Volumetric atlas of the rat inner ear from microCT and iDISCO+ cleared temporal bones.

Background: Volumetric atlases are an invaluable tool in neuroscience and otolaryngology, greatly aiding experiment planning and surgical interventions, as well as the interpretation of experimental and clinical data. The rat is a major animal model for hearing and balance studies, and a detailed volumetric atlas for the rat central auditory system (Waxholm) is available. However, the Waxholm rat atlas only contains a low-resolution inner ear featuring five structures. In the present work, we segmented and annotated 34 structures in the rat inner ear, yielding a detailed volumetric inner ear atlas which can be integrated with the Waxholm rat brain atlas.

Methods: We performed iodine-enhanced microCT and iDISCO+-based clearing and fluorescence lightsheet microscopy imaging on a sample of rat temporal bones. Image stacks were segmented in a semiautomated way, and 34 inner ear volumes were reconstructed from five samples. Using geometrical morphometry, high-resolution segmentations obtained from lightsheet and microCT stacks were registered into the coordinate system of the Waxholm rat atlas.

Results: Cleared sample autofluorescence was used for the reconstruction of most inner ear structures, including fluid-filled compartments, nerves and sensory epithelia, blood vessels, and connective tissue structures. Image resolution allowed reconstruction of thin ducts (reuniting, saccular and endolymphatic), and the utriculoendolymphatic valve. The vestibulocochlear artery coursing through bone was found to be associated to the reuniting duct, and to be visible both in cleared and microCT samples, thus allowing to infer duct location from microCT scans. Cleared labyrinths showed minimal shape distortions, as shown by alignment with microCT and Waxholm labyrinths. However, membranous labyrinths could display variable collapse of the superior division, especially the roof of canal ampullae, whereas the inferior division (saccule and cochlea) was well preserved, with the exception of Reissner's membrane that could display ruptures in the second cochlear turn. As an example of atlas use, the volumes reconstructed from segmentations were used to separate macrophage populations from the spiral ganglion, auditory neuron dendrites, and Organ of Corti.

Conclusion: We have reconstructed 34 structures from the rat temporal bone, which are available as both image stacks and printable 3D objects in a shared repository for download. These can be used for teaching, localizing cells or other features within the ear, modeling auditory and vestibular sensory physiology and training of automated segmentation machine learning tools.