The Evaluation of Endothelial Damage Following Corneal Storage: A Comparison of Staining Methods and the Value of Scanning Electron Microscopy

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Abstract
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.