改良角膜表面镜片术法建立兔曲霉菌性角膜炎动物模型

背景真菌性角膜感染动物模型是研究真菌性角膜炎发病机制的工具，目前的制作方法主要有划痕法、基质注射法和角膜表面镜片术法，但均有其不足之处。目的探讨一种简便、易操作的改良兔曲霉菌性角膜炎动物模型制作方法。方法成年新西兰白兔18只，采用烟曲霉菌孢子附贴滤纸片的改良角膜表面镜片术法制作真菌性角膜炎动物模型。将浸有10^8孢子／ml（10^8孢子／ml组，6只）或10^6孢子／ml（106孢子／ml组，6只）真菌混悬液的滤纸贴敷于去上皮的角膜基质并用角膜接触镜覆盖，将浸有生理盐水的滤纸贴敷于另6只兔眼角膜作为对照组。分别于造模后3、7、14d裂隙灯下观察眼前节症状，参照Dong的标准进行症状评分。制备角膜刮片并用质量分数10％KOH和荧光白染色在荧光显微镜下检测真菌菌丝，角膜组织切片分别行苏木精-伊红和过碘酸希夫染色，光学显微镜下观察角膜形态学改变和菌丝生长情况。对感染组织进行真菌培养以验证模型的质量。结果10^8孢子／ml组和10^6孢子／ml组真菌性角膜炎模型成功者分别为6眼和4眼，裂隙灯检查表明造模3d后10^8孢子／ml组眼前节症状明显重于10^6孢子／ml组，且随着时间的延长，炎性损伤逐渐转向增生期。造模后3d和7d，2组感染的兔眼症状评分明显高于对照组，差异均有统计学意义（P〈0．01），10^8孢子／ml组兔眼的症状评分均明显高于10^6孢子／ml组，差异有统计学意义（P〈0．01），造模后14d，10^8孢子／ml组兔眼的症状评分明显高于对照组，差异有统计学意义（P〈0．05）。造模后3d和7d，2组兔眼角膜刮片中均可见真菌菌丝。角膜组织病理学检查显示，造模3d和7d后10^8孢子／ml组可见炎性细胞浸润和角膜基质细胞坏死，并可见真菌菌丝，造模后14d可见新生血管长入。10^6孢子／ml组炎症轻于10^8孢子／ml组。真菌培养结果表明，造模后3d和7d时10^8孢子／ml组均见菌丝生长，而10^6孢子／ml组仅在造模3d时可见菌丝生长。结论改良角膜表面镜片术法可成功制备兔曲霉菌性角膜炎动物模型，是一种简便、易于操作的真菌性角膜炎动物模型制作方法。

Rabbit model of aspergillus keratitis induced by modified corneal surface lens method

Background Animal model of fungal keratitis is an available tool to the experimental study of the pathogenesis mechanism of fungal keratitis. Current modeling methods of fungal keratitis include corneal scratching, corneal stroma injection and corneal surface lens methods. But these methods still have their own shortages. Objective This experiment was to create a fungal keratitis animal model by modifying corneal surface lens method. Methods Modified animal models of fungal keratitis were created by modified corneal surface lens method in 12 general adult New Zealand white rabbits. The filter papers soaked 108 spores / ml or A106spores / ml of spergillus fumigatus suspension were attached on the de-epithelial cornea surface and fixed with contact lens and tarsorrhaphy for 2 days, and the filter paper with physiological saline was used as control group. The symptoms of anterior segment were examined under the slit lamp in 3 ,7 and 14 days after surgery and scored based on the criteria of Dong. Corneal scraping was stained with 10% potassium hydroxide and calcofluor white stain to observed mycelium under the fluorescence microscope. Corneal tissue sections were examined by hematoxylin-eosin staining and periodic acid Schiff staining under the light microscope. The use of animal followed the Standard of Association for Research in Vision and Ophthalmology. Results Fungal keratitis models were successfully established in 6 eyes and 4 eyes in 108 spores/ml group (6/6) and 106 spores/ml group respectively. The symptom was more severer and score was higher in the eyes of 108 spores/ml group than that in 106 spores/ml group. At 3 and 7 days after surgery,the symptom scores of fungal keratitis models were higher than those of control group from 3 through 7 days with the statistically significant difference (P<0. 01) and the symptom scores of 108 spores/ml group were significantly higher than those of 106 spores/ml group (P<0. 01). At 14 days after surgery, the symptom scores of 108 spores/ml group were still higher than those of control group (P<0. 05). Fungal hyphae was seen in the corneal scrapes in 108 spores/ml group and 106 spores/ml group respectively from 3 through 7 days after surgery. Inflammatory cell infiltration, stroma cells necrosis and fungal hyphae were presented in 108 spores/ml group, and the corneal neovascularization could be observed in 108 spores / ml group 14 days later. Fungal culture revealed the positive outcome in both 3 and 7 days after surgery in 108 spores/ml group,but in 106 spores/ml group,the positive result was only in the 3rd day. Conclusion Modified corneal surface lens method is more feasible and sample in the model of Aspergillus keratitis. This animal model of Aspergillus keratitis is practical for the further study of fungal keratitis.