Role of P450 Eicosanoids in Delayed Microvascular Spasm Following Subarachnoid He

描述(申请人提供)：今年美国将有近80万人罹患中风，成为第三大致死原因。蛛网膜下腔出血(SAH)是这种疾病的一种特别具有破坏性的形式，由于患者年龄相对较小，在所有形式的中风中，它占损失生命年的25%。当大脑中的大血管破裂并出血到大脑周围充满液体的空间时，就会发生蛛网膜下腔出血。从SAH中恢复的患者通常会因缺血而出现迟发性神经功能障碍，导致相当多的人死亡，并使幸存者永久残疾。这种损伤的发病机制尚不清楚，尽管对延迟性神经功能障碍的几种有希望的机制进行了非常详细的研究，但根据这项工作开发的治疗方法在临床试验中取得的成功有限。最近报道的一种与迟发性神经功能障碍相关的现象是迟发性微血管痉挛(DMVS)。到目前为止，可用于记录DMV的技术只能在非常低的分辨率下进行记录。使用一种名为光学微血管造影术(OMAG)的新型体内光学成像手段，我们已经能够直接显示小鼠皮质内的微血管，并明确识别SAH后的DMV。我们建议除了OMAG外，还可以使用神经行为测试、组织学和生化来探索预防DMV的潜在治疗靶点。脑微血管中的流动受细胞色素P450的花生四烯酸代谢产物环氧二十碳三烯酸(EETs)的控制。EET被酶可溶的环氧化物水解酶(SEH)灭活。我们的初步数据显示，sEH基因敲除小鼠在SAH后受到DMV的保护。我们将验证SAH诱导脑血管内皮细胞sEH上调，导致生物可用EET减少，DMV的发生和神经功能障碍的假说。在具体目标1中，我们将测试基因缺失和sEH活性的药物抑制是否能预防SAH后小鼠的DMV和神经功能障碍。我们将使用OMAG、神经行为检查和组织学进一步描述SAH后DMV和神经功能缺陷的特征。最后，我们将使用内皮细胞过表达sEH和EETs生物合成酶P450 2J2的转基因小鼠，以证实内皮sEH在SAH后调节DMV中的作用。在特定的目标2中，我们将通过Western blotting、实时定量聚合酶链式反应和质谱仪(LC-MS/MS)检测SAH后脑微血管内sEH表达和EETs的变化。此外，我们将测试SAH后sEH的上调是否依赖于血管紧张素信号，已知的血管紧张素信号在SAH后增加。本研究旨在寻找一种新的治疗靶点来预防蛛网膜下腔出血所致的DMVS和迟发性神经功能障碍。

DESCRIPTION (provided by applicant): This year in the U.S. close to 800,000 people will suffer a stroke, making it the third leading cause of death. A particularly devastating form of this illness, subarachnoid hemorrhage (SAH), represents 25% of life years lost among all forms of stroke due to the relatively young age of its victims. A SAH occurs when large vessels in the brain rupture and bleed into the fluid filled space surrounding the brain. Patients recovering from SAH often develop delayed neurological deficit due to ischemia, killing a significant number, and leaving survivors with permanent disability. The pathogenesis of this injury is not well understood, and even though several promising mechanisms of delayed neurological deficit have been studied in great detail, treatments developed from this work have had limited success in clinical trials. A recently documented phenomenon that correlates with delayed neurological deficit is delayed microvascular spasm (dMVS). Until now, the technology available to document dMVS has only been able to do so at very low resolution. Using a novel in vivo optical imaging modality called optical microangiography (OMAG), we have been able to directly visualize microvessels within the mouse cortex and definitively identify dMVS after SAH. We propose to use neurobehavioral tests, histology, and biochemistry in addition to OMAG to explore a potential therapeutic target to prevent dMVS. Flow in brain microvessels is controlled by cytochrome P450 metabolites of arachidonic acid called epoxyeicosatrienoic acids (EETs). EETs are inactivated by the enzyme soluble epoxide hydrolase (sEH). Our preliminary data show that sEH knockout mice are protected from dMVS after SAH. We will test the hypothesis that SAH induces upregulation of sEH in cerebrovascular endothelium, leading to a decrease in bioavailable EETs and the development of dMVS and neurological deficit. In Specific Aim 1, we will test whether genetic deletion and pharmacological inhibition of sEH activity prevent dMVS and neurological deficit in mice after SAH. We will further characterize dMVS and neurological deficit after SAH using OMAG, neurobehavioral examination and histology. Finally, we will use transgenic mice with endothelial overexpression of sEH and the EETs biosynthetic enzyme P450 2J2, to confirm the role of endothelial sEH in modulating dMVS after SAH. In Specific Aim 2, we will measure changes in sEH expression and EETs within brain microvessels after SAH by Western blotting, real-time quantitative PCR, and mass spectrometry (LC-MS/MS). Additionally, we will test whether the upregulation of sEH after SAH is dependent on angiotensin signaling, which is known to be increased following SAH. This research aims to identify a novel therapeutic target for prevention of dMVS and delayed neurological deficit caused by SAH.