Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Summary

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

Abstract

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) ^1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.

Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions ^{7, 8}. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium ^{9, 10}. Supporting cells are also important mediators of hair cell survival and death ¹¹. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Protocol

1. Utricle Dissection and Culture

1. Euthanize an adult (4 weeks of age or older) mouse using an approved protocol and decapitate.
2. Snip the external auditory canal on both sides of the head and pull the skin forward towards the nose. Bisect the head from back to front and remove the brain from both sides to reveal the bony labyrinth.
3. Trim the skull away from the bony cochlea and transfer the bony labyrinth (including the bulla) to a tissue culture hood equipped with a dissecting microscope (Note: Adenovirus work should be performed in a Class II biological safety cabinet).
4. In the hood, transfer each cochlea to a ....

Discussion

Sensory hair cells are susceptible to death caused by a variety of stresses, including aging, noise trauma, and exposure to ototoxic drugs, including the aminoglycoside antibiotics and the antineoplastic agent cisplatin. In mammals hair cell death results in permanent hearing loss and/or balance disturbance. In vitro model systems are critical tools for studies aimed at determining the cellular and molecular mechanisms underlying hair cell death, as well as those aimed at preventing or reversing hair cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medium 199	GIBCO, by Life Technologies	12350
DMEM/F12	GIBCO, by Life Technologies	11320
Fine forceps (#55)	Fine Science Tools	11255-20
Fine forceps (#5)	Fine Science Tools	11252-30
Forceps (#3)	Fine Science Tools	11231-30
Penicillin G	Sigma-Aldrich	P3032
Fetal bovine serum	Invitrogen	10082-139
Nunc mini-tray	Fisher Scientific	12-565-68
Adenovirus-GFP	Vector Biolabs	1060*
Cal-Ex decalcifier	Fisher Scientific	CS510-1D
sodium borohydride	Sigma-Aldrich	452882
Anti-Myosin 7a (polyclonal)	Proteus Biosciences	25-6790
Anti-Myosin 7a (monoclonal)	Developmental Studies Hybridoma Bank	MYO7A 138-1
Anti-Calmodulin	Sigma-Aldrich	C 3545
Anti-Calbindin	Chemicon International	AB1778
Fluoromount G	SouthernBiotech	0100-01
Table 1. Reagents and tools used in utricle culture and adenovirus infection
* Note: Ad-GFP is normally provided by Vector Biolabs at a stock titer of 1x1010 PFU/ml. We requested a custom amplification in order to provide the stock virus at a titer of 1.2×1011 PFU/ml.