Fetal ventral mesencephalon of human and rat origin maintained in vitro and transplanted to 6-hydroxydopamine-lesioned rats gives rise to grafts rich in dopaminergic neurons

Free-floating roller tube cultures of human fetal (embryonic age 6-10 weeks post-conception) and rat fetal (embryonic day 13) ventral mesencephalon were prepared. After 7-15 days in vitro, the mesencephalic tissue cultures were transplanted into the striatum of adult rats that had received unilateral injections of 6-hydroxydopamine into the nigrostriatal bundle 3-5 weeks prior to transplantation. Graft survival was assessed in tyrosine hydroxylase (TH)-immunostained serial sections of the grafted brains up to post-transplantation week 4 for the human fetal xenografts and post-transplantation week 11 for the rat fetal allografts. D-amphetamine-induced rotation was monitored up to 10 weeks after transplantation in the allografted animals and compared with that of lesioned-only control animals. All transplanted animals showed large, viable grafts containing TH-immunoreactive (ir) neurons. The density of TH-ir neurons in the human fetal xenografts and in rat fetal allografts was similar. A significant amelioration of the amphetamine-induced rotation was observed in the animals that received cultured tissue allografts. These results promote the feasibility of in vitro maintenance of fetal human and rat nigral tissue prior to transplantation using the free-floating roller tube technique.

Effects of GDNF pretreatment on function and survival of transplanted fetal ventral mesencephalic cells in the 6-OHDA rat model of Parkinson's disease

Transplantation of fetal dopaminergic (DA) neurons offers an experimental therapy for Parkinson's disease (PD). The low availability and the poor survival and integration of transplanted cells in the host brain are major obstacles in this approach. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with growth- and survival-promoting capabilities for developing DA neurons. In the present study, we examined whether pretreatment of ventral mesencephalic (VM) free-floating roller tube (FFRT) cultures with GDNF would improve graft survival and function. For that purpose organotypic cultures of E14 rat VM were grown for 2, 4 or 8 days in the absence (control) or presence of GDNF [10 ng/ml] and transplanted into the striatum of 6-hydroxydopamine-lesioned rats. While all groups of rats showed a significant reduction in d-amphetamine-induced rotations at 6 weeks posttransplantation a significantly improved graft function was observed only in the days in vitro (DIV) 4 GDNF pretreated group compared to the control group. In addition, no statistical significant differences between groups were found in the number of surviving tyrosine hydroxylase-immunoreactive (TH-ir) neurons assessed at 9 weeks posttransplantation. However, a tendency for higher TH-ir fiber outgrowth from the transplants in the GDNF pretreated groups as compared to corresponding controls was observed. Furthermore, GDNF pretreatment showed a tendency for a higher number of GIRK2 positive neurons in the grafts. In sum, our findings demonstrate that GDNF pretreatment was not disadvantageous for transplants of embryonic rat VM with the FFRT culture technique but only marginally improved graft survival and function.