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

使用体外和体内转基因模型评估兴奋性中间神经元在脊髓损伤后再生中的作用

• 批准号: 9119889
• 负责人: Nisha Iyer
• 金额: $ 0.58万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-11 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9119889
• 关键词: Ablation; Adult; Animal Model; Antibiotic Resistance; Appearance; Axon; Biological Factors; Brain-Derived Neurotrophic Factor; Bypass; Calcium; Cell Transplantation; Cells; Characteristics; Cicatrix; Clinical; Coculture Techniques; Complex; Cues; Development; Devices; Dorsal; ES Cell Line; Electric Stimulation; Environment; Event; Flow Cytometry; Future; Generations; Genes; Genetic; Glutamates; Goals; Grant; Growth; Healed; Health; In Vitro; Injury; Interneurons; Intervention; Knock-out; Knowledge; Lesion; Life; Literature; Locomotor Recovery; Mitotic; Modeling; Molecular Target; Motor; Motor Neurons; Mus; Natural regeneration; Neuraxis; Neurons; Neurotrophin 3; Outcome; Pathology; Patients; Pattern; Periodicity; Population; Proteins; Protocols documentation; Puromycin; Rattus; Reporter; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Site; Spinal; Spinal Cord; Spinal cord injury; Synapses; Techniques; Transgenic Animals; Transgenic Model; Transgenic Organisms; Transplantation; United States; Work; base; biomarker identification; cell type; central pattern generator; design; effective therapy; embryonic stem cell; gain of function; gray matter; healing; immunocytochemistry; improved; in vitro Model; in vivo; novel; partial recovery; prevent; promoter; protein expression; response; spinal cord regeneration; stem cell therapy; targeted treatment; therapeutic target; tool; transcription factor; white matter

 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.

 描述（由申请人提供）：脊髓损伤（SCI）是一种使人衰弱的疾病，会导致损伤部位以下运动功能不可逆地丧失。复杂的病理学 SCI 涉及一系列继发事件和抑制屏障的形成，可阻止病变部位的再生。然而，在自发运动恢复的极少数情况下，病变周围白质中幸存的神经元会萌发侧枝，绕过抑制性疤痕并形成功能性中继回路。这项研究的长期目标是了解损伤后脊髓的可塑性机制；识别参与这些机制的细胞类型、生物因素和药剂将有助于开发临床干预措施以改善运动结果。由于它们在中枢模式生成中的作用，有助于协调和节律，兴奋性谷氨酸能腹侧中间神经元 - V0、V2a 和 V3 - 是检查在重新布线事件中导致功能获得的作用的候选群体。虽然定义这些中间神经元的独特发育转录因子谱越来越明确，但缺乏成熟的识别标记使得对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将 V2a 和 V3 中间神经元与胚胎干细胞 (ESC) 区分开来的方案。通过使用重组工程技术生成转基因ESC，这些中间神经元的大量纯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对特定的中间神经元进行谱系追踪，从而能够研究 SCI 的内源性反应。第一个目标是生成并表征转基因 V2a ESC，用于体外研究和 SCI 动物模型移植。使用 BAC 重组工程，嘌呤霉素抗生素抗性或荧光蛋白将被插入 V2a 特异性 Chx10 基因下，当使用既定方案进行分化时，生成纯的或可追踪的 ESC 衍生的 V2a 中间神经元。第二个目标旨在应用一种新型体外微装置来研究来自转基因报告小鼠的分离和共培养的转基因ESC衍生的和初级中间神经元。我们假设，与其他细胞相比，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。第三个目标旨在通过评估轴突萌芽、突触重建以及中间神经元特异性萌芽与运动恢复的相关性，发现内源性腹侧脊髓中间神经元对转基因报告小鼠背侧半切脊髓损伤后再生的作用。这些目标共同开发体外和体内平台，以确定腹侧中间神经元在脊髓中的作用 SCI 后的重新布线事件。

项目成果

Author Correction: V2a interneuron differentiation from mouse and human pluripotent stem cells.

作者更正：小鼠和人类多能干细胞的 V2a 中间神经元分化。

• DOI: 10.1038/s41596-019-0266-z
• 发表时间: 2020
• 期刊: Nature protocols
• 影响因子: 14.8
• 作者: Butts,JessicaC;Iyer,Nisha;White,Nick;Thompson,Russell;Sakiyama-Elbert,Shelly;McDevitt,ToddC
• 通讯作者: McDevitt,ToddC