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Aminoglycoside antibiotics, loop diuretics, antineoplastic agents and other commonly used pharmacological drugs are ototoxic. Understanding of the cellular and molecular mechanisms underlying drug ototoxicity, however, has been hampered by the limited availability of inner ear tissues and drug side effects on laboratory animals. Immortalized cell lines derived from the auditory sensory organ, sensitive to ototoxic drugs and growing in environments that can be systematically manipulated, would facilitate the research directed at elucidating these mechanisms. Such immortalized cell lines could also be used to discover novel therapeutic agents for preventing drug-induced sensorineural hearing loss. Here, we report a conditionally immortalized organ of Corti-derived epithelial cell line, which shows evidence of activation of apoptosis when exposed to known ototoxic drugs. This cell line may be an excellent in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs.

Auditory outer hair cells can elongate and shorten at acoustic frequencies in response to changes ofplasma membrane potential. We show that this fast bidirectional contractile activity consists of an electromechanical transduction process that occurs at the lateral plasma membrane and can be activated and analyzed independently in small membrane patches inside a patch electrode. Bidirectional forces are generated by increases and decreases in membrane area in response to hyperpolarization and depolarization, respectively. We suggest that the force generation mechanism is driven by voltage-dependent conformational changes within a dense array of large transmembrane proteins associated with the site of electromechanical transduction.

The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-a, IL-1b, and IL-6, through the activation of ERK and NF-kB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-a. Immunohistochemical studies revealed that TNF-a expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-kB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-a, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

The recognition of invading microbes followed by the induction of effective innate immune response is crucial for host survival. Human surface epithelial cells are situated at host-environment boundaries and thus act as the first line of host defense against invading microbes. They recognize the microbial ligands via Toll-like receptors (TLRs) expressed on the surface of epithelial cells. TLR2 has gained importance as a major receptor for a variety of microbial ligands. In contrast to its high expression in lymphoid tissues, TLR2 is expressed at low level in epithelial cells. Thus, it remains unclear whether the low amount of TLR2 expressed in epithelial cells is sufficient for mediating bacteria-induced host defense and immune response and whether TLR2 expression can be upregulated by bacteria during infection. Here, we show that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by nontypeable Hemophilus influenzae (NTHi), an important human bacterial pathogen causing otitis media and chronic obstructive pulmonary diseases. Activation of an IKK␤-IB␣-dependent NF-B pathway is required for TLR2 induction, whereas inhibition of the MKK3/6-p38␣/␤ pathway leads to enhancement of NTHi-induced TLR2 up-regulation. Surprisingly, glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the p38 MAP kinase pathway. These studies may bring new insights into the role of bacteria and glucocorticoids in regulating host defense and immune response and lead to novel therapeutic strategies for modulating innate immune and inflammatory responses for otitis media and chronic obstructive pulmonary diseases.

The developing inner ears of mice (CBAKBA), ages ranging from gestational day 12 through postnatal day 21, were examined using scanning electron microscopy following the epoxy-embedding/ freeze-fracture technique. This technique provides unique three-dimensional views of surface and fractured structures of the developing inner ear, thus allowing excellent preservation of the relationships between the developing sensory epithelium and the overlying membranes (i.e. the tectorial membrane and cupula) during their development.The tectorial membrane is formed of two distinct parts: the major (medial) and the minor (distal). The major portion is produced by the cells of the greater epithelial ridge prior to the formation of the minor part, which is produced largely by the primordial supporting cells of the lesser epithelial ridge. The developing tectorial membrane has two types of fibers: radial (found mainly in the major part) and slanted (found mainly in the minor part). The slanted fibers become the cover net, which fuses with the marginal band. The marginal zone of the developing tectorial membrane is completely sealed during development by the third row of Deiters' cells. The surfaces of cells that produce the tectorial membrane are characterized by numerous long microvilli which are largely lost when the tectorial membrane completely forms and separates from the supporting cells.The surface of developing auditory sensory cells is initially covered with numerous microvilli, some of which become future stereocilia. Stereocilia form stepped rows in the shape of a "W", with the tallest row located at the periphery of the cell. As the sensory cell matures, the short transitional stereocilia gradually disappear. Kinocilia on hair cells are still seen in the 14-day-old mouse (even though the organ of Corti is morphologically mature) but not in the 21-day-old mouse, indicating that complete maturation of the sensory cells in all turns is attained by 21 days of age. The mouse has upper radial tunnel fibers and basal tunnel fibers. Neural contacts of the upper radial tunnel fibers with the outer hair cells at the apical portions are frequent in the developing organ of Corti.The external sulcus cells undergo drastic changes during development, forming numerous pits that are often covered with mucus-like droplets or grape-like spherical structures of varying sizes. This phenomenon was observed only during postnatal days 6 and 14.The developing cupula starts as a thin amorphous membrane, which later becomes compact and fibrotic-like as the mass increases. By the 6th postnatal day well-developed cupular canals occur. In some canals in the central zone of the crista, the tall stereociliary bundles are in contact with a part of the canal wall, but short stereociliary bundles are free-standing in the subcupular space. In the periphery of the crista, the cupular canals are smaller in diameter or are not seen at all at the extreme periphery. Consequently, only the tall ciliary bundles are directly in contact with the cupula. The on...

Background: Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and β defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable Haemophilus influenzae (NTHi) 12, Moraxella catarrhalis 035E, and Streptococcus pneumoniae 3, and 6B.