to simultaneously compare the effect of treatment on apical and basal turns after immunofluorescent staining.Hair cells are shown in green and neuronal structures appear red. Intact synapses can be assessed by labeling pre-synaptic proteins shown in red and post-synaptic proteins shown in green together with the staining of neuronal structures in blue. This figure shows representatives images of apical and basal cochlear explants after seven days in culture with either 3%FBS on the left or 1%FBS on the right.Cells incubated in 3%FBS start to migrate between days four and five as shown by the light gray arrows for hair cells and the red arrows for neurites. Explants in 1%FBS maintain organizational integrity until day seven. While attempting this procedure, it's important to remember to visually ensure the structural integrity and orientation of the cochlea tissue before adding the final culture media and substances of interest.Following this procedure, 3D reconstruction can be performed to confirm cell type specific localization of GFP expressing cells within the medialis.

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