Moderate Noise Exposure,Establishment Of Mouse Model Of Tinnitus With Normal Hearing And Its Accumulated Effect On The Plasticity Of Cochlear Ribbon Synapses

Objective: To investigate the characteristics of hearing impairment in 6 week old male C57BL/6J mice after being exposed to 100 d B SPL broadband white noise for 2 hours,and to establish model of tinnitus confirmed by the acoustic startle reflex and prepulse inhibition.Changes of the ribbon synapse in inner hair cells and neurotransmitter in auditory center are studied.Some of the mice suffered a second time noise exposure,to investigate the difference of hearing and cochlear pathologies between first and second noise exposure.Methods: Exposed to the 100 d B SPL broadband white noise for 2 hours,to investigate the characteristics of hearing impairment,and to establish model of tinnitus confirmed by the acoustic startle reflex and prepulse inhibition.The 6 week old male SPF C57BL/6J mice were used.ABR and acoustic behavioral tests were performed at 6 weeks(the same day post-exposure),7,8,10,14 weeks of age.Analysis the threshold,amplitude and latency of wave I of ABR;Analysis the data of ASR,NB-PPI and GAP-PPI.Mice were sacrificed for immunohistololgy and HPLC.Ribbon synapses of each inner hair cells were counted.The content of glutamic acid and GABA in inferior colliculus and auditory cortex were determined by liquid chromatography.Some of the mice suffered a second time noise exposure,changes of electrophysiological index,ribbon synapses,morphologies of inner and outer hair cells were observed.The data were analyzed by using one or two way ANOVA.Results:At 6 week of age(the same day post-exposure),threshold of noise exposure group: 4k Hz(39±4.59 d B)、8k Hz(26±3.16 d B)、16k Hz(21.5±5.79 d B)、32k Hz(22.5±5.89 d B);control group(6w): 4k Hz(31.5±5.29 d B)、8k Hz(22±3.49 d B)、16k Hz(16.5±3.37 d B)、32k Hz(18±3.49 d B),the difference between groups was statistically significant(P<0.05),one week after noise exposure(7w): 4k Hz(31.5±3.37 d B)、8k Hz(22±4.22 d B)、16k Hz(16.5±3.37 d B)、32k Hz(18±2.58 d B);control group(7w): 4k Hz(31.5±3.37)、8k Hz(22±4.22)、16k Hz(16.5±3.37)、32k Hz(18±2.58),the difference between groups was not statistically significant(P>0.05).Continued to observe the threshold at 8,10,14 w,the difference between groups was not statistically significant(P>0.05).At 80 d B the amplitude of wave I showed a similar trend,significantly reduced at the same day post-exposure(p<0.001),besides,one and two weeks after exposure,the difference between groups was still statistically significant(P<0.05): 6w(0.82±0.27 uv)、7w(0.98±0.39 uv)、8w(0.96±0.32 uv)、10w(0.97±0.34 uv)、14w(0.94±0.44 uv);control group:6w(1.12±0.29 uv)、7w(1.12±0.30 uv)、8w(1.10±0.28 uv)、10w(1.07±0.29 uv)、14w(0.99±0.28 uv).At 80 d B the latency of wave I was not significantly changed(P>0.05).The ASR was significantly increased after exposure.Compaired to control group ASR was significantly increased at 8 and 10 weeks of age,the difference at 10 w was more significant(p<0.01).The startle reflex increased within stimulation at level of 80,90,100 d B SPL,among which,ASR at 90 d B SPL showed the most significant differences(p<0.01).Analyzed by the frequencies,ASR were significantly increased at all four frequencies: 4k、8k、16k、32k Hz(p<0.05),8k Hz showed the most significant differences(p<0.01).For NB-PPI,as the intensity of prepulse signals gradually enhanced,the inhibition rate increased,however,there were no significant difference between the groups(p>0.05).Moreover,it was observed for 8 weeks after the noise exposure,and inhibition rate did not change with the growth of the age.There was no significant difference between the groups(p>0.05).For GAP-PPI,the startle reflex inhibition rate significantly decreased in the first week after noise exposure and continued until the mice at 14 weeks of age(p<0.05).Analyzed by the frequencies of background noise,the startle reflex inhibition rate was significantly lower than the control group after noise exposure at all frequencies.At 8k and 16 k The statistical significance was most significant(p<0.001),32 k Hz(p<0.01),4k Hz(p<0.05).Immunofluorescence staining of cochlear basal membrane was observed under confocal microscopy,pre-synaptic Ct BP2 and post-synaptic Glu R2/3 were specific expressed in intracellular cytoplasm of inner hair cells.The expression of pre-and post-synaptic structures in the inner hair cells showed a highly co-localization phenomenon.The number of ribbon synapses in the inner hair cells in the day after exposure was significantly lower than that of the control group(p<0.001).At 6 week of age(the same day post-exposure),ribbon counts were significantly decreased at all four frequencies: 4k、8k、16k、32k Hz,at 16 k and 32 k the statistical significance was more significant(p<0.001).At 7 weeks of age,there were no significant difference between the groups and continued until the mice at 14 weeks of age(p>0.05).The results of HPLC of normal C57 mice showed that GABA in the inferior colliculus was higher than in the auditory cortex;Glu in the auditory cortex was higher than in the inferior colliculus,the difference was statistically significant(p<0.001).In the auditory cortex,the excitatory neurotransmitter Glu was significantly increased at 10w(p<0.01),and the significant difference between the two groups was further increased at 14w(p<0.001);the inhibitory neurotransmitter GABA did not change significantly in the auditory cortex(p>0.05).In the inferior colliculus,Glu did not change significantly(p > 0.05);GABA was significantly decreased at 14w(p<0.05).After the second noise exposure,the threshold rose even more significantly(p<0.001),there were still very significant differences between groups at 14 weeks of age(p<0.001).The changes of amplitudes of wave I and the counts of ribbon synapse were parallel to the threshold.However,the morphology and count of hair cells were not effected.There was no significant difference in otoacoustic emission among the three groupsConclusion:(1)The establishment of tinnitus animal model without obvious hearing loss was successful by exposed to 100 d B SPL broadband white noise for 2 hours.(2)The counts of ribbon synapses decreased significantly at the day of noise exposure,suggesting there were deafferentation in the peripheral nerve.Because of the plasticity,the number can be restored to normal during the subsequent dynamic observation time.(3)The auditory center shows the imbalance of excitatory and inhibitory neurotransmitters,producing hyperexcitability.(4)The results of the second exposure were different from the first,the number of ribbon synapses in the cochlea decreased permanently,suggesting the limitations of the auditory nerve plasticity.