噪声对豚鼠耳蜗血管纹紧密连接蛋白claudin-5表达及血迷路屏障通透性的影响

目的研究噪声对豚鼠耳蜗血管纹紧密连接蛋白claudin-5表达及血迷路屏障功能的影响。方法40只豚鼠随机分为正常对照组(20只)和噪声暴露组(20只),噪声暴露组给予声强为115dB SPL白噪声暴露,每天6小时,连续两天,对照组不予噪声暴露。于实验前及噪声暴露后次日对两组豚鼠行ABR检测,第二次ABR检测后对两组豚鼠耳蜗基底膜行鬼笔环肽-异硫氰酸荧光素(Phalloidin-FITC)染色;用硝酸镧透射电镜和常规透射电镜观察两组豚鼠内耳血迷路屏障通透性和紧密连接的变化;Western blot和免疫组织化学染色观察两组豚鼠耳蜗血管纹和螺旋韧带处claudin-5的表达。结果噪声暴露组豚鼠ABR波Ⅲ反应阈较正常对照组阈移40dB以上,差异有统计学意义(P<0.05);基底膜铺片FITC染色示噪声暴露组底回基底膜毛细胞大量缺失,其余部分毛细胞纤毛排列紊乱并出现融合,而对照组毛细胞排列整齐,其纤毛呈V或W型,两组间外毛细胞计数比较差异有统计学意义(P<0.05);透射电镜下,可见噪声暴露组血管内皮细胞间和边缘细胞间的紧密连接均遭到破坏,细胞间隙增大;硝酸镧透射电镜下,大量镧颗粒从血管腔内渗漏到管腔外的组织间隙,而对照组镧颗粒仅局限于血管腔内;Western blot结果示,两组耳蜗外侧壁血管纹均有claudin-5表达,但噪声暴露组的表达量明显低于正常组;免疫组织化学染色也表明,两组豚鼠耳蜗外侧壁毛细血管内皮细胞、边缘细胞等处均有claudin-5阳性表达,但噪声组表达量显著低于正常组(P<0.05)。结论噪声通过下调紧密连接蛋白claudin-5的表达,破坏细胞间紧密连接,增加豚鼠血迷路屏障的通透性,从而导致内耳微环境的破坏,是噪声性聋可能的发病机制之一。

Noise-induced Alteration in the Claudin-5 Expression of Stria Vascularis and the Permeability of Blood-labyrinth Barrier in Guinea Pig Cochlea

Objective To investigate the expression of claudin-5 in guinea pig’s stria vascularis after noise exposure and the permeability of blood-labyrinth barrier along with its tight junction in noise-induced hearing loss. Methods Fourty guinea pigs were randomly divided into control and noise-exposed groups(20 guinea pigs in each group).In the noise-exposed group,all the animals were exposed to white noise at 115 dB SPL for 6 hours a day in 2 consecutive days.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.Then,we applied the tracer method of lanthanum nitrate and transmission electron microscope to investigate the noise-induced alteration in the permeability of blood-labyrinth barrier and its tight junction.Finally,we also used Western blot and Immunohistochemistry to detect the noise-induced alteration in the claudin-5 expression of stria vascularis and spiral ligament. Results Compared with the control group,the ABR threshold shift was more than 40 dB in the moise-exposed groups.There was statistically significant difference between these two groups(P<0.05).The results of stretched preparation of basilar membrane stained by FICT-phalloidin showed that there were obvious hair cell loss in base turn of basilar membrane in the noise-exposed group,along with some exfernal hairs’ disorder and fusion,while there were actually well-arranged hair cells whose external hairs shaped V or W type in the same areas of the control group.There was statistically significant difference of outer hair count between these two groups(P<0.05).The transmission electron microscope demonstrated the destruction of intercellular tight junction of vascular endothelial cells and marginal cells with the increase of intercellular space in stria vascularis after noise exposure.It also showed that a large number of lanthanum nitrate particles went through vessel wall from lumens to tissue space in the noise-exposed group,while they were all limited to vessel lumens in the control group.Western blot analysis revealed that both the control group and the noise-exposed group had strong expression of claudin-5 in stria vascularis of guinea pig cochlea lateral wall.In addition,the expression of claudin-5 in the noise-exposed group was obviously lower than that in the control group.Immunohistochemistry staining indicated that both two groups had claudin-5 presence in the tight junctions of stria vascularis endothelial cells and marginal cells and there was statistically significant difference of claudin-5 expression between these two groups(P<0.05). Conclusion Our data demonstrate that by down regulation of the claudin-5 expression in stria vascularis,noise exposure demolishes tight junction,increases the permeability of blood-labyrinth barrier in guinea pig cochlea,and finally results in the microenvironment disorder of inner ear.That is likely to be one of the mechanisms of noise-induced hearing loss.