TROPHIC ALTERATIONS IN DOPAMINE NEURON TRANSPLANTATION

早期临床试验的结果表明，胎儿黑质移植 可能提供一个令人兴奋的新的治疗策略， 帕金森病（PD）。然而，与存储相关的组织损失 移植前流产的胎儿组织，知识不足 关于患者年龄和/或疾病阶段对移植物存活的影响， 以及缺乏用于移植的连续组织来源， 在影响手术的因素中， procedure.我们实验室最近的工作表明，多巴胺（DA） 神经元的存活可以通过“预处理”DA神经元来增加， 营养分子在储存过程中。我们还发现， 随着年龄的增长，大鼠纹状体中的神经元数量减少， DA去神经支配的程度。这些数据表明，患者年龄和 帕金森病阶段可能对营养性 移植的组织被移植到其中的环境。具体目标1 因此，我们将系统地评估补充 含有营养分子（bFGF、BDNF、GDNF、EGF）、抗氧化剂的储存介质 （U83836 E和U 74389 G）和组织提取物（纹状体，中脑， 胎盘血清）。大鼠中脑将在以下条件下储存长达5天 这些条件，然后与纹状体细胞共培养。DA神经元 这些培养物中的存活率将通过评估K+诱发的DA来评估 释放和酪氨酸羟化酶免疫反应（THir）细胞计数。 最佳储存条件将通过植入 将“增强的”细胞植入单侧损伤（6 OHDA）大鼠。具体目标2 将重点关注移植受者年龄和DA程度的影响 对移植物存活的影响。增强型细胞将被植入 取2、9、17和22月龄大鼠纹状体， 将比较各组的植入部位。利用脑室内 6 OHDA模型，17月龄大鼠将双侧失神经支配35、50、75， 和90%，并移植了增强细胞。THir细胞计数将再次 在各组之间进行比较。尽管优化胎儿组织储存 条件和确定DA去神经支配的年龄和程度， 最大限度地提高DA神经元的存活率可能会立即产生积极的影响， 对目前正在进行的移植计划的影响，这些目标不 解决了围绕获得DA神经元的连续来源的问题 丰富的组织在这方面，我们最近表明，一个自然的， 多能祖细胞系可来源于胎儿和成人 中脑 与新鲜收获的中脑共培养 许多这些细胞进入DA神经元表型，而已知营养 因素没有。 这些文化中的许多文化都富含THir 细胞 因此，具体目标3将侧重于未来使用 多能祖细胞作为连续传代; 2-优化和 识别和鉴定中脑的粗成分 负责其向DA表型的转化;和3-移植 6 OHDA损伤大鼠的祖细胞。成功实施 该提案的具体目标涉及以下几个实际方面： 移植，可以很容易地转移到临床环境。

Results form early clinical trials suggest that fetal nigral transplants may offer an exciting new therapeutic strategy for the treatment of Parkinson's disease (PD). However, tissue loss associated with the storage of aborted fetal tissue prior to transplantation, inadequate knowledge about the impact of patient age and/or stage of disease on graft survival, and the lack of a continuous source of tissue for transplantation are among the factors that compromises an otherwise straight forward surgical procedure. Recent work in our laboratory suggests that dopamine (DA) neuron survival can be increased by "preconditioning" DA neurons with trophic molecules during storage. We have also shown that trophic activity in the rat striatum is reduced by aging and increased in direct proportion to the degree of DA denervation. These data suggest that patient age and Parkinsonian stage may have a significant impact on the trophic environment into which grafted tissue is transplanted. In Specific Aim 1 we will therefore systematically evaluate the effect of supplementing storage media with trophic molecules (bFGF, BDNF, GDNF, EGF), antioxidants (U83836E and U74389G), and tissue extracts (striatal, mesencephalic, placental serum). Rat mesencephalon will be stored for up to 5 days under these conditions and then co-cultured with striatal cells. DA neuron survival in these cultures will be evaluated by assessing K+-evoked DA release and tyrosine hydroxylase immunoreactive (THir) cell counts. Optimal storage conditions will be further evaluated by implanting the "enhanced" cells into unilaterally lesioned (6OHDA) rats. Specific Aim 2 will focus on the effects the transplant recipients age and degree of DA denervation has on graft survival. Enhanced cells will be implanted into the striata of 2,9, 17, and 22 month old rats and THir cell counts in the implant site will be compared across groups. Using the intraventricular 6OHDA model, 17 month old rats will be bilaterally denervated 35,50,75, and 90% and transplanted with enhanced cells. THir cell counts will again be compared across groups. Although optimizing fetal tissue storage conditions and identifying the age and degree of DA denervation that maximizes DA neuron survival is likely to have an immediate, positive impact on transplant programs currently under way, these Aims do not address the issues surrounding access to a continuous source of DA neuron enriched tissue. In this regard, we have recently shown that a natural, pluripotent, progenitor cell line can be derived from fetal and adult mesencephalon. Co-culture with freshly harvested mesencephalon converted many of these cells into the DA neuron phenotype whereas known trophic factors did not. Many of these cultures were highly enriched with THir cells. Specific Aim 3 will therefore focus on the future use of the pluripotent progenitor cell as a continuous passage; 2-optimizing and identifying and identifying the crude components of the mesencephalon responsible for its conversion to the DA phenotype; and 3- grafting the progenitor cells in 6OHDA lesioned rats. The successful implementation of the Specific Aims of this proposal addresses several practical aspects of transplantation that could be readily transferred to the clinical setting.