title: Expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and its roles in activated microglia in vivo and in vitro.
abstract: BACKGROUND
  We reported previously that amoeboid microglial cells in the postnatal rat brain expressed 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) both in vivo and in vitro; however, the functional role of CNPase in microglia has remained uncertain. This study extended the investigation to determine CNPase expression in activated microglia derived from cell culture and animal models of brain injury with the objective to clarify its putative functions.
  
  
  METHODS
  Three-day-old Wistar rats were given an intraperitoneal injection of lipopolysaccharide to induce microglial activation, and the rats were killed at different time points. Along with this, primary cultured microglial cells were subjected to lipopolysaccharide treatment, and expression of CNPase was analyzed by real-time reverse transcription PCR and immunofluorescence. Additionally, siRNA transfection was employed to downregulate CNPase in BV-2 cells. Following this, inducible nitric oxide synthase, IL-1β and TNF-α were determined at mRNA and protein levels. Reactive oxygen species and nitric oxide were also assessed by flow cytometry and colorimetric assay, respectively. In parallel to this, CNPase expression in activated microglia was also investigated in adult rats subjected to fluid percussion injury as well as middle cerebral artery occlusion.
  
  
  RESULTS
  In vivo, CNPase immunofluorescence in activated microglia was markedly enhanced after lipopolysaccharide treatment. A similar feature was observed in the rat brain after fluid percussion injury and middle cerebral artery occlusion. In vitro, CNPase protein and mRNA expression was increased in primary microglia with lipopolysaccharide stimulation. Remarkably, inducible nitric oxide synthase, IL-1β, TNF-α, reactive oxygen species and nitric oxide were significantly upregulated in activated BV-2 cells with CNPase knockdown. siRNA knockdown of CNPase increased microglia migration; on the other hand, microglial cells appeared to be arrested at G1 phase.
  
  
  CONCLUSIONS
  The present results have provided the first morphological and molecular evidence that CNPase expression is increased in activated microglia. CNPase knockdown resulted in increased expression of various inflammatory mediators. It is concluded that CNPase may play an important role as a putative anti-inflammatory gene both in normal and injured brain.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Yang
authors.forename: Enci Mary
authors.initials: EM
authors.lastname: Kan
authors.forename: Jia
authors.initials: J
authors.lastname: Lu
authors.forename: Chunyun
authors.initials: C
authors.lastname: Wu
authors.affiliation: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore. antlea@nus.edu.sg.
authors.forename: Eng-Ang
authors.initials: EA
authors.lastname: Ling
mesh_terms: D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001930:Brain Injuries / Q000209:etiology / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D004195:Disease Models, Animal; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000201:enzymology*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D020521:Stroke / Q000150:complications; D013997:Time Factors
chemical_list: D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D052247:Nitric Oxide Synthase Type II; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases