Evaluating the role of excitatory interneurons for regeneration after spinal cord injury using in vitro and in vivo transgenic models

 描述(申请人提供)：脊髓损伤(SCI)是一种衰弱的情况，导致损伤部位以下运动功能的不可逆转丧失。的复杂的病理学。 脊髓损伤涉及一连串的继发事件和抑制屏障的形成，阻止了损伤部位的再生。然而，在极少数自发运动恢复的情况下，病变周围白质中的神经元发芽，绕过抑制性瘢痕形成功能继而回路。这项研究的长期目标是了解损伤后脊髓可塑性的机制；识别参与这些机制的细胞类型、生物因素和药物将有助于临床干预措施的发展，以改善运动结果。由于兴奋性谷氨酸能腹侧中间神经元V0、V2a和V3在中枢模式生成中的作用，有助于协调和节律，因此它们是候选群体，可以研究它们在导致功能获得的重新连接事件中的作用。尽管定义这些中间神经元的不同发育转录因子谱越来越明确，但由于缺乏成熟的识别标记，使得对成年内源性群体的研究变得困难。我们的实验室最近开发了从胚胎干细胞(ESCs)中区分V2a和V3中间神经元的方案。通过使用重组工程技术来产生转基因ESCs，大量的、纯净的中间神经元群体将可用于研究治疗靶点和细胞替代策略。此外，最近转基因动物的出现使我们能够追踪特定的中间神经元，从而使研究脊髓损伤的内源性反应成为可能。第一个目标是建立和鉴定转基因的V2a胚胎干细胞，用于体外研究和移植到脊髓损伤动物模型中。利用BAC重组工程，扑霉素耐药性或荧光蛋白将被插入到V2a特异的Chx10基因下，当使用已建立的方案进行分化时，产生纯的或可追踪的ESC来源的V2a中间神经元。第二个目的是应用一种新的体外微设备来研究分离和共培养的转基因ESC来源的和转基因报告鼠的原代中间神经元。我们假设，与其他因素相比，某些生物因素的加入可能会显着促进神经元间群体的成熟和功能性突触的形成。第三个目的是通过评估轴突萌发、突触重建以及神经元间特异性萌发与运动恢复的相关性，来发现内源性腹侧脊髓中间神经元在转基因报告小鼠脊髓背侧横断损伤后再生中的作用。这些目标共同开发了体外和体内平台，以确定脊髓腹侧中间神经元的作用。 在SCI之后重新布线事件。

 DESCRIPTION (provided by applicant): Spinal cord injury (SCI) is a debilitating condition resulting in irreversible loss of motor function below the site of injury. The complex pathology of SCI, involving a cascade of secondary events and the formation of inhibitory barriers, prevent regeneration across the lesion site. However, in rare cases of spontaneous locomotor recovery, neurons spared in the white matter around the lesion sprout collaterals that bypass the inhibitory scar and form functional relay circuits. The long-term goal of this research is to understand mechanisms of plasticity in the spinal cord after injury; identifying cell types, biological factor, and pharmaceautical agents that are involved in these mechanisms will aid in the development of clinical interventions to improve locomotor outcomes. Because of their role in central pattern generation, contributing to coordination and rhythm, excitatory glutamatergic ventral interneurons-V0, V2a, and V3- are candidate populations to examine for roles in rewiring events resulting in gain of function. While the distinct developmental transcription factor profiles that define these interneurons are increasingly well defined, a lack of mature identification markers has made study of endogenous populations in adults difficult. Our lab has recently developed protocols to differentiate V2a and V3 interneurons from embryonic stem cells (ESCs). By using recombineering techniques to generate transgenic ESCs, large, pure populations of these interneurons will be available to study therapeutic targets and for cell replacement strategies. Furthermore, the recent availability of transgenic animals allowing us to lineage trace specific interneurons enables study of endogenous responses to SCI. The first aim seeks to generate and characterize transgenic V2a ESCs for in vitro study and for transplantation in animal models of SCI. Using BAC recombineering, puromycin antibiotic resistance or a fluorescent protein will be inserted under the V2a-specific Chx10 gene, generating pure or traceable ESC- derived V2a interneurons when differentiated using established protocols. The second aim seeks to apply a novel in vitro microdevice to study isolated and co-cultured transgenic ESC-derived and primary interneurons from transgenic reporter mice. We hypothesize that the addition of certain biological factors might significantly improve maturation and the formation of functional synapses in interneuron populations compared to others. The third aim seeks to discover the role of endogenous ventral spinal interneurons on regeneration after dorsal hemisection spinal cord injury in transgenic reporting mice by evaluating axon sprouting, reformation of synapses, and correlation of interneuron-specific sprouting to locomotor recovery. Together, these aims develop in vitro and in vivo platforms to determine the role of ventral interneurons in spinal cord rewiring events after SCI.