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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Immunofluorescence on cleared intact cochlea allows detailed analysis of the cochlear ultrastructure, while avoiding the problems of dissection and serial sections. Protocols have been developed for mice and Mongolian gerbils. This technical note proposes a detailed and optimised immunofluorescence protocol in the Mongolian gerbil comprising significant quantitative and qualitative improvements. This protocol sequentially comprises: fixation (1 day), decalcification (6 days), pre-treatment (7.5 hours), immunolabelling (42 hours), dehydration and clearing (23 hours), followed by mounting and laser scanning confocal microscopy acquisition. This protocol has been optimised in terms of duration (10 days versus 13 days) with a reduction of the number of steps, improvement of the specificity of immunolabelling and optimisation of the quality of the results obtained. This technical note provides a detailed description of this protocol. [ABSTRACT FROM AUTHOR]

Objectives To draw up a clearing protocol for Mongolian gerbil cochlea, and to assess the feasibility of quantifying and analyzing 3D cell architecture in the transparent cochleae. Materials and methods Freshly dissected inner ears were prepared on a 13-day protocol: fixation, microdissection, post-fixation, decalcification, pretreatment (signal enhancement, permeabilization and blocking), fluorescent labeling (indirect immunolabeling and direct labeling), dehydration, clearing in Spalteholz solution (MSBB: methyl salicylate and benzyl benzoate) and mounting. Image acquisition used laser scanning confocal microscopy. ImageJ software was used to measure the length of the organ of Corti thus available for analysis and to count inner and outer hair cells. Results Four cochleas underwent imaging. 3D reconstruction enabled organ of Corti length to be measured, at a mean 1269 ± 346 μm. Mean inner and outer hair-cell count per organ of Corti length was 142 ± 44 and 400 ± 122, respectively. Conclusion Cochlear clearing by MSBB was feasible in Mongolian gerbils and provided high-resolution immunofluorescence-labeled inner-ear images. To our knowledge, this was the first application of the technique in this species. Cell count could thus be performed along the organ of Corti length without traumatic dissection. [ABSTRACT FROM AUTHOR]