P. Brundin

Transplantation of Human Embryonic Stem Cell‐Derived Cells to a Rat Model of Parkinson's Disease: Effect of In Vitro Differentiation on Graft Survival and Teratoma Formation

Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinsons disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6‐OHDA (6‐hydroxydopamine)‐lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC‐derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion‐induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.

Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry

SummaryHuman fetal mesencephalic dopamine (DA) neurons, obtained from 6.5–9 week old aborted fetuses, were grafted to the striatum of immunosuppressed rats with 6-hydroxydopamine lesions of the ascending mesostriatal DA pathway. The effects on amphetamine-induced motor asymmetry were studied at various timepoints after grafting. At eight weeks, functional graft effects were not evident but after 11 weeks small effects on motor asymmetry could be monitored and rats tested 19–21 weeks after grafting exhibited full reversal of the lesion-induced rotational behaviour. Four rats were sacrificed at different timepoints between 8 and 20 weeks and the grafted DA neurons were studied in tyrosine hydroxylase (TH) immunocytochemically stained sections at the light and electronmicroscopic level. The grafts contained a total of 500–700 TH-positive neurons in each rat. In one rat sacrificed 8 weeks after grafting the grafted neurons were TH-positive but exhibited virtually no fiber outgrowth. In another rat, sacrificed after 11 weeks, a sparse TH-positive fiber plexus was seen to extend into the adjacent host neostriatum. Two rats sacrificed after 20 weeks both contained TH-positive neurons that gave rise to a rich fiber network throughout the entire host neostriatum, and this fiber network was also seen to extend into the globus pallidus and nucleus accumbens. Very coarse TH-positive processes, identified as dendrites in the electron microscope, projected up to 1.5–2.0 mm from the graft into the host striatum. Ultrastructural analysis revealed that the grafted neurons had formed no TH-positive synaptic contacts with host striatal neurons after 8 weeks, and at 11 weeks some few TH-positive synapses were identified. Twenty weeks after transplantation, abundant TH-positive synaptic contacts with host neurons were seen throughout the neostriatum, and such contacts were identified in the globus pallidus as well. Thus, the present study provides tentative evidence for a time-link between the development of synaptic contacts and the appearance of functional graft effects. Similar to the normal mesostriatal DA pathway, ingrowing TH-positive axons formed symmetric synapses and were mainly seen to contact dendritic shafts and spines. However, in comparison to the normal rat striatum there was a higher incidence of TH-immunoreactive boutons forming synapses onto neuronal perikarya. The TH-positive dendrites that extended into the host striatum were seen to receive non-TH-immunoreactive synaptic contacts, presumably arising from the host neurons. These results suggest that human fetal DA neurons are able to develop a reciprocal synaptic connectivity with the host rat when grafted to the adult brain. Grafting of human fetal DA neurons may therefore be expected to provide a means of restoring regulated synaptic DA release in patients with Parkinsons disease.

In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis.

Spontaneous release and metabolism of dopamine (DA) from intrastriatal grafts of fetal mesencephalic DA neurons was measured by intracerebral dialysis. Mesencephalic DA cell suspensions were implanted into the head of the caudate-putamen in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal DA pathway. Four months later, when tests for amphetamine-induced turning behaviour showed that the grafts had become functional, loops of dialysis tubing were implanted into the striatum on the grafted side and the contralateral non-lesioned side of the grafted rats, and in a similar position in the denervated caudate-putamen of 6-OHDA lesioned control rats. Dialysis perfusates collected from the 6-OHDA lesioned striata showed a reduction of about 95-98% in DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In the grafted animals these levels had recovered to about 40% of control for DA and to 12-16% of control for HVA and DOPAC. In addition, the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was increased in the grafted striata compared to both the lesioned and non-lesioned controls. Amphetamine had little or no effect on DA release in the 6-OHDA lesioned rats, but caused a marked increase in DA release in the grafted rats, this response being proportional to that seen in intact striata. Since the subsequent histochemical analysis showed that the dialysis probe had been located in the transplant-reinnervated part of the caudate-putamen, the results provide additional evidence that the grafted DA neurons exert their functional effects through a continuous active transmitter release from their newly-established terminals in the reinnervated host target.

Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis

The autoregulation of dopamine release and metabolism by intrastriatal grafts of mesencephalic dopamine neurons was examined in vivo using an intracerebral dialysis technique. Dopamine-rich cell suspension grafts were implanted into the head of the caudate putamen in rats with complete unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway. Six months later behavioural tests indicated that the grafts had reversed the lesion-induced rotational behaviour. Extracellular levels of striatal dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were monitored bilaterally in the halothane-anaesthetized grafted rat, both under basal conditions, and also following low (0.05 mg/kg) and high (0.5 mg/kg) doses of the dopamine receptor agonist apomorphine. The perfusate from the grafted striatum showed levels of dopamine which were not statistically different from those of the intact contralateral striatum, indicating that the baseline release of dopamine from the graft was close to normal. Similarly, 3-4-dihydroxyphenylacetic acid and homovanillic acid levels were well recovered on the grafted side (67% and 52%, respectively, of control values). Consistent with previous observations, levels of the serotonin metabolite 5-hydroxyindoleacetic acid measured in perfusate collected from the grafted side was elevated significantly above normal. Subsequent histological analysis revealed large grafts, rich in dopamine-containing neurons (mean +/- SEM number equalled 3138 +/- 630), giving rise to an approximately normal density of dopamine-containing fibres in the area of the host caudate putamen surrounding the probe. Treatment with 0.05 mg/kg (subcutaneous) apomorphine did not affect extracellular dopamine recovered from the grafted striatum, while extracellular DA decreased by a maximum of 30% on the intact side. However, a subsequent injection of 0.5 mg/kg apomorphine produced a large decrease of the dopamine recovered from both the grafted (maximum 40% decrease) and intact striata (maximum 80% decrease). Both the low and the high dose of apomorphine reduced extracellular dopamine metabolite levels, a response which was essentially similar for both the intact and grafted sides. Finally, the dopamine reuptake blocker nomifensine (10(-5) M) added to the perfusion medium produced similar large increases in dopamine in perfusates collected from both grafted and intact striata, while 3,4-dihydroxyphenylacetic acid and homovanillic acid did not change.(ABSTRACT TRUNCATED AT 400 WORDS)

Neural grafting in a rat model of huntington's disease: Striosomal-like organization of striatal grafts as revealed by acetylcholinesterase histochemistry, immunocytochemistry and receptor autoradiography

Grafts of fetal striatum were implanted in the form of a cell suspension into the brains of rats with prior ibotenic acid lesions of the caudate-putamen. The grafts were placed in three different sites: the lesioned caudate-putamen, or the denervated (but otherwise undamaged) globus pallidus and substantia nigra. After 3-6 months survival the grafts were investigated by means of immunohistochemistry and receptor autoradiography in combination with routine histology and acetylcholinesterase histochemistry. The grafts placed within the lesioned caudate-putamen were at least 10-fold larger larger than those placed in the substantia nigra region, with the grafts placed in the globus pallidus being of intermediate size. In all locations the acetylcholinesterase staining had an uneven, patchy distribution, which was most pronounced in the grafts located within the caudate-putamen. These patches did not bear any obvious relationship to variations in density of the neuronal perikarya within the grafted tissue. Many of the neuropeptide-immunoreactive neuron types present in the normal striatum, such as those containing substance P, [Met]enkephalin, somatostatin, cholecystokinin and neuropeptide Y were also detected in the grafted striatum along with acetylcholinesterase-positive staining. Acetylcholinesterase-positive, [Met]enkephalin-positive, substance P-positive and tyrosine hydroxylase-positive markers all showed uneven, patchy distributions in the grafts. This was also the case for the distribution of dopamine D2 and opiate receptors (as revealed by [3H]spiroperidol and [3H]diprenorphine autoradiography, respectively), whereas muscarinic receptor binding was even throughout the grafts. As is the case in the so-called striosomal patches (neurochemically defined compartments) in the immature intact striatum during the early postnatal period, patches of high acetylcholinesterase staining in the grafts showed partial correspondence with patches of high [Met]enkephalin fibre staining, and dopamine receptor density, and (although to a lesser degree) also with patches of high opiate receptor density and high substance P-immunoreactivity. This correspondence of patches also occurred between tyrosine hydroxylase fibre staining and acetylcholinesterase staining as revealed by grafts placed into the substantia nigra. These results suggest that the fetal striatal cell suspension grafts will give rise to a fairly normal range of striatal neuron and receptor types and that they develop at least some of the striosomal features characteristic for the normal striatum.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain-derived neurotrophic factor enhances function rather than survival of intrastriatal dopamine cell-rich grafts.

Brain-derived neurotrophic factor (BDNF) has been shown to promote the survival of dopaminergic neurons from the substantia nigra in cell culture. In order to assess whether a similar survival-promoting effect is present also in vivo, we grafted fetal nigral tissue to the dopamine-depleted striatum of 6-hydroxydopamine-lesioned rats receiving two-week intraventricular infusions or daily intrastriatal injections of BDNF, NGF, or vehicle. When infused chronically at a high dose (12 micrograms/day) into the lateral ventricle, BDNF caused a behavioral syndrome of reduced food and water intake, body weight loss, and locomotor hyperactivity in comparison to NGF- and vehicle-infused graft recipients. NGF-infused graft recipients displayed a transient weight loss during the first week of infusion. At 15 days, amphetamine-induced turning was significantly attenuated to 3% of pregraft values in BDNF-infused recipients, whereas functional graft effects were not present in NGF- or vehicle-infused animals. Survival of tyrosine hydroxylase-immunoreactive graft cells, however, was similar in all treatment groups. Notably, NGF- and BDNF-infusions led to a significant size increase of cholinergic host neurons in the medial septal nucleus and the vertical limb of the diagonal band ipsilateral to the infusion, whereas there was no cholinergic neuron hypertrophy in vehicle-infused animals. Daily intrastriatal injections of BDNF (2 micrograms) produced no weight loss or locomotor hyperactivity, but also enhanced functional graft effects in BDNF-injected, as compared to vehicle-injected animals. Survival rates of grafted tyrosine hydroxylase-immunoreactive cells were, however, similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

SummaryThe effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.

Amelioration of spatial memory impairment by intrahippocampal grafts of mixed septal and raphe tissue in rats with combined cholinergic and serotonergic denervation of the forebrain.

Previous studies in the rat have shown that a serotonergic depletion greatly potentiates the learning and memory impairments produced by pharmacological or lesion-induced cholinergic blockade in the forebrain. The impairment produced by combined serotonergic-cholinergic lesions is reminiscent of that seen in memory-impaired aged rats. In the present experiment, we investigated whether grafts of cholinergic septal tissue and serotonergic mesencephalic raphe tissue, placed in the hippocampus, could reverse the severe memory impairment produced by combined cholinergic-serotonergic lesions. Adult rats were given an intraventricular injection of 5,7-dihydroxytryptamine followed by a radiofrequency lesion of the septum 1-2 weeks later. Three weeks after lesion surgery, the rats were given bilateral intrahippocampal cell suspension grafts of either fetal septal or mesencephalic raphe tissue, or both. The rats were tested for spatial learning and memory in the Morris water maze task at 4 and 10 months after grafting. At 4 months, lesioned and grafted groups were all impaired compared to the normal controls in their swim time and distance swum to find the platform, and they did not show any spatially focussed search strategy in the spatial probe trial when the platform was removed from the tank. At 10 months, the rats with mixed cholinergic and serotonergic grafts were no longer impaired compared to normals in their swim time and distance to find the platform, and they were significantly improved compared to the other grafted groups. Moreover, in the spatial probe trial, the rats with mixed cholinergic and serotonergic grafts displayed a spatially focussed search behaviour over the previous platform site, which was not seen in the lesioned control rats or in the other graft groups. Morphological analysis of the hippocampus revealed that the septal grafts produced an acetylcholinesterase-positive innervation but were totally devoid of serotonin innervation. The raphe grafts produced mainly a serotonin innervation, of both acetylcholinesterase- and serotonin-positive fibres. The results suggest that a mixture of septal and raphe tissue is required when grafted to the hippocampal formation in order to ameliorate the severe spatial learning and memory impairments produced by a combined cholinergic and serotonergic denervation, and that each of these graft types separately are not sufficient to ameliorate such deficits.

Antioxidant treatment protects striatal neurons against excitotoxic insults

It has been suggested that oxidative stress plays an important role in mediating excitotoxic neuronal death. We have therefore investigated the protective effects of antioxidants against excitotoxic injury in the rat on striatal neurons both in vitro and in vivo. In the first part of the study, we determined whether two different types of antioxidants, the spin trapping agent, alpha-phenyl-tert-butyl nitrone and an inhibitor of lipid peroxidation, U-83836E, could protect cultured striatal neurons against either hypoglycemic injury or N-methyl-D-aspartate-induced excitotoxicity. Dopamine- and cyclic AMP-regulated phosphoprotein, which is enriched in medium-sized spiny neurons, was chosen as a marker for striatal neurons. alpha-Phenyl-t-butyl nitrone and U-83836E both significantly reduced cell death induced by these insults as indicated by an increased number of surviving dopamine- and cyclic AMP-regulated phospho-protein-positive neurons. The two antioxidants also promoted the survival of cultured striatal neurons grown at low cell density under serum-free culture conditions. In an in vivo experiment systemically administered alpha-phenyl-t-butyl nitrone exerted neuroprotective effects in the rat striatum following injection of the excitotoxin quinolinic acid. Apomorphine-induced rotation tests revealed that alpha-phenyl-t-butyl nitrone-treated animals were significantly less asymmetric in their motor behavior than control rats. Treatment with alpha-phenyl-t-butyl nitrone significantly reduced the size of the quinolinic acid-induced striatal lesions, as assessed by the degree of sparing of dopamine- and cyclic AMP-regulated phospho-protein-positive and nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons, and of microtubule-associated protein-2-immunorective areas. Furthermore, lesion-induced morphological changes in the substantia nigra pars reticulate, i.e. loss of dopamine- and cyclic AMP-regulated phosphoprotein-positive afferent fibers and atrophic changes due to transsynaptic degeneration, were also less extensive in the alpha-phenyl-t-butyl nitrone-treated animals. The results support the hypothesis that oxygen-free radicals contribute to excitotoxic neuronal injury. The in vivo cytoprotective effects of alpha-phenyl-t-butyl nitrone against striatal excitotoxic lesions suggest that antioxidants could be used as potential neuroprotective agents in Huntingtons disease, which has been suggested to involve excitotoxicity.

Effects of α-phenyl-tert-butyl nitrone on neuronal survival and motor function following intrastriatal injections of quinolinate or 3-nitropropionate

Abstract We have investigated the neuroprotective effects of the the spin-trapping agent α-phenyl-tert-butyl nitrone on striatal lesions produced by local injections of the excitotoxin quinolinate or the mitochondrial toxin 3-nitropropionate. We have assessed both the behavioural and morphological consequences of the lesion. Thus, we tested paw-reaching ability and amphetamine- and apomorphine-induced rotational behaviour in lesioned rats with or without α-phenyl-tert-butyl nitrone treatment, and also explored the relationship between the outcome of the behavioural studies and the extent of the lesion. In the morphological analysis, we chose dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32,000 as a specific marker for striatal neurons. The paw-reaching ability of rats with the quinolinate and 3-nitropropionate lesions was significantly impaired compared to normal control animals. Treatment with α-phenyl-tert-butyl nitrone significantly ameliorated the paw-reaching deficits produced by the quinolinate lesion, whereas the 3-nitropropionate-induced deficits were unaffected by α-phenyl-tert-butyl nitrone. Both quinolinate and 3-nitropropionate lesions resulted in a rotation asymmetry towards the lesioned side in response to both amphetamine and apomorphine. In the quinolinate lesion model, the α-phenyl-tert-butyl nitrone treatment resulted in a less marked motor asymmetry in response to both drugs. By contrast, α-phenyl-tert-butyl nitrone did not significantly reduce the drug-induced rotation asymmetry in rats with a 3-nitropropionate lesion. Morphological analyses disclosed that α-phenyl-tert-butyl nitrone significantly increased the size of the spared striatum in the quinolinate lesions, but only caused a non-significant trend towards an attenuation of the 3-nitropropionate lesions. The behavioural deficits were inversely correlated to the size of the spared residual striatum. The intrastriatal injection of 3-nitropropionate, unlike the injection of quinolinate, caused degeneration of the nigrostriatal dopamine system as well as of transverse fibre bundles of the internal capsule in the striatum, in addition to the striatal lesion. The behavioural studies revealed that the combination of multiple lesions seen in 3-nitropropionate-lesioned rats significantly exacerbated paw-reaching deficits and amphetamine-induced rotation asymmetry. In conclusion, α-phenyl-tert-butyl nitrone attenuated behavioural and morphological consequences of striatal lesions induced by local injections of quinolinate, but not of 3-nitropropionate. Deficits in behavioural tests of striatal function reflected well the extent of striatal lesion. The intrastriatal injection of 3-nitropropionate led to degeneration of both intrinsic striatal neurons and the nigrostriatal dopaminergic system, suggesting that this lesion may provide an animal model of a form of multiple system atrophy rather than Huntingtons disease.