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耦合（NVC）的信号机制与局部代谢需求紧密耦合。许多内源性NVC调节因子已被确定，包括星形胶质细胞衍生的P450血管扩张剂类二十烷酸，即环氧二十烷三烯酸（EETs）。在局部水平上，脑血流（CBF）部分由支配导管动脉（如大脑中动脉（MCA）和基底动脉（BAS））的外源性血管周围血管舒张神经和血管收缩神经调节。我们在此提供初步数据，证明EETs合成和代谢酶在副交感血管舒张血管周围神经中的表达。基于这些发现，我们建议验证eet是参与CBF神经源性调节的新型副交感神经源性放松因子的假设。我们将首先利用免疫荧光双标记、Western blot、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环氧化酶和eets -灭活酶可溶性环氧化水解酶（sEH）在脑血管神经及其起源神经节中的表达。然后，我们将利用活体颅窗准备来确定eet在中脑动脉对副交感神经节刺激的神经源性血管舒张反应中的功能作用。最后，我们将采用副交感神经元（PSN）和血管平滑肌细胞（VSMC）的区隔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。本研究将探索脑血流调节的新机制，进一步了解脑血流在生理条件下的调节，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.