Enhancing Plasticity in Damaged Spinal Cord to Repair Transmission and Function

描述（由申请人提供）： 许多士兵带着挫伤性脊髓损伤（SCI）回家，这主要是由于简易炸弹中使用的强力炸药，这些炸弹使重型装甲车内的美国军队感到不安。事实上，“六分之一的阿富汗战争伤员有脊髓损伤”（脊髓邮报，2009年11月11日）。目前，还没有治愈的方法，因此使用SCI动物模型的研究势在必行。最近的研究结果，包括我们自己的研究结果，揭示了至少有三个主要因素限制了成年人不完全SCI的功能恢复：（1）与胶质瘢痕相关的轴突生长/再生抑制剂的存在，（2）缺乏神经营养因子的支持，以及（3）受损脊髓中神经元预先存在的通路的兴奋性和可塑性降低。在试图改善受损脊髓的突触连接，并检查这是否会促进SCI后运动功能的恢复，我们最近利用了成年大鼠脊髓单侧半切（HX）模型。这是一个方便的实验模型，因为脊髓的一侧受损，另一侧保持完整。使用我们最近开发的HX模型（通过我们最初的优异奖资助的研究; 2008-2012），一种新的添加剂治疗，旨在中和轴突生长/再生抑制剂，提供神经营养因子并增强受损脊髓的可塑性。我们发现，这种联合治疗（与任何单一成分的治疗相比）可以在慢性半切脊髓损伤周围重建新的突触连接。这些生理学发现得到了观察到的损伤附近轴突的再生和分支增加以及功能恢复更好的支持。该组合治疗的组分是：（i）用酶软骨素酶-ABC（ChABC）降解瘢痕相关抑制分子（CSPG），（ii）递送神经营养因子NT-3（通过工程化成纤维细胞施用）和增加中间神经元和运动神经元中NMDA受体的功能（使用HSVl病毒载体介导的NMDA受体NR 2D调节亚基的表达）。 当前提案的目标是将这些原则转化为临床适用的应用。拟议的研究将是我们先前工作的延续，扩展到更现实的挫伤性SCI模型，并改进FDA批准或正在临床试验中的NT-3递送和增强脊髓兴奋性的方法。这种新的三重组合方法将联合收割机Ch-ABC治疗与（i）NT-3的AAV介导的转基因递送（AAV-NT 3;脊柱内和鞘内注射;已经发现AAV介导的基因递送在多个临床试验中是安全的）和（ii）施加在完整脊椎上的重复电磁刺激（FDA批准的并广泛用于治疗神经和精神疾病）组合。由于慢性SCI患者的数量不断增加，我们将在成年大鼠中使用急性和慢性胸中部挫伤SCI模型。为了评估这些治疗方法的疗效，我们将与该领域的顶尖科学家合作， 并进行多学科检查，包括体内生理学、解剖学、免疫化学、电子显微镜和行为学。利用这些方法，我们将研究三联疗法对以下方面的相加或协同作用：（1）加强通过损伤震中到腰运动神经元，然后到后肢肌肉的突触连接（电生理学）;（2）纤维的解剖可塑性解释了暴露于这种新治疗后突触反应的持续性（解剖示踪和共聚焦显微镜）;（3）联合治疗有益作用的细胞和分子机制（电子显微镜和免疫化学研究轴突髓鞘再生和离子通道分布）;和（4）多种行为测试中运动功能的恢复。正在进行的实验的初步结果显示，与对照组相比，接受新型三联治疗的大鼠在胸部挫伤性SCI后运动功能有显著改善。

DESCRIPTION (provided by applicant): Many soldiers return home with contusive spinal cord injuries (SCI), mostly due to the powerful explosives used in the improvised bombs that rattle U.S. troops inside heavily armored vehicles. In fact, "One in Six Wounded in Afghanistan War Have Spinal Cord Injuries" (The Spinal Post, Nov.11, 2009"). Currently, there is no cure and consequently studies using animal models of SCI are imperative. Results of recent studies, including our own, revealed that there are at least three major factors known to limit functional recovery from incomplete SCI in adults: (1) the presence of axonal growth/regeneration inhibitors associated with the glial scar, (2) the lack of neurotrophin support, and (3) the decreased excitability and plasticity in pre- existing pathways to neurons in damaged spinal cord. In attempts to improve synaptic connectivity in the damaged spinal cord and examine if this will facilitate recovery of motor function after SCI, we have recently utilized a model of unilateral hemisection (HX) of the spinal cord in adult rats. This is convenient experimental model because one side of the cord is lesioned and other remains intact. Using this HX model we have recently developed (through studies funded by our initial Merit Award; 2008-2012), a novel additive treatment designed to neutralize axonal growth/ regeneration inhibitors, deliver neurotrophins and enhance plasticity in damaged spinal cord. We found that this combination treatment (in contrast to treatment with any single component) can re-establish novel synaptic connections around a lesion in chronically hemisected spinal cord. These physiological findings were supported by the observation of increased regeneration and branching of axons near the injury and better functional recovery. Components of this combination treatment are: (i) degradation of scar-associated inhibitory molecules (CSPGs) with the enzyme chondroitinase-ABC (ChABC), (ii) delivery of neurotrophin NT-3 (administered via engineered fibroblasts) and increased function of NMDA receptors in interneurons and motoneurons (using HSV1 viral vector-mediated expression of NMDA receptor NR2D regulatory subunits). The goal of the current proposal is to translate these principles into a clinically amenable application. The proposed research will be a continuation of our previous work, extended to a more realistic contusive SCI model, with refinement of methods for NT-3 delivery and enhanced spinal cord excitability that are FDA- approved or under clinical trials. This novel triple combination approach will combine Ch-ABC treatment with (i) AAV-mediated transgene delivery of NT-3 (AAV-NT3; intraspinal and intrathecal injections; AAV-mediated gene delivery has been found to be safe in multiple clinical trials) and (ii) repetitive electro-magnetic stimulation applied over intact spinal vertebrae (FDA-approved and widely used for treatment of neurological and psychiatric disorders). Due to increasing numbers of individuals with chronic SCI, we will utilize both acute and chronic mid-thoracic contusion SCI models in adult rats. To evaluate the efficacy of these treatments, we will collaborate with leading scientists in the field and conduct a multidisciplinary examination, including in vivo physiology, anatomy, immunochemistry, electron microscopy and behavior. Using these methods we will study the additive or synergistic effects of the triple therapeutic treatment on (1) strengthening synaptic connections through the injury epicenter to lumbar motoneurons, and then to hindlimb muscles (electrophysiology); (2) anatomical plasticity of fibers accounting for the persistence of the synaptic response after exposure to this novel treatment (anatomical tracing and confocal microscopy); (3) cellular and molecular mechanisms underlying the beneficial effects of the combination treatment (electron microscopy and immunochemisctry to study axon remyelination and distribution of ion channels); and (4) recovery of locomotor function in multiple behavioral tests. Preliminary results of on-going experiments show significant improvements of motor function in rats that have received the novel triple treatment compared to controls after thoracic contusive SCI.