Investigating the Early Embryonic Murine Heart Using Optical Coherence Tomography

描述（申请人提供）：小鼠胚胎心脏具有与人类心脏相似的四腔结构，妊娠期短，基因组图谱完整，易于改变和操作，是人类心脏发育的重要模型。缺乏合适的成像技术阻碍了揭示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、研究和验证成像和分析工具，以便在以前不可能实现的空间和时间分辨率水平上快速彻底地研究活体胚胎小鼠心脏。这些工具基于光学相干断层扫描（OCT）的新兴技术，该技术能够对小生物组织样本进行微米级分辨率的无损实时成像。与鸟类和斑马鱼模型相比，小鼠胚胎在关键的早期循环阶段体外培养更具挑战性，因此呈现出独特的成像挑战。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。我们将通过提高成像速度、分辨率和扫描技术来开发技术，以充分实现这一潜力，从而完整地表征早期胚胎小鼠心脏的形态、收缩和血流动力学功能。数据解释是成像研究成功的关键。我们将开发一套先进的工具，用于二维/三维图像的预处理、分析和可视化，以方便对获取的图像数据进行检查和比较。这些工具，包括降噪、配准、强度非均匀性补偿、使用不透明度优化的可视化、半自动和监督3D图像分割、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发生阶段的有用性。我们将比较体外OCT获得的功能测量值与子宫内微超声获得的功能测量值。我们将研究NMHC-IIB转基因（和-/-）小鼠血液动力学改变对心脏环的影响。最后，我们将把基因表达与OCT数据的功能测量相关联。这项工作的最终成果将是一套基于OCT的新型成像工具，与改进的胚胎培养技术合作，该技术已被优化并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，而且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早期功能变化的关键开放问题。公共卫生相关性：只有当我们在心脏最初发育时了解缺陷的起源，才能开发先天性心脏缺陷的早期医学治疗。缺乏一种理想的方法来描绘这些微小的、几乎是微观的心脏的结构和功能，这限制了我们回答这些问题的能力。胚胎小鼠心脏的OCT成像可以满足这一需求，我们将开发和验证技术和方法来实现这一潜力。

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.