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。 血管内血栓清除为突破性的临床结果做出了贡献，尽管许多患者再也没有回到 发病前状态，特别是那些错过血栓清除治疗窗口的人。受损的修复 通过移植细胞建立神经元电路是非常可取的，这将是最终的解决方案。然而，只有少数人 研究表明，移植细胞与宿主细胞结构的任何整合，以及功能上的好处 如果有的话，也是谦虚的。这一努力的进展受到阻碍，因为这些研究依赖于静态的结果衡量标准 例如尸检评估，缺乏对细胞与 寄主神经元回路。因此，我们将使用活体成像来促进我们对移植物的了解- 脑梗塞后宿主在分子水平上的动态相互作用。双光子显微镜(2 PM) 越来越多地被用于研究活体动物的神经元回路，其优点是提供了高度的空间 以及单个细胞的时间分辨率图像以及对其功能的洞察。我们之前的工作 致力于开发使用2 PM的光学小区定位系统(OCPS)，以实现长期的单小区 追踪成年小鼠大脑中的神经前体细胞。在这里，我们提出两种新的方法来解决 新的细胞整合问题是目前神经系统疾病有效细胞替代治疗的瓶颈。 第一个是通过结合最先进的技术来升级OCPS，以实现长期功能的单细胞跟踪 功能传感器和2 PM成像系统。这将第一次揭示移植细胞在 移植。另一种是检验功能集成可以通过 同种异体供体细胞和支持环境在脑缺血皮质中的结合。我们将使用胚胎 新皮质神经元，从而获得拟替换的皮质神经元的表型同一性，以及 利用可塑性窗口的打开为中风亚急性期提供了机会 电路重组和新细胞的整合。第一种方法是一个强大的工具，可以解决 第二个。总体而言，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。与目前基于血栓清除的治疗策略不同 在中风发病后24小时内，对中风后亚急性期的关注大大扩大了 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.