title: RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: A glial in vivo-in vitro multi-hit paradigm in large mammalian brain.
abstract: The chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood.
  
  
  
  Here we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures.
  
  
  
  We validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1β) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro.
  
  
  
  This method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain.
  
  
  
  The presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro.
authors.affiliation: Animal Reproduction Research Centre (CRRA), Faculty of Veterinary Medicine, Université de Montréal, Montréal, QC, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Cortes
authors.affiliation: Department of Obstetrics and Gynaecology and Department of Neurosciences, CHU Ste.-Justine Research Centre, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Cao
authors.affiliation: Department of Obstetrics and Gynaecology and Department of Neurosciences, CHU Ste.-Justine Research Centre, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada.
authors.forename: H L
authors.initials: HL
authors.lastname: Liu
authors.affiliation: Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal, QC, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Burns
authors.affiliation: Neuroimmunology Unit, Montréal Neurological Institute, McGill University, Montréal, QC, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Moore
authors.affiliation: Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal, QC, Canada.
authors.forename: G
authors.initials: G
authors.lastname: Fecteau
authors.affiliation: Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal, QC, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Desrochers
authors.affiliation: Department of Pediatrics, CHU Ste.-Justine Research Centre, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada.
authors.forename: L B
authors.initials: LB
authors.lastname: Barreiro
authors.affiliation: Neuroimmunology Unit, Montréal Neurological Institute, McGill University, Montréal, QC, Canada.
authors.forename: J P
authors.initials: JP
authors.lastname: Antel
authors.affiliation: Animal Reproduction Research Centre (CRRA), Faculty of Veterinary Medicine, Université de Montréal, Montréal, QC, Canada; Department of Obstetrics and Gynaecology and Department of Neurosciences, CHU Ste.-Justine Research Centre, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada. Electronic address: mfrasch@uw.edu.
authors.forename: M G
authors.initials: MG
authors.lastname: Frasch
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001921:Brain / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods; D053583:Interleukin-1beta / Q000378:metabolism; D008070:Lipopolysaccharides; D017628:Microglia / Q000166:cytology / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods; D012756:Sheep; D059467:Transcriptome
chemical_list: D053583:Interleukin-1beta; D008070:Lipopolysaccharides
keywords: Astrocytes; Brain; Fetus; LPS; Microglia; Primary culture; RNAseq; in vitro; in vivo