Role of P450 Eicosanoids in Brain Microvasculature after Subarachnoid Hemorrhage

描述（由申请人提供）：今年在美国将有近80万人患中风，使其成为第三大死因。这种疾病的一种特别具有破坏性的形式是蛛网膜下腔出血（SAH），由于其受害者年龄相对较小，在所有形式的中风中，它占生命年损失的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，就会发生SAH。从SAH中恢复的患者往往因缺血而出现延迟性神经功能缺损，导致大量患者死亡，并使幸存者留下永久性残疾。这种损伤的发病机制尚不清楚，尽管对延迟性神经功能缺陷的几种有希望的机制已经进行了详细的研究，但从这项工作中开发的治疗方法在临床试验中取得的成功有限。最近发现的一种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，用于记录dMVS的技术只能在非常低的分辨率下完成。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层内的微血管，并明确识别SAH后的dMVS。我们建议除了OMAG外，还使用神经行为测试、组织学和生物化学来探索预防dMVS的潜在治疗靶点。脑微血管中的血流是由花生四烯酸的细胞色素P450代谢物控制的，这种代谢物被称为环氧二十碳三烯酸（EETs）。eet被酶溶环氧化物水解酶（sEH）灭活。我们的初步数据表明，敲除SAH的小鼠在SAH后可免受dMVS的侵害。我们将验证SAH诱导脑血管内皮中sEH的上调，导致生物可利用eet减少，dMVS和神经功能障碍的发展的假设。在Specific Aim 1中，我们将测试基因缺失和药物抑制sEH活性是否能预防小鼠SAH后的dMVS和神经功能缺损。我们将通过OMAG、神经行为检查和组织学进一步表征SAH后dMVS和神经功能缺损。最后，我们将使用内皮过表达sEH和EETs生物合成酶P450 2J2的转基因小鼠来证实内皮sEH在SAH后调节dMVS中的作用。在特异性目标2中，我们将通过Western blotting，实时定量PCR和质谱（LC-MS/MS）测量SAH后脑微血管内sEH表达和eet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，已知血管紧张素信号在SAH后会增加。本研究旨在寻找一种新的治疗靶点来预防由SAH引起的dMVS和延迟性神经功能缺损。

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.