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

 描述（由申请人提供）：脊髓损伤（SCI）是一种导致损伤部位以下运动功能不可逆丧失的衰弱性疾病。复杂的病理学 SCI涉及一系列继发性事件和抑制性屏障的形成，阻止损伤部位的再生。然而，在极少数自发性运动恢复的病例中，病变周围白色物质中保留的神经元会产生侧支，绕过抑制性瘢痕，形成功能性中继回路。这项研究的长期目标是了解脊髓损伤后的可塑性机制;识别参与这些机制的细胞类型，生物因子和药物将有助于临床干预措施的发展，以改善运动结果。由于它们在中央模式生成中的作用，有助于协调和节奏，兴奋性神经元腹侧中间神经元-V0，V2 a和V3-是候选人群，以检查在重新布线事件中的作用，从而获得功能。虽然定义这些中间神经元的不同发育转录因子谱越来越明确，但缺乏成熟的识别标记物使得成人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（ESC）分化V2 a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ESC，这些中间神经元的大而纯的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谱系，从而能够研究SCI的内源性反应。第一个目的是寻求产生和表征转基因V2 a ESCs用于体外研究和用于SCI动物模型中的移植。使用BAC重组工程，将嘌呤霉素抗生素抗性或荧光蛋白插入V2 a特异性ChxlO基因下，当使用已建立的方案分化时产生纯的或可追踪的ESC衍生的V2 a中间神经元。第二个目的是应用一种新的体外微装置来研究分离和共培养的转基因ESC衍生的和来自转基因报告小鼠的初级中间神经元。我们假设，与其他生物因子相比，添加某些生物因子可能会显着改善中间神经元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发芽、突触重建和神经元特异性发芽与运动恢复的相关性，发现内源性腹侧脊髓中间神经元在转基因小鼠脊髓背侧半横断损伤后再生中的作用。总之，这些目标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， SCI后的重接事件

 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.