Slow N-acetyltransferase 2 status leads to enhanced intrastriatal dopamine depletion in 6-hydroxydopamine-lesioned rats.

We previously reported an association between the N-acetyltransferase 2 (NAT2) slow acetylator status and Parkinson's disease (PD). We have now investigated the possible functional relevance of this association by treating Fischer 344 (F344) rapid and Wistar-Kyoto (WKY) slow NAT2 acetylator rat strains with the neurotoxin 6-hydroxydopamine (6-OHDA). Intrastriatal treatment with either 10 or 20 microg of 6-OHDA lead to a significantly greater reduction of striatal dopamine concentrations in the WKY slow acetylator rat strain than in the F344 rapid acetylator rat strain (P < 0.004), reflecting a more marked degree of dopaminergic denervation. Nigral dopaminergic cell counts were also lower in the WKY rats, but this difference failed to reach statistical significance, suggesting that slow acetylation is especially deleterious at the level of striatal nerve endings.

Monoamine oxidase-inhibition and MPTP-induced neurotoxicity in the non-human primate: comparison of rasagiline (TVP 1012) with selegiline.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to induce parkinsonism in man and non-human primates. Monoamine-oxidase B (MAO-B) has been reported to be implicated in both MPTP-induced parkinsonism and Parkinson's disease, since selegiline (L-deprenyl), an irreversible MAO-B inhibitor, prevents MPTP-induced neurotoxicity in numerous species including mice, goldfish and drosophyla. However, one disadvantage of this substance relates to its metabolism to (-)-methamphetamine and (-)-amphetamine. Rasagiline (R-(+)-N-propyl-1-aminoindane) is a novel irrevesible MAO-B-inhibitor, which is not metabolized to metamphetamine and/or amphetamine. The present study compared the effects of high doses of selegiline and rasagiline (10 mg/kg body weight s.c.) on MPTP-induced dopaminergic neurotoxicity in a non-human primate (Callithrix jacchus) model of PD. Groups of four monkeys were assigned to the following six experimental groups: Group I: Saline, Group II: Selegiline/Saline, Group III: Rasagiline/Saline, Group IV: MPTP/Saline, Group V: Rasagiline/MPTP, Group VI: Selegiline/MPTP. Daily treatment with MAO-B-inhibitors (either rasagiline or selegiline, 10 mg/kg body weight s.c.) was initiated four days prior to MPTP-exposure (MPTP-HCl, 2 mg/kg body weight subcutaneously, separated by an interval of 24 hours for a total of four days) and was continued until the end of the experiment, i.e. 7 days after the cessation of the MPTP-injections, when animals were sacrificed. MPTP-treatment caused distinct behavioural, histological, and biochemical alterations: 1. significant reduction of motor activity assessed by clinical rating and by computerized locomotor activity measurements; 2. substantial loss (approx. 40%) of dopaminergic (tyrosine-hydroxylase-positive) cells in the substantia nigra, pars compacta; and 3. putaminal dopamine depletion of 98% and its metabolites DOPAC (88%) and HVA (96%). Treatment with either rasagiline or selegiline markedly attenuated the neurotoxic effects of MPTP at the behavioural, histological, and at the biochemical levels. There were no significant differences between rasagiline/MPTP and selegiline/MPTP-treated animals in respect to signs of motor impairment, the number of dopaminergic cells in the substantia nigra, and striatal dopamine levels. As expected, both inhibitors decreased the metabolism of dopamine, leading to reduced levels of HVA and DOPAC (by >95% and 45% respectively). In conclusion, rasagiline and selegiline at the dosages employed equally protect against MPTP-toxicity in the common marmoset, suggesting that selegiline-derived metabolites are not important for the neuroprotective effects of high dose selegiline in the non-human MPTP-primate model in the experimental design employed. However, unexpectedly, high dose treatment with both MAO-inhibitors caused a decrease of the cell sizes of nigral tyrosine hydroxylase positive neurons. It remains to be determined, if this histological observation represents potential adverse effects of high dose treatment with monoamine oxidase inhibitors.

Liposome-mediated gene transfer to fetal human ventral mesencephalic explant cultures.

The feasibility of non-viral gene transfer using liposomes is described for human fetal nigral tissue. Ventral mesencephalic explants from 6 to 12 week old fetuses were grown as free-floating roller tube cultures. For the transfection, a vector coding for beta-galactosidase driven by the Rous Sarcoma Virus promoter was used. The developmental stage of the human tissue, time in vitro and the amount of vector DNA used significantly influenced the transfection efficiency. Optimal transfection results were obtained with tissue from a 10 week old fetus, cultured for 4 days and transfected with mixtures containing 4 microg vector DNA. Histological analysis suggested that a specific population of ventral mesencephalic precursor cells were the target for the gene transfer. This finding might have implications for gene delivery and cell replacement strategies in Parkinson's disease.

The development of dopamine overflow from foetal nigral grafts in the intact rat striatum and their influence on contralateral striatal dopamine overflow.

In this study, cell suspensions of foetal rat ventral mesencephalic dopaminergic tissue were grafted to the intact (non-lesioned) striatum of adult rats. Differential pulse voltammetry at carbon-fibre micro electrodes (12 microm diameter) was employed to first, monitor the development of dopamine overflow over a 20 week period within the grafts and secondly, their influence on contralateral striatal dopamine overflow. At 8 and 20 weeks, animals were pre-treated with pargyline and both striata were monitored for dopamine overflow for 90 min following d-amphetamine administration. Amphetamine led to a significant increase in dopamine overflow in both the grafted striatum and the contralateral striatum. The time course of dopamine overflow in both the grafted striatum and the striatum contralateral to the graft was similar in all groups of animals. Although the actual concentration of dopamine measured in 20 week old grafts was more (approximately 21%) than that measured in 8 week old grafts, there was no significant difference between the two time points. The concentration of dopamine measured in the striatum contralateral to 8 week old grafts was significantly lower (approximately 43%) than that measured in the striatum of a normal control rats. There was no significant difference between the concentration of dopamine measured in the striatum contralateral to 20 week old grafts and normal control rats. In conclusion, dopamine overflow from a ventral mesencephalic graft does not change significantly between 8 and 20 weeks following grafting. However, the grafted tissue causes a decrease of d-amphetamine-induced dopamine overflow in the contralateral side 8 weeks following grafting, which is restored 12 weeks later.

Influence of time in culture and BDNF pretreatment on survival and function of grafted embryonic rat ventral mesencephalon in the 6-OHDA rat model of Parkinson's disease.

Embryonic midbrain can be maintained as free-floating roller tube cultures prior to grafting in experimental Parkinson's disease. We examined the influence of pregrafting culture time and pretreatment with brain-derived neurotrophic factor on graft survival and function. Cultures were prepared from solid pieces of embryonic (E14) rat ventral mesencephalon and maintained 4, 8, or 12 days in vitro with or without brain-derived neurotrophic factor (100 ng/ml) and grafted into the striatum of 6-hydroxydopamine-lesioned rats. Graft survival and function were evaluated by amphetamine-induced rotation behavior, number of tyrosine hydroxylase-immunoreactive neurons, striatal reinnervation, and graft volume. Rats receiving untreated tissue cultured for 4 or 8 days displayed no differences in graft quality, while grafts from 12-day-old cultures contained significantly fewer (P < 0.05) tyrosine hydroxylase-immunoreactive neurons (340 +/- 97, 267 +/- 92, and 62 +/- 19) and displayed a lower survival rate (9.6 +/- 2.7, 7.9 +/- 2.7, and 2.6 +/- 0.8% for 4, 8, and 12 days in vitro, respectively). Only rats grafted with 4- and 8-day-old cultures recovered significantly (P < 0.05) from lesion-induced rotations (69.4 +/- 18.6, 70.3 +/- 13.9, and 23.2 +/- 12.1% for 4, 8, and 12 days in vitro, respectively). Striatal reinnervation decreased with increasing culture time (P < 0.05). Pretreatment of the cultures with brain-derived neurotrophic factor affected only graft-induced fiber reinnervation, which was reduced even after short culture times. We therefore suggest that a storage period of 8 days is well suited to maintain embryonic rat ventral mesencephalon with the free-floating roller tube culture technique prior to transplantation. BDNF pretreatment as a new strategy to improve graft survival and function, however, was not effective.

Systemic treatment with GM1 ganglioside improves survival and function of cryopreserved embryonic midbrain grafted to the 6-hydroxydopamine-lesioned rat striatum.

Cryopreservation may allow long-term storage of embryonic ventral mesencephalon (VM) for neural transplantation. We investigated whether the ganglioside GM1 or the lazaroid tirilazad mesylate (U-74006F) could improve survival of grafts derived from cryopreserved VM in a rat model of Parkinson's disease. VM was dissected from rat embryos (E14-E15), frozen and stored in liquid nitrogen under controlled conditions, thawed, dissociated, and then grafted into the 6-hydroxydopamine-lesioned rat striatum. In Experiment I, VM fragments were exposed in vitro either to GM1 (100 microM) or to lazaroid (0.3 microM) during all preparative steps. In Experiment II, rats receiving GM1-pretreated VM were, in addition, treated systematically with GM1 (30 mg/kg) daily for 3.5 weeks. Rats grafted with untreated cryopreserved or fresh VM were used as controls, respectively. Rats receiving fresh VM control grafts showed complete recovery from lesion-induced rotations after 6 weeks whereas rats grafted with cryopreserved VM (untreated or pretreated) did not recover. Cryografts contained significantly less (18%, control; 23%, GM1; and 12%, lazaroid) tyrosine hydroxylase-positive cells compared to fresh grafts (1415 +/- 153; mean +/- SEM). Graft volume was also significantly smaller after cryopreservation. In contrast, with additional systemic GM1 treatment cryografts contained almost the same number of tyrosine hydroxylase-positive cells (376 +/- 85) as fresh grafts (404 +/- 56), which was significantly more than that of untreated cryografts (147 +/- 20), showed a significantly larger volume (0.15 mm(3)) compared to that of untreated grafts (0.08 mm(3)) (fresh controls, 0.19 mm(3)), and induced significant and complete functional recovery in the rotation test. In conclusion, systemic treatment of rats with GM1 improved the low survival and functional inefficacy of grafts derived from cryopreserved VM whereas tissue pretreatment alone with either GM1 or lazaroid was not effective.

Temporo-spatial expression of bFGF and TGFbeta2 in embryonic dopaminergic grafts in a rat model of Parkinson's disease.

In the present study we analyzed the temporo-spatial expression pattern of basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in embryonic dopaminergic transplants in the 6-hydroxydopamine rat model of Parkinson's disease. The grafts differentiated for 1, 2, 4 and 8 weeks, respectively and were then analyzed using antibodies directed against tyrosine hydroxylase, bFGF and TGFbeta2. At all time points investigated, grafts contained tyrosine hydroxylase immunoreactive neurons. One week after transplantation the grafts displayed no immunoreactivity for bFGF and TGFbeta2. In more mature grafts (starting at 2 weeks post transplantation) bFGF and TGFbeta2 immunoreactivity became detectable within the graft and at the graft-host interface but was restricted only to astrocytes. In the striatum surrounding the graft, a transient increase of TGFbeta2 immunoreactive astrocytic processes was observed between 1 and 2 weeks after transplantation. This temporo-spatial expression pattern of TGFbeta2 immunoreactive astrocytes suggests that the upregulation of TGFbeta2 is more likely due to the trauma imposed by the transplantation procedure than to an intrinsic differentiation program. Lack of both bFGF and TGFbeta2 expression in grafted dopaminergic neurons compared to their normal expression in the adult rat substantia nigra indicates that these transplanted neurons do not develop their complete physiological phenotype. Together with the observed deficiency in astrocytic bFGF early after grafting this may be responsible for the poor survival of grafted embryonic dopaminergic cells.

Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function.

Free-floating roller tube (FFRT) cultures of fetal rat and human nigral tissue are a means for tissue storage prior to grafting in experimental Parkinson's disease. In the present study, FFRT cultures prepared from embryonic-day-14 rat ventral mesencephalon were maintained for 4, 8, 12, or 16 days in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4-5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase immunoreactive neurons was significantly higher for BDNF treated cultures (2729+/-300) at DIV 8, as compared to controls (1679+/-217). At DIV 12, the culture volume was significantly increased by BDNF (1.05+/-0.12 vs. 0.71+/-0.04 mm3). Similar results were obtained for total protein. Western blot analysis demonstrated increasing signals for GFAP with increasing time in culture, but levels for control and BDNF treated cultures did not differ at any time-point investigated. In conclusion, it is suggested that the time window for effective storage of dopaminergic tissue prior to grafting can be extended by using the FFRT culture technique and that the in vitro storage may be further prolonged by treatment with BDNF.

BDNF and TrkB expression in intrastriatal ventral mesencephalic grafts in a rat model of Parkinson's disease.

We investigated the expression of BDNF and its high affinity receptor trkB in fetal dopaminergic grafts in a rat model of Parkinson's disease. Grafts were allowed to differentiate for 7, 14, 28, or 56 days, respectively and were analyzed immunocytochemically thereafter with antibodies directed against tyrosine hydroxylase, BDNF and trkB. At all time points investigated, grafts contained tyrosine hydroxylase immunoreactive neurons. Immature grafts (7 days) displayed no immunoreactivity for BDNF which was restricted to glial cells at the graft-host interface. After longer differentiation periods BDNF-immunoreactivity was detectable in neurons and astrocytes within the grafts. No trkB immunoreactivity was found in immature grafts but a strong signal for trkB emerged in grafted neurons older than 14 days whereas glial cells remained unlabeled at all time points investigated. Expression of BDNF and trkB in grafted neurons and of BDNF in surrounding glial cells suggests an autocrine or paracrine action of BDNF on dopaminergic neurons possibly mediated by activated glia.

Chronic TVP-1012 (rasagiline) dose--activity response of monoamine oxidases A and B in the brain of the common marmoset.

The stereospecific form of the known acetylenic mechanism-based MAO-inhibitor AGN1135 (Rasagiline, TVP-1012) is devoid of sympathomimetic amphetamine-like properties. To evaluate the efficiency and selectivity of subcutaneous injections of TVP-1012 (dose range from 0.01 up to 10 mg/kg for 7 days) the activities of monoamine oxidases A and B (MAO-A,-B) were determined in different brain regions of the common marmoset. At a dose of 0.1 mg/kg TVP-1012, almost 80% of MAO-B activity is inhibited in all brain regions investigated (prefrontal and occipital cortex, cerebellum, caudate nucleus, putamen, nucleus accumbens). In contrast, MAO-A is not inhibited in putamen and nucleus accumbens. However, by increasing the TVP-1012 dose to 0.5 mg/kg, MAO-A is inhibited to a significant extent as well, concomitant to total inhibition of MAO-B. The results obtained indicate that TVP-1012 irreversibly inhibits both types of MAO in the common marmoset with selectivity for MAO-B at doses less than 0.5 mg/kg. TVP-1012 could be useful in studies requiring selective MAO-B inhibition without concomitant sympathomimetic amphetamine-like effects and could thus be of therapeutic interest for Parkinson's disease and retarded depression.

Effects of macrophage migration inhibitory factor and macrophage migration stimulatory factor on function and survival of foetal dopaminergic grafts in the 6-hydroxydopamine rat model of Parkinson's disease.

Activated microglia play an important role in the rejection of intracerebral grafts and the degeneration of axotomized neurones. We studied the effect of macrophage migration stimulatory factor (MSF) or macrophage migration inhibitory factor (MIF) on allogeneic foetal mesencephalic dopaminergic grafts transplanted into the striatum of 6-hydroxydopamine-lesioned rats. Rotation testing revealed a significant compensation of lesion-induced motor asymmetry 3 weeks post-grafting in animals treated with MIF and vehicle-treated controls compared with pre-graft values (Student's t-test, P < or = 0.005) and MSF-treated animals (ANOVA, post hoc Fisher PLSD test, P < or = 0.05). The MSF group showed no significant compensation. Graft recipients with MIF application (1452.06 +/- 164.32 tyrosine hydroxylase-positive ventral mesencephalic cells) and controls (1753.21 +/- 165.51 tyrosine hydroxylase-positive neurones) displayed good graft survival. Animals with MSF application showed a significant reduction of tyrosine hydroxylase-positive grafted cells (MSF 570.36 +/- 209.49 cells) and graft volumes compared with the MIF and the control group (ANOVA, post hoc Fisher PLSD test, P < or = 0.05). The proportional area of microglia was significantly reduced in MIF animals compared with control animals (ANOVA, post hoc Fisher PLSD test, P < or = 0.001). Activated microglia and macrophages were reduced by half in the MIF-treated group compared with MSF animals and controls. We conclude that intrastriatal injections of MSF result in impaired function and survival of allogeneic ventral mesencephalon (VM) grafts 3 weeks after transplantation. MIF can reduce the number of microglia and macrophages in allogeneic foetal VM grafts. A reduction of microglia via MIF application did not enhance graft function and survival.

Effects of combined BDNF and GDNF treatment on cultured dopaminergic midbrain neurons.

Neural transplantation is an experimental therapy for Parkinson's disease. Pretreatment of fetal donor tissue with neurotrophic factors may improve survival of grafted dopaminergic neurons. Free-floating roller tube cultures of fetal rat ventral mesencephalon were treated with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), or a combination of both. Dopamine content of the culture medium, the number of tyrosine hydroxylase-immunoreactive neurons, and culture volumes were moderately increased in the BDNF- and GDNF-treated cultures but significantly increased by 6.8-, 3.2- and 2.4-fold, respectively after treatment with the combination of both factors. We conclude that pretreatment of dopaminergic tissue in culture with a combination of BDNF and GDNF may be an effective means to improve the quality of tissue prior to grafting.

Implants of polymer-encapsulated genetically modified cells releasing glial cell line-derived neurotrophic factor improve survival, growth, and function of fetal dopaminergic grafts.

Neural transplantation as an experimental therapy for Parkinsonian patients has been shown to be effective in several clinical trials. Further benefit, however, may be expected if the grafting is combined with a treatment of neurotrophic factors thus improving the survival and growth of grafted embryonic dopaminergic neurons. Continuous trophic support may be needed and therefore requires the long-term delivery of neurotrophic factors to the brain. We demonstrate here that the implantation of polymer-encapsulated cells genetically engineered to continuously secrete glial cell line-derived neurotrophic factor to the adult rat striatum improves dopaminergic graft survival and function. Near complete compensation of 6-hydroxydopamine-induced rotation was already achieved within 3 weeks postgrafting in rats that received glial cell line-derived neurotrophic factor-releasing capsules in addition to dopaminergic cell grafts of cultured tissue. Rats without trophic factor supply showed only little recovery at the same time point and sham grafted rats showed no recovery. The number of tyrosine hydroxylase-immunoreactive cells per graft was increased 2.6-fold in the presence of glial cell line-derived neurotrophic factor 6 weeks postgrafting. Similarly, tyrosine hydroxylase-immunoreactive fibers around the graft were increased by 53%. Moreover, these fibers showed a preferential growth towards the trophic factor-releasing capsule. Taken together, these results provide evidence that encapsulated genetically engineered cells are an effective means of long-term trophic factor supply into the adult rat brain and that the delivery of glial cell line-derived neurotrophic factor can sustain dopaminergic graft function and survival.

Pharmacological characterisation of dopamine overflow in the striatum of the normal and MPTP-treated common marmoset, studied in vivo using fast cyclic voltammetry, nomifensine and sulpiride.

The in vivo measurement of electrically-evoked dopamine overflow was measured for the first time in the striatum of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets using fast cyclic voltammetry at untreated carbon-fibre microelectrodes, (7 microm, o.d.). The identity of dopamine was confirmed using electrochemical, pharmacological and histological criteria and complied with rat data from earlier studies. Dopamine overflow depended on the intensity, number of pulses, and frequency of the applied stimuli. Maximum dopamine overflow occurred using 1.0-2.0 mA, 200 micros pulse width, 150-200 pulses at 80-120 Hz stimulation of the medial forebrain bundle. Evoked dopamine overflow in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated marmosets showed a similar electrochemical and pharmacological profile compared to healthy controls, albeit the concentration detected was significantly reduced. The catecholamine uptake blocker, nomifensine, significantly increased the dopamine signal in control marmosets. However, in contrast, nomifensine had no significant effect on evoked dopamine overflow in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated marmosets. Apart from demonstrating that fast cyclic voltammetry with electrical stimulation can be reliably used to monitor dopamine overflow within the primate brain, our results describe for the first time the technical prerequisites for the fast cyclic voltammetric technique in the non-human primate brain.

Degeneration of pre-labelled nigral neurons induced by intrastriatal 6-hydroxydopamine in the rat: behavioural and biochemical changes and pretreatment with the calcium-entry blocker nimodipine.

Intrastriatal application of 6-hydroxydopamine (6-OHDA) initiates a delayed and progressive loss of nigral dopaminergic neurons and therefore may better resemble the slowly developing neuropathology of Parkinson's disease. We investigated the anatomical, behavioural and biochemical consequences of intrastriatal 6-OHDA after prior labelling of nigral dopaminergic neurons in rats and whether the dihydropyridine L-type calcium channel blocker nimodipine protected from the induced deficits. Adult rats received bilateral intrastriatal injections of the retrograde fluorescence tracer fluorogold and nimodipine (n=12) or placebo (n=9) pellets implanted subcutaneously. One week later all rats were injected unilaterally with 6-OHDA (20 microg) at the same intrastriatal site. Placebo-treated rats displayed relatively few d-amphetamine-induced ipsilateral net rotations (R) (1.3+/-1.4 R/min; mean+/-SEM) 1 week after the lesion with a slight but non-significant decline thereafter (after 2, 3 and 4 weeks). In nimodipine-treated rats the rotation behaviour after 1 week was more prominent (3.5+/-0.8 R/min; mean+/-SEM) with a similar slight decline until week 4. Fluorescent and immunocytochemical analysis of the midbrain after 4 weeks revealed a 35% and 39% loss of tyrosine hydroxylase positive cells and a 62% and 56% (placebo and nimodipine, respectively) loss of fluorogold-labelled cells in the ipsilateral substantia nigra pars compacta. Striatal dopamine levels were reduced to 47% (placebo) and 43% (nimodipine) of the control side and the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid to about 50%. Pretreatment with nimodipine failed to antagonize or to ameliorate any of the lesion-induced deficits. We conclude that pretreatment with 80 mg nimodipine pellets does not prevent nigrostriatal damage induced by intrastriatal 6-OHDA.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in non-human primates is antagonized by pretreatment with nimodipine at the nigral, but not at the striatal level.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to induce parkinsonism in man and non-human primates. Hypotheses concerning the mechanism of action of MPTP have been related to the pathogenesis of nigral cell death in Parkinson's disease. For instance, alterations of calcium influxes have been reported to be implicated in both MPTP-induced parkinsonism and Parkinson's disease. Recently, we reported that nimodipine, a blocker of L-type calcium channels, prevents dopaminergic MPTP-induced neurotoxicity in C57B1/6 black mice. The present study extended these rodent findings to the non-human primate model of Parkinson's disease and assessed the effects of nimodipine, continuously applied by pellet for 18 days, on behavioural, biochemical and histological parameters, following systemic application of MPTP in common marmosets (Callithrix jacchus). The experimental design involved five groups of common marmosets and a total of 24 animals. Monkeys assigned to group I (n = 4) received subcutaneously implanted vehicle pellets 7 days prior to subcutaneous saline injections (control). Monkeys of group II (n = 4) were treated with nimodipine pellets (80 mg) and saline injections. Marmosets in group III (n = 8) were treated with vehicle pellets and received 4 times MPTP (MPTP-HCl, 2 mg/kg body weight subcutaneously, separated by an interval of 24 h for a total of 4 days). Monkeys in group IV (n = 4) and V (n = 4) were treated as group-III animals except for the implantation of nimodipine pellets (80 mg and 120 mg, respectively) 7 days prior to toxin exposure. In common marmosets MPTP induced severe parkinsonian symptoms, a pronounced dopamine depletion in the caudate-putamen (more than 99% of control) and a loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra (50% percent of control) 7 days after MPTP-administration. Pretreatment with nimodipine (120 mg pellets) did neither attenuate the behavioural impairments in MPTP-treated animals nor antagonize the striatal neurotoxin-induced dopamine depletion, but almost completely prevented (in a dose-dependent manner) the MPTP-induced decrease of nigral tyrosine hydroxylase immunoreactive cells. These data suggest that application of nimodipine, during the observation period of 7 days, protects against MPTP-induced neurotoxicity in common marmosets at the cellular nigral level, but not at the synaptic striatal level, implicating differential mechanisms of actions of MPTP-induced neurotoxicity at the nigral versus the striatal level.

Foetal ventral mesencephalic cell suspension grafts to the 6-hydroxydopamine-lesioned rat reduce the rate of dopamine uptake in the contralateral striatum.

The present study employed fast cyclic voltammetry, at carbon-fibre microelectrodes, to monitor and compare the rate of dopamine uptake in the rat striatum contralateral to (a) the 6-hydroxydopamine (6-OHDA)-lesioned/grafted striatum and (b) the 6-OHDA-lesioned/ sham grafted striatum. Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted striatum of unilaterally 6-OHDA-lesioned rats. Six weeks after grafting, animals with functional, mature grafts were monitored for dopamine elimination in the contralateral striatum following electrical stimulation of the median forebrain bundle, before and after treatment with the dopamine uptake inhibitor GBR 12909. Compared to animals with sham grafts, amphetamine-amplified rotational behaviour was significantly reduced in animals with grafts of foetal ventral mesencephalic tissue. Fast cyclic voltammetric measurements followed by evaluation with the aid of a kinetic model revealed that in grafted animals, the rate of dopamine uptake via the high affinity uptake mechanism, following treatment with GBR 12909, was significantly reduced when compared to sham grafted animals.

Foetal nigral cell suspension grafts influence dopamine release in the non-grafted side in the 6-hydroxydopamine rat model of Parkinson's disease: in vivo voltammetric data.

The present study employed differential-pulse voltammetry to assess the influence of foetal ventral mesencephalic grafts on dopamine overflow in the contralateral caudate putamen of the 6-hydroxydopamine rat model of Parkinson's disease. The experimental design involved measurements of dopamine overflow in the grafted and contralateral striatum. Control measurements of dopamine overflow were performed in 6-hydroxydopamine-lesioned rats only and the caudate putamen of normal control rats. Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted caudate putamen of unilaterally 6-hydroxydopamine-lesioned rats. At 6 weeks, animals with functional, mature grafts (as assessed by amphetamine-amplified behavioural asymmetry), were pretreated with pargyline (75 mg/kg i.p.), and both striatal sides were monitored for dopamine overflow for 90 min following amphetamine sulphate administration (5 mg/kg i.p.). The time course of dopamine overflow inside the graft was similar to that in the contralateral caudate putamen of the same animal, the normal control animal and the contralateral caudate putamen of 6-hydroxydopamine-lesioned animals. However, in grafted animals the mean dopamine overflow detected in the contralateral caudate putamen was approximately 34% lower than the concentration of dopamine detected in the contralateral caudate putamen of 6-hydroxydopamine-lesioned control animals and approximately 39% lower than the concentration of dopamine detected in the caudate putamen of the normal control animal. There was no statistical difference in the concentration of amphetamine-induced dopamine overflow between the caudate putamen contralateral to the 6-hydroxydopamine lesion and the caudate putamen of the normal control animal. These data suggest that intrastriatal foetal ventral mesencephalic suspension grafts reduce amphetamine-induced dopamine release in the contralateral non-grafted caudate putamen.

Methylcellulose during cryopreservation of ventral mesencephalic tissue fragments fails to improve survival and function of cell suspension grafts.

Cryopreservation may allow long-term storage of fetal ventral mesencephalon (VM) for transplantation in patients suffering from Parkinson's disease (PD). We investigated whether the polymer methylcellulose protects fetal rat VM during cryopreservation in liquid nitrogen and improves survival and function of this tissue as intrastriatal suspension grafts in the 6-hydroxydopamine (6-OHDA) rat model. VM tissue fragments (E14-E15) were either immediately dissociated and grafted as a cell suspension (FRESH) or cryopreserved under controlled conditions for 7 days in a conventional cryoprotective medium (CRYO) or a medium containing 0.1% methylcellulose (mCRYO) and then dissociated and grafted. Rats from the cryo-groups showed only limited behavioral compensation in contrast to complete compensation observed in rats from the FRESH group. Cryopreservation of fetal rat VM decreased the viability of cell suspensions in vitro to about 70%, survival of grafted tyrosine hydroxylase-immunoreactive (TH-IR) neurons to 11% and 20%, and transplant volume to 8% and 17% (mCRYO and CRYO, respectively, compared to FRESH). The addition of 0.1% methylcellulose to tissue fragments during freezing did neither improve in vitro viability nor survival of TH-IR neurons nor behavioral compensation when compared to the control CRYO group. These results suggest that methylcellulose failed to improve survival of cryopreserved dopaminergic ventral mesencephalic neurons.

Acute MPTP treatment produces no changes in mitochondrial complex activities and indices of oxidative damage in the common marmoset ex vivo one week after exposure to the toxin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to cause a Parkinsonian syndrome in man and non-human primates. Hypotheses concerning the pathogenetic mechanisms of MPTP toxicity on nigro-striatal dopaminergic neurons relate to impairment of mitochondrial function and oxidative stress. However, surprisingly few primate studies addressed these issues ex vivo. Thus, the present study assessed the enzyme activities of the respiratory chain, GSH/GSSG and ubiquinol/ubiquinone content in the MPTP primate model (common marmoset, Callithrix jacchus; 2 mg MPTP-hydrochloride/kg body wt were injected subcutaneously (s.c.) on four consecutive days; animals were sacrificed 7 days after last MPTP exposure). Activities of respiratory chain enzymes were measured in crude homogenates of the caudate nucleus, because the probable toxic metabolite of MPTP, MPP+, is transported into dopaminergic neurons via the dopamine uptake system in striatal synapses and mitochondria are concentrated in axonal terminals. Since MPP+ can damage membranes of axonal terminals of nigro-striatal neurons we measured GSH/GSSG contents in the putamen and ubiquinol/ubiquinone concentrations in the substantia nigra and putamen as indices of oxidative damage. At the time of sacrifice MPTP-induced deficits comprised severe behavioural Parkinsonian symptoms, profound depletion of striatal dopamine and its major metabolites as well as pronounced loss of nigro-striatal neurons. Despite these severe lesions, acute MPTP treatment had no effect on any of the enzymes of the respiratory chain in the caudate nucleus and indices of oxidative damage in both the substantia nigra and putamen. These results suggest that factors other than mitochondrial impairment and/or oxidative stress may be involved in MPTP neurotoxicity in primates. Alternatively, early compensatory mechanisms and/or transient effects could account for the reported results and will be discussed.