角膜穿通伤早期兔眼角膜上皮基底膜的修复和再生过程

Procedure for early corneal basement membrane repair and regeneration in corneal penetrating injury in rabbits

目的观察角膜穿通伤早期兔眼角膜上皮基底膜(EBM)的修复和再生过程。方法采用随机数字表法将42只新西兰白兔分为建模后1、3、5、7、14、21和30 d组,每组6只,均取右眼建模作为实验眼。另取6只未经任何处理的新西兰白兔作为正常对照组。应用2.0 mm环钻建立兔角膜环钻穿通伤模型。建模后各组于相应时间点在裂隙灯显微镜下观察角膜,Image J软件测定角膜荧光素着色面积评估上皮愈合情况,Fantes分级量表评估角膜混浊程度,苏木精-伊红染色观察角膜上皮和基质修复情况,透射电子显微镜下观察EBM再生情况。结果兔角膜穿通伤后各组角膜荧光素染色面积总体比较,差异有统计学意义(F=3398.88,P<0.01),其中建模后1、3、5、7和14 d组角膜上皮荧光素着色面积分别为(4.00±0.10)、(3.11±0.10)、(2.00±0.06)、(0.90±0.04)和(0.67±0.03)mm2,角膜上皮荧光素着色面积逐渐缩小,两两比较差异均有统计学意义(均P<0.05);建模后21 d和30 d组均未见角膜上皮荧光素着色。建模后1、3、5、7、14、21和30 d组角膜混浊程度评分分别为3.44±0.53、0.67±0.25、1.33±0.50、2.11±0.60、2.44±0.53、3.22±0.44和3.78±0.44,建模后1~5 d角膜混浊评分下降,建模后5~30 d角膜混浊评分逐渐升高,各组角膜混浊评分总体比较,差异有统计学意义(F=51.182,P<0.01)。苏木精-伊红染色结果显示角膜伤口区开始由纤维斑块填充,建模后3 d组单层上皮覆盖,建模后5 d组大量成纤维细胞及其分泌的细胞外基质填充,建模后21 d及30 d组前部基质中胶原纤维排列紧密。透射电子显微镜下观察结果显示,伤口区由肌成纤维细胞及不规则胶原纤维填充,建模后21 d角膜基质开始重塑,EBM于建模后21 d和30 d开始不完全再生。结论兔角膜穿通伤后角膜纤维化启动并逐渐加重,EBM不完全再生。

Objective To describe the procedure for early corneal epithelial basement membrane(EBM)repair and regeneration in rabbits with corneal penetrating injury.Methods Forty-two New Zealand white rabbits were divided into modeling 1-,3-,5-,7-,14-,21-,and 30-day groups using a random number table method,with 6 rabbits in each group;the right eyes were selected as the experimental eyes.Another 6 New Zealand white rabbits without any treatment were taken as the normal control group.A 2.0-mm trephine was used to ablate a full-thickness button of the central corneal tissue of each rabbit.The corneas were observed by slit lamp biomicroscopy at the respective time points after the trephined injury.Corneal epithelial fluorescein staining was used to evaluate re-epithelialization with Image J software and haze grading was evaluated with the Fantes classification.Hematoxylin-eosin staining was used to observe the healing process of the cornea.Transmission electron microscopy was conducted to assess the regeneration of the EBM and the reconstruction of the cornea.The study protocol was approved by the Ethics Committee of Guangxi Medical University(No.201811031).The use and care of the experimental animals complied with the Statement for the Use of Animals in Ophthalmic and Vision Research.Results The corneal epithelial fluorescein areas in modeling 1-,3-,5-,7-,and 14-day group were(4.00±0.10),(3.11±0.10),(2.00±0.06),(0.90±0.04)and(0.67±0.03)mm2,respectively,with a significant difference among them(F=3398.88,P<0.01).With the increasing of time after modeling,the corneal epithelium fluorescein area was gradually reduced,showing significant differences between any two groups(all at P<0.05),and the staining was disappeared at 21 and 30 days after modeling.The corneal haze grades were 3.44±0.53,0.67±0.25,1.33±0.50,2.11±0.60,2.44±0.53,3.22±0.44 and 3.78±0.44 in modeling 1-,3-,5-,7-,14-,21-,and 30-day group,respectively.The corneal opacity score gradually decreased during 1-5 days after modeling and gradually increased during 5-30 days after modeling,with a significant difference among them(F=51.182,P<0.01).Hematoxylin-eosin staining revealed that a fibrin clot formed in the wound area,and a single layer of epithelium covered the area at initial 3 days after modeling,and a large number of fibroblasts and some extracellular matrix were found at 5 days after modeling.At 21 and 30 days after modeling,the collagen fibers were tightly arranged in the anterior stroma.Transmission electron microscopy showed that the wound was filled with irregular collagen fibers and myofibroblasts.The stroma was remodeled at 21 days after modeling,and defective regeneration of the EBM was detected at 21 and 30 days after modeling.Conclusions Corneal fibrosis initiates after corneal penetrating injury in rabbits and gradually aggravates,the EBM regenerates defectively.