| Objective:Acoustic trauma is one of the main occupation hazardsin the world. Sound conditioning is the process of body adaptation inthe lower training noise. Given the modest stimulus, should obtain thegreater tolerance to acoustic overstimulation, and protect the hearingfunction. The mechanism of sound conditioning for hearing protectionis not clear. In recent years, with the further development of humangenome project, miRNAs has been paid more and more attention. ThemiR-183/Taok1target pair is likely to play a role in regulation of thedegenerative process of the cochlea following acoustic overstimulation.The study is to observe the expression of miRNA-183(miR-183) incochlea of guinea pigs after low level sound conditioning.Methods: With normal Preyer’s reflex,forty male healthy guineapigs were used. The animals were randomly divided into four groups。NG group （normal group), CG group (conditioning noise group), CHGgroup （conditioning noise followed by high-level noise group）, HGgroup (high-level noise group). The animals of NG group were in thereverberation chamber with the background noise being less than35dB.The guinea pigs were numbered and each one was fixed in the wire cagewith loud speaker above animals’ heads. The noise exposure system wascomposed of signal generator, power amplifier and loudspeaker. Thenoise signal from dynamic signal analyzer was inputted to theloudspeaker after amplified by the power amplifier. Based on the literature and our previous experience, acclimatization exposure wasused the center frequency of1000Hz, and intensity of (90±l)dB SPL,daily exposure to6h, for7d. The CHG group rested2d after soundconditioning, and then accepted the high intensity noise with the samefrequency as CG group and intensity of (112±l)dB SPL, exposure to4h. The HG group was exposed the same conditions as the CHG group.The signal generator, power amplifier and loudspeaker were warmed upfor10minutes before test. The noise intensity was calibrated by usingsound level meter before each test and1h interval. The hearingthreshold shift was detected by the auditory brainstem response (ABR).Animals in the waking state were fixed on the audiometry table in asound insulation shielding box and earphone diaphragm from theexternal auditory canal opening of3cm. The results were determined bythe needle electrodes. The acoustic stimulation was for click. The testwas begun from the high intensity determination, gradually decay, untilthe lowest strength graphics for its response threshold appearing. Thethreshold intensity was tested twice. Groups of guinea pigs werebeheaded, and the bilateral cochlear tissues were removed quickly, oneside put into the0.5%silver nitrate solution(AgNO3) to stain,4%PFA fixed, exposure, peeling, color stretched observation andcounting. Outer hair cell loss of the cochlear basilar membrane wasmeasured by surface preparation technique. The total RNA wasextracted from the other side of cochlear tissue stored in liquid nitrogenwith Trizol. The whole process of extraction was in the situationwithout RNA enzyme. The extract was assayed by spectrophotometryand denaturing gel. The total RNA was transcribed intocDNAwithmiR-183reverse primers. The expression of miR-183inguinea pigs cochleae were examined by Real-time quantitative PCR(qRT-PCR) with miR-183primers and the cDNA as the template. TheU6molecule as the control gene, every sample repeated3holes. ΔCt=CtmiR-183-CtU6, expression of miR-183=2-△Ct. The amplificationefficiency of PCR must be greater more than90%is the necessaryfactor to use the formula for calculating. PCR efficiency could beobtained by the slope of calibration curve. The system would automatically give the standard curve of miR-183and U6and the slopeof linear regression test after the datum of detectedgene(miR-183)cDNA and control gene(U6)cDNA with10timesgradient dilution being input. The slope should represent the efficiencyof PCR.Results:1.The impaction of sound conditioning on hearingthreshold. The threshold shift gradually reduced daily in the process ofsound conditioning. It showed that the sound conditioning was efficient.The hearing injury caused by noise exposure in four groups were shownthat the hearing threshold shift of CG group (8.7±1.4)dB was less thanCHG (29.3±6.4) dB and HG group（42.8±9.2）dB. That of HG group wasthe most serious. The difference between CHG group and HG groupwas significant(P<0.01).2.The impaction of sound conditioning on haircell morphology and outer hair cell loss rate. The cochlear basilarmembrane was observed by the optical microscope (×400). It was foundthat a certain number of outer hair cells(OHC) were lack of normalguinea pig cochlea basilar membrane every circle, and the deletion of Ito Ⅲ ring had the trend to increase gradually. After noise exposure, thestereocilia of cochlea outer hair cells were injured obviously,manifested as stereocilia arranged scattered, fusion, degenerative anddeletion. The HG group was the most serious. The loss of stereociliamainly concentrated in the basilar membrane Ⅱ, Ⅲ ring. IHC was notfound obvious injury. The damage of outer hair cells between the CGgroup （%，T1：0.89±0.18、T2：2.08±0.44、T3：2.26±0.84）was slightlymore serious than NG group（%，T1：0.64±0.16、T2：1.64±0.34、T3：1.81±0.38）, but the difference was not significant. The CHG group（%，T1：1.24±0.21、T2：4.68±0.67、T3：6.81±0.98）and HG group（%，T1：4.01±0.84、T2：6.78±1.18、T3：8.94±1.89）compared withthe NG group, the significant reduction of the outer hair cells in everyring were observed（P＜0.05）. The significant reduction of the outerhair cells was observed between CHG group and HG group （P＜0.05）.It was suggested that sound conditioning should reduce the outerhair cell loss induced by strong noise exposure.3. The expression ofmiR-183in the cochlea of guinea pigs. The expression of miR-183in guinea pigs cochlea was detected by QRT-PCR. The results showed that,the expression of miR-183in CG group （13.04±0.86） was highercompared with that in NG group（6.19±0.88）increased（P＜0.05）;theexpression of miR-183in CHG group （4.13±0.66） was lowersignificantly than that of NG group (P＜0.05); the expression ofmiR-183in HG group（1.55±0.21）was lower than that of NG group (P＜0.05); the expression of miR-183in HG group was lower than that ofCHG group (P＜0.05). The results suggested that sound conditioningshould promote the expression of miR-183in the cochlea, and strongnoise damage should reduce the expression of miR-183. There was nosignificant difference of outer hair cell damage in NG and CG group,but the expression of miR-183in the CG group was higher than NGgroup. The outer hair cells injury were more serious and the expressionof miR-183was lower. The expression of miR-183in CHG group（4.13±0.66） and HG group （1.55±0.21） showed a significantreduction compared with NG group(P <0.05). The level of miR-183inHG group was lower than CHG group(P＜0.05).Conclusion: Sound conditioning should cause some changes ofcochlea outer hair cells and protect the outer hair cells from damage ofacoustic overstimulation. The protection maybe achieved by promotingthe expression of miR-183. miR-183in cochlea induced by soundconditioning is higher than that in normal cochlea organ, soundconditioning can drop off outer hair cells loss arised from the followinghigh-level noise exposure. The high expression of miR-183shouldinhibit the injury and loss of outer hair cells’ by repressing itsdownstream target genes, and result in hearing protection.ThereforemiR-183in cochlea organ induced by sound conditioningmight provide protective effect on auditory system. |
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