噪声损伤引起耳蜗外毛细胞凋亡时细胞核内单链DNA的产生及EndoG的转移

目的观察噪声损伤后耳蜗外毛细胞内单链DNA和EndoG的变化,探讨耳蜗外毛细胞的死亡机制。方法豚鼠随机分为噪声暴露组、MNNG耳蜗灌流组和对照组(每组各12只);小鼠随机分为噪声暴露组和对照组(每组12只)。分离解剖耳蜗后,用碘化丙啶(PI)染色细胞核、Pholloidin染色F-actin,免疫荧光抗体分别染色单链DNA(ssDNA)、核酸内切酶G(Endonuclease G EndoG)和凋亡诱导因子(Apoptosis inducing factors,AIF),制备耳蜗铺片,激光共聚焦显微镜下观察凋亡和坏死毛细胞内的荧光信号变化。结果(1)暴露于120dBSPL的白噪声环境中每天4小时,连续2天后引起豚鼠和小鼠耳蜗外毛细胞凋亡时,其细胞核内产生ssDNA,而在正常细胞内没有三ssDNA;(2)在正常情况下,EndoG分布于耳蜗毛细胞的细胞核外,在暴露于上述噪声后发生凋亡和坏死的豚鼠耳蜗外毛细胞中,EndoG从细胞核外转移到细胞核内,细胞核中的EndoG显著增加;(3)豚鼠耳蜗外淋巴灌流烷化剂MNNG后发生耳蜗外毛细胞凋亡和坏死,在凋亡和坏死的耳蜗外毛细胞中,AIF自线粒体转移到细胞核,其变化与噪声损伤引起耳蜗外毛细胞凋亡和坏死时一致。结论噪声刺激或烷化剂MNNG灌流后,造成耳蜗外毛细胞DNA损伤,产生ssDNA,引起AIF和EndoG自线粒体释放,激活Caspase-3,AIF和EndoG进一步向细胞核转移,最终使细胞核内的DNA降解,导致耳蜗毛细胞的死亡。

Noise exposure induced cochlear hair cell death pathways in guinea pig

Objective To understand the mechanism of noise exposure induced outer hair cells(OHCs) death pathways. Methods Thirty two guinea pigs were used in this study. The animals were either exposed for 4 h/day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused with MNNG. After auditory test, the cochleae of animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. F-actin staining was used to determine missing OHCs. Caspase-3 was detected in living organ of Corti whole mounts using the fluorescent probe. The single strand DNA (ssDNA) in apoptotic OHCs in guinea pigs and apoptosis inducing factor (AIF) in hair cells in guinea pigs were examined by immunohistology method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. Results (1) Both apoptotic and necrotic hair cells appeared following noise exposure. (2) Noise exposure induced single strand DNA in apoptotic OHCs but not in the normal OHCs. (3) Either after noise exposure or after MNNG perfusion, apoptotic OHCs were featured by nuclear condensation or fragmentation with caspase-3 activation, whereas necrotic OHCs were characterized by nuclear swelling without caspase-3 activation. (4) In normal organ of Corti, AIF was located in the mitochondria areas. After noise exposure, AIF was translocated from mitochondria in apoptotic and necrotic OHCs. Conclusion These findings indicate that noise exposure damages DNA in the OHC, which triggers action of Caspase-3. Subsequently, AIF is translocated to the nucleus, leading to DNA damage and OHCs death.

Phosphatidylserine-positive particles in the apical domain of sensory hair cells

Apical membrane recycling has been proposed to be important for normal hair cell function. The current study reports an in vitro work that demonstrates the presence of phosphatidylserine (PS) and PS-positive vesicles labeled by Annexin V in the apical portion of hair cells. The following characteristics of the PS-positive vesicles were noticed using scanning confocal fluorescence microscopy:(1) variable sizes around 200 nm; (2) variable distribution patterns (either uniformly along individual stereocilia in the hair bundle or irregular) in the stereocilia from cell to cell; (3) variable sizes and numbers at locations along the border of the cuticular plate (CP), with a large number of them located at the vestigal kinocilial location; (4) motility with some of the vesicles during the observation period; (5) increase in PS labeling and the number of PS-positive vesicles after loud sound stimulation; and (6) decreased PS labeling and PS-positive vesicle numbers following treatment with LY-294002, a PI3 -kinase inhibitor. These results suggest that the presence of PS-positive vesicles at the apical area of hair cells may be indicative of vesicle shedding or transportation of a protein or rafts.

噪声刺激致豚鼠外毛细胞凋亡时半胱氨酸天冬氨酸蛋白酶3激活和凋亡诱导因子转移

目的探讨噪声损伤后豚鼠外毛细胞发生凋亡的通路及其机制。方法分离解剖对照组(12只)和噪声暴露组(12只)豚鼠耳蜗,利用免疫荧光抗体和免疫荧光染料,分别染色细胞核、线粒体、凋亡诱导因子(apoptosis inducing factor,AIF)和半胱氨酸天冬氨酸蛋白酶3(Caspase 3),按表面制备法行耳蜗铺片,观察凋亡和坏死毛细胞内的荧光信号。结果对照组正常耳蜗毛细胞内没有激活的 Caspase 3,AIF 分布在毛细胞的线粒体内。实验组动物暴露于声压级120 dB 的白噪声环境中每天4 h,连续2 d 后,耳蜗外毛细胞出现死亡,表现为坏死和凋亡两种方式,但以凋亡为主。在凋亡的外毛细胞中,Caspase 3被激活,AIF 从线粒体转移到细胞核中;而在坏死的外毛细胞中,没有 Caspase3的激活,只有 AIF 自线粒体向细胞核的转移。结论噪声损伤后耳蜗外毛细胞的凋亡以 Caspase 依赖型的通路为主;线粒体释放的 AIF 在外毛细胞死亡通路中起重要作用。