Pathophysiology of Chronic Cerebral Vasospasm

描述（申请人提供）：我们正在研究脑血管痉挛是蛛网膜下腔出血（SAH）后脑缺血的重要原因。该资助的长期目标是确定SAH后血管痉挛的机制，从而开发预防和/或逆转血管痉挛的治疗方法。我们已经证明血红蛋白引起血管痉挛，血管痉挛与动脉舒张受损有关。血红蛋白诱导血管痉挛的一个机制可能是一氧化氮（NO）的结合和清除。我们利用电子顺磁共振（EPR）光谱检测了SAH后蛛网膜下腔的亚硝基血红蛋白，证明了这一机制的发生。因此，我们将通过以下方法验证血管痉挛存在NO可逆成分的假设：1)在SAH猴模型中定义NO供体血管痉挛可逆的程度；2)用EPR光谱测定SAH后不同时间从猴蛛网膜下腔取出的血块中血红素- no加合物（亚硝基血红蛋白）的变化；3)测定猴子SAH后不同时间血管周围空间NO含量；4)确定猴SAH后脑动脉和血管周围血凝块中NOS 3种亚型的变化时间和免疫组化位置。其次，由于一氧化氮供体似乎不能完全预防血管痉挛，我们将通过以下方法研究一氧化氮非依赖性血管痉挛的机制：1)测量猴子血管痉挛时间过程中的蛋白激酶G信使核糖核酸、蛋白和活性；2)观察血管痉挛时间过程中猴子脑动脉钙敏感性的变化。在大鼠模型中，我们将通过以下方法评估其他下游效应物对no诱导弛缓的贡献：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通过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）；2)在大鼠血管痉挛期间测量全细胞钙电流，因为评估钾通道功能需要了解细胞内钙，并且因为SAH后血管痉挛期间平滑肌钙稳态可能被改变。

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.