内质网应激反应（ERS）参与强噪声诱导豚鼠耳蜗细胞凋亡的实验研究
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摘要
细胞凋亡是一个复杂的病理生理过程,其过程归纳为两条途径:一是细胞外途径,即死亡受体途径。细胞外死亡信号蛋白与其受体结合,以激活Caspase-8为特征。二是细胞内途径,研究比较多是线粒体途径。通透性改变的线粒体,释放细胞色素C,激活Caspase-9为特征。近年来研究表明内质网(endoplasmic reticulum, ER)可能是细胞内诱导凋亡的一个新场所,因此提出内质网应激反应性凋亡通道。最近研究表明,细胞凋亡也是强噪声暴露诱导耳蜗细胞死亡的一种方式。本课题通过用中心频率4kHz的窄带噪声、给声强度120dB SPL、暴露时间4h的噪声刺激建立豚鼠噪声性耳聋(noise-induced hearing loss,NIHL)的动物模型,探讨噪声性耳聋是否存在细胞凋亡,以及内质网应激反应(endoplasmic reticulum stress,ERS)是否参与了强噪声诱导耳蜗细胞凋亡以及其调控机制的研究,为进一步阐明噪声性耳聋的发病机制奠定基础,为将来治疗噪声性耳聋提供新的思路。本文共分为三个部分。
第一部分噪声性耳聋动物模型的建立
[目的]建立噪声性耳聋的动物模型,为下一步研究噪声性耳聋过程中是否存在细胞凋亡奠定基础。
[方法]选用雄性白色豚鼠80只,随机分为实验组60只,对照组20只。实验组用中心频率4kHz的窄带噪声,给声强度为120dB SPL,噪声暴露时间为4h的噪声条件进行声暴露。健康对照组不接触噪声。分别在噪声暴露前及噪声暴露后1、4和14d后测动物ABR阈值。
[结果]以Ⅲ波作为判断ABR阈值的指征。噪声暴露后1d、4d、14d组ABR平均阈值分别为90.00±3.33dB,64.50±6.85dB,60.00±6.67dB,对照组ABR平均阈值33.00±5.38dB。统计学分析各组之间差异有统计学意义(P<0.01),实验组与对照组ABR阈值相比差异有统计学意义(P<0.01),1d与4d、14d组差异有统计学意义(P<0.01),4d与14d组差异没有统计学意义(P>0.05)。
[结论]本噪声刺激条件可以引起豚鼠永久性听阈位移,成功地建立了一种噪声性听力损害的动物模型,此模型可用于噪声性耳聋发病机制的研究。
第二部分噪声性耳聋耳蜗细胞凋亡的研究
[目的]强噪声诱发豚鼠耳蜗细胞凋亡的研究。
[方法]通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal- deoxynucleotidyl transferase mediated nick end labeling,TUNEL),耳蜗基底膜铺片、Hoechst33342荧光染色及透射电镜观察Corti/s器及螺旋神经节细胞的超微结构来检测耳蜗凋亡细胞。
[结果]光镜下,噪声暴露组,耳蜗Corti/s器、螺旋神经节及侧壁出现明显的TUNEL染色阳性反应,表现为细胞核呈棕黄色,并伴有核固缩及核不规则,每高倍视野以1d组最多,而正常对照组耳蜗Corti/s器、螺旋神经节及侧壁的染色极少见阳性细胞,阴性对照切片中未见阳性细胞。Corti/s器的TUNEL阳性细胞主要为外毛细胞及其周围的支持细胞,偶见内毛细胞。经统计学分析噪声暴露后1d、4d、14d组Corti/s器、螺旋神经节及侧壁的TUNEL染色平均吸光度值与对照组相比差异有统计学意义(P<0.01),1d组Corti/s器、螺旋神经节及侧壁的平均吸光度值与4d、14d组相比差异有统计学意义(P<0.01),4d组与14d组相比差异没有统计学意义(P>0.05)。耳蜗基底膜铺片、Hoechst33342荧光染色正常对照组毛细胞核为淡蓝色,细胞核大小一致,三排外毛细胞及一排内毛细胞排列整齐,噪声刺激后1d、4d、14d组Hoechst33342荧光染色的内、外毛细胞均呈现3种病理改变:①核肿胀;②核固缩;③核消失;即出现坏死、凋亡、缺失三种病理改变,以底回末端及第二回起始外毛细胞最多见;损伤的外毛细胞定量分析,在噪声暴露后1d、4d、14d总的趋势是缺失的外毛细胞逐渐增加,坏死和凋亡的细胞数逐渐减少,14d总的损伤外毛细胞细胞数(坏死、凋亡、缺失)明显上升。当比较凋亡和坏死细胞数在不同的时间点是不同的,在1d、4d组凋亡的细胞数明显多于坏死(P<0.05),14d组凋亡与坏死的细胞数没有明显差别(P>0.05)。通过透射电镜观察发现实验组1d耳蜗外毛细胞、螺旋神经节细胞均可见散在的早期凋亡特征性改变:细胞核染色质固缩、边集,在细胞核膜周边聚集成块,线粒体轻度增多,其他细胞器变化不大。健康对照组外毛细胞、螺旋神经节细胞未发现上述特征的细胞。
[结论] 1、说明强噪声可以引起耳蜗细胞广泛性的凋亡。
2、说明凋亡可能是强噪声引起豚鼠耳蜗细胞早期死亡的主要方式。
3、说明凋亡过程至少可以持续到14天。
第三部分内质网应激反应(ERS)参与噪声性耳聋的耳蜗细胞凋亡及其信号调控的机制研究
[目的]通过检测Grp78/Bip(内质网伴侣蛋白,内质网应激反应的标志)的表达,Caspase-12的激活,JNK(C-Jun氨基末端激酶)信号通道的激活,CHOP/Gadd153(生长停滞、DNA损伤诱导的凋亡前期信号),Bcl-2蛋白的表达,来探讨内质网应激反应是否参与了强噪声诱导耳蜗细胞凋亡以及凋亡发生的调控机制。
[方法]用免疫组化、Weatern Blot方法检测耳蜗细胞Grp78/Bip、Caspase-12、P-JNK、P-c-Jun、CHOP/Gadd153、Bcl-2蛋白的表达。
[结果]免疫组化、Western Blot检测实验组Grp78/Bip蛋白明显高于正常对照组(P<0.01),实验组1d、4d、14d的蛋白表达量没有明显差异(P>0.05);光镜下Grp78/Bip免疫组织化学染色,噪声暴露后1d、4d、14d均可见Corti/s器、螺旋神经节及侧壁的细胞质上有染色程度不等的棕黄色颗粒分布,正常对照组染色弱阳性;Caspase-12蛋白含量在噪声刺激后迅速增高,1d、4d达到高峰,14d减少;Western Blot检测Caspase-12免疫组化染色,实验组Corti/s器、螺旋神经节及侧壁的细胞质上有染色程度不等的棕黄色颗粒分布,正常对照组表达阴性。免疫组织化学染色观察到实验组P-JNK、P-c-Jun有免疫反应阳性,定位于胞核,对照组未见阳性细胞。Western Blot检测P-JNK、P-c-Jun含量在噪声刺激后迅速增高并快速活化,1d、4d达到高峰,随后逐渐下降,但在14天仍然维持较高水平。免疫组化、Western Blot检测实验组CHOP/Gadd153表达明显高于对照组(P<0.01),实验组1d、4d、14d组CHOP/Gadd153表达没有差异(P>0.05);免疫组织化学观察到实验组CHOP/Gadd153在Corti/s器、螺旋神经节及侧壁有免疫反应阳性,定位于胞核,对照组表达阴性。Western Blot检测实验组Bcl-2蛋白无表达,而对照组Bcl-2的表达明显高于实验组(P<0.01);免疫组织化学染色,对照组Corti/s器、螺旋神经节及侧壁的细胞质上有染色程度不等的棕黄色颗粒分布,实验组表达阴性。
[结论]
1、实验组Grp78/Bip高表达,Caspase-12的活化说明强噪声刺激引起耳蜗组织细胞的内质网应激性凋亡。
2、强噪声引起内质网应激性凋亡,可能通过下面三条途径激活:
(1)通过激活Caspase-12,诱导细胞凋亡。
(2)通过激活JNK信号通道,诱导细胞凋亡。
(3)通过上调CHOP/Gadd153蛋白、下调Bcl-2蛋白的表达,诱导细胞凋亡。
Cell apoptosis is a complicated pathophysiological process. It has been confirmed that there are two pathways leading to cell apoptosis: extracellular pathway and intracellular pathway. Extracellular pathway is characterized by activation of Caspase-8 which is triggered by combination of specific death ligands and death receptors. In intracellular pathway, involves the participation of mitochondria. It is characterized by the release of cyto c and activaction of Caspase-9. In recent years, it has been reported that endoplasmic reticulum(ER) may be a new location which participates in intracellular pathway of cell apoptosis. That is called ER stress-mediated apoptosis pathway. Recent studies indicate that apoptosis is an important way leading to cell death in cochlear induced by intense noise exposure. In this study, we established an animal model of noise-induced hearing loss(NIHL) induced by 4KHz narrow band noise at 120dB SPL for 4h, and investigated the effect of apoptosis in inner ear cells after exposure to intense noise, and assessed the effect of ER stress in the procedure of cochlea cell apoptosis induced by intense noise. This study provides the foundation for the further research in noise-induced hearing loss and provides a new thinking for the therapy of noise-induced hearing loss in the future. This paper is divided into following three parts.
PartⅠEstablishment of an animal model of noise-induced hearing loss
Objective To establish an animal model of noise-induced hearing loss, and to lay the foundation for further research in noise-induced hearing loss.
Methods 80 guinea pigs were randomly divided into 2 groups. In the experimental group, 60 guinea pigs were exposed to 4KHz narrow band noise at 120dB SPL for 4h. The rest 20 guinea pigs receive no noise exposure for control. After noise exposure for1, 4, 14days , animals were killed, and auditory brainstem response(ABR) of the guinea pigs were tested to assess hearing threshold shift of before and after noise exposure.
Results The threshold of waveⅢwas used to assess ABR threshold. After noise exposure for1, 4, 14 days, the average ABR threshold were 90.00±3.33dB,64.50±6.85dB,60.00±6.67dB respectively, while that of control group was 33.00±5.38dB. Statistical analysis revealed that there were statistically significant differences(P<0.01) among the groups. There was statistically significant difference (P<0.01) in the average ABR threshold between the experimental group and the control. There were statistically significant differences (P<0.01) in the average ABR threshold among the group of 1 day after noise exposure and the group of 4 days or 14 days, while there was no statistically difference(P>0.05)between the group of 4 days and 14 days(P>0.05).
Conclusion The intense noise used in our study can cause permanent hearing threshold shift in the guinea pig. We successfully established an animal model of noise-induced hearing loss. It can be used to study the pathogenesis of noise-induced hearing loss.
PartⅡThe study of cell apoptosis in noise-induced hearing loss
Objective To investigate the effect of apoptosis in Cochlea cells after exposure to intense noise.
Methods Terminal deoxynucleotidyl Transferase(TdT)-mediated deoxyuridine triphosphate (d-UTP) nick and labeling method (TUNEL), Hochest 33342 staining, and transmission electron microscopy were used to investigate changes of ultrastructure in the organ of Corti and spiral ganglion cells, and to assess the effect of apoptosis in noise induced hearing loss.
Results After noise exposure, there were a lot of Tunel-positive cells in the organs of Corti , SGC and SV of experimental groups, with a performance of brown nuclei, nuclear condensation and rregular, especially in the group of 1day after noise exposure. However, no Tunel-positive cells was observed in the organ of Corti , SGC and SV of the control group and the negative control. Tunel-positive cells in the organ of Corti were always outer hair cells and supporting cells around, sometimes inner hair cells. Statistical analysis revealed that there were statistically significant differences (P<0.01) in the average absorbance of Tunel-positive cells between the experimental group and the control. There were statistically significant differences (P<0.01) in the average absorbance among the group of 1day after noise exposure and the group of 4 days or 14 days, while there was no statistically difference between the group of 4 days and 14 days(P>0.05). Surface preparation of organ of Corti and hochest 33342 staining of the control group show that nuclei of hair cells was light blue, and had the same size. Three rows of outer hair cells and one row of inner cells ranked neatly. While in the groups of 1day, 4 days and 14 days after noise exposure, three kinds of pathological changes were observed:swollen nuclei, condensed nuclei and the loss of nuclei, especially in outer hair cells of the bottom circle and the second circle. There were significantly more apoptotic OHCs than necrotic OHCs in the cochleas examined at days1 and 4 after the noise exposure(P<0.05),whereas at day 14, the difference in the numbers of apoptotic and necrotic OHCs becomes statistically insignificant (P>0.05).That was the most of the total number of missing and apoptotic and necrotic OHCs at day 14 after the noise exposure.In the group of 1 day after noise exposure, the observation by electron microscopy showed the characteristic changes of apoptotic cells in the organ of Corti and SGC. Nuclear chromatin condensed and aggregated surrounding nuclear membrane. The number of mitochondria was slightly increased, other cell organelle was little altered. In the control group, there was no characteristic changes of apoptotic cells in the organ of Corti and SGC.
Conclusion The intense noise used in our study can cause extensive apoptosis in cochlear cells. Apoptosis may be the primaryevent in the early stage of cochlear lesion induced by intense noise. Apoptosis proceeds more than 14 days.
PartⅢInvolvement of endoplasmic reticulum (ER) stress -mediated apoptosis in intense noise-induced cochlea cells death
Objective By detecting the expression of Grp78/Bip, Caspase-12, CHOP/Gadd153, Bcl-2 and the activation of JNK signal transduction pathway, to investigate the effect and regulation mechanism of ER stress in the procedure of cochlea cell apoptosis induced by intense noise.
Methods Expression of Grp78/Bip、Caspase-12、P-JNK、P-c-Jun、CHOP/Gadd153 and Bcl-2 were tested by immunohistochemistry and western blot analysis. Results Both immunohistochemistry and western blot analysis revealed that Grp78/Bip was highly expressed after noise exposure, and there was significant differences compared with the control group(P<0.01). Immunohistochemistry of Grp78/ Bip showed that brown granules were observed in the organ of Corti , SGC and SV in the groups of 1 day, 4 days and 14 days after noise exposure, while in the seations of the control group,weakly staining was observed in those cells. The protein level of Caspase-12 increased rapidly after noise exposure, and it peaked in 1 day and 4 day, and it reduced until 14 day. Protein levels of P-JNK, and P-c-Jun increased quickly after noise exposure, and achieved peak in 1d,4d, then fell down, but still maintained in high level within 14d. CHOP/Gadd153 was much higher expressed in 1 day, 4 days and 14 days groups than that of the control, which has significant differences(P<0.01).However there was no statistically difference (P>0.05) among 1day, 4days and 14days groups. In the experimental groups Bcl-2 was little expressed ,while it had significantly high expression(P<0.01)in the control group.
Conclusion Intense noise causes apoptosis in cochlea cells and endoplasmic reticulum (ER) stress-related apoptosis plays an important role in the procedure of apoptosis. ER stress-related apoptosis maybe mainly induced through the following three ways: activating of Caspase-12, activating of JNK signal transduction pathway, up-regulating the expression of CHOP /Gadd153 and reducing the expression of Bcl-2.
第一部分噪声性耳聋动物模型的建立
[目的]建立噪声性耳聋的动物模型,为下一步研究噪声性耳聋过程中是否存在细胞凋亡奠定基础。
[方法]选用雄性白色豚鼠80只,随机分为实验组60只,对照组20只。实验组用中心频率4kHz的窄带噪声,给声强度为120dB SPL,噪声暴露时间为4h的噪声条件进行声暴露。健康对照组不接触噪声。分别在噪声暴露前及噪声暴露后1、4和14d后测动物ABR阈值。
[结果]以Ⅲ波作为判断ABR阈值的指征。噪声暴露后1d、4d、14d组ABR平均阈值分别为90.00±3.33dB,64.50±6.85dB,60.00±6.67dB,对照组ABR平均阈值33.00±5.38dB。统计学分析各组之间差异有统计学意义(P<0.01),实验组与对照组ABR阈值相比差异有统计学意义(P<0.01),1d与4d、14d组差异有统计学意义(P<0.01),4d与14d组差异没有统计学意义(P>0.05)。
[结论]本噪声刺激条件可以引起豚鼠永久性听阈位移,成功地建立了一种噪声性听力损害的动物模型,此模型可用于噪声性耳聋发病机制的研究。
第二部分噪声性耳聋耳蜗细胞凋亡的研究
[目的]强噪声诱发豚鼠耳蜗细胞凋亡的研究。
[方法]通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal- deoxynucleotidyl transferase mediated nick end labeling,TUNEL),耳蜗基底膜铺片、Hoechst33342荧光染色及透射电镜观察Corti/s器及螺旋神经节细胞的超微结构来检测耳蜗凋亡细胞。
[结果]光镜下,噪声暴露组,耳蜗Corti/s器、螺旋神经节及侧壁出现明显的TUNEL染色阳性反应,表现为细胞核呈棕黄色,并伴有核固缩及核不规则,每高倍视野以1d组最多,而正常对照组耳蜗Corti/s器、螺旋神经节及侧壁的染色极少见阳性细胞,阴性对照切片中未见阳性细胞。Corti/s器的TUNEL阳性细胞主要为外毛细胞及其周围的支持细胞,偶见内毛细胞。经统计学分析噪声暴露后1d、4d、14d组Corti/s器、螺旋神经节及侧壁的TUNEL染色平均吸光度值与对照组相比差异有统计学意义(P<0.01),1d组Corti/s器、螺旋神经节及侧壁的平均吸光度值与4d、14d组相比差异有统计学意义(P<0.01),4d组与14d组相比差异没有统计学意义(P>0.05)。耳蜗基底膜铺片、Hoechst33342荧光染色正常对照组毛细胞核为淡蓝色,细胞核大小一致,三排外毛细胞及一排内毛细胞排列整齐,噪声刺激后1d、4d、14d组Hoechst33342荧光染色的内、外毛细胞均呈现3种病理改变:①核肿胀;②核固缩;③核消失;即出现坏死、凋亡、缺失三种病理改变,以底回末端及第二回起始外毛细胞最多见;损伤的外毛细胞定量分析,在噪声暴露后1d、4d、14d总的趋势是缺失的外毛细胞逐渐增加,坏死和凋亡的细胞数逐渐减少,14d总的损伤外毛细胞细胞数(坏死、凋亡、缺失)明显上升。当比较凋亡和坏死细胞数在不同的时间点是不同的,在1d、4d组凋亡的细胞数明显多于坏死(P<0.05),14d组凋亡与坏死的细胞数没有明显差别(P>0.05)。通过透射电镜观察发现实验组1d耳蜗外毛细胞、螺旋神经节细胞均可见散在的早期凋亡特征性改变:细胞核染色质固缩、边集,在细胞核膜周边聚集成块,线粒体轻度增多,其他细胞器变化不大。健康对照组外毛细胞、螺旋神经节细胞未发现上述特征的细胞。
[结论] 1、说明强噪声可以引起耳蜗细胞广泛性的凋亡。
2、说明凋亡可能是强噪声引起豚鼠耳蜗细胞早期死亡的主要方式。
3、说明凋亡过程至少可以持续到14天。
第三部分内质网应激反应(ERS)参与噪声性耳聋的耳蜗细胞凋亡及其信号调控的机制研究
[目的]通过检测Grp78/Bip(内质网伴侣蛋白,内质网应激反应的标志)的表达,Caspase-12的激活,JNK(C-Jun氨基末端激酶)信号通道的激活,CHOP/Gadd153(生长停滞、DNA损伤诱导的凋亡前期信号),Bcl-2蛋白的表达,来探讨内质网应激反应是否参与了强噪声诱导耳蜗细胞凋亡以及凋亡发生的调控机制。
[方法]用免疫组化、Weatern Blot方法检测耳蜗细胞Grp78/Bip、Caspase-12、P-JNK、P-c-Jun、CHOP/Gadd153、Bcl-2蛋白的表达。
[结果]免疫组化、Western Blot检测实验组Grp78/Bip蛋白明显高于正常对照组(P<0.01),实验组1d、4d、14d的蛋白表达量没有明显差异(P>0.05);光镜下Grp78/Bip免疫组织化学染色,噪声暴露后1d、4d、14d均可见Corti/s器、螺旋神经节及侧壁的细胞质上有染色程度不等的棕黄色颗粒分布,正常对照组染色弱阳性;Caspase-12蛋白含量在噪声刺激后迅速增高,1d、4d达到高峰,14d减少;Western Blot检测Caspase-12免疫组化染色,实验组Corti/s器、螺旋神经节及侧壁的细胞质上有染色程度不等的棕黄色颗粒分布,正常对照组表达阴性。免疫组织化学染色观察到实验组P-JNK、P-c-Jun有免疫反应阳性,定位于胞核,对照组未见阳性细胞。Western Blot检测P-JNK、P-c-Jun含量在噪声刺激后迅速增高并快速活化,1d、4d达到高峰,随后逐渐下降,但在14天仍然维持较高水平。免疫组化、Western Blot检测实验组CHOP/Gadd153表达明显高于对照组(P<0.01),实验组1d、4d、14d组CHOP/Gadd153表达没有差异(P>0.05);免疫组织化学观察到实验组CHOP/Gadd153在Corti/s器、螺旋神经节及侧壁有免疫反应阳性,定位于胞核,对照组表达阴性。Western Blot检测实验组Bcl-2蛋白无表达,而对照组Bcl-2的表达明显高于实验组(P<0.01);免疫组织化学染色,对照组Corti/s器、螺旋神经节及侧壁的细胞质上有染色程度不等的棕黄色颗粒分布,实验组表达阴性。
[结论]
1、实验组Grp78/Bip高表达,Caspase-12的活化说明强噪声刺激引起耳蜗组织细胞的内质网应激性凋亡。
2、强噪声引起内质网应激性凋亡,可能通过下面三条途径激活:
(1)通过激活Caspase-12,诱导细胞凋亡。
(2)通过激活JNK信号通道,诱导细胞凋亡。
(3)通过上调CHOP/Gadd153蛋白、下调Bcl-2蛋白的表达,诱导细胞凋亡。
Cell apoptosis is a complicated pathophysiological process. It has been confirmed that there are two pathways leading to cell apoptosis: extracellular pathway and intracellular pathway. Extracellular pathway is characterized by activation of Caspase-8 which is triggered by combination of specific death ligands and death receptors. In intracellular pathway, involves the participation of mitochondria. It is characterized by the release of cyto c and activaction of Caspase-9. In recent years, it has been reported that endoplasmic reticulum(ER) may be a new location which participates in intracellular pathway of cell apoptosis. That is called ER stress-mediated apoptosis pathway. Recent studies indicate that apoptosis is an important way leading to cell death in cochlear induced by intense noise exposure. In this study, we established an animal model of noise-induced hearing loss(NIHL) induced by 4KHz narrow band noise at 120dB SPL for 4h, and investigated the effect of apoptosis in inner ear cells after exposure to intense noise, and assessed the effect of ER stress in the procedure of cochlea cell apoptosis induced by intense noise. This study provides the foundation for the further research in noise-induced hearing loss and provides a new thinking for the therapy of noise-induced hearing loss in the future. This paper is divided into following three parts.
PartⅠEstablishment of an animal model of noise-induced hearing loss
Objective To establish an animal model of noise-induced hearing loss, and to lay the foundation for further research in noise-induced hearing loss.
Methods 80 guinea pigs were randomly divided into 2 groups. In the experimental group, 60 guinea pigs were exposed to 4KHz narrow band noise at 120dB SPL for 4h. The rest 20 guinea pigs receive no noise exposure for control. After noise exposure for1, 4, 14days , animals were killed, and auditory brainstem response(ABR) of the guinea pigs were tested to assess hearing threshold shift of before and after noise exposure.
Results The threshold of waveⅢwas used to assess ABR threshold. After noise exposure for1, 4, 14 days, the average ABR threshold were 90.00±3.33dB,64.50±6.85dB,60.00±6.67dB respectively, while that of control group was 33.00±5.38dB. Statistical analysis revealed that there were statistically significant differences(P<0.01) among the groups. There was statistically significant difference (P<0.01) in the average ABR threshold between the experimental group and the control. There were statistically significant differences (P<0.01) in the average ABR threshold among the group of 1 day after noise exposure and the group of 4 days or 14 days, while there was no statistically difference(P>0.05)between the group of 4 days and 14 days(P>0.05).
Conclusion The intense noise used in our study can cause permanent hearing threshold shift in the guinea pig. We successfully established an animal model of noise-induced hearing loss. It can be used to study the pathogenesis of noise-induced hearing loss.
PartⅡThe study of cell apoptosis in noise-induced hearing loss
Objective To investigate the effect of apoptosis in Cochlea cells after exposure to intense noise.
Methods Terminal deoxynucleotidyl Transferase(TdT)-mediated deoxyuridine triphosphate (d-UTP) nick and labeling method (TUNEL), Hochest 33342 staining, and transmission electron microscopy were used to investigate changes of ultrastructure in the organ of Corti and spiral ganglion cells, and to assess the effect of apoptosis in noise induced hearing loss.
Results After noise exposure, there were a lot of Tunel-positive cells in the organs of Corti , SGC and SV of experimental groups, with a performance of brown nuclei, nuclear condensation and rregular, especially in the group of 1day after noise exposure. However, no Tunel-positive cells was observed in the organ of Corti , SGC and SV of the control group and the negative control. Tunel-positive cells in the organ of Corti were always outer hair cells and supporting cells around, sometimes inner hair cells. Statistical analysis revealed that there were statistically significant differences (P<0.01) in the average absorbance of Tunel-positive cells between the experimental group and the control. There were statistically significant differences (P<0.01) in the average absorbance among the group of 1day after noise exposure and the group of 4 days or 14 days, while there was no statistically difference between the group of 4 days and 14 days(P>0.05). Surface preparation of organ of Corti and hochest 33342 staining of the control group show that nuclei of hair cells was light blue, and had the same size. Three rows of outer hair cells and one row of inner cells ranked neatly. While in the groups of 1day, 4 days and 14 days after noise exposure, three kinds of pathological changes were observed:swollen nuclei, condensed nuclei and the loss of nuclei, especially in outer hair cells of the bottom circle and the second circle. There were significantly more apoptotic OHCs than necrotic OHCs in the cochleas examined at days1 and 4 after the noise exposure(P<0.05),whereas at day 14, the difference in the numbers of apoptotic and necrotic OHCs becomes statistically insignificant (P>0.05).That was the most of the total number of missing and apoptotic and necrotic OHCs at day 14 after the noise exposure.In the group of 1 day after noise exposure, the observation by electron microscopy showed the characteristic changes of apoptotic cells in the organ of Corti and SGC. Nuclear chromatin condensed and aggregated surrounding nuclear membrane. The number of mitochondria was slightly increased, other cell organelle was little altered. In the control group, there was no characteristic changes of apoptotic cells in the organ of Corti and SGC.
Conclusion The intense noise used in our study can cause extensive apoptosis in cochlear cells. Apoptosis may be the primaryevent in the early stage of cochlear lesion induced by intense noise. Apoptosis proceeds more than 14 days.
PartⅢInvolvement of endoplasmic reticulum (ER) stress -mediated apoptosis in intense noise-induced cochlea cells death
Objective By detecting the expression of Grp78/Bip, Caspase-12, CHOP/Gadd153, Bcl-2 and the activation of JNK signal transduction pathway, to investigate the effect and regulation mechanism of ER stress in the procedure of cochlea cell apoptosis induced by intense noise.
Methods Expression of Grp78/Bip、Caspase-12、P-JNK、P-c-Jun、CHOP/Gadd153 and Bcl-2 were tested by immunohistochemistry and western blot analysis. Results Both immunohistochemistry and western blot analysis revealed that Grp78/Bip was highly expressed after noise exposure, and there was significant differences compared with the control group(P<0.01). Immunohistochemistry of Grp78/ Bip showed that brown granules were observed in the organ of Corti , SGC and SV in the groups of 1 day, 4 days and 14 days after noise exposure, while in the seations of the control group,weakly staining was observed in those cells. The protein level of Caspase-12 increased rapidly after noise exposure, and it peaked in 1 day and 4 day, and it reduced until 14 day. Protein levels of P-JNK, and P-c-Jun increased quickly after noise exposure, and achieved peak in 1d,4d, then fell down, but still maintained in high level within 14d. CHOP/Gadd153 was much higher expressed in 1 day, 4 days and 14 days groups than that of the control, which has significant differences(P<0.01).However there was no statistically difference (P>0.05) among 1day, 4days and 14days groups. In the experimental groups Bcl-2 was little expressed ,while it had significantly high expression(P<0.01)in the control group.
Conclusion Intense noise causes apoptosis in cochlea cells and endoplasmic reticulum (ER) stress-related apoptosis plays an important role in the procedure of apoptosis. ER stress-related apoptosis maybe mainly induced through the following three ways: activating of Caspase-12, activating of JNK signal transduction pathway, up-regulating the expression of CHOP /Gadd153 and reducing the expression of Bcl-2.
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9. Chen YS,Tseng FY, Liu TC, et al. Involvement of nitric oxide generation in noise-induced temporary threshold shift in guinea pigs. Hear Res,2005,203(1-2):94-100.
10. Shi X ,Dai C,Nuttall AL. Altered expression of inducible nitric oxide s ynthase (iNOS) in the cochlea . Hear Res,2003,177(1-2):43-52
11. Ohlemiller KK, Wright JS, Dugan LL. Early elevation of cochlear reactive oxygen species following noise exposure. Audiol. Neuro-Otol. 1999,4 (5):229– 236.
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13. Karlidag T, Yalcin S,Ozturk A,et al. The role of free oxygen radicals in noise induced hearing loss: effects of melatonin and methylprednisolone. Auris, Nasus, Larynx,2002,29 (2):147– 152.
14.候粉霞,王生,胡吟燕..噪声对豚鼠耳蜗抗氧化酶防御体系的影响.中华劳动卫生职业病杂志,2003, 21(2):121-123.
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1. Glowatzki E, Fuchs PA.Transmitter release at the hair cell ribbon synapse. Nature Neurosci,2002,5(2):147–154.
2. Ehrenberger K, Felix D. Glutamate receptors in afferent cochlear neurotransmission in guinea pigs.Hear Res,1991 ,52(1):73-80.
3. Duan ML, Ulfendahl M, Laurell G, et al. Protection and treatment of sensorineural hearing disorders caused by exogenous factors: experimental findings and potential clinical application. Hear. Re,2002,169(1-2):169–178.
4. Puel JL, Pujol R, Tribillac F, et al. Excitatory amino acid antagonists protect cochlear auditory neurons from excitotoxicity.J Comp Neurol ,1994,341(2):241–256.
5. Chen Z, Ulfendahl M, Ruan R, et al. Protection auditory function against noise trauma with local caroverine administration in guinea pig. Hear Res ,2004, 197(1-2):131-136.
6. Fessenden JD, Shacht J.The nitric oxide/cyclic GMP pathway: A potential major regulator of cochlear physiology . Hear Res , 1998,118(1-2):168-176.
7. Shi X,Ren T,Nuttall AL.The electrochemical and fluorescence detection of nitric oxide in the cochlea and its increase following loud sound. Hear Res, 2002 ,164(1-2):49-58.
8. Canlon B,Agerman K, Dauman R, et al. Pharmacological strategies for preventing cochlear damage induced by noise trauma. Noise Health,1998,1(1):13-23
9. Chen YS,Tseng FY, Liu TC, et al. Involvement of nitric oxide generation in noise-induced temporary threshold shift in guinea pigs. Hear Res,2005,203(1-2):94-100.
10. Shi X ,Dai C,Nuttall AL. Altered expression of inducible nitric oxide s ynthase (iNOS) in the cochlea . Hear Res,2003,177(1-2):43-52
11. Ohlemiller KK, Wright JS, Dugan LL. Early elevation of cochlear reactive oxygen species following noise exposure. Audiol. Neuro-Otol. 1999,4 (5):229– 236.
12. Dehne N,Lautermann J, ten Cate WJ, Rauen U, et al. In vitro effects of hydrogenperoxide on the cochlear neurosensory epitheliumof the guinea pig. Hear Res,2000 , 143(1-2):162– 170.
13. Karlidag T, Yalcin S,Ozturk A,et al. The role of free oxygen radicals in noise induced hearing loss: effects of melatonin and methylprednisolone. Auris, Nasus, Larynx,2002,29 (2):147– 152.
14.候粉霞,王生,胡吟燕..噪声对豚鼠耳蜗抗氧化酶防御体系的影响.中华劳动卫生职业病杂志,2003, 21(2):121-123.
15. Ohinata Y, Yamasoba T, Schacht J, et al. Glutathione limits noise-induced hearing loss . Hear Res,2000,146(1-2):28-34.
16. Jacono AA,Hu B,Kopke RD,et al. Changes in cochlear antioxidant enzyme activity after sound conditioning and noise exposure in the chinchilla. Hear Res,1998,117 (1-2):31-38.
17. Ising H, Handrock M, Gunther T, et al. Increased noise trauma in guinea pigs through magnesium deficiency. Arch Otorhinolaryngol,1982,236(2):139-146.
18. Gunther T ,Ising H ,Joachims Z. Biochemical mechanisms affecting susceptibility to noise-induced hearing loss. Am J Otol,1989 ,10(1):36-41.
19. Joachims Z, Babisch W, Ising H, et al. Dependence of noise-induced hearing loss upon perilymph magnesium concentration. J Acoust Soc Am,1983 ,74(1):104-108.
20. Walden BE, Henselman LW, Morris ER. The role of magnesium in the susceptibility of soldiers to noise-induced hearing loss. J Acoust Soc Am, 2000,108(1):453-456.
21. Attias J,Sapir S, Bresloff I,et al. Reduction in noise-induced temporary threshold shift in humans following oral magnesium intake. Clin Otolaryngol Allied Sci,2004 Dec,29(6):635-641.
22. Lynch ED,Kil J.Compounds for the prevention and treatment of noise-induced hearing loss.Drug Discov Today,2005 Oct 1,10(19):1291-1298.
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