Microtransplantation of dopaminergic cell suspensions: further characterization and optimization of grafting parameters.

Intracerebral transplantation of dopaminergic (DA) cells is currently further explored as a potential restorative therapy for Parkinson's disease (PD). However, before they can be considered for a more widespread clinical use a number of critical issues have to be resolved, including an optimized transplantation protocol. This study has been performed in a rat 6-hydroxydopamine model of PD and is based on the microtransplantation approach. The results demonstrate a reduced survival (threefold) for a single cell suspension of E14 rat ventral mesencephalon compared to a fragment suspension when a metal cannula is used for implantation. However, fragment suspensions result in a more variable graft survival and ectopically placed cells along the implantation tract. When a glass capillary is used for implantation, the survival of the single cell suspension (so-called "micrograft") improved by fourfold (vs. single cells/metal cannula) and is superior to the combination of the metal cannula and fragment suspension (+40%). The micrografts show a reduced variability in DA neuron survival as well as fewer ectopically placed cells. Moreover, the implantation time can significantly be reduced from 19 to 7 min in micrografted animals without a compromise in DA graft survival and functional behavioral outcome. Using the microtransplantation approach graft size can be tailored effectively by varying the density of the final cell suspension at least between 11,000 and 320,000 cells/microl, resulting in comparable survival of tyrosine hydroxylase (TH)-positive neurons in the range of 2-4%. With this approach no more than 100 surviving TH-positive neurons are necessary to produce functional effects in the amphetamine-induced rotation test. Interestingly, we found that DA micrografts into lesion striatum present 20% higher survival rates of TH neurons in comparison to the intact striatum. In summary, these results provide further evidence for the usefulness of the microtransplantation approach and allow for a more precise and tailored adaptation of the implantation parameters for further studies on DA, and possibly also other neural-, glial-, and stem cell-derived grafts.

Pattern of long-term sensorimotor recovery following intrastriatal and--accumbens DA micrografts in a rat model of Parkinson's disease.

The functional restorative capacity of fetal dopaminergic (DA) transplants is governed by a number of critical parameters including graft location, survival of DA neurons, and transplantation technique. In addition, there is an ongoing controversy whether "too much" or "too little" survival of DA neurons is responsible for the incomplete functional recovery observed in some transplanted Parkinson's disease (PD) patients. Here we investigated two implantation sites, the nucleus accumbens (NAc) and the caudate-putamen unit (CPU), and two different graft distributions within the CPU, i.e., two 0.75 microL deposits (CPU-2) versus six 0.25 microL deposits (CPU-6) in a rat model of PD. Grafts were derived from E14 rat ventral mesencephalon and the long-term functional outcome was evaluated with a wide range of complex-sensorimotor behavioral tests. The data show that forelimb stepping, balancing behavior, and skilled forelimb reaching behavior was more restored in CPU-6-grafted animals as compared to CPU-2 animals, although the number surviving dopaminergic neurons and dopamine release were similar in the two groups. Furthermore, a correlation analysis revealed a number of inverse relationships between the rate of DA neuron survival and sensorimotor performances, e.g., for skilled forelimb use. DA grafts placed into the NAc induced a partial recovery in drug-induced rotation tests but failed to restore any of the other sensorimotor behaviors tested. Taken together, these data have important implications both for a better understanding of the complex functional graft-host interactions as well as for the further optimization of clinical neural transplantation strategies in neurodegenerative diseases.