Intravital 2-photon imaging the integration of transplanted embryonic neurons in a mouse model of cerebral ischemia at the subacute phase

项目摘要 大脑血液供应中断会导致中风，这是严重慢性残疾的主要来源。 血管内凝块清除有助于突破性的临床结局，尽管许多患者从未返回 病前状态，尤其是错过血栓清除治疗窗的患者。修复受损 移植细胞的神经元回路是非常需要的，并且将是最终的解决方案。然而，只有少数 研究表明，移植细胞与宿主细胞结构的任何整合， 是谦虚的，如果有的话。由于这些研究依赖于静态的结果测量， 例如尸检评估，缺乏对细胞整合的动态和功能特征的了解， 宿主神经元回路因此，我们将使用活体成像来加深我们对移植物的理解- 在分子水平上动态地研究梗死脑中的宿主相互作用。双光子显微镜（2PM） 越来越多地用于研究活体动物的神经元回路，其优点是提供高空间分辨率， 和单细胞的时间分辨率图像以及对其功能的见解。我们以前的工作 有助于开发一个光学细胞定位系统（oCPS）使用2PM，以实现长期的单细胞 追踪成年小鼠大脑中的神经祖细胞。在这里，我们提出了两种新的方法来解决 新的细胞整合问题是神经系统疾病的有效细胞替代疗法的当前瓶颈。 第一个是升级oCPS，通过结合最先进的 功能传感器和2PM成像系统。这将首次揭示移植后细胞的行为， 移植另一个是检验功能整合可以通过 在缺血皮质中，同源供体细胞和支持性环境的组合。我们将使用胚胎 - 新皮层神经元，从而获得意图用于替代的皮层神经元的表型身份，以及 利用中风亚急性期可塑性窗口的开放， 电路重组和新细胞的整合。第一种方法是一个强大的工具来解决 第二个。总的来说，我们的研究将解决再生医学中最紧迫的问题：功能 将移植细胞整合到成人神经回路中。与目前基于凝块清除的治疗策略不同， 在中风发作后的24小时内，对中风后亚急性期的关注实质上扩大了 中风的治疗窗口

Project Summary Interruption in the blood supply to the brain causes stroke, the leading source of severe chronic disability. Endovascular clot removal contributed to breakthrough clinical outcomes, though many patients never return to premorbid status, especially those who miss the clot removal treatment window. Restoration of damaged neuronal circuits by transplanted cells is highly desirable and would be an ultimate solution. However, only a few studies demonstrated any integration of transplanted cells with host cytoarchitecture, and the functional benefit was modest, if any. Progress in this effort is hindered because these studies rely on static outcome measures such as post-mortem assessment, lacking insight into cell integration's dynamic and functional features with the host neuronal circuits. Therefore, we will employ intravital imaging to advance our understanding of the graft- host interactions in the infarcted brain dynamically at the molecular level. Two-photon microscopy (2PM) has been increasingly used to study neuronal circuits in live animals, with the advantage of providing high spatial and temporal resolution images of single cells as well as insights into their function. Our previous work contributed to developing an optical cell positioning system (oCPS) using 2PM to achieve long-term single-cell tracking of neural progenitors in the adult mouse brain. Here, we propose two novel approaches to address the new cell integration issue as a current bottleneck for effective cell replacement therapy of neurological disorders. The first one is to upgrade the oCPS for long-term functional single-cell tracking by combining state-of-the-art functional sensors and 2PM imaging system. This will, for the first time, shed light on grafted cell behaviors after transplantation. The other is to test the hypothesis that functional integration can be achieved through the combination of homotopic donor cells and a supportive environment in ischemic cortex. We will use embryonic neocortical neurons, thus obtaining the phenotypic identity of the cortical neurons intended for replacement, and take advantage of the opening of a plasticity window in the subacute phase of stroke provides an opportunity for circuit re-organization and new cells' integration. The first approach serves as a powerful tool to address the second one. Overall, our study will address the most burning issue in regenerative medicine: functional integration of grafted cells into adult neural circuits. Unlike current therapeutic strategies based on clot removal within 24 hours after the onset of stroke, the focus on the subacute phase after stroke substantially widens the treatment window for stroke.