title: Isolation and cultivation of canine corneal cells for in vitro studies on the anti-inflammatory effects of dexamethasone.
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.
  
  
  METHODS
  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 E(2) (PGE(2)). The concentration of PGE(2) 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 PGE(2)-production in epithelial cells and fibroblasts, which was significant for epithelial cells. The PGE(2)-concentration was decreased in a dose dependent manner by the addition of dexamethasone.
  
  
  CONCLUSIONS
  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 PGE(2)-production following dexamethasone treatment.
authors.affiliation: Institute of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany. Anke.Werner@tihohannover.de
authors.forename: Anke
authors.initials: A
authors.lastname: Werner
authors.forename: Michael
authors.initials: M
authors.lastname: Braun
authors.forename: Manfred
authors.initials: M
authors.lastname: Kietzmann
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D015153:Blotting, Western / Q000662:veterinary; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology* / Q000187:drug effects*; D003907:Dexamethasone / Q000494:pharmacology*; D015232:Dinoprostone / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects; D015353:Eye Enucleation; D005455:Fluorescent Antibody Technique / Q000662:veterinary; D008070:Lipopolysaccharides / Q000494:pharmacology; D008858:Microscopy, Phase-Contrast / Q000662:veterinary; D011965:Receptors, Glucocorticoid; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D015232:Dinoprostone