P450 Eicosannoids: Novel Nerve-Deerived Relaxing Factors in the Brain

描述(由申请人提供)：流向大脑的血液通过统称为神经血管偶联(NVC)的信号机制与局部代谢需求紧密耦合。许多NVC的内源性调节因子已被确定，包括星形胶质细胞来源的P450血管扩张剂，被称为环氧二十碳三烯酸(EETs)的二十烷基类化合物。在区域水平上，脑血流量(CBF)部分由外源性血管周围血管扩张剂和血管收缩神经调节，这些神经支配管道动脉，如大脑中动脉(MCA)和基底动脉(BAS)。我们在此提供的初步数据显示，EETs合成酶和代谢酶在副交感血管扩张血管周围神经中的表达。基于这些发现，我们建议检验EETs是新的副交感神经衍生的松弛因子参与CBF的神经源性调节的假设。我们将首先利用免疫荧光双标记、Western印迹、实时定量RT-PCR(rtqRT-PCR)和顺行神经示踪来表征EETS合成酶细胞色素P450 2C11环氧合酶和EETS失活酶可溶性环氧化物水解酶(SEH)在脑血管神经及其起源神经节中的表达。然后，我们将利用体内颅窗准备来确定EETs在大脑中动脉对副交感神经节刺激的神经源性血管扩张反应中的功能作用。最后，我们将利用副交感神经元(PSN)和血管平滑肌细胞(VSMC)的分室共培养模型来确定副交感神经元释放EETs的机制及其对VSMC的超极化作用。这些研究将探索一种新的脑血流调节机制，加深我们对生理条件下脑血流调节的理解，并可能对蛛网膜下腔出血后血管痉挛、血管性痴呆、偏头痛、中风和创伤性脑损伤等疾病状态下的神经血管功能障碍具有重要的临床意义。

DESCRIPTION (provided by applicant): Blood flow to the brain is tightly coupled to local metabolic demand through signaling mechanisms collectively termed neurovascular coupling (NVC). A number of endogenous regulators of NVC have been identified including astrocyte-derived P450 vasodilator eicosanoids referred to as epoxyeicosatrienoic acids (EETs). At the regional level, cerebral blood flow (CBF) is regulated in part by extrinsic perivascular vasodilator and vasoconstrictor nerves that innervate conduit arteries such as the middle cerebral (MCA) and basilar arteries (BAS). We provide herein preliminary data demonstrating the expression of EETs synthetic and metabolizing enzymes within parasympathetic vasodilator perivascular nerves. Based on these findings, we propose to test the hypothesis that EETs are novel parasympathetic nerve-derived relaxing factors involved in the neurogenic regulation of CBF. We will first utilize immunofluorescent double-labeling, Western blot, real-time quantitative RT-PCR (rtqRT-PCR) and anterograde nerve tracing to characterize the expression of EETs-synthetic enzyme cytochrome P450 2C11 epoxygenase and EETs-inactivating enzyme soluble epoxide hydrolase (sEH) within the cerebral vascular nerves and their ganglia of origin. We will then utilize an in vivo cranial window preparation to determine the functional role of EETs in the neurogenic vasodilator response of the MCA to parasympathetic ganglia stimulation. Lastly, we will employ compartmented co-culture model of parasympathetic neurons (PSN) and vascular smooth muscle cells (VSMC) to determine the mechanism of EETs release by parasympathetic neurons and their hyperpolarizing action upon VSMC. The proposed studies will explore a novel mechanism of CBF regulation, which will further our understanding of CBF regulation under physiological conditions, and may have important clinical implications relevant to neurovascular dysfunction in such disease states as vasospasm after subarachnoid hemorrhage, vascular dementia, migraine, stroke and traumatic brain injury.