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).

Representative 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 requires fracturing and removal prior to the extraction of the stapes superstructure. Histological analysis (Figure 3B) of the representative RWM shows a clear three-layered epithelial structure with an adjacent, intact round window niche.

From a technique perspective, there are two critical steps. Firstly, when making the cochectomy in step 2.7, it is important to use the equator of the drill bit, rather than the tip, to make the cut, as jitters at the burr tip can lead to traumatic fractures in the cochlear bone that may extend to the round window membrane. Secondly, it is important to fully drill out the internal auditory canal, as this allows for the complete removal of the cochlear nerve and easier dissection of the osseous spiral lamina, resulting in a unified cavity on the intracochlear side for experimental sampling (Figure 2C).

After the RWM has been successfully extracted, our group utilized a modified Ussing chamber to evaluate pharmacokinetic properties of the membrane. The modified Ussing chamber has been validated by other groups in tympanic and round window membranes as well as retinal tissue to evaluate transport properties of epithelial membranes^14,15. This 3D printed device was constructed using Poly-Jet Vero and consists of two triangular base pieces, each with a 400 µL fluid chamber (Figure 3). The bony rim of the RWM is affixed to the base using 2-part epoxy (Gorilla, see Table of Materials), followed by silicon sealant (see Table of Materials) to ensure a water-tight seal (Figure 4). Meticulous care is taken to avoid any epoxy or sealant contacting the membrane. With the RWM sandwiched in place, the two bases are then glued together with epoxy. During transport experiments, the loading (middle-ear facing) is filled with phosphate-buffered saline (PBS) containing the delivery vehicle (or any molecule of interest), and the sampling chamber (scala tympani facing) is filled with PBS only. At regular time intervals, fluid from the sampling chamber is completely aspirated and replaced with fresh PBS.

Quality control measures are taken during each experiment to ensure that there is both a water-tight seal around the specimen within the apparatus, as well as a fully intact membrane without microperforations. Representative results with a brown, iron-core nanoparticle solution are shown below. Quantitative verification of a water-tight seal is accomplished by aspirating the full volume of the sampling chamber during each sampling interval; fluid leaking out of the sampling chamber would result in less than the full aspiration volume. At the conclusion of each experiment, the fluid volume in the loading chamber should also remain the same. Additionally, as our target fluid is brown in color, it is also readily visible in the event of a leak.

To ensure no leak within the membrane, several approaches were taken. First, RWM specimens used in this study are all immediately extracted, and light, confocal, and electron microscopy images confirm intact cellular and membrane structures without microperforations (Figure 3A,B). Second, microperforations were deliberately created in a subset of RWM specimens to compare the effect on nanoparticle delivery. Visual inspection of the sampling and loading chamber aspirate serves as further confirmation of specimen integrity and watertight attachment. In RWM with perforations, there is rapid equilibration of the loading and sampling chamber, which can be observed via a distinct color change (Supplementary Figure 1). Additionally, it was found that transport in microperforated RWMs was higher than the maximal variance observed in intact membranes (Supplementary Figure 2). Together, these serve as quality control mechanisms for the integrity of the water-tight seal surrounding the specimen, as well as the RWM.

Figure 1: Preoperative image of the guinea pig temporal bone. The tympanic bulla, external auditory canal, and facial canal (*) are shown in this preoperative image of the guinea pig temporal bone. Scale bar = 1 cm. Please click here to view a larger version of this figure.

Figure 2: Intraoperative images. Intraoperative images demonstrating the relationship between the round window membrane and the incus (*), cochlea (**), stapes (†), and decompressed internal auditory canal (‡). Shaded areas indicate portions of the specimen that will be removed. (A) Guinea pig middle ear cavity after removal of tympanic membrane and facial nerve (step 2.3). (B) Middle ear cavity after decompression of the tensor tympanic canal (step 2.6). (C) Basal view of RWM after cochleostomy and removal of inner ear contents (step 2.11). (D) View of RWM after removal of stapes and identification of the vestibule (step 2.13). Scale bar = A,B (1 cm); C,D (5 mm). Please click here to view a larger version of this figure.

Figure 3: Final RWM specimen and histology. (A) Guinea pig RWM gross specimen with a bony annulus. Scale bar = 1 cm. (B) Histology of the explanted guinea pig round window membrane demonstrates an intact three-layered structure. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 4: Microfluidic device for transmembrane transport experiments. (A) Diagram of the microfluidic device used for transmembrane transport experiments. (B,C) Printed microfluidic device for the experiments. Please click here to view a larger version of this figure.

Supplementary Figure 1: Loading and sampling chamber contents over a 5 h iron-core nanoparticle delivery experiment. (A) Loading chamber of intact RWM shows a gradual decrease in color intensity as the concentration of nanoparticles decreases. (B) Sampling chamber of intact RWM shows stable coloration over time, consistent with low levels of nanoparticle delivery. (C) Loading chamber of perforated RWM demonstrates a rapid decrease in color intensity as the contents equilibrate with the sampling chamber. (D) Sampling chamber of perforated RWM shows a gradual increase in color over time, consistent with high levels of nanoparticle delivery. Please click here to download this File.

Supplementary Figure 2: Representative transport results for delivery of nanoparticles. Representative transport results for the delivery of nanoparticles (Fe₃O₄ core with polyethylene glycol coating, mean radius of 77 nm) across guinea pig RWM, both intact and with perforations (mean ± SD, n ≥ 3 RWM experiments). Please click here to download this File.

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 RWM permeability model^2,16; however, the specific steps of the surgical extraction have not been detailed until now, and the fragility of the cochlear bone and complex anatomy have posed a challenge for the consistent, en bloc harvest of intact RWMs.

The guinea pig RWM is located at the end of the scala tympani of the cochlea and is surrounded by a thin layer of cochlear bone. Fracture of this osseous structure during the explantation process can result in an unusable specimen, as the RWM tends to tear away if the surrounding cochlear bone does not remain intact. Our group noted that fractures most often occurred when drilling near the junction between the highly-dense skull base bone and the brittle cochlea, as the transition in bone density increased the likelihood of fracture propagation through the round window. For this reason, it is suggested that maintaining the denser bone of the tensor tympani fossa, which is attached to the cochlea in close proximity to the RW, will increase the yield of specimen harvest, particularly in older animals. Disruption of the bone can also occur during the removal of the cochlear contents; care must be taken to gently remove the osseous spiral lamina with minimal manipulation of the surrounding cochlear bone.

An important detail to consider with this model is the viability of the RWM after explantation. Prior groups have suggested that mammalian RWM remains viable for 24-48 h after extraction¹⁷. The present study has reflected these findings; consistent transport studies and histological analyses demonstrating intact cell structures (Figure 2A) have both supported the viability of the guinea pig RWM at the time of experiments. To maintain the overall health of the extracted specimen, the RWM is extracted and embedded within 3 h of euthanasia.

In the study of drug pharmacokinetics across the RWM in vivo, there remains considerable technical difficulty in making measurements of perilymphatic distribution and concentration¹. Changes in intra-tympanic delivery methods and applied amount have resulted in varied therapeutic results. These difficulties are compounded by the complex fluid dynamics within the labyrinth, as well as irregular egress of injected material through the eustachian tube. The described method of explantation allows for the isolated study of the RWM and factors that affect its permeability as an ex vivo model. Furthermore, the explant also allows for the direct interrogation and visualization of various methods currently employed to increase RWM permeability, such as ultrasound microbubbles¹⁸ and chemically induced junctional modulation¹⁹. Future studies on specific endocytosis mechanisms implicated in drug delivery would also benefit from this benchtop model.

Materials

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