Neurotransplantation and Training to Promote Recovery of Chronic SCI Cats

描述（由申请人提供）：外周神经移植物（PNG）为轴突再生提供了极好的基质;它们可以将再生轴突导向特定靶点，并且它们有助于电生理学实验以检测再生轴突和远端脊髓神经元之间的突触连接。脊髓损伤后这种和所有其他移植方法的主要障碍是轴突从移植物中返回宿主脊髓的生长不良。我们在大鼠中将软骨素酶（ChABC，消化抑制性硫酸软骨素蛋白聚糖分子）与PN移植相结合，并在急性和延迟（慢性损伤）治疗模式中通过损伤再生轴突形成功能性突触有解剖学和电生理学证据。最近，我们在猫身上复制了这种大鼠急性PNG方法，我们观察到数千个轴突再生到移植物中，其中一小部分从移植物延伸到损伤远端的脊髓中，并且在神经移植物的电刺激后脊髓神经元突触激活（由c-Fos免疫反应性确定）。虽然我们将继续使用大鼠模型来扩展我们的治疗方案，但本研究的目的是将我们的治疗策略应用于慢性损伤的猫，作为转化为人类研究之前的必要临床前步骤。猫模型使我们能够调查与移植模型的放大相关的问题，猫很容易训练来执行运动任务，并且可以通过运动学和电生理学措施来评估功能的恢复。运动的生物力学在猫中得到更好的定义，猫的后肢步态比大鼠更接近人类。拟议的工作还将提供有关有效治疗大型动物神经胶质瘢痕形成的能力，促进慢性损伤大型动物结构和功能再生的能力以及康复训练促进再生和功能恢复的潜力的信息。该项目有两个具体目标。1)我们将确定慢性损伤后轴突再生成PNG的来源和程度，并测试这些轴突是否在损伤处形成功能性连接。2)我们将测试物理康复的开始时间是否会影响再生轴突的生长、整合和/或突触活动。将使用治疗策略的组合，包括移植、ChABC治疗和跑步机训练，以促进活动依赖性可塑性。结构修复将通过解剖追踪和免疫细胞化学标记进行评估;前肢-后肢协调将通过运动学和肌电图（EMG）分析进行评估;功能重新连接将在移植物的电生理刺激期间和通过突触激活神经元中的c-fos表达进行测量。SCI后的手术干预通常在患者稳定之前是不可行的，因此大多数SCI患者在开始修复治疗策略之前可能会受到慢性损伤。我们对慢性损伤大鼠的研究证明了通过在损伤远端形成功能活跃的突触来促进长距离再生的能力。这项拟议的研究将利用已经（和正在）开发的慢性损伤大鼠的治疗方法，但将其应用于大型SCI动物模型。这一临床前进展是转化为临床应用的关键一步。我们提出了一种独特的方法来解决SCI的一个非常重要的方面，即在大型动物模型中的慢性损伤。许多实验室已经对受伤的猫进行了运动训练，但不是在促进轴突再生的情况下。这将是神经再生和神经康复技术的新应用，以增加我们对SCI后修复潜力的理解。

DESCRIPTION (provided by applicant): Peripheral nerve grafts (PNGs) provide an excellent substratum for axonal regrowth; they can direct regenerating axons towards a specific target and they facilitate electrophysiological experimentation to detect synaptic connectivity between regenerating axons and distal spinal cord neurons. A major impediment to this and all other transplantation approaches after spinal cord injury is the poor growth of axons out of the graft back into the host spinal cord. We have combined Chondroitinase (ChABC, to digest inhibitory chondroitin sulfate proteoglycan molecules) with PN grafting in rats and have anatomical and electrophysiological evidence for functional synapse formation by injured, regenerating axons in both acute and delayed (chronic injury) treatment paradigms. Recently we replicated this rat acute PNG approach in cats where we observed thousands of axons regenerating into the graft, a small percentage of which extended from the graft into the spinal cord distal to the injury, and spinal neurons synaptically activated (determined by c-Fos immunoreactivity) after electrical stimulation of the nerve graft. While we will continue to use rat models for expanding our treatment repertoire, the objective of this study is to focus on application of our treatment strategies to chronically injured cats as a necessary preclinical step before translation into human research. The cat model permits us to investigate issues related to the scaling up of a transplantation model, cats are easily trained to perform locomotor tasks, and recovery of function can be assessed by kinematic and electrophysiological measures. The biomechanics of locomotion are better defined in cats and cats have a hindlimb gait that is close to human than is the rat. The proposed work also will provide information about the ability to effectively treat glial scarring in a large animal, the ability to promote structural and functional regeneration in a large animal with a chronic injury and the potential for rehabilitation training to foster regeneration and functional recovery. There are 2 Specific Aims for this project. 1) We will identify the source and extent of axonal regeneration into a PNG after chronic injury and test whether these axons form functional connections across the lesion. 2) We will test whether the start time of physical rehabilitation affects outgrowth, integration and/or synaptic activity of regenerating axons. A combination of treatment strategies will be used, including transplantation, ChABC treatments and treadmill training to promote activity dependent plasticity. Structural repair will be assessed by anatomical tract tracing and immunocytochemical labeling; forelimb-hindlimb coordination will be assessed by kinematic and electromyogram (EMG) analysis; functional reconnection will be measured during electrophysiological stimulation of the graft and by c-fos expression in synaptically activated neurons. Surgical intervention after SCI usually is not an option until the patient is stabilized, thus the majority of individuals with SCI likely will be chronically injured before a treatment strategy for repair is initiated. Our work with chronically injured rats demonstrates the ability to promote long distance regeneration with formation of functionally active synapses distal to an injury. The proposed study will take advantage of the treatment approaches that have been (and are being) developed with chronically injured rats, but will apply them to a large animal model of SCI. This preclinical advancement is a crucial step towards translation to a clinical application. We propose a unique approach to address a very important aspect of SCI, i.e. chronic injury in a large animal model. Locomotor training of injured cats has been carried out by numerous labs, but not in a situation where axon regeneration is facilitated. This will be a novel application of neuroregeneration and neurorehabilitation techniques to increase our understanding of the potential for repair after SCI.