Abstract
The avian inner ear can regenerate sensory hair cells after damage and has served as a model for the study of hearing regeneration for more than 30 years. Here we present a detailed surgical protocol to induce rapid apoptosis of all hair cells in the chicken cochlea and utricle with a single, local infusion of the aminoglycoside sisomicin. S-phase entry of supporting cells engaged in proliferative regeneration peaks at 48 h and newly regenerated hair cells emerge as early as 4–5 days post-sisomicin. We provide reliable read-outs for hair cell loss, such as overt manifestations of vestibular deficiencies, and quick validation of regeneration using reliable markers that can be detected with commercial antibodies. Titrating down the dose of sisomicin reveals differential susceptibilities of hair cell subtypes: cochlea versus utricle, cochlear tall versus cochlear short hair cells, vestibular type I versus type II hair cells, and proximal versus distal location along the cochlea. We provide a method to quantitate cells within the sensory epithelium in 3D, leveraging vibratome sectioning and imaging methods that are presented in a companion chapter. Finally, we present the technique of cold-peeling the cochlear sensory epithelium for the purposes of RNA or protein extraction, and single-cell dissociation in preparation for RNA-seq.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Notes
- 1.
Note that new hair cells can also be specifically marked by strong expression of CALB2 and TUBB3, which are not expressed in controls. By contrast, antibodies to OTOF do not label new hair cells as intensely as controls (Fig. 4c).
References
Benkafadar N et al (2021) Transcriptomic characterization of dying hair cells in the avian cochlea. Cell Rep 34(12):108902
Scheibinger M, Janesick A, Diaz GH, Heller S (2021) Immunohistochemistry and in situ mRNA detection using inner ear vibratome sections. Neuromethods (submitted)
Ellwanger DC et al (2018) Transcriptional dynamics of hair-bundle morphogenesis revealed with CellTrails. Cell Rep 23(10):2901–2914.e14
Janesick AS et al (2021) Cell type identity of the avian cochlea. Cell Rep 34(12):108900
Goodyear RJ et al (2010) Identification of the hair cell soma-1 antigen, HCS-1, as otoferlin. J Assoc Res Otolaryngol 11(4):573–586
Peng HB (1991) Xenopus laevis: practical uses in cell and molecular biology. Solutions and protocols. Methods Cell Biol 36:102
Suzuki J et al (2017) Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction. Sci Rep 7:45524
Talaei S et al (2019) Dye tracking following posterior semicircular canal or round window membrane injections suggests a role for the cochlea aqueduct in modulating distribution. Front Cell Neurosci 13:471
Bissonnette JP, Fekete DM (1996) Standard atlas of the gross anatomy of the developing inner ear of the chicken. J Comp Neurol 368(4):620–630
Skrzat J, Wrobel A, Walocha J (2013) A preliminary study of three-dimensional reconstruction of the human osseous labyrinth from micro-computed tomography scans. Folia Morphol (Warsz) 72(1):17–21
Grahek R, Zupancic-Kralj L (2009) Identification of gentamicin impurities by liquid chromatography tandem mass spectrometry. J Pharm Biomed Anal 50(5):1037–1043
O’Sullivan ME et al (2020) Dissociating antibacterial from ototoxic effects of gentamicin C-subtypes. Proc Natl Acad Sci U S A 117(51):32423–32432
Kitasato I et al (1990) Comparative ototoxicity of ribostamycin, dactimicin, dibekacin, kanamycin, amikacin, tobramycin, gentamicin, sisomicin and netilmicin in the inner ear of guinea pigs. Chemotherapy 36(2):155–168
Zakir M, Dickman JD (2006) Regeneration of vestibular otolith afferents after ototoxic damage. J Neurosci 26(11):2881–2893
Cafaro J, Lee GS, Stone JS (2007) Atoh1 expression defines activated progenitors and differentiating hair cells during avian hair cell regeneration. Dev Dyn 236(1):156–170
Scheibinger M et al (2018) Aminoglycoside damage and hair cell regeneration in the chicken utricle. J Assoc Res Otolaryngol 19(1):17–29
Bhave SA et al (1995) Cell cycle progression in gentamicin-damaged avian cochleas. J Neurosci 15(6):4618–4628
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Janesick, A., Scheibinger, M., Heller, S. (2022). Molecular Tools to Study Regeneration of the Avian Cochlea and Utricle. In: Groves, A.K. (eds) Developmental, Physiological, and Functional Neurobiology of the Inner Ear. Neuromethods, vol 176. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2022-9_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2022-9_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2021-2
Online ISBN: 978-1-0716-2022-9
eBook Packages: Springer Protocols