Role of P450 Eicosanoids in Delayed Microvascular Spasm Following Subarachnoid He

描述（由申请人提供）：今年在美国，近80万人将遭受中风，使其成为第三大死亡原因。这种疾病的一种特别具有破坏性的形式，蛛网膜下腔出血（SAH），由于其受害者相对年轻，在所有形式的中风中占生命年损失的25%。当脑中的大血管破裂并出血进入脑周围充满液体的空间时，SAH发生。从SAH中恢复的患者通常由于缺血而发展为迟发性神经功能缺损，导致大量死亡，并使幸存者永久性残疾。这种损伤的发病机制还不清楚，尽管已经详细研究了延迟性神经功能缺损的几种有希望的机制，但从这项工作中开发的治疗方法在临床试验中取得了有限的成功。 最近记录的与迟发性神经功能缺损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dMVS的技术只能在非常低的分辨率下进行记录。使用一种称为光学微血管造影（OMAG）的新型体内光学成像方式，我们已经能够直接可视化小鼠皮质内的微血管，并明确识别SAH后的dMVS。 我们建议使用神经行为测试，组织学和生物化学，除了OMAG探索潜在的治疗靶点，以防止dMVS。脑微血管中的流动由花生四烯酸的细胞色素P450代谢产物（称为环氧二十碳三烯酸（ESTs））控制。可溶性环氧化物水解酶（sEH）可使Eglycoprotein失活。我们的初步数据表明，sEH基因敲除小鼠在SAH后免受dMVS的影响。我们将检验SAH诱导脑血管内皮细胞sEH上调，导致生物可利用的Ehrs减少以及dMVS和神经功能缺损的发展的假设。 在具体目标1中，我们将测试基因缺失和sEH活性的药理学抑制是否可以预防SAH后小鼠的dMVS和神经功能缺陷。我们将使用OMAG、神经行为检查和组织学进一步表征SAH后的dMVS和神经功能缺损。最后，我们将使用内皮过表达sEH和Escherichia coli生物合成酶P450 2 J2的转基因小鼠，以证实内皮sEH在SAH后调节dMVS中的作用。在具体目标2中，我们将通过蛋白质印迹、实时定量PCR和质谱法（LC-MS/MS）测量SAH后脑微血管内sEH表达和Ehrs的变化。此外，我们将检测SAH后sEH的上调是否依赖于血管紧张素信号传导，已知血管紧张素信号传导在SAH后增加。本研究旨在确定一种新的治疗靶点，用于预防SAH引起的dMVS和迟发性神经功能缺损。 公共卫生相关性：从蛛网膜下腔出血（中风的一种形式）中恢复的个体，由于在初始出血后很久出现的延迟性神经功能缺损，通常生活在严重残疾中。迟发性微血管痉挛是一种原因不明的脑内小阻力血管和毛细血管收缩，与大量发生迟发性神经功能缺损的患者相关。本研究的目的是研究蛛网膜下腔出血后微血管扩张通路的中断是如何引起迟发性微血管痉挛和随后的迟发性神经功能缺损的。

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. PUBLIC HEALTH RELEVANCE: Individuals recovering from subarachnoid hemorrhage, a form of stroke, often live with significant disability due to delayed neurological deficit that arises well after the initial hemorrhage. Delayed microvascular spasm, a constriction of small resistance vessels and capillaries in the brain by an unknown cause, is associated with a significant number of patients who experience delayed neurological deficit. Here the aim is to study how disruption of a microvascular vasodilator pathway could cause delayed microvascular spasm after subarachnoid hemorrhage and subsequently, delayed neurological deficit.