Role Of Tight Junction Protein In Noise-induced Acoustic Trauma And The Mechanism Of Its Regulation

1Objective: To investigate the expression of claudin-5in guinea pig’s striavascularis after noise exposure and the permeability of blood-labyrinth barrieralong with its tight junction in noise-induced acoustic trauma.2Methods:60healthy, adult, albino guinea pigs of only male were randomlydivided into control，noise-exposed4h and noise-exposed2d groups（20guineapigs in each group）. In the noise-exposed groups, all the animals were exposedto white noise at115dB SPL for6hours a day twice in2consecutive days andonce for4hours respectively; while the animals in control group were given thesham-exposure. We respectively adopted auditory brainstem response （ABR）and stretched preparation of basilar membrane stained by FICT-phalloidin to confirm the acoustic trauma functionally and structurally after noise exposureThen, we applied the tracer method of lanthanum nitrate and transmissionelectron microscope to investigate the noise-induced alteration in thepermeability of blood-labyrinth barrier and its tight junction. Finally, we alsoused Western blot and Immunohistochemistry to detect the noise-inducedalteration in the claudin-5expression of stria vascularis and spiral ligament.3Results: After click sound stimulus, the data of auditory brainstem response（ABR） indicated that there were both statistically different between the hearingthresholds of pre-exposure and post-exposure in the noise exposed2d and4hgroups (^*P<0.05;^**P<0.05); The difference of hearing threshold between thecontrol and the noise exposed2d groups was statistically significant after noiseexposure (^***P<0.05). The results of stretched preparation of basilar membranestained by FICT-phalloidin showed that there were obviously hair cell losses inbase turn of basilar membrane of the noise-exposed group, along with someexternal hairs’ disorder and fusion, while there were actually well-arranged haircells whose external hairs shaped V or W type in the same areas of the controlgroup. There was statistically significant difference of outer hair cell countbetween these two groups （P<0.05）. The transmission electron microscopedemonstrated the destruction of intercellular tight junction of vascularendothelial cells and marginal cells with the increase of intercellular space instria vascularis after noise exposure. It also showed that a large number oflanthanum nitrate particles went through vessel wall from lumens to tissue spacein the noise-exposed groups, while they were all limited to vessel lumens in thecontrol group. Western blot analysis revealed that both the control group and thenoise-exposed groups had strong expression of occluding and claudin-5in cochlea lateral wall of guinea pig. In addition, the expression of occluding （andclaudin-5） in the noise-exposed2d group was obviously lower than that in thecontrol group. Immunohistochemistry staining also indicated that both thenoise-exposed2d and control groups had occluding （and claudin-5） presence inthe tight junctions of stria vascularis endothelial cells and marginal cells andthere was statistically significant difference of occluding （and claudin-5）expressions between these two groups （P<0.05;*P<0.05）.4Conclusion: These data demonstrate that by down regulation of the claudin-5expression in stria vascularis, noise exposure demolishes tight junction,increases the permeability of blood-labyrinth barrier in guinea pig cochlea, andfinally results in the microenvironment disorder of inner ear. That is likely to beone of the mechanisms of noise-induced acoustic trauma. 1Objective: To further explore molecular mechanisms of regulation of tightjunction proteins after noise exposure, and hopefully develop new therapies andsupply new theories for prevention of noise-induced acoustic trauma in clinic.2Methods: Ninety healthy, male and adult albino guinea pigs were randomizedequally into six groups （15guinea pigs in each group）, control group, noiseexposure for4h （NE4h）group, noise exposure for2d （NE2d） group, PD+NE2d （PD） group, SU+NE2d （SU） group, and DMSO+NE2d （DMSO） group,respectively. The posterior four groups were exposed to white noise at115dBSPL for6hours a day in2consecutive days. The animals in NE4h group weretreated with white noise at115dB SPL for4hours. The PD, SU and DMSOgroups were given PD98059（0.3mg/kg）, SU1498（50mg/kg）, and DMSO（50ml/kg） by intraperitoneal injection before noise exposure in30mins,respectively. The control group was given no noise exposure and un-injection.We adopted auditory brainstem response （ABR） at pre-exposure andpost-exposure to confirm the acoustic trauma functionally for all experimentalgroups. Then, we detected protein alteration of occludin, claudin-5, erk andP-erk by Western blot from lateral well of cochleae. In addition, we appliedco-focus immunofluorescence to investigate the expression of VEGF and P-erkin stria vascularis.3Results: The data of auditory brainstem response （ABR） indicated that afternoise exposure there were both statistically different between the hearingthresholds of PD group and the noise exposed2d group or DMSO group（*P<0.05;***P<0.05）; The difference of hearing threshold between SU groupand the noise exposed2d groups or DMSO group was also statisticallysignificant after noise exposure （**P<0.05;****P<0.05）. It implied that both ofthe inhibitors might be protective for noise-induced acoustic trauma. Westernblot analysis revealed that the expression of occludin and claudin-5decreasedgradually from control to NE2d group; While the expression of P-erk graduallyincreased in above three groups （the amount of T-erk is invariable）. It suggeststhat noise exposure downregulates the expression of tight junction proteins,meanwhile, it upregulates the phosphorylated ERK, which both depend on the intensity and length of noise exposure. Treated with PD98059before noiseexposure, the expression of tight junction proteins in PD group was superior tothat in NE2d and DMSO group, and the results had a resemblance in SU group,which indicate VEGF and phosphorylated ERK are involved in the regulation oftight junction proteins. by noise. The data from co-focus immunofluorescencedemonstrated that the expression of phosphorylated ERK in NE2d group wasobviously more than that in control group in stria vascularis, and the greenfluorescence of phosphorylated ERK was coincided with the red in the nucleus.All of these imply that noise exposure upregulates phosphorylation of ERK, andphosphorylated ERK is found to translocate nucleus. Phosphorylated ERK in thePD and SU group was manifestly inferior to that in NE2d group, which alsosuggests that noise exposure activates the pathway of ERK by VEGF. Theexpression of VEGF in NE2d and DMSO group is obviously higher than that incontrol group in marginal cells. It indicates that noise upregulates the expressionof VEGF.4Conclusion: By up regulation of the expression of VEGF in marginal cells,noise exposure activates the pathway of ERK, and finally downregulates theexpression of tight junction proteins occludin and claudin-5in cochlea lateralwall.