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 the inner ear. Ideally, the surgical approach would be easy to perform, the anatomic landmarks would be consistent and easy to identify, and the resulting transduction of targeted cell types would be high.

In a recent study, we showed that when viral gene therapy was injected through the posterior semicircular canal of the whirler mutant mouse (a model of hearing loss and vestibular dysfunction), efficient transduction of sensory hair cells was seen in the vestibular organs as well as in the cochlea⁵. The high efficiency of sensory hair cell transduction resulted in improvement of auditory and vestibular functions in these mutant mice.

In this article, we describe in detail the posterior semicircular canal approach to access the neonatal mouse inner ear.

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 into the micropipette.

2. Anesthesia

NOTE: The mouse strain used in this study is the whirler mouse. Both homozygous mutants (Whrn^wi/wi) and heterozygous littermates (Whrn^+/wi) were used.

1. Place the mother in a separate cage (separate from the litter).
2. Place the home cage containing the litter (P0 - P5 pups) on a recirculating heat pad (set at 37.5 °C) to keep the mice warm.
3. Cut out the thumb portion of a latex glove, and place a pup in it.
4. Place the pup in latex glove thumb into a bucket of ice for ~2 min.
5. Place the anesthetized pup on a large square commercial plastic freeze pack with a 4" x 4" gauze between the pup and the pack's surface.
6. Fill a heavy-duty latex glove with crushed ice, and place the ice glove around the pup.
7. Check to see if the pup is adequately anesthetized by the complete lack of any response to various stimuli (including a firm toe pinch). Leave the pup on the ice pack for the duration of the surgery (approximately 5 - 10 min).
   NOTE: We recommend leaving the pups on the ice pack for no more than 15 min during the surgery.

3. Surgical Approach (Figure 1)

1. Clean the skin behind the ear with an iodine wipe and an alcohol wipe once the animal is anesthetized.
2. Make a postauricular incision ~2 mm behind the ear using micro-scissors, and divide the sternocleidomastoid muscle with micro-scissors.
3. Identify the facial nerve and the bulla. The bulla is cartilaginous and semi-transparent at this age and it lies medial to the facial nerve. The stapedial artery can be seen through the bulla at this age, which is a useful landmark.
4. Follow the facial nerve superiorly and posteriorly to locate the posterior semicircular canal (PSCC). Remove the muscle fibers and soft tissue overlying the posterior semicircular canal using micro-scissors.
   NOTE: The PSCC is cartilaginous at this age.
5. Penetrate the PSCC using a glass micropipette (~10 μm in diameter) on the micro-injector.
6. Inject viral gene therapy into the inner ear.
   NOTE: Typically, a total of 20 injections of 49 nL of the gene therapy are delivered into the posterior semicircular canal over ~40 s (total volume ~1 µL). The viral titer used was 1 x 10¹³ genome copies per mL.
7. Close the skin incision using a 5-0 polyglactin suture.

4. Postoperative Care

1. Place the pup on a warming pad to restore a normal body temperature during recovery from anesthesia with constant manual stimulation/rolling with gloved human fingers.
2. Once the pup is awake, place it back into its home cage.
3. Caress each pup with a cotton swab that has been exposed to the home cage bedding.
   NOTE: The purpose of this is to have the mice smell as they did prior to surgery, which increases the likelihood of the mother re-accepting her litter post-surgery. If possible, urine from the mother can be collected and rubbed on the pups using a cotton swab to further decrease the likelihood of rejection.
4. Apply mineral oil to the mother's nose to desensitize her⁸, and reintroduce the mother into the home cage.

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)

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

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