The avian auditory epithelium is capable of complete regeneration after hair cell (HC) loss. Most new HCs arise via cell division, but approximately one-third of new HCs arise via direct transdifferentiation (DT), in which supporting cells (SCs) alter their phenotype without dividing. In this study, we used synchronous, gentamicin-induced near-total HC loss in the basal end of the epithelium and continuous infusion of the cell division marker bromodeoxyuridine (BrdU) to identify the origin of each individual regenerating HC. Early new HCs were identified by immunolabeling for the HC-specific marker myosin-VIIa, and mitotic cells with BrdU immunolabeling. The first new HCs arising via DT appear 72-96 hr after gentamicin, 24-48 hr earlier than the first new mitotic HCs. After Day 6, however, most new HCs are mitotic. The "intermediate" morphology that has been suggested to be characteristic of DT is seen in HCs arising via both pathways. These findings suggest that DT is a simpler, more rapid process that produces the first new HCs, and that mitotic regeneration is somewhat slower but ultimately produces most new HCs. The identical morphology of regenerating HCs from both pathways suggests that once HC fate is established, all new HCs follow similar cellular processes during differentiation and reorganization into the regenerated epithelium.

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

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