Guinea Pig Round Window Membrane Explantation for Ex Vivo Studies

Summary

This protocol outlines a method for the explantation of the round window membrane from guinea pig temporal bones, providing a valuable resource for ex vivo studies.

Abstract

Efficient and minimally invasive drug delivery to the inner ear is a significant challenge. The round window membrane (RWM), being one of the few entry points to the inner ear, has become a vital focus of investigation. However, due to the complexities of isolating the RWM, our understanding of its pharmacokinetics remains limited. The RWM comprises three distinct layers: the outer epithelium, the middle connective tissue layer, and the inner epithelial layer, each potentially possessing unique delivery properties.

Current models for investigating transport across the RWM utilize in vivo animal models or ex vivo RWM models which rely on cell cultures or membrane fragments. Guinea pigs serve as a validated preclinical model for the investigation of drug pharmacokinetics within the inner ear and are an important animal model for the translational development of delivery vehicles to the cochlea. In this study, we describe an approach for explantation of a guinea pig RWM with surrounding cochlear bone for benchtop drug delivery experiments. This method allows for preservation of native RWM architecture and may provide a more realistic representation of barriers to transport than current benchtop models.

Introduction

Novel classes of therapeutics have emerged for the treatment of sensorineural hearing loss. The translation of these therapeutics to clinical populations is limited by safe and efficacious routes of transport into the inner ear. Current methods of in vivo delivery in animal studies rely on either fenestration into the inner ear or diffusion through the round window membrane (RWM), a non-osseous barrier that separates the middle ear space from the cochlea¹.

Surgical fenestration and microinjection into the inner ear are both invasive and can pose risks to residual inner ear function². Therefore, the RWM is an important route for local drug delivery, and guinea pigs are the primary preclinical animal model used to study local drug pharmacokinetics across the RWM and in the inner ear for pharmaceutical development^3,4. Although thinner than the human RWM, the guinea pig RWM shares an identical three-layered structure. It is approximately 1 mm in diameter, 15-25 µm thick, and comprised of two epithelial cell layers sandwiching a connective tissue layer⁵. The epithelial layer facing the middle ear is densely packed and connected via tight junctions, while the layer facing the inner ear and scala tympani has looser architecture and does not have significant intercellular adhesions.

Current pre-clinical studies investigating drug permeability in the guinea pig RWM rely on in vivo middle ear injections followed by the sampling of the perilymph fluid within the inner ear, which does not allow for the specific study of RWM transport^6,7. Fragments of RWM explants have been used in preclinical studies, but due to their fragility and small size, they are not suitable for systematic, microfluidic investigations of drug and vehicle transport requiring a watertight seal across the RWM². Other groups have employed in vitro models with cultured human epithelial cells to approximate the RWM^8,9,10. However, the majority of these constructs focus solely on the outer epithelial layer and do not capture the complexity of native tissue architecture. For a more detailed understanding of transport mechanisms across the RWM, targeted, ex vivo studies are required.

In this study, we demonstrate the explantation of a guinea pig RWM with surrounding bony support to preserve membrane integrity and illustrate their use in an experimental paradigm designed for the specific study of the RWM transport of drug delivery vehicles.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (GP18M226). Hartley albino guinea pigs (both male and female, weighing 500-700 g) were used in the present study.

1. Procedure setup and preparation

1. Sterilize all instruments with ethylene oxide before beginning the experiment.
2. Euthanize the animals following the institutionally approved protocol.
   NOTE: In the current study, a non-precharged chamber was employed to release 100% carbon dioxide (CO₂) from a commercial cylinder. An inline restrictor was used to regulate the gas flow, maintaining it within the range of 30% to 70% of the chamber's volume per minute in accordance with the 2020 AVMA Guidelines¹¹.
3. Place the animal in the chamber and dispense carbon dioxide for 5 min. CO₂ flow is maintained for 1 min following respiratory arrest.
4. Perform decapitation following respiratory arrest to ensure euthanasia.

2. Surgical approach and explantation

1. Extract the temporal bone from the guinea pig skull in the usual fashion¹². Remove the excess soft tissue with a rongeur. Identify the external acoustic meatus, the temporal bulla, and the facial canal¹³ (Figure 1).
2. Drill away the ventral aspects of the temporal bulla with a 6 mm diamond bit (see Table of Materials), exposing the middle ear space and the external auditory canal circumferentially.
3. Using rongeurs, gently remove the external auditory canal and the tympanic ring, simultaneously separating the incudomalleolar joint. Identify the incus, incudostapedial joint, cochlea, horizontal semicircular canal, and facial canal¹³ (Figure 2A).
4. Separate the incudostapedial joint and remove the incus using forceps. Identify the bony niche of the round window.
5. Use a 6 mm diamond bit to drill away the bony lamina connecting the cochlea with the medial wall of the tympanic cavity toward the tensor tympani canal. Carefully decompress the bony channel of the tensor tympani and remove the tensor tympani muscle using a 28 G needle.
   NOTE: The medial wall of the tensor tympani fossa connects directly with cochlear bone around the RWM, and care is taken not to cause fractures that can extend to the round window.
6. Drill away the bony lamina connecting the cochlea to the inferior wall of the tympanic cavity until there is 1 mm of bony ledge abutting the cochlea remaining (Figure 2B).
7. Using a 2 mm diamond bit (see Table of Materials), make a cochleostomy at the basal turn of the cochlea, leaving approximately 2 mm of bone to the round window. Continue the cochleostomy inferiorly in a plane parallel to the round window membrane to separate the base from the apex of the cochlea.
8. Extend the cochleostomy cut through the skull base, which is much denser, resulting in a cross-sectional view of the basal turn of the cochlea.
   NOTE: Aiming the drill toward the meatus of the internal auditory canal results in a trajectory that maximizes bone removal while avoiding traversing too close to the round window.
9. Examine the specimen from the skull base side and, if not already done, identify the internal auditory canal and drill to the cochlear aperture. Remove the cochlear nerve with a 28 G needle.
10. Examine the specimen from the intracochlear side. Identify and remove the osseous spiral lamina in the basal turn and the remaining modiolus with forceps or a 28 G needle.
11. Irrigate the unified scala tympani-scala vestibuli cavity copiously to remove debris. The round window should be clearly visible from the cochectomy without any overlying debris (Figure 2C).
12. Next, examine the specimen from the middle ear side. Drill the lateral semicircular canal and facial canal to the level of the oval window. Gently remove the stapes using forceps, exposing the oval window niche. Of note, there is a bony bridge between the crura of the stapes known as the crista stapedis.
13. Using a 1 mm diamond drill (see Table of Materials), open the vestibule further by extending the oval window along the face of the round window, taking care to maintain 1-2 mm of cochlear bone abutting the round window niche (Figure 2D).
14. Complete the temporal bone cuts by connecting the oval window cuts with the cochlectomy cuts on each side of the round window.
    NOTE: Due to the fragility of the cochlear bone, preserving the tensor tympani fossa in the specimen and avoiding cuts through it will help prevent cochlear bone fractures that extend to the RWM and compromise its integrity.
15. Make the final attachments to the dense bone of the skull base adjacent to the internal auditory canal and gently shave down to result in an excised RWM specimen (Figure 3A).

Results

As demonstrated in Figure 3A, this method allows for the explantation of the intact guinea pig round window membrane with a surrounding ring of rigid bone. The RWM should be fully connected to the bony annulus circumferentially. No fractures of the cochlear bone should be appreciated. In comparison with human round window specimens, guinea pig RWM does not have an overlying pseudomembrane. Additionally, unlike humans, there is a bony bridge between the crura of the guinea pig stapes, which r...

Discussion

In local drug delivery to the ear, the RWM is the primary route of passage for therapeutics to reach the inner ear. An accurate and reliable benchtop model is needed to better understand transport mechanisms and permeability across novel delivery vehicles and for drug development. In this study, we demonstrate that guinea pig RWM explantation is a feasible and dependable procedure to allow for systematic investigations of drug-membrane interactions. Lundman et al. and Kelso et al. previously described utilizing a similar...

Materials

Name	Company	Catalog Number	Comments
1 mm Diamond Ball Drill Bit	Anspach	1SD-G1
2 mm Diamond Ball Drill Bit	Anspach	2SD-G1
6 mm Diamond Ball Drill Bit	Anspach	6D-G1
ANSPACH EMAX 2 Plus System	Anspach	EMAX2PLUS	Any bone cutting drilling system will work
BD Eclipse Needle 27 G x 1/2 in. with detachable 1 mL BD Luer-Lok Syringe	Becton, Dickinson, and Co.	382903057894	Any 27-28 G needle
Gorilla Epoxy	Gorilla	4200101
Kwik-CAST	World Precision Instruments	KWIK-CAST