Cochlear Surface Preparation in the Adult Mouse

Summary

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

Abstract

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

Introduction

The cochlea is dedicated to the detection of sound and responsible for hearing. The cochlear duct is coiled in a spiral shape in the bony labyrinth and holds the auditory sensory end organ, the organ of Corti (OC). The OC rests on the basilar membrane, making up the cochlear epithelium, with a length of about 5.7 mm when uncoiled in adult CBA/CaJ mice^1,2. Because the OC is tonotopically organized with high frequencies detected in the base and low frequencies in the apex, the cochlear epithelium is often divided into three parts for analytical comparisons: the apical, middle, and basal turns corresponding to lo....

Protocol

All research protocols involving male adult CBA/J mice at the ages of 10–12 weeks and C57BL/6J mice at the ages of 6–8 weeks were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical University of South Carolina (MUSC). Animal care was under the supervision of the Division of Laboratory Animal Resources at MUSC.

NOTE: For the procedures presented below, mice are anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injections. Mice are decapitated after the animal no longer responds to painful stimuli, such as a toe pinch.

1. Ex....

Results

Surface preparations of the cochlear epithelium, in combination with immunolabeling and confocal imaging, have been used broadly in hearing science for the investigation of cochlear pathologies, such as quantification of ribbon synapses, sensory hair cells, and protein expression in sensory hair cells^5,6,7,8. Although the dissection of adult mouse cochleae for surface preparations is not simple.......

Discussion

Cochlear microdissection of whole-mount surface preparations in combination with immunolabeling provides a basic tool for investigation of inner ear pathologies and molecular mechanisms. This modified adult mouse cochlear dissection method using the cell and tissue adhesive simplifies this difficult procedure.

Although this modified cochlear surface preparation method is relatively easy and accessible, it still requires practice in order to achieve proficiency. To make the correct cuts, the di.......

Materials

List of materials used in this article

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