Quantitative assessment of anteroposterior keratocyte density in the normal rabbit cornea

The anteroposterior keratocyte density distribution in the rabbit cornea was measured. Unsectioned tissue blocks from the central cornea of five rabbits were stained with propidium iodide and imaged using a Leica laser scanning confocal microscope. A z-series of images was acquired in each sample, from anterior to posterior stroma in either 3- or 8-μm steps. Software was developed to allow interactive marking of the keratocyte nuclei within each section of the z-series and for calculating cell density. For convenience, cell density was expressed as the number of cells per corneal volume element (CVE), where CVE is a newly defined volume unit with x, y, and z dimensions of 250, 250, and 10 μm, respectively. The calculated keratocyte density was 20.2 ± 1.0 cells/CVE (n = 5), which is equivalent to 32,360 ± 1,660 cells/mm³. The greatest density was underneath the epithelium (26.3 ± 2.5 cells/ CVE), the density then decreased linearly with depth to 15.2 ± 1.4 cells/CVE; there was a slight increase in density pre-Descemets membrane to 18.5 ± 3.5 cells/CVE. A 30% decrease in cell density over the entire anteroposterior stromal thickness was observed. To facilitate statistical analysis, the cell density was averaged over 5% thickness intervals from anterior to posterior cornea. A significant difference in mean cell density of these intervals was found (ANOVA, n = 20, p < 0.01). To further assess the density distribution, linear regression analysis was performed. A significant correlation was found between keratocyte density and stromal depth (R = - 0.94, n = 20, p < 0.05). We conclude that in the rabbit, keratocyte cell density is maximal underlying the epithelium and progressively decreases from anterior to posterior cornea.