Biomedical subjects
Publications and source records attributed to S Sperling.
Scanning electron microscopic observations of human corneal endothelium revealed a binding of ruthenium red-osmium tetroxide to a material on the inner surface of the endothelial cells on the cornea and the trabecular meshwork. The coating material was partially digested by proteolytic enzymes.
In 16 whole flat corneal mounts stained by alizarine red and trypane blue it was investigated whether estimates of central numerical endothelial cell density could be based on peripheral cell density, on age or on morphological cell characteristics. With certain precautions estimates of central cell density could be based on peripheral cell density, on age and on the occurrence of large central or peripheral cells. Estimates could not be based on the occurence of other cellular morphological characteristics.
Nineteen human cadaver corneas with few damaged endothelial cells were incubated under tissue culture conditions for time periods ranging from five min to 48 h. Morphological alterations of the endothelial cells were studied in whole wet mounts stained by alizarine red-alkohol-trypane blue and by scanning electron microscopy. Joint meetings of three cells are characteristic for normal corneal endothelium. After 15--60 min of incubation, damaged cells were expelled from the coherent cell sheet by expanding neighbouring cells. Joint meetings of 5--8 expanding cells were formed. After 24 h of incubation, joint meetings of four cells were the dominating morphological abnormality. Morphological changes during reduction of the numbers of cells in joint meetings are described.
The precision of estimates of central corneal endothelial density was studied in 16 human corneas stained by alizarine red and trypane blue. Estimates based on central counts and estimates based on peripheral counts were considered separately. The numerical density was estimated employing an unbiased sampling technique. From central counts estimates with an error of less than five per cent could be obtained. From peripheral counts a maximum precision of mean +/- 12.2 per cent could be obtained. The theoretical maximum precision was calculated by application of a variance component model. The precision of estimates was calculated for 1, 2 and 4 test areas of different sizes. Economy of sampling was evaluated by comparison of the actual precision to the theoretical maximum precision of estimates.
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Combined staining by alizarine red and trypane blue is an easy and reliable method for in vitro visualization of corneal endothelial nuclei, cellular borders and uncovered parts of the membrane of Descemet. A stained full thickness wet mount can be prepared in less than five min. The staining method and preliminary findings concerning endothelial morphology in the centre and in the periphery of normal corneas are presented.
The construction and operation of an apparatus for controlled rate corneal freezing is described. The cooling is obtained by close thermal contact between a vial holding the cornea and a metal core. A predetermined cooling rate is achieved by partial immersion of the core in a fixed amount of liquid nitrogen. In a preliminary series greater than or equal to 95% endothelial survival was obtained.
Electron microscopy revealed the presence of a 600-1500 A thick layer of polysaccharide on the surface of human corneal endothelial cells. The surface layer was visualized by combined fixation and staining in a mixture of ruthenium red and osmium tetroxide. The coating material was stable for at least 39 h post mortem and was retained on disintegrating cells.
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Corneal endothelial cells were visualized in intact bovine eyes by specular microscopy of unstained and stained cell borders. The corneas were excised, and flat corneal preparations were studied by reflected and transmitted light. In the excised corneas, the cell borders were visualized by osmotically induced dilation of intercellular spaces, alizarin red staining, and a combination of alizarin red and trypan blue staining. In whole eyes and in excised corneas, the estimates of endothelial cell densities varied by less than 3% from one method of visualization to the next. Estimates of endothelial cell densities obtained in intact eyes at 1 mm Hg were highly correlated to, but 13.6% lower than, estimates in excised corneas. Estimates of endothelial cell density obtained at intraocular pressures of 50 mm Hg were 7.7% lower than estimates obtained at 1 mm Hg.