PURPOSE: To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison. METHODS: The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air-liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically. Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E(2) (PGE(2)) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry. RESULTS: A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE(2) production, which could be reduced by dexamethasone. CONCLUSIONS: The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.
PURPOSE: To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison. METHODS: The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air-liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically. Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E(2) (PGE(2)) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry. RESULTS: A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE(2) production, which could be reduced by dexamethasone. CONCLUSIONS: The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.