Characterization of Damage and Regeneration in Cultured Avian Utricles

JONATHAN I. MATSUI 0 ELIZABETH C. OESTERLE 0 JENNIFER S. STONE 0 EDWIN W. RUBEL 0 0 Virginia Merrill Bloedel Hearing Research Center and the Department of Otolaryngology-HNS, University of Washington , Seattle, WA 98195 USA Hair cell regeneration occurs spontaneously throughout life and following hair cell injury in the vestibular epithelia of mature birds and other nonmammalian vertebrates. We examined hair cell regeneration in post-hatch chick utricles that were cultured in media with or without the ototoxin, streptomycin, for various periods. The goal of our study was to characterize the dose- and time-dependent effects of streptomycin on hair cell loss and regeneration in vitro. Utricles that were cultured with streptomycin for 1 day displayed a dose-dependent loss of hair cells in spatial patterns and levels that were consistent with those observed in comparable experimental paradigms in vivo. Incorporation of the nucleotide analog bromodeoxyuridine (BrdU) demonstrated that supporting cell proliferation is decreased during the first day of culture in the presence of streptomycin, but it increases over time when cultures are subsequently placed in streptomycin-free media. Utricles 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 culturing 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 and 5 days in vitro, in a dose-dependent manner. Keywords: hair cells, vestibular, chicken, tissue culture, differentiation, proliferation INTRODUCTION Hair cells are the mechanoreceptors of the auditory, vestibular, and lateral-line end organs that are involved in the detection of sound, balance, orientation, and head movements. Disease, aging, infection, and exposure to noise or ototoxic drugs cause hair cell loss in the inner ear. Birds and other nonmammalian species are capable of regenerating hair cells and thereby repairing their receptor epithelium such that function is regained (reviewed in Cotanche et al. 1994; Carey et al. 1996; Stone et al. 1998; Cotanche 1999; Smolders 1999). In contrast, damage to the mammalian inner ear results in permanent hearing and/or balance deficits. Insights gained from studying hair cell regeneration in nonmammalian species may shed light onto mechanisms that can be used to trigger hair cell regeneration in mammals. In the mature vestibular epithelia of birds, hair cells die spontaneously and are continually replaced via mitosis throughout the life of the animal (Jrgensen 1991; Jrgensen and Mathiesen 1988; Roberson et al. 1992; Weisleder and Rubel 1993). Hair cell production in this system is highly upregulated over control levels when large numbers of hair cells are killed by experimental exposure to the ototoxic aminoglycoside antibiotic, streptomycin (Weisleder and Rubel 1992, 1993; Weisleder et al. 1995; Oesterle et al. 1993; Carey et al. 1996; Bhave et al. 1998). On the other hand, continual postembryonic production of hair cells does not occur in the auditory receptor epithelium, the basilar papilla, of birds (Ryals and Rubel 1988; Corwin and Cotanche 1988; Oesterle and Rubel 1993). However, restoration of the auditory hair cell population does occur after hair cell loss is experimentally induced by ototoxic drugs or intense noise (Cotanche 1987; Cruz et al. 1987; Corwin and Cotanche 1988; Ryals and Rubel 1988; Lippe et al., 1991). Regenerated hair cells in both the avian auditory and vestibular systems achieve sufficient function to restore balance and hearing function (for review, see Cotanche et al. 1994; Stone et al. 1998; Cotanche 1999; Smolders 1999). In birds, the progenitors to the new hair cells appear to be a subpopulation of the resident supporting cell population (Corwin and Cotanche 1988; Girod et al. 1989; Raphael 1992; Roberson et al. 1992; Hashino and Salvi 1993; Stone and Cotanche 1994; Tsue et al. 1994; Warchol and Corwin 1996). Recent studies also suggest that the mature mammalian vestibular epithelium has a limited capability for the mitotic replacement of hair cells (Warchol et al. 1993; Lambert 1994; Tanyeri et al. 1995; Yamashita and Oesterle 1995; Li and Forge 1997; Lopez et al. 1997; Zheng et al. 1997; Zheng and Gao 1997; Kuntz and Oesterle 1998). Cell proliferation and hair cell regeneration occur in in vitro preparations of the hair cell epithelia of many nonmammalian species. In mature birds, new hair cells are formed in cultures of the utricle (e.g., Oesterle et al. 1993; Warchol and Corwin 1993) and the basilar papilla (Navaratnam et al. 1996; Stone et al. 1996). None of these studies, however, has provided an in-depth analysis of the extent of hair cell loss, mitotic activity, and hair cell replacement in culture. This project had two objectives. First, we wished to develop and standardize an in vitro preparation of the avian utricle for studies of streptomycin-induced hair cell loss and of subsequent events leading to hair cell regeneration. Second, we sought to determine the extent to which newly produced cells in vitro display hair cell phenotypes by using a mitotic tracer in combination with a hair cell-specific marker. Chick utricles were cultured in media supplemented with various concentrations of streptomycin. Some utricles were examined immediately after the streptomycin treatment (short-term cultures) to define the patterns and extent of hair cell loss, whereas other utricles were cultured in streptomycin-free media following the drug exposure (long-term cultures) to allow hair cell regeneration to occur. Hair cell loss was directly related to streptomycin concentration and was confined to the striolar region in short-term cultures but extended beyond the striolar region in long-term cultures. Sensory epithelial cell proliferation, as assessed by labeling with the nucleotide analog, bromodeoxyuridine (BrdU), decreased immediately after exposure to low and moderate doses of streptomycin and increased significantly by 1 day after removal of the drug. High doses of streptomycin inhibited supporting cell proliferation at all times. Scanning electron microscopy and the use of a cell proliferation tracer in combination with the hair cell-specific marker, TuJ1, demonstrated that many cells that are generated mitotically in culture goon to differentiate as hair cells. Preliminary reports of portions of these data were presented previously (Matsui et al. 1997a,b). MATERIALS AND METHODS White Leghorn chickens (Gallus domesticus) were obtained from a local distributor (H & N International, Redmond, WA) and were housed in brooders with ample food and water in the University of Washingtons ani (...truncated)