title: Primary mixed glial cultures from fetal ovine forebrain are a valid model of inflammation-mediated white matter injury.
abstract: Astrocytes, microglial cells and oligodendrocytes (OLs) have been employed separately in vitro to assess cellular pathways following a variety of stimuli. Mixed glial cell cultures, however, have not been utilized to the same extent, despite the observed discrepancy in outcomes resulting from cell-to-cell contact of different glia in culture. Our objective was to standardize and morphologically characterize a primary culture of preterm ovine glial cells in order to attain a relevant in vitro model to assess the intracellular effects of infection and inflammation. This would provide a high-throughput model necessary for in-depth studies on the various pathophysiological mechanisms of white matter injury (WMI), which may occur in the preterm infant as a consequence of maternal infection or the fetal inflammatory response. Glial cells from the forebrains of 0.65-gestation ovine fetuses (comparable to 24- to 26-week human fetal brain development) were mechanically and enzymatically isolated and plated at a final density of 250,000 cells per well. When reaching confluence at 5 days after plating, the cultures contained astrocytes, microglial cells, as well as progenitor, precursor and immature OLs. Glial cell morphology and phenotypic immunoreactivity were characteristic of and consistent with previous observations of separately cultured cell types. To determine the effects of infection or inflammation in our in vitro model, we then treated mixed glial cultures with tumour necrosis factor-α (TNF-α; 50 or 100 ng/ml) or lipopolysaccharide (LPS; 1 µg/ml) for a period of 48 h. Cytokine levels were measured by ELISA and cell numbers for specific glial cell types were determined along with OL proliferation and apoptosis by Ki67 and caspase-3 immunocytochemistry, respectively. Our results showed that exposure to TNF-α or LPS resulted in a characteristic inflammatory response entailed by up-regulation of pro-inflammatory cytokines, a lack of astrogliosis and a marked reduction in OLs attributable to increased apoptosis. In LPS-treated cultures, there was a marked increase in the pro-inflammatory cytokine TNF-α at both 24 and 48 h. In conclusion, this is the first report of the immunocytochemical description and characterization of fetal ovine-derived mixed glial cell primary cultures. This in vitro model provides a novel and efficient system to explore the mechanisms of infection/inflammation-mediated WMI at the cellular level and for screening candidate therapeutic strategies.
authors.affiliation: The Liggins Institute, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Luke
authors.initials: L
authors.lastname: Weaver-Mikaere
authors.forename: Hannah M
authors.initials: HM
authors.lastname: Gibbons
authors.forename: Devaki
authors.initials: D
authors.lastname: De Silva
authors.forename: Mhoyra
authors.initials: M
authors.lastname: Fraser
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D053148:Caspase 3 / Q000032:analysis / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000473:pathology; D016207:Cytokines / Q000032:analysis / Q000378:metabolism; D004195:Disease Models, Animal; D004660:Encephalitis / Q000150:complications* / Q000473:pathology; D005260:Female; D005333:Fetus; D006801:Humans; D007239:Infections / Q000378:metabolism; D019394:Ki-67 Antigen / Q000032:analysis / Q000378:metabolism; D056784:Leukoencephalopathies / Q000209:etiology* / Q000378:metabolism / Q000473:pathology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008954:Models, Biological; D009457:Neuroglia / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D009836:Oligodendroglia / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D011247:Pregnancy; D016548:Prosencephalon / Q000473:pathology*; D034561:Sheep, Domestic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016207:Cytokines; D019394:Ki-67 Antigen; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D053148:Caspase 3