Pathophysiology of Chronic Cerebral Vasospasm

描述(申请人提供)：我们正在研究脑血管痉挛，这是蛛网膜下腔出血(SAH)后脑缺血的一个重要原因。这笔赠款的长期目标是确定SAH后血管痉挛的机制，从而开发预防和/或逆转它的治疗方法。我们已经证明，血红蛋白导致血管痉挛，血管痉挛与动脉松弛受损有关。血红蛋白诱发血管痉挛的一个机制可能是结合和清除一氧化氮(NO)。我们用电子顺磁共振(EPR)谱检测了蛛网膜下腔出血后蛛网膜下腔中的亚硝基血红蛋白，证明了这一机制的发生。因此，我们将通过以下方法检验血管痉挛的不可逆成分：1)定义在无供体的情况下，血管痉挛的可逆程度；2)用EPR光谱分析SAH后不同时间从猕猴蛛网膜下腔取出的血块中的血红素-NO加合物(亚硝基血红蛋白)；3)定量测定猕猴SAH后不同时间血管周围间隙中的NO；以及4)确定SAH后脑动脉和血管周围血凝块中NOS三种亚型的变化及其免疫组织化学定位。第二，由于血管痉挛似乎不是没有供体就能完全预防的，我们将通过以下几个方面来研究血管痉挛的机制：1)测定猴血管痉挛过程中的蛋白激酶G信使核糖核酸、蛋白质和活性；2)评估血管痉挛时间过程中猴脑动脉的钙敏感性。在一个大鼠模型中，我们将通过以下方式评估NO诱导的松弛的其他下游效应因子的作用：1)评估血管痉挛期间的钾通道功能(钙激活的钾通道密度、单通道电导和开放概率将使用分离的血管痉挛大鼠脑血管平滑肌细胞的全细胞和单通道膜片钳记录来评估)；以及2)测量大鼠血管痉挛期间的全细胞钙电流，因为评估钾通道功能需要了解细胞内钙离子，而且SAH后血管痉挛期间平滑肌钙稳态可能发生改变。

DESCRIPTION (provided by applicant): We are investigating cerebral vasospasm which is an important cause of cerebral ischemia after subarachnoid hemorrhage (SAH). The long-term objective of this grant is to determine the mechanism of vasospasm after SAH and to thereby develop treatments that will prevent and/or reverse it. We have shown that hemoglobin causes vasospasm and that vasospasm is associated with impaired arterial relaxation. One mechanism of hemoglobin-induced vasospasm may be the binding and removal of nitric oxide (NO). We have used electron paramagnetic resonance (EPR) spectroscopy to detect nitrosyl hemoglobin in the subarachnoid space after SAH, proving that this mechanism occurs. We will therefore test the hypothesis that there is an NO-reversible component of vasospasm by: 1) defining the extent to which vasospasm is reversible with NO donors in a monkey model of SAH; 2) measuring heme-NO adducts (nitrosyl hemoglobin) by EPR spectroscopy in clots removed from the subarachnoid space of monkeys at different times after SAH; 3) quantifying NO in the perivascular space at different times after SAH in monkeys; and 4) defining the time course of changes in and the immunohistochemical locations of the 3 isoforms of NOS in cerebral arteries and perivascular blood clot after SAH in monkeys. Second, because vasospasm does not seem to be completely preventable by NO donors, we will investigate mechanisms of NO-independent vasospasm by: 1) measuring protein kinase G messenger ribonucleic acid, protein and activity during the time course of vasospasm in monkeys; and 2) assessing calcium sensitivity of monkey cerebral arteries during the time course of vasospasm. In a rat model, we will assess the contribution of other downstream effectors of NO-induced relaxation by: 1) assessing potassium channel function during vasospasm (calcium-activated potassium channel density, single channel conductance, and open probability will be assessed using whole cell and single channel patch clamp recordings of isolated vasospastic rat cerebrovascular smooth muscle cells); and 2) measuring whole cell calcium currents during vasospasm in rats because assessment of potassium channel function requires knowledge of intracellular calcium and because smooth muscle calcium homeostasis may be altered during vasospasm after SAH.