title: The review of the methods to obtain non-neuronal cells to study glial influence on Amyotrophic Lateral Sclerosis pathophysiology at molecular level in vitro.
abstract: OBJECTIVE
  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that displays a rapid evolution. Current treatments have failed to revert clinical symptoms because the mechanisms involved in the death of motoneuron are still unknown. Recent publications have put non-neuronal cells, particularly, astrocyte and microglia, in the scenario of pathophisiology of the disease. Animal models for ALS, particularly transgenic mice expressing the human SOD1 gene with a G93A mutation (hSOD1), are available and display the phenotype of the disease at cellular and clinical levels. However, it is a lack of detailed information regarding the methods to study the disease in vitro to better understand the contribution of non-neuronal cells in the onset and progression of the pathology.
  
  
  METHODS
  Colonies of Swiss mice and transgenic mice expressing hSOD1 mutation as well as non-transgenic controls (wild-type) were amplified after a genotyping evaluation. Disease progression was followed behaviorally and mortality was registered. Highly purified primary cultures of astrocytes and microglia from mouse spinal cord were obtained. Cells were identified by means of GFAP and CD11B immunocytochemistry. The purity of astroglial and microglial cell cultures was also accompanied by means of Western blot and RT-PCR analyses employing a number of markers.
  
  
  RESULTS
  The disease onset was about 105 days and the majority of transgenic mice displayed the disease symptoms by 125 days of age and reached the endpoint 20 days later. A substantial motor weakens was registered in the transgenic mice compared to wild-type at the end point. Immunocytochemical, biochemical and RT-PCR analyses demonstrated a highly purified primary cultures of spinal cord astrocytes and microglia.
  
  
  CONCLUSIONS
  It is possible to achieve highly purified primary cultures of spinal cord astrocytes and microglia to be employed in cellular and molecular analyses of the influence of such non-neuronal cells in the pathophysiology of ALS.
authors.affiliation: Department of Neurology, USP, São Paulo, Brazil.
authors.forename: Juliana Milani
authors.initials: JM
authors.lastname: Scorisa
authors.forename: Tatiana
authors.initials: T
authors.lastname: Duobles
authors.forename: Gabriela Pintar de
authors.initials: GP
authors.lastname: Oliveira
authors.forename: Jessica Ruivo
authors.initials: JR
authors.lastname: Maximino
authors.forename: Gerson
authors.initials: G
authors.lastname: Chadi
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000473:pathology* / Q000503:physiopathology; D000818:Animals; D001253:Astrocytes / Q000473:pathology*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D017628:Microglia / Q000473:pathology*; D009457:Neuroglia / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000166:cytology; D013482:Superoxide Dismutase / Q000235:genetics*; D000072105:Superoxide Dismutase-1
chemical_list: C000606290:SOD1 protein, human; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1