Division of Neurosurgical Research, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
PLoS One. 2018 Jun 18;13(6):e0198553. doi: 10.1371/journal.pone.0198553. eCollection 2018.
Focal brain ischemia markedly affects cerebrovascular reactivity. So far, these changes have mainly been related to alterations in the level of smooth muscle cell function while alterations of the endothelial lining have not yet been studied in detail. We have, therefore, investigated the effects of ischemia/reperfusion injury on bradykinin (BK)-induced relaxation since BK is an important mediator of tissue inflammation and affects vascular function in an endothelium-dependent manner. Focal brain ischemia was induced in rats by endovascular filament occlusion (2h) of the middle cerebral artery (MCA). After 22h reperfusion, both MCAs were harvested and the response to BK studied in organ bath experiments. Expression of the BK receptor subtypes 1 and 2 (B1, B2) was determined by real-time semi-quantitative RT-qPCR methodology, and whole mount immunofluorescence staining was performed to show the B2 receptor protein expression. In control animals, BK did not induce significant vasomotor effects despite a functionally intact endothelium and robust expression of B2 mRNA. After ischemia/reperfusion injury, BK induced a concentration-related sustained relaxation in all arteries studied, more pronounced in the ipsilateral than in the contralateral MCA. The B2 mRNA was significantly upregulated and the B1 mRNA displayed de novo expression, again more pronounced ipsi- than contralaterally. Endothelial cells displaying B2 receptor immunofluorescence were observed scattered or clustered in previously occluded MCAs. Relaxation to BK was mediated by B2 receptor activation, abolished after endothelium denudation, and largely diminished by blocking nitric oxide (NO) release or soluble guanylyl cyclase activity. Relaxation to BK was partially inhibited by charybdotoxin (ChTx), but not apamin or iberiotoxin suggesting activation of an endothelium-dependent hyperpolarization pathway. When the NO-cGMP pathway was blocked, BK induced a transient relaxation which was suppressed by ChTx. After ischemia/reperfusion injury BK elicits endothelium-dependent relaxation which was not detectable in control MCAs. This gain of function is mediated by B2 receptor activation and involves the release of NO and activation of an endothelium-dependent hyperpolarization. It goes along with increased B2 mRNA and protein expression, leaving the functional role of the de novo B1 receptor expression still open.
局部脑缺血显著影响脑血管反应性。到目前为止，这些变化主要与平滑肌细胞功能水平的改变有关，而内皮衬里的改变尚未得到详细研究。因此，我们研究了缺血/再灌注损伤对缓激肽（BK）诱导的松弛的影响，因为 BK 是组织炎症的重要介质，并且以依赖内皮的方式影响血管功能。通过血管内细丝闭塞（2 小时）大脑中动脉（MCA）诱导局部脑缺血。再灌注 22 小时后，收获双侧 MCA，并在器官浴实验中研究 BK 的反应。通过实时半定量 RT-qPCR 方法确定 BK 受体亚型 1 和 2（B1、B2）的表达，并进行全贴壁免疫荧光染色以显示 B2 受体蛋白表达。在对照动物中，尽管内皮功能完整且 B2 mRNA 表达丰富，但 BK 并未诱导明显的血管运动效应。缺血/再灌注损伤后，BK 在所有研究的动脉中诱导浓度相关的持续松弛，在同侧 MCA 中比在对侧 MCA 中更为明显。B2 mRNA 显著上调，B1 mRNA 显示新表达，再次在同侧比在对侧更为明显。在先前闭塞的 MCA 中观察到显示 B2 受体免疫荧光的内皮细胞呈散在或簇状分布。BK 引起的松弛是通过 B2 受体激活介导的，在内皮剥脱后被消除，并且通过阻断一氧化氮（NO）释放或可溶性鸟苷酸环化酶活性而大大减少。BK 的松弛部分被芋螺毒素（ChTx）抑制，但不被蜂毒素或 Iberiotoxin 抑制，提示激活了一种依赖内皮的超极化途径。当 NO-cGMP 途径被阻断时，BK 诱导短暂的松弛，该松弛被 ChTx 抑制。缺血/再灌注损伤后，BK 引起的内皮依赖性松弛在对照 MCA 中无法检测到。这种功能获得是通过 B2 受体激活介导的，涉及 NO 的释放和依赖内皮的超极化的激活。它伴随着 B2 mRNA 和蛋白表达的增加，而新表达的 B1 受体的功能作用仍不清楚。
