Frodl E M, Duan W M, Sauer H, Kupsch A, Brundin P
Department of Neurology, University Hospital, Lund, Sweden.
Brain Res. 1994 Jun 6;647(2):286-98. doi: 10.1016/0006-8993(94)91328-5.
When grafting human mesencephalic tissue to patients suffering from Parkinson's disease, the number of surviving dopamine (DA) neurons in the graft is probably crucial. It may be possible to increase the number of DA neurons available for grafting to a patient by pooling tissue from many human embryos collected over several days or by obtaining more DA neurons from each embryo. We have addressed these issues by cryopreserving human mesencephalic DA neurons prior to transplantation and also by grafting human embryonic diencephalic DA neurons. The effects of cryopreservation were assessed 4-15 weeks after xenografting ventral mesencephalic tissue into the DA-depleted striatum of immunosuppressed rats with unilateral 6-hydroxydopamine lesions of the mesostriatal pathway. Control rats grafted with fresh mesencephalic tissue displayed robust reductions in amphetamine-induced turning following transplantation. Functional effects of the cryopreserved mesencephalic grafts were only observed in the one rat out of nine which contained the largest graft in this group. The number of tyrosine hydroxylase immunoreactive neurons in animals transplanted with cryopreserved tissue was significantly reduced to 9% of fresh tissue control grafts. Morphological analysis showed that cryopreserved DA neurons were approximately 22% and 28% smaller regarding the length of the long and short axis, respectively, when compared to the neurons found in fresh grafts. In the second part of the study, the survival and function of human embryonic diencephalic DA neurons were examined following xenografting into the DA-depleted rat striatum. A reduction of motor asymmetry was observed in two out of seven diencephalon-grafted rats. This finding was consistent with a good graft survival in these particular rats, which both contained large grafts rich in tyrosine hydroxylase immunoreactive neurons. Moreover, there was immunopositive staining for graft-derived fibers in the rat striatum containing tyrosine hydroxylase and human neurofilament, both in rats grafted with mesencephalic and diencephalic DA neurons. These findings suggest that cryopreservation, using the current technique, is not a suitable storage method for use in clinical trials of DA neuron grafting in Parkinson's disease. On the other hand, the application of alternative sources of DA neurons may in the future develop into a strategy which can increase the number of neurons obtainable from each human embryo.
将人脑中脑组移植到帕金森病患者体内时，移植组织中存活的多巴胺（DA）神经元数量可能至关重要。通过汇集几天内收集的多个人类胚胎的组织，或者从每个胚胎中获取更多DA神经元，有可能增加可供移植到患者体内的DA神经元数量。我们通过在移植前冷冻保存人脑中脑DA神经元以及移植人胚胎间脑DA神经元来解决这些问题。将腹侧中脑组织异种移植到免疫抑制的大鼠纹状体内，该大鼠具有单侧中脑纹状体通路6-羟基多巴胺损伤，在异种移植后4-15周评估冷冻保存的效果。移植新鲜中脑组织的对照大鼠在移植后苯丙胺诱导的旋转行为有显著减少。仅在该组中九个大鼠中最大移植体的那只大鼠中观察到冷冻保存的中脑移植体的功能效应。移植冷冻保存组织的动物中酪氨酸羟化酶免疫反应性神经元的数量显著减少至新鲜组织对照移植体的9%。形态学分析表明，与新鲜移植体中的神经元相比，冷冻保存的DA神经元的长轴和短轴长度分别约小22%和28%。在研究的第二部分，将人胚胎间脑DA神经元异种移植到大鼠纹状体内后，检测其存活和功能。七只间脑移植大鼠中有两只观察到运动不对称性降低。这一发现与这些特定大鼠中良好的移植体存活情况一致，这两只大鼠都含有富含酪氨酸羟化酶免疫反应性神经元的大移植体。此外，在移植中脑和间脑DA神经元的大鼠纹状体内，含有酪氨酸羟化酶和人神经丝的移植衍生纤维均有免疫阳性染色。这些发现表明，使用当前技术进行冷冻保存，不是帕金森病DA神经元移植临床试验中合适的储存方法。另一方面，DA神经元替代来源的应用未来可能发展成为一种可以增加从每个人类胚胎获得的神经元数量的策略。
