摘要
目的通过比较凋亡坏死的细胞数量,揭示苯乙烯致大鼠耳蜗外毛细胞死亡的主要途径。方法成年Long Evens大鼠14只,随机分为实验组和对照组。实验组8只(16耳),胃饲苯乙烯400mg/kg(2g苯乙烯溶于1ml橄榄油),每日1次,每周5d,共3周。对照组6只(12耳),胃饲相同剂量的橄榄油,胃饲次数和时间与实验组相同。胃饲前及3周后应用电位反应测听仪检测短声诱发的动物双侧听性脑干反应阈值(ABR)。大鼠听觉功能检测后,断头处死,取出双耳听泡,固定耳蜗组织,分离耳蜗基底膜。分别用碘化丙锭(PI)和原位末端标记(TUNEL)染色细胞核,异硫氰酸荧光素标记的鬼笔环肽染色细胞的丝状肌动蛋白,鉴别凋亡、坏死和缺失的毛细胞,荧光显微镜下计数损伤(凋亡、坏死和缺失)的外毛细胞。结果对照组动物ABR阈值及耳蜗毛细胞形态未见异常改变。实验组大鼠胃饲苯乙烯3周后,短声诱发的平均ABR阈值(44.5±4.3dBSPL)较胃饲苯乙烯前(29.2±2.6dB SPL)提高约15dB SPL(P=0.001)。荧光显微镜下丝状肌动蛋白染色发现,实验组大鼠毛细胞主要损伤区域位于耳蜗中回,第三排外毛细胞损伤最为严重,其次为第二排、第一排外毛细胞。PI染色的外毛细胞核呈现核固缩、核肿胀和核缺失三种形态学变化,TUNEL强绿色荧光标记物存在于固缩的细胞核内。定量观察耳蜗外毛细胞死亡方式发现,固缩的外毛细胞核均数(42.2±40.4)为肿胀外毛细胞核(13.4±11.8)的3倍(P=0.01)。结论凋亡为胃饲苯乙烯3周后大鼠耳蜗外毛细胞死亡的主要途径。
Objective To compare the prevalence of apoptosis and necrosis, and investigate the primary death pathway of outer hair cells of rat cochlea following styrene exposure. Methods Fourteen adult Long Evans rats were used in the present study. The animals were randomly assigned into test group （n=8） and control group （n=6）. Animals in test group were exposed to styrene by garage at 400 mg/kg （2g styrene was mixed with 1ml olive oil）. Treatment was performed once a day, 5 days per week for 3 weeks. Animals in control group were fed by gavage the same volume of olive oil on an identical time schedule used for the test group. The auditory brainstem response （ABR） thresholds of both ears elicited with clicks were measured before and at the end of the 3-week styrene or olive oil treatment. After hearing was re-assessed, animals were sacrificed and cochleae were quickly removed from the skull. Following fixation, whole specimens comprising the basilar membrane with Corti＇s organ were separated from the modiolus. Apoptotic, necrotic and missing outer hair cells （OHCs） were distinguished by combined assays of nuclear staining with propidium iodide （PI）, TUNEL assay and filamentous actin （F-actin）staining with FITC-phalloidin. Each Corti＇s organ was thoroughly examined by fluorescence microscopy. The numbers of damaged OHCs （apoptotic, necrotic and missing OHCs） were documented. Results Neither threshold shift of ABR nor sign of hair cell （HC） damage was found in the cochlea of control animals. The anirnals of test group showed both physiological and pathological changes in the cochleae following the 3-week styrene treatment. ABR testing revealed an average of 15 dB of threshold shifts. F-actin staining exhibited the maximal level of OHCs damage in the middle portion of Corti＇s organ. The major damage occurred in the third row of OHCs, followed by the second and first rows of OHCs. Three types of morphological changes in damaged OHC nuclei were revealed by PI labeling： nuclear condensation, nuclear swelling and nuclear missing. Strong TUNEL green fluorescence appeared in the OHCs with condensed nuclei. Quantitative analysis showed that the average number of apoptotic OHCs was approximately three times greater than the number of necrotic OHCs （P=0. 01）. Conclusion It is indicated that apoptosis is the primary death pathway of OHCs leading to generation of the cochlear lesion following styrene exposure.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第7期855-857,共3页
Medical Journal of Chinese People's Liberation Army
基金
美国NIH资助项目(NIDCD 1 R 01 OH 008113-01A1)
