Caspase-Mediated Apoptosis In The Cochleae Contributes To The Early Onset Of Hearing Loss In A/J Mice

Objective:The purpose of this study was to observate the change of the function、morphology and structure of cochleae in the process of age-related hearing loss and apoptosis related gene transcription and expression levels in the cochleae of A/J mice. By knockdowning the level of Cdh23 and/or Cs of HEI-OC1 cells,to analysis the mechanisms of age-related hearing loss. Finally,through giving antiapoptotic drugs to A/J mice, the curative effect was observed.Methods:A total of 157 A/J mice (83 male and 74 female) and 137 B6 mice (75 male and 62 female) with ages from 2 to 46 weeks were used in this study.① ABR and DPOAE were measured at various intervals at 3,4,10,12,28, and 42 weeks for B6 and A/J mouse strains.② The time points selected were 2 weeks,4 weeks,10 weeks, and 46 weeks. Sections (5 μm) mounted on glass slides were counterstained in hematoxylinand eosin (HE)and immunostaining for active caspase-3.③ Observing of OHC in the cochleae at 3 weeks,10 weeks,16 weeks, and 28 weeks.④ Assays for gene transcription of apoptosis related genes at 2 and 8 weeks.⑤ Through transfecting with shRNA in HEI-OC1 cells, to observe theexpression of caspase-3 by western blotting.⑥ Twenty-one A/J mice at the age of 7 days were divided into three groups:a test group, a dimethyl sulfoxide (DMSO) group, and an untreated group. In the test group, the mice were injected intraperitoneally under sterile conditions with Z-VAD-FMK or DMSO. ABR and DPOAE were tested at 3,4,6, and 8 weeks for the mice in each group, and then all of the mice from each group were sacrificed for histological investigation.Results:Hearing loss occurs much earlier in A/J mice at about 4 weeks of age. We firstly showed that A/J mice displayed more severe degeneration of hair cells, spiral ganglion neuron (SGNs) and stria vascularis in the cochleae compared with B6 mice. Moreover, mRNA accumulation levels of Caspase-3 and Caspase-9 in inner ears of A/J mice were significantly higher than those in B6 mice at 2 and 8 weeks of age. Immunohistochemistry localized Caspase-3 expression mainly to hair cells, SGNs and stria vascularis in cochleae. In vitro transfection with Cs shRNA alone or cotransfection with Cs shRNA and Cdh23 shRNA significantly increased the levels of Caspase-3 in an inner ear cell line (HEI-OC1). Finally, a pan-caspase inhibitor Z-VAD-FMK could preserve the hearing of A/J mice by lowing about 15 decibel (dB) sound pressure level (SPL) of the ABR thresholds.Conclusion:Taking together, we conclude that apoptosis in the cochleae, which may be related to mutation of Cs, contributes to the early onset of hearing loss in A/J mice.