The Keratocyte Network of Human Cornea: A Three-Dimensional Study Using Confocal Laser Scanning Fluorescence Microscopy

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Abstract
Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of ≤270 μm in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 ± 5.05%) and lowest in the central stroma (9.31 ± 1.09%). In the anterior stroma, volume density was found to be 10.19 ± 4.37%. Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease.