Integrated Microfluidics for Nerve Regeneration Studies using Laser Nano-Axotomy

描述（由申请人提供）：了解神经再生和变性的生物学机制是开发人类神经变性疾病（如阿尔茨海默氏症、帕金森氏症、亨廷顿氏症和影响数百万人的其他神经系统疾病）的新疗法的重要一步。再生过程可以通过以受控的方式切断轴突，然后观察其再生长和功能恢复来研究。为了在体内切断轴突，需要高精度和非侵入性的切割工具。由于缺乏切断轴突（轴突切断术）的精确技术，迄今为止的研究仅限于复杂的生物体（小鼠和斑马鱼）。就在最近，我们证明了飞秒激光脉冲可以用于线虫秀丽隐杆线虫（C。elegans）并且那些轴突在功能上可以在手术后再生。这些超短激光脉冲的优点是它们能够蒸发极少量的组织，而不会加热或损伤周围的细胞。这种精确的外科技术的应用现在使得神经再生能够在具有简单神经系统的生物体内进行研究。由于简单的生物如C.由于秀丽线虫具有顺从的遗传学，飞秒激光轴突切断技术的应用将有助于快速鉴定影响神经再生和变性的基因和分子。本研究的目标是建立一个高通量的激光纳米手术平台，用于C。elegans in vivo.具体而言，将开发一种集成的微流体装置来捕获蠕虫。这种微流体陷阱将促进动物的适当固定，以使用飞秒激光脉冲对轴突进行精确的纳米手术，并对纳米手术后受损轴突的再生长进行成像。高通量筛选平台的开发需要集成用于纳米外科手术、喂养和成像的不同模块，以及通过计算机控制的自动化实现它们的同步。一个具有100个用于喂养单个蠕虫的腔室的平台将有助于对多个蠕虫进行自动化手术和筛选研究，从而大大减少时间和成本。为了成功开发用于体内神经再生/变性研究的高通量纳米手术平台，我们组建了一个多学科团队，该团队在三个关键领域具有专业知识：（1）生物材料的超快激光消融（Ben-Yakar博士），（2）微流体系统的制造和组装（Chronis博士），以及（3）C. elegans（Dr. Bargmann）.与高通量微流体平台的开发同时，将研究两个重要的神经再生/变性问题：（1）轴突再生长和损伤轴突变性的延时体内成像和（2）Sir 2基因家族和药物白藜芦醇在损伤轴突再生中的作用。了解神经再生和退化的生物学机制是开发人类神经退行性疾病（如阿尔茨海默氏症、帕金森氏症、亨廷顿氏症和影响数百万人的其他神经系统疾病）的新疗法的重要一步。建立了一种适用于简单生物体如C. elegans在体内将有助于快速识别影响神经再生和退化的基因和分子。

DESCRIPTION (provided by applicant): Understanding the biological mechanisms of nerve regeneration and degeneration is an important step towards the development of novel therapies for human neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and other neurological disorders affecting millions of people. The regeneration processes can be studied by severing an axon in a controlled manner and then observing its re-growth and functional recovery. To sever an axon in-vivo, a high precision and non-intrusive cutting tool is required. In the absence of precision techniques for severing axons (axotomy), investigations have so far been limited to complex organisms (mouse and zebrafish). Just recently we demonstrated that femtosecond laser pulses can be used for axotomy in the roundworm Caenorhabditis elegans (C. elegans) and those axons functionally can regenerate after the operation. The advantage of these ultrashort laser pulses is their ability to evaporate an extremely small volume of tissue without heating or damaging the surrounding cells. Application of this precise surgical technique now enables nerve regeneration to be studied in-vivo in organisms with simple nervous systems. Since simple organisms such as C. elegans have amenable genetics, application of the femtosecond laser axotomy technique will help in the rapid identification of genes and molecules that affect nerve regeneration and degeneration. The goal of this research project is to develop a high-throughput laser nano-surgery platform for axon regeneration & degeneration studies in C. elegans in-vivo. Specifically, an integrated microfluidic device will be developed to trap the worms. This microfluidic trap will facilitate the appropriate immobilization of the animals for precision nanosurgery of axons using femtosecond laser pulses and for imaging the re-growth of the injured axons following nanosurgery. Development of a high throughput screening platform requires integration of different modules for nanosurgery, feeding, and imaging and their synchronization through computer controlled automation. A platform having 100's of chambers for feeding of individual worms will facilitate automated surgical and screening studies of multiple worms, thus greatly reducing time and cost. For successful development of a high-throughout nanosurgery platform for in-vivo nerve regeneration/degeneration studies, we have assembled a multi-disciplinary team with expertise in three critical areas (1) ultrafast laser ablation of bio-materials (Dr. Ben-Yakar), (2) fabrication and assembly of microfluidic systems (Dr. Chronis), and (3) developmental neurobiology of C. elegans (Dr. Bargmann). Concurrently with the development of a high-throughput microfluidic platform, two important nerve regeneration/degeneration problems will be investigated: (1) time-lapse in-vivo imaging of axon re-growth and degeneration of injured axons and (2) the role of the Sir2 family of genes and the drug resveratrol in regeneration of injured axons. Understanding the biological mechanisms of nerve regeneration and degeneration is an important step towards the development of novel therapies for human neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and other neurological disorders affecting millions of people. Development of a high-throughput laser nano-surgery platform applicable to simple organism such as C. elegans in-vivo will help in the rapid identification of genes and molecules that affect nerve regeneration and degeneration.