Protective Effects Of MiRNA-182against Drug-induced Hearing Loss In Rats

Part One Establishment of Acute Drug-induced Hearing Loss byCo-administrating Loop Diuretic Furosemide and Kanamycin in RatsObjectsTo establish an acute drug-induced hearing loss modal by co-administrating loopdiuretic furosemide (Fur) and kanamycin (KM) in rats.Methods32adult female SD rats were randomly divided into four groups:1day group,7daygroup,14day group and control group. All rats received intraperitoneal injections of KM,followed by an intraperitoneal injection of Fur30minutes later. The control groupreceived no treatment. Auditory brain responses (ABR) the day before and1,7and14 after drug administration were recorded. Histopathological and immunohistochemicalchanges were examined by fluorescence microscopy and scanning electron microscopy.ResultsThe ABR threshold in1,7,14day drug administration groups were significant higherthan control group. The ABR threshold shift between the three drug administration groupshad no significant differences. Immunofluorescence and scanning electron microscopyshowed obvious outer hair cell loss in the basal turn in each drug administration groups,with cilia disarrangement and fusion. The expression of cleaved caspase-3was stronger inthe drug administration groups than control group.ConclusionsCo-administration of furosemide and kanamycin by intraperitoneal injection caninduce stable and profound hearing loss in rats and is an effective method to establishacute drug-induced hearing loss model in animal experiments.Part Two The Study of Distribution and Effect of miRNA-182in Cochlearthrough Round Window AdministrationObjectsTo evaluate the distribution of miRNA-182in cochlear when administrated throughround window path and its effect on hair cells.Methods24female S-D rats were randomly divided into three groups: operation+miRNA-182administration group; operation+RNase-free water administration; operation only. Bothears of each rat accepted operation respectively. ABR were record at12,24,48hour afteroperation. The distribution of mi-RNA-18was observed by immunofluorescence at24,48h. The changes of hair cell were observed by scanning electron microscopy (SEM) andconfocal microscopy at24h, respectively. ResultsThe ABR threshold shift had no significant difference among the three groups (P＞0.05). The immunofluorescence of miRNA-182can be obviously observed in the corti,stria vascularis at24hours after operation, much stronger than48h. No outer hair cellloss and microstructure changes were observed.ConclusionmiRNA-182can distribute into corti, stria vascularis when administrated withstronger immunofluorescence at24hour than48h. miRNA-182administration throughround window had no effect on the audiology and structure of rat cochlear.Part Three The Protective Role of miRNA-182via Antiapoptotic Mechanismin Rats of Drug-induced Hearing LossObjectsTo evaluate the protective effect of miRNA-182in rats of drug-induced hearing loss.Methods24female S-D rats were divided into three groups: miRNA-182group, RNase-freewater group and control group. Each rat accepted operation and administrated ofmiRNA-182or RNase-free water through round window, the control group acceptedoperation only. Then Fur and KM were administered i.p.24hours after operation. TheABR threshold shift of each rat was recorded at day1,7,14after drug administration. Therate of outer hair cell loss was calculated. The expression of cleaved caspase3wasdetected by confocal microscopy and the microstructure changes were evaluated underSEM.ResultsThe ABR threshold shift of miRNA-182group was significantly reduced than theother two groups. There was no significant difference of ABR threshold shift betweenRNase-free water group and control group. Immunofluorescence and scanning electronmicroscopy showed obvious outer hair cell loss with cilia disarrangement and fusion in RNase-free water group and control group with the rate of outer hair cell loss rate of40.47%and47.06%, respectively. The outer cell loss and disarrangement in miRNA-182group was significantly reduced with lower outer hair cell loss rate (21.57%). Theexpression of cleaved caspase-3was significantly stronger in the RNase-free water groupand control group than miRNA-182group.ConclusionmiRNA-182administrated through round window can distribute into the cochleareffectively. Exogenous miRNA-182protected the outer hair cell from apoptosis andreduced drug-induced hearing loss, for which the mechanism needs further investigation.