Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Summary

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

Abstract

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

Introduction

Inner ear gene therapy is a rapidly developing field of investigation. It has been applied in various animals models to combat ototoxicity, noise trauma, and hereditary hearing loss¹. Several recent studies have shown functional recovery of hearing and balance functions in mutant mice after inner ear gene therapy delivery^2,3,4,5,6,7. One of the key factors in determining the success of inner ear gene therapy is the surgical approach used to access t....

Protocol

All animal procedures were approved by the Animal Care and Use Committee at the National Institute on Deafness and Other Communication Disorders (NIDCD ASP1378-15).

1. Procedure Setup and Preparation

1. Sterilize all instruments by ethylene oxide in the beginning of the experiment. Between animals, clean instruments using bead sterilization.
2. Load the solution containing viral gene therapy into a micropipette on the micro-injector. The viral vector used in this study was AAV2/8-whirlin (1 x 10¹³ genome copies per milliliter, see the table of materials).
   NOTE: Typically, 1.1 µL total volume is loaded ....

Results

Injection of AAV8-whirlin gene therapy into neonatal whirler mice through the posterior semicircular canal resulted in whirlin expression (green) in utricular hair cells (Figure 2), with the overall infection efficiency of 53.1% (SD 38.1, n = 28)⁵. Transduced hair cells had elongated stereocilia (red) compared to hair cells from contralateral non-injected ears (5.35 ± 2.11 µm vs. 3.20 ± 0.34 µm, respectively)⁵.<.......

Discussion

Several surgical approaches have been described to access rodent inner ears. Cochleostomy and round window approaches are most commonly used to access the cochlea, whereas the posterior semicircular canal and endolymphatic sac approaches are typically used to access the vestibular organs¹. In a recent study, we showed that posterior semicircular canal injections of viral gene therapy resulted in high efficiency of hair cell transduction in both the vestibular organs and the cochlea

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Operating microscope	Zeiss		OPMI Pico ENT microscope. Other dissection microscopes would also work.
Micro-forcepts	Fine Science Tools	11251-10, 11295-51	#5 and #55 Dumont
Micro-scissors	Fine Science Tools	15002-08
Nanoliter2000 microinjector	World Precision Instruments
Heating pad	Mastex	Model 500/600
5-0 vicryl sutures	Ethicon
AAV8-whirlin	Vector Biolabs
Glass pipette	Sutter Instruments	B100-75-10	Borosilicate glass