Multiplexed Microfluidic Gradients for Axon Guidance

用于轴突引导的多重微流体梯度

基本信息

批准号：
8109748
负责人：
ALBERT FOLCH
金额：
$ 34.18万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-06-15 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8109748
关键词：
Affect Anatomy Anterior Axon Benign Binding Blindness Blood Vessels Cell Count Cell Culture Techniques Cell Separation Cells Complex Computer software Cues Culture Media Defect Development Embryo Environment Ephrins Erinaceidae Extracellular Matrix Eye Family Goals Growth Growth Factor Image Image Analysis In Vitro Individual Lasers Length Measurement Measures Microfluidics Molecular Mus Nerve Regeneration Nervous System Physiology Neurons Optic Nerve Pathology Pattern Phototoxicity Play Population Procedures Process Proteins Retina Retinal Retinal Ganglion Cells Screening procedure Shapes Signal Transduction Solutions Source Speed System Techniques Technology Testing Time Visual Pathways axon growth axon guidance base brain tissue cell type combinatorial design human NTN1 protein in vivo insight mimicry molecular imaging movie nervous system development nervous system disorder netrin-1 neurodevelopment neuronal cell body research study response spatiotemporal tool user-friendly

项目摘要

DESCRIPTION (provided by applicant): During development of the nervous system the response of growing axons to their environment is critical to the formation of the complex wiring pattern between neurons. Growth and guidance factors combined with extracellular matrices influence the speed and direction of axonal growth. Although much progress has been made in identifying the factors that influence axonal growth, as well as how axons respond to these factors individually, much less is known about how axons behave in response to the combined effects of multiple factors. As a complementary approach to present in vivo molecular imaging approaches, we propose to develop an in vitro environment that potentially mimics some of the complexity found in vivo, in particular the development of the anterior visual pathway. In this system, the axon trajectories are simple, multiple relevant guidance molecules have been identified already (many tested with explants in vitro), and a common cause of blindness (Optic Nerve Hypoplasia) is associated with defects in this process. Additionally, the patterns of guidance molecules found on the flat anatomy of the retina are ideally suited to mimicking by micropatterning and microfluidics techniques. This mimicry will be accomplished by combining microfluidics patterning of diffusible gradients and laser patterning of substrate-bound axon pathfinding cues, including axon guidance factors and extracellular matrix molecules. As a source of highly homogeneous cell populations, we will isolate mouse retinal ganglion cells (RGCs), a cell type that responds to Netrin-1 gradients. For experiments designed to maximize the integrity of the cells (isolation procedures are damaging to cells), we will use retinal explants and we will microfluidically isolate the axons from their somas. RGCs (or their axons) will be exposed to various soluble factors that have previously been shown to affect their axon growth in vivo. The new microfluidic systems will allow us to test the combinatorial effects of multiple factors on the direction and speed of axonal growth of RGCs. These experiments will allow us to quantitatively examine the basic principles that govern axon pathfinding in the development of the anterior visual pathway. This information will help to better understand the basis of developmental defects in axon growth that alter the organization and function of the nervous system. PUBLIC HEALTH RELEVANCE: The study of axon guidance has been limited to a single signal gradient. In vivo, neurons encounter multiple signals (both bound to substrate and in solution) and must make choices in an information rich environment. The proposed study will probe axon guidance interactions of unprecedented complexity as well as with unprecedented measurement precision, which will significantly further our understanding of axon growth on a cellular and molecular level, neural development as a whole, and may provide insight on treating neurological disorders, nerve regeneration, and vascular pathologies.

描述（由申请人提供）：在神经系统的发展过程中，生长轴突对环境的反应对于神经元之间的复杂布线模式的形成至关重要。生长和引导因素与细胞外矩阵结合影响轴突生长的速度和方向。尽管在确定影响轴突生长的因素以及轴突如何对这些因素响应的因素方面取得了很多进展，但对轴突如何对多种因素的综合作用进行响应的了解少了。作为呈现体内分子成像方法的互补方法，我们建议开发一种体外环境，该环境可能模仿体内发现的某些复杂性，尤其是前视觉途径的发展。在该系统中，轴突轨迹很简单，已经确定了多个相关的引导分子（许多在体外测试了epplants），并且在此过程中，盲目的常见原因（视神经下降症）与缺陷有关。此外，在视网膜平坦解剖结构上发现的引导分子的模式非常适合模仿微型图案和微流体技术。这种模仿将通过结合可扩散梯度的微流体图案和基质结合的轴突探索线索的激光模式，包括轴突引导因子和细胞外基质分子。作为高度均匀细胞群体的来源，我们将分离小鼠视网膜神经节细胞（RGC），这是对Netrin-1梯度响应的细胞类型。对于旨在最大化细胞完整性的实验（隔离程序对细胞造成了损害），我们将使用视网膜外植体，并将微流体从其somas中分离出。 RGC（或其轴突）将暴露于以前已证明会影响其体内轴突生长的各种可溶性因子。新的微流体系统将使我们能够测试多种因素对RGC轴突生长的方向和速度的组合效应。这些实验将使我们能够定量检查在前视觉途径发展中控制轴突途径的基本原理。这些信息将有助于更好地理解轴突生长中改变神经系统组织和功能的轴突生长中的发展基础。公共卫生相关性：轴突指导的研究仅限于单个信号梯度。在体内，神经元遇到多个信号（均与底物和解决方案绑定），并且必须在信息丰富的环境中做出选择。拟议的研究将探测前所未有的复杂性以及前所未有的测量精度的轴突指导相互作用，这将显着进一步进一步了解我们对细胞和分子水平上轴突生长的理解，整个神经发育，并可能提供有关治疗神经系统疾病，NERVERENER RENEVERENER RENEVERANES和VASSENERANE和血管病理学的洞察力。