Investigating the Early Embryonic Murine Heart Using Optical Coherence Tomography

描述(申请人提供)：胚胎小鼠心脏是人类心脏发育的一个非常重要的模型，因为它具有与人类心脏相似的四腔结构，妊娠期短，并且基因组图谱完整，易于修改和操作。此前，缺乏适当的成像技术阻碍了揭示控制小鼠早期心脏发育的正常/异常机制的进展。这个五年计划中描述的多学科项目将开发、调查和验证成像和分析工具，以快速和彻底地研究活的胚胎小鼠心脏，其空间和时间分辨率以前是不可能的。这些工具是基于新兴的光学相干层析成像(OCT)技术，该技术能够非破坏性和实时地对小生物组织样本进行微米级分辨率成像。与鸟类和斑马鱼模型相比，小鼠胚胎在关键的早期成环阶段的体外培养方面面临着更大的挑战，因此提出了独特的成像挑战。我们将开发培养小鼠胚胎的方法和技术，使其能够正常发育并促进光学成像。OCT在胚胎成像方面的巨大潜力是显而易见的。我们将开发这项技术，通过提高成像速度、分辨率和扫描技术来充分实现这一潜力，以实现对早期胚胎小鼠心脏的形态以及收缩和血流动力学功能的完整表征。数据解释是利用成像技术成功进行调查的关键。我们将开发一套先进的2D/3D图像预处理、分析和可视化工具，以便于对采集的图像数据进行检查和比较。这些工具包括降噪、配准、强度不均匀补偿、使用不透明度优化的可视化、半自动和监督3D图像分割以及组织位移和血流映射，将有助于从这些独特的数据中进行新的观察和发现。一组基线实验将确定我们的OCT技术和图像分析工具在最戏剧性的心脏形态形成阶段的有用性。我们将比较OCT在体外获得的功能测量结果与在子宫中使用显微超声获得的结果。我们将研究NMHC-IIB转基因(和-/-)小鼠血流动力学改变对心跳的影响。最后，我们将根据OCT数据将基因表达与功能测量相关联。这项工作的最终产品将是一套基于OCT的新型成像工具，与改进的胚胎培养技术合作，这些工具已经过优化和验证，用于研究发育中的小鼠心脏。所开发的技术的适用性不仅将扩展到其他心脏缺陷模型，还将扩展到发育生物学的许多其他领域。在验证目标中测试的假设将解决有关导致先天性心脏缺陷的最早功能变化的关键开放问题。公共卫生相关性：只有在心脏首次发育时了解缺陷的来源，才能开发出先天性心脏缺陷的早期医学治疗方法。缺乏一种理想的方式来成像这些微小的、几乎是微观的心脏的结构和功能，限制了我们回答这些问题的能力。小鼠胚胎心脏的OCT成像可以满足这一需求，我们将开发和验证实现这一潜力的技术和方法。

DESCRIPTION (provided by applicant): The embryonic murine heart is a very important model of human heart development due to its four- chambered structure similar to the human heart, a short gestation period and a completely mapped genome which is easy to alter and manipulate. Lack of an appropriate imaging technology has previously hindered progress in uncovering the normal/abnormal mechanisms that govern early heart development in mice. The multi-disciplinary project described in this five-year proposal will develop, investigate and validate imaging and analysis tools to rapidly and thoroughly investigate the living embryonic murine heart at a level of spatial and temporal resolution previously not possible. These tools are based on the emerging technology of optical coherence tomography (OCT), which is capable of micrometer-scale resolution imaging of small biological tissue samples non-destructively and in real time. Compared to avian and zebrafish models, the murine embryo is far more challenging to culture in vitro in critical early looping stages and thus presents unique imaging challenges. We will develop methods and technology to culture mouse embryos that enable normal development and facilitate optical imaging. The significant potential of OCT for embryonic imaging is clear. We will develop the technology to fully realize this potential by improving imaging speed, resolution and scanning technology to enable complete characterization of the morphology and contractile and hemodynamic function of the early embryonic murine heart. Data interpretation is a key to the success of investigations using imaging. We will develop a suite of advanced tools for 2D/3D image preprocessing, analysis and visualization that will facilitate examination and comparison of the acquired image data. These tools, including noise reduction, registration, intensity inhomogeneity compensation, visualization using opacity optimization, semi-automatic and supervised 3D image segmentation, and tissue displacement and blood flow mapping, will facilitate new observations and discovery from these unique data. A set of baseline experiments will establish the usefulness of our OCT technology and image analysis tools during the stages of most dramatic cardiac morphogenesis. We will compare functional measurements obtained in vitro from OCT with those obtained in utero using micro ultrasound. We will investigate the effect of altered hemodynamics in NMHC-IIB transgenic ( and -/-) mice on heart looping. Finally, we will correlate gene expression with functional measurements made from OCT data. The end product of this effort will be a novel set of imaging tools based on OCT, partnered with improved embryo culturing technology, which have been optimized and validated for investigating the developing murine heart. The applicability of the developed technology will not only extend to other models of heart defects, but also to many other fields of developmental biology. The hypothesis tested in the validation aim will address critical open questions regarding the earliest functional changes leading to congenital heart defects. PUBLIC HEALTH RELEVANCE: Early medical treatments of congenital heart defects can only be developed if we understand the origins of the defects while the heart is first developing. The lack of an ideal way to image the structure and function of these tiny, almost microscopic hearts has limited our ability answer these questions. OCT imaging of embryonic mouse heart can fill this need and we will develop and validate the technology and methods to reach this potential.