Our group is investigating drugs and drug delivery vehicles for the treatment of inner ear diseases, such as hearing loss. The round window membrane is the critical barrier that prevents locally applied drugs from getting into the cochlear, and we're currently working to understand how nano particle delivery vehicles can be engineered to pass through this barrier. Understanding how the round window membrane works in terms of drug transport is crucial in developing effective drug delivery strategies to the ear.
Guinea pig is an important preclinical animal model for studying drug delivery to the ear. However, isolating this fragile membrane, which is only 17 microns thick, for drug transport studies remains a significant challenge. Here we describe a method for extracting the Guinea pig round window membrane that allows for efficient bench top drug transport studies.
Current investigations of drug delivery to the inner ear rely primarily on in vivo experimental models, followed by perilymph sampling, which do not allow for a specific and targeted study of round window membrane transport mechanisms. In this study, we demonstrate the extraction of the guinea pig round window membrane with surrounding bony support. This method preserves native tissue architecture integrity and allows for precise and targeted studies of drug delivery across the round window membrane.
To begin, take the temporal bone extracted from the skull of a properly euthanized Guinea pig and remove the excess soft tissue. Next, using the six millimeter diamond bit, drill away the ventral aspects of the temporal bulla, exposing the middle ear space and the external auditory canal circumferentially. With the help of rongeurs, gently remove the external auditory canal and the tympanic ring.
Simultaneously separating the incudomalleolar joint. Using forceps, separate the incudostapedial joint and remove the incus to identify the bony niche of the round window. Then use a six millimeter diamond bit to drill away the bony lamina, connecting the cochlea with a medial wall of the tympanic cavity, toward the tensor tympani canal.
Carefully decompress the bony channel of the tensor tympani, and with forceps, remove the tensor tympani muscle. Next, drill away the bony lamina connecting the cochlea to the inferior wall of the tympanic cavity until there is one millimeter of bony ledge abutting the cochlea remaining. Using a two millimeter diamond bit, make a cochleostomy at the basal turn of the cochlea, leaving about two millimeters 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. 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. Examine the specimen from the skull base side and identify the internal auditory canal.
Drill to the cochlear aperture. With the 28 gauge needle, remove the cochlear nerve. Examine the specimen from the intraocular side.
Using a 28 gauge needle or forceps, identify and remove the osseous spiral lamina in the basil turn and the remaining modiolus. Irrigate the unified scalar tympani scalar vestibuli cavity copiously to remove debris. Examine the specimen from the middle ear side, drill the lateral semi-circular canal and facial canal to the level of the oval window.
Gently remove the stapes using a 28 gauge needle, exposing the oval window niche. Using a one millimeter diamond drill, open the vestibule further by extending the oval window along the face of the round window. Taking care to maintain one to two millimeters of cochlear bone, abutting the round window niche.
Complete the temporal bone cuts by connecting the oval window cuts with the ectomy cuts on each side of the round window. Drill away the final attachments to the bone of the skull base adjacent to the internal auditory canal and gently shaved down to result in an excised round window membrane specimen. This method resulted in the explanation of an intact Guinea pig round window membrane with a surrounding ring of rigid bone.
Histological analysis of the specimen showed a clear three layered epithelial structure with an adjacent intact round window niche.
