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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.

Background . Autologous neural cell ecosystem (ANCE) transplantation improves motor recovery in MPTP monkeys. These motor symptoms were assessed using semi-quantitative clinical rating scales, widely used in many studies. However, limitations in terms of sensitivity, combined with relatively subjective assessment of their different items, make inter-study comparisons difficult to achieve. Objective . The aim of this study was to quantify the impact of MPTP intoxication in macaque monkeys on manual dexterity and assess whether ANCE can contribute to functional recovery. Methods . Four animals were trained to perform 2 manual dexterity tasks. After reaching a motor performance plateau, the animals were subjected to an MPTP lesion. After the occurrence of a spontaneous functional recovery plateau, all 4 animals were subjected to ANCE transplantation. Results . Two of 4 animals underwent a full spontaneous recovery before the ANCE transplantation, whereas the 2 other animals (symptomatic) presented moderate to severe Parkinson's disease (PD)-like symptoms affecting manual dexterity. The time to grasp small objects using the precision grip increased in these 2 animals. After ANCE transplantation, the 2 symptomatic animals underwent a significant functional recovery, reflected by a decrease in time to execute the different tasks, as compared with the post-lesion phase. Conclusions . Manual dexterity is affected in symptomatic MPTP monkeys. The 2 manual dexterity tasks reported here as pilot are pertinent to quantify PD symptoms and reliably assess a treatment in MPTP monkeys, such as the present ANCE transplantation, to be confirmed in a larger cohort of animals before future clinical applications.

Objective: The purpose of this study was to investigate the role of deep brain stimulation (DBS) of the globus pallidus internus (GPi) in dopamine and dopamine transporter metabolism and to explore the regulatory role of DBS on dopaminergic neurons in Tourette syndrome by constructing an autoimmune model., Methods: Serum with high concentrations of antinuclear antibodies or phosphate-buffered saline solution was injected into the striatum of rats by a stereotactic technique and micropump. Then, electrodes were planted in the rats' globus pallidus internus. Concentrations of dopamine and dopamine transporter in the striatum were detected by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot analysis after 7 days of high-frequency stimulation (130 Hz)., Results: The tic behavior score of rats in the Tourette syndrome group was higher than that of rats in the control group (P < 0.01). After high-frequency stimulation, the scores of the Tourette syndrome model group and the control group significantly decreased. The concentration of dopamine in the Tourette syndrome model group and the control group also significantly decreased after electric stimulation (P < 0.05). In addition, immunohistochemical analysis and Western blot test results showed that dopamine transporter in the Tourette syndrome model nonstimulation group was lower than in the Tourette syndrome model stimulation group, and that dopamine transporter in the control nonstimulation group was lower than in the control stimulation group (P < 0.05)., Conclusions: The results of this study show that the mechanism of DBS of the GPi in the treatment of Tourette syndrome involved monoamine neurotransmitters, especially the dopamine system, that affected the metabolism and transport of corresponding neurotransmitters, playing an important role in regulating the concentration of synaptic neurotransmitters and changing the biologic activity of basal ganglia nerve circuits., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.)

Objective: To describe a technique to approach the anterior inferior basal ganglia., Case Description: A 29-year-old man presented with several episodes of facial droop and a cavernous malformation anterior, inferior, and lateral to the head of the caudate nucleus. Several possible surgical approaches were considered, including a transsylvian approach and a contralateral interhemispheric approach. Ultimately, the patient underwent a contralateral interhemispheric trans-striatocapsular approach to the lesion and was discharged without neurologic deficit within 72 hours., Conclusion: The approach described here is a novel alternative to transsylvian or supracarotid approaches to the anterior inferior basal ganglia and in this patient provided a well-tolerated surgical corridor that allowed complete resection of his cavernoma. We discuss several advantages and disadvantages of the various approaches to the anterior inferior basal ganglia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum(1). Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra(2). The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions(3,4). These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Recent discoveries have brought mitochondria functions in focus of the neuroscience research community and greatly stimulated the demand for approaches to study mitochondria dysfunction in neurodegenerative diseases. Many mouse disease models have been generated, but studying mitochondria isolated from individual mouse brain regions is a challenge because of small amount of the available brain tissue. Conventional techniques for isolation and purification of mitochondria from mouse brain subregions, such as ventral midbrain, hippocampus, or striatum, require pooling brain tissue from six to nine animals for a single mitochondrial preparation. Working with pooled tissue significantly decreases the quality of data because of the time required to dissect several brains. It also greatly increases the labor intensity and the cost of experiments as several animals are required per single data point. We describe a method for isolation of brain mitochondria from mouse striata or other 7-12 mg brain samples. The method utilizes a refrigerated table-top microtube centrifuge, and produces research grade quality mitochondria in amounts sufficient for performing multiple enzymatic and functional assays, thereby eliminating the necessity for pooling mouse brain tissue. We also include a method of measuring ADP-ATP exchange rate as a function of mitochondrial membrane potential (ΔΨm) in small amounts of isolated mitochondria, adapted to a plate reader format.

Huntington's disease (HD) is an inherited neurodegenerative disorder that usually occurs in the third or fourth decades of life. Stem cell therapy is one of the approaches for HD treatment. Since mesenchymal stem cells (MSCs) have the ability to migrate into the lesioned site, we transplanted rat bone marrow-derived MSCs intravenously, following unilateral intrastriatal lesion made by quinolinic acid (QA) in Wistar rats. QA administration caused widespread neuropathological deficits similar to those found in HD, including impairments in motor and cognitive functions. Animals receiving MSCs exhibited significant improvement in motor and cognitive performance compared with sham group animals that did not receive cells. Animals were tested by apomorphine-induced rotations, beam walk, cylinder and hang wire tests at different times after cell transplantation. Results indicate that systemic transplantation of MSCs can significantly reduce the behavioral abnormalities of these animals. This method of systemic injection has a great advantage over invasive surgical techniques for transplantation of cells at the lesioned site.

Aims: This study was aimed to elucidate if bone marrow stromal cells (BMSC) could ameliorate cognitive dysfunction due to chronic cerebral ischemia when transplanted into the brain., Methods: The BMSC were harvested from green fluorescence protein (GFP)-expressing mice. Wistar rats were subjected to bilateral common carotid artery (CCA) ligation. The BMSC (4 × 10⁵ cells) or vehicle were stereotactically injected into the right striatum 24 h after the insult. Cognitive function was evaluated with the Morris water maze task after 3 and 5 weeks. Histological analysis was performed after 6 weeks., Results: Cognitive function was significantly impaired in the vehicle-transplanted animals, when compared with the non-CCA-ligation animals. BMSC transplantation significantly improved it. The BMSC were widely distributed in the ischemic brain, including the neocortex, white matter and hippocampus, and some of them expressed the phenotypes of neurons, astrocytes and endothelium. They also significantly ameliorated white matter damage., Conclusions: These findings strongly suggest that the BMSC may have the potential to attenuate white matter injury and improve cognitive dysfunction due to chronic cerebral ischemia. The present results would shed light on the potential of a novel strategy, cell therapy against ischemia-related cognitive dysfunction., (Copyright © 2010 S. Karger AG, Basel.)

Stem cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months). NPs were derived from adherent monolayer cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity and an avid differentiation into neurons that projected via major white matter tracts to a variety of forebrain targets. A third of NP-derived neurons expressed the basal forebrain-neostriatal marker dopamine-regulated and cyclic AMP-regulated phosphoprotein. Graft-derived neurons formed mature synapses with host postsynaptic structures, including dendrite shafts and spines. NPs inoculated in white matter tracts showed a tendency toward glial (primarily astrocytic) differentiation, whereas NPs inoculated in the ventricular epithelium persisted as nestin(+) precursors. Our findings demonstrate the long-term ability of noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while maintaining some cell fate plasticity that is strongly influenced by particular central nervous system (CNS) niches.

Ventral mesencephalic (VM) precursor cells are of interest in the search for transplantable dopaminergic neurons for cell therapy in Parkinson's disease (PD). In the present study we investigated the survival and functional capacity of in vitro expanded, primary VM precursor cells after intrastriatal grafting to a rat model of PD. Embryonic day 12 rat VM tissue was mechanically dissociated and cultured for 4 or 8 days in vitro (DIV) in the presence of FGF2 (20 ng/ml), FGF8 (20 ng/ml) or without mitogens (control). Cells were thereafter differentiated for 6 DIV by mitogen withdrawal and addition of serum. After differentiation, significantly more tyrosine hydroxylase-immunoreactive (TH-ir), dopamine-producing neurons were found in FGF2- and FGF8-expanded cultures compared to controls. Moreover, expansion for 4 DIV resulted in significantly more TH-ir cells than expansion for 8 DIV both for FGF2 (2.4 fold; P<0.001) and FGF8 (3.8 fold; P<0.001) treated cultures. The functional potential of the expanded cells (4 DIV) was examined after grafting into striatum of aged 6-hydroxydopamine-lesioned rats. Amphetamine-induced rotations performed 3, 6 and 9 weeks postgrafting revealed that grafts of FGF2-expanded cells induced a significantly faster and better functional recovery than grafts of FGF8-expanded cells or control cells (P<0.05 for both). Grafts of FGF2-expanded cells also contained significantly more TH-ir cells than grafts of FGF8-expanded cells (P<0.05) or control cells (P<0.01). In conclusion, FGF2-mediated pregrafting expansion of primary VM precursor cells considerably improves dopaminergic cell survival and functional restoration in a rat model of PD.

Transplantation of bone marrow stromal cells (BMSC) has recently been demonstrated to provide neuroprotection in animal models of brain injuries such as ischemia and trauma. The present study was undertaken to explore whether BMSC can promote the survival of dopamine (DA) neurons in neuronal insult models in vitro. We also examined whether BMSC can increase the survival rate of embryonic DA neurons grafted into the striatum of a rat model of Parkinson's disease (PD). Treatment with conditioned media derived from BMSC cultures was found to significantly prevent the death of DA neurons in in vitro cell injury models such as serum deprivation and exposure to the neurotoxin 6-OHDA. In a transplantation study, we also found that the survival of grafted DA cells was significantly enhanced by treating donor cells with the conditioned media at the steps of both cell dissociation and implantation. The results suggest that BMSC may secrete diffusible factors able to protect DA neurons against neuronal injuries. Indeed, BMSC expressed mRNA encoding brain-derived neurotrophic factor, fibroblast growth factor-2 and glial cell line-derived neurotrophic factor, all of which have previously been shown to exhibit potent neurotrophic effects on DA cells. Enzyme-linked immunosorbent assay revealed that the cells release these growth factors into culture media. The present data indicate that BMSC may be a potential donor source of cell-based regenerative therapy for PD where the progressive loss of the midbrain DA neurons takes place.

The present study analyzed whether grafts of the mesencephalic progenitor cell line CSM14.1 into the neonatal rat caudate putamen (CPu) differentiate into neurons and whether this is accompanied by a functional improvement in 6-hydroxydopamine (6-OHDA)-lesioned animals. As in previous studies, a neuronal differentiation of CSM14.1 cells transplanted into the CPu of adult animals could not be observed, so we here used neonatal rats, because graft location and host age seemingly are crucial parameters for neural transplant differentiation and integration. Rats bilaterally lesioned at postnatal day 1 by intraventricular 6-OHDA-injections 2 days later received 100,000 CSM14.1 cells prelabelled with the fluorescent dye PKH26 into the right CPu. Five weeks after grafting, the cylinder test was performed, and the data compared with data from age-matched intact controls and bilaterally lesioned-only animals. Brain slices immunostained for tyrosine hydroxylase (TH) were quantified by optical densitometry. We observed a significant preference of left forelimb use exclusively in transplanted animals. In these rats, TH-containing perikarya were found in the grafted CPu, presumedly leading to the significant increase of TH-immunoreactive fibers in this region. Moreover, confocal laser microscopy revealed a differentiation of transplanted PKH26-labelled CSM14.1 cells into neuronal nuclei antigen or TH-immunoreactive cells. Thus, CSM14.1 cells differentiate into TH-containing neurons, which most probably contribute to the preferred forelimb use, indicating a functional integration of CSM14.1 cells into the host basal ganglia loops during early postnatal development. These findings that are in contrast to observations in adult rats suggest instructive cues for neuronal differentiation and integration given by the neonatal microenvironment.

For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.

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.)

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra projecting to the striatum. One therapeutic approach to this disease has been the intrastriatal transplantation of dopamine-secreting cells. We have investigated the suitability of glomus cells of the carotid body for dopamine-cell replacement in animal models of Parkinson's disease. Carotid body glomus cells are physiologic arterial oxygen sensors that release large amounts of dopamine in response to hypoxia. We have used hemi-Parkinsonian rats, induced by injection of 6-hydroxydopamine into the substantia nigra, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated monkeys with chronic Parkinsonism. In both cases we made transplants of carotid body cell aggregates into the putamen. Functional recovery of the grafted animals was observed after the surgery and was stable for several months. Although the study was more detailed in the rat, in the two animal models the amelioration of the motor deficits was paralleled by striatal dopaminergic reinnervation and survival of grafted glomus cells. Our results suggest that intrastriatal autotransplants of carotid body tissue could be a feasible technique to treat some cases of Parkinson's disease in humans.

Excitotoxicity has been involved in the pathogenesis of several neurodegenerative disorders. Using intrastriatal quinolinic acid (QUIN) injection as an animal model of Huntington's disease, we attempt to identify the neurotransmitter phenotype of striatal projection neurons protected by neurturin (NRTN). Control or NRTN-secreting cell lines were grafted in the striatum before QUIN injection and striatal projection neurons were examined by retrograde Fluorogold labeling and in situ hybridization. Intrastriatal grafting of NRTN-secreting cell line selectively prevented the loss of striatopallidal neurons and also the decrease in the mRNA levels for their markers (glutamic acid decarboxylase 67 and preproenkephalin) induced by QUIN, without affecting striatonigral neurons. Thus, our findings show that NRTN is a selective neuroprotective factor for striatopallidal neurons, suggesting that it might be a candidate for the treatment of movement disorders in which this neuronal population is affected.

Current ex vivo gene therapy for Parkinson's disease using glial cell line-derived neurotrophic factor (GDNF) is limited by the lack of a monitoring mechanism to determine the expression of GDNF once the cells or other vehicles are transferred into animal models. The purpose of this study was to test whether a Renilla luciferase (RUC)-GDNF fusion protein secreted by the genetically engineered glial cell line RG-1 could be measured photometrically in cerebrospinal fluid (CSF). RG-1 was constructed by permanent transformation with a plasmid DNA construct that contains a GDNF cDNA (gdnf) fused to a RUC cDNA (ruc). The fusion protein secreted by RG-1 was shown to retain both GDNF and RUC activity. The concentration of GDNF determined by enzyme-linked immunoadsorbent assay (ELISA) was correlated with the light emission detected by assaying for RUC bioluminescence in RG-1 culture medium, indicating that RUC can be used as a reporter for GDNF in vitro. The cells were then implanted into rat brain (n=20), and the cisternal CSF was analyzed. Bioluminescence was successfully detected in the CSF samples, and was quantified over a period of 25 days, while Western blotting and ELISA failed to detect GDNF in CSF, presumably because the concentration of the RUC-GDNF fusion was too low. This study demonstrates that the transformed glial cell line RG-1 offers a sensitive self-reporting assay for GDNF expression.

Adult C57BL/6 mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Intrastriatal transplantation of bone marrow stromal cells (MSCs) was performed 1 week after MPTP administration. MSCs were harvested from donor adult mice, and then cultured and prelabeled with bromodeoxyuridine (BrdU). MPTP-Parkinson's disease (PD) mice treated with intrastriatal injection of phosphate-buffered saline (PBS), and normal non-MPTP mice were used as controls. MPTP-PD mice with MSC intrastriatal transplantation exhibit significant improvement on the rotarod test (P<0.05) at day 35 compared with PBS controls. Immunohistochemistry shows that BrdU reactive cells survive in the transplanted areas in the MPTP-PD striatum at least 4 weeks after administration. Scattered BrdU reactive cells express tyrosine hydroxylase (TH) immunoreactivity. Our findings suggest that MSCs injected intrastriatally survive, express dopaminergic protein TH immunoreactivity, and promote functional recovery.

In this study we investigated whether CNS axons regenerate following attenuation of scar formation using a combination of antibodies against two isoforms of transforming growth factor beta (TGFbeta). Anaesthetized adult rats were given unilateral mechanical lesions of the nigrostriatal tract. Implantation of transcranial cannulae allowed wounds to be treated with a combination of antibodies against TGFbeta1 and TGFbeta2 once daily for 10 days postaxotomy. Eleven days post-transection brains from animals under terminal anaesthesia were recovered for histological evaluation. Gliosis, inflammation and the response of dopaminergic nigral axons were assessed by immunolabelling. Treatment with antibodies against TGFbeta1 and TGFbeta2 attenuated (but did not abolish) the response of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and of NG2-immunoreactive glia but did not attenuate the response of CR3-immunoreactive microglia and macrophages. However, this reduction in scar formation was not accompanied by growth of cut dopaminergic nigral axons. We conclude that treatment of injured adult rat brain with a combination of antibodies against TGFbeta1 and TGFbeta2 results in a reduction of scar formation but that this is not sufficient to enhance spontaneous long distance CNS axon regeneration.

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.

Bilateral astroglial transplantation into the neostriatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys resulted in significant performance improvement in a spatial delayed response task, but failed to modify perseveration in an object retrieval detour task, or to improve motor clinical rating. Results suggest that brain circuits subserving various motor and cognitive performances can be functionally dissociated, and that remaining resources for the reorganization of neural circuits involved in spatial working memory performance in parkinsonian monkeys, appear to be responsive to striatal transplantation of subcultured, fetal striatal astroglial cells.

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.

Intracerebral neural xenografts elicit a host immune response that results in their rapid rejection. This forms a key barrier to the therapeutic use of xenogeneic tissue transplantation for conditions such as Parkinson's disease. The current study sought to provide insight into the cellular components of donor cell suspensions that are important in stimulating the host rejection response and thereby to suggest rational manipulations of xenogeneic donor tissue that might ultimately enhance its clinical utility. The neural stem cell mitogens, epidermal growth factor and fibroblast growth factor-2, have been used to isolate and expand populations of primordial neural precursor cells from the embryonic pig brain. The immune response elicited by these cells on transplantation into the non-immunosuppressed rat has been fully characterised. In the first experiments, expanded neural precursors were grafted into the hemi-parkinsonian, non-immunosuppressed Sprague-Dawley rat and graft status and host response examined 10, 21, 35 and 60 days post-transplantation. While equivalent primary tissue grafts were completely eliminated at 35 days, grafts of expanded neural precursors with healthy neurofilament-positive projections were present at all time-points, and two large grafts remained even at 60 days. Some grafts appeared to elicit minimal host immune responses at the time-points they were examined, although most did appear to be undergoing a rejection process since a co-ordinated response involving host cytotoxic T-lymphocytes, microglia/macrophages, immunoglobulin M and complement could be demonstrated to varying degrees. Subsequent experiments went on to demonstrate further that expanded precursor populations and primary tissue suspensions differed in their immunogenic profile. Firstly, when primary tissue was injected intraperitoneally into immunocompetent rats a vigorous primary humoral response was generated. No such response was detected following injection of expanded neural precursors. Secondly, flow cytometric analysis revealed small but significant levels of class II porcine major histocompatibility complex expression in primary cell suspensions but no such expression in expanded precursor populations.The results of this study therefore demonstrate that the immunogenicity of porcine neural cell suspensions used for intracerebral grafting is reduced when neural stem cell mitogens are used to expand precursor cells. The implications of these findings in the development of novel xenogeneic cellular therapies for neurodegenerative conditions such as Parkinson's disease are discussed.

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.

After striatal injury, sprouting dopaminergic fibres grow towards and intimately surround wound macrophages which, together with microglia, express the dopaminergic neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF). To evaluate the importance of these endogenously secreted neurotrophic factors in generating striatal peri-wound dopaminergic sprouting, the peri-wound expression of BDNF or GDNF was inhibited by intrastriatal infusion of antisense oligonucleotides for 2 weeks in mice. Knock-down of both BDNF and GDNF mRNA and protein levels in the wounded striatum were confirmed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Dopamine transporter immunohisto-chemistry revealed that inhibition of either BDNF or GDNF expression resulted in a marked decrease in the intensity of peri-wound sprouting. Quantification of this effect using [H3]-mazindol autoradiography confirmed that peri-wound sprouting was significantly reduced in mice receiving BDNF or GDNF antisense infusions whilst control infusions of buffered saline or sense oligonucleotides resulted in the pronounced peri-wound sprouting response normally associated with striatal injury. BDNF and GDNF thus appear to be important neurotrophic factors inducing dopaminergic sprouting after striatal injury.

Approaches to ex vivo gene therapy hold great promise for the treatment of central nervous system disorders where there is currently no care or enough treatment for suffering patients. In this review we have focused on the application of ex vivo gene therapy techniques in Parkinson's disease models, however, the issues and approaches are applicable to other neurodegenerative disorders. In utilizing the ex vivo strategy two considerations are critical for delivery of therapeutic levels of transgene product to the target organ: (i) the vector system and (ii) the cell type for grafting. We describe different vector systems that are currently available and review the various cell types that have been genetically modified and grafting into the striatum of animal models with experimental Parkinson's disease. The strategies for application of gene therapy techniques to the treatment for Parkinson's disease have expanded beyond the classical dopamine replacement toward the use of neurotrophic factors in enhancing cell function or preventing cell death. In addition, we explore the utility of autologus primary skin fibroblasts as alternative cell type for ex vivo gene therapy in the animal model of Parkinson's disease. These results have general implications for the application of ex vivo gene therapy to human neurological diseases and specific implications for Parkinson's disease.

Intracerebral grafting combined with gene transfer may provide a powerful technique for local delivery of therapeutic agents into the CNS. The present study was undertaken to: (i) develop a reliable and reproducible automated cell implantation system, (ii) determine optimal implantation parameters of cells into the striatum, (iii) determine upper safe limits of cellular implantation into the neostriatum of monkeys. Autologous fibroblasts were infused into six sites of the striatum in nonhuman primates (Macaca mulatta, n = 11). Twenty-six-gauge cannulae were inserted vertically through cortical entry sites into the striatum (two sites in the caudate nucleus and four sites in the putamen) at predefined coordinates based on magnetic resonance imaging (MRI). The cannulae were guided by an electronically operated, hydraulic micropositioner and withdrawn at controlled rates, while cells (5, 10, 20, 40, or 80 microl/site) were infused simultaneously. Varying infusion rates and cell concentrations were also evaluated. Visualization and evaluation of graft placement were performed using contrast MRI at 3-5 days postsurgery. Animals were monitored for signs of clinical complications and sacrificed 2 weeks following surgery. Postimplantation MRI revealed a tissue mass effect of the implant with shifting of midline, edema, and infiltration of the white tracts at 40 and 80 microl/site. In addition, these animals developed transient hemiparesis contralateral to the implant site. MRI of animals grafted with 20 microl/site exhibited columnar-shaped implants and evidence of infiltration into white matter tracts possibly due to a volume effect. No clinical side effects were seen in this group. At 14 days postsurgery, MRI scans showed consistent columnar grafts (measuring approximately 5 mm in height) throughout the striatum in animals implanted with 5 or 10 microl/site. No signs of clinical side effects were associated with these volumes and postmortem histological examination confirmed MRI observations. Optimal surgical parameters for delivery of cells into the striatum consist of a graft volume of 10 microl/site, an infusion rate of 1.6 microl/min, a cell concentration of 2.0 x 10(5) cells/microl, and a cannula withdrawal rate of 0.75 mm/min. These results show that infusion of cells into the striatum can be done in a safe and routine manner.

We investigated the role of dopamine neurons in the manifestation of levodopa-induced dyskinesia in a rat model of Parkinson's disease. Daily treatment with a subthreshold dose of levodopa gradually induced abnormal involuntary movements (AIM) in 6-hydroxydopamine-lesioned rats, which included stereotypy and contraversive rotation. After 4 weeks of levodopa treatment, rats with mild and severe AIM were assigned to two treatment subgroups. The graft subgroup received embryonic ventral mesencephalic tissue into the striatum, whilst the sham-graft subgroup received vehicle only. Rats continued to receive levodopa treatment for 3 months post-graft. Brain sections at the level of the basal ganglia were processed for autoradiography using a ligand for dopamine transporter, and in situ hybridization histochemistry for mRNAs encoding postsynaptic markers. Levodopa-induced AIM significantly improved in grafted rats. The severity of AIM correlated inversely with the density of dopamine nerve terminals in the striatum (P < 0. 001), with almost no AIM when the density of dopamine nerve terminals was >10-20% of normal. Embryonic dopamine neuronal grafts normalized not only mRNA expression for preproenkephalin (PPE) in the indirect pathway, but also mRNA expression for prodynorphin (PDyn) in the direct pathway, which was upregulated by levodopa treatment. AIM scores correlated linearly with expression of PPE mRNA in the indirect pathway (P < 0.001) and also with PDyn mRNA in the direct pathway (P < 0.001). We conclude that embryonic dopamine neuronal grafts may improve levodopa-induced dyskinesia by restoring altered activities of postsynaptic neurons, resulting not only from dopamine denervation, but also from levodopa therapy, provided that the density of striatal dopaminergic nerve terminals is restored above a 'threshold' level.

This study examines the effects of xenografts of microencapsulated bovine chromaffin cells (BCCs) on the rotational behavior of hemiparkinsonian monkey recipients. In addition, it determines the content of monoamine neurotransmitters and their major metabolites in the neostriatum in hemiparkinsonian monkeys. The hemiparkinsonian model in monkeys was induced by a unilateral intracarotid injection of methyl-phenyl-tetrahydropyridine (MPTP). Unencapsulated BCCs, BCCs microencapsulated in alginate-polylysine-alginate (ALA) membranes as well as empty microencapsules were grafted into the neostriatum of the hemiparkinsonian monkeys. Following the transplantation the hemiparkinsonian symptoms subsided and the number of rotations induced by apomorphine decreased for up to nine months in the group of recipients grafted with microencapsulated BCCs, while only a temporary improvement (one month) was detected in the recipients of the unencapsulated BCCs. No change was observed in the recipients of empty microencapsules. Dopamine and its metabolites were found considerably depleted in the MPTP-lesioned side versus the unlesioned side of the neostriatum in the hemiparkinsonian monkeys(P<0.05).

The caudate nucleus and putamen form part of a complex but topographically connected circuitry that links the cortex, the basal ganglia and the thalamus. Within this complex system lie a series of functionally and anatomically segregated loops that allow the concurrent processing of a wide range of cognitive and motor information (Alexander et al., 1986; Alexander and Crutcher, 1990). As a constituent of these loops it has been shown that the striatum is involved in movement initiation, response selection and attentional processes (Robbins and Brown, 1990; Alexander, 1994; Lawrence et al., 1998). Although it is the medium spiny GABAergic projection neurones that are primarily lost in HD, it is not sufficient merely to replace the GABA. Instead it is crucial for striatal tissue transplants to integrate with the host tissue in such a way that the cortico-striatal-thalamic circuitry is restored and is functional. Rodent studies have progressed a long way in establishing the principle that striatal grafts can, at least partially, restore function and integrate appropriately with the host (Dunnett and Svendsen, 1993; Björklund et al., 1994; Sanberg et al., 1998) but the limited behavioural repertoire and the undifferentiated striatum meant that it was inevitable that studies should progress into primate models. Anatomical tracing studies have demonstrated that motor, premotor and somatosensory cortical areas send corticostriatal projections primarily to the putamen region in primates, whereas the head and body of the caudate nucleus mostly receive efferent input from associative cortical areas (Kemp and Powell, 1970; Kunzle, 1975, 1977, 1978; Selemon and Goldman-Rakic, 1985). Based on such anatomical, and functional, studies Alexander and colleagues have proposed the existence of at least five cortico-striatal-thalamic loops including a motor, a dorsolateral-prefrontal and an orbito-frontal loop (Alexander et al., 1986). The concentration of motor inputs to the putamen region suggests a particular involvement of this structure in the motor loop. Indeed, unilateral lesions of the putamen disrupt motor performance in the marmoset and generate apomorphine-induced dyskinesias in larger primates (Burns et al., 1995; Kendall et al., 2000). The implantation of striatal grafts into marmosets that had previously received unilateral putamen lesions ameliorated some of the motor impairments, which suggested at least partial restoration of the motor loop. In support of this we found direct evidence of host-graft cortico-striatal connectivity using an anterograde tracer injected in the primary motor cortical region (Kendall et al., 1998a). In larger primates, with lesions of the caudate and putamen, striatal [figure: see text] allografts and xenografts have been shown to reduce apomorphine-induced dyskinesias (Isacson et al., 1989; Hantraye et al., 1992; Palfi et al., 1998). The mechanism by which dyskinesias are elicited is not fully understood but alterations in firing patterns within both segments of the globus pallidus have been identified during dyskinetic movements (Matsumura et al., 1995). It seems likely that it would actually require re-establishment of afferent connections between the implanted putamen and the globus pallidus as well as of functioning dopamine receptors within the graft for the reduction in the dyskinetic profile to be observed. Certainly there is evidence, from rodent studies and the marmoset study described here, that close proximity of the graft to the globus pallidus yields better functional recovery (Isacson et al., 1986). In addition, anatomical tracing studies in rats have demonstrated connections between the implanted tissue and the host globus pallidus (Wictorin et al., 1989b, 1990) However, the relationship between graft placement and functional recovery remains to be fully substantiated.

Stroke remains a major brain disorder that often renders patients severely impaired and permanently disabled. There is no available treatment for reversing these deficits. Hippocampal, striatal and cortical grafting studies demonstrate that fetal cells/tissues, immortalized cells, and engineered cell lines can survive grafting into the ischemic adult brain, correct neurotransmitter release, establish both afferent and efferent connections with the host brain, and restore functional and cognitive deficits in specific models of stroke. The success of neural transplantation depends on several factors: the stroke model (location, extent, and degree of infarction), the donor cell viability and survival at pre- and post-transplantation, and the surgical technique, among others. Further exploitation of knowledge of neural transplantation therapy already available from our experience in treating Parkinson's disease needs to be critically considered for stroke therapy. While the consensus is to create a functional neuronal circuitry in the damaged host brain, there is growing evidence that trophic action of the grafts and host, as well as exogenous application of trophic factors may facilitate functional recovery in stroke. Current treatment modules, specifically that of rehabilitative medicine, should also be explored with neural transplantation therapy. However, validation of neural transplantation and any other treatment for stroke should be critically assessed in laboratory experiments and limited clinical trials. No direct treatment is recognized as safe and effective for reversing the stroke-induced brain damage and functional/cognitive deficits. The first clinical trial of neural transplantation in stroke patients is a mile-stone in stroke therapy, but subsequent large-scale trials should be approached with caution.

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.