Cochlear isolation in neonatal mice for in vitro and ex vivo models: an anatomical landmark-based protocol

Ex vivo and in vitro models utilizing inner ear tissue from neonatal rodents are vital for developing treatments for hearing loss caused by damage to hair cells or auditory neurons. The cochlear isolation from the skull is associated with a high risk of mechanical damage to the soft tissues enclosed in the otic capsule. Such damage may result in artifactual hair cell loss and morphological alterations of SGNs. This stage of cochlear dissection requires more detailed description. To overcome technical difficulties, we described a protocol based on the use of key anatomic lines of the skull during dissection.

We propose an optimized stepwise technique for cochlear isolation based on dissection of skull bones along specific anatomical skull lines-the spheno-occipital, spheno-petrosal, and petro-occipital synchondroses, along with the petrosquamosal suture. This approach utilizes targeted dissection along natural anatomical skull lines to carefully separate skull bones, reducing excess tissue and unnecessary manipulation of the cochlea. Sample preservation was assessed through immunofluorescence staining for hair cells (phalloidin) and spiral ganglion neurons (β-III tubulin) in both ex vivo and in vitro models.

The technique significantly reduced superfluous tissue adherence, thereby facilitating subsequent processing of the cochlear surface. Intact cochleae were consistently obtained with preserved auditory capsules and the structure of the membranous labyrinth. Immunofluorescence analysis confirmed the preservation of inner ear samples obtained for creating in vitro and ex vivo models. The protocol allows cochlear isolation in approximately 3 min, depending on the researcher's skill, with a success rate of 92.9% (based on 70 dissections). Modiolus isolation takes approximately 7 min.

Cochlear isolation is a technically challenging procedure that requires both practiced skill and careful execution. A key feature of our protocol lies in the emphasis on specific anatomical landmarks, which facilitates a clearer understanding of the cochlear isolation process and prevents actions that would compromise the integrity of the cochlea. This is important for researchers who are developing the skills necessary to create in vitro or ex vivo cochlear models. The described technique may potentially accelerate cochlear isolation, although its effectiveness largely depends on the researcher's skill and experience.