Branton R L, Clarke D J
Department of Human Anatomy and Genetics, University of Oxford, United Kingdom.
Exp Neurol. 1999 Nov;160(1):88-98. doi: 10.1006/exnr.1999.7207.
Transplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum. However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery. A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear. Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival. Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media. After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically. Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h. Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons. There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h. We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.
世界各地的多个研究团队已对200多名帕金森病（PD）患者进行了胎儿黑质移植，试图恢复纹状体的多巴胺能（DA）输入。然而，无论使用悬浮移植、部分解离移植还是实体移植，植入的DA神经元存活比例都很小，通常低至5%至10%，不足以实现完全功能恢复。在帕金森病动物模型中，很大一部分移植神经元已被证明通过凋亡死亡，但其原因尚不清楚。由于用于神经移植供体组织制备和体外培养的方法相同，我们使用体外检测系统研究了细胞悬浮液制备后DA神经元损失的时间进程，并考虑所采用的程序是否可能部分导致了DA神经元存活率低。将E14大鼠腹侧中脑的原代解离培养物在含血清和无血清培养基中孵育不同时间。固定后，使用TUNEL法以及溴化乙锭和吖啶橙检测凋亡，并通过免疫细胞化学方法定位DA神经元。结果显示，大多数凋亡发生在最初的24小时内，且50%的DA神经元在最初的8小时内丢失。双重免疫荧光标记证实DA神经元内存在TUNEL阳性核。在最初的32小时内，含血清和无血清培养基中的损失程度和速率均无差异。因此，我们认为，现有的细胞悬浮液制备方法可能会诱导凋亡，可能需要进行改进以提高DA神经元的存活率。
