Visualization of In Vivo Myelin Architecture Using Nonlinear Microscopy

描述(由申请人提供)：SC2试点项目奖申请的这一修订版来自年轻的研究人员，他建议开发一种非侵入性的方法，以足够的分辨率成像组织中的神经元，以研究髓鞘结构。如果成功，这项研究 可能会对神经再生和神经退行性疾病的研究产生重大影响。 评论者真的很喜欢这个写得很好的应用程序。它的优势包括新奇的 成像方法，项目的意义和潜在影响，PI及其导师的杰出资历，以及强大的发展和指导计划。其他优势是创新的方法，深思熟虑的目标，以及已经为研究开发的成像系统。也注意到了一些次要的弱点。其中包括对成像领域的相关发展、获得足够分辨率的可能性以及光在组织中传输的限制的讨论不足。有人认为，特派对前几位审查员的大多数批评都作出了很好的回应。 总体而言，审查小组认为，这是一位训练有素的年轻调查员提出的一份出色的申请。尽管该项目有一定的风险，但结果的潜在影响是重大的。建议的研究非常适合SC2机制，而且优秀的指导计划表明，PI将从SC2奖中受益匪浅。 摘要：髓鞘是轴突周围的一种特殊膜，它不仅在神经元信号转导中发挥重要作用，而且与S多发性硬化症、夏科-玛丽-图思病等多种临床疾病有关。轴突和神经胶质细胞之间的复杂相互作用是维持髓鞘厚度相对于轴突直径或G比率的最佳水平的关键机制。然而，由于缺乏体内测量髓鞘厚度的技术，对髓鞘形成的动态过程还没有很好的了解。在这里，我们建议开发一种非侵入性的、基于成像的方法来确定髓鞘厚度和G比率，而不需要外源性标记。我们的方法依赖于髓鞘薄层周围产生的非线性光学信号。与以前的方法不同，我们的新技术将在活细胞和组织中提供微米级的结构信息，因此适合于髓鞘形成、脱髓鞘和再髓鞘形成动力学的纵向研究。我们将进行适当的初步实验，以验证新方法与髓鞘结构之间的相关性。首先，我们将使用髓鞘共培养系统测试我们成像的敏感性。其次，我们将研究极化敏感性作为识别髓鞘结构域的一种途径。最后，我们将调查两者之间的相关性 在我们的度量和g比率之间使用一个发育中的动物模型。我们预计，我们提出的研究将有助于在活体内研究神经胶质细胞之间的动态相互作用，这是一种以最小动物牺牲为代价的轴突。此外，作为脱髓鞘疾病的诊断，我们的发展也可能具有临床意义。

DESCRIPTION (provided by applicant): This revision of an application for a SC2 Pilot Project Award is from young investigator who proposes to develop a non-invasive method for imaging neurons in tissue at sufficient resolution to study myelin structure. If successful, this research could potentially have a major impact on the study of neuro-regeneration and neuro-degenerative diseases. The reviewers really liked this well-written application. Its strengths include the novelty of the imaging method, the project's significance and potential impact, the outstanding qualifications of the PI and his mentors, and the strong development and mentoring plans. Additional strengths were the innovative approach, the well thought out aims, and the already developed imaging system for the studies. A few minor weaknesses were also noted. These included inadequate discussion of relevant developments in the imaging field, of the likelihood that sufficient resolution can be attained, and of the limits of light transmission in tissues. It was felt that te PI had responded well to most of the criticisms of the previous reviewers. Overall, the review panel felt that this is an excellent application from a well-trained young investigator. Although the project is somewhat risky, the potential impact of the results is significant. The proposed research is highly suited to the SC2 mechanism, and the excellent mentoring plan suggests that the PI will significantly benefit from an SC2 Award. ABSTRACT: Myelin is a specialized membrane around axon, which does not only play a significant role in neuronal signaling but is also implicated in several clinical conditions, such s multiple sclerosis and Charcot-Marie- Tooth disease. The complex interaction between axon and glial cells is the crucial mechanism that maintains an optimal level of the myelin thickness with respect to the axon diameter, or g-ratio. However, the dynamic process of myelin formation is not well understood partly because of the lack of technology for measuring the myelin thickness in vivo. Here we propose to develop a non-invasive, imaging-based methodology to determine the thickness of myelin and the g-ratio without exogenous labeling. Our method relies on a nonlinear optical signal generated around the lamellae of myelin. Unlike previous methods, our novel technique will provide micrometer-resolution structural information in live cells and tissue, therefore suited for longitudinal studies of the dynamics of myelination, demyelination, and remyelination. We will undertake appropriate initial experiments in order to validate the correlation between the new method and the myelin structure. First we will test the sensitivity of our imaging using a myelinating co-culture system. Secondly, we will study the polarization-sensitivity as a route to identify the myelin domains. Finally, we will investigate the correlation between our metric and g-ratio using a developing animal model. We anticipate that our proposed research will facilitate in vivo studies of the dynamic interaction between glial cells an axon with minimum animal sacrifice. Moreover, our development may also have a clinical relevance as a diagnostic for demyelination diseases.