Neurovascular Mechanisns of Brain Function and Disease

描述(申请人提供)：大脑中内源性基因表达的改变经常伴随着神经疾病。基因或细胞被用来纠正基因转译，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。移除组织样本的侵入性手术程序严重限制了好处，特别是对我们试图治愈的细胞。我们的目标是开发利用磁共振(MR)在活体动物或人类受试者的转录水平上对内源性基因表达进行成像和定量比较的方法。我们使用短的硫代修饰的寡核苷酸(SODN)和超顺磁性的氧化铁纳米颗粒(Spion，一种MR T2试剂)制成了一种新型的MR探针。本申请中概述的工作调查了这种对比探针作为活动物中mRNA转录本的生物标记物的实用性。我们设计了三个用于磁共振成像的探针：两个是与基质金属蛋白酶-9(sODN-MMP9)或β-肌动蛋白(sODN-bactin)序列互补的探针，另一个是随机的S-寡核苷酸(sODN-RAN)，没有与mR NA序列互补的序列。Spion-Ran探测器将作为控制装置。我们将评估使用核磁共振成像内源性基因表达的最佳条件。具体动物将演示：目标1：在小鼠大脑的高分辨率磁共振成像中使用Spion-bactin最大化MR对比度增强。假设sODN连接的Spion将被脑细胞保留，以便在活体动物中使用MRI进行检测，并在死后样本中使用组织学(氧化铁)和结合分析(Spion-bactin)进行验证。我们选择β-肌动蛋白mRNA作为靶点是因为β-肌动蛋白mRNA是恒定的，对应激是惰性的。为了支持这一假说，我们将：(A)在活体动物中输注不同剂量的Spion-bactin后，通过磁共振成像选择最佳的Spion保留率；我们将证明Spion在脑中的摄取是sODN连接依赖的；(B)证明在最佳剂量的Spion-bactin输注后细胞内存在氧化铁；以及(C)证明内化的Spion-bactin与其靶mRNA结合。目的：脑内Spion-MMP9在活体C57Black6小鼠体内的滞留预示着双侧颈动脉结扎60分钟或90分钟所致卒中后的脑水肿。假设大脑中的基质金属蛋白酶-9mRNA转录本报告基质金属蛋白酶-9的表达。为了支持这一假说，我们将证明：(A)卒中后脑水肿与MMP-9mRNA表达的升高呈正相关；(B)卒中治疗组小鼠脑内Spion-MMP9的保留率高于假手术组。具体地说，我们将通过减去中风治疗动物和假手术动物之间的R2*MAP来确定中风治疗动物中Spion-MMP9保留的热点。此外，我们还将比较Spion-MMP9保留的热点与中风引起的脑损伤，野生型和MMP9基因敲除株。R21NS05755601-A1 Pi：Liu，Philip K.博士项目简介：脑水肿是一种神经疾病，使人类几种疾病的康复变得复杂。尽管使用各种方法恢复循环，人类的心脏骤停仍可导致脑水肿(Roine等人。1993年；Fujioka et al.1994；肖2002)；脑损伤预示着不良的神经结局。目前，很少有模型模拟心脏骤停引起的脑水肿，以便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了解，我们提出了一种应用全脑缺血-再灌注模型来研究模拟心脏骤停的雄性C57Black/6小鼠脑内的病理生理学(Liu等人。1996年)。我们的长期目标是建立使用MRI造影剂的新方法，以了解活体受试者基因表达的病理变化，以便进一步评估该疗法的疗效和毒性。目前的目标是证明我们的探针可以报告脑缺血后基因转录本的上升。

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.