角膜胶原交联治疗小鼠真菌性角膜炎的疗效

Antifungal efficacy of corneal collagen cross-linking on treating experimentally induced fungal keratitis of mice

目的研究角膜胶原交联术（CXL）在治疗真菌性角膜炎过程中的疗效。方法选择6～8周龄雄性SPF级C57BL/6小鼠80只，制作腐皮镰孢菌性角膜炎模型，其中40只用于角膜真菌活性的检测，40只用于疗效观察，均以左眼为实验眼。角膜真菌活性的检测中将40只小鼠按照随机数字表法分为假手术组（不接种菌丝）、模型对照组、去上皮组（造模后刮除角膜上皮）和交联治疗组（去上皮联合交联治疗），各组小鼠分别于造模后3 d用裂隙灯显微镜对术眼角膜炎症进行评分，并行角膜组织的载菌量计数。交联疗效观察实验中，应用随机数字表法将40只小鼠随机分为假手术组（不接种菌丝）、模型对照组、去上皮组和交联治疗组（去上皮联合交联治疗）。4个组小鼠分别于造模后连续7 d行裂隙灯显微镜下术眼角膜炎症评分及眼前节数码照相。造模后14 d，裂隙灯显微镜下对术眼角膜炎症单项评分后，行组织病理学检查及炎症细胞计数。结果造模后交联治疗组小鼠角膜载菌量最低，各组角膜载菌量的总体比较，差异有统计学意义（F＝11.97，P＝0.00），交联治疗组角膜载菌量平板计数与角膜炎症评分呈明显正相关（r＝0.723，P＝0.043）。交联治疗组各时间点小鼠的角膜炎症评分明显低于模型对照组，3个组间和不同时间点角膜炎症评分的总体比较，差异均有统计学意义（F分组＝34.44，P＝0.00；F时间＝17.49，P＝0.00）。造模后14 d，去上皮组和交联治疗组小鼠角膜病灶面积和溃疡程度评分均明显低于模型对照组，差异均有统计学意义（均P〈0.05）。角膜组织病理学检查显示，模型对照组小鼠角膜水肿，部分角膜基质溶解坏死，去上皮组于造模后角膜炎症表现较轻。交联治疗组小鼠角膜炎症反应最轻微，造模后7 d角膜炎症接近痊愈。假手术组角膜基质内细胞很少；模型对照组基质增厚，基质内炎症细胞增多，可以确认的中性粒细胞占基质所有细胞总数的73.65%；去上皮组中性粒细胞百分比为59.33%；交联治疗组中性粒细胞百分比为11.29%。结论CXL可以有效抑制小鼠角膜真菌活性，减轻真菌诱导的角膜炎症反应。

Objective This study was to assess the efficacy of corneal collagen cross-linking treatment on fungal keratitis.Methods Eighty SPF male C57BL/6 mice aged 6-8 weeks were selected for the experiment. Fusarium solani infected model was established on the left eyes of all 80 mice.Forty mice were distributed randomly into sham operation group, model control group, scraped epithelium group and corneal collagen cross-linking （CXL） group （treated with epithelium scraped and CXL）. Three days after modeling, the levels of the corneal disease severity were scored by slit lamp microscopy.The fungal activity was confirmed by plate counts.The left 40 mice were divided randomly into sham operation group, model control group, scraped epithelium group and CXL group （treated with epithelium scraped and CXL）. In 1 day and 2, 3, 4, 5, 6, 7, 14 days after modeling, the corneas were examined under the slit lamp microscope.The corneal pathological examination of each group were conducted with hematoxylin and eosin staining at postoperative 14 days.The animal feeding and use was in accordance with the standards set by the ARVO, and the experiment was approved by the Ethic Committee for Experimental Animal of Henan Eye Institute.ResultsThe colony-forming units （CFUs） of fungal solutions in culture significantly decreased with CXL treatment （F=11.97, P=0.00）. The Pearson correlation analysis of CFU and clinical scores in CXL group showed that inflammatory cells infiltration was positively correlated with corneal disease severity （r=0.723, P=0.043）. Corneal inflammatory score was significantly lower in the CXL group in various time points, with a significant differences among the groups and time points （Fgroup=34.44, P=0.00; Ftime=17.49, P=0.00）. Corneal lesion and the depth of ulceration in scraped epithelium group and CXL group were remarkably lower than that in the model control group （all at P〈0.05）. Histopathology revealed that the degree of corneal collagen fibers destruction and the ratio of inflammatory cells infiltration in scraped epithelium group （59.33%） and CXL group （11.29%） were much lower than that in the model control group （73.65%）.Conclusions CXL can inhibit the fungal activity effectively in the cornea of mice, and reduce the fungal induced keratitis reaction.