The assessment of endothelial integrity by scanning electron microscopy and fluorescein diacetate staining following treatment with cryoprotective additives

Abstract

As part of the development of methods of corneal cryostorage for transplantation, a toxicity study was carried out on the rabbit corneal endothelium using four cryoprotective additives (CPA's) 1) dimethyl sulphoxide (Me2SO), 2) propane-1,2-diol (PG), 3) glycerol (GLY), 4) polyvinylpyrrolidone (PVP). A fifth group, based upon a CPA combination of Me2SO and PVP, was used to characterize both the assays, and the response of the endothelial layer to osmotic stress. The effect upon the cell membrane was assessed using scanning electron microscopy (SEM) and fluorescein diacetate with ethidium bromide staining (FDA/EB). Two sampling points were used, one immediately after treatment and the other following an incubation period. Calculations were performed to predict the maximum relative volume of cells during CPA exchange. Immediately following serial addition and removal of 2 or 3 mol/L (M) PG or GLY, the cells exhibited adverse morphological changes shown with SEM, and the proportion of intact cells judged by FDA/EB staining was significantly reduced when CPA equilibration was performed at 37 degrees C rather than at 20 degrees C. A 3M Me2SO concentration gave less morphological change than 3M PG or GLY, but even after treatment with 4M Me2SO more than 95% cells were judged intact by FDA/EB staining. PVP at 40% w/v showed minimal damage with both assays, and the fifth experimental group suggested that PVP may protect from injury during hypotonic stress. With all groups, the integrity of the cell layer recovered during incubation, so that for each sample the percentage of intact cells was high. However, although confluency was often restored following incubation, total cell density was usually reduced. The results indicate that serial addition and removal of 3M Me2SO is tolerated by the cornea, whereas PG or GLY cannot be used at 2 or 3M without inducing osmotic damage. There was low toxicity to PVP, and it was an effective osmotic buffer.