Neurovascular Mechanisns of Brain Function and Disease

脑功能和疾病的神经血管机制

• 批准号: 7448456
• 负责人: Philip K Liu
• 金额: $ 19.08万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-16 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7448456
• 关键词: A Mouse; Actins; Affect; Animals; Applications Grants; Autopsy; Bilateral; Binding; Biological Assay; Biological Markers; Biopsy; Blood Circulation; Blood Platelets; Blood Vessels; Brain; Brain Edema; Brain Injuries; Cardiac Edema; Cell membrane; Cells; Cerebral Edema; Cerebral Ischemia; Cerebrum; Condition; Contrast Media; Contrast Sensitivity; Control Groups; DNA; DNA Probes; Detection; Development; Disease; Doctor of Philosophy; Dose; Edema; Elevation; Endothelium; Evaluation; Extravasation; Functional disorder; Future; Gelatinase B; Gene Expression; Genes; Genetic Transcription; Goals; Heart Arrest; Histology; Human; Image; Imaging technology; In Situ; Infusion procedures; Injury; Intraperitoneal Injections; Invasive; Investigation; Knock-out; Label; Life; Link; Magnetic Resonance; Magnetic Resonance Imaging; Maps; Matrix Metalloproteinases; Messenger RNA; Metalloproteases; Methods; Modeling; Modification; Mus; Myocardial Infarction; Nervous System Trauma; Neurological outcome; Oligonucleotides; Operative Surgical Procedures; Organ; Oxidative Stress; Physiological reperfusion; Polymerase Chain Reaction; RNA; Radioactive; Randomized; Recovery; Reperfusion Therapy; Reporter; Reporting; Resolution; Reverse Transcription; Running; Sampling; Signal Transduction; Simulate; Stress; Stroke; Techniques; Testing; Therapeutic Agents; Tissue Sample; Tissues; Toxic effect; Transcript; Transcriptional Activation; Up-Regulation; Validation; Work; beta Actin; brain cell; design; dextran-coated SPIO; gene correction; gene therapy; human subject; improved; iron oxide; mRNA Expression; male; nanoparticle; nervous system disorder; neurogenesis; neutrophil; novel; phosphorothioate; preclinical study; repaired; restoration; uptake

DESCRIPTION (provided by applicant): Altered expression of endogenous genes in the brain often accompanies neurological disorders. Genes or cells have been used to correct gene trancription so that brain can repair itself. Currently, most detection techniques of gene transcription require biopsy or autopsy samples. Invasive surgical procedures for removing tissue samples severely limit the benefits, especially to the cells we try to cure. Our goals are to develop methods to image and quantitatively compare endogenous gene expression at the transcript level using magnetic resonance (MR) in live animal or human subjects. We made a novel MR probe using short phosphorothioate-modified oligodeoxynucleotides (sODN) with SuperParamagnetic Iron Oxide Nanoparticles (SPION, an MR T2 agent). The work outlined in this application investigates the utility of this contrast probe as an biomarker for mRNA transcripts in live animals. We designed three probes for MRI: two are with sequence complementary to matrix metalloprotease-9 (sODN-mmp9) or beta- actin (sODN-bactin) mRNA and a randomized s-ODN (sODN-Ran) with no sequence complementary to mRNA. The SPION-Ran probes will serve as controls. We will evaluate conditions that allow optimal imaging endogenous gene expression using MR.The specific anims are to demonstrate: Aim 1: Maximize MR Contrast Enhancement using SPION-bactin in High-resolution MRI in Mouse Brains. The hypothesis is that sODN-linked SPION will be retained by brain cells for detection using MRI in live animals, and for validation using histology (iron oxide) and binding assay (SPION-bactin) in postmortem samples. We select beta-actin mRNA as a target because beta-actin mRNA is constant and is inert to stress. To support this hypothesis, we will: (a) select an optimal SPION-retention using MRI in live animals after infusion with various doses of SPION-bactin; We will demonstrate that the uptake of SPION in the brain is sODN-linkage dependent; (b) demonstrate the presence of intracellular iron oxide after infusion of SPION-bactin at the optimal dose, and (c) show that the internalized SPION-bactin binds to its target mRNA. Aim 2: Retention of cerebral SPION-mmp9 in live C57black6 mice predicts brain edema after stroke inducted by 60 or 90 minutes bilateral carotid occlusion. The hypothesis is that cerebral mmp-9 mRNA transcript reports MMP-9 expression. To support this hypothesis, we will demonstrate:(a) elevation of mmp-9 mRNA expression is positively correlated with cerebral edema after stroke, (b) retention of SPION-mmp9 is higher in the stroke-treated than in the sham-operated mice. Specifically, we will identify the hotspot of SPION-mmp9 retention in stroke-treated animals using subtraction of R2* map between stroke-treated and sham-operated animals. In addition, we will compare the hotspots of SPION-mmp9 retention to stroke-induced damage in the brain of wild type and mmp-9 knockout strains. R21NS05755601-A1 PI: Liu, Philip K. PhD Project Narratives: Brain edema is one neurological disorder that complicates the recovery of several diseases in the humans. Cardiac arrest in humans can induce brain edema despite restoration of circulation using various means (Roine et al. 1993; Fujioka et al. 1994; Xiao 2002); brain damage is predictive of a poor neurological outcome. Currently few models simulate brain edema from cardiac arrest for us to test therapeutic agents that can reduce neurological damage. To improve our understanding of cerebral injury caused by heart attack, we proposed this grant application a global cerebral ischemia-reperfusion model to investigate the pathophysiology in the brain of male C57black/6 mice simulating cardiac arrest (Liu et al. 1996). Our long-term goal is to establish new methods using MRI contrast agent to gain understanding on the pathological change in gene expression in live subjects, which can be saved for further evaluation on efficacy and toxicity of the therapy. The immediate goal is to show our probe can report the elevation of gene transcripts after cerebral ischemia.

描述（由申请人提供）：脑中内源性基因表达的改变通常伴随神经系统疾病。基因或细胞已被用来纠正基因转录，以便大脑能够自我修复。目前，大多数基因转录检测技术需要活检或尸检样本。用于移除组织样本的侵入性外科手术严重限制了益处，特别是对于我们试图治愈的细胞。我们的目标是开发方法，图像和定量比较内源性基因表达的转录水平，使用磁共振（MR）在活的动物或人类受试者。我们使用短的硫代磷酸酯修饰的寡脱氧核苷酸（sODN）与超顺磁性氧化铁纳米颗粒（SPION，MR T2剂）制成了一种新的MR探针。本申请中概述的工作研究了这种对比探针作为活动物中mRNA转录物的生物标志物的效用。我们设计了三种用于MRI的探针：两种具有与基质金属蛋白酶-9（sODN-mmp 9）或β-肌动蛋白（sODN-bactin）mRNA互补的序列，以及不具有与mRNA互补的序列的随机s-ODN（sODN-Ran）。SPION-Ran探头将作为对照。我们将评估条件，允许最佳的成像内源性基因表达使用MR。具体的动画是为了证明：目的1：最大限度地提高MR对比度增强使用SPION-杆菌素在高分辨率MRI在小鼠大脑。假设sODN连接的SPION将被脑细胞保留，用于在活体动物中使用MRI进行检测，并用于在死后样本中使用组织学（氧化铁）和结合试验（SPION-杆菌素）进行验证。我们选择β-肌动蛋白mRNA作为靶点，因为β-肌动蛋白mRNA是恒定的，对压力是惰性的。为了支持这一假设，我们将：（a）在输注不同剂量的SPION-杆菌素后，在活体动物中使用MRI选择最佳SPION保留;我们将证明脑中SPION的摄取是sODN-连接依赖性的;（B）证明以最佳剂量输注SPION-杆菌素后细胞内氧化铁的存在，以及（c）证明内化的SPION-杆菌素与其靶mRNA结合。目标二：C57 black 6小鼠脑SPION-mmp 9的保留预测了双侧颈动脉闭塞60或90分钟诱导的中风后的脑水肿。假设脑MMP-9 mRNA转录报告MMP-9表达。为了支持这一假设，我们将证明：（a）MMP-9 mRNA表达的升高与中风后的脑水肿呈正相关，（B）SPION-MMP 9在中风治疗小鼠中的保留高于假手术小鼠。具体而言，我们将使用中风治疗和假手术动物之间的R2* 图的减法来鉴定中风治疗动物中SPION-mmp 9保留的热点。此外，我们将比较SPION-mmp-9保留的热点与野生型和mmp-9敲除菌株脑中中风诱导的损伤。R21 NS 05755601-A1主要研究者：Liu，Philip K.博士项目叙述：脑水肿是一种神经系统疾病，使人类多种疾病的恢复复杂化。尽管使用各种方法恢复了循环，但人体心脏骤停仍可能诱发脑水肿（Roine et al. 1993; Fujioka et al. 1994; Xiao 2002）;脑损伤预示着不良的神经学结局。目前，很少有模型模拟心脏骤停引起的脑水肿，以供我们测试可以减少神经损伤的治疗药物。为了提高我们对心脏病发作引起的脑损伤的理解，我们提出了这项赠款申请，即全脑缺血-再灌注模型，以研究模拟心脏骤停的雄性C57 black/6小鼠脑中的病理生理学（Liu et al. 1996）。我们的长期目标是建立使用MRI造影剂的新方法，以了解活体受试者中基因表达的病理变化，从而可以保存用于进一步评估治疗的疗效和毒性。近期的目标是证明我们的探针可以报告脑缺血后基因转录的升高。