To assess a clearing protocol using ethyl cinnamate, an organic substance which is non-toxic for humans, and its value in light-sheet microscopy study of post-implantation cochlear damage in the Mongolian gerbil.

The animals underwent right cochlear implantation in the round window by a retroauricular approach. They were then euthanized 10 weeks after implantation (electrode array in place). The cochleae were prepared according to a 29-day protocol including steps of fixation, microdissection, decalcification, permeabilization, blocking, fluorescent immunolabeling, dehydration and finally clearing in ethyl cinnamate solution. Acquisition of transparent cochleae was performed by light-sheet microscopy. Imaris software was then used for 3D analysis.

The transparent cochleae had not undergone any shrinkage or any significant architectural changes. Six cochleae were acquired by light-sheet microscopy, allowing good visibility of the whole cochlea. 3D immunofluorescence analysis of the cochlea provided sufficient image resolution for analysis of the spiral ganglion neurons and assessment of the fibrotic tissue reaction surrounding the electrode array.

The ethyl cinnamate clearing protocol was effective for light-sheet microscopy analysis of the whole Mongolian gerbil cochlea with the implant left in situ. This technique is suitable for the study of post-implantation cell and tissue damage in the same sample, without the potential toxicity of other methods described to date.