共聚焦显微镜观察圆锥角膜行角膜胶原交联术后早期组织形态学变化

目的探讨进展期圆锥角膜行角膜胶原交联术后早期共聚焦显微镜下的组织形态学改变。方法回顾性病例系列研究。应用共聚焦显微镜观察2017年9月至2019年3月于解放军总医院眼科行角膜胶原交联术的进展期圆锥角膜患者23例(32只眼), 其中男性15例(24只眼), 女性8例(8只眼);年龄(26±10)岁。所有患者于术前和术后1周、1个月、3个月应用共聚焦显微镜观察角膜组织结构改变、定量记录角膜上皮下神经纤维、基质细胞、内皮细胞密度和基质结构改变的深度并进行对比分析。不同时间的总体差异比较采用重复测量资料的方差分析或Friedman检验, 不同时间的差异的多重比较采用LSD-t检验或Bonferroni校正。结果角膜胶原交联术后1周, 角膜上皮细胞处于修复期, 表现为上皮基底细胞核增大、反射增强, 上皮下可见活化细胞, 浅基质肿胀呈海绵状, 深基质肿胀呈不均匀斑块状或条索状, 反射强。术后1个月, 上皮基底细胞恢复, 上皮下神经开始生长, 浅基质持续呈海绵状肿胀, 反射进一步增强, 深基质肿胀呈更粗大的斑块或条索样结构并持续至术后3个月。术前与术后3个月内, 角膜上皮下神经纤维密度差异有统计学意义(F=233.30, P<0.001), 术后1周、1个月、3个月角膜上皮下神经纤维密度分别为(0.51±0.31)、(3.65±2.21)、(8.50±4.02)mm/mm², 与术前的(14.60±2.57)mm/mm²相比均明显降低(均P<0.05);不同时间点角膜前部基质细胞密度比较, 差异有统计学意义(χ²=92.48, P<0.001), 术后1周、1个月、3个月角膜前部基质细胞密度分别为2.00(1.00, 5.75)、2.50(1.00, 5.75)、79.00(64.25, 94.00)个/mm², 与术前的347.00(345.00, 395.75)个/mm²相比均明显降低(均P<0.05)。术后3个月内角膜基质结构改变的深度范围为(400.56±86.12)μm:术后1周、1个月、3个月分别为(402.13±89.20)、(399.88±85.92)、(399.69±85.94)μm, 差异无统计学意义(F=0.80, P=0.455)。术前和术后不同时间角膜后部基质细胞密度[术前为(260.6±33.2)个/mm², 术后1周、1个月、3个月分别为(264.4±44.5)、(263.9±37.6)、(266.3±40.2)个/mm²]和内皮细胞密度[术前为(2 707.1±152.6)个/mm², 术后1周、1个月、3个月分别为(2 704.2±148.5)、(2 705.6±152.6)、(2 704.5±150.1)个/mm²]比较, 差异均无统计学意义(F=1.38, 1.01;P=0.259, 0.351)。结论圆锥角膜行角膜胶原交联术后早期, 共聚焦显微镜下可见角膜上皮和上皮下神经的改变呈现损伤修复的过程;角膜浅基质呈均匀的蜂窝状肿胀, 深基质呈不均匀斑块状或条索状肿胀;术后早期角膜基质结构改变的深度相对稳定。

Early histological changes detected by confocal microscopy in patients with advanced keratoconus receiving collagen cross-linking therapy

Objective To investigate the early histological changes by confocal microscopy of patients with advanced keratoconus receiving collagen cross-linking therapy.Methods In this prospective case series study,confocal microscopy was used to observe 23 patients(32 eyes)who were diagnosed with advanced keratoconus and treated with collagen cross-linking at the Department of Ophthalmology,Chinese PLA General Hospital from September 2017 to March 2019,aged(26±10)year.All patients were examined before and at 1 week,1 month and 3 months after the therapy.The tissue structure changes,the density of nerve fibers,stromal cells and endothelial cells,and the depth of the corneal stroma were recorded and compared.The overall differences at different times were compared by repeated measurement analysis of variance or Friedman test,and the pairwise comparison was corrected by LSD-t test or Bonferroni test.Results One week after collagen cross-linking,the epithelial cells were in the repair stage,showing an increased nucleolar size and an enhanced reflection,and the activated cells could be detected under the epithelium.The superficial corneal stroma was swollen and spongiform,while the deep corneal stroma was patchy or cord-like,scattered and with a strong reflection.One month after the therapy,epithelial cells recovered,subepithelial nerves began to grow,the superficial corneal stroma still showed a spongy structure,and the reflection was further enhanced.The activation of the deep corneal stroma exhibited as thicker plaques or cord-like structure.Three months after the therapy,the continuous elongation of single nerve fibers could be detected occasionally.There was statistically significant difference in the density of nerve fibers before and early after the therapy(F=233.30,P<0.001).Compared with the preoperative value[(14.60±2.57)mm/mm²],the density of subepithelial nerve fibers decreased significantly in the early postoperative period,which was(0.51±0.31),(3.65±2.21)and(8.50±4.02)mm/mm²,respectively,at 1 week,1 month and 3 months,and there were significant differences between different time points(all P<0.05).There was also statistically significant differences in the density of anterior stromal cells before and early after the therapy(χ²=92.48,P<0.001).Compared with the preoperative value[347.00(345.00,395.75)cells/mm²]the density of anterior stromal cells decreased significantly in the early postoperative period,which was 2.00(1.00,5.75),2.50(1.00,5.75)and 79.00(64.25,94.00)cells/mm²,respectively,at 1 week,1 month and 3 months,and there were significant differences between different time points(all P<0.05).Within 3 months after the therapy,the depth of the corneal stroma observed by confocal microscopy ranged from 245 to 536μm,with an average of(400.56±86.12)μm.Histologically,the depth of the corneal stroma ranged from 245 to 536μm[average,(402.13±89.20)μm],from 251 to 527μm[average,(399.88±85.92)μm]and from 259 to 530μm[average,(399.69±85.94)μm]at 1 week,1 month and 3 months,respectively,with no significant difference(F=0.797,P=0.455).There was no significant difference in the density of posterior stromal cells[(260.6±33.2)cells/mm² preoperatively,(264.4±44.5)cells/mm² at 1 week,(263.9±37.6)cells/mm² at 1 month and(266.3±40.2)cells/mm² at 3 months]and endothelial cells[(2707±152.6)cells/mm² preoperatively,(2704±148.5)cells/mm² at 1 week,(2705±152.6)cells/mm² at 1 month and(2704±150.1)cells/mm² at 3 months]between different time points(F=1.380,1.011;P=0.259,0.351).Conclusions Confocal microscopy is able to clearly document the early morphological characteristics after collagen cross-linking in the treatment of keratoconus,including the epithelial and subepithelial nerve injury repair,the spongiform superficial corneal stroma,the patchy or cord-like deep corneal stroma,and the relatively stable stromal depth change.