MRI contrast for molecular and cellular imaging of the brain

用于大脑分子和细胞成像的 MRI 对比

• 批准号: 7735334
• 负责人: Alan Koretsky
• 金额: $ 280.79万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7735334
• 关键词: Actins; Animals; Area; Binding; Binding Proteins; Binding Sites; Biological; Biological Process; Blood - brain barrier anatomy; Brain; Calcium; Calcium Channel; Cell physiology; Cells; Chromosome Pairing; Clinical; Complex; Contrast Media; Coupling; Cytoskeleton; Detection; Development; Disease; Disorder by Site; Disruption; Dyes; Effectiveness; Endocytosis; Engineering; Enzymes; Ferritin; Gene Expression; Goals; Hepatocyte; Image; Imaging Techniques; In Transferrin; In Vitro; Injection of therapeutic agent; Ions; Label; Ligands; Magnetic Resonance Imaging; Manganese; Measures; Metals; Microfabrication; Molecular; Molecular Weight; Monitor; Neurons; Nickel; Particle Size; Personal Satisfaction; Phase; Pliability; Pre-Clinical Model; Process; Production; Range; Rattus; Reporter; Reporting; Rodent; Shapes; Signal Transduction; Simulate; Solutions; Specificity; Stream; Stroke; Structure; Synapses; Techniques; Testing; Tissues; Transferrin; Transplantation; Water; Work; base; cell motility; cell type; design; in vivo; interest; iron oxide; migration; molecular imaging; molecular/cellular imaging; nanosized; nerve stem cell; novel; olfactory bulb; particle; polymerization; precursor cell; receptor; relating to nervous system; research study; simulation; size; success; tumor; voltage

There is rapidly increasing interest in developing molecular imaging approaches that enable traditional radiological imaging techniques to obtain a wide range of information about molecular and cellular processes that occur in normal and diseased tissue. A range of information is considered important such as the ability to monitor cell migration, the development of reporters that enable imaging of gene expression, the development of robust strategies to image receptors, and the development of environmentally sensitive agents that can be used to detect the presence of specific enzymes or monitor changes in ion status such as increases in intracellular calcium. The long term goals of this work are to develop strategies that enable MRI contrast that is sensitive to a wide range of molecular and cellular processes. This work builds on over 15 years of work where we have demonstrated the first MRI strategy for detecting gene expression, the first MRI approach for monitoring a surrogate of calcium influx, the first MRI approach for performing neuronal track tracing, and the first MRI approach for monitoring the migration of single cells in vivo. These all represented initial reports by any radiological imaging technique which enabled these processes to be measured. These techniques are finding widespread application to imaging pre-clinical models of a broad range of diseases. Based on this past track record of success we continue to look for novel ways to develop MRI contrast specific for biological processes. Over the past year we have made progress in all of the specific aims. Aim 1: Develop iron oxide based contrast for labeling and imaging the migration of endogenous neural stem cells. Over the past few years we have demonstrated the unique advantages of micron sized iron oxide particles for MRI of specific cells. Single cells can be detected and indeed, single particles within single cells can be detected. The main paradigm for MRI of cell migration is to label cells ex vivo and monitor migration after transplantation into an animal. These studies have traditionally required very efficient labeling using nano sized particles. The ability to detect a single particle enables inefficient labeling strategies. In particular we have demonstrated that injection of particles into the ventricles of the rat brain enables particles to be taken up by neural precursors in the subventricular zone and MRI can monitor the migration of cells to the oflactory bulb. Over the past year we have demonstrated that the micron particles can be used as carriers of fluorescent molecules that enable cells to be labeled for morphological studies or with calcium sensitive dyes to enable monitoring of calcium transients. Furthemore, we have quantitated the distribution of cells that are labeled and have found all expected cell types in the rostral migratory stream labeled to about 30% efficiency. Inhibition of neuronal precursor production demonstrated that contrast detected in MRI was due to migration of precursor cells. In the coming year we will develop techniques for more efficiently delivering particles to precursor cells. We will determine if single cells can be detected and we will develop techniques that enable quantitation of the distribution of new cells in the olfactory bulb. Furthermore we will test if this labeling strategy enables monitoring cell migration to sites of disease (tumors), damage (stroke), or areas where plasticity is occuring. Aim 2: Apply microfabrication techniques to manufacture unique metal structures that may be valuable for MRI contrast. Iron oxide particles commonly used for MRI are very potent contrast agents enabling detection of single mciron sized particles. However, due to bulk phase manufacture of particles they are not very uniform and they do not contain very high content of metal. A solution to this problem is to use modern microfabrication techniques to manufacture metal based, micron sized contrast agents. To begin this work we have explored a variety of approachs to microfabrication of MRI contrast agents. We have demonstrated that precise definition of shapes and spacing of microfabricated structures leads to novel MRI agents. As expected mciron sized microfabricated nickel structures are very potent MRI contrast agents. Microfabrication gives us a great deal of flexibility to make structures that may have novel uses. For example, particles spaced at distances much smaller than an MRI voxel can be distinguished and water associated with properly designed structures can be distinguished. These initial results are all in phantoms and over the coming year we will develop strategies that enable us to label cells with these microfabricated particles. Aim 3: Develop novel delivery mechanisms to extend the applicability of manganese enhanced MRI. Over the past ten years we have demonstrated the remarkable utility of the manganese ion for MRI contrast. Manganese ion enters cells on ligand or voltage gated calcium channels and so can be used as an MRI agent to monitor calcium influx. Once inside of neurons, manganese will move in an anterograde direction and cross functional synapses enabling neuronal networks to be imaged with MRI. Finally, manganese given systemically gives cytoarchitectural information about the rodent brain. These successed have us interested in broadening the ways in which manganese ion can be delivered to cells. Over the past year we have made transferrin-manganese complexes. When bound to transferrin manganese is a poor MRI contrast agent. However, when transferrin is taken up by cells it can release manganese which is then trapped intracellularly. Thus, transferrin manganese is an agent that monitors the successful endocytosis of the transferrin by its receptor. Experiments in hepatocytes and in brain demonstrate that this strategy is succesful and gives efficient contrast. We have begun to hunt for small molecular weight chelates that mimic transferrin in that they are poor MRI contrast agents until endocytosis occurs after which they would activate. This would enable coupling to any molecule that can recognize a target and trigger endocytosis. It is hypothesized that the combined targetting plus effective endoctosis will add a level of specificity to targeted contrast agents. In addition, the fact that manganese has a final intracellular distribution increases its efficacy as a contrast agent. Furthermore, we will begin to engineer the transferrin binding site so that it can be readily fused to other domains to recognize other receptors. Aim 4: Develop strategies that enable cellular processes to alter the relaxivity of MRI contrast agents. In specific aim 3 we demonstrated a way in which a normal biological process (endocytosis of transferrin-Mn) can alter the effectiveness of an MRI contrast agent. It would be very exciting to find ways in which this can occur which are sensitive to other biological processes. To this end we have simulated the effects of changing spacing of ferritin, a known biologically occuring iron oxide particle. These simulations show that the effects of ferritin on MRI signal are very sensitive to the specific spacing of ferritin molecules. This opens the possibility of coupling ferritin to molecules that change aggregation state to make MRI reporters of cellular processes. In particular, we have demonstrated in vitro that ferritn-actin fusions can make MRI sensitive to the state of actin polymerization. It is well known that the state of the cytoskeleton reports on a wide range of biological states. The results with ferritin open the possibility of having an MRI reporter of cytoskeleton or of cytoskeleton binding proteins.

人们对开发分子成像方法的兴趣迅速增加，这些方法使传统的放射学成像技术能够获得有关正常组织和患病组织中发生的分子和细胞过程的广泛信息。 一系列信息被认为是重要的，例如监测细胞迁移的能力，能够成像的记者的发展，可用于形象受体的强大策略的发展以及可用于检测特定酶的存在或监测离子状态（例如细胞内钙的增加）的变化的环境敏感剂的发展。 这项工作的长期目标是制定策略，以使MRI对比对广泛的分子和细胞过程敏感。 这项工作建立在超过15年的工作基础上，我们证明了第一种检测基因表达的MRI策略，这是监测钙涌入的替代物的第一种MRI方法，这是执行神经元跟踪的第一种MRI方法，也是监测VIVO中单细胞迁移的第一种MRI方法。 所有这些都代表了任何放射成像技术的初始报告，该技术使这些过程得以测量。 这些技术正在发现广泛应用多种疾病的临床前模型。 基于过去的成功记录，我们继续寻找新的方法来开发针对生物过程的特定的MRI对比度。 在过去的一年中，我们在所有具体目标方面取得了进步。 目标1：开发基于氧化铁的对比度，用于标记和成像内源性神经干细胞的迁移。 在过去的几年中，我们证明了特定细胞MRI的微米大小氧化铁颗粒的独特优势。 可以检测到单个细胞，实际上，可以检测到单个细胞内的单个颗粒。 细胞迁移MRI的主要范式是将细胞在体内标记并监测移植到动物后的迁移。 传统上，这些研究需要使用纳米尺寸颗粒进行非常有效的标记。 检测单个粒子的能力可实现效率低下的标记策略。特别是我们已经证明，将颗粒注射到大鼠脑的心室中，使颗粒可以被脑室下区域的神经前体吸收，MRI可以监测细胞向乳酸鳞茎的迁移。 在过去的一年中，我们已经证明，微米颗粒可以用作荧光分子的载体，使细胞能够标记用于形态学研究或使用钙敏感染料来启用钙瞬变。 此外，我们已经量化了标记的细胞分布，并在标记为约30％的效率约为30％的Rostral迁移流中发现了所有预期的细胞类型。 神经元前体产生的抑制表明，在MRI中检测到的对比是由于前体细胞的迁移。在来年，我们将开发技术，以更有效地将颗粒传递到前体细胞。 我们将确定是否可以检测到单个细胞，并将开发能够定量嗅球中新细胞的分布的技术。 此外，我们将测试该标签策略是否可以监测细胞迁移到疾病部位（肿瘤），损伤（中风）或发生可塑性的区域。 AIM 2：应用微型制造技术来制造可能对MRI对比有价值的独特金属结构。 通常用于MRI的氧化铁颗粒是非常有效的对比剂，可检测单个McIron尺寸颗粒。 但是，由于粒子的散装相生产，它们不是很均匀，并且不包含很高的金属含量。 解决此问题的一种解决方案是使用现代的微加工技术来生产基于金属的微米大小对比剂。 为了开始这项工作，我们探讨了MRI对比剂微加工的多种方法。 我们已经证明了形状的精确定义和微生物结构的间距会导致新型MRI剂。 正如预期的那样，McIron尺寸的微生物镍结构是非常有效的MRI对比剂。微分化为我们提供了很大的灵活性来制造可能具有新颖用途的结构。 例如，可以区分比MRI体素小得多的距离的颗粒可以区分与正确设计的结构相关的水。 这些最初的结果全都在幻象上，在来年，我们将制定策略，使我们能够用这些微生物颗粒标记细胞。 AIM 3：开发新型的输送机制，以扩展锰增强MRI的适用性。 在过去的十年中，我们证明了MRI对比度的锰离子具有出色的效用。 锰离子进入配体或电压门控钙通道上的细胞，因此可以用作MRI剂来监测钙涌入。 一旦进入神经元，锰将沿顺行向移动，并交叉功能突触，使神经元网络能够使用MRI成像。 最后，锰从系统地提供有关啮齿动物大脑的细胞结构信息。 这些成功的使我们有兴趣扩大锰离子可以传递到细胞的方式。在过去的一年中，我们制作了转铁蛋白 - 曼加尼综合体。 当绑定到转铁蛋白时，MRI对比剂较差。 但是，当细胞吸收转铁蛋白时，它会释放锰，然后将其细胞内捕获。 因此，转铁蛋白锰是一种通过其受体成功的转铁蛋白内吞作用的药物。 肝细胞和大脑中的实验表明，这种策略是成功的，并提供了有效的对比度。 我们已经开始寻找小分子量螯合物，因为它们模仿了较差的MRI对比剂，直到发生内吞作用，之后它们会激活。 这将使能够耦合到可以识别靶标并触发内吞作用的任何分子。假设合并的靶向加上有效的内吞噬作用将为靶向对比剂增加一定程度的特异性。 此外，锰具有最终的细胞内分布这一事实增加了其作为对比剂的功效。 此外，我们将开始设计转铁蛋白结合位点，以便可以轻松地将其融合到其他域以识别其他受体。 目标4：制定使细胞过程改变MRI对比剂的松弛性的策略。 在特定目标3中，我们证明了一种正常生物学过程（转铁蛋白-MN的内吞作用）可以改变MRI对比剂的有效性。 找到可能发生对其他生物学过程敏感的方法将非常令人兴奋。 为此，我们模拟了铁蛋白的间距变化的影响，铁蛋白是一种已知的生物学上发生的氧化铁颗粒。 这些模拟表明铁蛋白对MRI信号的影响非常敏感铁蛋白分子的特定间距。 这打开了将铁蛋白偶联到改变聚集态以使MRI记者成为细胞过程的可能性。 特别是，我们在体外证明了Ferritn-Actin融合可以使MRI对肌动蛋白聚合状态敏感。 众所周知，细胞骨架的状态报告了广泛的生物态。 铁蛋白的结果开放了具有细胞骨架或细胞骨架结合蛋白的MRI报告基因的可能性。