Supporting cell characteristics in long-deafened aged mouse ears.

Significant sensory hair cell loss leads to irreversible hearing and balance deficits in humans and other mammals. Future therapeutic strategies to repair damaged mammalian auditory epithelium may involve inserting stem cells into the damaged epithelium, inducing non-sensory cells remaining in the epithelium to transdifferentiate into replacement hair cells via gene therapy, or applying growth factors. Little is currently known regarding the status and characteristics of the non-sensory cells that remain in the deafened auditory epithelium, yet this information is integral to the development of therapeutic treatments. A single high-dose injection of the aminoglycoside kanamycin coupled with a single injection of the loop diuretic furosemide was used to kill hair cells in adult mice, and the mice were examined 1 year after the drug insult. Outer hair cells are lost throughout the entire length of the cochlea and less than a third of the inner hair cells remain in the apical turn. Over 20% and 55% of apical organ of Corti support cells and spiral ganglion cells are lost, respectively. We examined the expression of several known support cell markers to investigate for possible support cell dedifferentiation in the damaged ears. The support cell markers investigated included the microtubule protein acetylated tubulin, the transcription factor Sox2, and the Notch signaling ligand Jagged1. Non-sensory epithelial cells remaining in the organ of Corti retain acetylated tubulin, Sox2 and Jagged1 expression, even when the epithelium has a monolayer-like appearance. These results suggest a lack of marked SC dedifferentiation in these aged and badly damaged ears.

Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear.

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.