Exercise Promotes Neurite Extensions from Grafted Dopaminergic Neurons in the Direction of the Dorsolateral Striatum in Parkinson's Disease Model Rats.

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.

Fine Manual Dexterity Assessment After Autologous Neural Cell Ecosystem (ANCE) Transplantation in a Non-human Primate Model of Parkinson's Disease.

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.

Globus Pallidus Internus Electric High-Frequency Stimulation Modulates Dopaminergic Activity in the Striatum of a Rat Model of Tourette Syndrome.

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.

Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after intrastriatal transplantation in hemi-Parkinsonian rats.

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.

Trans-striatocapsular contralateral interhemispheric resection of anterior inferior basal ganglia cavernous malformation.

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.

Dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays.

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 ßIII 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.

Isolation and functional assessment of mitochondria from small amounts of mouse brain tissue.

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.

Bone marrow stromal cell transplantation attenuates cognitive dysfunction due to chronic cerebral ischemia in rats.

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.

Systemic transplantation of mesenchymal stem cells can reduce cognitive and motor deficits in rats with unilateral lesions of the neostriatum.

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.

Long-term, stable differentiation of human embryonic stem cell-derived neural precursors grafted into the adult mammalian neostriatum.

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.

Functional effect of FGF2- and FGF8-expanded ventral mesencephalic precursor cells in a rat model of Parkinson's disease.

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.

Protection of dopamine neurons by bone marrow stromal cells.

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.

Transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the neonatal parkinsonian rat caudate putamen.

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.

Iris pigment epithelial cells: a possible cell source for the future treatment of neurodegenerative diseases.

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 enrichment affects striatal graft morphology and functional recovery.

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 exposure enhances survival of NT2N cells (hNT neurons) in the hemiparkinsonian rat.

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.

Long-term fate of human telencephalic progenitor cells grafted into the adult mouse brain: effects of previous amplification in vitro.

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.

Dopaminergic cells of the carotid body: physiological significance and possible therapeutic applications in Parkinson's disease.

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.