Effect And Mechanisms Of Aβ Toxicity Accelerating Age-related Hearing Loss

Objectives:Alzheimer’s disease（AD）and Age-related hearing loss（ARHL）are both neurodegenerative diseases.However,the correlation between the two diseases and whether they can be used as biomarkers for each other has not been thoroughly explored.The purpose of this study was to determine whether there was clinically consistent ARHL in 9-month-old C57BL/6J mice,and to trace the changes in cognitive function and auditory function in elderly mice by constructing Aβ-toxic AD model,to explore the progress of AD and ARHL,and to provide a theoretical basis for the application of hearing tests in the early diagnosis of AD.Methods:Part I ARHL in C57BL/6J miceAuditory brainstem response（ABR）was used to detect changes in the auditory thresholds of click,4k Hz,8k Hz,16k Hz,24k Hz,and 32k Hz in the ears of 2-month and 9-month healthy male C57BL/6J mice.Changes in auditory function,loss of outer hair cells（OHCs）in apex,middle and base cochlear were studied by surface preparation.Part II Aβtoxicity on auditory and cognitive function in ARHL miceThe 9-month C57BL/6J mice were randomly divided into 2 groups:saline group and AD-like model group(Aβ_1-42)after baseline tests of auditory function and recognition memory using ABR（click）and novel object recognition（NOR）test.Sterile0.9%Nacl and 0.1μg/μl Aβ_1-42 solution（5μl/piece）were injected into the lateral ventricle using a stereotaxic instrument.ABR test and NOR test were conducted at 1,2,3 and 4 weeks after injection,and the changes of auditory system and cognitive system were continuously observed.The number of outer hair cells in the apex,middle and basal of cochlea was observed by the staining technique of basement membrane.Western blot was used to detect expression levels of Aβ,P-tau,cell metabolism associated protein P-AMPK,apoptosis associated protein Bcl2 and Bax in hippocampus,auditory cortex and brain stem.Results:Part I ARHL in C57BL/6J miceCompared with 2-month-old mice,the ABR thresholds of click,4k Hz,8k Hz,16k Hz,24k Hz and 32k Hz were increased in 9-month-old mice（P﹤0.05）,particularly at high frequency（24k Hz、32k Hz）,from（51.43±4.76）（62.86±6.36）at 2 months to（84.29±4.50）and（91.43±3.78）at 9 months,respectively.Cochlear surface preparation results showed the number of outer hair cells in the apex,middle and basal cochlear was decreased in 9-month mice（P﹤0.05）,in comparison to 2-month mice,suggesting that C57BL/6J mice started to manifest ARHL at 9-month and lose of cochlear outer hair cells,which is consistent with clinical data.Part II Aβtoxicity on auditory and cognitive function in ARHL miceAuditory and cognitive function baseline showed that the there were no significant differences in ABR（click）and NOR between saline group and with Aβ_1-42 group（P>0.05）.There were no significant differences in ABR and NOR of Saline group at 0,1,2,3,4 weeks after injection（P>0.05）,despite the ABR threshold（click）present a rising trend.Compared with 0 week,ABR threshold in Aβ_1-42 group at 1 week after injection is rising dramatically,and still rising at 2,3,4 weeks after injection,with statistically significant（P﹤0.05）.NOR results showed significant difference in preference index（number）and recognition index（time）of new objects since 3 weeks after injection.These results suggested that auditory dysfunction manifested earlier than cognitive dysfunction after exposure of Aβ.Cochlear surface preparation results showed there were no statistical significance in OHCs counts at apex,middle and basal cochlear in Aβ_1-42 group at 0,1,2,3,4 weeks after injection,indicating Aβhad no significant toxic effect on peripheral auditory system.WB results showed that compared with baseline of the Aβ_1-42 group,the protein of Aβ,P-tau and Bax in hippocampus increased at 1 week after injection（P﹤0.05）,Bcl2decreased at 1 week after injection（P﹤0.05）,and P-AMPK increased at 2 weeks after injection（P﹤0.05）.In the auditory cortex,the expression levels of Aβ,P-tau and Bax were increased at 1 week after injection（P﹤0.05）,Bcl2 was decreased at 1 week after injection（P﹤0.05）,and P-AMPK was increased at 3 weeks after injection（P﹤0.05）.WB results showed that compare to baseline,Aβ,P-tau and Bax protein in hippocampus started to increase from 1 week after injection（P﹤0.05）;Bcl2 started to decrease since1 week after injection（P﹤0.05）;and P-AMPK started to increase from 2 weeks after injection（P﹤0.05）.In auditory cortex,Aβ,P-tau and Bax expression started to increase from 1 week after injection（P﹤0.05）;Bcl2 started to decrease since 1 week after injection（P﹤0.05）;and P-AMPK started to increase from 2 weeks after injection（P﹤0.05）.In brain stem,Aβ,P-tau and Bax expression started to increase from 1 week after injection（P﹤0.05）;Bcl2 started to decrease since 1 week after injection（P﹤0.05）;and P-AMPK started to increase from 3 weeks after injection（P﹤0.05）.These results suggested Aβhas significant toxic effects on central auditory system and cognitive system.Conclusions:1.C57BL/6J mice at 9 months showed clinically consistent ARHL,ie full-frequency hearing loss with an increased threshold of high frequency,accompanied by loss of cochlear outer hair cells.2.Aβhad toxic effects on both auditory system and cognitive function in ARHL（9-month-old C57BL/6J）mice,characterized by increased auditory threshold and impaired recognition memory.Hearing impairment manifested earlier than cognitive impairment.The elevated auditory threshold can be used as an early biomarker of AD.3.Age and Aβcan both increase the auditory threshold.ARHL is associated with the loss of outer hair cells in the cochlea,and Aβcan accelerate the increase of the auditory threshold in ARHL mice,which may be related to the damage of central auditory nervous system caused by Aβtoxicity.