Protective Effect Of 17-DMAG From Nicotine Ototoxicity In Rat Cochlear Organtypic Culture

Part one:Culturing of organs in inner ear in vitro Objective:To establish a stable method for culturing of organs in inner ear in vitro.Methods:The basilar membrane was dissected from postnatal two or three-day-time Sprague-Dawley rat. The basilar membrane were cut into three parts according to the three turns:the basal, middle and apical one, then the cultures were flatted on culture dishes by the surface tension. The species were stained with fluorescent and observed by laser scanning confocal microscope and fluorescence microscope.Results:The cultures were flattened by the surface tension of medium in 24 hours. The spieces were examined by immunofluorescent staining. The light microscopy and fluorescence microscope showed the integrity of the basic structures of cultures, the normal structure of the hairs, spiral ganglion cells, and nerve fibers. There was no hair cell loss or defect. The stereocilia on hair cells was arranged orderly without significant disorder.Conclusion:The integrity of the basic structures of cultures meets the demands of Ototoxicity experiment.Part two:Nicotine ototoxicity in rat cochlear organotypic culturesObjective:To establish the animal model of nicotine ototoxicity and investigate the toxic effect of nicotine on cochlear hair cells, spiral ganglion cells, and nerve fibers in cochlear organ cultures as well as its dose response relationship.Metholds:Cochlear basilar membrane of two-or three-day-old Sprague-Dawley rat was cultured and treated with various doses of nicotine for 24 hours. Cochlear hair cells and the spiral ganglion neurons were observed by immunofluorescent staining. In addition, the ultrastructure of the stereocilia bundle, hair cells, and supporting cells were examined by scanning and transmission electron microscopy.Result:Increased in hair cell loss was concomitanted with the increase in nicotine concentrations. Nicotine impaired both the inner and outer hair cells at a similar degree. The damage to hair cells was more severe at basal turn than apical turn. The treatment of nicotine resulted in a fusion and loss of stereocilia bundles in hair cells, decrease of mitochondria, and vacuolation and degranulation of the endoplasmic reticulum. In contrast, the spiral ganglion neurons and auditory nerve fibers were intact.Conclusion:Nicotine primarily caused hair cell loss, but not significant impairment the neural structure in vitro. Hair cell damage induced by nicotine was initiated at the basal turn and extended towards the apical turn. Nicotine ototoxicity was dose-dependent in vitro.Part three:Protective effects of 17-DMAG on nicotine induced ototoxicity in cochlear organotypic cultures of neonatal rat through upregulation of heat shock protein 70Objective:To evaluate the effectiveness of 17-DMAG in treatment of Nicotine induced ototoxicity.Methods:Cochlear basilar membrane of two-or three-day-old Sprague-Dawley rat was dissected and cultured. Specimens were exposed to Nicotine in the presence or absence of pre-incubation with 17-DMAG. The protein and mRNA expressions of HSP70 in all groups were detected by reverse transcription-quantitative polymerase chain reaction (RT-PCR) and ELISA. Cellular localization was obtained by immunofluorescent staining. The surviving hair cells, spiral ganglion cells, and nerve fibers were counted.Results:Induced mRNA and protein expressions of HSP70 were significantly elevated in pre-incubation of 17-DMAG. Immunofluorescent staining showed HSP70 were mainly expressed in the cytoplasm of hair cells. In comparison with nicotine group,17-DMAG significantly prevented nicotine on cochlear culture in treatment group.Conclusion:Our study demonstrated that ototoxicity of nicotine in cochlear basilar membrane was partly due to HSP70 induced by 17-DMAG in hair cells, and 17-DMAG could be a potential protective agent against nicotine ototoxicity in vitro.Part four:Downregulation of the large conductance Ca2+-activated K+ channel expression in C57BL/6J cochlea AbstractObjective:Age-related hearing loss (AHL) is the most common among elderly persons. The large conductance Ca2+-activated K+channels (BK), act as sensors for membrane voltage and intracellular Ca2+, are essential for hearing. To investigate the distribution of BK channel in the C57BL/6J cochlea, and the relationship between the expression of BK channel and the etiology of AHL.Methods:We studied BK channels expression in the cochleae of C57BL/6J mice at various ages (4,12,26,52 weeks). The expressions of BK at the protein and mRNA levels were detected by Immunofluorescence technique, western blot and quantitative real-time PCR.Results:In comparison with 4-week-old mice, BK expressions in the cochlea at 12, 26, and 52 weeks of age were significantly and gradually decreased at both the protein and the mRNA levels. Immunofluorescence technique showed BK was located in the hair cells and cells of the spiral ganglion, spiral ligament and stria vascularis, and its expression was also decreased with aging.Conclusion:Our findings indicate that BK channel expression in the cochlea is decreased in the AHL mice. BK channel may be associated with AHL.