Neurotransplantation and Training to Promote Recovery of Chronic SCI Cats

描述（申请人提供）：周围神经移植物（png）为轴突再生提供了良好的基质；它们可以引导再生轴突指向特定目标，并促进电生理实验以检测再生轴突与远端脊髓神经元之间的突触连通性。脊髓损伤后，这种方法和所有其他移植方法的主要障碍是移植物轴突生长不佳，无法回到宿主脊髓。我们将软骨素酶（ChABC，用于消化抑制性硫酸软骨素蛋白多糖分子）与PN移植结合在大鼠身上，并获得了解剖学和电生理学证据，证明在急性和延迟（慢性）损伤治疗范式中，受损的、再生的轴突形成功能性突触。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，脊髓神经元在神经移植物电刺激后突触激活（由c-Fos免疫反应性决定）。虽然我们将继续使用大鼠模型来扩大我们的治疗范围，但本研究的目的是将我们的治疗策略应用于慢性损伤猫，作为转化为人类研究之前的必要临床前步骤。猫模型允许我们研究与移植模型扩展相关的问题，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测量来评估功能恢复。运动的生物力学在猫身上得到了更好的定义，猫的后肢步态比老鼠更接近人类。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促进再生和功能恢复的康复训练潜力的信息。这个项目有两个具体的目标。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病变处形成功能性连接。2)我们将测试物理康复的开始时间是否影响再生轴突的生长、整合和/或突触活动。将采用多种治疗策略，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道示踪和免疫细胞化学标记进行评估；通过运动学和肌电图（EMG）分析评估前肢-后肢协调性；通过电生理刺激和突触激活神经元中c-fos的表达来测量移植物的功能重连。脊髓损伤后的手术干预通常在患者稳定之前是不可行的，因此大多数脊髓损伤患者可能会在开始修复治疗策略之前遭受慢性损伤。我们对慢性损伤大鼠的研究表明，通过在损伤远端形成功能活跃的突触，可以促进远距离再生。拟议的研究将利用已经（和正在）开发的治疗慢性损伤大鼠的方法，但将它们应用于大型脊髓损伤动物模型。这一临床前进展是迈向临床应用的关键一步。我们提出了一种独特的方法来解决脊髓损伤的一个非常重要的方面，即大型动物模型中的慢性损伤。许多实验室已经进行了受伤猫的运动训练，但不是在轴突再生促进的情况下进行的。这将是神经再生和神经康复技术的一个新应用，以增加我们对脊髓损伤后修复潜力的理解。

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.