| Objective: Cochlear ribbon synapses between inner hair cells(IHCs)and auditory nerve endings are important structures in the nerve conduction pathway,which play key role in sound coding.Cochlear ribbon synapses are vulnerable to noise damage,and the cumulative damage caused by repeated stimulation can lead to irreversible lesions and abnormal hearing function.The mechanism of cochlear ribbon synapses sensitivity to noise and the specific regulation mechanism of plasticity are not very clear.The neuroprotective effect of brain-derived nerve growth factor(BDNF)on the inner ear has attracted increasing attention,but the regulatory mechanism of BDNF involved in noise sensitivity is still unclear.Studies have shown that autophagy plays an important role in synaptic remodeling in the nervous system.This study aims to investigate the regulatory effect of autophagy on the plasticity of cochlear ribbon synapses after noise exposure,and to analyze the temporal significance of autophagy expression in the ribbon synapses of inner hair cells in the cochlea.Brain-derived nerve growth factor(BDNF)was used to protect cochlear ribbon synapses from noise-induced damage,and to explore the molecular mechanism of BDNF’s protective effect on cochlear ribbon synapses and hearing function in mice,and to explore the protective mechanism of BDNF on cochlear high-frequency hearing.Methods: In this study,6-week-old male C57BL/6J mice were exposed to broadband white noise at 100 d B SPL for 2 hours in single and secondary noise exposures(NE).At D1,D14,D15 and D28 after noise exposure,the auditory brainstem response(ABR)threshold and I wave amplitude at different frequencies were detected,and the auditory function and synaptic function were evaluated by immunofluorescence staining.The expression changes of autophagosome LC3 B and lysosome Lamp1 were observed by immunofluorescence staining.The relationship between the changes of autophagy flux and the changes of cochlear ribbon synapses was further observed.BDNF was introduced into the posterior semicircular canal.Based on the animal models of TTS and PTS,the protective effect of BDNF on cochlear hearing function and synaptic function was further evaluated by detecting the changes of ABR threshold,I wave amplitude and the number of ribbon synapses.The spatial specificity of the number of immunofluorescence ribbon synapses among the apical,middle and basal axis of the cochlea and the expression of autophagosome LC3 B and lysosomal Lamp1 were observed.Western blot was used to detect the expression of autophagy markers LC3 B and p62 in the cochlear tissues,and the autophagic flow protein expression were analyzed.At the same time,an in vitro mouse basement membrane tissue culture model was established to simulate the excitotoxic effect of glutamate,and autophagy enhancer RAP and PI3 K specific inhibitor LY294002 were used to regulate the effect.Immunofluorescence staining was used to observe the amount of Ct BP2 in ribbon synapses of inner hair cells,and the expression of autophagosome LC3 B and lysosomal Lamp1.Western blot was used to observe the expression of LC3 B,p62,PI3 K,p-AKT,AKT,p-mTOR and mTOR pathway-related proteins.Results: 1.ABR threshold: in the TTS model,there was no significant difference between the BDNF+NE group and the control group.In the PTS model,there were significant differences in the frequency of click,4k Hz and 8k Hz between the BDNF+NE group and the control group on D15,and there were significant statistical differences in the frequency of 16 k Hz and 32 k Hz between the BDNF+NE group and the NE group on D15 and D28.In the TTS model,the amplitude of ABR I in the BDNF+NE group was significantly lower than that in the control group at D1 and D7,but there was no significant difference at D14.The D1 amplitude was higher in the BDNF+NE group than in the NE group,and the difference was statistically significant.In the PTS model,the amplitudes of the BDNF+NE group and the NE group were lower than those of the control group,but the amplitudes of the BDNF+NE group were higher than those of the NE group on D15,D21 and D28.(3)Changes in morphology and number of cochlear ribbon synapses: in the TTS model,the number of ribbon synapses in BDNF+NE group was lower than that in control group at D1,but the difference was not statistically significant,and was statistically significant compared with NE group.There was no statistically significant difference among the three groups on D14.In the PTS model,the number of ribbon synapses in the BDNF+NE group and the NE group on D15 and D28 was significantly reduced,but the BDNF+NE group was significantly more than the NE group.The loss of ribbon synapses in the parietal and middle axis was more significant in the BDNF+NE group on D28.(4)The change of autophagic flow: in the TTS model,the autophagic flow in the NE group increased rapidly and then returned to normal,and there was no significant difference between the BDNF+NE group and the control group on D1.In the PTS model,the autophagic flow in the NE group increased gradually,and there was no significant difference between the BDNF+NE group and the control group on D15,and the autophagic flow increased slowly on D28.5.In vitro basement membrane culture: Glutamate excitotoxicity in vitro simulated noise-induced damage to cochlear ribbon synapses.The NK group significantly increased the expression of LC3 B and Lamp1 in the ribbon synapses of inner hair cells,and the number of ribbon synapses was significantly decreased in the NK group.The BDNF+NK group inhibited the expression of autophagy,and the number of ribbon synapses was significantly increased compared with the NK group.Western blot showed that BDNF+NK inhibited the expression of LC3 B and increased the expression of p62 compared with the NK group.The expressions of PI3 K,p-AKT/AKT and p-mTOR/mTOR in BDNF+NK group were significantly increased,and the expressions of PI3K/AKT/mTOR pathway related proteins in BDNF+NK group treated with LY294002 were significantly inhibited.The number of ribbon synapses was significantly reduced in the BDNF+NK+LY294002group compared with the BDNF+NK group.Conclusion: The early cochlear ribbon synapse remodeling after moderate noise exposure is regulated by the level of autophagy.Repeated noise exposure leads to abnormal enhancement of ribbon synapse autophagy activity in inner hair cells,reducing the plasticity of ribbon synapses,and further leading to the decline of hearing function.BDNF can reduce noise-induced cochlear ribbon synapse damage in animal models of TTS and PTS,protect the auditory function,and regulate the high-frequency susceptibility of cochlear ribbon synapses through autophagy.BDNF regulates the remodeling process of cochlear ribbon synapses induced by noise by inhibiting autophagy.Moreover,inhibition of autophagy through PI3K/AKT/mTOR pathway can protect cochlear ribbon synapses from noise-induced damage. |
