S-Nitrosylated Hemoglobin and Ischemic Brain Injury

描述(由申请人提供)：缺血性中风是由于脑血流(CBF)不足而导致脑组织存活。从接受组织纤溶酶原激活剂治疗的人类中得知，通过溶栓后脑血流量的恢复，可以从脑梗塞中挽救缺血组织。这会改善结果。因此，改善脑血流量是治疗的目标。已知的各种化合物，既可以提供一氧化氮(NO)，也可以上调eNOS的表达(如辛伐他汀)，增加缺血组织的脑血流量，改善实验性中风的预后。虽然常见的机制似乎是增加无生物活性和血管扩张，但大多数此类化合物作为中风治疗药物是不切实际的，因为要么延迟起效，要么缺乏对缺血组织的特异性，导致剂量受到全身低血压的限制。选择性血管扩张的一种新机制是变构调节血红蛋白携带的一氧化氮生物活性的传递。S-硝化血红蛋白(SnO-Hb)在有氧状态下是稳定的，但在氧气卸载时不稳定。这提供了在维持血流动力学稳定性的同时，有选择地增加血液和缺血组织之间的PO2梯度上的NO生物活性的机会。亚硝酸乙酯是一种可吸入的气体，可显著增加SNO-Hb。亚硝酸乙酯可降低成人和婴儿肺动脉高压的严重程度，并选择性地减轻气腹、脑血管痉挛和游离肌瓣模型中的终末器官缺血，对血压没有影响。我们建议研究调节SNO-Hb对脑缺血结局的影响。利用已建立的生理控制的大鼠大脑中动脉阻塞(MCAO)模型，我们将1)定义在细丝状MCAO期间及之后吸入20ppm乙酸乙酯对脑血流量的急性影响，并测量随吸入氧分数的变化而发生脑梗塞的组织数量(Fi02)；2)定义乙基亚硝酸乙酯在永久性局灶性脑中风大鼠模型中的疗效与Fi02的函数关系；3)定义乙基亚硝酸乙酯在暂时性局灶性脑中风大鼠模型中的疗效与Fi02的函数关系。对该模型的初步研究表明，在短暂性局灶性脑缺血期间或之后使用亚硝酸乙酯可引起激光多普勒脑血流的快速和可逆的增加。初步数据显示，该药对血小板功能没有不良影响。因此，我们预测暂时性和永久性MCAO的结果都会有所改善。公共卫生相关性：该项目的目标是确定吸入气体亚硝酸乙酯在改善实验性中风大鼠血液流动方面的有效性。我们有证据表明，亚硝酸乙酯将选择性地改善流向大脑中风部位的血液流动，但对流向身体其他部位的血液影响很小或没有影响。然后，我们将研究这一效应在减少中风引起的组织损伤和神经损伤方面是否重要。

DESCRIPTION (provided by applicant): Ischemic stroke is caused by cerebral blood flow (CBF) inadequate to allow brain tissue survival. It is known from humans treated with tissue plasminogen activator that ischemic tissue can be salvaged from infarction by thrombolytic CBF restoration. This improves outcome. Thus, improved CBF is a therapeutic target. Various compounds, known to either donate nitric oxide (NO) or upregulate eNOS expression (e.g., simvastatin), increase CBF in ischemic tissue and improve experimental stroke outcome. While the common mechanism appears to be increased NO bioactivity and vasodilation, most such compounds are impractical as stroke therapeutics because of either delayed onset of action, or lack of specificity for ischemic tissue causing the dose to be limited by systemic hypotension. A novel mechanism for selective vasodilation is allosterically- modulated delivery of hemoglobin borne nitric oxide bioactivity. S-nitrosylated hemoglobin (SNO-Hb) is stable in the oxygenated form but unstable when O2 is offloaded. This offers opportunity to selectively increase NO bioactivity along PO2 gradients between blood and ischemic tissue while maintaining hemodynamic stability. Ethyl nitrite is an inhaled gas that markedly increases SNO-Hb. Ethyl nitrite decreases severity of pulmonary artery hypertension in adult and infant humans, and selectively decreases end-organ ischemia in models of pneumoperitoneum, cerebral vasospasm and free muscle flaps and has no effect on blood pressure. We propose projects to investigate effects of modulating SNO-Hb on cerebral ischemic outcome. Using an established, physiologically-controlled rat middle cerebral artery occlusion (MCAO) model, we will 1) Define acute effects of 20 ppm ethyl nitrite inhalation on absolute CBF during and after filament MCAO and measure amount of tissue at risk for cerebral infarction as a function of the fraction of inspired oxygen (Fi02), 2) Define ethyl nitrite efficacy in a long-term outcome rat model of permanent focal ischemic stroke as a function of Fi02, and 3) Define ethyl nitrite efficacy in a long-term outcome rat model of temporary focal ischemic stroke as a function of Fi02. Preliminary studies in this model have shown that treatment with ethyl nitrite either during or after transient focal ischemia causes rapid and reversible increases in laser Doppler CBF. Preliminary data indicates lack of an adverse effect on platelet function. We, therefore, predict improved outcome in both temporary and permanent MCAO. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine the effectiveness of an inhaled gas, ethyl nitrite, in improving blood flow during an experimental stroke in rats. We have evidence that indicates that ethyl nitrite will selectively improve blood flow to the part of the brain that is experiencing a stroke, but that will do so with little or no effect on blood flow to other parts of the body. We will then examine whether this effect is important in reducing the amount of tissue damage and neurologic impairment caused by the stroke.