Corneal epithelial proliferation and thickness in a mouse model of dry eye

Abstract

Although several studies have previously focused on the conjunctival epithelial response to surface dryness, little is known about the effect of a dry environment on corneal epithelium, which is the most clinically significant tissue affected in dry eye. The aim of this study was to quantitatively evaluate the effect of desiccating stress on the number of proliferating corneal epithelial cells and corneal epithelial thickness in mice placed in a controlled-environment chamber (CEC) that induces dry eye. Corneal epithelial cell proliferation and thickness were studied in 8- to 12-week-old female BALB/c mice placed in the CEC (temperature: 22.3+/-0.7 degrees C; relative humidity: 22.5+/-4.5%; airflow: 15 L/min) for 7 days and compared to a control group of mice with no dry eye. Actively proliferating cells were identified by immunofluorescence using a FITC-conjugated antibody against the Ki-67 protein, a cell proliferation marker expressed during active phases of the cell cycle. To detect the spatial distribution of proliferative cells, Ki-67(+) cells were counted in three areas of the epithelium: center, periphery, and limbus. Corneal epithelial thickness was evaluated in the central cornea after staining with hematoxylin-eosin. Results from each experimental group were compared using the Mann-Whitney test. The number of Ki-67(+) cells observed in the corneal epithelium of mice exposed to the CEC was significantly higher in each area (center: 32.1+/-1.1; periphery: 94.2+/-5.3; limbus: 4.0+/-1.5) than in the control group (center: 13.2+/-1.0, p=0.02; periphery: 42.9+/-2.3, p=0.02; limbus: 0.0, p=0.01). In mice subjected to desiccating stress, a significant number of Ki-67(+) positive cells were detected in the basal and suprabasal cell layers (central area 46%; periphery 30.8%: limbus 0%), whereas in the control group the cells were exclusively distributed through the basal cell layer. Ki-67(+) cells were not found in the corneal stroma or endothelium in any group. The corneal epithelium was found to be significantly thicker in dry eye mice (54.94+/-6.09 microm) as compared to the controls (43.9+/-6.23 microm, p<0.0001) by a mean of 25%. These results demonstrate that desiccating stress increases corneal epithelial turnover and thickness, similar to what is observed in other chronic inflammatory states of other epithelialized surfaces. The CEC can facilitate the study of the regulation of epithelial cell function and turnover at the molecular and cellular levels under desiccating stress conditions.

Figures

Figure 1

Immunofluorescence staining of central area of normal (top) and dry (bottom) eyes. Actively proliferating cells were identified by immunofluorescence using a FITC-conjugated antibody against the Ki-67 protein (green); coverslips were mounted with DAPI to mark the nuclei of all cells (red); photographs were taken and merged to recognize the cycling cells (orange). Ki-67+ cells were not found in the corneal stroma or endothelium in any group. All photographs were taken at the same magnification.

Figure 2

Proliferating cells in central, peripheral and limbal corneal epithelium in normal (left) and dry (right) eye mice. The number of Ki-67+ cells was significantly higher in each individual area (center; periphery; limbus), as compared to the control group. The peripheral epithelium had the highest degree of labelling in both groups. Ki-67+ cells were found in the limbus of dry eye corneas, but not in the normal epithelium. Data show median ± interquartile; Mann-Whitney test.

Figure 3

Ki-67+ cells distribution among the corneal epithelium in mice subjected to desiccating stress (green). A significant number of Ki-67+ cells (34.2%) were detected in the suprabasal cell layers of the central and peripheral corneal epithelium (white arrows). No suprabasal cell was detected in the limbus of dry eye mice.

Figure 4

Corneal epithelial thickness: comparison between normal and dry eye mice. A. Central corneal sections of 3 μm were stained with hematoxylin-eosin (H&E). Normal control cornea had a stratified epithelium of 4 to 6 cell layers and an avascular stroma with cells aligned in a lamellar manner. After 7 days of exposure to the CEC epithelial stratification was marked in the central regions. Besides marked stratification, the most striking finding was the increased cellularity and the general hyperplastic appearance of cells, evidenced by the cuboidal shape of postmitotic suprabasal cells (photographs were taken at the same magnification). B. Measurement of the central part of the corneal epithelium revealed that the epithelium was significantly thicker in eyes of dry eye mice as compared to the controls. Data show median ± interquartile; Mann-Whitney test.