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Objectives: to study the apoptotic response of auditory hair cells in the organ of Corti after exposure to neomycin within the first 6 hours. Materials and methods: auditory hair cells harvested on the postnatal day 7 from the cochleae of C57BL6 wild type mice were cultured in vitro in the presence (neomycin group, n= 59 cochleas) or absence of neomycin (control group, n= 31 cochleas). Apoptotic cells were evaluated within 6 hours of incubation for the expression of active caspases (8, 9 or 3) in the cytoplasm and counterstained using propidium iodide to highlight apoptotic nuclei. Cells were statistically quantified using average and standard deviation. The differences between the averages were tested with the help of Student T test (threshold p< 0,05). Results: The statistic comparison between the different caspase types (caspase 3, 8 and 9), the average and dispersion inside the cell groups, permitted us to detect the first step of apoptosis taking place in the cytoplasm. The comparison of the cell group where propidium iodide was accumulated in the nucleus with the cell group where both the caspase and propidium iodide appeared, permitted us to detect the initiation and development of the second step of apoptosis, the one where the nucleus has already been affected. Conclusions: Neomycin is initiating, right after 6 hours of incubation, the process of apoptosis in auditory hair cells, determining the activation of cytoplasm caspases.

In vitro expansion of chondrocytes for tissue-engineering applications is limited by forms of growth arrest known as quiescence and replicative senescence. At the molecular level cyclin-dependent kinase inhibitors (CDKIs) are involved in mediating growth arrest in the G1 phase of the cell cycle. Using ribonuclease protection assays and immunocytochemical staining methods, we quantitatively analyzed expression profiles of G1 cell cycle inhibitors at the mRNA and protein levels. These inhibitors included the CDKIs of the CIP/KIP family (p21CIP1 p27KIP1, and p57KIP2) and the INK4 family (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) as well as the retinoblastoma protein-family (pRb, p107, and p130) and the tumor suppressor p53. Analysis was carried out in proliferating, quiescent, and senescent states of primary cultures of adult human nasoseptal chondrocytes. The most pronounced effect (p0.0001) between cultures in proliferation and cultures in growth arrest was an increased expression of the CDKIs p57KIP2 and p15INK4b for quiescent growth arrest, and of p16INK4a, p15INK4b, and p57KIP2 for senescent growth arrest. Thus, these cell cycle inhibitors represent potential candidates for selective intervention to promote cellular multiplication of chondrocytes undergoing in vitro expansion for tissue-engineering applications. Possible methods of modulation include the targeted elimination of specifically identified cell cycle inhibitors by antisense technologies.

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al . (1995) Science 267, 1024–1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27 Kip1 is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27 Kip1 -gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27 Kip1 -induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

The spatial-temporal course of hair cell degeneration and hair cell death was examined in the mammalian cochlea following aminoglycoside treatment. Organotypic cultures were established from postnatal rats (P3) and treated with 1 mM neomycin sulfate for 12-48 h and analyzed using a live/dead assay under epifluorescence microscopy. Live hair cells were labeled with calcein, a probe whose fluorescence and cellular retention depends upon intracellular esterase activity and cell-membrane integrity, respectively. Hair cell death was determined by ethidium homodimer-1, a probe that can enter cells with compromised cell membranes only. Inside the cell it binds to DNA. Hair cell morphology was also examined using phalloidin labeling, scanning electron microscopy and semi-thin section analysis. Results showed that hair cell degeneration and hair cell death occurred in a time dependent gradient from base to apex. After 48 h of neomycin treatment, most apical hair cells survived while most basal hair cells died. Calcein labeling provides a sensitive functional assay for measuring hair cell survival.