Intranigral transplants of GABA-rich striatal tissue induce behavioral recovery in the rat Parkinson model and promote the effects obtained by intrastriatal dopaminergic transplants

Intrastriatal transplantation of fetal ventral mesencephalon (VM) is currently explored as a potential clinical therapy in Parkinson's disease (PD). Although providing substantial benefit for the patient, behavioral recovery so far obtained with intrastriatal VM grafts is not complete. Using the 6-hydroxydopamine lesion model of PD, we show here that near-complete restoration of the striatal dopamine (DA) innervation can be achieved by multiple intrastriatal microtransplants of fetal DA cells; nevertheless, complete recovery in complex sensorimotor behaviors was not obtained in these animals. In line with the current model of basal ganglia function, this suggests that the lesion-induced overactivity of the basal ganglia output structures, i.e., the substantia nigra (SN) and the entopeduncular nucleus, may not be completely reversed by intrastriatal VM grafts. In the present study, we have transplanted fetal VM tissue or fetal striatal tissue, as a source of DA and GABA neurons, respectively, into the SN of DA-depleted rats. Intranigral VM grafts induced behavioral recovery in some sensorimotor behaviors (forelimb akinesia and balance tests), but the effect did not exceed the recovery observed after intrastriatal VM grafts. Intranigral grafts of striatal tissue induced a pattern of functional recovery which was distinctly different from that observed after intranigral VM grafts, and recovery in coordinated forelimb use in the paw-reaching test was even more pronounced than after intrastriatal transplantation of VM cells. Combined transplantation of DA neurons into the striatum and GABA-rich striatal neurons into the SN induced additive effects of behavioral recovery observed in the forelimb akinesia test. We propose that intranigral striatal transplants, by a GABA-mediated inhibitory action, can reduce the overactivity of the host SN projection neurons and can induce significant recovery in complex motor behavior in the rat PD model and that such grafts may be used to increase the overall functional efficacy of intrastriatal VM grafts.     

Survival and growth of fetal catecholamine neurons transplanted into primate brain

Dopamine and norepinephrine neuroblasts of the ventral mesencephalon, hypothalamus, and dorsolateral pons were transplanted from fetal African green monkeys into multiple brain sites in adult (host) African green monkeys. Tissue was grafted from both early and late gestational age fetuses. Immunohistochemical analysis, with antibodies to tyrosine hydroxylase, a marker of catecholamine-containing neurons, showed large numbers of transplanted catecholamine neurons in host cerebral cortex, corpus striatum and lateral ventricles up to 69 days after transplantation. Serial reconstructions revealed extensive outgrowth of neuronal processes from large numbers of transplanted neurons as well as expansion of the size of transplanted (solid) grafts of fetal brain tissue in the host brain. Some grafts extended from the caudate nucleus into the adjacent lateral ventricles or from the cerebral cortex into the underlying corpus callosum and ventricle. There were dense networks of varicose fibers emanating from the tyrosine hydroxylase positive neurons within intraparenchymal and intraventricular grafts. The size and shape of transplanted neurons retained characteristics common to catecholaminergic neurons from the dissected regions of fetal brain. Thus, a variety of fetal, catecholamine-containing neurons survive transplantation to primate brain and produce extensive neuritic outgrowths. Moreover, rejection of transplanted tissue was not apparent. These findings provide essential information on nerve cell grafting in a species closely related to humans as a prerequisite in the consideration of neural transplants as therapeutic measures in neurological disease.     

Zuckerkandl's organ improves long-term survival and function of neural stem cell derived dopaminergic neurons in Parkinsonian rats

Transplantation of neural stem cells (NSC) derived dopamine (DA) neurons has emerged as an alternative approach to fetal neural cell transplantation in Parkinson's disease (PD). However, similar to fetal neural cell, survival of these neurons following transplantation is also limited due to limited striatal reinnervation (graft with dense neuronal core), limited host-graft interaction, poor axonal outgrowth, lack of continuous neurotrophic factors supply and principally an absence of cell adhesion molecules mediated appropriate developmental cues. In the present study, an attempt has been made to increase survival and function of NSC derived DA neurons, by co-grafting with Zuckerkandl's organ (a paraneural organ that expresses neurotrophic factors as well as cell adhesion molecules); to provide continuous NTF support and developmental cues to transplanted DA neurons in the rat model of PD. 24 weeks post transplantation, a significant number of surviving functional NSC derived DA neurons were observed in the co-transplanted group as evident by an increase in the number of tyrosine hydroxylase immunoreactive (TH-IR) neurons, TH-IR fiber density, TH-mRNA expression and TH-protein level at the transplantation site (striatum). Significant behavioral recovery (amphetamine induced stereotypy and locomotor activity) and neurochemical recovery (DA-D2 receptor binding and DA and DOPAC levels at the transplant site) were also observed in the NSC+ZKO co-transplanted group as compared to the NSC or ZKO alone transplanted group. In vivo results were further substantiated by in vitro studies, which suggest that ZKO increases the NSC derived DA neuronal survival, differentiation, DA release and neurite outgrowth as well as protects against 6-OHDA toxicity in co-culture condition. The present study suggests that long-term and continuous NTF support provided by ZKO to the transplanted NSC derived DA neurons, helped in their better survival, axonal arborization and integration with host cells, leading to long-term functional restoration in the rat model of PD.     

Differential effects of Bcl-2 overexpression on fibre outgrowth and survival of embryonic dopaminergic neurons in intracerebral transplants

The causes of death of transplanted neurons are not known in detail, but apoptotic mechanisms involving caspase activation are likely to play a role. We examined whether overexpression of the anti-apoptotic protein Bcl-2 may enhance the survival of dopaminergic [tyrosine hydroxylase (TH)-immunoreactive] grafted neurons. For this purpose, we prepared cells from embryonic day 13 ventral mesencephalon (VM) of mice overexpressing human Bcl-2, or from their wild-type littermates. The bcl-2 transgene was strongly expressed in these cells, and resulted in protection of neuronal cultures from death triggered by serum deprivation or exposure to staurosporine. To model pretransplantation stress more closely in vitro, we stored dissociated embryonic mesencephalic cells for 8 h in the same type of medium used for intracerebral transplantation. This resulted in massive cell death as quantified by lactate dehydrogenase (LDH) release, and increased DNA fragmentation. Although this cell loss was strongly reduced by a caspase inhibitor, Bcl-2 had no significant protective effect. Finally, mesencephalic cell suspensions were xenografted into the striatum of immunosuppressed hemiparkinsonian rats. Neither the survival of TH-immunopositive transplanted neurons nor the functional recovery of the rats was improved by Bcl-2, although the Bcl-2 protein was strongly expressed in transgenic grafts 5 weeks after implantation, and dopaminergic fibre outgrowth from the grafts was significantly improved. These data suggest that cell death in neuronal transplants involves apoptotic mechanisms that can bypass negative regulation by Bcl-2.     

Pretreatment of donor cells with FGF-2 enhances survival of fetal hippocampal CA3 cell transplants in the chronically lesioned young adult hippocampus

The lesioned CA3 region of the young adult hippocampus is very conducive for robust survival and integration of fetal hippocampal CA3 cell grafts when transplanted at an early postlesion delay of 4 days. However, similar CA3 cell grafts placed at 45 days postlesion display significantly diminished cell survival, implying that the receptivity of the lesioned young adult host hippocampus to grafts decreases considerably with a prolonged postlesion transplantation delay. We hypothesize that decreased cell survival in grafts placed into the chronically lesioned hippocampus is due to a reduced level of host neurotrophic factors that support fetal hippocampal cells; hence, pretreatment and grafting of donor fetal CA3 cells with fibroblast growth factor-2 (FGF-2) considerably enhances graft neuronal integration into the chronically lesioned young adult hippocampus. We employed the optical fractionator cell counting method and rigorously quantified the number of surviving cells and neurons derived from 5'-bromodeoxyuridine-labeled Embryonic Day 19 CA3 cell grafts pretreated and transplanted with FGF-2 into the lesioned CA3 region of the young adult rat hippocampus, at a delay of 60 days after a unilateral intracerebroventricular administration of the kainic acid. For comparison, we also analyzed the survival of standard fetal CA3 cell grafts (i.e., without FGF-2 treatment) after similar transplantation. Pre treatment and transplantation of CA3 cell grafts with FGF-2 resulted in a robust yield of surviving cells (115% of injected cells) and neurons (100% of injected cells) from grafts. In contrast, standard CA3 cell grafts exhibited a reduced yield of surviving cells (29%) and neurons (25%). Thus, the yield of neurons from fetal hippocampal CA3 cell grafts placed into the chronically lesioned young adult hippocampus can be greatly enhanced by a simple pretreatment and grafting of donor fetal CA3 cells with FGF-2. These results have significance toward advancement of clinically feasible cell grafting strategies for repair of the damaged young adult hippocampus, particularly at extended periods after the injury or the onset of neurodegenerative diseases.     

Fibroblast growth factor-20 promotes the differentiation of Nurr1-overexpressing neural stem cells into tyrosine hydroxylase-positive neurons

Stem cells are currently considered as alternative cell resources for restorative transplantation strategies in Parkinson's disease. However, the mechanisms that induce differentiation of a stem cell toward the dopaminergic phenotype are still partly unknown thus hampering the production of dopaminergic neurons from stem cells. In the past, FGF-20 has been found to promote the survival of ventral mesencephalic (VM) dopaminergic (DA) neurons in culture. We hereby provide evidence that FGF-20, a growth factor of the FGF family, is expressed in the adult and 6-OHDA-lesioned striatum and substantia nigra, but is not expressed by VM glia or DA neurons, suggesting that FGF-20 may work on DA neurons in a paracrine- or target-derived manner. We also found that co-culture of Nurr1-NSCs with Schwann cells overexpressing FGF-20 induced the acquisition of a neuronal morphology by the NSCs and the expression of tyrosine hydroxylase (TH) as assessed by immunocytochemistry, cell ELISA, and Western blot analysis. RT-PCR showed, that both, Schwann cells and Nurr1-NSCs (differentiated or not), expressed the FGF-1 receptor suggesting that both direct and indirect actions of FGF-20 are possible. We show that differentiated Nurr1 cells retained both neuronal morphology and TH expression after transplantation into the striatum of 6-OHDA-lesioned postnatal or adult rats, but that neuritogenesis was only observed after postnatal grafts. Thus, our results suggest that FGF-20 promotes the differentiation of Nurr1-NSCs into TH-positive neurons and that additional factors are required for the efficient differentiation of DA neurons in the adult brain.     

Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation

Worldwideattention is presently focused on proliferating populations of neural precursor cells as an in vitro source of tissue for neural transplantation and brain repair. However, successful neuroreconstruction is contingent upon their capacity to integrate within the host CNS and the absence of tumorigenesis. Here we show that human neural precursor cells express very low levels of telomerase at early passages (less than 20 population doublings), but that this decreases to undetectable levels at later passages. In contrast, rodent neural precursors express high levels of telomerase at both early and late passages. The human neural precursors also have telomeres (approximately 12 kbp) significantly shorter than those of their rodent counterparts (approximately 40 kbp). Human neural precursors were then expanded 100-fold prior to intrastriatal transplantation in a rodent model of Parkinson's disease. To establish the effects of implanted cell number on survival and integration, precursors were transplanted at either 200,000, 1 million, or 2 million cells per animal. Interestingly, the smaller transplants were more likely to extend neuronal fibers and less likely to provoke immune rejection than the largest transplants in this xenograft model. Cellular proliferation continued immediately post-transplantation, but by 20 weeks there were virtually no dividing cells within any of the grafts. In contrast, fiber outgrowth increased gradually over time and often occupied the entire striatum at 20 weeks postgrafting. Transient expression of tyrosine hydroxylase-positive cells within the grafts was found in some animals, but this was not sustained at 20 weeks and had no functional effects. For Parkinson's disease, the principal aim now is to induce the dopaminergic phenotype in these cells prior to transplantation. However, given the relative safety profile for these human cells and their capacity to extend fibers into the adult rodent brain, they may provide the ideal basis for the repair of other lesions of the CNS where extensive axonal outgrowth is required.     

Influence of cell preparation and target location on the behavioral recovery after striatal transplantation of fetal dopaminergic neurons in a primate model of Parkinson's disease

Surgeries involving transplantation of fetal dopamine (DA) neurons into the caudate-putamen of patients with Parkinson's disease (PD) have been performed in various clinical trials to examine a potential restoration of motor function. The absence of studies in non-human primates to define the best transplantation protocols have lead to the use of a broad variety of techniques that potentially could have a major impact on the clinical outcome. The effects of using different cell and tissue preparation, and surgical targets, remain unknown. For this purpose, 20 St. Kitts African Green Monkeys (AFG) rendered parkinsonian by i.m. injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were balanced into 4 groups and unilaterally grafted in the (a) caudate or (b) putamen with fetal ventral mesencephalic (VM) tissue as (c) solid pieces or as a (d) cell suspension. By 9 months post-transplantation all animals showed significant and similar behavioral improvement as determined by a UPDRS based PD scale. Postmortem analyses showed that VM transplants survived in all animals. They were located in both surgical target sites, producing a broad DA reinnervation of the targeted nuclei that could also extend to the non-grafted nucleus on the ipsilateral side. Although no differences between groups were found in survival of DA neurons or degree of DA reinnervation, there was a significant correlation between striatal reinnervation and behavioral recovery only in animals transplanted in the putamen surgical target. Additionally, there was in general a stronger glial reaction to solid grafts than to cell suspensions. These studies provide data for the optimal time course, cell preparation and surgical targets for systematic examinations of both potential benefits and side effects of dopamine neuron cell transplantation in primate models of PD.     

Fetal neural transplants into an area of neurodegeneration in the spinal cord of the adult rat

Lesioning the spinal cord with an excitotoxic agent provides a model of neuronal degeneration while sparing afferent axons. The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension were able to rebuild a neural circuitry in the neuron-depleted adult cord. Fetal spinal cords, taken from rat embryos (gestational day E12-13), were transplanted as cell suspensions into an area of the lumbar cord previously depleted of neurons using kainic acid. The excitotoxic lesion extended over ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb. During the first month after injection, the damaged cord was characterized by proliferation and recruitment of various glial cell and Schwann cell populations. First to appear were activated microglia/macrophages and next reactive astrocytes which entered the lesion from its borders with the intact tissue. Schwann cells also ensheathed central axons. Differential sensitivity of various afferents to loss of postsynaptic target neurons was observed: rubrospinal and corticospinal afferents decreased in density while no conspicuous changes were observed for immunostained CGRP-containing or monoaminergic fibers. Two to fourteen months after surgery, transplants occupied most of the neuron-depleted area. The grafts did not display a laminar organization. Monoaminergic afferents grew for a long distance and formed a network within transplants. Similarly, primary sensory CGRP-immunoreactive fibers entering in the dorsal roots penetrated deeply into transplants. In contrast, cortico- and rubrospinal afferents entered only the most peripheral portion of transplants. Our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord. Host-to-graft connections can be formed, although their spatial extent in the transplants may depend upon features of the afferent fiber systems.     

A role for tumor necrosis factor alpha in death of dopaminergic neurons following neural transplantation

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease (PD). We have examined the temporal release profile of an inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of TNFalpha released in vivo when added to cultures of embryonic DA neurons, significantly reduced the survival of DA neurons in vitro, and this cell death could be prevented by the inclusion of an antibody to the TNFalpha receptor type 1. Inclusion of this antibody in cell suspensions during transplantation also increased the survival of transplanted fetal DA neurons by approximately 250%. Use of this therapeutic antibody approach may offer significant improvements to neural transplantation as a treatment for PD.     

Homotypic fetal transplants into an experimental model of spinal cord neurodegeneration

Many neurotransplantation studies have dealt with the ability of solid fetal spinal grafts to develop in the previously traumatized spinal cord of a host. In neurodegenerative spinal diseases, however, motoneuronal death occurs in the absence of a trauma, i.e., in the absence of axotomy of afferent fibers. Lesioning the spinal cord with an excitotoxic agent may provide a useful neurodegenerative model. The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension are able to rebuild a neural circuitry. Emphasis is given here to the analysis of the development of transplanted motoneurons and host-graft connectivity. The lesion was made by kainic acid on the right side of the lumbar enlargement 1 week before transplantation. The fetal spinal cords were taken from rat embryos (gestational day E12-13) and transplanted as cell suspensions. Light- and electron-microscopic analysis demonstrated that the excitotoxic lesion extended over the entire spinal segment and was confined primarily to the ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb. The lesion was characterized by a lack of neurons, glial proliferation, and sparing of fibers of passage and afferents. Two to fourteen months after surgery, the transplants were generally large, occupying most of the neuron-depleted area. The boundaries between the transplant and host tissue were clearly delineated by the higher cellular density of the graft and the particular cytoarchitecture, i.e., the cell suspension grafts did not display a laminar organization. Among the different neuronal populations within the transplant, one resembled motoneurons: large, typically Nissl-stained and immunoreactive for calcitonin gene-related peptide (CGRP). No grafted neuron, however, extended an axon into the host ventral roots. Monoaminergic afferents from the host were studied using immunostaining for serotonin, noradrenaline, and tyrosine hydroxylase. These afferent fibers, thin and varicose, grew for a long distance and formed a network within transplants. Similarly, primary sensory CGRP-immunoreactive fibers (entering the graft from the dorsal host-graft interface) penetrated deeply into transplants. The response of cortico- and rubro-spinal afferents to the implantation of fetal tissue was different. After injection of WGA-HRP, a few anterogradely labeled cortical and rubral fibers entered only the most peripheral portion of transplants. In conclusion, our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lineage-restricted neural precursors survive, migrate, and differentiate following transplantation into the injured adult spinal cord

Fetal spinal cord from embryonic day 14 (E14/FSC) has been used for numerous transplantation studies of injured spinal cord. E14/FSC consists primarily of neuronal (NRP)- and glial (GRP)-restricted precursors. Therefore, we reasoned that comparing the fate of E14/FSC with defined populations of lineage-restricted precursors will test the in vivo properties of these precursors in CNS and allow us to define the sequence of events following their grafting into the injured spinal cord. Using tissue derived from transgenic rats expressing the alkaline phosphatase (AP) marker, we found that E14/FSC exhibited early cell loss at 4 days following acute transplantation into a partial hemisection injury, but the surviving cells expanded to fill the entire injury cavity by 3 weeks. E14/FSC grafts integrated into host tissue, differentiated into neurons, astrocytes, and oligodendrocytes, and demonstrated variability in process extension and migration out of the transplant site. Under similar grafting conditions, defined NRP/GRP cells showed excellent survival, consistent migration out of the injury site and robust differentiation into mature CNS phenotypes, including many neurons. Few immature cells remained at 3 weeks in either grafts. These results suggest that by combining neuronal and glial restricted precursors, it is possible to generate a microenvironmental niche where emerging glial cells, derived from GRPs, support survival and neuronal differentiation of NRPs within the non-neurogenic and non-permissive injured adult spinal cord, even when grafted into acute injury. Furthermore, the NRP/GRP grafts have practical advantages over fetal transplants, making them attractive candidates for neural cell replacement.     

Diminished survival of mesencephalic dopamine neurons grafted into aged hosts occurs during the immediate postgrafting interval

The survival rate of dopamine (DA) neurons in mesencephalic grafts to young adult rats is poor, estimated at 5-20%, and even poorer in grafts to the aged striatum. Grafted cells die in young adult rats during the first 4 days after implantation. The present study was undertaken to determine whether the decreased survival of DA neurons in grafts to aged rats is (1) due to additional cell death during the immediate postgrafting interval or (2) due to protracted cell loss during longer postgrafting intervals. We compared survival rates of tyrosine hydroxylase-immunoreactive (THir) neurons in cell suspension grafts to young adult (3 months) and aged (24 months) male Fischer 344 rats at 4 days and 2 weeks after transplantation. At 4 days after grafting, mesencephalic grafts within the aged rat striatum contain approximately 25% of the number of THir neurons in the same mesencephalic cell suspension grafted to young adult rats. This corroborates the decreased survival of grafted DA neurons we have demonstrated previously at 10 weeks postgrafting. THir neurons in grafts to the intact striatum possessed a significantly shorter "long axis" than their counterparts on the lesioned side. No significant differences in the number of apoptotic nuclear profiles or total alkaline phosphatase staining between mesencephalic grafts to young and aged rats were detectable at 4 days postgrafting. In summary, the present study indicates that the exaggerated cell death of grafted DA neurons that occurs following implantation to the aged striatum occurs during the immediate postgrafting interval, timing identical to that documented for young adult hosts.     

Cellular distribution of the NMDA receptor subunit NMDAR1 in fetal ventral mesencephalon transplants in the dopamine-depleted striatum of a rat

Immunohistochemistry was performed to demonstrate the cellular distribution of N-methyl-D-aspartate (NMDA) receptor subunit NMDAR1 in the intrastriatal grafts of a rat model of Parkinson's disease. Unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal pathway were produced in young adult female rats. Neural transplantation was performed with fetal ventral mesencephalon (VM) tissue (at embryonic day 15) 3 weeks after the 6-OHDA lesions. In the fetal VM in which the tyrosine hydroxylase (TH) immunoreactivity was intensely observed, no NMDAR1 subunit immunoreactivity was detected. Immunopositive cells of NMDAR1 were densely distributed in the intact SNc contralateral to the lesions, in which intense immunoreactivity for TH was observed. In contrast, the cells positive for NMDAR1 in the SNr were scattered. The immunoreactivity for NMDAR1 was markedly decreased in the SNc, but not in the SNr on the lesioned side. Double immunostaining revealed that most TH-positive cells in the SNc showed moderate NMDAR1 immunoreactivity. Within the intrastriatal fetal VM grafts containing TH-positive cells, NMDAR1-positive cells tended to locate homogeneously within the grafts. These were composed of various cell sizes and shapes, but they were mainly medium-sized and aspiny cells. Double immunostaining revealed that a part of the TH-positive cells in the grafts was also immunopositive for NMDAR1. Taken together with our previous studies, it is suggested that both dopaminergic neurons and nondopaminergic neurons in the VM transplants appear to be modified functionally by glutamatergic afferents via various glutamate receptors, including NMDAR1.     

Transplantation of cultured human adrenal chromaffin cells into 6-hydroxydopamine-lesioned rat brain

Adult young rats were subjected to a unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway and then given intrastriatal grafts of human fetal adrenal chromaffin cell cultures. Amphetamine-induced ipsiversive turning behavior in the lesioned rats was largely reversed in four of eight rats given such transplants when tested at 1.5 and 4.5 months post-transplantation. Two rats showed a transient recovery at 1.5 months followed by deterioration at 4.5 months, while two other rats showed continuous deterioration. Six rats given sciatic nerve grafts as controls all showed deterioration from the pretransplantation levels. Catecholamine fluorescent and immunohistochemical examination of chromaffin-cell-transplanted brains demonstrated neurons and neuronal processes positive for catecholamines or tyrosine hydroxylase in the transplanted area. This transplantation of cultured human fetal cells to an animal model may provide the necessary basic experimental system for assessing the possible utility of human neuronal transplants.     

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons

Neural transplantation offers the potential of treating Parkinson's disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson's disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced overexpression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson's disease.     

Comparison of pure and mixed populations of human fetal-derived neural progenitors transplanted into intact adult rat brain

We examined the influence of initial graft composition on the number, type, and distribution of human progenitor cells after transplantation into the anterior subventricular zone (SVZa) of normal adult rats. The grafted populations were derived from 19-week-old human cortical tissue grown under adherent conditions in the presence of fibroblast growth factor (FGF) and from a subpopulation of nestin-expressing cells, isolated using negative immunoselection methods, which exhibited properties of neural progenitors. Identical numbers of each were transplanted and the number and location of engrafted cells were compared 4 weeks later. We found a significantly greater number of presumptive neurons and astrocytes in animals that received mixed grafts compared to those enriched for progenitors. In addition, the number of human cells undergoing division was significantly greater in animals that received mixed grafts. The spatial distribution of grafted cells was not significantly different, suggesting that the patterns of cell migration were unaffected by transplant composition, whereas, a greater proportion of neurons was observed in the neurogenic areas of animals that received progenitor-enriched grafts. From a clinical perspective, our results suggest that the cellular composition of human fetal-derived transplants may be an important parameter that influences the number and pattern of differentiation of engrafted cells following transplantation in the mature CNS.     

Intranigral transplantation of solid tissue ventral mesencephalon or striatal grafts induces behavioral recovery in 6-OHDA-lesioned rats

Parkinson's disease (PD) is characterized by a degeneration of the dopamine (DA) pathway from the substantia nigra (SN) to the basal forebrain. Prior studies in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats have primarily concentrated on the implantation of fetal ventral mesencephalon (VM) into the striatum in attempts to restore DA function in the target. We implanted solid blocks of fetal VM or fetal striatal tissue into the SN to investigate whether intra-nigral grafts would restore motor function in unilaterally 6-OHDA-lesioned rats. Intra-nigral fetal striatal and VM grafts elicited a significant and long-lasting reduction in apomorphine-induced rotational behavior. Lesioned animals with ectopic grafts or sham surgery as well as animals that received intra-nigral grafts of fetal cerebellar cortex showed no recovery of motor symmetry. Subsequent immunohistochemical studies demonstrated that VM grafts, but not cerebellar grafted tissue expressed tyrosine hydroxylase (TH)-positive cell bodies and were associated with the innervation by TH-positive fibers into the lesioned SN as well as adjacent brain areas. Striatal grafts were also associated with the expression of TH-positive cell bodies and fibers extending into the lesioned SN and an induction of TH-immunolabeling in endogenous SN cell bodies. This finding suggests that trophic influences of transplanted fetal striatal tissue can stimulate the re-expression of dopaminergic phenotype in SN neurons following a 6-OHDA lesion. Our data support the hypothesis that a dopaminergic re-innervation of the SN and surrounding tissue by a single solid tissue graft is sufficient to improve motor asymmetry in unilateral 6-OHDA-lesioned rats.     

Toward full restoration of synaptic and terminal function of the dopaminergic system in Parkinson's disease by stem cells

New therapeutic nonpharmacological methodology in Parkinson's disease (PD) involves cell and synaptic renewal or replacement to restore function of neuronal systems, including the dopaminergic (DA) system. Using fetal DA cell therapy in PD patients and laboratory models, it has been demonstrated that functional motor deficits associated with parkinsonism can be reduced. Similar results have been observed in animal models with stem cell-derived DA neurons. Evidence obtained from transplanted PD patients further shows that the underlying disease process does not destroy transplanted fetal DA cells, although degeneration of the host nigrostriatal system continues. The optimal DA cell regeneration system would reconstitute a normal neuronal network capable of restoring feedback-controlled release of DA in the nigrostriatal system. The success of cell therapy for PD is limited by access to preparation and development of highly specialized dopaminergic neurons found in the A9 and A10 region of the substantia nigra pars compacta as well as the technical and surgical steps associated with the transplantation procedure. Recent laboratory work has focused on using stem cells as a starting point for deriving the optimal DA cells to restore the nigrostriatal system. Ultimately, understanding the cell biological principles necessary for generating functional DA neurons can provide many new avenues for better treatment of patients with PD.     

Intrastriatal and intranigral grafting of hNT neurons in the 6-OHDA rat model of Parkinson's disease

The clinical findings on neural transplantation for Parkinson's disease (PD) reported thus far are promising but many issues must be addressed before neural transplantation can be considered a routine therapeutic option for PD. The future of neural transplantation for the treatment of neurological disorders may rest in the discovery of a suitable alternative cell type for fetal tissue. One such alternative may be neurons derived from a human teratocarcinoma (hNT). hNT neurons have been shown to survive and integrate within the host brain following transplantation and provide functional recovery in animal models of stroke and Huntington's disease. In this study, we describe the transplantation of hNT neurons in the substantia nigra (SN) and striatum of the rat model for PD. Twenty-seven rats were grafted with one of three hNT neuronal products; hNT neurons, hNT-DA neurons, or lithium chloride (LiCl) pretreated hNT-DA neurons. Robust hNT grafts could be seen with anti-neural cell adhesion molecule and anti-neuron-specific enolase immunostaining. Immunostaining for tyrosine hydroxylase (TH) expression revealed no TH-immunoreactive (THir) neurons in any animals with hNT neuronal grafts. THir cells were observed in 43% of animals with hNT-DA neuronal grafts and all animals with LiCl pretreated hNT-DA neuronal grafts (100%). The number of THir neurons in these animals was low and not sufficient to produce significant functional recovery. In summary, this study has demonstrated that hNT neurons survive transplantation and express TH in the striatum and SN. Although hNT neurons are promising as an alternative to fetal tissue and may have potential clinical applications in the future, further improvements in enhancing TH expression are needed.