The Study Of Endoplasmic Reticulum (ER) Stress-related Apoptosis In Intense Noise-induced Cochlea Cells Death Of Guinea Pig

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 lossObjective 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 lossObjective 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 deathObjective 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.