Combination strategies to improve outcome after SCI

这些研究的最终目标是制定有效的策略来改善人类脊髓损伤的结果。大鼠中度挫伤模型，其特征与大多数人类损伤一样，即控制病变的轴突中断，不进行治疗就不会发生。该建议首先描述了一系列的三个目标，比较了促进病变部位和远端脊髓再生的逐步复杂的策略。使再生最大化的策略将在延迟的时间点进行测试。所有大鼠受伤后立即注射一种神经保护剂。在Aim 1中，损伤部位将在损伤后1周通过移植进行修饰，培养基中含有纤维蛋白原和成纤维细胞生长因子- 1，或许旺细胞或脱鞘胶质细胞（EG），或这些促进再生的细胞类型的1:1混合物。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轴突再生到病变部位。在损伤后12周，将通过计数适当标记的神经元来确定导致轴突再生最多的移植类型。在Aim 2中，最有效的Aim 1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）水平升高的情况下进行测试。1)移植细胞感染编码BDNF和NT-3的腺相关病毒（AAV）构建物诱导轴突再生，2)未感染的细胞移植携带BDNF/NT-3的可生物降解微球，或3)胚胎干细胞（未感染或星形胶质细胞限制性）感染编码具有BDNF和NT-3活性的双功能神经营养因子的逆转录病毒构建物，将进行比较。在Aim 3中，这些策略中最有效的将与治疗相结合，以诱导轴突生长出病变并进入远端脊髓。EG将被移植到病变远端，单独或与更有效的AAV-BDNF/AAV-NT-3或BDNF/NT-3负载微球一起注射到病变远端mm处。治疗导致轴突再生到远端脊髓最多将被确定。在所有的研究中，再生也将通过对皮层后肢区域、前庭外侧核和网状结构神经元轴突的顺行追踪来评估。免疫细胞化学方法将用于对再生轴突进行分类，并确定trkB和trkC受体在响应轴突上的表达。Basso-Beattie-Bresnahan野外运动测试和电生理分析也将进行。重要的是，在第四个也是最后一个Aim中，当细胞移植和神经营养药物治疗延迟到损伤后4周和4周时，将确定切断的运动相关轴突对Aim 3中定义的最有效策略的反应。进一步了解损伤后轴突再生潜能随时间的变化对于预测任何促进再生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.