耳蜗局部应用NR2B受体阻滞剂对水杨酸钠致豚鼠螺旋神经节细胞损伤的拮抗作用

Blocking NR2B Receptor Reversed the Process of Cytotoxicity to Spiral Ganglion Neurons Induced by Sodium Salicylate in Guinea Pig

目的通过圆窗龛局部给予特异性NR2B受体阻滞剂艾芬地尔,探讨其拮抗水杨酸钠致豚鼠螺旋神经节细胞损伤的作用。方法将ABR阈值小于40dB SPL的健康花色豚鼠48只随机分为4组,每组12只,Ⅰ组为空白对照组;Ⅱ组为人工外淋巴液(artificial perilymph,APL)组,左耳圆窗龛注人APL 60μl;Ⅲ组为水杨酸钠组,左耳圆窗龛注入APL 60μl,腹腔注射水杨酸钠;IV组为艾芬地尔组,左耳圆窗龛注入溶于APL的艾芬地尔(10μmol/L)60μl后,腹腔注射水杨酸钠。水杨酸钠注射量为400mg·kg-1·d-1,连续注射7天。停药并行ABR测试后将动物处死,取出左侧耳蜗,每组随机取6只动物进行免疫组织化学染色观察耳蜗螺旋神经节(SGN)caspase-3表达情况,6只行原位末端转移酶标记技术(terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling assay,TUNEL)法检测螺旋神经节细胞凋亡率。结果给药后,I、II、III、IV组动物左耳ABR反应阈分别为31.67±5.16、33.33±5.17、64.17±7.36、49.17±5.85dB SPL;免疫组织化学染色显示,Ⅰ、Ⅱ组螺旋神经节细胞中caspase-3的表达量差异无统计学意义,Ⅲ、IV组螺旋神经节细胞中caspase-3的表达量较Ⅰ、Ⅱ组显著增高(P<0.01),IV组螺旋神经节细胞中caspase-3的表达量较Ⅲ组明显降低(P<0.01)。TUNEL法检测结果示,Ⅰ、Ⅱ组SGN细胞凋亡率无显著差异,Ⅲ、IV组SGN细胞凋亡率较Ⅰ、Ⅱ组显著增高(P<0.01),IV组SGN细胞凋亡率较Ⅲ组明显降低(P<0.01)。结论耳蜗局部特异性阻断NR2B受体可以拮抗水杨酸钠致豚鼠螺旋神经节细胞的损伤。

Objective To investigate whether blocking NR2B receptor can reverse the process of cytotoxicity to spiral ganglion neurons induced by sodium salicylate in guinea pig by applying ifenprodil （a NR2B antagonist） at the round window niche .Methods Sixty healthy guinea pigs provided by the experimental animal center of Guangxi medical university were randomly and evenly divided into a control group （Group Ⅰ ,no treatment） ,an APL group （Group Ⅱ ,60μl APL directly applied to the round window ） ,a sodium salicylate group （Group Ⅲ ,60 μl APL di-rectly applied to the round window and then be given intraperitoneal sodium salicylate injection ） ,and an ifenprodil group （Group Ⅳ ,60μl of 10μmol/l ifenprodil in APL directly applied to the round window and then be given intra-peritoneal sodium salicylate injection ） .Sodium salicylate was given at 400 mg · kg ^-1 · d^-1 for 7 days .Auditory brainstem responses （ABRs） were recorded before animal sacrifice by decapitation .The left cochlea was removed and prepared for detection of caspase -3 expression in spiral ganglion neurons via immunohistochemistry .From each group ,6 cochleae were used to test apoptosis index in spiral ganglion neurons using the TUNEL technique .Results Before salicylate administration ,the ABR threshold was less than 40 dB SPL in all animals .After salicylate ad-ministration ,the ABR threshold was 33 .33 ± 5 .17 dB SPL in Group Ⅱ ,64 .17 ± 7 .36 dB SPL in Group Ⅲ and 49 .17 ± 5 .85 dB SPL in Group Ⅳ ,in contrast to 31 .67 ± 5 .16 dB SPL in Group I （controls） .The caspase -3 ex-pression was not changed obviously in Group Ⅰ and Group Ⅱ ,but was significantly changed in Group Ⅲ and Group Ⅳ （P〈0 .01） .The caspase-3 expression appeared to be decreased in Group Ⅳ compared to those in Groups Ⅲ （P〈0 .05） ,but still increased compared to those of in Group I and Ⅱ （P〈0 .05） .The apoptosis index among spiral ganglion neurons in Groups Ⅲ and Ⅳ increased significantly compared to those of in Group Ⅰ and Ⅱ （P〈0 .001） .It was however ,lower in Group Ⅳ than in Group Ⅲ （P〈0 .01） .Conclusion Blocking NR2B receptor with specificity can reverse the process of cytotoxicity to spiral ganglion neurons induced by sodium salicylate in guinea pig .