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Isolation and cultivation of canine corneal cells for in vitro studies on the anti-inflammatory effects of dexamethasone

Authors


Address communications to: Anke Werner Tel.: +49 511 953 8737 Fax: +49 511 953 8731 e-mail: Anke.Werner@tiho-hannover.de

Abstract

Objective  This study was conducted to establish a protocol for the isolation and culture of canine corneal cells (i.e. endothelium, keratocytes, and epithelium) to be used for in vitro studies on the effects of dexamethasone in corneal inflammation.

Animal material  Corneal endothelial cells, epithelial cells and keratocytes from enucleated eyes of dogs (euthanized not related to this study) were isolated and cultured.

Procedure  Canine corneal cells were isolated using a combined enzymatic and mechanical technique and separately taken into culture. The three different cell types were verified by phase contrast microscopy, immunofluorescence, and Western blot using antivimentin and anticytokeratin antibodies. The mRNA for the glucocorticoid receptor (GR) was detected using reverse transcriptase polymerase chain reaction (RT-PCR).

To study dexamethasone effects, primary cells were stimulated with lipopolysaccharides (LPS) to induce production of inflammatory mediators, particularly prostaglandin E2 (PGE2). The concentration of PGE2 in cell culture supernatant was determined utilizing an ELISA assay. Results were compared between control, stimulated as well as stimulated and dexamethasone treated cells.

Results  A protocol for the isolation and culture of canine corneal endothelium, keratocytes, and epithelium was successfully established. Using morphological criteria as well as immunocytochemistry and Western blotting the identity of the cells could be verified. RT-PCR of the primary cells showed mRNA for the GR in all three cell types of the canine cornea. Furthermore, stimulation with LPS led to an increased PGE2-production in epithelial cells and fibroblasts, which was significant for epithelial cells. The PGE2-concentration was decreased in a dose dependent manner by the addition of dexamethasone.

Conclusion  The three major cell types of the canine cornea (i.e. endothelium, keratocytes, and epithelium) can be isolated and cultured in vitro. The mRNA for the GR is shown in all three cell types, its functionality is demonstrated by the dose dependent reduction of PGE2-production following dexamethasone treatment.

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