title: Characterization of canine microglial cells isolated ex vivo.
abstract: Microglia are the principal immune effector elements of the brain sharing immunophenotypic and functional characteristics of macrophages as well as of antigen presenting cells (APCs). The purpose of this study was to isolate canine microglial cells and make them available for ex vivo characterizations of their functions and immunophenotype. After isolation, carried out by density gradient centrifugation, microglial cells accumulated on distinct interfaces of 1.077 and 1.066 g/ml of a Percoll gradient. Identification of microglial cells in other species is realized by their specific immunophenotype of CD11b/c+ and CD45low. Our results indicate, that expression of CD45 is very low or even absent in canine microglial cells. In addition, they expressed CD18 and CD11b/c+, as determined by flow cytometry and immunohistochemistry. Fourteen additional monoclonal antibodies (mAbs) were used to characterize and compare canine microglial cells with monocytes. Microglia and monocytes can be clearly distinguished by their differential expression intensity of surface antigens (CD45, CD44, CD14). Functional characterization was assessed by a reactive oxygen species (ROS)-generation test and phagocytosis assay using flow cytometry. In conclusion, ex vivo examination of microglia is possible in dogs and most probably reflects the conditions in vivo. The measurement of tissue culture artifacts can be largely avoided using this method.
authors.affiliation: Department of Small Animal Medicine and Surgery, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany. veronika.stein@tiho-hannover.de
authors.forename: Veronika M
authors.initials: VM
authors.lastname: Stein
authors.forename: Markus
authors.initials: M
authors.lastname: Czub
authors.forename: Regine
authors.initials: R
authors.lastname: Hansen
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Leibold
authors.forename: Peter F
authors.initials: PF
authors.lastname: Moore
authors.forename: Andreas
authors.initials: A
authors.lastname: Zurbriggen
authors.forename: Andrea
authors.initials: A
authors.lastname: Tipold
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000662:veterinary; D004285:Dogs / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D016130:Immunophenotyping / Q000662:veterinary; D066298:In Vitro Techniques; D008297:Male; D017628:Microglia / Q000276:immunology* / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology; D011205:Povidone; D017382:Reactive Oxygen Species / Q000276:immunology; D012822:Silicon Dioxide; D013047:Specific Pathogen-Free Organisms
chemical_list: D017382:Reactive Oxygen Species; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone