More than four centuries have passed, since the early descriptions of the inner ear by Andreas Vesalius, Bartolome Eustachi and Galen were picturing the inner ear filled with a type of purified air, “aer ingenitus” (Water, 2012). During all this time, the inner ear has become a living yearbook of the anatomist community. Profesor Cotugno termed “liquor Cotunni” what we know today as perilymph in 1775. Some years later, Professor Antonio Scarpa named the endolymph and the peripheral ganglion of the vestibular system as the Scarpa´s fluid and ganglion, respectively. Before committing to the noble life after the death of his father, the
50 Marquis Alphonse Corti, later Baron Corti, published the first histological description of the hearing epithelium in 1851, which later his mentor, Professor Kölliker from Wurzburg, would name the organ of Corti, containing the rods of Corti (currently known as pillar cells) and the tunnel of Corti. His work was followed up by a series of professors that described many cells and spaces in the cochlea and named after themselves: Dieters, Claudius, Hensen, Boetstscher, Nuel and Huscke (for a historical review of the inner ear histology and anatomy, see Water, 2012).
All these anatomists, and many others, contributed to the description of one of the most elegant and still intriguing organs of the human body, the inner ear and specially the cochlea. Until the advent of the of computerized techniques such as CT or MRI in the seventies, most description were limited to exquisite anatomic dissections, to describe the coarse structure of the tissue, or to histological physical sections of different thicknesses, if cellular resolution was needed.
It was not until the work of Voie in 1993, that both preparations and aims could start to merge since it orthogonal-plane fluorescence optical sectioning microscopy (OPFOS) imaging allowed to image a whole intact cochlea and achieving even cellular resolution.
After the work of (Voie et al., 1993), it seems that the cochlea clearing have not flourished in the auditory field. To my knowledge, in these almost 30 years, only 27 papers have used this technique with little or none modifications to the initials protocols. A summary of these efforts is presented in Table 4. Together with the axial resolution limitations inherent to the imaging technology, some of the reasons might be 1) the lack of a robust screening of antibodies, 2) the lack of an accessible, standardized, easy-to-implement analysis workflow, 3) the use of highly toxic reagents (such as BABB or MSBB, whose damages to the imaging setup are not covered by the product warranty, for example, of LaVision Ultramiscope II), 4) the lack of access to a lightsheet microscope.
Given than our estimates of transduction efficiency in our previous papers on cochlear optogenetics were based on cryosections, only a very rough location classification could be done in Apical, Mid and Basal turn. In order to increase our spatial resolution and to comprehensively study the transduction levels as a function of the tonotopy position, we wanted to have a method that would allow us to study the expression levels of GFP in the whole-intact cochlea. Given the lack of such studies in the literature, we had to gather parts of different protocols to engineer an adaptation of iDisco+ and to create an image analysis workflow that would allow retrieving numerical data from the image stacks. Since this kind of tool will not only be useful for studying the tonotopic distribution of the expression of GFP in mouse, we also screened its compatibility with other stainings and with other species.
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Table 4. Cochlea clearing in the literature
Publication Clearing
method Staining Imaging method Specie Analysis Comment
Voie et al., 1993 MSBB RITC OPFOS Guinea Pig Rough reconstruction
of ST
First use of OPFOS with biological
specimens Voie and
Spelman, 1995 MSBB RITC OPFOS Guinea Pig Rough reconstruction
of ST, CA, RWM -
Dirckx, 2007 MSBB RITC HR-OPFOS Gerbil Reconstruction of
stapes and adjacent
DNA: DAPI, TO-PRO-3 LCSM Mouse - Comparison with
Spurr’s Resin Hardie et al., 2004) Santi et al., 2008 MSBB Autoflourescence, RITC OPFOS Mouse 3D reconstruction of
cochlear structures
Development of the Mouse Cochlear
Database Buytaert and
Dirckx, 2009 MSBB RITC HR-OPFOS Gerbil Reconstruction of
stapes and adjacent Santi et al., 2009 MSBB Autoflourescence, RITC TSLIM Mouse 3D reconstruction of
inner ear Development of
TSLIM Mouse 3D reconstruction of inner ear
2013 MSBB Autoflourescence LCSM Guinea Pig SGN density counts per mm2;manually + ICTN
Myo6a sTSLIM Mouse Manual counting of IHC
and SGN
Actin: Phalloidin-TRITC LCSM Gerbil IHC and OHC density -
Tinne et al.,
2017 MSBB Extinction and
Autofluorescence SLOT Human - Comparison with
µCT; Imaging with cochlear implant Nolte et al.,
2017 MSBB Primary: NF200, OTOF SLOT Mouse Reconstruction of
neurofilament labeling
7-AAD; Vessel: Lectin Ultramicroscope Mouse, Rat, Gerbil, Marmoset
SGN and IHC counting, GFP distribution,
tonotopy mapping -
OPFOS: Orthogonal-plane fluorescence optical sectioning; MSBB: Methyl Salicylate Benzyl Benzoate Spalteholz’s fluid; RITC: Rhodamine-B isothiocyanate; ST: Scala Tympani, CA: Cochear Aqueduct, RW: Round Window; BM: Basilar membranesTSLIM: scanning Thin-Sheet Laser Imaging Microscopy; SLOT: Scanning Laser Optical Tomography; LCSM: Laser Confocal Scanning Microscopy; Ab: Antibody for details on the concentration and manufacturer information of the antibody, see original publication
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