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

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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.