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

基本信息

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
项目类别：
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

项目摘要

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（通过应用程序提供）：脊髓损伤（SCI）是一种使人衰弱的状况，导致损伤部位下方运动功能的不可逆转丧失。复杂的病理 SCI涉及一系列次要事件和抑制性屏障的形成，可防止整个病变部位的再生。然而，在极少数赞助的运动恢复的情况下，神经元在病变芽孔孔子周围的白质中幸免于难，这些神经元绕过抑制性疤痕并形成功能中继电路。这项研究的长期目标是了解受伤后脊髓中可塑性的机制。鉴定这些机制涉及的细胞类型，生物学因子和药物，将有助于开发临床干预措施，以改善运动型的结局。由于它们在中央模式产生中的作用，有助于协调和节奏，兴奋性谷氨酸腹膜间中间神经元-V0，V2A和V3-是候选人群，以检查导致功能增长的重新接线事件中的作用。虽然定义这些中间神经元的独特发育转录因子曲线的定义越来越明确，但缺乏成熟的鉴定标记使成人内源性种群的研究变得困难。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）区分开的方案。通过使用重组技术来产生转基因ESC，这些中间神经元的较大，纯纯种将用于研究治疗靶标和细胞替代策略。此外，最近的转基因动物的可用性使我们能够痕迹特定的中间神经元可以研究内源性对SCI的反应。第一个目的旨在生成和表征转基因V2A ESC进行体外研究和在SCI动物模型中移植。使用BAC重组，将在V2A特异性CHX10基因下插入紫霉素抗生素耐药性或荧光蛋白，当使用既定方案进行区分时，会产生纯或可追踪的ESC衍生的V2A中间神经元。第二个目的旨在将新型的体外微电位应用于研究转基因报告基因小鼠的孤立和共培养的转基因ESC衍生和原代神经元。我们假设与其他生物学因素相比，增加某些生物学因素可能会显着改善成熟度和中间神经元种群中功能突触的形成。第三个目的旨在通过评估轴突发芽，突触的识别和间隔特定的特异性发芽与运动型的相关性，以发现转基因报告中内源性腹侧脊柱中间神经元对再生的作用。这些目的共同发展体外和体内平台，以确定腹膜中间神经元在脊髓中的作用科幻后重新布线事件。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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