Madden P W
Department of Surgery, Cambridge University, UK.
Curr Eye Res. 1987 Dec;6(12):1441-51. doi: 10.3109/02713688709044508.
Staining techniques and scanning electron microscopy (SEM) were used to assess damage to the endothelial cells following corneal storage. The aim was to establish whether these assays are useful in the assessment of endothelial integrity following corneal storage. Four groups ensured a range of normal and damaged endothelial cells: 1) fresh; 2) stored for 7 days in a moist chamber; 3) stored by the cryopreservation method of Capella and Kaufman; 4) damaged by rapid freezing with a cryoprobe. Trypan blue (TB), nitroblue tetrazolium (NBT), acridine orange (AO), fluorescein diacetate (FDA) and ethidium bromide (EB), were used to stain the endothelial layer. SEM was carried out on duplicate samples. Staining with NBT resulted in low cell counts due to loss of cells. There was no significant difference between the extent of damage measured by TB, AO or FDA, but it was shown that staining with FDA and EB can distinguish between damaged and intact cells. Tissue stored for 7 days in a moist chamber had a reduced number of intact cells compared to fresh tissue, and tissue stored by the Capella and Kaufman technique gave a reduced number of intact cells compared to both these control and storage groups. SEM showed surface perforation was characteristic of injured cells, rather than complete disruption. FDA has a theoretical advantage over the other stains, and should provide a more accurate appraisal of defects of cell membrane integrity. For this reason, the use of FDA with EB to stain the endothelium, with SEM carried out on duplicate samples, were preferred as assays to use in the development of corneal storage.
采用染色技术和扫描电子显微镜（SEM）评估角膜储存后内皮细胞的损伤情况。目的是确定这些检测方法在评估角膜储存后内皮完整性方面是否有用。四组确保了一系列正常和受损的内皮细胞：1）新鲜的；2）在潮湿环境中储存7天；3）采用卡佩拉和考夫曼的冷冻保存方法储存；4）用冷冻探头快速冷冻损伤。使用台盼蓝（TB）、硝基蓝四氮唑（NBT）、吖啶橙（AO）、荧光素二乙酸酯（FDA）和溴化乙锭（EB）对内皮细胞层进行染色。对重复样本进行扫描电子显微镜检查。由于细胞丢失，NBT染色导致细胞计数较低。TB、AO或FDA测量的损伤程度之间没有显著差异，但结果表明，FDA和EB染色可以区分受损细胞和完整细胞。与新鲜组织相比，在潮湿环境中储存7天的组织中完整细胞数量减少，与这两个对照组和储存组相比，采用卡佩拉和考夫曼技术储存的组织中完整细胞数量减少。扫描电子显微镜显示，表面穿孔是受损细胞的特征，而非完全破坏。FDA在理论上优于其他染色剂，应该能更准确地评估细胞膜完整性缺陷。因此，在角膜储存研究中，首选使用FDA和EB对内皮细胞进行染色，并对重复样本进行扫描电子显微镜检查作为检测方法。
