两种内淋巴给药方式对豚鼠耳蜗功能和形态的影响

Influences of two different endolymphatic infusion ways to cochlear morphology and function in guinea pigs

目的观察经耳蜗侧壁打孔（侧壁径路）和经圆窗膜、基底膜穿刺（双膜径路）两种内淋巴系统给药方式对豚鼠耳蜗整体形态结构和功能的影响并比较两种方式的优劣。方法40只正常健康杂色豚鼠分为A、B两组（每组20只），所有动物左侧为给药耳，右侧为非给药耳。A组采用侧壁径路进入中阶灌注携带增强型绿色荧光蛋白基因的5型重组腺病毒（adenovims5-enhanced green fluorescence protein，AdS—EGFP）5μl；B组采用双膜径路进入中阶灌注AdS-EGFP5μ1。给药前后行听眭脑干反应（ABR）测试，观察听功能改变。耳蜗冰冻切片直接荧光观察腺病毒分布，HE染色观察手术径路的愈合情况。基底膜铺片鬼笔环肽染色观察毛细胞受损情况，扫描电镜观察局部损害情况。结果所有动物术后均存活。穿刺部位修复良好，耳蜗的完整性得以保持。EGFP在Corti器和血管纹内壁细胞内标记明显，表明两种给药径路都可以将药物成功注入内淋巴系统。A组证实成功14只（70％），手术前后ABR反应阈（声压级）变化[（33．1±10．3）dB]与对侧非给药耳[（9．4±3．9）dB]比较差异具有统计学意义（F=46．34，P=0．0005）；B组证实成功8只（40％）手术前后阈值改变[（2．5±3．8）dB]与对侧耳[（2．5±3．8）dB]比较差异无统计学意义（F=0．00，P=1．000）。两种方法在部分动物中都有药物渗漏入外淋巴的现象，给药局部产生炎性反应，侧壁径路对毛细胞的损害范围大于双膜径路。结论两种手术径路都可将药物成功注入豚鼠耳蜗的内淋巴系统中，局部有炎性反应，术后耳蜗的完整性可以获得完全恢复。侧壁径路对豚鼠耳蜗毛细胞缺失和ABR反应阈的影响大于双膜径路，但是经侧壁径路进入中阶的手术成功率高于双膜径路，选择何种灌注径路需要根据实验要求来定。

Objective To explore the influence of two different endolymphatic infusion ways on cochlear morphology and function. Methods Forty healthy pigment guinea pigs （250 -350 g）with normal Prey＇ s reflex were divided into A and B group with 20 animals respectively. The right ears served as treated ears and the left ones as control ones. In group A, 5 μl of adenovirus 5-enhanced green fluorescence protein （AdS-EGFP） suspension was infused into the scala media through an opened cochleostomy on the lateral wall of the scala media （LWS）. In group B, the same volume of AdS-EGFP suspension was infused into the scala media through punctured round window membrane and the basilar membrane （RBM）. Cochlear morphology was examined under scan electric microscope and phalloidin staining was used to observe the hair cells in the infused ears after the animals were sacrificed. Auditory brainstem thresholds of the ears of all the animals were measured before and after treatment. Results All the animals recovered well after operation. The holes on the lateral wall of the scala media and punctures on the round window membrane were healed completely. EGFP labeling appeared in the organ of Corti and lining wall of the stria vascularis indicated that adenovirus suspension was injected into the scala media using LWS （ succeed in 14 animals accounted for 70% ） and RBM （ in 8 animals accounted for 40% ） ways. Viruses were inoculated into the scala media with only locally inflammation reaction. In group A the hearing threshold decreased significantly in the treated ears compared with the control ears after the operation [ （33.1 ± 10. 3） dB, （9. 4 ±3. 9） dB, F = 46. 34 ,P = 0. 0005 ]. However, in group B there was no significantly different between the treated ears and the control ears after the operation [ （ 2. 5 ± 3.8 ） dB, （ 2. 5 ±3.8 ） dB, F = 0.00, P = 1. 000 ]. Phalloidin staining indicated that in group A the extension of hair cells loss was bigger than in group B. In some animals of two groups, EGFP labeling appeared in the extra-lymphatic system indicating that some of the injected suspension leaked out of the scala media. Conclusions AdS-EGFP could be infused into the scala media through LWS or RBM and adenovirus could infect the lining cells of scala media and supporting ceils in the basal membrane successfully without causing immunoreaction in the whole cochlea. LWS caused more hair cell loss and hearing loss than RBM. However, the cochlear morphology could be recovered completely after surgery. The positive inoculation rate was relatively higher that through LWS than that through RBM.