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Notizen: Summary Human 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 Parkinson's disease.

Notizen: Summary A recently introduced quantitative radioautographic technique was used to characterize the striatal dopaminergic deficit in weaver mutant mice and to evaluate the extent of DA reinnervation resulting from cell suspension grafts of fetal ventral mesencephalic tissue. Brain slices from normal mice and unilaterally grafted weaver mice were incubated in [3H]DA, in the presence of desipramine and pargyline, 3–5 months after graft surgery. Semi-thin sections from the fixed and resin-embedded slices were subsequently exposed on tritium sensitive film and afterwards dipped in nuclear emulsion for light microscope radioautography. Alternate slices were embedded in Epon for post-embedding tyrosine hydroxylase (TH) immunocytochemistry. The grain density of the film radioautographs matched well the distribution of TH positive fibers. Both methods revealed an almost complete absence of DA axons in the dorsomedial quadrant of the weaver neostriatum and an increasing density of DA innervation towards the ventrolateral areas. In the light microscope radioautographs, only the ventral striatum (i.e. nucleus accumbens and olfactory tubercle) and a narrow ventral and periventricular zone of the caudate-putamen were covered by silver grain clusters typical of DA varicosity labeling. Such labeled varicosities were nevertheless found in reduced numbers in the lateral portion of both nucleus accumbens and the olfactory tubercle. The remaining neostriatum was overlaid by diffuse silver grains, suggesting a deficient DA uptake and storage mechanism in the residual DA fibers in this region. Immunocytochemistry using antibodies specific for DA or TH provided further evidence that the residual DA innervation in the weaver neostriatum was biochemically defective. Weaver mice with grafts of ventral mesencephalic tissue in the right neostriatum showed an amphetamine-induced rotational bias to the contralateral side, which was not seen in the sham-operated animals. In contrast to the intrinsic weaver neostriatal DA innervation, DA fibers of graft origin exhibited the normal, clustered type of varicosity labeling. The computerized image analysis of silver grain density in film radioautographs was calibrated by counting these labeled varicosities in selected areas of light microscope radioautographs from the same sections. Results showed a mean DA reinnervation of neostriatal tissue surrounding the graft of about 20%, in some cases up to 80%, of the density seen in wild type mice, with a gradual decrease with distance up to 1–1.4 mm from the graft. The ventral parts of the neostriatum, which contained higher numbers of residual intrinsic DA fibers, were much more sparsely reinnervated than the dorsal and dorsomedial areas. These data show that a quantitatively significant DA reinnervation of the weaver neostriatum can be provided by fetal mesencephalic cell suspension grafts and that these DA fibers become functional, at least with respect to their DA uptake and storage mechanisms, in a neostriatal environment where intrinsic weaver DA axons are strongly deficient. However, observations in long-term weaver mice (9 months after transplantation) suggested that the graft-derived DA fiber outgrowth was reduced with time in the affected striatum, in spite of good survival of the grafted neurones.

Notizen: Summary The ventral mesencephalon, containing the developing dopaminergic neurons of the substantia nigra-ventral tegmental region, was obtained from aborted human fetuses of 9–19 weeks of gestation. The tissue was grafted into the striatum of rats previously subjected to a 6-hydroxydopamine lesion of the mesostriatal dopamine pathway. The graft recipients were immunosuppressed by daily injections of Cyclosporin A. Amphetamine-induced motor asymmetry was reduced, and finally totally reversed, only in rats receiving grafts from the 9-week old fetal donor. The fluorescence microscopic analysis revealed large numbers of surviving dopamine neurons, and extensive fiber outgrowth into the host striatum, in these rats. By contrast, rats receiving grafts from 11–19 week old donors had at most only few surviving dopamine neurons. These results indicate that human fetal mesencephalic tissue may be an efficient source of dopamine neurons for functional intracerebral grafting in patients with Parkinson's disease.

Notizen: Summary We have used a rat model of Parkinson's disease (PD) to address issues of importance for a future clinical application of dopamine (DA) neuron grafting in patients with PD. Human mesencephalic DA neurons, obtained from 6.5–8 week old fetuses, were found to survive intracerebral cell suspension xenografting to the striatum of rats immunosup-pressed with Cyclosporin A. The grafts produced an extensive new DA-containing terminal network in the previously denervated caudate-putamen, and they normalized amphetamine-induced, apomorphine-induced and spontaneous motor asymmetry in rats with unilateral lesions of the mesostriatal DA pathway. Grafts from an 11.5-week old donor exhibited a lower survival rate and smaller functional effects. As assessed with the intracerebral dialysis technique the grafted DA neurons were found to restore spontaneous DA release in the reinnervated host striatum to normal levels. The neurons responded with large increases in extracellular striatal DA levels after the intrastriatal administration of the DA-releasing agent d-amphetamine and the DA-reuptake blocker nomifensine, although not to the same extent as seen in striata with an intact mesostriatal DA system. DA fiber outgrowth from the grafts was dependent on the localization of the graft tissue. Thus, grafts located within the striatum gave rise to an extensive axonal network throughout the whole host striatum, whereas grafted DA neurons localized in the neocortex had their outgrowing fibers confined within the grafts themselves. In contrast to the good graft survival and behavioural effects obtained in immunosuppressed rats, there was no survival, or behavioural effects, of human DA neurons implanted in rats that did not receive immunosuppression. In addition, we found that all the graft recipients were immunized, having formed antibodies against antigens present on human T-cells. This supports the notion that the human neurons grafted to the non-immunosuppressed rats underwent immunological rejection. Based on an estimation of the survival rate and extent of fiber outgrowth from the grafted human fetal DA neurons, we suggest that DA neurons that can be obtained from one fetus may be sufficient to restore significant DA neurotransmission unilaterally, in one putamen, in an immunosuppressed PD patient.

Notizen: Summary The purpose of the present study was to investigate the amplifying function of the nucleus accumbens septi region (NAS) in 6-hydroxydopamine (6-OHDA)-induced rotational behaviour by implanting fetal dopamine (DA)-rich mesencephalic cell suspensions unilaterally in the NAS of rats previously subjected to combined mesostriatal (MS) and NAS 6-OHDA lesions. First, all the rats received a unilateral 6-OHDA lesion of the ascending MS DA pathway, which produced an amphetamine-induced rotational asymmetry towards the lesioned side. In a second step, the rats received a local bilateral 6-OHDA lesion of the NAS which, as previously shown, caused a significant attenuation of the amphetamine-induced locomotor (1.5 mg/kg) and rotational (5 mg/kg) behaviour. Finally, some of these MS + NAS lesioned rats received a unilateral mesencephalic DA graft into the NAS ipsilateral to the original MS lesion. The unilateral DA-rich grafts in the NAS significantly elevated the amphetamine-induced locomotion and ipsilateral circling (opposite to the direction of rotation produced when a graft is placed in the ipsilateral caudate-putamen), suggesting that the NAS plays only an amplifier role in locomotor behaviour and not a directional role. In addition, these grafts significantly attenuated the supersensitive locomotor response observed in lesioned rats when given apomorphine (0.05 mg/kg). The findings emphasize the amplifying role of the NAS in locomotion and circling behaviour and they extend previous findings demonstrating the functional heterogeneity of the striatal complex as well as the regional specificity of the graft-derived functional effects. Moreover, the results argue against the notion that DA grafts can function through a diffusion of transmitter over large distances since, despite the large size of the grafts, the functional graft effects were well localized to the reinnervated NAS and ventromedial striatal regions. We conclude, therefore, that graft-induced amelioration of postural and locomotor deficits are affected through different parts of the striatal complex, and that multiple graft placements are required to produce more complete recovery of motoric behaviour in the DA-depleted brain.

Notizen: Summary The ability to maintain tissue in culture prior to grafting would greatly facilitate the widespread application of graft therapy to neurological diseases such as Parkinson's disease. However, neurons cultured on planar substrata can be easily damaged when they are removed from the substrata and redissociated for use in grafting procedures. To overcome this limitation we utilized aggregate tissue culture methods, which allowed dopamine (DA)-rich neuronal tissue to be grafted directly following culture, without an additional redissociation. Fetal rat dopamine-neuron-containing ventral mesencephalon was cultured for 9 days in rotating flasks. The cells formed many small spheres (280 μm mean diameter), each estimated to contain about 10000 cells. Forty such aggregate spheres were injected via a 22G needle into the DA-denervated striata of host Parkinsonian rats. A significant reduction of amphetamine-induced rotation was seen onward from 6 weeks post-transplantation, with a complete reversal of rotational asymmetry by 15 weeks post-transplantation. Well placed, surviving grafts were found in all behaviorally compensated rats (N = 6). Grafts contained an average of 517 tyrosine hydroxylase (TH)-positive neurons, as well as TH-positive fibers seen extending into the host striatum. These results suggest that aggregate culture methods are a promising means to maintain and deliver tissue for transplant therapy.

Notizen: Summary Fetal mesencephalic mouse tissue, rich in dopamine neurons, was xenografted as a dissociated cell suspension into the striatum of rats with unilateral 6-hydroxydopamine induced lesions of the mesostriatal pathway. The rats were either assigned to a 10-day, 21-day or 42-day Cyclosporin A (CyA) immunosuppression scheme, or given no immunosuppression. The functional effects of the grafts were followed over 6 months by monitoring changes in the recipient rats' amphetamine-induced turning behaviour. Without immunosuppression no grafts were functional at the end of the experiment. In the 10-, 21-and 42-day CyA treatment groups there was a significant reduction of rotational asymmetry at some timepoint following grafting in 26 of the 33 rats. However, by 6 months only 8 grafts remained functional suggesting that in several rats an immunological rejection took place following the termination of immunosuppression. This was supported by catecholamine histofluorescence analysis which revealed evidence of surviving grafts only in the few rats which had shown sustained functional graft effects at 6 months after grafting. In animals in which the grafts had undergone rejection, there was scarlike tissue in the striatum which appeared more extensive in rats that had lost their grafts after several weeks compared to rats in which the grafts were rejected at an early time-point. In a subgroup of the grafted animals the humoral antibody response against major transplantation antigens present on the grafted cells was investigated. All the studied rats were found to be immunized against the grafted mouse tissue following the intrastriatal implantation. This occurred irrespective of prior immunosuppressive treatment. In a parallel group of rats, the leakage of the blood-brain barrier was studied following intrastriatal implantation of a syngeneic fetal neural cell suspension. Evans Blue was infused into rats 3–12 days following transplantation surgery. At the early time-points there was a marked barrier leakage at the implantation site. This subsided with time such that there was minor leakage after 7–8 days and no leakage after 12 days. In summary, the results indicate the CyA is effective in promoting survival of intracerebral xenografts of fetal neural tissue, but that cessation of immunosuppressive treatment in most cases results in rejection of the grafted tissue. Temporary CyA treatment, even exceeding the time it takes for the blood-brain barrier to reform after transplantation surgery, is thus not sufficient to reliably support long term survival of xenografted dopamine neurons.