离子导入角膜胶原交联术治疗兔眼金黄色葡萄球菌性角膜炎

Experimental study of iontophoresis-mediated corneal collagen crosslinking for rabbit Staphylococcus aureus keratitis

背景 研究表明,核黄素联合紫外线A照射产生的交联效果能够提高角膜的抗酶解能力,并且能够抑制多种细菌的生长.目前胶原交联用于治疗圆锥角膜的效果已得到证实,但能否用于治疗细菌性角膜炎国内尚未见相关报道. 目的 评价离子导入角膜胶原交联法单独应用或联合药物治疗兔眼金黄色葡萄球菌性角膜炎的效果.方法 选取40只成年家兔,右眼角膜基质内注射2×10^9个/ml金黄色葡萄球菌悬液（比浊法）制备金黄色葡萄球菌角膜炎模型,然后采用随机数字表法将实验兔随机分为模型组、加替沙星滴眼液点眼组、核黄素-紫外线角膜胶原交联组和药物＋交联治疗组,右眼为实验眼,左眼为空白对照.于造模后24 h行实验眼角膜分泌物涂片进行细菌培养和鉴定,核黄素-紫外线角膜胶原交联组和药物＋交联治疗组行离子导入角膜胶原交联治疗;加替沙星滴眼液点眼组和药物＋交联治疗组用加替沙星滴眼液点眼,每日7次,连续14 d.治疗前及治疗后每天于裂隙灯显微镜下检查实验眼并进行炎症分级;分别于治疗前和治疗后14d行眼前节光学相干断层扫瞄（AS-OCT）检查,观察实验眼病变区角膜厚度变化;于治疗后14 d摘出实验眼分别行角膜的组织病理学观察和透射电子显微镜超微结构观察.实验动物的使用和喂养按照潍坊医学院实验动物伦理委员会（AEEC）的指导方针进行.本研究方案得到AEEC批准. 结果 裂隙灯显微镜检查可见,所有实验眼造模后24 h均形成角膜溃疡,各组角膜炎症分级的差异无统计学意义（χ^2=0.293,P＞0.05）;至治疗后14 d,药物＋交联治疗组实验眼角膜病变程度1只眼为2级、9只眼为3级,模型组、加替沙星滴眼液点眼组和核黄素-紫外线角膜胶原交联组均为4级,4个组间不同病变分级眼数分布的差异有统计学意义（χ^2=38.710,P＜0.001）.AS-OCT显示,治疗后14 d,药物＋交联治疗组实验眼病变区角膜厚度为（428.1±146.2）μm,明显厚于模型组、加替沙星滴眼液点眼组和核黄素-紫外线角膜胶原交联组,4个组间差异有统计学意义（F=8.310,P＜0.001）.角膜组织病理学检查显示,药物＋交联治疗组实验眼基质胶原纤维破坏程度最轻,炎性细胞浸润少;角膜透射电子显微镜观察显示,药物＋交联治疗组实验眼亚细胞结构（即细胞器）接近正常的角膜基质细胞明显多于模型组、加替沙星滴眼液点眼组和核黄素-紫外线角膜胶原交联组.结论 离子导入角膜胶原交联术可用于兔金黄色葡萄球菌性角膜炎的治疗,角膜胶原交联术联合局部抗生素应用的治疗效果优于单纯局部药物疗法和单纯角膜胶原交联法.

Background It is determined that riboflavin/ultraviolet A （UVA）-induced corneal collagen crosslinking is able to increase resistance of cornea against enzymatic digestion and has antimicrobial efficacy for various kinds of bacteria in vitro.However,its in vivo study is less now.Objective This study aimed to evaluate the efficacy of iontophoresis-mediated corneal collagen crosslinking combined with or without drugs for Staphylococcus aureus keratitis.Methods Bacterial keratitis models were induced by the interstromaly injection of Staphylococcus aureus suspension with concentration 2× 10^9/ml in the right eyes of 40 rabbits,and then the rabbits were randomly classified into the model group,gatifloxacin eye drops group,riboflavin/UVA corneal crosslinking group and drugs＋ crosslinking group.The smearing of corneal surface was performed for the identification of bacteria 24 hours after injection.Iontophoresis-mediated riboflavin/UVA crosslinking was applied on the eyes of the riboflavin/UVA corneal crosslinking group and drugs＋crosslinking group,and gatifloxacin eye drops was topically used 7 times per day on the eyes of the gatifloxacin eye drops group and drugs＋crosslinking group.The corneal inflammation was examined and graded under the slit lamp biomicroscope before and after treatment.Ocular anterior segment optical coherence tomography（AS-OCT）,corneal histopathology and ultrastructure were examined 14 days after treatment.The living environment of the experimental animals was maintained at 21 ℃ with a 12-hour light and dark cycle.Animals used in this study were treated in accordance with the Weifang Medical College Animal Experimentation Ethic Committee （AEEC） guidelines.The study protocol was approved by the AEEC.Results Corneal inflammation and ulcer were observed,but no significant difference was found in the inflammatory grade among the 4 groups 24 hours after injection （χ^2=0.293,P〉0.05）.In the 14th day after injection,the corneal ulcer area was smaller and corneal edema was milder in the drugs＋crosslinking group compared with the model group,gatifloxacin eye drops group and riboflavin/ UVA corneal crosslinking group,showing a significant difference in the inflammatory grade among them （χ^2=38.710,P〈0.001）.The cornea thickness values of ulcer zone were （428.1 ± 146.2） μm on the 14th postinjected day in the drugs＋crosslinking group,which was evidently higher than those in the model group,gatifloxacin eye drops group and riboflavin/UVA corneal crosslinking group,with a significant difference among the 4 groups （F =8.310,P〈0.001）.A lower degree of destruction of cornea collagen and less inflammatory cells were seen in the cornea tissue of the drugs＋ crosslinking group by haematoxylin and eosin staining in comparison with other 3 groups,and normal keratocytes were much more in the drugs ＋ crosslinking group than those in other treated groups.Conclusions Iontophoresismediated corneal collagen crosslinking can alleviate Staphylococcus aureus keratitis.The combination of crosslinking with drugs has a better effectiveness than the administration of gatifloxacin eye drops only or riboflavin/UVA corneal crosslinking only.