Light and Electron Microscopy Methods for Examination of Cochlear Morphology in Mouse Models of Deafness

Abstract

Mice are an invaluable model organism for the study of auditory function. Even though there are differences in size and frequency response, the anatomy and physiology of the mouse and human ear are remarkably similar. In addition, the tools available for genetic manipulation in the mouse have enabled the generation of models carrying mutations in orthologous human deafness-causing genes, helping to validate these lesions and assess their functional consequence. Reciprocally, novel gene mutations discovered to cause auditory deficits in the mouse highlight potential new loci for human hearing loss, and expand our basic knowledge of the mechanisms and pathways important for the function of the mammalian ear. Microscopy and imaging are invaluable techniques that allow detailed characterization of cochlear pathologies associated with particular gene mutations. However, the highly organized, delicate, and intricate structures responsible for transduction of sound waves into nerve impulses are encapsulated in one of the hardest bones in the body - the temporal bone. This makes sample preparation without damage to the soft tissue, be it from dissection or processing, somewhat challenging. Fortunately, there are numerous methods for achieving high-quality images of the mouse cochlea. Reported in this article are a selection of sample preparation and imaging techniques that can be used routinely to assess cochlear morphology. Several protocols are also described for immunodetection of proteins in the cochlea. In addition, the advantages and disadvantages between different imaging platforms and their suitability for different types of microscopic examination are highlighted. © 2016 by John Wiley & Sons, Inc.

Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

“…SEM was performed following the protocol reported previously ( Parker et al, 2016 ). Briefly, we made holes at the cochlear apex.…”

Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles

“…In practice, the best test is a comparison of the mutant sample with a control sample of normal morphology prepared in parallel. Following recommendations available in the literature (Bozzolla and Russell, 1998) and protocols described by other groups (Reynolds, 1963;Heywood and Resnick, 1981;Pickles et al, 1984;Furness and Hackney, 1985;Glauert and Lewis, 1998;Verpy et al, 2001Verpy et al, , 2011Goodyear et al, 2005;Michel et al, 2005;Ahmed et al, 2006;Harris et al, 2006;Kazmierczak et al, 2007;Grillet et al, 2009;Fischer et al, 2012;Indzhykulian et al, 2013;Kizilyaprak et al, 2014;Fang et al, 2015;Miranda et al, 2015;Jones, 2016;Parker et al, 2016;Velez-Ortega et al, 2017;Velez-Ortega and Frolenkov, 2019), we used fixation, staining, immunolabeling, dehydration, embedding reagents, and conditions that minimize these factors.…”

“…For many years, electron microscopy (EM) techniques such as transmission electron microscopy (TEM; Furness and Hackney, 1985;Kachar et al, 2000;Goodyear et al, 2005;Mogensen et al, 2007;Grillet et al, 2009;Karavitaki and Corey, 2010;Mahendrasingam et al, 2017) and scanning electron microscopy (SEM; Furness and Hackney, 1985;Kachar et al, 2000;Mogensen et al, 2007;Grillet et al, 2009;Verpy et al, 2011;Indzhykulian et al, 2013;Fang et al, 2015;Parker et al, 2016;Mahendrasingam et al, 2017;Velez-Ortega et al, 2017;Ivanchenko et al, 2020bIvanchenko et al, , 2021 have been used to study hair-cell bundles, and EM has proven to be essential for understanding stereocilia bundle development, maintenance, normal function, and dysfunction in disease. Immunogold electron microscopy techniques have been increasingly used in hearing research, providing excellent insight into structure-function relationships and protein distribution within the cell (Hasson et al, 1997;Garcia et al, 1998;Siemens et al, 2004;Furness et al, 2005;Michel et al, 2005;Goodyear et al, 2010;Verpy et al, 2011;Chen et al, 2012;Indzhykulian et al, 2013;Fang et al, 2015;Mahendrasingam et al, 2017;Wu et al, 2019;Han et al, 2020;Ivanchenko et al, 2020a).…”

Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

“…SEM was performed following the protocol reported previously (Parker et al, 2016). Briefly, we made holes at the cochlear apex and then washed the samples gently with 0.9% NaCl (Cat#10019318, Sinopharm Chemical Reagent Co, Ltd.) and fixed them with 2.5% glutaraldehyde (Cat# G5882, Sigma) overnight at 4°C.…”