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Background: Cell transplantation is expected to be a promising treatment for Parkinson's disease (PD), in which re-innervation of the host striatum by grafted dopamine (DA) neurons is essential. In particular, the dorsolateral part of the striatum is important because it is the target of midbrain A9 DA neurons, which are degenerated in PD pathology. The effect of exercise on the survival and maturation of grafted neurons has been reported in several neurological disease models, but never in PD models., Objective: We investigated how exercise influences cell transplantation for PD, especially from the viewpoint of cell survival and neurite extensions., Methods: Ventral mesencephalic neurons from embryonic (E12.5) rats were transplanted into the striatum of adult 6-OHDA-lesioned rats. The host rats then underwent treadmill training as exercise after the transplantation. Six weeks after the transplantation, they were sacrificed, and the grafts in the striatum were analyzed., Results: The addition of exercise post-transplantation significantly increased the number of surviving DA neurons. Moreover, it promoted neurite extensions from the graft toward the dorsolateral part of the striatum., Conclusions: This study indicates a beneficial effect of exercise after cell transplantation in PD.

Biomaterials have been shown to significantly improve the outcome of cellular reparative approaches for Parkinson's disease in experimental studies because of their ability to provide transplanted cells with a supportive microenvironment and shielding from the host immune system. However, given that the margin for improvement in such reparative therapies is considerable, further studies are required to fully investigate and harness the potential of biomaterials in this context. Given that several recent studies have demonstrated improved brain repair in Parkinsonian models when using dopaminergic grafts derived from younger foetal donors, we hypothesized that encapsulating these cells in a supportive biomaterial would further improve their reparative efficacy. Thus, this study aimed to determine the impact of a GDNF-loaded collagen hydrogel on the survival, reinnervation, and functional efficacy of dopaminergic neurons derived from young donors. To do so, hemi-Parkinsonian (6-hydroxydopamine-lesioned) rats received intrastriatal transplants of embryonic day 12 cells extracted from the rat ventral mesencephalon either alone, in a collagen hydrogel, with GDNF, or in a GDNF-loaded collagen hydrogel. Methamphetamine-induced rotational behaviour was assessed at three weekly intervals for a total of 12 weeks, after which rats were sacrificed for postmortem assessment of graft survival. We found that, following intrastriatal transplantation to the lesioned striatum, the GDNF-loaded collagen hydrogel significantly increased the survival (4-fold), reinnervation (5.4-fold), and functional efficacy of the embryonic day 12 dopaminergic neurons. In conclusion, this study further demonstrates the significant potential of biomaterial hydrogel scaffolds for cellular brain repair approaches in neurodegenerative diseases such as Parkinson's disease., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Environmental conditions and behavioural experience can affect neuronal function and morphology. It is less well known whether such factors also influence the growth, integration and functional recovery provided by neural grafts placed within the damaged brain. Here we report on the effects of differential housing conditions on striatal graft morphology and functional recovery after striatal lesions. Rats were pretrained on a skilled bilateral forelimb task, the staircase test, and lesioned unilaterally in the lateral dorsal striatum with quinolinic acid. One group of lesioned animals was given suspension grafts of E15 whole ganglionic eminence implanted into the lesioned striatum. Following transplantation, the animals were housed either in standard cages (four per cage) or in enriched environment housing conditions (10 per cage) with tunnels, ladders and increased living space available for exploration, social interaction and play. The differentially housed animals were retested on the skilled staircase test at two separate time points. Repeated testing, environmental enrichment and transplantation positively influenced behavioural recovery. Partial recovery was observed bilaterally amongst the grafted animals in both housing conditions. Nevertheless, the grafted animals housed in the enriched environment performed significantly better in the final test compared with all of the other experimental groups. The grafts survived equally well under both housing conditions but the grafts of animals housed in the enriched environment contained larger projection neurons and were somewhat better reinnervated by dopaminergic afferents. An increased level of striatal brain-derived neurotrophic factor was observed in the control animals housed under the enriched compared with the standard conditions. The results indicate that an enriched environment can affect both graft function and graft morphology through as yet unknown mechanisms.

Lithium (Li +) treatment of NTera2/D1 (or hNT Neurons) in culture increases tyrosine hydroxylase (TH) expression in this cell-line [Zigova et al., (1999) Exp. Neurol., 157, 251-258]. It is not known if these Li + treated cells maintain TH expression once transplanted into the striatum of the hemiparkinsonian rats. hNT neurons were either treated with 1 mm LiCl or left untreated and then transplanted into the striatum of Sprague-Dawley rats. Some cells were exposed to the lithium for 24 h in culture while others were exposed only briefly (2-3 h) just prior to transplantation. We also examined whether Li + treatment of the animal after transplantation (0.24% w/w lithium carbonate in chow) was effective in increasing neuronal survival. One week after transplantation, the animals were perfused with 4% paraformaldehyde and immunocytochemistry was performed on 30 micro m sections through the transplant. Human nuclear matrix antigen immunostaining demonstrated that there was significantly better survival of cells in the group treated briefly with lithium compared to all other groups. Brief exposure to lithium resulted in a greater expression of TH in situ as well. Neuron specific enolase immunohistochemistry showed that there was extensive fibre outgrowth in all groups. These results suggest that brief Li + exposure may enhance survival to over 60% and increase TH expression of hNT Neurons transplanted in the hemiparkinsonian rat nearly three-fold.

We assessed the developmental potential of human telencephalic progenitor cells, with and without previous amplification in vitro, following grafting into the nonlesioned adult mouse CNS. Cell suspensions, shown to contain neuroepithelium-like and neuroblast-like cells, were injected into the subventricular zone (SVZ) and the striatum. These two regions were selected for comparative studies because one, the SVZ, is mitotically active, whereas the other, the striatum, is mitotically inactive. In situ hybridization with a human-specific Alu probe showed that the cells survived for up to 30 weeks in both targets and migrated away from the injection site. Fresh cells continued to proliferate and gave rise to very extended grafts before differentiating into neurons and glia. We further show that, when grown in vitro prior to grafting, human cells acquired new properties: Their proliferation was very limited, and they differentiated more rapidly. This study therefore provides new information about the use of these cells, which are a potential tool for both cellular and gene therapy., (Copyright 2002 Wiley-Liss, Inc.)

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.

Embryonic neurons transplanted to the adult CNS extend axons only for a developmentally defined period. There are certain intercellular factors that control the axonal extension, one of which may be the expression of the bcl-2 protein. In this study, rats with complete striatal dopamine fiber denervation received embryonic day 14 mouse ventral mesencephalon cells overexpressing human bcl-2 or control wild-type ventral mesencephalon cells. All rats were treated with cyclosporine to prevent rejection and the surviving grafts were analyzed for cell survival and outgrowth of dopaminergic fibers. The results demonstrate that bcl-2 overexpression does not enhance neuronal graft survival. However, the bcl-2 overexpressing neurons had a higher number of dopaminergic fibers that grew longer distances. These results show that overexpression of bcl-2 can result in longer distance axonal growth of transplanted fetal dopaminergic neurons and that genetic modification of embryonic donor cells may enhance their ability to reinnervate a neuronal target territory.

Transplantation of embryonic dopaminergic neurons is an experimental therapy for Parkinson's disease, but limited tissue availability and suboptimal survival of grafted dopaminergic neurons impede more widespread clinical application. Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4/5 (NT-4/5) exert neurotrophic effects on dopaminergic neurons via different receptor systems. In this study, we investigated possible additive or synergistic effects of combined GDNF and NT-4/5 treatment on rat embryonic (embryonic day 14) nigral explant cultures grown for 8 days. Contrary to cultures treated with GDNF alone, cultures exposed to NT-4/5 and GDNF+NT-4/5 were significantly larger than controls (1.6- and 2.0-fold, respectively) and contained significantly more protein (1.6-fold). Treatment with GDNF, NT-4/5 and GDNF+NT-4/5 significantly increased dopamine levels in the culture medium by 1.5-, 2.5- and 4.7-fold, respectively, compared to control levels, and the numbers of surviving tyrosine hydroxylase-immunoreactive neurons increased by 1.7-, 2.1-, and 3.4-fold, respectively. Tyrosine hydroxylase enzyme activity was moderately increased in all treatment groups compared to controls. Counts of nigral neurons containing the calcium-binding protein, calbindin-D28k, revealed a marked increase in these cells by combined GDNF and NT-4/5 treatment. Western blots for neuron-specific enolase suggested an enhanced neuronal content in cultures after combination treatment, whereas the expression of glial markers was unaffected. The release of lactate dehydrogenase into the culture medium was significantly reduced for GDNF+NT-4/5-treated cultures only. These results indicate that combined treatment with GDNF and NT4/5 may be beneficial for embryonic nigral donor tissue either prior to, or in conjunction with, intrastriatal transplantation in Parkinson's disease.

The transplantation of foetal mesencephalic cells (FMC) into the brain striatal system is an emerging treatment for Parkinson's disease, despite of the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate neurone survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We analyzed the effect of pre-treatment of FMC suspensions with bone morphogenetic protein-2 (BMP-2) (50 ng/ml) prior to grafting into the striatum of 6-hydroxydopamine lesioned rats. The viability of a FMC suspension was enhanced in vitro by BMP-2. Four weeks after transplantation, the number of dopaminergic neurones was higher and their morphology more developed in grafts pre-treated with BMP-2, compared with non-pre-treated grafts and rats showed a significant reduction in the turning behaviour test. Thus, the pre-treatment of FMCs with BMP-2 should be considered, together with other neurotrophic factors, as a procedure for transplantational treatment of Parkinson's disease.

We investigated here the effect of the novel glial cell line-derived neurotrophic factor (GDNF)-family member neurturin (NTN) on transplanted fetal dopamine (DA) neurons. Three groups of rats with complete unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal DA system received intrastriatal grafts of embryonic ventral mesencephalic tissue. Following transplantation animals received repeated injections of vehicle or NTN (0.3 microg or 3.0 microg) over three weeks posttransplantation. NTN-treated animals had significantly (1.8-fold) more tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Graft volume, TH-IR cell volume and overall dopaminergic host reinnervation remained unchanged. Amphetamine-induced rotation was rapidly compensated in all grafted rats. We conclude that administration of NTN may be a powerful way to increase survival of transplanted fetal DA neurons.

Cell transplantation therapy for Parkinson's patients, although seen to bring benefit to some patients during first clinical trials, remains impracticable on a large scale, in part because of the poor survival of the dopaminergic neurones transplanted. The loss of dopaminergic neurones occurs rapidly over the first 1-2 days after transplantation, in response to factors intrinsic to the host brain. Here we investigated whether contamination of the grafted cell suspension with blood during the transplantation procedure may be one factor responsible for the poor survival of DA neurons within the graft, possibly through factors such as free iron or complement. 6-Hydroxydopamine lesioned rats were grafted with 2 microl suspension of dissociated E14 ventral mesencephalon to which 1 microl blood or 1 microl grafting medium was added. After 6 weeks, there was no significant difference in the number of surviving DA neurones in the two groups. We conclude that contamination of grafts with blood is not a major factor responsible for the extensive death of dopaminergic neurones within them., (Copyright 1999 Academic Press.)