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

Summary

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.

Abstract

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.

Introduction

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 auditor....

Protocol

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

1. Preparing the surgical table
   1. Use 70% ethanol to disinfect the surgical table.
   2. Place two nonsterile prep pads next to the microscope.
   3. Prepare the instrument tray, including heat-sterilized operating scissors, a scalpel handle, a micro knife, and forceps (two each of #4 & #55). Place the instrument tray and a 50 mm, clear-walled, glass-b....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 x 20 mm	Greiner Bio-One	664160
CELLSTAR Cell Culture Dish, 35 x 10 mm, 4 inner rings	Greiner Bio-One	627170
CELLSTAR Cell Culture Dish, 60 x 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 x 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
Dulbecco's Modified Eagle's Medium (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 (EtOH), Pure, 200 proof, anhydrous, ≥99.5%	Sigma-Aldrich	459836-1L
Fetal Bovine Serum (FBS), qualified, USDA-approved regions, 500 mL	Thermo Fisher Scientific	10437-028	Aliquot in 50 mL tubes and store in -20°C freezer
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, 500 mL	EMD Millipore	TMS-006-B
Hank's Balanced Salt Solution (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 (PFA), 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 mm thick	Zipline	0609132541599