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成像技术能够获得有关正常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、能够对基因表达进行成像的报告基因的开发、对受体成像的稳健策略的开发以及可用于检测细胞迁移的环境敏感剂的开发。特定酶的存在或监测离子状态的变化，例如细胞内钙的增加。 这项工作的长期目标是开发策略，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，我们已经展示了第一个用于检测基因表达的 MRI 策略、第一个用于监测钙流入替代物的 MRI 方法、第一个用于执行神经元轨迹追踪的 MRI 方法以及第一个用于监测体内单细胞的迁移。 这些都代表了任何能够测量这些过程的放射成像技术的初步报告。 这些技术广泛应用于多种疾病的临床前模型成像。 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 MRI 造影剂。 过去一年，我们在所有具体目标上都取得了进展。 目标 1：开发基于氧化铁的造影剂，用于标记内源性神经干细胞的迁移并对其进行成像。 在过去的几年中，我们已经展示了微米级氧化铁颗粒用于特定细胞 MRI 的独特优势。 可以检测单个细胞，并且实际上可以检测单个细胞内的单个颗粒。 细胞迁移 MRI 的主要范例是离体标记细胞并监测移植到动物体内后的迁移。 传统上，这些研究需要使用纳米尺寸的颗粒进行非常有效的标记。 检测单个粒子的能力使得标记策略效率低下。特别是，我们已经证明，将颗粒注射到大鼠脑室中，可以使颗粒被脑室下区的神经前体细胞吸收，并且 MRI 可以监测细胞向泌乳球的迁移。 在过去的一年里，我们已经证明微米颗粒可以用作荧光分子的载体，使细胞能够被标记以进行形态学研究，或使用钙敏感染料来监测钙瞬变。 此外，我们还对标记细胞的分布进行了定量，并发现头端迁移流中所有预期的细胞类型都被标记为约 30% 的效率。 神经元前体产生的抑制表明，MRI 中检测到的对比度是由于前体细胞的迁移所致。在接下来的一年中，我们将开发更有效地将颗粒递送至前体细胞的技术。 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分布的技术。 此外，我们将测试这种标记策略是否能够监测细胞迁移到疾病部位（肿瘤）、损伤部位（中风）或发生可塑性的区域。 目标 2：应用微加工技术来制造可能对 MRI 对比有价值的独特金属结构。 常用于 MRI 的氧化铁颗粒是非常有效的造影剂，能够检测单个微米大小的颗粒。 然而，由于颗粒的本体相制造，它们不是很均匀，并且它们不包含非常高含量的金属。 该问题的解决方案是使用现代微加工技术来制造基于金属的微米级造影剂。 为了开始这项工作，我们探索了各种 MRI 造影剂微加工方法。 我们已经证明，精确定义微加工结构的形状和间距可以产生新型 MRI 试剂。 正如预期的那样，mciron 尺寸的微加工镍结构是非常有效的 MRI 造影剂。微加工为我们提供了很大的灵活性来制造可能具有新用途的结构。 例如，可以区分间隔距离远小于 MRI 体素的颗粒，并且可以区分与正确设计的结构相关的水。 这些初步结果都是幻影，在未来的一年里，我们将制定策略，使我们能够用这些微加工颗粒标记细胞。 目标 3：开发新的传递机制以扩展锰增强 MRI 的适用性。 在过去的十年中，我们已经证明了锰离子在 MRI 对比方面的显着效用。 锰离子通过配体或电压门控钙通道进入细胞，因此可用作 MRI 试剂来监测钙流入。 一旦进入神经元内部，锰将沿顺行方向移动并跨功能突触，使神经元网络能够通过 MRI 进行成像。 最后，系统地给予锰可以提供有关啮齿动物大脑的细胞结构信息。 这些成功使我们对拓宽将锰离子输送到细胞的方式感兴趣。在过去的一年里，我们制备了转铁蛋白-锰复合物。 当与转铁蛋白结合时，锰是一种较差的 MRI 造影剂。 然而，当转铁蛋白被细胞吸收时，它可以释放锰，然后将其捕获在细胞内。 因此，转铁蛋白锰是一种通过其受体监测转铁蛋白成功内吞作用的试剂。 肝细胞和大脑中的实验证明这种策略是成功的并且提供了有效的对比。 我们已经开始寻找模拟转铁蛋白的小分子量螯合物，因为它们是较差的 MRI 造影剂，直到发生内吞作用，然后它们才会被激活。 这将能够与任何能够识别靶标并触发内吞作用的分子偶联。据推测，联合靶向加上有效的内吞作用将增加靶向造影剂的特异性水平。 此外，锰具有最终的细胞内分布这一事实增加了其作为造影剂的功效。 此外，我们将开始设计转铁蛋白结合位点，以便它可以轻松地与其他结构域融合以识别其他受体。 目标 4：制定策略，使细胞过程能够改变 MRI 造影剂的弛豫度。 在具体目标 3 中，我们展示了一种正常生物过程（转铁蛋白-Mn 的内吞作用）可以改变 MRI 造影剂有效性的方法。 找到对其他生物过程敏感的实现这种情况的方法将是非常令人兴奋的。 为此，我们模拟了改变铁蛋白（一种已知的生物氧化铁颗粒）间距的影响。 这些模拟表明，铁蛋白对 MRI 信号的影响对铁蛋白分子的特定间距非常敏感。 这开启了将铁蛋白与改变聚集状态的分子偶联以形成细胞过程的 MRI 报告剂的可能性。 特别是，我们在体外证明了铁蛋白-肌动蛋白融合可以使 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状态。 铁蛋白的结果开启了细胞骨架或细胞骨架结合蛋白的 MRI 报告器的可能性。