Characterization of damage and regeneration in cultured avian utricles

Hair cell regeneration occurs spontaneously throughout life and following h air cell injury in the vestibular epithelia of mature birds and other nonma mmalian vertebrates. We examined hair cell regeneration in post-hatch chick utricles that were cultured in media with or without the ototoxin, strepto mycin, for various periods. The goal of our study was to characterize the d ose- and time-dependent effects of streptomycin on hair cell loss and regen eration in vitro. Utricles that were cultured with streptomycin for 1 day d isplayed a dose-dependent loss of hair cells in spatial patterns and levels that were consistent with those observed in comparable experimental paradi gms in vivo. Incorporation of the nucleotide analog bromodeoxyuridine (BrdU ) demonstrated that supporting cell proliferation is decreased during the f irst day of culture in the presence of streptomycin, but it increases over time when cultures are subsequently placed in streptomycin-free media. Utri cles cultured for 1 day with streptomycin followed by 2-4 more days without streptomycin had numerous bundles of immature stereocilia, suggesting that new hair cells were generated in vitro. We tested this hypothesis by cultu ring utricles with BrdU for 3 or 5 days and double-labeling them to detect BrdU and the hair cell-specific antigen, TuJ1. Numerous BrdU-positive/TuJ1- positive cells with phenotypic characteristics of immature hair cells were present in the cultures, and the number of such cells increased between 3 a nd 5 days in vitro, in a dose-dependent manner.