激光共聚焦显微镜观察春季角结膜炎患者的角膜形态变化

Morphological characteristics of cornea in patients with vernal keratoconjunctivitis by in vivo laser scanning confocal microscopy

目的探讨应用活体激光共聚焦显微镜观察春季角结膜炎（VKC）患者的角膜形态变化。方法观察型系列病例研究。选择2008年10月至2009年8月在复旦大学附属眼耳鼻喉科医院眼科就诊的26例双眼VKC患者，其中眼睑型13例、角膜缘型5例、混合型8例；另选择26名年龄、性别匹配的正常志愿者作为对照。使用活体激光共聚焦显微镜对患者右眼角膜进行检查，分别取中央角膜和上方周边部角膜为检查点，对所得图像进行记录和分析。利用内置细胞计数软件计算角膜各层组织的细胞密度，ImageJ软件分析神经密度、直径、分支数量及弯曲度。使用独立t检验比较VKC患者与正常人群角膜各层细胞密度的差异以及VKC患者周边与中央角膜细胞密度的差异；Fisher精确卡方检验比较VKC患者和对照组角膜上皮内郎格罕细胞浸润情况差异；方差分析比较VKC3种亚型之间和不同病程之间各层细胞密度的差异；独立t检验和卡方检验分析VKC患者与正常人群之间的神经差异。结果VKC患者的角膜形态学变化包括角膜上皮最表层的高亮多角形细胞缺失、上皮内和上皮下大量郎格罕细胞浸润及浅基质层内大量基质细胞活化。共焦显微镜下，角膜出现新生血管翳的患者表现为角膜上皮结膜化，基质内可见新生血管；合并圆锥角膜的患者，深基质内可见大量粗大的斜形或纵形暗纹。正常对照组的中央和周边部角膜上皮细胞密度分别为（6033．1±998．7）个／mm^2和（6098．4±298．3）个／mm^2，VKC患者分别为（5972．2±1148．2）个/mm^2和（6178．5±318．9）个／mm^2，差异无统计学意义（t=1．191，1．011；P=0．238，0．318）；但VKC患者周边部上皮细胞密度显著高于中央部（t=2．249，P=0．03）。正常对照组的中央和周边浅基质细胞密度分别（1001．4±125．3）个／mm。和（924．6±201．4）个／mm^2，VKC患者分别为（1184．5±115．3）个／mm^2和（1101．4±151．1）个／mm^2，均显著高于正常对照（t=6．617，3．439；均P=0．001）。正常对照与VKC患者中央角膜深基质层细胞密度分别为（537．7±42．6）个／mm^2和（548．7±79．8）个／mm^2，内皮细胞层细胞密度分别为（2985．7±401．2）个／mm^2和（3021．5±383．3）个／mm^2，两者比较均无明显差异（t=0．174，1．112；P=0．864，0．282）。61．5％0（16例）VKC患者的角膜上皮内发现郎格罕细胞浸润，显著高于正常人群（2例，7．7％）（X^2=12．49，P=0．001）。3种不同亚型VKC患者中，角膜缘型和混合型患者上皮内郎格罕细胞浸润情况较为严重。与正常对照组相比，VKC患者的上皮下神经密度和直径下降、弯曲度增加。结论VKC主要累及角膜上皮层、上皮下神经及浅基质层。共焦显微镜对于VKC的分型诊断有一定辅助价值。

Objective To explore the morphological characteristics on cornea in patients with vernal keratoconjunctivitis （VKC） by the application of in vivo laser scanning confocal microscopy （LSCM）. Methods The experimental design was retrospective observation case series （case control study）. Twenty-six patients, each diagnosed as bilateral VKC, were enrolled in the study, among which 13 were tarsal form, 5 were bulbar form and the rest were mixed form. Nine patients had the clinical course less than one year, eight subjects longer than three years, and the rest between them. Another twenty-six healthy volunteers with matching age and gender were selected as normal control. All participants had their right eyes examined with the in vivo confocal microscopy （ HRT II/RCM）. Central cornea and superior peripheral cornea were chosen as the examination points. The images were recorded automatically and cellular density of each layer was analyzed by installed software. Software ImageJ was utilized to analyze the density, diameter, branch number and tortuosity of subbasal nerve fiber in VKC patients. Independent t test was performed to assess the differences on cellular density between VKC patients and normal control, as well as those between central and peripheral cornea in VKC patients. Fisher chi-square test was used to compare the infiltration rate of Langerhans cells in corneal epithelium between VKC patients and controls. ANOVA was applied to assess the differences in cellular density among three subtypes, as well as among different duration of VKC. Independent t-test and chi-square test were applied to analyze the parameters of subbasal nerve fiber. Results The morphological changes in cornea included the absence of superficial hyperreflective polygonal epithelial cells, infiltration of Langerhans cells in and（or） underneath corneal epithelium and activation of keratocytes in anterior stroma. Corneal epithelium conjunctivalization and stromal neovascularization could be identified in patients with corneal neovascular epithelium. Longitudinal or oblique dark striae could be found in the posterior stroma in patients with complicated keratoconus. The density of epithelial cells at central and peripheral cornea in healthy controls were （ 6033.1 ＋ 998.7 ） cells/mm^2 and （ 6098.4 ＋ 298.3 ） cells/mm^2, while that in VKC patients were （ 5972. 2 ＋ 1148.2） cells/mm^2 and （ 6178.5 ＋ 318.9 ） cells/mm^2 respectively, the differences being no statistical significant between them（t = 1. 191,1. 011 ;P = 0. 238, 0. 318）. However, it＇s found in VKC patients that cellular density at peripheral cornea was significantly higher than that at central area（ t = 2. 249, P = 0. 03 ）. The density of anterior stromal ceils at central and peripheral cornea in healthy controls was （ 1001.4 ＋ 125.3 ） cells/mm^2 and （ 924. 6 ＋ 201. 4 ） cells/mm^2, while that in VKC patients was （1184. 5 ＋ 115.3） cells/mm^2 and （1101.4 ＋ 151.1 ） cells/mm^2, the difference bearing no statistical significance（ t = 6. 617,3.439 ;P = 0. 001 ）. The density of posterior stromal cells in normal subjects and VKC patients was （ 537.7 ＋ 42.6 ） cells/mm^2 and （ 548.7 ＋ 79.8 ） cells/mm^2, that of endothelial cells was （ 2985.7 ＋ 401.2 ） cells/mm^2 and （ 3021.5 ＋ 383.3 ） cells/mm^2, respectively, neither difference had statistical significance （t = 0. 174, 1. 112; P = 0. 864,0. 282 ） . Langerhans cell infiltration could be identified in 61.5% （ 16 cases ） VKC patients, which was significantly higher than normal control （2 cases, 7.7% ） （X^2 = 12.49, P =0. 001 ）. Furthermore, much intense Langerhans cells infiltration was found in bulbar form and mix form than tarsal form. （ t = 6. 617, P = 0. 001 ）. The density and diameter of subbasal nerve fiber in VKC patients decreased significantly than those in normal subjects, whereas the tortuosity increased significantly. Conclusions The morphological changes of cornea in VKC patients mainly involve corneal epithelium, subbasal nerve fiber and anterior stroma. In vivo LSCM is helpful in discriminating the subtypes of VKC.