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 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.
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 "natural" 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 "precision medicine" 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.
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
2. Murine Cochlear Explant Dissection
3. Transfer of Specimens to the Culture Plates
4. Adding Final Culture Media and Substances of Interest
NOTE: Cochlear explants will be ready for use 10-16 h later.
5. Immunofluorescence – Day 1
6. Immunofluorescence – Day 2
7. Confocal Imaging
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.
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 µm. (B–E) Zoomed-in views of hair cells and neurites. Scale bar = 50 µm. Green = Myosin 7A (Myo7A), stains hair cells; red = class III beta-tubulin (Tuj1), stains neuronal structures. Please click here to view a larger version of this figure.
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 µm. (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 µm. (C) Close-up of separate synapses (2 pre- and postsynaptic pairs marked with white arrowheads). Scale bar = 1 µm. Please click here to view a larger version of this figure.
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 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 µm, applied to all panels. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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 ("floating" 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.
The authors have nothing to disclose.
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.
Ampicillin, Sodium Salt | Invitrogen | 11593-027 | |
anti-CtBP2 antibody, mouse(IgG1) | BD Transduction Laboratories | 612044 | |
anti-Myo7A antibody, rabbit | Proteus Biosciences | 25-6790 | |
anti-NF-H antibody, chicken | EMD Millipore | AB5539 | |
anti-PSD95 antibody, mouse(IgG2a) | Antibodies Inc. | 75-028 | |
anti-TuJ1 antibody, mouse | BioLegend | 801202 | |
Cell-Tak Cell and Tissue Adhesive, 5 mg | Corning | 354241 | |
CELLSTAR 15 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile | Greiner Bio-One | 188161 | |
CELLSTAR Cell Culture Dish, 100×20 mm | Greiner Bio-One | 664160 | |
CELLSTAR Cell Culture Dish, 35×10 mm, four inner rings | Greiner Bio-One | 627170 | |
CELLSTAR Cell Culture Dish, 60×15 mm | Greiner Bio-One | 628160 | |
CELLSTAR 50 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile | Greiner Bio-One | 227261 | |
Clear Nail Polish | Electron Microscopy Sciences | 72180 | |
Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO®, Sleeve/20, 50×7 mm | Ted Pella Inc. | 14027-20 | |
Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13-0.16mm | Ted Pella Inc. | 260368 | |
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | |
Distilled water, 500 ml | Thermo Fisher Scientific | 15230-162 | |
DMEM, high glucose, pyruvate, no glutamine, 500 ml | Thermo Fisher Scientific | 10313-039 | |
Dumont #4 Forceps | Fine Science Tools | 11241-30 | |
Dumont #55 Forceps (Dumostar) | Fine Science Tools | 11295-51 | |
Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5% | Sigma-Aldrich | 459836-1L | |
Fetal Bovine Serum, qualified, USDA-approved regions, 500 ml | Thermo Fisher Scientific | 10437-028 | Aliquot in 50 ml tubes and store in -20°C freezer |
Glutamate – GlutaMAX supplement, 100 ml | Thermo Fisher Scientific | 35050-061 | |
goat anti-chicken-647 secondary antibody | Thermo Fisher Scientific | A-21469 | |
goat anti-mouse(IgG)-568 secondary antibody | Thermo Fisher Scientific | A-11004 | |
goat anti-mouse(IgG1)-568 secondary antibody | Thermo Fisher Scientific | A-21124 | |
goat anti-mouse(IgG2a)-488 secondary antibody | Thermo Fisher Scientific | A-21131 | |
goat anti-rabbit-488 secondary antibody | Thermo Fisher Scientific | R37116 | |
H2O, sterile, EmbryoMax Ultra Pure Water, 500ml | EMD Millipore | TMS-006-B | |
HBSS, calcium, magnesium, no phenol red, 500 ml | Thermo Fisher Scientific | 14025-092 | |
Instrument Tray with Lid Stainless Steel | Mountainside Medical | TechMed4255 | |
Micro (dissecting) knife – angled 30° | Fine Science Tools | 10056-12 | |
Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1"), 1 mm thick, white, pack of 72 | VWR | 16004-382 | |
N-2 Supplement (100X), 5 ml | Thermo Fisher Scientific | 17502-048 | |
NaHCO3, Sodium Bicarbonate 7.5% solution, 100 ml | Thermo Fisher Scientific | 25080-094 | |
NaOH, sodium hydroxide solution, 1 l | Thermo Fisher Scientific | SS266-1 | |
Normal Horse Serum (NHS) | Invitrogen | 16050130 | |
Operating scissors | Roboz Surgical Instruments Co. | RS-6806 | |
Paraformaldehyde, Reagent Grade, Crystalline | Sigma-Aldrich | P6148 | Prior to use: Establish Standard Operating Procedures based on protocols available online |
PBS, pH 7.4, 500 ml | Thermo Fisher Scientific | 10010-023 | Autoclave prior to use |
Phalloidin, Alexa Fluor 568 | Thermo Fisher Scientific | A12380 | |
Prep Pad, Non Sterile | Medline | 05136CS | |
Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 ml | Eppendorf | 022363719 | Autoclave prior to use |
Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 ml | Eppendorf | 022363204 | Autoclave prior to use |
Scalpel Blades – #15 | Fine Science Tools | 10015-00 | |
Scalpel Handle – #4 | Fine Science Tools | 10004-13 | |
Stemi 2000-C Stereo Microscope | Zeiss | 000000-1106-133 | |
TCS SP5 confocal microscope | Leica | N/A | |
Triton-X (non-ionic surfactant) | Integra | T756.30.30 | |
VectaShield antifade mounting medium for fluorescence | Vector Laboratories, Inc. | H-1000 | |
Zipper Bag, Reclosable, 4'' x 6'' – 2 mil. thick | Zipline | 0609132541599 |