Neurovascular Mechanisns of Brain Function and Disease

描述（由申请人提供）：大脑中内源性基因表达的改变通常伴随着神经系统疾病。基因或细胞被用来纠正基因转录，这样大脑就可以自我修复。目前，大多数基因转录检测技术需要活检或尸检样本。切除组织样本的侵入性外科手术严重限制了其益处，尤其是对我们试图治愈的细胞。我们的目标是开发方法成像和定量比较内源性基因表达在转录水平上使用磁共振（MR）在活体动物或人类受试者。我们使用短硫代修饰的寡脱氧核苷酸（sODN）和超顺磁性氧化铁纳米颗粒（SPION，一种MR T2试剂）制作了一种新型MR探针。本应用程序中概述的工作研究了这种对比探针作为活体动物mRNA转录物的生物标志物的效用。我们设计了三种MRI探针：两种探针与基质金属蛋白酶-9 （sODN-mmp9）或β -肌动蛋白（sODN-bactin） mRNA序列互补，一种随机s-ODN （sODN-Ran）不与mRNA序列互补。SPION-Ran探测器将作为控制装置。我们将评估使用MR实现最佳成像内源性基因表达的条件。具体的动物将演示：目的1：在小鼠大脑的高分辨率MRI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，用于活体动物的MRI检测，并用于尸检样本的组织学（氧化铁）和结合试验（SPION-bactin）验证。我们选择β -肌动蛋白mRNA作为靶标，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)在活体动物中注射不同剂量的SPION-bactin后，使用MRI选择最佳的SPION-retention；我们将证明大脑中SPION的摄取依赖于sodn连锁；(b)以最佳剂量输注SPION-bactin后，细胞内存在氧化铁，(c)内化的SPION-bactin与其靶mRNA结合。目的2：活C57black6小鼠脑SPION-mmp9的保留可预测60或90分钟双侧颈动脉闭塞引起的脑水肿。假设是脑mmp-9 mRNA转录报告mmp-9的表达。为了支持这一假设，我们将证明：(a)脑卒中后mmp-9 mRNA表达的升高与脑水肿呈正相关；(b)脑卒中治疗小鼠中SPION-mmp9的保留率高于假手术小鼠。具体来说，我们将利用脑卒中治疗动物和假手术动物之间的R2*图相减，确定脑卒中治疗动物SPION-mmp9保留的热点。此外，我们将比较SPION-mmp9保留在野生型和mmp-9敲除菌株脑卒中诱导损伤中的热点。PI: Liu, Philip K. PhD项目描述：脑水肿是一种神经系统疾病，使人类多种疾病的恢复变得复杂。人类心脏骤停可引起脑水肿，尽管通过各种手段恢复循环（Roine et al. 1993; Fujioka et al. 1994; Xiao 2002）；脑损伤预示着不良的神经预后。目前，很少有模型可以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脑损伤的认识，我们提出了一项全球脑缺血再灌注模型的申请，以研究雄性C57black/6小鼠模拟心脏骤停的脑病理生理（Liu et al. 1996）。我们的长期目标是建立新的方法，利用MRI造影剂来了解活体患者基因表达的病理变化，为进一步评估治疗的疗效和毒性提供依据。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的升高。

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.