听泡穿刺注射法构建C57BL/6小鼠急性中耳炎模型

A mouse model for acute otitis media via transbullar injection

目的 探讨听泡穿刺注射法建立小鼠急性中耳炎模型的可行性和实用性.方法 采用听泡穿刺注射的方法建立C57BL/6小鼠急性中耳炎模型,造模组注射5μl肺炎链球菌19F悬液（1×10^7 CFU/ml）,对照组注射等量无菌磷酸盐缓冲液.观察注射后小鼠行为变化,分别于建模后12h、1d、2d、3d、5d、7d处死小鼠,分离中耳组织HE染色观察病理改变,同时收集中耳灌洗液检测炎性细胞数量、细菌载量及相关炎性因子含量.结果 接种1d后,中耳腔内细菌迅速增殖,产生大量肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）和白细胞介素1β（IL-1β）,中耳黏膜基质内血管扩张充血,以中性粒细胞为主的炎性细胞逐渐渗出;接种2～3d后细菌载量开始降低,炎性因子表达水平下降,但中耳腔内炎性细胞浸润及黏膜增厚达最高水平;接种5～7d后,细菌基本被清除,中耳炎性反应逐渐消退.结论 听泡穿刺注射方法可成功构建C57BL/6小鼠急性中耳炎模型,具有较好的可行性和实用性。

Objective To develop a mouse model for acute otitis media （AOM） via transbullar injection method and evaluate its feasibility and practicability.Methods The middle ears （ME） of C57BL/6 mice were inoculated via transbullar injection method with 5 μl streptococcus pneumoniae （S.pn）19F suspension （1 × 10^7 CFU/ml）,and the control group was inoculated equivalent phosphate buffered solution（PBS）.Behavior changes were observed daily following inoculation.The ME tissues for histological examination and the middle ear lavage fluid （MELF） for total cells quantification,S.pn load determination and cytokines measurement were collected at 12 h,day 1,2,3,5,7 after inoculation,respectively.Results Within 24 hours after instillation,the density of S.pn and the level of acute inflammatory cytokines in ME cavity increased rapidly,some mucosal hyperplasia was evident and leukocytic infiltration （primarily neutrophils） began.The level of ME inflammatory response reached maximal at 2-3 days after inoculation,with extensive effusion,leukocytic infiltration and mucosal thickening.Meanwhile,the density of S.pn decreased gradually.Bacterial clearance was completed by day 5 with extensive resolution of ME inflammation,although mucosal hyperplasia did not resolute until day 7.Conclusion A mouse model for AOM is successfully established via transbullar injection method,laying foundation for future study of AOM.