Atoh1 Overexpression Regulate Cochlear Hair Cell Regeneration In Neonatal Mammals In Vitro

OBJECTIVES:Overexpression of Atohl inducing HCLCs has been confirmed both in vivo and in vitro. Meanwhile, inhibition of Notch signaling generates supernumerary hair cells in embryonic or neonatal cochleae. However, the effects of Atohl overexpression and DAPT, both simultaneously and separately, on neomycin-treated cochleae were previously unknown. Proper Atohl expression levels and durations are required for inner and outer hair cell differentiation in vivo,whether Atohl affect EHCLCs formation in the same way is undiscoverd. There is no research whether ectopic regenerated hair cells will occur CE movement which are similar to the developmental hair cells and the regulatory factors. The following questions need to be solved:1) To compare the different effect of Atohl overexpression and Notch signaling inhibitor DAPT simultaneously and separately on neomycin-treated cochlea. 2) Whether different Atohl titres has an effect on the formation and cilia of EHCLCs in LER of the neonatal rat cochleae.3) Does the EHCLCs induced by Atohl overexpression have CE movement and the what’s the regulatory factors.METHODS:Cochlear basement membrane cultured in vitro to induced EHCLCs was the model.1). only the middle turns cultured in the special foetal bovine serum (FBS) condition after neomycin treatment, then treated with adenovirus-EGFP-Atohl and DAPT (an y-secretase inhibitor), both simultaneously and separately.8 days post tranfection, counted the number of myosin7a positive cells along the SE. Immuno-fluorescence technique and T-test were used to prove and analysis the effect of Notch signaling inhibitor to the EHCLCs induced by Atohl overexpression.2). Cultured explants were infected with several virus titres, and EHCLCs expression was detected in the LER at different time points. Observe EHCLCs number and cilia morphology at different time points and virus titres.3). Measure the width of the EHCLCs to the SE and the gap between the EHCLCs at different time points and virus titres. Assess whether EHCLCs have group CE phenomenon, and analyze the related regulation factors.RESULTS:1)、① basilar membrane cultured in DMEM/F-12 containing 10% foetal bovine serum(FBS) for 48 h leads to large gap between supporting cells and adenovirus vector highly transfecting the SE cells.② Atohl overexpression induced the appearance of HCLCs in the organ of corti. ③ Combined application of DAPT and Atohl overexpression dramatically increased the number of myosin7a-positive cells with hair cell features. EGFP、DAPT% DMSO alone as control group did not induce HCLCs. 2)、①24h after the titre of 0.1 x 108, 0.4x 108,0.8x 108,1.6x 108 PFU/ml Ad5-EGFP- Atohl tranfected the cochlear explants, higher titres leads to greater Atohl expression.However, when 2.4x10 PFU/ml was used, the cultured explants disintegrated. ?Very low titres of Ad5-EGFP-Atohl such as 0.16xi08PFU/ml induced only weak Atohl expression without inducing EHCLCs formation at different time pointin O.4xio8,0.8xio8,1.6xio8 PFU/ml titres, the number of EHCLCs increased with Atohl expression level and time. ?Higher Atohl expression cells early than lower expression cells to become EHCLCs. Nosensory cells in the LER which failed to become EHCLCs gradually transformed into the precursor cells. ?0.4x108 tol.6 ×108 PFU/ml concentration range, EHCLCs cilia become more mature with Atohl expression level and time prolonged. 3X ?EHCLCs induced by Atohl overexpression can occur spontaneously covergent in group migration, the most lateral cells gradually and slowly move to Corti’s area, finally merge with the inner and outer hair cells into an organic whole, the same phenomenon was not observed in control group. ? 0.4 xlO8 tol.6xl08 PFU/ml concentration range, EHCLCs group gathering migration velocity increases with the prolonged Atohl expression level and time. ? surface cell adhesion protein E-cadherin expression level in EHCLCs is high, and which in the control group significantly decreased.CONCLUSIONS: IX ?Joint application of neomycin and FBS helped adenovirus infection. ? Atohl overexpression induced HCLCs in the organ of corti. DAPT promote the hair cell regeneration induced by Atohl. 2X ?Different virus titer leads to different transfection efficiency after EGFP-Atohl infection in the LER. ?Higher Atohl expression level and time induced more EHCLCs and cilia more mature. 3)> EHCLCs are coverge spontaneously in group migration, and positively correlated with Atohl expression level and time.