Meyer M, Widmer H R, Wagner B, Guzman R, Evtouchenko L, Seiler R W, Spenger C
Department of Neurosurgery, University of Bern, Inselspital, Switzerland.
Exp Brain Res. 1998 Apr;119(3):345-55. doi: 10.1007/s002210050350.
Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.
源自胎鼠和人类的腹侧中脑（VM）在自由漂浮滚管（FFRT）培养中生长，随后移植到成年大鼠纹状体后能够存活。为了进一步探究此类移植的功能效果，将胚胎第13天的腹侧中脑组织在培养7天后进行移植，或者直接作为解离细胞悬液进行移植，并比较成年单侧6-羟基多巴胺（6-OHDA）损伤大鼠的神经元存活情况以及使旋转行为正常化的能力。其他损伤大鼠注射无细胞培养基作为对照。在移植前18天至移植后80天的不同时间点监测所有6-OHDA损伤动物的苯丙胺诱导的旋转行为。对组织学处理后的大脑进行酪氨酸羟化酶（TH）免疫染色，以评估移植的多巴胺能神经元的长期存活情况，并将其与行为效应相关联。还固定并保存了额外的培养物和急性制备的外植体用于组织学研究，以估计培养中和移植后多巴胺能神经元的损失。移植FFRT培养物的大鼠（旋转减少127%）和移植细胞悬液的大鼠（旋转减少122%）在苯丙胺诱导的旋转显著减少方面观察到类似的行为改善，而对照动物的旋转行为未恢复正常。移植后84天，培养组织移植（775±98，平均值±标准误）和新鲜解离细胞悬液移植（806±105，平均值±标准误）中的TH免疫反应性（TH-ir）神经元数量相似。新鲜外植体、7日龄培养物和移植培养物中的细胞计数显示，外植后7天TH-ir细胞损失68.2%，移植后额外损失23.1%，脑内移植中仅留存原始TH-ir细胞数量的8.7%。与之相比，细胞悬液移植中TH-ir细胞的存活率为9.1%。对钙结合蛋白钙视网膜蛋白、钙结合蛋白和小白蛋白的免疫染色显示，两种移植类型之间这些蛋白的神经元表达没有差异。总之，我们发现组织培养移植和细胞悬液移植的多巴胺能细胞存活情况和功能效果相当，而细胞悬液移植是目前实验和临床移植中最常用的移植类型。从这个意义上讲，该结果对于开发有效的胎儿黑质组织体外保存方法很有前景，同时这也将允许在脑内移植前进行神经保护和神经营养治疗。
