Experimental Study Of Apoptosis And The Related Mechanisms In Cochlea Of Rats After Noise Exposure

NIHL is a common occupational disease and its incidence is high. For manyyears, researchers have been going in for the mechanisms of NIHL. HC loss ismajor cause of permanent hearing loss. For the reason that the mammaliancochlea is unable to regenerate HCs, the mode of cell death and the relatedmechanisms are the key points for the research of NIHL. Though much evidence has indicated there is noise induced apoptosis incochlea, the phase regularity and the related mechanisms are rarely reported. So,we put the experimental emphasis on this aspect. Objective: In our study, to investigate the phase regularity and mechanismsand to make a theoretical basis for the intervention of NIHL, we observedapoptosis,expression of caspase-9,caspase-3,P-JNK and interfered with JNKinhibitor CEP-1347 in a stable and reliable animal model by advanced practicaltechnique methods. Methods: Healthy male wistar rats were exposed to 118 dB SPL,4KHz for6h and divided into groups according to time course. We detected cochlearapoptosis with TUNEL, expression of caspase-9,caspase-3,P-JNK with RT-PCR,immunohistochemistry and Western blot. We also observed the change of ABRthreshold and apoptosis after the animals were subcutaneous injected withCEP-1347. Meanwhile, we probed the relation between OHC apoptosis and ABRthreshold, expression of caspase-9,caspase-3,P-JNK and OHC apoptosis,expression of P-JNK and expression of caspase-9 , caspase-3 with linearregression.Results:1,Apoptosis and expression of caspase-9 and caspase-3 in cochlea afternoise exposure.① Results of ABR threshold:There were significant difference between noiseexposed groups and control group(P<0.01);There were no significant differencebetween 3h,6h and 24h group(P>0.05);There were no significant differencebetween 7d and 14d group(P>0.05);There were significant difference amongother noise exposed groups(P<0.01).②Results of apoptosis with TUNEL: Apoptosis were detected after noiseexposure. The positive cells mainly distributed in OHC,SG and SV. A fewapoptotic cells were observed at 3h, the positive cells increased to peak at 24hwhile some OHCs were lost, then decreased gradually. At 14d there werethimbleful positive cells. There were significant difference between 3h and controlgroup (P<0.05). The differences between other noise exposed groups and controlgroup were more significant (P<0.01).③R esults of RT-PCR: There were slight caspase-9 and caspase-3mRNA incontrol group. Up regulations were detected at 3h (P<0.05 compared to controlgroup) and increased at 6h. The peak point was at 24h. Then the expressiondecreased gradually up to 14d (P<0.01 compared to control).④R esults of immunohistochemistry and Western blot: The positive granuleswere brown-yellow or dark brown, located in cytoplasm and mainly distributed inOHC,SG and SV. There were slight expressions of caspase-9 and caspase-3protein in control group. The expressions increased after noise exposure.Expression reached at peak point at 24h and decreased gradually up to 14d. Thepositive OHC indicated there were significant difference between 3h and controlgroup (P<0.05) while the differences between other noise exposed groups andcontrol group were more significant (P<0.01).The results of western blot were expressed by the gradations of protein band.There were slight expressions of caspase-9 and caspase-3 protein in control group.The expression increased after noise exposure. Expression reached at peak pointat 24h and decreased gradually up to 14d. The value of gradation indicated therewere significant difference between 3h and control group (P<0.05 compared tocontrol group) while the differences between other noise exposed groups andcontrol group were more significant (P<0.01).⑤Results of linear regression: Linear regression indicated that the relationbetween OHC apoptosis and ABR threshold was positive straight correlationwhich meant ABR threshold increased when apoptosis increased (r=0.4876,P<0.01), They had a close correlation;The relation between OHC apoptosis andexpression of caspase-9 protein was positive straight correlation which meantOHC apoptosis increased when expression of caspase-9 increased (r=0.9810,P<0.001).They had a close correlation;The relation between OHC apoptosis andexpression of caspase-3 protein was positive straight correlation which meantOHC apoptosis increased when expression of caspase-3 increased (r=0.9594,P<0.001). They also had a close correlation.2,The expression of P-JNK in cochlea after noise exposure.① Results of RT-PCR: 422bp (JNK) and 621bp (β-actin) electrophoresisproductions were seen in cochlea of all groups with RT-PCR amplifaction.Statistical analysis indicated that there were slight expressions in control group.Up regulation of expressions were detected in all groups after noise exposure(significant difference compared to control group P<0.01). The expressionreached to peak at 6h and decreased slightly up to 14d while the expression wasstill at a high level.②Results of immunohistochemistry and Western blot: Positive granuleswere brown-yellow or dark brown and located mainly in cell nucleus. There wereslight expressions of P-JNK protein in control group. Positive cells were detectedin all noise exposed groups and mainly distributed in OHC,SG and SV. Thecounts of positive OHC indicated there were significant difference between noisegroup and control group at all time point (P<0.01). Higher expressions weredetected at 3h and reached to peak at 6h, then decreased slightly up to 14d whilethe expression was still at a high level.The results of western blot were expressed by the gradations of protein band.There were slight expressions of P-JNK protein in control group. The value ofgradation indicated there were significant difference between all noise exposedgroups and control group (P<0.01). High expression was detected at 3h .The peakpoint was at 6h, then expression decreased slightly up to 14d while the expressionwas still at a high level.③Results of linear regression: Linear regression indicated that the relationbetween OHC apoptosis and expression of P-JNK protein was positive straightcorrelation which meant OHC apoptosis increased when expression of P-JNKincreased (r=0.6120, P<0.001). They had a close correlation;The relation betweenexpression of P-JNK and expression of caspase-9 protein was positive straightcorrelation which meant expression of caspase-9 increased when expression ofP-JNK increased (r=0.5594, P<0.001). They had a close correlation;The relationbetween expression of P-JNK and expression of caspase-9 protein was positivestraight correlation which meant expression of caspase-9 increased whenexpression of P-JNK increased (r=0.5694, P<0.001). They had a close correlation,too.3,The protection of CEP-1347 to noise damaged cochlea.①R esults of ABR threshold: There were no significant difference betweennoise plus CEP-1347 group and noise group at 1d (P>0.05). At 4d the ABRthresholds of noise plus CEP-1347 group were significant compared to that of thenoise group (P<0.05) and at 14d the difference were more significant (P<0.01).There were no significant difference between saline plus noise group and noisegroup at all time points (P>0.05).②Results of apoptosis with TUNEL: At all time point after noise exposure,apoptosis were detected in noise plus CEP-1347 group, but the staining attenuatedcompared to the noise group and OHC loss were not detected. The peak point wasat 1h and decreased gradually to 14d in noise plus saline group.The counts of apoptotic OHC indicated that there were no significantdifference between noise plus CEP-1347 group and noise group at 1d (P>0.05). At4d the apoptotic OHC of noise plus CEP-1347 group were significant compared tothat of the noise group (P<0.05) and at 14d the difference were more significant(P<0.01). There were no significant difference between saline plus noise groupand noise group at all time points (P>0.05).Conclusions:1,Noise exposure can induced apoptosis in cochlea;apoptotic cells mainlydistribute in OHC,SG and SV;The peak of apoptosis appears at early phase;Apoptosis lasts for a long time (can last to 14d).2,Noise induced apoptosis in cochlea reach to peak point early and continuefor a long time up to 14d.3,From 24h to 7d after noise exposure, apoptosis is perhaps the primary celldeath pathway of OHC and the major cause of hearing loss.4,There are noise induced expressions of caspase-9,caspase-3 and P-JNK incochlea. Positive granules mainly distribute in OHC,SG and SV.5,The expressions of caspase-9,caspase-3 and P-JNK increase to peak atearly phase and the expressions may last to a long time (can last to 14d).6,The expressions of caspase-9,caspase-3 and P-JNK are closely related toOHC apoptosis after noise exposure.7,The expression of P-JNK are closely related to the expressions ofcaspase-9 and caspase-3;The mitochondrion pathway and JNK pathway make akey contribution to noise induced OHC apoptosis.8,The pathway that noise exposure-oxygen free radical insult-JNK pathwayactivation-mitochondrion pathway-apoptosis is perhaps one of the mechanisms ofnoise induced apoptosis in cochlea.9,Inhibitor of JNK pathway CEP-1347 may diminish the noise inducedapoptosis and hearing loss at some extent which indicate that JNK pathwayparticipate in noise induced apoptosis and CEP-1347 may protect cochlea fromnoise insult.10,JNK pathway may become a new target and direction for NIHL research.New ideas of our research1,Observe noise induced apoptosis at time phase and preliminary explore therelation between apoptosis and hearing loss .2,Identify the expression regularity of caspase-9,caspase-3 and P-JNK,theircorrelation with apoptosis;Confirm the key roles of mitochondrion pathway andJNK pathway to noise induced apoptosis in cochlea.3,Propose the pathway that noise exposure-oxygen free radical insult-JNKpathway activation-mitochondrion pathway-apoptosis is perhaps one of themechanisms of noise induced apoptosis in cochlea.4,Certify the protection of CEP-1347, the inhibitor of JNK pathway, fornoise damaged cochlea;Make the application for inhibitor of JNK pathwaypossible and provide a new perspective for therapeutics of NIHL.