Ikebe H

Age-dependent changes in nuclear DNA content and cell size of presumably normal human corneal endothelium

In order to investigate a relationship between cellular DNA-content and enlargement in cell size of human corneal endothelium in healthy population, both nuclear DNA-cytofluorometry and cell morphometry were performed on individual cells of presumably normal corneas taken from six autopsy cases. The cornea was treated in 0.2 M phosphate buffer containing 60 mM Na2EDTA and fixed with absolute methanol. The endothelial-cell layer was separated from Descemets membrane by placing the tissue in distilled water. The specimen was marked with rhodamine-labelled wheatgerm agglutinin (WGA)-lectin for identifying cell boundaries and subsequently stained with DAPI for measurement of DNA contents. The area of each cell was measured by means of a color image analyser and compared with its cytofluorometric nuclear DNA content. The enlarged endothelial cells which appeared with age contained diploid, polyploid or multiple nuclei. The polyploid and multinucleated cells had larger mean and maximum cell sizes and more irregular and polymorphous shapes than diploid cells. There were neither small polyploid nor small multinucleated cells. It was suggested that the loss of human corneal endothelium was compensated in vivo by cell size enlargement with or without doubling of nuclear DNA.

Cytofluorometric nuclear DNA determinations on human corneal endothelial cells

In order to investigate polyploidization of human corneal endothelium, nuclear DNA cytofluorometry was performed. Touch smears of 31 human corneal endothelium taken from 21 autopsies and 10 donor eyes were fixed with absolute methanol and subsequently stained by the pararosaniline-Feulgen nuclear reaction. The optimum condition for Feulgen hydrolysis of the specimens was determined, and all smears were hydrolysed in 1 N HCl for 5 min at 60 degrees C. After the Feulgen reaction, the pararosaniline-DNA fluorescence was measured with an epi-illumination cytofluorometer (Olympus AH-QRFL). Polyploid nuclei, which are the result of abortive cell division after DNA synthesis, appeared with DNA contents of tetraploid (19 cases) and octaploid (two cases) classes. All other nuclei had DNA quantities within the range of the diploid class. Nuclei containing DNA between diploid and tetraploid amounts were not found. It was concluded that most human corneal endothelial cells are of post mitotic Gl population but that a few are in the generative G0 phase.