Objective To analyze the morphology on the ocular surface of severe alkali burns patients by in vivo laser scanning confocal microscopy.Methods This research was a retrospective observation case series.From February to November 2008 in Eye Ear Nose and Throat Hospital of Fudan University, 39 alkali burns patients who classified as Ⅲ or IV according to Roper-Hall classification were enrolled in this study.They were divided into four groups according to the course of disease: A (less than 3 months) , B (3-6 months) , C (6-12 months) and D (over 12 months).In vivo laser scanning confocal microscopic examinations were performed on the injured cornea, the limbus and the bulbar conjunctiva and the images were recorded.The morphology of the injured cornea, the limbus and the bulbar conjunctiva was analyzed and the densities of the inflammatory cells and dendritic cells in the limbus were calculated.Oneway analysis of variance was used to compare the means of the inflammatory cells and dendritic cells.Subsequently the data between two groups were analyzed by least significant difference.Results The corneal epitheliums of the patients in Group A manifested large irregular features with hyperreflective cytoplasm and hyporeflective nuclei, sometimes losing cell features.There were numerous small hyperreflective inflammatory cells in groups beneath the superficial epitheliums.Shallow corneal stroma was edema, and it was hard to discriminate the morphology of the stromal cells.Deep stromal cells were in the activated state.The images of the endothelial layer were unclear.In Croup B and Group C, there were the same manifestation of the superficial epitheliums as the group A and it disappeared in Group D.The inflammatory cells beneath the superficial epitheliums reduced and some residual basal epitheliums and hyperreflective conjunctiva-like epitheliums were visible in Group B and Group C.In Group D, there were small oval tight-arranged cells with punctiform hyperreflective nuclei instead of normal comeal basal epitheliums.In Group B, it was still hard to discriminate the morphology of the shallow stroma cells.Deep stromal cells were still in the activated state.In Group C and Group D, comeal stroma was replaced by the fibrous tissues.The images of the endothelial layer were still unclear in the other groups.The Vogt palisades in the limbus of the severe alkali bums patients were destroyed in all groups.There were rich vascular nets in the limbus.The densities of the limbal inflammatory cells in four groups were (4023 ± 343 ) , (2975 ± 246), (2652 ± 375), (2679±299) cells/mm~2, respectively.Significant difference in inflammatory cell density was found among groups (F= 40.001, P = 0.000).The densities of the limbal dendritic cells in four groups were (106±19),(132±35) , (141±26), (98±24) cells/mm~2, respectively.Significant difference in dendritic cell density was found among groups (F=8.053 ,P=0.000).When the injured area of the conjunctiva was limited, it was hard to discriminate the morphology of the conjunctival epitheliums in both Group A and Group B.Numerous inflammatory cells infiltrated in the conjunctival lamina propria and goblet cells were invisible.In Group C and Group D, the conjunctival epitheliums were almost normal.There were stall some inflammatory cells and dendritic cells in the conjunctival lamina propria, and there were residual goblet cells visible in parts of the patients.When the injured area of the conjunctiva was large, the conjunctivas in four groups displayed hyperreflective stripy fibrous tissues instead of normal conjunctival epitheliums.Conclusions The application of laser scanning confocal microscopy indicates that there is much difference on the cellular morphology of the ocular surface of severe alkali bums patients among diverse courses of the disease.The technique is a useful tool to the observation on the ocular surface of severe alkali burns patients.%目的 探讨利用激光共焦显微镜观察严重碱烧伤患者眼表的形态结构.方法 系列病例研究.于2008年2月至11月期间在复旦大学附属眼耳鼻喉科医院就诊的眼表碱烧伤患者中,选择39例Roper-Hall分度为Ⅲ度或Ⅳ度的患者,根据眼部受伤时间分组,A组(9例,12只眼)为受伤3个月以内,B组(8例,11只眼)为受伤3～6个月,C组(11例,14只眼)为受伤6～12个月,D组(11例,14只眼)为受伤12个月以上.使用激光共焦显微镜对其受伤角膜、角膜缘以及球结膜进行检查,各层图像均被记录,分析角膜各层细胞形态,角膜缘栅栏结构形态,角膜缘炎性细胞与树突状细胞密度,以及球结膜各层细胞形态.运用单因素方差分析对角膜缘炎性细胞与树突状细胞密度进行统计学分析,运用最小显著性差异分析组间差异.结果 A组的碱烧伤患者角膜上皮细胞体积增大,形态不一,细胞质高反光,细胞核低反光,有时可见部分细胞缺失;表层细胞下见大量高亮小圆的炎性细胞浸润,聚集成团;浅基质层水肿明显,难以分辨细胞形态;深基质层细胞呈激活态;内皮层模糊不清.随着病程的延长,表层上皮细胞仍表现为上述异常结构,D组碱烧伤患者中,此类异常细胞消失;表层细胞下炎性细胞减少,可见部分残存的角膜上皮基底层细胞,存在散在或成团分布的中高亮反光的结模样细胞生长,随着病程的延长,角膜上皮基底层细胞大面积消失,代之以卵圆形,体积较小,排列紧密,细胞核呈点状高反光的结模样细胞;B组碱烧伤患者浅基质层仍难以分辨细胞形态,深基质层细胞呈激活态,C组和D组的碱烧伤患者中,角膜基质被纤维组织所替代;内皮层始终模糊不清.碱烧伤患者角膜缘Vogt栅栏结构始终被破坏;角膜缘血管网密集;A～D组角膜缘炎性细胞密度分别为(4023±343)、(2975±246)、(2652±375)、(2679±299)个/mm~2,经统计学分析差异有统计学意义(F=40.001,P=O.000);角膜缘树突状细胞密度分别为(106±19)、(132±35)、(141±26)、(98±24)个/mm~2,经统计学分析差异有统计学意义(F=8.053,P=0.000).碱烧伤患者的结膜损伤面积较小时,A组与B组碱烧伤患者结膜上皮细胞形态较难分辨,结膜固有层可见大量炎性细胞浸润,杯状细胞难以见到;C组和D组的碱烧伤患者中,结膜上皮形态基本正常,结膜固有层仍有部分炎性细胞与树突状细胞浸润,部分患者可见残存的杯状细胞.而当结膜受到大面积损伤时,各组碱烧伤患者的结膜均表现为上皮大面积缺失,代之以条索状高反光的纤维组织.结论 在激光共焦显微镜下,不同病程的严重碱烧伤患者的眼表组织细胞形态存在一定差异,激光共焦显微镜是观察严重碱烧伤患者眼表形态的有效工具.
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