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量（CBF）不足以使脑组织存活而引起的。从使用组织型纤溶酶原激活剂治疗的人类中可以得知，通过溶栓性脑血流恢复可以挽救梗塞后的缺血组织。这改善了结果。因此，改善脑血流是一个治疗靶点。各种已知的化合物，要么提供一氧化氮（NO），要么上调eNOS表达（如辛伐他汀），增加缺血性组织的CBF，改善实验性卒中结局。虽然常见的机制似乎是增加一氧化氮的生物活性和血管舒张，但由于延迟起效，或对缺血组织缺乏特异性，导致剂量受到全身性低血压的限制，大多数此类化合物作为中风治疗药物是不切实际的。选择性血管舒张的新机制是变构性调节血红蛋白携带的一氧化氮生物活性的递送。s -亚硝基化血红蛋白（SNO-Hb）在氧合形式下是稳定的，但在氧卸载时不稳定。这为在维持血流动力学稳定性的同时，选择性地增加血液和缺血组织之间沿PO2梯度的NO生物活性提供了机会。亚硝酸盐乙酯是一种吸入气体，可显著增加SNO-Hb。亚硝酸盐乙酯可降低成人和婴儿肺动脉高压的严重程度，选择性降低气腹、脑血管痉挛和游离肌瓣模型的终末器官缺血，对血压无影响。我们计划研究调节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闭塞（MCAO）模型，我们将1)确定吸入20ppm亚硝酸乙酯在长丝MCAO期间和之后对绝对CBF的急性影响，并测量脑梗死危险组织的数量作为吸入氧分数（Fi02）的函数；2)确定亚硝酸乙酯在长期结局大鼠永久性局灶性缺血性卒中模型中的疗效作为Fi02的函数。3)确定亚硝酸盐乙酯在暂时性局灶性脑卒中大鼠长期预后模型中的疗效与Fi02的关系。该模型的初步研究表明，在短暂局灶性缺血期间或之后用亚硝酸盐乙酯治疗可引起激光多普勒CBF快速和可逆的增加。初步数据显示对血小板功能无不良影响。因此，我们预测暂时性和永久性MCAO的预后都会得到改善。公共卫生相关性：该项目的目的是确定吸入气体亚硝酸盐乙酯在实验性中风大鼠中改善血液流动的有效性。我们有证据表明，亚硝酸盐乙酯会选择性地改善大脑中正在经历中风的部分的血液流动，但这对身体其他部位的血液流动几乎没有影响。然后，我们将检查这种作用是否对减少中风引起的组织损伤和神经损伤的数量很重要。

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.