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Grafts of fetal neural tissue, rich in dopamine (DA) neurons, have previously been shown to improve the symptoms of parkinsonism, both in humans and in animal models. In order to circumvent some of the problems associated with cell transplant therapy, such as the limited availability of transplant tissue, we have established a reaggregate (three-dimensional) tissue culture system that can be used to proliferate normal mammalian neuronal precursors. We demonstrate the in vitro growth of DA-neuronal precursors derived from embryonic porcine ventral mesencephalon, Carnegie stages 15-18. Cultures of DA-neuronal precursors were maintained in F12 medium supplemented with Chang C Supplement for 5 days and switched to serum-free N2 medium for an additional 10 days. Cultures labeled with tritiated thymidine on Days 5-7 in vitro revealed that 43.5% of the DA neurons had incorporated the label, indicative of cell division. Histological examination of the cultured cells demonstrated rosette-like structures, similar to developing neuroepithelium in vivo. Neuronal maturation in vitro was stimulated by dibutyryl cyclic AMP (dbcAMP). Exposure to 5 mM dbcAMP for 7 days stimulated tyrosine hydroxylase (TH), neuron-specific enolase, and 200-kDa neurofilament accumulation three- to sixfold above control levels. After 15 days in vitro, cultured cells reversed amphetamine-induced rotation when grafted into the striata of hemiparkinsonian rats. Successful transplants of cultured neurons were dependent upon a minimum density of DA neurons within the graft (greater than 100 DA neurons/mm3 of graft volume). Data suggest that the percentage of TH neurons can be increased about threefold by culturing the aggregates in tyrosine-free medium, which selects for TH-positive cells. The ability to cultivate mammalian neuronal precursor cells in vitro may eventually make graft therapy a more practical approach to treatment of neurological diseases.

The blink reflex abnormalities present in the 6 hydroxydopamine (6-OHDA) lesioned rat model of parkinsonism mimicked those of the human with Parkinson's disease. In alert rats, we monitored the long and short latency components of the orbicularis oculi electromyographic (OOemg) response evoked by electrical stimulation of the supraorbital branch of the trigeminal nerve (SO). Two paradigms, habituation and double pulse, provided a measure of blink reflex excitability. In normal rats, repeated stimulation of the SO produced habituation of the R2 component of the blink. In the double pulse paradigm, presentation of two identical SO stimuli resulted in a reduced or suppressed OOemg response to the second stimulus relative to the first. In rats with complete, unilateral lesions of midbrain dopamine neurons, repeated SO stimulation produced facilitation rather than habituation of the R2 component of the blink reflex. This facilitation occurred only with the eyelid contralateral to the lesion. In the double pulse paradigm, the lesioned rats showed increased excitability rather than suppression. This effect occurred bilaterally, although the increased excitability was strongest contralateral to the lesion. Rats with partial lesions of midbrain dopamine neurons exhibited qualitatively similar, but less pronounced blink reflex abnormalities. The R1 component of the blink reflex was unaffected by either the complete or partial lesions. Thus, modification of the blink reflex by 6-OHDA lesions provides a reproducible parkinsonian-like symptom which is amenable to investigations of increases in reflex excitability.

A suspension of cells from embryonic day 21 fetal pig ventral mesencephalon was transplanted into the striatum of 20 immunosuppressed rats with 6-hydroxydopamine-induced lesions of the nigrostriatal dopamine pathway. Of these rats, 15 showed reduction of amphetamine-induced ipsilateral rotation by 9 weeks and complete reversal of rotation by 14-17 weeks. Animals maintained stable reversal of rotations (contralateral direction) until cessation of Cyclosporin A (CyA) treatment at 15-20 weeks. Within 4-9 weeks after CyA removal, these rats showed exclusively ipsilateral rotations during behavioral testing which were comparable to pre-transplant levels, suggesting that the grafts were rejected upon cessation of CyA treatment. Rats were sacrificed and tyrosine hydroxylase (TH) immunohistochemistry was performed at several time points, both on and off CyA, to examine a possible correlation between the degree of rotational behavior and the number of TH-positive surviving grafted cells. Staining showed large numbers (230-12,329) of TH-positive surviving cells in animals displaying a high degree of rotational correction (1.6 to -9.6 net ipsilateral rotations/min) after cessation of CyA treatment. Two control groups, those transplanted with non-neuronal cells from the pig ventral mesencephalon (n = 5) and those receiving only daily CyA injections (n = 4) showed no significant reduction of net ipsilateral rotations throughout the experiment. No TH-positive surviving cells were seen in the one non-neuronal transplant analyzed. This data demonstrates long-term retention of xenografted tissue with immunosuppression and its concomitant restoration of normal motor behavior in the rat model of Parkinson's disease.

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.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo voltammetry was used to monitor dopamine (DA) neuron activity during the course of reinnervation of the initially denervated caudate-putamen by grafted mesencephalic neurons. Fetal DA neurons were implanted as a cell suspension into the depth of the caudate-putamen in adult 6-hydroxy-dopamine-lesioned recipient rats. Recordings were performed over a period of 2.5-4 months, starting within a week after transplantation, using chronically implanted surface-treated multifiber carbon electrodes. The voltammetric method used in this study has generated considerable discussion centred on the ability of the multifiber electrodes to measure DA alone in vivo, but the results of previous studies have led to the conclusion that changes in the voltammetric signal most probably reflect dopaminergic terminal activity. It seems therefore possible to follow the time-course of changes in the voltammetric signal amplitude during the process of dopaminergic reinnervation of the host striatum from the grafts. A 6-hydroxydopamine lesion of the mesostriatal dopamine pathway caused a substantial (greater than 80%) reduction of the voltammetric signal within 8-10 days, and the low residual signal remained essentially unchanged for time periods up to at least 5 months in the non-grafted control rats. In 7 of 11 rats with DA-rich grafts there was a recovery of the signal amplitude to levels within, or close to, the range recorded from the striatum of normal intact rats. The increase was observed 6-8 weeks after grafting in the rats which had received the largest transplants, and at about 13-14 weeks after grafting in the rats which had received the smallest ones. The recovery of the signal amplitude, from baseline to maximal response, was quite rapid and typically developed between two or three recording sessions, i.e. over a period of one to two weeks. In contrast to the intact striatum, the recovered signal in the graft-reinnervated striata showed a progressive decline within one hour of sampling time at high sampling frequencies (1 per min to 1 per 3 min). Grafted striata also showed a larger response to systemically administered amphetamine than did intact striata. Since the changes in the voltammetric signal recorded with the multifiber electrode mainly reflect dopaminergic terminal activity, the results provide evidence that the intrastriatal DA-rich grafts are spontaneously active, and that the grafted DA neurons can restore DA neuro-transmission in the reinnervated part of the host caudate-putamen to levels which are within the normal range.(ABSTRACT TRUNCATED AT 400 WORDS)

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 resinembedded slices were subsequently exposed on tritium sensitive film and afterwards dipped in nuclear emulsion for light microscope radioautography. Alternative 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 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.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

The in vitro culture approach was combined with the cell suspension grafting technique to examine whether the maturation of dopamine (DA) neurones in vitro imposed similar limitations on their ability to survive grafting as when they are allowed to develop in situ in the fetus. The functional capacity, survival and growth of DA neurones from 2.5- and 7-day-old cultures, grafted to rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway, was compared with similar grafts freshly prepared from fetal donors of embryonic days 14, 16 and 20. Grafts of freshly dissociated mesencephalic DA neurones, taken from embryonic day 14-16 donors and 2.5-day-old cultures, generally survived well and markedly reduced amphetamine-induced rotational asymmetry in the recipient rats. However, when cultured for 7 days prior to grafting, or when taken from 20-day-old fetuses, the mesencephalic DA neurones survived very poorly and the grafts did not have any functional effects. Plating of aliquots of cell suspension used for grafting indicated that the survival rate of dissociated DA neurones is in the same order of magnitude when grown in vitro (about 2 DA neurones per 1000 cells) as when grafted in vivo to the rat striatum (about 1-5 DA neurones per 1000 cells). When the number of surviving grafted DA neurones was plotted against the behavioural effects of the grafts, a threshold number of around 100-200 DA neurones was found necessary to obtain a marked reduction (greater than 50%) in amphetamine-induced rotational asymmetry. Moreover, the survival of 300-500 DA neurones seemed to produce a 'ceiling effect' beyond which additional surviving DA neurones gave rise to little or no further effect on the amphetamine-induced rotational behaviour.

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 microns mean diameter), each estimated to contain about 10,000 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.

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 scar-like 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.

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.