Shrinkage and distortion of the rabbit corneal endothelial cell mosaic caused by a high osmolality glutaraldehyde-formaldehyde fixative compared to glutaraldehyde

The purpose of this study was to quantitatively compare cell dimensions and cell layer organization of the corneal endothelium after chemical fixation. Rabbit corneas (9-10 weeks of age) were prepared immediately postmortem for transmission electron microscopy (TEM) and scanning electron microscopy (SEM) using either a widely used high osmolality fixative (glutaraldehyde-formaldehyde, after Karnovsky; 1% formaldehyde, 2.5% glutaraldehyde, 0.1 M cacodylate, pH 7.6, 850 mOsm/kg) or a glutaraldehyde fixative (2% glutaraldehyde in 80 mM cacodylate, pH 7.4, 330 mOsm/kg). With the glutaraldehyde-formaldehyde fixative, TEM revealed gross shrinkage (up to 40%) and distortion of the cytoplasm and organelles, while the regions of the cell-cell junctions were not attenuated but included dilated extracellular space. With the glutaraldehyde fixative, TEM also revealed shrinkage but the cytoplasm was less compact than with the high osmolality fixative. The overall cell shrinkage and relative accentuation of the cell-cell borders was confirmed by SEM, which also revealed that the 11 to 20% area shrinkage was also related to the number of cell sides.