Lee Ji Eun, Nakagawa Takayuki, Kim Tae Soo, Iguchi Fukuichiro, Endo Tsuyoshi, Dong Youyi, Yuki Kazuo, Naito Yasushi, Lee Sang Heun, Ito Juichi
Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Japan.
Laryngoscope. 2003 Jun;113(6):994-9. doi: 10.1097/00005537-200306000-00015.
OBJECTIVES/HYPOTHESIS: The survival of the spiral ganglion (SG) is a critical issue in preservation of hearing. Research on topics related to this issue requires a mouse experimental model because such a model has advantages including use of genetic information and knockout or "knockin" mice. Thus, the aim of the study was to establish a mouse model for induction of apoptosis of SG neurons with a definite time course.
Laboratory study using experimental animals.
C57BL/6 mice were used as experimental animals and were subjected to direct application of cisplatin into the inner ear. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunostaining for Neurofilament 200-kD (NF) and peripherin were used for analysis of SG degeneration. In addition, generation of peroxynitrite in affected spiral ganglions was examined by immunostaining for nitrotyrosine. Cellular location of activated caspase-9 and cytochrome-c in dying SG neurons were examined for analysis of cell death pathway.
The TUNEL assay and immunohistochemical analysis for NF and peripherin indicated that type I neurons in spiral ganglions were deleted through the apoptotic pathway over time. Spiral ganglion neurons treated with cisplatin exhibited expression of nitrotyrosine, indicating induction of peroxynitrite by cisplatin. In dying SG neurons, expression of activated caspase-9 and translocation of cytochrome-c from mitochondria to cytoplasm were observed, indicating the mitochondrial pathway of apoptosis.
The predictable fashion of induction of apoptosis in SG neurons over a well-defined time course in the model in the study will aid studies of the molecular mechanism of cell death and elucidation of a strategy for prevention of SG degeneration.
目的/假设：螺旋神经节（SG）的存活是听力保存中的关键问题。关于该问题的相关研究需要小鼠实验模型，因为这种模型具有诸多优势，包括可利用遗传信息以及使用基因敲除或“基因敲入”小鼠。因此，本研究的目的是建立一个具有明确时间进程的诱导SG神经元凋亡的小鼠模型。
使用实验动物的实验室研究。
将C57BL/6小鼠作为实验动物，向内耳直接注射顺铂。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法以及针对神经丝200-kD（NF）和外周蛋白的免疫染色来分析SG的退变情况。此外，通过对硝基酪氨酸的免疫染色检测受影响螺旋神经节中过氧亚硝酸盐的生成。检测死亡SG神经元中活化的半胱天冬酶-9和细胞色素c的细胞定位，以分析细胞死亡途径。
TUNEL法以及针对NF和外周蛋白的免疫组化分析表明，随着时间推移，螺旋神经节中的I型神经元通过凋亡途径被清除。用顺铂处理的螺旋神经节神经元呈现硝基酪氨酸表达，表明顺铂诱导了过氧亚硝酸盐的产生。在死亡的SG神经元中，观察到活化的半胱天冬酶-9的表达以及细胞色素c从线粒体向细胞质的转位，表明存在凋亡的线粒体途径。
本研究模型中SG神经元在明确时间进程内可预测的凋亡诱导方式，将有助于细胞死亡分子机制的研究以及阐明预防SG退变的策略。
