BACKGROUND Gap junctions are involved in the development of cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). However, the specific roles and regulatory functions of related connexin isoforms remain unknown. The aim of this study was to investigate the importance of connexin 43 (Cx43) in CVS and determine whether Cx43 alterations are modulated via the protein kinase C (PKC) signaling transduction pathway. METHODS Oxyhemoglobin (OxyHb)-induced smooth muscle cells of basilar arterial and second-injection model in rat were used as CVS models in vitro and in vivo. In addition, dye transfer assays were used for gap junction-mediated intercellular communication (GJIC) observation in vitro and delayed cerebral ischemia (DCI) was observed in vivo by perfusion-weighted imaging (PWI) and intravital fluorescence microscopy. RESULTS Increase in Cx43 mediated the development of SAH-induced CVS was found in both in vitro and in vivo CVS models. Enhanced GJIC was observed in vitro CVS model, this effect and increased Cx43 were reversed by preincubation with specific PKC inhibitors (chelerythrine or GF 109203X). DCI was observed in vivo on day 7 after SAH. However, DCI was attenuated by pretreatment with Cx43 siRNA or PKC inhibitors, and the increased Cx43 expression in vivo was also reversed by Cx43 siRNA or PKC inhibitors. CONCLUSIONS These data provide strong evidence that Cx43 plays an important role in CVS and indicate that changes in Cx43 expression may be mediated by the PKC pathway. The current findings suggest that Cx43 and the PKC pathway are novel targets for developing treatments for SAH-induced CVS.

中文翻译：

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连接蛋白43在蛛网膜下腔出血引起的脑血管痉挛中的重要作用。

背景技术蛛网膜下腔出血（SAH）后，间隙连接参与了脑血管痉挛（CVS）的发展。但是，相关的连接蛋白同工型的具体作用和调节功能仍然未知。这项研究的目的是调查连接蛋白43（Cx43）在CVS中的重要性，并确定是否通过蛋白激酶C（PKC）信号转导途径调节Cx43的改变。方法采用氧合血红蛋白（OxyHb）诱导的大鼠基底动脉平滑肌细胞和第二次注射模型作为体内和体外CVS模型。此外，染料转移测定用于体外间隙连接介导的细胞间通讯（GJIC）观察，并通过灌注加权成像（PWI）和活体荧光显微镜观察体内延迟性脑缺血（DCI）。结果在体外和体内CVS模型中均发现Cx43介导SAH诱导的CVS的发展。在体外CVS模型中观察到GJIC增强，通过与特定PKC抑制剂（白屈菜红碱或GF 109203X）预孵育可逆转这种作用和Cx43升高。SAH后第7天在体内观察到DCI。但是，DCI通过用Cx43 siRNA或PKC抑制剂预处理而减弱，并且体内Cx43表达的增加也被Cx43 siRNA或PKC抑制剂逆转。结论这些数据提供了有力的证据，表明Cx43在CVS中起重要作用，并表明Cx43表达的改变可能是由PKC途径介导的。目前的发现表明，Cx43和PKC途径是开发SAH诱导的CVS治疗的新靶标。