Mechanisms of axonal pathfinding in three dimensional matrices

描述(由申请人提供)：对控制轴突生长和指导的过程的详细了解对于了解神经系统的发育和在脊髓损伤后成功地设计切断神经元的再生是必不可少的。虽然许多负责促进或抑制生长以及吸引和排斥引导的分子已经被确定，但我们对轴突运动机制的大部分知识来自于对平坦、无特征、僵硬的底物的研究。然而，越来越多的证据表明，细胞的运动性对结构和机械环境很敏感。相反，在创造在体外和体内复杂环境中控制轴突生长的结构特征方面已经取得了重大进展，但在大多数情况下，负责运动调节的机制尚不清楚。这项拟议的研究的目标是利用成像和分析工具研究严格控制的机械和结构环境中的轴突引导，以前所未有的细节揭示亚细胞的形态和动力学。这些研究将阐明丝状伪足、局部粘连、机械力和引导因素在受控环境中的作用，并为以可预测的方式影响轴突运动的体内细胞外环境的操作提供关键信息，并为植入材料促进损伤后神经再生提供重要信息。我们的具体目标是：(1)确定丝状伪足-胶原纤维相互作用促进3D轴突运动和引导的机制；(2)评估基质成分对3D胶原-I基质中生长锥形态、轴突生长和轴突引导的影响，以及(3)检验限制在狭窄通道(模拟纤维限制)的轴突对非定向引导线索的反应较差的假设。这项研究将利用PI实验室开发的专用高速、高灵敏度旋转盘共焦显微镜系统，以及定制的图像分析软件。这项研究是一项跨学科的合作努力，涉及具有细胞生物学、物理学、材料科学和工程学专业知识的研究人员。这一努力将对轴突运动产生一个全新的视角，并将极大地提高我们理解和控制复杂环境中轴突生长的能力。 与公共卫生相关：目前还没有有效的治疗中枢神经系统损伤的方法。这项提案中描述的研究将为指导干预措施提供关键信息，以便在脊髓损伤后通过治疗损伤区域或设计能够为重新连接提供桥梁的植入物来允许神经再生。

DESCRIPTION (provided by applicant): A detailed understanding of the processes that control axon growth and guidance is essential for understanding the development of the nervous system and for engineering successful regrowth of severed neurons following spinal cord injury. While many of the molecules responsible for enhancing or inhibiting growth and for attractive and repulsive guidance have been identified, the majority of our knowledge of the mechanisms of axon motility comes from studies on flat, featureless, stiff substrates. There is accumulating evidence, however, that cell motility is sensitive to the structural and mechanical environ- ment. Conversely, there has been significant progress in creating structural features that control axon growth in complex environments in vitro and in vivo, but in most cases the mechanisms responsible for the modulation of motility are not known. The goal of the proposed research is to investigate axonal guidance in rigorously controlled mechanical and structural environments using imaging and analysis tools that will reveal subcellular morphology and dynamics with unprecedented detail. These studies will elucidate the roles of filopodia, focal adhesions, mechanical forces and guidance factors in controlled environments, and provides crucial information for the development of manipulations of the extracellular environment in vivo that impact axon motility in predictable ways, and for the engineering of implant materials to promote nerve regeneration after injury. Our specific aims are to: (1) Determine the mechanisms by which filopodia-collagen fibril interactions promote axon motility and guidance in 3D (2) Assess the effects of matrix composition on growth cone morphology, axon outgrowth, and axon guidance in 3D collagen-I matri- ces, and (3) Test the hypothesis that axons confined to narrow lanes, which mimic fibrillar confinement, are less responsive to non-directed guidance cues. This research will utilize a specialized high-speed, high sensitivity spinning disk confocal microscope system developed in the laboratory of the PI, as well as customized image analysis software. The research is an interdisciplinary collaborative effort involving re- searchers with expertise in cell biology, physics, materials science, and engineering. This effort will produce an entirely new perspective on axon motility and should significantly advance our ability to understand and control axon growth in complex environments. PUBLIC HEALTH RELEVANCE: There is currently no effective treatment for injuries to the central nervous system. The research described in this proposal will provide critical information for guiding interventions to allow nerves to regenerate after spinal injury either through treatment of the injured area or the design of implants that can provide bridges for reconnection.