Cell-specific roles of ephrin-B2 signaling in neurovascular repair and regeneration after ischemic injury of the brain

中风后的主要并发症是血脑屏障的破坏和恶性脑肿胀的发展。中风后，大脑能够修复其神经血管单元；然而，往往太慢和不够。我们的目标是更好地理解这种修复的潜在机制/治疗优化。在第一个资助期，我们发现ephrin-B2是细胞特异性参与多种修复过程的，并且ephrin-B2的激活增强了中风后神经血管单位的再生。在第二个资助期，我们将进一步加强我们对ephrin-B2在缺血性脑卒中中的作用的认识。为此，我们将更现实地模拟临床情景，老龄动物和具有共病表型的动物。我们将测试内皮ephrin-B2在脑卒中后介导血管完整性和血脑屏障修复中的相关性是否在这些动物中保守。其次，我们将利用细胞特异性缺失Efnb2的遗传模型，阐明ephrin-B2依赖的信号通路及其对中风后神经血管修复和再生的影响。我们将应用RNA-seq技术，并在RNA、蛋白质和功能水平上验证结果。第三，我们将揭示正常和缺血条件下神经元ephrin-B2在脊柱生物学中的作用。为此，我们将采用离体（器官型切片培养）和体内（2P-LSCM）方法来观察神经元特异性缺失Efnb2后脊柱形态/数量的变化。第四，我们将运用我们在缺血脑的活体成像方面的知识，使用2P-LSCM和细胞特异性体内标记技术，了解细胞特异性ephrin-B2在神经血管单元的解剖和功能重组过程中对细胞相互作用的作用。综上所述，这一建议将显著有助于我们理解脑卒中后神经血管单元的内源性修复机制以及ephrin-B2在脑卒中后的功能/治疗作用。

A major complication after stroke is the disruption of the blood-brain barrier and development of malignant brain swelling. The brain is capable of repairing its neurovascular unit after stroke; however, often too slow and insufficient. We aim at a better understanding of the underlying mechanisms/therapeutic optimization of this repair. In the 1st funding period, we have revealed that ephrin-B2 is cell-specifically involved in diverse repair processes and that ephrin-B2 activation enhances regeneration of the neurovascular unit after stroke. In the 2nd funding period, we will further strengthen our knowledge on the role of ephrin-B2 in ischemic stroke. To this end, we will more realistically model the clinical scenario, with aged animals and animals with comorbidity phenotypes. We will test whether the relevance of endothelial ephrin-B2 in mediating vascular integrity and BBB repair after stroke is conserved in these animals. Second, we will elucidate the ephrin-B2 dependent signaling pathways and their influence on neurovascular repair and regeneration after stroke, using our genetic models with cell-specific deletion of Efnb2. We will apply RNA-seq technologies and validate the results on the RNA, protein, and functional level. Third, we will unravel the role of neuronal ephrin-B2 in spine biology under normal and ischemic conditions. For this, we will take ex vivo (organotypic slice cultures) and in vivo (2P-LSCM) approaches to visualize changes in spine morphology/number after neuron-specific deletion of Efnb2. Forth, we will apply our knowledge in intravital imaging of the ischemic brain, using 2P-LSCM and cell specific in vivo labeling techniques, to understand the role of cell-specific ephrin-B2 for cell interactions during the anatomical and functional re-assembly of the neurovascular unit. Taken together, this proposal will significantly contribute to our understanding of the endogenous repair mechanisms of the neurovascular unit and the functional/therapeutic role of ephrin-B2 in this context after stroke.