Abstract
Purpose: :
To evaluate quantitatively 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.
Methods: :
Cell proliferation and thickness of the corneal epithelium were studied in 8–9 week old female Balb/C mice placed in a CEC (temperature: 22.3±0.7ºC; relative humidity: 22.5±4.5%; airflow: 15 L/min) for 7 days and compared to a control group, each experimental group consisting of 4 animals (4 eyes). Actively proliferating cells were identified by immunofluorescence using a FITC–conjugated antibody against the Ki–67 protein, a cell proliferation marker expressed during all active phases of the cell cycle. To detect spatial alterations in cell proliferation, Ki–67 positive cells were counted in three areas of the epithelium: (i) center, (ii) periphery, and (iii) limbus. Corneal epithelial thickness was evaluated in the central cornea after staining with hematoxylin–eosin. Results from each experimental group were compared using the Student–t test.
Results: :
The number of Ki–67 positive cells observed in the corneal epithelium of mice exposed to the CEC was in each area significantly higher (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<0.0001; periphery: 42.9±2.3, p<0.0001; limbus: 0.0, p<0.002). In mice subjected to desiccating stress, a significant number of Ki–67 positive cells were detected at the basal and suprabasal cell layers, whereas in the control group, cells were exclusively distributed through the basal cell layer. Ki–67 positive cells were not found in the corneal stroma or endothelium in any group. The corneal epithelium was significantly thicker in dry eye mice (44.2±0.4 µm) as compared to the control group (51.2±1.5 µm, p<0.0001).
Conclusions: :
These results show that desiccating stress in a mouse model of dry eye induces epithelial turnover and increased thickness of the corneal epithelium similar to what is observed in chronic inflammatory states of other epithelialized surfaces. The controlled–environment chamber should facilitate the study of the regulation of epithelial cell function at the molecular and cellular levels under stress conditions.
Keywords: cornea: tears/tear film/dry eye • cornea: epithelium • proliferation