The lazaroid U-83836E improves the survival of rat embryonic mesencephalic tissue stored at 4°C and subsequently used for cultures or intracerebral transplantation

We assessed the effects of addition of the lazaroid U-83836E to a preservation medium on the survival of rat dopamine neurons stored before culturing or intracerebral transplantation. Embryonic ventral mesencephalic tissue was preserved at 4°C for 8 days with or without the addition of 0.3 μM of U-83836E to a chemically defined “hibernation” medium. Freshly dissected mesencephalic tissue was used in control groups. For culture experiments, the mesencephalic tissue was dissociated and grown in serum-containing medium. Following 24–48 h in vitro, the number of dopamine neurons in cultures derived from tissue hibernated without the lazaroid was 40% of fresh control, compared with 67% of control in cultures prepared from tissue stored in the presence of U-83836E. When mesencephalic tissue was transplanted to the dopamine-depleted striatum of hemiparkinsonian rats following 8 days storage at 4°C in a medium without U-83836E, the mean number of surviving dopamine neurons in the grafts was significantly reduced to 40% of control. In contrast, grafts of tissue which had been hibernated in U-83836E-containing medium contained as many dopamine neurons as transplants of freshly dissected tissue. High yields of surviving grafted dopamine neurons were correlated to a significantly faster onset of functional recovery of amphetamine-induced motor asymmetry. We conclude that the storage period for rat mesencephalic tissue can be prolonged up to 8 days when using lazaroid-supplemented hibernation medium. As lazaroids have undergone clinical safety testing, the application of lazaroids for tissue storage in clinical transplantation trials can be envisaged.     

The response of human and rat fetal ventral mesencephalon in culture to the brain-derived neurotrophic factor treatment

Brain-derived neurotrophic factor (BDNF) has been shown to increase the survival of dopaminergic neurons in rodent mesencephalic cultures. The mRNAs of BDNF and trk B receptor have been found to be expressed in the substantia nigra of rat. In this study, the action of BDNF was studied on the survival and transmitter-specific differentiation of dopaminergic neurons of fetal human CNS aged 9–10-week in vitro. Dopaminergic neuron viability and phenotypic expression were monitored by tyrosine hydroxylase (TH) immunohistochemistry and measurement of dopamine (DA) content with HPLC, respectively. After seven days of treatment with BDNF there were 2.2-fold greater number of TH⁺ neurons surviving than in untreated cultures. Although very low levels of DA were detectable in human tissue, considerable amounts of DA was found in the culture medium from around 13 days in vitro (DIV), indicating that DA in human fetal tissue tended to be synthesised and released into the incubation medium more readily than from cultured rat fetal tissue during the same period. The content of DA in the BDNF-treated cultures was approximately double that of untreated cultures after 7 days. In rat fetal tissue, the capacity of each TH⁺ neuron to produce DA was not changed in the BDNF-treated cultures (7 DIV) compared with control cultures, suggesting that BDNF does not up-regulate the production of DA but rather acts to reduce cell death rates. Ciliary neurotrophic factor (CNTF) treatment of rat mesencephalic culture failed to improve the period of survival of fetal dopaminergic neurons and had no effect on the production of DA in cultures. Taken together, our results suggest that BDNF has potent trophic effect on both rat and human fetal mesencephalic dopaminergic neurons in culture and has a potential application in the treatment of Parkinson's disease.     

An in vitro interval before transplantation of mesencephalic reaggregates does not compromise survival or functionality

Grafts of primary ventral mesencephalic tissue and cell suspensions to the denervated striatum are currently utilized as a treatment strategy for Parkinson's disease. Survival rates of grafted dopamine (DA) neurons are extremely poor (5-20%) and is even poorer in grafts to the aged striatum. Short pretreatment of grafted cells with various survival-promoting agents has elicited 2- to 3-fold improvements in these survival rates. However, the duration of pretreatment is limited by the necessity of implanting the embryonic cells within a critical period after tissue harvest, potentially limiting the beneficial effects of these interventions. This study details the use of a modified mesencephalic reaggregate culture system combined with striatal-derived trophic factor support to provide an extended ex vivo cell culture interval before grafting. Mesencephalic cell suspension grafts implanted immediately following dissociation were compared to grafts of an equivalent number of cells reaggregated in the presence of striatal oligodendrocyte-type-2 astrocyte (SO2A) conditioned medium for 3 or 7 days. All grafts were placed in the denervated striatum of young adult male Fischer 344 rats. Rotational assessment of amphetamine-induced rotations indicates that aggregates maintained for 3 days in culture present statistically similar functional recovery profiles as compared to cell suspension grafts. Grafts of mesencephalic reaggregates maintained in vitro for 7 days did not display significant improvements in functional recovery. Immunohistochemical analysis for tyrosine hydroxylase immunoreactive (THir) neurons conducted at 10 weeks post-grafting revealed equivalent survival rates of THir neurons in grafts of fresh cell suspensions and aggregates held in culture for 3 days. Grafts of reaggregates held in culture for 7 days possessed significantly fewer THir neurons, about 25% of the cell suspension or 3-day aggregate grafts. This ex vivo reaggregate system allows for extended pretreatment (3 days) of mesencephalic cells with survival-promoting agents and immunological screening of tissue before transplantation.     

Tirilazad Mesylate Improves Survival of Rat and Human Embryonic Mesencephalic Neuronsin Vitro

The survival rate of embryonic dopamine (DA) neurons after transplantation to the striatum is only 5–20%. Therefore, mesencephalic tissue from several donors needs to be implanted in a parkinsonian patient to induce a therapeutic improvement. Lazaroids are a group of neuroprotective compounds which inhibit lipid peroxidation. Previously, two lazaroids (U-74389G and U-83836F) have been found to improve the survival of both cultured and grafted rat DA neurons. The only lazaroid approved for human use is tirilazad mesylate. The objective of the present study was to explore the effects of tirilazad mesylate on DA neuron survival in cultures of rat ventral mesencephalon and its capacity to promote thein vitrocell viability of embryonic rat and human mesencephalic tissue, treated and dissociated in the same way as in clinical trials. After 7 daysin vitro,the number of tyrosine hydroxylase-immunopositive, presumed DA neurons was 140% higher in rat cultures treated with 0.3 μMtirilazad mesylate than that in control cultures. Rat and human cell suspensions supplemented with tirilazad mesylate maintained a high degree of viability for several hours longer than control suspensions. These results indicate that tirilazad mesylate promotes the survival of both rat and human embryonic mesencephalic neuronsin vitro.Tirilazad mesylate can be administered clinically and may become a useful tool for increasing survival of grafted DA neurons in patients, thereby reducing the needed quantity of human donor tissue.     

Tumor Necrosis Factor α Is Toxic to Embryonic Mesencephalic Dopamine Neurons

Levels of the proinflammatory cytokine tumor necrosis factor α (TNFα) are increased in postmortem brain and cerebral spinal fluid from patients with Parkinson's disease (PD). This observation provides a basis for associating TNFα with neurodegeneration, but a specific toxicity in dopamine (DA) neurons has not been firmly established. Therefore, we investigated TNFα-induced toxicity in DA neurons by utilizing primary cultures of embryonic rat mesencephalon. Exposure to TNFα resulted in a dose-dependent decrease in DA neurons as evidenced by decreased numbers of tyrosine hydroxylase-immunoreactive (THir) cells. TNFα toxicity was selective for DA neurons in that neither glial cell counts nor the total number of neurons was decreased and no general cytotoxicity was evidenced by lactate dehydrogenase assay. Many of the cells which remained immunoreactive for TH had shrunken and rounded cell bodies with broken, blunted, or absent processes. However, TNFα-treated cultures also contained some THir cells which appeared to be undamaged and possibly resistant to TNFα-induced toxicity. Additionally, immunocytochemistry revealed basal expression of TNFα receptor 1 (p55, R1) and TNFα receptor 2 (p75, R2) on all cells within the mesencephalic cultures to some degree, even though only DA neurons were affected by TNFα treatment. These data strongly suggest that TNFα mediates cell death in a sensitive population of DA neurons and support the potential involvement of proinflammatory cytokines in the degeneration of DA neurons in PD.     

Diminished viability, growth, and behavioral efficacy of fetal dopamine neuron grafts in aging rats with long-term dopamine depletion: an argument for neurotrophic supplementation.

We examined the behavioral and morphological correlates of the response to a single intrastriatal dispersed cell graft of fetal rat ventral mesencephalic tissue in male Fischer-344 rats of varying age (4, 17, and 24-26 months old) and history of mesostriatal dopamine (DA) depletion (1 or 14 months). Our goal was to determine the impact of advancing age and duration of DA depletion in the host on DA graft viability and function. The findings can be summarized as follows. (1) Fetal DA neuron grafts that were effective in completely ameliorating amphetamine-induced rotational behavior in young rats with short-term lesions were virtually without effect in aged rats with long-term lesions. Middle-aged rats with long-term lesions responded to these grafts with partial behavioral recovery. (2) Age of the host at the time of transplantation, and not duration of DA depletion, was the primary determinant of response to DA grafts. (3) Diminished efficacy of grafts in lesioned aging rats was related to decreased survival and neurite extension of transplanted DA neurons. (4) Co-grafts of DA neurons with Schwann cells as a source of neurotrophic support improved the behavioral outcome of grafts in aged lesioned rats. These findings support the view that the DA-depleted striatum of aged rats is an impoverished environment for survival, growth, and function of DA grafts. Consistent with this view, local supplementation of the neurotrophic environment of grafted DA neurons with products of co-grafted Schwann cells, a demonstrated source of neurotrophic activity for embryonic DA neurons, improved graft outcome.     

Dopamine content and metabolism in mesencephalic and diencephalic cell cultures: sex differences and effects of sex steroids.

Sexual differentiation of dopaminergic neurons was studied in gender-specific cultures. Dissociated cell cultures were prepared from di- or mesencephalon of gestational day 14 rat embryos and raised in the absence or presence of 17 beta-estradiol or testosterone for up to 13 days in vitro (DIV). Developmental profiles of levels of dopamine (DA) and metabolites as well as capacity for vesicular storage of the transmitter were determined by HPLC. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted. Higher levels of DA were measured in female than in male cultures of both brain regions. In mesencephalic cultures, the differences in DA levels were fully accounted for by sex differences in numbers of TH-IR cells, whereas no sex differences in cell numbers were found in diencephalic cultures. Dihydroxyphenylacetic acid (DOPAC) levels and vesicular storage capacity matured faster in mesencephalic than in diencephalic cultures, but no sex differences were observed. Homovanillic acid (HVA) could not be detected except in 13-DIV mesencephalic cultures. Hormonal treatment did not erase sexual differentiation of dopaminergic neurons. Irrespective of the gender, however, both steroids decreased DA and DOPAC contents in diencephalic cultures but not in mesencephalic cultures. It is proposed that sexual differentiation of dopaminergic systems proceeds in a region-specific fashion and that neurogenesis and development of various parameters of dopaminergic activity may be differentially affected. Sexual differentiation of dopaminergic neurons may be initiated independently of the action of gonadal steroid hormones and may subsequently be modified by differences in hormonal environment.     

Toxicity of Dieldrin for Dopaminergic Neurons in Mesencephalic Cultures

Dieldrin can be retained for decades in lipid-rich tissue and has been measured in some postmortem PD brains. Dieldrin has been reported to deplete brain monoamines in several species and has been shown to inhibit mitochondrial respiration. To further investigate the possibility that it may be involved in the pathogenesis of parkinsonism, its toxicity for dopaminergic (DA) neurons was assessed in a mesencephalic cell culture model. Primary neuronal cultures of mesencephalic neurons were prepared from fetal rats or fetal mice, grown for 1 week and incubated with Dieldrin (0.01–100 μM) for 24 or 48 h. Toxicity for DA neurons was determined by measuring density of surviving tyrosine hydroxylase immunoreactive (TH-ir) cells. Toxicity for gamma-aminobutyric acid (GABA)-ergic neurons was determined by measuring survival of glutamate decarboxylase (GAD)-ir neurons. General, nonselective cytotoxicity was determined by counting cells visualized by phase contrast microscopy or by DAPI-stained cells with fluorescence microscopy. Dieldrin exposure for 24 h resulted in a dose-dependent decrease in survival of TH-IR cells (DA neurons) with a 50% decrease (EC₅₀) produced by 12 μM in rat mesencephalic cultures. Dieldrin also produced a dose- and time-dependent decrease in mouse DA-ergic and GABA-ergic neurons in mouse mesencephalic cultures. GABA-ergic neurons were less sensitive to the toxin compared to DA-ergic neurons. Cellular uptake of³H-DA was also affected by lower concentrations of Dieldrin (EC₅₀ = 7.98 μM) than uptake of³H-GABA (EC₅₀ = 43 μM). Thus, Dieldrin appears to be a relatively selective DA-ergic neurotoxin in mesencephalic cultures. Dieldrin, which may be ubiquitous in the environment, is proposed as an agent which can initiate and promote dopaminergic neurodegeneration in susceptible individuals.     

Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson's disease

One promising therapy for the treatment of Parkinson's disease is transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted cells die, many via apoptosis. In this study we attempted to prevent anoikis-induced cell death, which is triggered during the preparation of cells for grafting, and examine the impact on graft viability and function. We utilized the extracellular matrix molecule tenascin-C (tenascin) and an antibody (Ab) to the cell adhesion molecule L1 to specifically mimic survival signals induced by cell-matrix and cell-cell interactions. In vitro, both tenascin- and L1 Ab-treated cultures doubled the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to control. Additionally, cell survival assays determined that tenascin and L1 Ab-treated cell suspensions yielded more metabolically active and fewer dead cells than control suspensions. In contrast to the culture results, tenascin- and L1 Ab-treated mesencephalic grafts did not yield an increase in the number of THir neurons using our standard grafting paradigm (3 microl of 100,000 cells/microl). However, under low-density conditions (3 microl of 3,000 cells/microl), tenascin augmented grafted THir neuron survival. These findings are consistent with the view that cell density can dramatically influence the degree of stress placed on THir neurons and consequently affect the success of survival strategies in vivo. In conclusion, pretreatment with tenascin may prove beneficial to prevent anoikis in dilute cell suspension grafts, while long-term in vivo delivery methods need to be explored to determine if L1 can prevent anoikis in grafts of mesencephalic dopamine neurons after transplantation.     

联合培养胚鼠的黑质及纹状体LGE细胞脑内移植治疗帕金森病大鼠的实验研究

目的　观察并检测胚鼠纹状体外侧节突 (LGE)对多巴胺能 (DA)细胞存活性的促进和营养导向作用。方法　将帕金森病 (PD)模型随机分成四组 :Co -culture组 (n =12 ) ;Cograft组 (n =12 ) ;Solo -VM组 (n =12 ) ;Con trol组 (n =8)。将胚鼠LGE细胞和腹侧中脑组织 (VM )制成细胞悬液 ,植入Control组外的其他各组动物的尾壳核。 2周后进行PD鼠行为学检测 ,连续观察 2 4周 ,继之将各组大鼠处死 ,进行免疫组化染色。结果　Co -culture组和Co - graft组大鼠移植后旋转行为较Solo -VM组大鼠明显减少。CO -culture组和CO - graft组之间大鼠的旋转行为比较 ,无统计学差异。免疫组化观察证实LGE和VM离体培养移植和新鲜移植均能提高DA细胞的存活性 ,增加宿主纹状体内DA纤维重新支配的密度 ,并形成明显的DA细胞团。结论　LGE细胞对VM移植物有明显的营养导向作用 ,并可增强DA细胞的存活 ,促进移值后DA细胞功能持久维持 ,并增加DA细胞再支配的密度     

Trophic and tropic effects of striatal astrocytes on cografted mesencephalic dopamine neurons and their axons

Astrocytes from the ventral mesencephalon and from the striatum respectively promote the dendritic and axonal arborization of dopamine (DA) neurons in vitro. To test this response in vivo, astrocytes in primary cultures from the neonatal cerebral cortex, ventral mesencephalon, or striatum were coimplanted with fetal ventral mesencephalic tissue into the intact or DA-denervated striatum of adult rats and these cografts examined after 3–6 months by tyrosine hydroxylase (TH) immunohistochemistry (intact recipients) or after 5–6 months by in vitro [H]DA-uptake autoradiography (DA-denervated recipients). In contrast with single ventral mesencephalic grafts, all types of cograft displayed a rather uniform distribution of TH-immunoreactive perikarya. The average size of TH-immunoreactive cell bodies was not significantly different in cografts containing cortical or mesencephalic astrocytes and in single ventral mesencephalic grafts, but it was significantly larger in cografts containing striatal astrocytes. Nevertheless, the number of [H]DA-labeled terminals in the DA-lesioned host striatum was clearly smaller with cografts of striatal astrocytes than with single mesencephalic grafts or with cografts containing cortical astrocytes. On the other hand, cografts of striatal astrocytes contained much higher numbers of [H]DA-labeled terminals than the other types of graft or cograft. Thus, while cografted astrocytes in general influence the distribution of DA neurons within the graft, astrocytes from the neonatal striatum have a trophic effect on DA perikarya and a tropic effect on DA axons, keeping the latter within the graft.     

Stimulant-induced alterations in dopaminergic and serotonergic function in fetal raphe neurons

Methamphetamine and its structural analogues have been demonstrated to be neurotoxic to CNS dopamine (DA) and serotonin (5-HT) neurons both in vivo and in vitro. Our laboratory has been actively characterizing mesencephalic cultures and the effects of methamphetamine exposure on neurochemical and immunochemical indices. The purpose of the present studies was to extend our findings with mesencephalic cultures and compare them with methamphetamine-induced alterations in fetal raphe cultures that contain both DA and 5-HT cells. Methamphetamine (10 and 100 μM) was added to the cultures 24 h after plating and fresh daily thereafter. The effects of chronic methamphetamine exposure on [³H]-DA and [³H]-5-HT uptake were determined after 5 days of drug treatment. Additional cultures were immunochemically analyzed for the presence of DA- and 5-HT-containing cells and total neuronal density. Results indicate that repeated methamphetamine exposure decreased DA and 5-HT uptake. Furthermore, repeated exposure to the higher concentration of methamphetamine (100 μM) caused a significant reduction in the number of DA and 5-HT cells as well as reducing the total neuronal density. This would suggest that this higher concentration of methamphetamine results in generalized neurotoxicity. Exposure to 10 μM methamphetamine resulted in more specific effects on dopaminergic function. These findings indicate that repeated methamphetamine administration can induce similar alterations in both dopaminergic and serotonergic neurons in raphe cultures.     

Attenuation of levodopa-induced toxicity in mesencephalic cultures by pramipexole

Summary The direct-acting dopamine (DA) agonist pramipexole (2 amino-4,5,6,7-tetrahydro-6-propyl-amino-benzthiazole-dihydrocfiloride) was evaluated for its ability to attenuate levodopa-induced loss of tyrosine hydroxylase immunoreactive (THir, a marker for dopamine neurons) cells in mesencephalic cultures. Pramipexole reduced levodopa-induced THir cell loss in a dosedependent and saturable fashion (ED₅₀=500 pM), its inactive stereoisomer was significantly less potent in this regard and pergolide and bromocriptine had negligible cytoprotective effects. Culture media from mesencephalic cultures incubated with pramipexole for 6 days increased THir cell counts in freshly harvested recipient cultures. The magnitude of this effect was directly proportional to the amount of pramipexole in the donor cultures and heatinactivation of the media abolished the growth promoting effect. The results from this exploratory set of experiments suggest that pramipexole may be cytoprotective to dopamine neurons in tissue culture. Pramipexole's affinity for DA receptors, its antioxidant action or its ability to enhance mesencephalic trophic activity could be responsible for this effect.     

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.     

Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.     

Cryopreserved GABAergic neurons in cultures of rat cerebral cortex and mesencephalon: a comparative morphometric study with anti-GABA antibodies

Blocks of embryonic rat cerebral cortex and mesencephalon were cryopreserved and stored for up to 1 year in liquid nitrogen at − 196°C with 7.5% dimethylsulfoxide (DMSO) as cryoprotectant. After thawing, these tissues were only mechanically dissociated and the cells were cultured for 2–7 weeks before immunocytochemical staining with anti-GABA (γ-aminobutyric acid) antibodies. The freeze-stored GABA-immunoreactive (IR) mesencephalic neurons were compared, with computerized morphometry, to fresh mesencephalic cells and to their fresh and frozen cerebral cortical counterparts. A part of the cortical cells was treated with thienyl-phencyclidine (TCP) in order to assess the potential morphological effects of this neuroprotective agent upon these cortical neurons. Two types of GABA-IR neurons (small and large neuritic field cells) could be evidenced in both structures without any difference between fresh and frozen materials, but with significant quantitative morphological differences linked to their anatomical source. GABAergic phenotype is expressed similarly in fresh and frozen cultured neurons with intrinsically programmed morphological features and only minor influences of epigenetic factors. Small and large neuritic field GABA-IR neurons represent, respectively, local and long-range circuits of inhibition, strongly reminiscent of those described in vivo and which remain unchanged in culture even after freeze-storage.     

Co-grafts of embryonic dopamine neurons and adult sciatic nerve into the denervated striatum enhance behavioral and morphological recovery in rats

We have recently demonstrated that a diffusible factor(s) derived from explanted adult rat sciatic nerve can increase the number and neurite outgrowth of embryonic rat dopamine (DA) neurons in culture. The present study extends this finding to compare DA neuron-sciatic nerve co-grafts to grafts of DA-rich neural tissue alone for behavioral and morphological effects in rats with unilateral nigrostriatal lesions of the DA pathway. Our results indicate that the presence of a co-grafted segment of sciatic nerve increased the likelihood of rapid behavioral recovery and promoted complete recovery mediated by small grafts that yielded only modest behavioral changes in the absence of co-grafted nerve. These behavioral effects were accompanied by a modest increase in survival of grafted tyrosine hydroxylase-positive neurons in the striatum and a more pronounced increase in the area and density of striatal reinnervation provided by grafted DA neurons in co-grafted animals. This evidence supports the view that a diffusible product of explanted peripheral nerve acts as a growth-promoting factor for embryonic DA neurons and that the presence of this factor augments the behavioral efficacy of grafted DA neurons.     

Isolation and transplantation of dopaminergic neurons and neural stem cells

Although transplantation of mesencephalic tissue is considered a promising therapy for Parkinson's disease (PD), its clinical use is still restricted to a very few cases. A major limiting factor of this therapy is the difficulty of obtaining sufficient quantities of viable embryonic mesencephalic tissue. To overcome this limitation, techniques to produce dopaminergic (DA) neurons in vitro have been developed. However, these cultures are likely to contain a variety of unidentified cells, which must be removed before implantation. Specific cell-surface markers to sort DA neurons or their precursors are not available. We have developed an alternative strategy, by which these cells can be labeled with green fluorescent protein and isolated with fluorescent activated cell sorter. Transplantation of the sorted cells resulted in recovery of a rat model of the PD. This strategy should be useful for developing new therapies for PD.     

Increased cell suspension concentration augments the survival rate of grafted tyrosine hydroxylase immunoreactive neurons

The poor survival rate (5-20%) of grafted embryonic dopamine (DA) neurons is one of the primary factors preventing cell replacement from becoming a viable treatment for Parkinson's disease. Previous studies have demonstrated that graft volume impacts grafted DA neuron survival, indicating that transplant parameters influence survival rates. However, the effects of mesencephalic cell concentration on grafted DA neuron survival have not been investigated. The current study compares the survival rates of DA neurons in grafts of varying concentrations. Mesencephalic cell suspensions derived from E14 Fisher 344 rat pups were concentrated to 25,000, 50,000, 100,000 and 200,000 cells/microl and transplanted into two 0.5 microl sites in the 6-OHDA-denervated rat striatum. Animals were sacrificed 10 days and 6 weeks post-transplantation for histochemical analysis of striatal grafts. The absolute number of DA neurons per graft increased proportionally to the total number of cells transplanted. However, our results show that the 200,000 cells/microl group exhibited significantly higher survival rates (5.48+/-0.83%) compared to the 25,000 cells/microl (2.81+/-0.39%) and 50,000 cells/microl (3.36+/-0.51%) groups (p=0.02 and 0.03, respectively). Soma size of grafted DA neurons in the 200,000 cells/microl group was significantly larger than that of the 25,000 cells/microl (p<0.0001) and 50,000 cells/microl groups (p=0.004). In conclusion, increasing the concentration of mesencephalic cells prior to transplantation, augments the survival and functionality of grafted DA neurons. These data have the potential to identify optimal transplantation parameters that can be applied to procedures utilizing stem cells, neural progenitors, and primary mesencephalic cells.     

Cell Therapeutics in Parkinson’s Disease

The main pathology underlying motor symptoms in Parkinson’s disease (PD) is a rather selective degeneration of nigrostriatal dopamine (DA) neurons. Intrastriatal transplantation of immature DA neurons, which replace those neurons that have died, leads to functional restoration in animal models of PD. Here we describe how far the clinical translation of the DA neuron replacement strategy has advanced. We briefly summarize the lessons learned from the early clinical trials with grafts of human fetal mesencephalic tissue, and discuss recent findings suggesting susceptibility of these grafts to the disease process long-term after implantation. Mechanisms underlying graft-induced dyskinesias, which constitute the only significant adverse event observed after neural transplantation, and how they should be prevented and treated are described. We summarize the attempts to generate DA neurons from stem cells of various sources and patient-specific DA neurons from fully differentiated somatic cells, with particular emphasis on the requirements of these cells to be useful in the clinical setting. The rationale for the new clinical trial with transplantation of fetal mesencephalic tissue is described. Finally, we discuss the scientific and clinical advancements that will be necessary to develop a competitive cell therapy for PD patients.