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Journal of Neuroinflammation

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Open Access 01-12-2014 | Research

Expression of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and its roles in activated microglia in vivo and in vitro

Authors: Lijuan Yang, Enci Mary Kan, Jia Lu, Chunyun Wu, Eng-Ang Ling

Published in: Journal of Neuroinflammation | Issue 1/2014

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.

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Title: Expression of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and its roles in activated microglia in vivo and in vitro
Authors: Lijuan Yang
Enci Mary Kan
Jia Lu
Chunyun Wu
Eng-Ang Ling
Publication date: 01-12-2014
Publisher: BioMed Central
Published in: Journal of Neuroinflammation / Issue 1/2014
Electronic ISSN: 1742-2094
DOI: https://doi.org/10.1186/s12974-014-0148-9

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Expression of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and its roles in activated microglia in vivo and in vitro

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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