长波紫外线照射小鼠角膜构建Fuchs角膜内皮营养不良疾病模型的可行性研究

Feasibility of constructing Fuchs endothelial corneal dystrophy model in mice by ultraviolet A irradiation

背景Fuchs角膜内皮营养不良(Fuchs endothelial corneal dystrophy,FECD)是一种常见的角膜内皮疾病,其具体发病机制尚不明确,临床治疗效果不佳。目的研究长波紫外线(ultraviolet A,UVA)照射小鼠角膜构建迟发型FECD动物模型的可靠性和稳定性。方法取雌性和雄性小鼠各36只,使用波长为365 nm的紫外光照射小鼠右眼角膜作为UVA组,左眼不做处理作为对照组。于建模后1周、2周、4周进行观察并采集样本,分别采用免疫荧光染色、苏木精-伊红(hematoxylin-eosin,HE)染色和透射电子显微镜进行观察,每组4个样本。裂隙灯显微镜观察角膜透明度、前节OCT观察角膜厚度、活体共聚焦显微镜观察角膜内皮细胞数量及形态。结果裂隙灯照相及前节OCT显示UVA组较对照组小鼠角膜水肿混浊、增厚(P<0.05);活体共聚焦显微镜结果显示小鼠角膜内皮细胞扩大变形伴数量减少(P<0.05),此外在第4周时观察到赘生物“Guttae”的形成,这是FECD的显著病理特征之一;免疫荧光染色(ZO-1/DAPI)结果显示小鼠角膜内皮细胞扩大变形且胞间紧密连接程度下降,DAPI染色细胞核计数下降(P<0.05);HE染色可见UVA照射后小鼠角膜基质胶原纤维疏松水肿伴间隙增大、角膜增厚,内皮细胞受损;透射电子显微镜显示UVA组小鼠角膜内皮细胞中线粒体结构遭到破坏,后弹力层较对照组显著增厚(P<0.05)。结论本研究证实了UVA照射小鼠角膜诱导迟发型FECD动物模型的可行性;该模型呈现的角膜后弹力层增厚和内皮细胞损伤等特征与FECD病理特征相吻合。

Background Fuchs endothelial corneal dystrophy(FECD)is a common corneal endothelial disease.Its specific pathogenesis is still unclear,which limits the effect of clinical treatment.Objective To explore the reliability and stability of establishing a delayed onset FECD animal model by ultraviolet A(UVA)irradiation of mouse cornea.Methods A total of 36 female and 36 male mice were selected,with the right eyes exposed to 365 nm ultraviolet light to irradiate the cornea as the UVA group,while the left eyes served as the control group.Observations and sample collection were conducted at 1 week,2 weeks,and 4 weeks post-modeling.The samples were used for immunofluorescence staining,hematoxylin-eosin(HE)staining,and transmission electron microscopy,with four samples in each group.The observation methods included the use of a slit-lamp microscope to assess corneal transparency,anterior segment optical coherence tomography(OCT)to measure corneal thickness,and in vivo confocal microscopy to examine the number and morphology of corneal endothelial cells.Results Slit lamp photography and anterior segment OCT showed that the cornea of the mice in the UVA group was edematous,cloudy,and thickened(P<0.05).In vivo confocal microscopy results showed that the corneal endothelial cells of the mice were expanded,deformed,and decreased in number(P<0.05).In addition,the formation of vegetation“Guttae”was observed at the 4th week,which was one of the significant pathological features of FECD.The immunofluorescence staining(ZO-1/DAPI)results showed that mouse corneal endothelial cells were enlarged and deformed,and the junctions between cells were loosening.The decrease in the nuclear count of DAPI staining was significant(P<0.05);HE staining revealed that the corneal stroma collagen fibers of the UVA group were loose and edematous with enlarged gaps and corneal thickening,and endothelial cells were damaged.Transmission electron microscopy showed that the mitochondrial structure in the corneal endothelial cells of the UVA group was damaged,and the Descemet's membrane was thicker than that of the control group(P<0.05).Conclusion This study confirms the feasibility of the animal model of late-onset FECD induced by UVA irradiation of mouse cornea,and the characteristics of corneal Descemet layer thickening and endothelial cell damage presented by this model are consistent with the pathological characteristics of FECDs.