Abstract
BackgroundAn increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia. A key [Ca2+]i-regulated signaling molecule is the phosphatase calcineurin (CN), which plays important roles in the modulation of inflammatory cascades. Here, we have analyzed the role of endogenous regulator of CN 1 (Rcan1) in response to experimental ischemic stroke induced by middle cerebral artery occlusion.MethodsAnimals were subjected to focal cerebral ischemia with reperfusion. To assess the role of Rcan1 after stroke, we measured infarct volume after 48 h of reperfusion in Rcan1 knockout (KO) and wild-type (WT) mice. In vitro studies were performed in astrocyte-enriched cortical primary cultures subjected to 3% oxygen (hypoxia) and glucose deprivation (HGD). Adenoviral vectors were used to analyze the effect of overexpression of Rcan1-4 protein. Protein expression was examined by immunohistochemistry and immunoblotting and expression of mRNA by quantitative real-time Reverse-Transcription Polymerase Chain Reaction (real time qRT-PCR).ResultsBrain ischemia/reperfusion (I/R) injury in vivo increased mRNA and protein expression of the calcium-inducible Rcan1 isoform (Rcan1-4). I/R-inducible expression of Rcan1 protein occurred mainly in astroglial cells, and in an in vitro model of ischemia, HGD treatment of primary murine astrocyte cultures induced Rcan1-4 mRNA and protein expression. Exogenous Rcan1-4 overexpression inhibited production of the inflammatory marker cyclo-oxygenase 2. Mice lacking Rcan1 had higher expression of inflammation associated genes, resulting in larger infarct volumes.ConclusionsOur results support a protective role for Rcan1 during the inflammatory response to stroke, and underline the importance of the glial compartment in the inflammatory reaction that takes place after ischemia. Improved understanding of non-neuronal mechanisms in ischemic injury promises novel approaches to the treatment of acute ischemic stroke.
Highlights
An increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia
We report that Rcan1-4 protein and mRNA accumulate in brain cortex early after ischemia/reperfusion, mainly in glial fibrillary acidic protein (GFAP)-positive cells, and that Rcan1 deficiency worsens stroke outcome and increases expression of inflammation-associated genes
Rcan1-4 protein is induced in murine models of brain ischemia/reperfusion injury The possible accumulation of Rcan1-4 protein in response to ischemia/reperfusion (I/R) brain injury was examined in a rat model
Summary
An increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia. A key [Ca2+]i-regulated signaling molecule is the phosphatase calcineurin (CN), which plays important roles in the modulation of inflammatory cascades. We have analyzed the role of endogenous regulator of CN 1 (Rcan1) in response to experimental ischemic stroke induced by middle cerebral artery occlusion. Cerebral ischemia triggers a marked inflammatory reaction that involves local cellular activation in the brain and production of inflammatory mediators, including cytokines, chemokines, are not halted by endogenous or exogenous anti-inflammatory agents. Endogenous regulation of CN is mediated by members of the regulator of calcineurin (Rcan) family, previously named Down syndrome critical region (DSCR), modulatory calcineurin interacting protein (MCIP), calcipressin and Adapt in mammals [10,11]. Rcan is upregulated by increases in [Ca2+]i in several cell types, including brain cells, via a CN/NFAT-dependent pathway [12,13,14,15,16]
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