Genetically modified fibroblast grafts into spinal cord

描述(由申请人提供) 我们之前的研究表明，转基因成纤维细胞可以 表达脑源性神经营养因子促进长途旅行 红核脊髓轴突再生与运动功能部分恢复 移植入成年大鼠脊髓次全横断损伤模型。这个 拟议的实验将测试这样一种想法，即额外的种群 对运动很重要的脊髓上神经元将会再生 表达BDNF和神经营养因子-3(NT3)的成纤维细胞的移植。 我们的目标是证明这些移植不仅有效 在受伤时提供，但也在延迟后提供，因此可以 适用于慢性脊髓损伤模型。第一组实验 将检验这样一种假设，即成纤维细胞经过基因改造后可以表达BDNF 或NT3或这些成纤维细胞的组合将诱导再生 皮质脊髓、前庭脊髓和红核脊髓轴突的急性移植 变成了子宫颈的半切。顺行和逆行追踪技术将 提供再生的定量和定性表征， 包括再生神经元的数量、它们的路径、长度和 目标。免疫细胞化学方法将鉴定生长的宿主背根 移植和5-羟色胺能和去甲肾上腺素能轴突 长入移植物和尾侧宿主脊髓的脑干。这个 第二组实验将测试这样一种想法，即基因工程成纤维细胞 最大限度地促进急性偏瘫大鼠的再生， 据推测，表达BDNF和NT3的成纤维细胞的组合也将 脊髓上轴突移植入半横断后可诱导再生 延迟时间长达12周(慢性损伤)。我们将最大化 基因导入抗细胞凋亡基因对轴突损伤神经元存活的影响 在最初受伤时和在受伤时注射质粒 移植。逆行和顺行追踪研究和 免疫细胞化学方法将使再生分析成为可能。第三 一系列实验将检验这些移植在急性和慢性疾病中的效果。 慢性脊髓横断症。横切术是最具挑战性的，但也是 最不明确的实验性脊髓损伤和再生 模型将令人信服地确认这些措施的有效性 移植。一系列测试将评估患者的运动和感觉功能 每一系列的实验，都会重新损伤吻端至初始损伤 指示再生轴突是否有助于恢复。生理学 对恢复的功能的评估将作为以下内容进行研究 两个项目。这些战略的成功将是重要的一步。 在人类身上开发有效的治疗方法。

DESCRIPTION (provided by applicant) Our previous studies have shown that fibroblasts genetically modified to express Brain Derived Neurotrophic Factor (BDNF) promote long distance regeneration of rubrospinal axons and partial recovery of motor function when transplanted into a subtotal hemisection spinal cord injury in adult rats. The proposed experiments will test the idea that additional populations of supraspinal neurons important for locomotion will regenerate in response to transplants of fibroblasts engineered to express BDNF and neurotrophin-3 (NT3). Our aim is to demonstrate that these transplants will be effective not only when provided at the time of injury but also after a delay and can therefore be applied to models of chronic spinal cord injury. The first set of experiments will test the hypothesis that fibroblasts genetically modified to express BDNF or NT3 or a combination of these fibroblasts will elicit regeneration of corticospinal, vestibulospinal, and rubrospinal axons when transplanted acutely into a cervical hemisection. Anterograde and retrograde tracing techniques will provide a quantitative and qualitative characterization of the regeneration, including the numbers of neurons that regenerate, their path, length and targets. Immunocytochemical methods will identify host dorsal roots that grow into the transplants and serotonergic and noradrenergic axons originating in the brainstem that grow into the transplants and caudal host spinal cord. The second set of experiments will test the idea that engineered fibroblasts that promote the greatest amount of regeneration in rats with acute hemisections, presumably a combination of BDNF- and NT3-expressing fibroblasts, will also elicit regeneration of supraspinal axons when transplanted into a hemisection after delays as long as 12 weeks (chronic injury). We will maximize the survival of axotomized neurons by introducing the antiapoptotic Bcl-2 gene by injection of plasmid both at the time of the initial injury and at the time of transplantation. Retrograde and anterograde tracing studies and immunocytochemical methods will permit analysis of the regeneration. The third set of experiments will examine the effects of these transplants in acute and chronic spinal cord transections. Transection is the most challenging but also the least ambiguous experimental spinal cord injury, and regeneration in this model will represent compelling confirmation of the effectiveness of these transplants. A battery of tests will evaluate motor and sensory function in each series of experiments, and re-lesion rostral to the initial injury will indicate whether regenerated axons contribute to recovery. Physiological assessment of the recovered functions will be studied as a component of two Projects. The success of these strategies will be an important step toward developing an effective treatment in humans.