Unidad de Investigación Neurovascular, Departamento de Farmacología, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
J Neuroinflammation. 2012 Mar 7;9:48. doi: 10.1186/1742-2094-9-48.
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. 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.
Animals 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).
Brain 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.
Our 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.
细胞内钙离子浓度 [Ca2+]i 的增加是脑缺血后发生的最早事件之一。钙调磷酸酶（CN）是一种关键的 [Ca2+]i 调节信号分子，在炎症级联的调节中发挥重要作用。在这里，我们分析了内源性 CN 1 （Rcan1）调节剂在大脑中动脉闭塞引起的实验性缺血性中风中的作用。
动物进行大脑中动脉闭塞伴再灌注的局灶性脑缺血。为了评估中风后 Rcan1 的作用，我们在 Rcan1 敲除（KO）和野生型（WT）小鼠中测量了再灌注 48 小时后的梗死体积。在富含星形胶质细胞的皮质原代培养物中进行体外研究，使其处于 3%氧气（缺氧）和葡萄糖剥夺（HGD）状态。腺病毒载体用于分析 Rcan1-4 蛋白过表达的影响。通过免疫组织化学和免疫印迹法检查蛋白质表达，并通过实时定量逆转录聚合酶链反应（实时 qRT-PCR）检查 mRNA 表达。
体内脑缺血/再灌注（I/R）损伤增加了钙诱导的 Rcan1 同工型（Rcan1-4）的 mRNA 和蛋白表达。Rcan1 蛋白的 I/R 诱导表达主要发生在星形胶质细胞中，在体外缺血模型中，HGD 处理原代鼠星形胶质细胞培养物诱导 Rcan1-4 mRNA 和蛋白表达。外源性 Rcan1-4 过表达抑制了炎症标志物环加氧酶 2 的产生。缺乏 Rcan1 的小鼠具有更高的炎症相关基因表达，导致更大的梗死体积。
我们的结果支持 Rcan1 在中风后炎症反应中具有保护作用，并强调了胶质细胞在缺血后发生的炎症反应中的重要性。对缺血性损伤中非神经元机制的深入了解有望为急性缺血性中风的治疗提供新的方法。
