17-DMAG Induces HSP70and Protects The Auditory Hair Cells From Kanamycin Ototoxicity In Vitro

Part one:The study on the culturing of corti’s organ in vitroObjective:To establish a stable method for culturing of the organ of Corti and staining of cochlear hair cells in vitro.Methods:The post natal3-5days mice were used. The basal membrane was separated away from the cochlea under the anatomical microscope and cut into three segments: the basal turn, the middle turn and the apical turn. The segments were positioned on the bottom of the dishes by the surface tension of culture medium. The segments were stained by fluorescent labeled phalloidin and observed under fluorescence microscope.Results:After24hours the basal membrane segments were held in place by the surface tension of culture medium. There were some newborn epithelial cells and fibrocytes at peripheral region. After2-4days the newborn cells increased. The cochlea segments were examined under fluorescence microscope. The stereocilia arranged as an inverted’V without significant disorder. The one-row inner hair cells and three-row outer hair cells were all in a well arranged order without marked defect or loss.Conclusion:The method for culturing of the organ of Corti by the surface tension of culture medium was successful. And the growing status of cochlear hair cells could be observed by phalloidin staining method. Part two:The effects of17-DMAG and kanamycin on the audtory hair cells in vitroObjective:To investigate whether geldanamycin derivative17-(Dimethy lamino ethylamino)-17-demethoxygeldanamycin (17-DMAG) and kanamycin have any effect on the audtory hair cells in vitro.Methods:To assess possible cytotoxic effect of17-DMAG on hair cells,17-DMAG of four concentrations was performed (0.5μM,1μM,2μM and5μM) for24h. To assess the toxic effect of kanamycin sulfate on the cochlear hair cells, three concentrations were performed (0.2mM,0.4mM and1.0mM) for24h. The surviving hair cells were examined by phalloidin labeling and were counted.Results:No visible negative effects of17-DMAG (0.5μM,1μM,2μM and5μM) on the hair cells were observed. At the lowest concentration of kanamycin slight deficiency of hair cells was observed. At the concentration of0.4mM the survived hair cells reduced further. At the highest concentration the most serious loss was observed. A dose-dependent deleterious effect of kanamycin on hair cells was showed. The damage of outer hair cells (OHCs) was more serious than that of inner hair cells (IHCs).Conclusion:17-DMAG is not toxic to the hair cells at the concentration of0.5μM,1μM,2μM and5μM. A dose-dependent deleterious effect of kanamycin on hair cells was showed. The results provide the drug concentration for the next study. Part three:17-DMAG induces HSP70and protects the auditory hair cells from kanamycin ototoxicity in vitroObjective:The aim of this study was to investigate whether geldanamycin derivative17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) has the ability to induce heat shock protein70in the cultured organ of Corti and to protect hair cells from kanamycin ototoxicity in vitro.Methods:The organ of Corti (OC) explants were isolated from mice at postnatal day3-5. Then, the explants were exposed to kanamycin with or without pre-incubation with17-DMAG The expression of HSP70was assessed by reverse transcription-quantitative polymerase chain reaction, ELISA, and immunofluorescent staining. The surviving hair cells were examined by phalloidin labeling and were counted.Results:We found that HSP70expression in the explants after pre-incubation with17-DMAG was significantly increased at both mRNA and protein levels. Immunofluorescent staining showed that HSP70was mainly located in the auditory hair cells. Compared with kanamycin group, the loss of hair cells was inhibited significantly in17-DMAG+kanamycin group.Conclusion:Our study demonstrated that17-DMAG induces HSP70in the hair cells, and has a significant protective effect against kanamycin ototoxicity in vitro.17-DMAG has the possibility to be a safe and effective anti-ototoxic drug.