Transforming growth factor alpha with insulin stimulates cell proliferation in vivo in adult rat vestibular sensory epithelium.

Hair cells, the sensory receptors of the mammalian inner ear, have long been thought to be produced only during embryogenesis, and postnatal hair cell loss is considered to be irreversible and is associated with permanent hearing and balance deficits. Little is known about the factors that regulate hair cell genesis and differentiation. The mitogenic effects of insulin and transforming growth factor alpha (TGFalpha) were assayed in vivo in normal and drug-damaged rat inner ear. Tritiated thymidine and autoradiographic techniques were used to identify cells synthesizing DNA. Simultaneous infusion of TGFalpha and insulin directly into the inner ear of adult rats stimulated DNA synthesis in the vestibular sensory receptor epithelium. New supporting cells and putative new hair cells were produced. Infusion of insulin alone or TGFalpha alone failed to stimulate significant DNA synthesis. These results suggest that exogenous growth factors may have utility for therapeutic treatment of hearing and balance disorders in vivo.

Ultrastructure of hyaline, border, and vacuole cells in chick inner ear.

The sense organ for hearing in birds, the basilar papilla, is capable of replacing lost or damaged hair cells and supporting cells through regeneration. Potential candidates for precursor-cell populations include cells within the auditory receptor epithelium and nonsensory cells inferior to the sensory epithelium. Ultrastructural characteristics of hyaline cells, border cells, and vacuole cells, nonsensory cells which border or lie inferior to the receptor epithelium proper, were studied with transmission electron microscopy. Data were obtained from normal neonatal and adult chickens. Several rows of epithelial cells separate hyaline cells from inferiorly located organ supporting cells and hair cells. Ultrastructural characteristics and location of these epithelial cells differentiate them from organ supporting cells and hyaline cells; consequently, we have termed them "border cells." Synaptic specializations are observed between neural elements and border cells, and gap junctions are found between adjacent border cells, between border cells and neighboring organ supporting cells, and between juxtaposed border and hyaline cells. Hyaline cells, in contrast to border cells, are highly specialized. Dense bundles of filaments are present in hyaline cells from the basal one-half of the papilla, and an unusual structure, a rough tubular aggregate, is present in hyaline-cell cytoplasm. Pre- and postsynaptic specializations are observed between neural elements and hyaline cells, and gap-junctional complexes link neighboring hyaline cells. Vacuole cells lie inferior to the hyaline cells and rest on the inferior fibrocartilaginous plate. They are unspecialized morphologically. Their only remarkable morphological feature is the abundance of spherical vacuoles within their cytoplasmic matrix.

Induction of cell proliferation in avian inner ear sensory epithelia by insulin-like growth factor-I and insulin.

Postembryonic production of inner-ear hair cells occurs both normally and after insult in lower vertebrates and avians. To determine how this proliferation is controlled, several growth factors were tested for effects on progenitor-cell division in cultured avian vestibular sensory epithelium. Mitogenic effects of bombesin, epidermal growth factor, insulin-like growth factor-I (IGF-I), insulin, and transforming growth factor-alpha were assayed in organo-typic cultures of utricles from the mature, undamaged (normal) chicken inner ear. Tritiated thymidine and autoradiographic techniques and 5-bromo-2'-deoxyuridine (BrdU) immunocytochemistry were used to identify cells synthesizing DNA. IGF-I stimulated DNA synthesis in the vestibular sensory receptor epithelium in a dose-dependent manner. DNA synthesis was also stimulated by insulin. These results suggest that stimulation of the IGF-I receptors by IGF-I or insulin binding stimulates cell proliferation in the mature avian vestibular sensory epithelium.

Neurofilament proteins in avian auditory hair cells.

The distribution of middle-weight neurofilament protein (NF-M), an intermediate filament of neurons, was examined in the developing and mature avian inner ear by using immunocytochemical techniques. NF-M was detected in auditory hair cells and VIIIth cranial nerve neurons. NF-M-positive hair cells are first detected at embryonic day 11 (E11) in superior hair cells in the mid-proximal (mid-frequency) region of the chicken basilar papilla. With time, increasing numbers of hair cells express NF-M. Two developmental gradients occur: 1) a radial gradient, in which superior hair cells are labeled first, and progressively more inferiorly located hair cells are labeled during ontogeny, and 2) a longitudinal gradient, in which hair cells in the mid-proximal region are labeled first, and then progressively more distal (low-frequency) hair cells are labeled. There is also a small proximally directed progression of NF-M expression. By E19, NF-M-positive hair cells are found throughout the distal and mid-proximal regions, and this expression is maintained through 3 weeks posthatching. By 22 weeks posthatching, NF-M staining in hair cells is markedly diminished; staining is seen in only a few tall hair cells in the distal one-fourth of the papilla and in short hair cells in the distal one-half of the papilla. NF-M is never expressed by hair cells at the proximal (high-frequency) end of the papilla at any time examined. These findings suggest that some cell types that have traditionally been classified as nonneural may express neurofilament and that the basilar papilla of the neonatal chicken is not morphologically mature.

Macrophage and microglia-like cells in the avian inner ear.

Recent studies suggest that macrophages may influence early stages of the process of hair cell regeneration in lateral line neuromasts; numbers of macrophages were observed to increase prior to increases in hair cell progenitor proliferation, and macrophages have the potential to secrete mitogenic growth factors. We examined whether increases in the number of leukocytes present in the in vivo avian inner ear precede the proliferation of hair cell precursors following aminoglycoside insult. Bromodeoxyuridine (BrdU) immunohistochemistry was used to identify proliferating cells in chicken auditory and vestibular sensory receptor epithelia. LT40, an antibody to the avian homologue of common leukocyte antigen CD45, was used to label leukocytes within the receptor epithelia. Macrophages and, surprisingly, microglia-like cells are present in normal auditory and vestibular sensory epithelia. After hair cell loss caused by treatment with aminoglycosides, numbers of macrophage and microglia-like cells increase in the sensory epithelium. The increase in macrophage and microglia-like cell numbers precedes a significant increase in sensory epithelial cell proliferation. The results suggest that macrophage and microglia-like cells may play a role in releasing early signals for cell cycle progression in damaged inner ear sensory epithelium.

Basic fibroblast growth factor inhibits cell proliferation in cultured avian inner ear sensory epithelia.

Postembryonic production of inner ear hair cells occurs after insult in nonmammalian vertebrates. Recent studies suggest that the fibroblast family of growth factors may play a role in stimulating cell proliferation in mature inner ear sensory epithelium. Effects of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) were tested on progenitor cell division in cultured auditory and vestibular sensory epithelia taken from posthatch chickens. The effects of heparin, a glycosaminoglycan that often potentiates the effects of the FGFs, were also assessed. Tritiated-thymidine autoradiographic techniques and 5-bromo-2;-deoxyuridine (BrdU) immunocytochemistry were used to identify cells synthesizing DNA. The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end-label (TUNEL) method was used to identify apoptotic cells. TUNEL and overall counts of sensory epithelial cell density were used to assess possible cytotoxic effects of the growth factors. FGF-2 inhibited DNA synthesis in vestibular and auditory sensory epithelia and was not cytotoxic at the concentrations employed. FGF-1 did not significantly alter sensory epithelial cell proliferation. Heparin by itself inhibited DNA synthesis in the vestibular sensory epithelia and failed to potentiate the effects of FGF-1 or FGF-2. Heparin was not cytotoxic at the concentrations employed. Results presented here suggest that FGF-2 may be involved in inhibiting cell proliferation or stimulating precursor cell differentiation in avian inner ear sensory epithelia.

Characterization of damage and regeneration in cultured avian utricles.

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.

Postnatal production of supporting cells in the chick cochlea.

The auditory receptor organ in birds, the basilar papilla, is mitotically active after acoustic overstimulation or pharmacological insult and is capable of self-repair. The damaged epithelium is repopulated with new hair cells and supporting cells. The cell production that underlies this regenerative self-repair is believed to be a response evoked by damage in populations of cells that normally become mitotically quiescent even before hatching. In contrast, regeneration in the vertebrate nervous system is often correlated with continued or recent neurogenesis in the tissue concerned. The hypothesis that there may be ongoing postnatal production of cells in the normal avian basilar papilla was investigated. Autoradiographic analysis of tritiated-thymidine-injected animals was used to look for the existence of newly formed cells in the basilar papilla of normal posthatch chickens. Several types of supporting cells, namely, organ supporting cells, border cells and hyaline cells, are produced postnatally in the normal chicken. Typically, they are added interstitially to the apical (distal) half of the basilar papilla.

Intermediate filaments in the inner ear of normal and experimentally damaged guinea pigs.

The hypothesis that proteins known to occur in glial cells in the central nervous system may be present in inner-ear supporting cells was investigated. Immunocytochemical techniques were used to look for the existence of two classes of intermediate filaments, vimentin and glial fibrillary acidic protein (GFAP), in cellular elements of the inner-ear epithelium in normal and experimentally damaged guinea-pig cochleas. Vimentin is present in two types of supporting cells in the normal organ of Corti: Deiters' cells and inner pillar cells. Differences in intensity and distribution of vimentin immunostaining are observed across the three rows of Deiters' cells. GFAP immunoreactivity was not detected in any supporting-cell type in the organ. Cochlear hair cells were not labeled for either GFAP or vimentin. Following hair-cell destruction by exposure to noise or the administration of aminoglycosides, GFAP and vimentin are not present in phalangeal scars replacing lost hair cells.

Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.

The sensory epithelium of the avian inner ear retains into adulthood progenitor cells for inner-ear hair cells and other cell types in the epithelium. Hair cells are produced normally on an ongoing basis in the vestibular sensory epithelium, and hair-cell production is increased after insult in both auditory and vestibular sensory epithelia. The details of postnatal hair-cell production are not understood. In particular, molecular factors involved in the initiation and regulation of hair-cell genesis and differentiation are not known. Studies of this phenomena have been hampered by the lack of cell culture models. An organ culture system was developed which encourages generation and differentiation of hair cells in mature inner-ear sensory epithelia. Continuous labeling with tritiated thymidine showed genesis of both supporting cells and hair cells in normal vestibular epithelia grown in culture, and an increase in hair-cell and supporting-cell proliferation in damaged sensory epithelia grown in culture as compared to undamaged controls. This demonstrates, in vitro, both the division and differentiation of hair-cell progenitor cells in normal vestibular epithelia, and the maintenance of the hair-cell regeneration process in damaged inner-ear epithelia. This culture system should be useful for studies of hair-cell genesis and differentiation as well as studies of hair-cell and supporting-cell functioning in general.

Transforming growth factor-alpha with insulin induces proliferation in rat utricular extrasensory epithelia.

Hair cell loss in the human inner ear leads to sensorineural hearing loss and vestibular dysfunction. Recent studies suggest that exogenous addition of growth factors, for example, transforming growth factor-alpha with insulin, may stimulate the production of new supporting cells and hair cells in the mature mammalian vestibular sensory epithelium. Before any growth factor can be seriously considered for the treatment of clinical problems related to hair cell loss, its effects on the extrasensory epithelia must also be fully explored. The aim of this study was to determine whether transforming growth factor-alpha and insulin stimulate cell proliferation in rodent vestibular extrasensory epithelia. The cell proliferation marker, tritiated thymidine, was infused along with transforming growth factor-alpha, insulin, or transforming growth factor-alpha plus insulin into the inner ears of adult rats via osmotic pumps. Effects of the test agents were assessed on normal and drug-damaged utricles. Drug damage was produced by delivering gentamicin directly into the inner ear before the infusion of test agent. Animals were killed 4 or 10 days after pump placement. Utricles were sectioned, processed for autoradiography, and examined for labeled cells within the extrasensory epithelia. In normal animals, transforming growth factor-alpha plus insulin stimulated DNA synthesis in all regions of the extrasensory epithelia, suggesting that these agents are mitogenic for these tissues.

Hair cell regeneration in the inner ear.

Hearing and balance disorders caused by the loss of inner ear hair cells is a common problem encountered in otolaryngology-head and neck surgery. The postembryonic production of hair cells in cold-blooded vertebrates has been known for several decades, and recent studies in the avian inner ear after ototoxic drug and noise damage have demonstrated a remarkable capacity for both anatomic and functional recovery. The regeneration of sensory hair cells has been shown to be integral to this repair process. Current work is focusing on the cellular progenitor source of new hair cells and the trigger mechanism responsible for inducing hair cell regeneration. Preliminary studies suggest that reparative proliferation may also occur in the mammalian inner ear. Work in this field is moving at a rapid pace. The results thus far have yielded optimism that direct stimulation of hair cell production or transplantation of living hair cells may eventually become treatment modalities for the damaged human inner ear. These proposals would have been considered unrealistic less than 10 years ago, but they now have caught the full attention of both clinician and researcher.

Intracellular recordings from supporting cells in the guinea pig cochlea: DC potentials.

1. Supporting cells and hair cells from the low-frequency region of the guinea pig cochlea were studied in vivo using intracellular recording and horseradish-peroxidase (HRP) marking techniques. 2. The response of third- and fourth-turn support cells to tone bursts is composed of a number of components: an AC component at the frequency of the stimulating tone, harmonic components, a DC component present at the onset of the stimulating tone (the early DC), a slowly developing depolarization, and a slowly decaying afterpotential. 3. The early DC of support-cell responses is generally less than or equal to that in the adjacent organ of Corti fluids [at the best frequency (BF) for an 80 or 90 dB sound pressure level (SPL) stimulus the average early DC of support-cell responses is 0.9 times that of the adjacent fluids; n = 71], and both are less than that seen in the hair cells [average early DC of inner hair-cell (IHC) responses at the same sound levels is 14.2 times that in the adjacent organ fluids, n = 15; average early DC of outer hair-cell (OHC) responses is 11.5 times that in nearby organ fluids, n = 2)]. 4. The end DC, magnitude of the DC response shortly before signal end, in responses of support cells deep into Corti's organ [e.g., pillar, inner phalangeal, border cells] is often greater than that recorded in the potentials of the adjacent organ fluids (e.g., for an 80 or 90 dB SPL stimulus at the BF with a 30 ms steady-state time, the average end DC of the support cells deep into the organ is 2 times that of the adjacent organ fluids, n = 42). In contrast, the end DC for the responses of peripheral support cells--the Hensen's cells--generally equals, or is smaller than, the extracellular-fluid counterpart (for an 80 or 90 dB SPL stimulus, the average end DC of Hensen's cells is 0.9 times that of the nearby, outer-tunnel fluids, n = 29). Thus a difference exists across support-cell type with respect to support-cell end DC vis-à-vis that of the adjacent organ of Corti fluids. 5. A slowly increasing depolarization is often present in moderate and high-level support-cell responses. It is not normally present in IHC or OHC responses. Magnitude of the slowly increasing depolarization, the slow DC, is dependent on stimulus duration and stimulus level.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular recordings from supporting cells in the guinea-pig cochlea: AC potentials.

Supporting cells and hair cells from the low-frequency region of the guinea-pig cochlea were studied in vivo using intracellular recording and horseradish peroxidase marking techniques. Response characteristics of the support cells to tone bursts at various sound levels, frequencies, and durations were compared to hair-cell responses and potentials recorded in the organ of Corti fluid spaces. Findings suggest that the fundamental component of the support-cell response accrues from hair-cell-generated currents. This component of the support-cell response probably results from the flow of receptor currents across support-cell membranes through nonspecialized membrane patches, or across nonspecialized membranes and through gap junctions that couple adjacent support cells. Findings suggest a lack of electrotonic coupling between hair cells and neighboring support cells.

Induction of cell proliferation in mammalian inner-ear sensory epithelia by transforming growth factor alpha and epidermal growth factor.

Regenerative proliferation occurs in the inner-ear sensory epithelial of warm-blooded vertebrates after insult. To determine how this proliferation is controlled in the mature mammalian inner ear, several growth factors were tested for effects on progenitor-cell division in cultured mouse vestibular sensory epithelia. Cell proliferation was induced in the sensory epithelium by transforming growth factor alpha (TGF-alpha) in a dose-dependent manner. Proliferation was also induced by epidermal growth factor (EGF) when supplemented with insulin, but not EGF alone. These observations suggest that stimulation of the EGF receptors by TGF-alpha binding, or EGF (plus insulin) binding, stimulates cell proliferation in the mature mammalian vestibular sensory epithelium.

Growth factor regulation of the cell cycle in developing and mature inner ear sensory epithelia.

Growth factors and other extracellular signals regulate cell division in many tissues. Consequently, growth factors may have therapeutic uses to stimulate the production of replacement sensory hair cells in damaged human inner ears, thereby assisting in alleviating hearing loss and vestibular dysfunction. Assessment of the ability of growth factors to stimulate cell proliferation in inner ear sensory epithelia is at an early stage. This paper provides a brief account of what we know regarding growth factor regulation of cell proliferation in developing and mature inner ear sensory epithelia.

Hair cell regeneration in the avian inner ear.

The postembryonic production of hair cells in fish and reptiles has been known for several decades. Until recently it was assumed that this capacity was absent in the more highly specialized inner ears of birds and mammals. Recent research has shown, however, that birds have the capacity to rebuild a damaged inner ear. Summarized here are studies conducted in our laboratory which address the following questions: (1) Which are the precursors of the regenerated hair cells? (2) Are the new hair cells functional? (3) What are the ultrastructural properties of regenerated hair cells? and (4) Can the level of proliferation be regulated? Both the auditory and the vestibular systems of the avian inner ear were studied. Our results provide some answers to these questions. The implications of the results are discussed.

Diffusible factors regulate hair cell regeneration in the avian inner ear.

Damage to the avian inner ear results in up-regulation of mitotic activity resulting in regeneration of hair cells. The objective of this investigation was to determine whether the damaged inner ear epithelium releases a soluble mitogen that is responsible for the up-regulation of proliferation. The sensory epithelium from normal and drug-damaged avian inner ears was cultured alone or in the presence of other cultures. As previously shown in vivo and in vitro, damaged organs displayed increased supporting cell proliferation compared with undamaged organs, leading to eventual morphologic and functional recovery. When damaged organs were cocultured with an undamaged organ, proliferation was increased in the undamaged tissue. When undamaged organs were cultured together, proliferation was decreased. These results indicate that a soluble factor released from the damaged inner ear epithelium stimulates proliferation and suggest the release of a factor from normal tissue that suppressed mitotic activity. Thus, reparative hair cell regeneration in the inner ear appears to be regulated by a balance between proliferative and antiproliferative paracrine factors.

Recent insights into regeneration of auditory and vestibular hair cells.

Advances in hair cell regeneration are progressing at a rapid rate. This review will highlight and critique recent attempts to understand some of the cellular and molecular mechanisms underlying hair cell regeneration in non-mammalian vertebrates and efforts to induce regeneration in the mammalian inner ear sensory epithelium.