Central Rho-Kinase Inhibition and Ang II-induced Sympatho-excitation

描述(由申请人提供)：本申请描述了作为内布拉斯加大学医学中心医学博士/博士项目的一部分，获得博士学位的研究计划和课程。该研究计划的主要假设是，血管紧张素II(Ang II)激活中枢Rho-Kinase有助于在疾病状态(如心力衰竭、高血压)中产生交感神经兴奋，Rho-Kinase抑制可能是这些疾病的治疗靶点。为了解决这一问题，提出了三个具体目标。具体目标1：确定中枢Rho-Kinase抑制对慢性全身注射Ang II反应中自主神经张力调节的影响。这些实验将在长期使用仪器的清醒新西兰大白兔身上进行。在Ang II输注期间，Rho-Kinase抑制剂法舒地尔将被注入脑室，并将通过肾交感神经活动、压力反射敏感性、心率变异性、美托洛尔和阿托品血浆去甲肾上腺素后心率的变化以及神经节阻断后动脉压的变化来评估服用法舒地尔和赋形剂的兔的自主神经张力。特异性目的2：研究全身注射血管紧张素Ⅱ后，中枢抑制Rho-Kinase对交感神经核内交感兴奋性活性氧(ROS)和交感抑制性一氧化氮(NO)平衡的影响。我们将使用几种方法来量化氧化应激以及脑干和下丘脑自主神经中心中与ROS和NO生物利用度相关的各种蛋白质。这些研究将在用于特定目的1的兔组织上进行。特定目的3：确定体外过表达RhoA是否会增强Ang II介导的ROS增加和NO生物利用度的降低。在神经细胞系中，我们将使用与特定目标2相同的分子终点来评估RhoA的过度表达对Ang II刺激下ROS和NO之间平衡的影响。总体而言，这些研究将为交感兴奋疾病状态下可能成为治疗靶点的神经通路提供新的信息。我的培训的教学部分也被描述，包括期刊俱乐部，研讨会报告，以及高级生理学、统计学、工程学和科学写作的课程。我的综合考试将安排在2013年底或2014年初。我将得到资助参加科学会议，展示我的研究成果。在我的博士培训期间，我将继续接触临床医学，在一家免费诊所协助，跟踪UNMC医生，参加临床研讨会。

DESCRIPTION (provided by applicant): This application describes a research plan and course of study leading to the Ph.D. degree as part of the M.D./Ph.D. Program at The University of Nebraska Medical Center. The overarching hypothesis of the research plan is that activation of central Rho-kinase by Angiotensin II (Ang II) contributes to sympathoexcitation in disease states (like heart failure, hypertension) and that Rho-kinase inhibition may be a therapeutic target in these diseases. To address this issue, three specific aims are proposed. Specific Aim 1: Determine the effect of central Rho-kinase inhibition on the regulation of autonomic tone in response to chronic systemic Ang II infusion. These experiments will be carried out in chronically instrumented, conscious New Zealand White rabbits. The Rho-kinase inhibitor, fasudil, will be administered intracerebroventricularly during Ang II infusion, and autonomic tone will be assessed in fasudil- and vehicle-treated rabbits by renal sympathetic nerve activity, baroreflex sensitivity, heart rate variability, changes in heart rate after metoprolol and atropine plasma norepinephrine, and changes in arterial pressure in response to ganglionic blockade. Specific Aim 2: Determine the effects of central Rho-kinase inhibition on the balance between sympatho-excitatory reactive oxygen species (ROS) and sympatho-inhibitory nitric oxide (NO) in sympathetic nuclei in response to systemic Ang II infusion. We will use several methods to quantify oxidative stress and a variety of proteins relevant to ROS and NO bioavailability in autonomic centers of the brainstem and hypothalamus. These studies will be performed on tissue from the rabbits used in Specific Aim 1. Specific Aim 3: Determine if overexpression of RhoA in vitro potentiates Ang II-mediated increases in ROS and decreases in NO bioavailability. In a neuronal cell line, we will evaluate the effects of overexpression of RhoA, the activator of Rho-kinase, on the balance between ROS and NO in response to Ang II stimulation, using the same molecular endpoints as Specific Aim 2. Overall, these studies will provide novel information on a neuronal pathway that could be a therapeutic target in sympathoexcitatory disease states. The didactic component of my training is also described, including journal club, seminar presentations, and courses in advanced physiology, statistics, engineering, and scientific writing. My comprehensive exam will be scheduled for late 2013 or early 2014. I will be supported to attend scientific conferences to present the results of my research. During my Ph.D. training, I will continue to be exposed to clinical medicine by assisting at a free clinic, shadowing UNMC physicians, and attending clinical seminars.