Combination strategies to improve outcome after SCI

这些研究的最终目标是开发有效的策略来改善人类脊髓损伤的结果。一种中等严重程度的大鼠挫伤模型，其特点与人类大多数损伤一样，是由于控制损伤的轴突中断，如果不治疗，就不会发生损伤。这项建议首先描述了一系列三个目标，比较了逐步更复杂的策略，以促进跨病变部位和远端脊髓的再生。然后，将在延迟的时间点测试最大化再生的策略。受伤后立即向所有大鼠注射神经保护剂。在目标1中，损伤部位将在损伤后1周通过移植在含有纤维蛋白原和成纤维细胞生长因子-I的培养液中、或雪旺细胞或脱鞘胶质细胞(EG)、或这些促进再生细胞类型的1：1混合物中进行修改。一种双标记逆行追踪技术将用于检测大脑和脑干运动中心的神经元，这些神经元将轴突再生到病变中。在损伤后12周，将通过对适当标记的神经元进行计数来确定导致损伤中轴突再生数量最多的移植类型。在目标2中，最有效的移植将在病变中脑源性神经营养因子(BDNF)和神经营养因子-3(NT-3)水平增加的情况下进行测试。1)感染编码BDNF和NT-3的腺相关病毒(AAV)的移植细胞，2)移植BDNF/NT-3可生物降解的微球的未感染细胞，或3)感染具有BDNF和NT-3活性的双功能神经营养因子的逆转录病毒构建体感染的胚胎干细胞(幼稚或星形胶质细胞受限)诱导的轴突再生将被比较。在目标3中，这些策略中最有效的将与诱导轴突生长的治疗相结合，使轴突从病变中长出，进入远端脊髓。Eg将被单独或与更有效的AAV-BDNF/AAV-NT-3或负载BDNF/NT-3的微球一起移植到病变远端。最大程度的轴突再生进入远端脊髓的治疗将被确定。在所有研究中，还将通过顺行追踪皮质后肢区域、前庭外侧核和网状结构中的神经元的轴突来评估再生。将使用免疫细胞化学方法对再生轴突进行分类，并确定TrkB和trkC受体在反应性轴突上的表达。还将进行Basso-Beattie-Bresna han开阔场地运动测试和电生理分析。重要的是，在第四个也是最后一个目标中，当细胞移植和神经营养素治疗被推迟到损伤后4周和4周时，将确定切断的运动相关轴突对目标3中定义的最有效策略的反应。更好地了解损伤后轴突再生潜力随时间的变化，对于预测任何促进再生的策略的临床价值至关重要。

The ultimate goal of these studies is to develop effective strategies for improving the outcome of injuries to the spinal cord in humans. A moderate-severity contusion injury model in rats, characterize as are most injuries in humans by the interruption of axons that control lesion will not occur without treatment. This proposal first describes a series of three Aims comparing progressively more complex strategies to promote regeneration across a lesion site and into distal spinal cord. The strategy that maximizes regeneration will then be tested at delayed time points. A neuroprotective agent will be injected into all rats immediately following injury. In Aim 1, the lesion site will be modified at 1 week after injury by the transplantation, in medium containing fibrinogen and fibroblast growth factor-I, or Schwann cells or unsheathing glia (EG), or a 1:1 mixture of these regeneration-promoting cell types. A double-labeling retrograde tracing technique will be used to detect neurons in locomotion centers in the brain and brainstem which regenerate their axons into the lesion. At 12 weeks after injury the transplant type resulting in the regeneration of the highest number of axons into the lesion will be determined by counting the appropriately labeled neurons. In Aim 2, the most effective transplant from Aim 1 will be tested in the presence of increased levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the lesion. Axonal regeneration induced by 1) transplanted cells infected with adeno-associated viral (AAV) constructs coding for BDNF and NT-3, 2) uninfected cells transplanted with BDNF/NT-3 laden biodegradable microspheres, or 3) embryonic stem cells (naive or astrocyte-restricted) infected with a retroviral construct coding for a bi-functional neurotrophin with BDNF and NT-3 activity will be compared. In Aim 3, the most effective of these strategies will be combined with treatments to induce growth of the axons out of the lesion and into the distal cord. EG will be transplanted just distal to the lesion, alone or with the more effective of AAV-BDNF/AAV-NT-3 or BDNF/NT-3 laden microspheres injected mm distal to the lesion. The treatment resulting in the most axonal regeneration into the distal cord will be determined. In all studies, regeneration will also be assessed using anterograde tracing of axons from neurons in the hindlimb region of the cortex, in the lateral vestibular nucleus, and the reticular formation. Immunocytochemical methods will be used to classify the regenerating axons and to determine the expression of trkB and trkC receptors on responsive axons. Basso-Beattie-Bresnahan open field locomotion tests and electrophysiological analysis will also be performed. Importantly, in the fourth and final Aim, the response of severed locomotion-related axons to the most effective strategy defined in Aim 3 will be determined when cellular transplantation and neurotrophin treatments are delayed until 4 and 4 weeks after injury. An improved understanding of the change in potential of axons to regenerate with time after injury is critical for predicting the clinical merit of any regeneration-promoting strategy.