Effects of growth factors on the hair cells after ototoxic treatment of the neonatal mammalian cochlea in vitro.

The aim of this study was to test the possible regenerative potential of several molecules and growth factors such as retinoic acid (RA), insulin, epidermal growth factor (EGF) and transforming growth factors alpha (TGFalpha) and beta (TGFbeta) on the neonatal cochlea in vitro after neomycin intoxication. Our studies show that cochlear sensory epithelium behaves differently while maintained in various culture conditions, although we did not observe regeneration whatever the molecules or growth factors tested. The ototoxic action of neomycin in vitro produced a specific death of hair cells, except in the apical region. Organ of Corti of rats 3 days after birth always presented two regions that responded differently to the antibiotic: a widespread scar region extending from the basal cochlea up to the beginning of the apical turn, where most hair cells had disappeared, and a second region called the resistance region localized in the apex, and which was more or less developed depending on culture conditions. The length of the resistance region was modulated by molecules or growth factors added to the feeding solution suggesting that some of them could produce a protective action on hair cells against neomycin. Slight protection effects may be found with RA and insulin, however, the most definite protection results from the combination of insulin with TGFalpha as shown by the large increase in the length of the resistance region compared to organ of Corti treated with antibiotic alone. The tested molecules and growth factors did not promote cochlear hair cell regeneration in vitro after neomycin treatment, however some of them may offer a protective action against ototoxicity.

Factors modulating supernumerary hair cell production in the postnatal rat cochlea in vitro.

It has been shown in the past that extra hair cells or supernumerary cells can be produced when neonatal cochleae are maintained in vitro. In this report, we investigated the effects of the culture methods, molecules and growth factors that are thought to be involved in cell proliferation. Quantitative studies of supernumerary hair cells were made by measuring the cell density over the entire spiral lamina at two postnatal stages: birth and 3 days after birth. With a standard feeding solution without serum, a difference in cell density was observed between the two methods of culture. Cochlear explants in a standard feeding solution supplemented with serum showed an increase of cell density only when the explantation is made at birth. Retinoic acid added to the standard feeding solution did not increase the hair cell density, while insulin induced an increase, especially at 5 micrograms/ml. Several growth factors were tested. Epidermal growth factor (EGF) presented a dose dependent effect with an increase of up to 30% of hair cell density that was observed in the basal region when the explantation was made at birth. Transforming growth factor-alpha did not induce an increase of cell density, whereas transforming growth factor-beta presented an effect on hair cell density, with a dose dependent effect reaching 37.4% for the basal inner hair cells. Interpretation of these results is limited because of the lack of data concerning the presence of specific membrane receptors. One possibility is that insulin stimulates hair cell differentiation from existing undifferentiated cells. Another hypothesis may be related to the EGF and transforming growth factor-beta, where these molecules might induce transdifferentiation of cells by acting on the transmembrane molecules and the extracellular matrix.

Ontogenesis of rat cochlea. A quantitative study of the organ of Corti.

A systematic quantitative set of data concerning the organ of Corti in developing Sprague-Dawley rats at intervals from 18 days of gestation to 10 days after birth (DAB) is provided in this study. Using phalloidin staining, the total number of inner and outer hair cells, the whole length of cochlea, as well as the diameter of inner and outer hair cells and the intercellular space between inner hair cells were determined in order to analyze the quantitative change of inner and outer hair cells during development and to explore some roles of the factors regulating the growth of cochlea. The results show that: (1) The length of cochlea approached its adult size by 7DAB. (2) The growth of the extreme part of the apex was responsible for the delayed elongation of the cochlea. (3) Growth in the cochlear length mainly results from an increase of cell diameter tempered by a decrease of intercellular space. (4) The adult size of inner and outer hair cells was obtained by 7-14DAB. (5) The final number of inner and outer hair cells was reached at 3DAB and remained constant through adulthood. No significant hair cell overproduction and cell death were observed during ontogenesis of the cochlea. The negligible importance of overproduction and missing hair cells during hair cell differentiation suggest that there is a precise regulation phenomenon for producing the right spatial organization of the organ of Corti.

The spontaneous appearance of hair cell-like cells in the mammalian cochlea following aminoglycoside ototoxicity.

Young rats (in vivo) and cochleas from neonatal rats (in vitro) were treated with ototoxic antibiotics. Scanning electron microscope observations of the cicatricial epithelium of the former outer hair cell region revealed cells with a tuft of microvilli at their apical surface that could contain actin filaments, as observed by phalloidin staining. The apical organization of these hair cell-like cells was reminiscent of fetal hair cells topped with a bundle of microvilli. During both in vivo and in vitro observations, and despite the use of several growth factors in vitro, these hair cell-like cells did not differentiate into mature sensory cells. These hair cell-like cells might represent an attempt by the former sensory epithelium to regenerate.