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)回国，主要是由于简易炸弹中使用的强大炸药，使美军在重型装甲车中发出嘎嘎声。事实上，“在阿富汗战争中，每六个伤员中就有一个有脊髓损伤”(《脊椎邮报》，2009年11月11日)。目前，尚无治愈方法，因此利用脊髓损伤动物模型进行研究势在必行。最近的研究结果，包括我们自己的研究结果显示，至少有三个已知的主要因素限制了成人不完全性脊髓损伤后的功能恢复：(1)与胶质瘢痕相关的轴突生长/再生抑制物的存在；(2)缺乏神经营养素的支持；(3)损伤脊髓神经元原有通路的兴奋性和可塑性降低。为了改善受损脊髓的突触连接，并研究这是否有助于脊髓损伤后运动功能的恢复，我们最近利用了一种成年大鼠脊髓单侧半横断(HX)的模型。这是一个方便的实验模型，因为脐带的一侧受损，另一侧完好无损。使用这个HX模型，我们最近开发了(通过我们的初始功绩奖资助的研究；2008-2012)，一种新的添加剂治疗，旨在中和轴突生长/再生抑制物，输送神经营养因子，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新建立新的突触连接。这些生理学发现得到了损伤附近轴突再生和分支增加以及更好的功能恢复的观察支持。这种联合治疗的组成部分包括：(I)软骨素酶ABC(ChABC)降解瘢痕相关抑制分子(CSPGs)，(Ii)神经营养因子NT-3的输送(通过工程成纤维细胞)，以及增加中间神经元和运动神经元中NMDA受体的功能(使用HSV1病毒载体介导的NMDA受体NR2D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上可接受的应用。这项拟议的研究将是我们先前工作的继续，扩展到更现实的挫伤SCI模型，改进NT-3的传递方法，并增强FDA批准或正在进行的临床试验的脊髓兴奋性。这种新的三联疗法将结合CH-ABC治疗和(I)AAV介导的NT-3转基因输送(AAV-NT3；脊髓和鞘内注射；AAV介导的基因输送在多个临床试验中被发现是安全的)和(Ii)在完整的脊椎上应用重复电磁刺激(FDA批准并广泛用于神经和精神疾病的治疗)。由于慢性脊髓损伤的个体数量不断增加，我们将在成年大鼠中同时使用急性和慢性中胸挫伤模型。为了评估这些疗法的疗效，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在以下方面的相加或协同作用：(1)通过损伤中心加强突触连接到腰椎运动神经元，然后到后肢肌肉(电生理学)；(2)暴露于这种新疗法后(解剖追踪和共聚焦显微镜)导致突触反应持续的纤维的解剖可塑性；(3)联合疗法有益效果的细胞和分子机制(电子显微镜和免疫化学试图研究轴突再髓鞘形成和离子通道的分布)；以及(4)在多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，接受新型三联疗法的大鼠在胸部挫伤后的运动功能有了显著的改善。

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.