No functional effects of embryonic neuronal grafts on motor deficits in a 3-nitropropionic acid rat model of advanced striatonigral degeneration (multiple system atrophy)

Neurotransplantation in a model of advanced MSA

Pergamon

PII: S0306-4522(00)00500-5

Neuroscience Vol. 102, No. 3, pp. 581±592, 2001 581 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00

www.elsevier.com/locate/neuroscience

NO FUNCTIONAL EFFECTS OF EMBRYONIC NEURONAL GRAFTS ON MOTOR DEFICITS IN A 3-NITROPROPIONIC ACID RAT MODEL OF ADVANCED STRIATONIGRAL DEGENERATION (MULTIPLE SYSTEM ATROPHY) R. WALDNER, a Z. PUSCHBAN, a C. SCHERFLER, a K. SEPPI, a K. JELLINGER, b W. POEWE a and G. K. WENNING a* a

Neurological Research Laboratory, Department of Neurology, University Hospital Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria b

Ludwig Boltzmann Institute for Clinical Neurobiology, Baumgartner Hoehe 1, A-1140 Vienna, Austria

AbstractÐIntrastriatal injection of 3-nitropropionic acid results in secondary excitotoxic local damage and retrograde neuronal cell loss in substantia nigra pars compacta, thus mimicking salient features of striatonigral degeneration, the core pathology underlying Parkinsonism associated with multiple system atrophy. We used 3-nitropropionic acid to create a rat model of advanced striatonigral degeneration in order to assess the effects of embryonic allografts upon rotational and complex-motor behavioural abnormalities. Following stereotaxic intrastriatal administration of 500 nmol 3-nitropropionic acid in male Wistar rats we observed consistent amphetamine- and apomorphine-induced ipsiversive rotation. Furthermore, there were marked de®cits of contralateral paw reaching. Subsequently, animals received intrastriatal implantations of either E14 mesencephalic or striatal or mixed embryonic cell suspensions. In addition, one group received sham injections. Grafted rats were followed for up to 21 weeks and repeated behavioural tests were obtained during this period. Drug-induced rotation asymmetries and complex motor de®cits measured by paw reaching tests were not compensated by embryonic grafts. Persistence of drug-induced rotations and of paw reaching de®cits following transplantation probably re¯ects severe atrophy of adult striatum, additional nigral degeneration as well as glial demarcation of embryonic grafts. We suggest that dopamine rich embryonic grafts fail to induce functional recovery in a novel 3-nitropropionic acid rat model of advanced striatonigral degeneration (multiple system atrophy). q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: mesencephalic graft, striatal graft, co-graft, rotation, staircase test.

implanted into the CNS have been widely studied experimentally. It has become apparent that dopaminergic grafts can ameliorate a variety of behavioural de®cits induced by the 6-hydroxydopamine (6-OHDA) lesion of substantia nigra pars compacta (SNc) in the rat. Mesencephalic grafts are capable of abolishing druginduced rotational asymmetry, most commonly tested by injection of the dopamine releasing drug amphetamine or the directly acting dopamine-receptor agonist apomorphine. 3,34 However, more complex sensorimotor functions, such as skilled forelimb use and disengagement behaviour remain impaired following grafting of mesencephalic tissue. 17 Similar studies in monkeys with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineinduced Parkinsonism have also demonstrated graft survival, ®ber outgrowth, restoration of dopaminergic neurotransmission, and amelioration of akinesia. 1 Based on these studies, worldwide more than 300 PD patients have received mesencephalic grafts during this decade. 28 Partial clinical improvement has occurred in many patients in some correlating with a signi®cant increase of 6-L-[18F]¯uoro-dopa uptake con®ned to the grafted putamen. 23,47 After PD, multiple system atrophy (MSA) is the

Neurodegenerative movement disorders are characterized by selective and anatomically restricted neuronal damage resulting in characteristic and often severe motor de®cits. Experimental work in rodent models of such disorders, particularly Parkinson's disease (PD), suggests that intracerebral implantation of embryonic neurons can at least partially substitute, both anatomically and functionally, for lost intrinsic pathways in the brain. The functional effects of dopamine-rich grafts *Corresponding author. Tel.: 143-512-504-4292; fax: 143-512-5043852. E-mail address: [email protected] (G. K. Wenning). Abbreviations: AChE, acetylcholineesterase; ANOVA, analysis of variance; AP, anterior±posterior; CRL, crown-to-rump-length; DAB, diaminobenzidine tetrahydrochloride; DARPP-32, dopamine and cyclic adenosine 3`5`-monophosphate-regulated phosphoprotein; GCI, glial cytoplasmic inclusion; GFAP, glial ®brillary acidic protein; HE, haematoxylin- eosin; HD, Huntington's disease; MSA, multiple system atrophy; MSA±P, multiple system atrophy±Parkinsonism; NHS, normal horse serum; NP, non-P zones; 3-NP, 3-nitropropionic acid; 6-OHDA, 6-hydroxydopamine; PBS, phosphate-buffered saline; PD, Parkinson's disease; QA, quinolinic acid; SNc, substantia nigra pars compacta; SND, striatonigral degeneration; TH, tyrosine hydroxylase. 581

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Table 1. Experimental rationale: Drugs were given in following doses: 3-NP (500 nmol), amphetamine (2.5 mg/ kg i.p.) and apomorphine (1.0 mg/ kg s.c.). Transplantation groups: striatal (n ˆ 8), mesencephalic (n ˆ 6), mesencephalic and striatal co-graft (n ˆ 8), sham graft (n ˆ 6).

commonest degenerative disease causing Parkinsonism. Epidemiological studies suggest an incidence ratio of 3.0 per 100,000 6 and prevalence ratio of 4.4 per 100,000. 40 MSA±Parkinsonism (MSA±P) is characterized neuropathologically by striatonigral degeneration (SND), i.e. cell loss and gliosis in somatotopically related areas of the SNc and of the putamen. 19,26 Furthermore, oligodendroglial cytoplasmic inclusions (GCIs) are observed throughout the cortico±striato±pallido±cortical loops and may contribute to the basal ganglia dysfunction. 27 Progressive l-dopa-unresponsive Parkinsonism due to underlying SND dominates the clinical syndrome of MSA±P in the majority of cases. 45 In early stages of MSA±P many patients are misdiagnosed as PD. However, in MSA±P there is more rapid progression of motor disabilities than in PD, autonomic failure is seen in almost all cases, and cerebellar and/or pyramidal signs are present in about half of these patients at some stage of their illness. 45,48 At the time of clinical diagnosis, on

average four to ®ve years after disease onset, most MSA patients suffer from advanced disabling Parkinsonism. A median survival of 9.3 years has been reported in a series of 100 patients with probable MSA. 45 The treatment of MSA is disappointing. l-dopa substitution usually causes no persistent improvement, only up to 30% of the patients may transiently bene®t from ldopa. 45 Due to suboptimal diagnostic accuracy 29 it is likely that a number of MSA±P patients misdiagnosed as PD have undergone mesencephalic transplantation. Indeed, two such patients (one post mortem con®rmed, the other clinically possible) have been reported whose clinical course following transplantation indicated incomplete functional bene®t, suggesting that mesencephalic grafts alone may be insuf®cient for the treatment of MSA±P. 42,47 Since both substantia nigra and striatum degenerate in MSA, embryonic mesencephalic and striatal tissue would seem to be required for transplantation. 48,49 So far, no studies have been reported on the effect of embryonic transplants in animals with experimental lesions of both substantia nigra and striatum except for two rodent studies showing partial functional bene®ts induced by embryonic grafts. 37,46 The clinical application of neural transplantation in MSA±P requires experimental evidence of major graft-induced functional improvement in appropriate animal models. The diagnosis of MSA±P is mostly made in advanced stages of the disease, dominated by severe striatal degeneration. 19 We therefore aimed to explore the functional effects of embryonic neuronal grafts in an animal model of advanced SND. The pathology of SND can be modelled by the ªdouble toxin±double lesionº approach by a sequential unilateral injection of 6-OHDA into the medial forebrain bundle and of quinolinic acid (QA) into the ipsilateral striatum. 46,48 The ªsingle toxin± double lesionº approach refers to a single intrastriatal injection of mitochondrial neurotoxins such as 3-nitropropionic acid (3-NP) that produce local striatal damage as well as retrograde loss of dopaminergic projection neurons with corresponding depletion of striatal dopamine and respective metabolites. 32,48,49 Therefore intrastriatal administration of 3-NP generates a rat model corresponding to advanced MSA±P with prominant striatal degeneration. In contrast, systemic administration of 3-NP leads to excitotoxic-like lesions, selective for the dorsolateral striatum, resembling the striatal pathology of Huntington's disease (HD) with sparing of striatal dopaminergic afferent projections. 2,22 In the present experiment we evaluated the behavioural effects of embryonic striatal and mesencephalic grafts in a unilateral 3-NP rat model of MSA±P. EXPERIMENTAL PROCEDURES

Time-course (the protocol of the experiments is shown in Table 1) Animals. Twenty-eight male Wistar rats (Harlan Winkelmann) aged seven weeks were used. At the time of lesion surgery animals weighed between 213 and 277 g. All animals were housed on a 12-h light/dark cycle, with free access to food and water throughout the experiment, excluding the time during the paw reaching testing, when they were maintained on food restriction (8 g/d/animal). All efforts were made to minimize the

Neurotransplantation in a model of advanced MSA

number of animals used and their suffering. The following in vivo protocols were approved by the Federal Ministry of Science and Transport of Austria. Apomorphine- and amphetamine-induced rotation. To assess drug-induced rotation asymmetries, rats were injected with 2.5 mg/kg d-amphetamine-sulfate (i. p., dissolved in 0.9% saline; Sigma) or with 1.0 mg/kg apomorphine hydrochloride (s.c., dissolved in 0.2% ascorbate/saline; Sigma). 32 The turning behaviour was monitored over 90 and 60 min, respectively, using automated rotometer bowls (San Diego Instruments, Sandown Scienti®c). Motor asymmetry score was calculated as net ipsiversive turns (turns contraversive to the lesion subtracted from those towards the lesioned side). Paw reaching test. In order to test skilled paw function, a slightly modi®ed version of the staircase test of Montoya et al. was employed. 31 Plexiglass test boxes (Dr E. Torres, MRC Cambridge Centre for Brain Repair, Cambridge, UK) with a central platform and a removable staircase on each side were used. Rats were deprived of food for 48 h before the test and allowed to train their grasping abilities during these two days. Six wells on each staircase were used (including the ®rst to the sixth step) and each well was baited with two food pellets (Noyes precision food pellets, formula: P, puri®ed rodent diet, size 45 mg; Sandown Scienti®c). The animals were tested over 12 consecutive days. For each test the rats were placed in the test box for 15 min. Then the staircase was removed, and the number of pellets remaining in each well on the two sides was counted, from which the total number of pellets retrieved on each side was calculated. Lesion surgery. 3-NP (Sigma; N-5636) was dissolved in 0.1 M phosphate-buffered saline (PBS, pH 7.4) at a concentration of 250 nmol/ml. For surgery each rat was anaesthetized with halothane (Halothan; Hoechst; Z. Nr.: 11.812, starting with a ¯ow of 4 l of 4% halothane and subsequent reduction to 1.5 l of 1.5%). The animals were placed in a stereotaxic frame (Kopf instruments) with the incisor bar set at 3.3 mm below the interaural line. They received a unilateral injection of 500 nmol of 3NP using a 5-ml Hamilton syringe (26S-gauge stainless steel cannula) at the following stereotaxic coordinates, measured in millimetres from bregma in the anterior±posterior (AP) and lateral (L) planes, and ventral from dura in the vertical (V) plane: AP 10.48, L 13.0, V 24.5. 33 The toxin was infused over a period of 2 min, and thereafter the injection cannula was left in place for an additional 5 min to prevent the injected solution from diffusing along the cannula track. The cannula was retracted over a period of 2 min. Transplantation procedure. Embryonic transplantation was performed according to standard protocols. 4,46,53 Six weeks after the lesion, graft tissue was obtained from gestational day 14 embryos (Wistar; 9±11 mm crown-to-rump length, CRL). Embryonic stage was estimated by palpation of the uterine horns of pregnant female rats. Pregnant rats were anaesthetized with an excessive i.p. dose of thiopental (Tyrol Pharma, Z. Nr.: 5.134), the embryos removed by Caesarian section and gestational stage was con®rmed by CRL measurement. For dissecting the mesencephalon we used a conventional razor breaker and holder. Whole ganglionic eminences were dissected bilaterally. Dissected embryonic tissue was collected in working medium comprising 0.6% glucose in 0.9% sterile saline at room temperature. Pieces of embryonic tissue were mechanically snipped into smaller pieces and incubated in 300 ml of 0.1% trypsin (Sigma; T-1005) in working medium for 20 min at 378C. Incubation was carried out in Eppendorf tubes in a tissue incubator. The trypsin was removed and the tissue was carefully washed (®ve to eight times) with working medium containing 0.05% DNAse using a Pasteur pipette. Following centrifugation at 1000 r.p.m. for 4 min, tissue pieces were mechanically dissociated at a ®nal volume of 3 ml/striatal primordium and 4 ml/ventral mesencephalon by trituration through the tip of four ¯ame-polished Pasteur

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pipettes with decreasing internal tip diameters. The cell suspension was once again centrifugated at 1000 r.p.m. for 4 min and triturated using a Pasteur pipette with a smaller tip aperture. The Eppendorf tubes were covered and stored on ice. The standard procedure of conventional embryonic transplantation is also described in detail elsewhere. 4 Viability of the embryonic cells was assessed by Trypan Blue exclusion just prior to grafting using a haemocytometer (Neubauer improved) and was always more than 90% for both mesencephalic and striatal cell suspensions. The average cell concentrations were 66,000/ml for the mesencephalic and 110,000/ml for the striatal cell suspension. Cell viability was again monitored at the end of the transplantation procedure (approximately 4 h after dissection) and was more than 80% for both mesencephalic and striatal cell suspensions. The animals were randomly assigned to four transplantation groups based on their pre-grafting rotometer scores, so that each group comprised animals with comparable amphetamine- (range: 10.33±10.99) and apomorphine- (range: 4.67±5.34) induced rotation rates. Graft volumes and stereotaxic implantation coordinates were as follows (tooth bar set 3.3 mm below the interaural line): striatal graft: 1 £ 3 ml; AP 10.48, L 14.0, V 25.0; n ˆ 8 mesencephalic graft: 2 £ 2 ml; AP 11.0/11.0, L 13.6/13.6, V 25.5/24.5; n ˆ 6 striatal and mesencephalic separate co-grafts: separate implantation of one striatal graft (1 £ 3 ml; coordinates as above) and two mesencephalic grafts (2 £ 2 ml, coordinates as above); nˆ8 sham graft: working medium containing 0.05% DNAse, stereotaxic coordinates as above; n ˆ 6 The cell suspensions were grafted into host animals by stereotaxic injection via a 5 ml Hamilton syringe, using a stainless steel cannula (26 gauge, internal diameter of 0.26 mm, external diameter of 0.46 mm). Cells were injected over 3 min for striatal cell suspensions or 2 min for mesencephalic cell suspension (both 1 ml/min) and a further 4 min was allowed for diffusion for every single deposit before retraction of the syringe over a period of 2 min. Tissue processing. Six months after grafting, the animals were deeply anaesthetized with thiopental (Tyrol Pharma) and perfused transcardially with ice-cold PBS for 2 min, followed by ice-cold 4% paraformaldehyde in 0.1 M PBS (pH adjusted to 7.4). Rats were decapitated and the brains removed and post®xed for three days in the same ®xative at 48C. For dehydration they were transferred to a solution containing 25% sucrose in 0.1 M PBS and left at 48C until they had sunk. The brains were frozen at 2708C using methylbutan (Sigma) and dry ice. Sections were cut on a freezing microtome at a thickness of 40 mm through both the striatum and substantia nigra. For immunohistochemistry sections were stored in assorters in 0.25 M Tris-buffer (500 ml 1 M Tris-buffer in 1500 ml distilled water and 2 ml 10% NaN3 added; Sigma; pH 7.4). Sections for haematoxylin-eosin and acetylcholinesterase-stain were directly mounted onto gelatincoated microscope slides. Haematoxylin-Eosin stain. For haematoxylin-eosin (HE)staining a series of sections (1:10 in striatum, 1:8 in substantia nigra) were mounted onto gelatin-coated microscope slides. Sections were dehydrated in an ascending series of alcohol (30, 70, 96 and 100%) in each alcohol for 3 min. Then they were placed in a descending series of alcohol (100, 96, 70 and 30%) for another 3 min in each solution. The slides were dipped in distilled water and stained for 8 min in haematoxylin, again dipped in distilled water, placed for 10 min in warm water and dipped once more in distilled water. The sections were stained for 3 min with eosin and differentiated in ascending alcohols before being cleared for 10 min in butylacetat (Scharlau) and coverslipped with entellan (Merck). Acetylcholinesterase stain. Activity of acetylcholinesterase (AChE) was visualized according to Jensen±Blackstad. 20 One series of striatal sections (1:10) was mounted onto gelatin-coated

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Table 2. Amphetamine-induced rotation behaviour. Amphetamine-induced rotation behaviour was assessed three weeks following 3-nitropropionic acid injection and three, eight and 18 weeks after grafting procedure. Mean amphetamine-induced rotation ^ S.D. Time-point Three weeks post 3-NP lesion Three weeks post grafting Eight weeks post grafting 18 weeks post grafting

Mesencephalic (n ˆ 6)

Striatal (n ˆ 8)

Co-graft (n ˆ 8)

Sham (n ˆ 6)

10.33 ^ 0.94 5.48 ^ 1.59 5.38 ^ 2.25 6.27 ^ 1.78

10.58 ^ 2.45 8.91 ^ 1.62 7.93 ^ 2.05 6.68 ^ 2.93

10.55 ^ 2.7 7.87 ^ 3.43 7.07 ^ 3.23 6.97 ^ 4.38

10.99 ^ 4.95 8.3 ^ 2.52 7.99 ^ 4.58 9.46 ^ 7.19

The net mean rotations (^ S.D.) per minute obtained over 90 min following amphetamine administration (2.5 mg/kg) are shown. All rats were subjected to unilateral intrastriatal injections of 500 nmol 3-NP followed by grafting of either pure mesencephalic (n ˆ 6), pure striatal (n ˆ 8), mesencephalic-striatal co-grafts (n ˆ 8) or sham grafts (n ˆ 6; 0.6% glucose-saline and 0.05% DNAse). Values represent ipsiversive turns.

microscope slides. They were incubated overnight in Jensen± Blackstad AChE-medium at 48C. After washing for 10 min with distilled water they were incubated for 10 min in 10% KFeCN (in distilled water, Merck) at room temperature. The sections were further washed for 10 min in distilled water and differentiated in ascending alcohols, cleared in butylacetate and coverslipped with entellan. Immunohistochemistry. Immunohistochemical staining was conducted on free ¯oating sections. Sections were washed in 0.1 M PBS (2 £ 10 min), quenched for 10 min in 0.3% H2O2 (dissolved in PBS) in order to block the endogenous peroxidase, washed again twice in PBS and incubated in 10% normal horse serum (NHS; Sigma) as blocking agent. Antibodies were diluted in PBS containing 0.1% Triton X-100. Series of sections were incubated overnight in the primary antibody against dopamine and cyclic adenosine 3 0 5 0 -monophosphate-regulated phosphoprotein (DARPP-32, 1:20 000; kindly donated by Dr Hugh C. Hemmings, The New York Hospital, Cornell Medical Center, New York, USA), tyrosine hydroxylase (TH, 1:250, Sigma), glial ®brillary acidic protein (GFAP, 1:100, Boehringer Mannheim Biochemica) and OX 18 (1:1000, Serotec Ltd.). Following washing in 0.1 M PBS (2 £ 10 min) the sections were incubated in biotinylated secondary horse-anti-mouseantibody (1:200, Vector Laboratories) for 1.5 h. After further washing in PBS, sections were incubated in ABC reagent (Vectastain) for 1 h. Sections were washed three times in PBS. Peroxidase visualization of immunocytochemical markers was shown by using diaminobenzidine tetrahydrochloride (DAB, Sigma) as substrate (1 ml DAB with 50 ml PBS and 8 ml H2O2). After washing in PBS (2 £ 10 min), the free ¯oating sections were mounted onto gelatin-coated microscope slides and left to air dry overnight, counterstained with haematoxylin, dehydrated in alcohols, cleared in butylacetat and coverslipped using entellan. Morphological analysis. For evaluating the extent of the striatal lesion we chose to measure the size of spared striatum. The residual, spared striatum was de®ned as the area exhibiting DARPP-32 immunoreactivity. Morphometric analysis was performed using computerized image analysis. DARPP-32immunostained sections were digitized using a high resolution video camera (SONY 3CCD color video camera) and the software package Image-Pro Plus (version 1.0, Media Cybernetics). Sections of three predetermined anatomical levels (AP 11.2 mm, 10.2 mm, 20.3 mm) were chosen according to Nakao et al. 32 The residual striatum exhibiting DARPP-32-immunoreactivity was measured and then expressed as a percentage of the area of the striatum on the intact side at the same anatomical levels. The outer borders of the striatum were de®ned by the lateral ventricle and the graft border medially, the corpus callosum dorsolaterally and the anterior commissure ventrally. The shell of the accumbens nuclei was not included into the measurement. An average of the values obtained from the three sections was calculated for each animal. To estimate the survival of substantia nigra dopaminergic neurons, we chose three different levels through the substantia

nigra: rostral (AP 24.8mm), middle (25.3 mm) and caudal (26.0 mm). 32,33 On each section, the numbers of TH-immunostained neurons in the SNc were counted bilaterally at £ 200 magni®cation with the aid of a superimposed grid. The number of TH-positive SNc cells was expressed as the mean number of the count per section calculated from these three sections. In the rostral sections the medial boundary of the SNc was de®ned as a line extending dorsally from the most medial part of the cerebral peduncle. In the middle and caudal sections, the SNc and the ventral tegmental area were separated by the accessory optic tract and the medial lemniscus, respectively. The total graft volume of striatal and co-grafts was measured using DARPP-32-stained sections. Borders of the grafts were clearly de®ned and outlined on every tenth section through the graft and the graft area was measured after calibration of the area units into millimetres squared. The sum of the measured areas was multiplied with the inter-section distance of 400 mm to calculate the total graft volume. In order to calculate P-zone volume, DARPP-32 positive areas in the graft were encircled using a threshold function. P-zonevolume was calculated as above. Surviving TH-positive neurons in mesencephalic and striatalmesencephalic co-grafts were detected on TH-immunostained sections. Neurons in the graft exhibiting TH-immunoreactivity were counted at £ 200 magni®cation with the aid of a superimposed grid. Statistical analysis. All data are expressed as mean value ^ S.D. For analysis of morphometrical parameters two-tailed paired Student's t-tests were used to compare numbers of surviving THpositive neurons in the SNc on the two sides of the brain, unpaired t-test were used to compare graft parameters as graft volume, volume of P-zones and number of surviving dopaminergic neurons between the graft groups. For the analysis of behavioural test results repeated measures of analysis of variance (ANOVA) were employed considering in¯uence of time and group as well as group £ time interactions for rotational data and in¯uence of time, group and side (right versus left paw) as well as group £ time, group £ side, time £ side and group £ time £ side interactions for paw reaching data. Correlation analyses were performed using Pearson's test. A probability value of less than 0.05 was considered signi®cant, when multiple tests were performed, the Bonferroni±Holmes correction was applied. RESULTS

Drug-induced rotation tests Amphetamine-induced rotation tests. Three weeks post 3-NP-lesion placement amphetamine provoked a robust turning ipsiversive to the lesioned side in all groups. The number of net ipsiversive turns/min was 10.33 in the mesencephalic, 10.58 in the striatal, 10.55 in the co-graft and 10.99 in the sham graft group (see Table 2). There was no group difference of pre-grafting

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Neurotransplantation in a model of advanced MSA

Table 3. Apomorphine-induced rotation behaviour. Apomorphine-induced rotation behaviour was assessed three weeks following 3-nitropropionic acid injection and three, nine and 19 weeks after grafting procedure. Mean apomorphine-induced rotation ^ S.D. Time-point Four weeks post 3-NP lesion Four weeks post grafting Nine weeks post grafting 19 weeks post grafting

Mesencephalic (n ˆ 6)

Striatal (n ˆ 8)

Co-graft (n ˆ 8)

Sham (n ˆ 6)

4.67 ^ 0.71 4.35 ^ 1.25 6.78 ^ 2.38 6.73 ^ 1.81

4.76 ^ 1.04 3.38 ^ 1.41 6.45 ^ 2.41 6.82 ^ 2.05

4.92 ^ 1.44 4.1 ^ 1.89 6.61 ^ 3.02 7.12 ^ 3.72

5.34 ^ 0.94 6.01 ^ 1.61 7.87 ^ 2.28 7.62 ^ 2.35

The net mean rotations (^ S.D.) per minute obtained over 60 min following apomorphine administration (1.0 mg/kg) are shown. All rats were subjected to unilateral intrastriatal injections of 500 nmol 3-NP followed by grafting of either pure mesencephalic (n ˆ 6), pure striatal (n ˆ 8), mesencephalic-striatal co-grafts (n ˆ 8) or sham grafts (n ˆ 6; 0.6% glucose-saline and 0.05% DNAse). Values represent ipsiversive turns. Table 4. Mean numbers of pellets taken (^ S.D.) for both paws are shown. All rats were subjected to unilateral intrastriatal injections of 500 nmol 3nitropropionic acid followed by grafting of either pure mesencephalic (n ˆ 6), pure striatal (n ˆ 8), mesencephalic-striatal co-grafts (n ˆ 8) or sham grafts (n ˆ 6; 0.6% glucose-saline and 0.05% DNAse). Skilled forelimb use was tested following 3-nitropropionic acid lesion placement and up to 20 weeks post-grafting. Skilled forelimb use: number of pellets taken Time-point

Mesencephalic (n ˆ 6) left

Post 3-NP lesion Five weeks post grafting 10 weeks post grafting 20 weeks post grafting

6.4 ^ 2.8 9.5 ^ 2.8 8.6 ^ 2.8 10.1 ^ 2.9

right 4.4 ^ 1.5 4.3 ^ 0.8 3.8 ^ 1.1 3.7 ^ 0.8

Striatal (n ˆ 8)

Co-graft (n ˆ 8)

Sham (n ˆ 6)

left

right

left

right

left

right

4.5 ^ 2.6 9.0 ^ 2.3 10.0 ^ 2.7 10.9 ^ 1.8

4.5 ^ 1.6 3.8 ^ 1.3 3.9 ^ 1.9 4.2 ^ 1.6

8.3 ^ 4.1 8.7 ^ 4.2 9.9 ^ 3.9 10.2 ^ 3.5

5.2 ^ 1.8 5.1 ^ 2.0 4.9 ^ 2.2 4.2 ^ 1.6

7.2 ^ 3.1 11.3 ^ 1.5 12.8 ^ 1.0 12.2 ^ 1.6

4.3 ^ 1.2 4.6 ^ 1.5 4.5 ^ 1.8 4.1 ^ 1.3

rotation scores. Repeated measures of ANOVA revealed a signi®cant impact of time on the rotational tests (P , 0.001) but no signi®cant in¯uence of group assignment and no signi®cant group £ time interaction. Apomorphine-induced rotation tests. Four weeks post 3-NP-lesion placement apomorphine induced ipsiversive rotation behaviour in all animals, with a mean ipsiversive rotation rate of 4.67 in the mesencephalic, 4.76 in the striatal, 4.92 in the co-graft and 5.34 in the sham graft group. (Table 3). There was no group difference of pre-grafting rotation scores. Using repeated measures of ANOVA there was a signi®cant in¯uence of time on the rotation scores (P , 0.001), however, no signi®cant impact of group assignment and no group £ time interaction. Paw reaching As animals show a learning effect in the skilled forelimb use with reaching plateau levels after day 7 on both sides 32 we analysed only results of the last ®ve days of each testing period. The mean number of pellets taken and eaten during this period was calculated. After the lesion and the grafting procedures, a clear separation in the performance of the two limbs was visible. Pellets taken. At every time-point tested rats took more pellets with their left ªgoodº paw (ipsilateral to the lesion). During the post-grafting-period all groups showed an increase in number of pellets taken ipsilaterally compared to pre-grafting scores (Table 4). Repeated measures of ANOVA revealed a signi®cant impact of test

time-point, side (left versus right paw; P , 0.001), but not of group assignment on the pellets taken parameter. There was a signi®cant time £ group (P , 0.05) and time £ side (P , 0.001) interaction, however, side £ group and time £ side £ group interactions did not reach signi®cance. Pellets eaten. Repeated measures of ANOVA revealed a signi®cant impact of test time-point, side (left versus right paw) (P , 0.001), but not of group assignment on the pellets eaten parameter. There was a signi®cant time £ group (P , 0.05) and time £ side (P , 0.001) interaction, however, side £ group and time £ side £ group interactions did not reach signi®cance (Table 5). Success rate. The success rate was expressed in terms of number of pellets eaten divided by number of pellets taken. Repeated measures of ANOVA revealed a signi®cant impact of test time-point, side (left versus right paw) (P , 0.001), but not of group assignment on the success rate parameter. There was a signi®cant time £ side (P , 0.01) and time £ side £ group (P , 0.05) interaction, however, time £ group and side £ group interactions did not reach signi®cance (Table 6). Histological analysis Morphometry of the lesioned adult nigrostriatal system. SNc. Loss of dopaminergic neurons ipsilateral to the striatal lesion was found. Paired t-test revealed a signi®cant reduction of TH-positive cells compared to the intact side in all treatment groups (P , 0.05; P , 0.01 in striatal graft group; Fig. 1b). The mean

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Table 5. Mean numbers of pellets eaten (^ S.D.) for both paws are shown. All rats were subjected to unilateral intrastriatal injections of 500 nmol 3nitropropionic acid followed by grafting of either pure mesencephalic (n ˆ 6), pure striatal (n ˆ 8), mesencephalic-striatal co-grafts (n ˆ 8) or sham grafts (n ˆ 6; 0.6% glucose-saline and 0.05% DNAse). Skilled forelimb use was tested following 3-nitropropionic acid lesion placement and up to 20 weeks post-grafting. Skilled forelimb use: number of pellets eaten Time-point

Post 3-NP lesion Five weeks post grafting 10 weeks post grafting 20 weeks post grafting

Mesencephalic (n ˆ 6)

Striatal (n ˆ 8)

Co-graft (n ˆ 8)

Sham (n ˆ 6)

left

right

left

right

left

right

left

right

3.0 ^ 1.8 4.7 ^ 2.5 4.6 ^ 2.1 5.9 ^ 2.6

1.1 ^ 0.9 1.5 ^ 0.8 1.8 ^ 0.8 2.3 ^ 0.5

2.9 ^ 1.5 5.0 ^ 2.0 6.2 ^ 3.0 6.7 ^ 2.4

1.1 ^ 1.0 1.7 ^ 1.4 2.2 ^ 1.3 2.3 ^ 0.9

4.8 ^ 2.7 4.4 ^ 2.5 6.3 ^ 2.8 6.8 ^ 2.2

2.0 ^ 0.5 2.2 ^ 0.8 2.2 ^ 0.9 2.2 ^ 0.5

3.5 ^ 2.6 6.8 ^ 2.1 8.8 ^ 1.6 7.8 ^ 1.6

1.5 ^ 0.7 2.0 ^ 0.6 2.5 ^ 0.7 2.8 ^ 0.6

Table 6. Success rate was calculated by dividing the number of pellets eaten by the number of pellets taken. Mean values ^ S.D. are shown. All rats were subjected to unilateral intrastriatal injections of 500 nmol 3-nitropropionic acid followed by grafting of either pure mesencephalic (n ˆ 6), pure striatal (n ˆ 8), mesencephalic-striatal co-grafts (n ˆ 8) or sham grafts (n ˆ 6; 0.6% glucose-saline and 0.05% DNAse). Skilled forelimb use was tested following 3-nitropropionic acid lesion placement and up to 20 weeks post-grafting. Skilled forelimb use: success rate Time-point

Post 3-NP lesion Five weeks post grafting 10 weeks post grafting 20 weeks post grafting

Mesencephalic (n ˆ 6)

Striatal (n ˆ 8)

Co-graft (n ˆ 8)

Sham (n ˆ 6)

left

right

left

right

left

right

left

right

0.4 ^ 0.2 0.5 ^ 0.2 0.5 ^ 0.1 0.5 ^ 0.2

0.2 ^ 0.2 0.4 ^ 0.2 0.5 ^ 0.2 0.6 ^ 0.1

0.4 ^ 0.1 0.5 ^ 0.1 0.6 ^ 0.2 0.6 ^ 0.1

0.2 ^ 0.2 0.4 ^ 0.2 0.6 ^ 0.1 0.5 ^ 0.1

0.6 ^ 0.2 0.5 ^ 0.2 0.7 ^ 0.2 0.7 ^ 0.1

0.4 ^ 0.2 0.5 ^ 0.2 0.5 ^ 0.2 0.6 ^ 0.2

0.4 ^ 0.2 0.6 ^ 0.2 0.7 ^ 0.1 0.6 ^ 0.1

0.4 ^ 0.2 0.4 ^ 0.2 0.5 ^ 0.1 0.7 ^ 0.1

Fig. 1. (A) Photomicrograph showing a representative coronal striatal section of a sham grafted animal histochemically stained with AChE. Injection of 500 nmol 3-NP resulted in a severe striatal atrophy and dilatation of the lateral ventricle (V) on the operated side (indicated by arrow). (B) Photomicrograph showing a representative coronal section of a sham grafted animal immunohistotochemically stained with TH- antibody. Intrastriatal injection of 500 nmol 3-NP resulted in retrograde neuronal loss in the substantia nigra pars compacta (lesioned side indicated by arrow).

remaining number of dopaminergic neurons was 55% ^ 10.2 of the intact side. No signi®cant group difference in dopaminergic cell numbers of both sides could be detected. Striatum. The remaining striatum exhibited severe shrinkage with remarkably enlarged ventricles. DARPP32 immunoreactivity as well as AchE staining (Fig. 1a) was detected over the cross-sectional area of the spared striatum. Mean area of residual striatum exhibiting DARPP-32 immunoreactivity, expressed as percentage of the DARPP-32 positive striatal area on the intact side, was 23.9% ^ 8.6. There was no signi®cant difference in the size of the spared striatal tissue between

groups. Medial parts of striatum were not preserved in any animal. GFAP-immunoreactivity was found in the spared striatum of all groups, including the sham graft group. In sham-grafted animals the staining was pronounced at the medial borders of remaining striatal tissue adjacent to the enlarged lateral ventricle. Rats receiving embryonic grafts showed most intensive GFAP-staining at the graft-host border. The distribution of OX 18 immunopositivity as a marker for microglial activation showed a similar pattern to GFAP. Morphometry of the embryonic grafts. Mesencephalic grafts. Surviving TH-positive neurons were found in all

587

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mesencephalic grafts. Although two deposits had been placed separately one above the other, in only one animal could two separate grafts be distinguished. TH-positive cell clusters were seen in the periphery at the graft/host border. In one animal surviving dopaminergic neurons were located along the needle track. Positive staining for GFAP and OX 18 was found at the outer borders of the graft, OX 18-immunoreactivity also inside the graft. Striatal grafts. The mean total graft volume was 8.16 ^ 8.13 mm 3 (range 0.97 to 22.65 mm 3). Seven of eight rats receiving pure striatal grafts showed speci®c DARPP-32 immunoreactivity within the graft area. Pzones as a marker for surviving striatal tissue were present throughout the graft. In order to calculate the proportion of striatal-like tissue within the graft, volume of P-zones was analysed and found to be 1.11 ^ 1.52 mm 3 (range 0.02 to 4.26 mm 3). Every single striatal graft was surrounded by a rather thick GFAP-positive capsule. In all striatal grafts TH-immunoreactive ®bres could be detected, which however did not penetrate the surrounding. OX 18 immunoreactivity was detected at the borders of the graft as well as inside the graft. Striatal and mesencephalic co-grafts. In most animals the mesencephalic and the striatal deposits had merged to form a single mass. Due to severe atrophy of remaining adult striatum the position of the grafts was adjacent to the ventral border of striatum. The mean total graft volume was 12.17 ^ 10.3 mm 3, ranging from 0.71 to 29.96 mm 3. Six of eight (75%) rats receiving co-grafts showed DARPP-32 positive P-zones (Fig. 2a) within the striatal graft area, indicating survival of embryonic striatal tissue, interspersed with areas of more diffuse lighter staining non-P-(NP)zones. 21 P-zone volume was found to be 2.77 ^ 2.56 mm 3 (range 0.07±5.99). The expression of DARPP-32 co-localized with positive staining for AChE (Fig. 2b) as well as for TH, which was con®rmed using a double-labelling immunohistochemistry-technique (data not shown). TH-positive embryonic (Fig. 2c) neurons were identi®ed in all animals indicating survival of the mesencephalic graft component. TH-positive neurons and ®bres were usually distributed in the periphery of mesencephalic grafts. Total cell numbers per mesencephalic graft were not obtained due to limited numbers of TH-stained sections. In two co-grafted animals mesencephalic grafts could be distinguished whereas no striatal tissue had survived. Grafts were surrounded by a capsule staining positive with GFAP and OX 18 (Fig. 3a, b). Group comparison. Surviving embryonic TH-positive neurons. Due to the inter-section distance of 400 mm mean cross-sectional rather than total graft counts of TH positive neurons were obtained. The mean crosssectional number of TH-positive neurons was signi®cantly higher in animals receiving co-grafts (84.3 ^ 41.7) compared to pure mesencephalic grafts (22.7 ^ 29.8; P , 0.05). Total graft volume and P-zone-volume. Mean total graft volume of animals receiving co-grafts was 49.1% larger compared to pure striatal graft volume. Despite this fact, statistical analysis of the graft volumes showed

no signi®cant difference between the two groups (P . 0.05) as graft volumes in both treatment groups were very variable. Additionally, absolute P-zonevolume was found to be non-signi®cantly larger in cografted animals compared to those with embryonic striatal grafts. In order to assess correlation between total graft volume and proportion of striatal-like tissue within the grafts, Pearson's tests were performed. These analyses revealed a highly signi®cant correlation between these parameters in the pure striatal graft group (r ˆ 0.959; P , 0.01). Correlation of behavioural and morphological parameters. We explored the relationship between size of spared striatum and results of drug-induced rotation tests performed before grafting. Inverse, non-signi®cant correlations between amphetamine- and apomorphineinduced rotation scores and spared striatal area were observed. In order to assess the in¯uence of the number of spared dopaminergic neurons in SNc ipsilateral to the lesioned side on rotation scores Pearson's tests were performed. This analysis detected a signi®cant inverse correlation (r ˆ 20.492; P , 0.01) between apomorphineinduced net ipsiversive turns/min at the latest time-point tested (19 weeks after grafting) and the relative number of remaining TH-positive SNc neurons. No signi®cant correlation between amphetamine-induced rotational behaviour and dopaminergic nigral cell counts was found. We next studied the correlation between surviving TH-positive cells in the graft and amphetamine-induced circling behaviour 18 weeks post grafting compared to the pre-grafting results. In all but two of eight (75%) cografted animals and one of six (83%) animals in the pure mesencephalic graft group a reduction of amphetamineinduced rotation rates was observed. Surviving dopaminergic cells within the graft were found in all animals. The degree of reduction of amphetamine-induced rotation failed to correlate with the numbers of TH-positive cells per section. Nineteen weeks post grafting an increase of apomorphine-induced rotation scores in comparison to the post-3-NP-lesion level was found in six of eight (75%) animals of both striatal and co-graft group as well as ®ve of six (83%) animals of both sham and mesencephalic graft group. The relative changes of ipsiversive turns/min at each time-point following transplantation did not correlate with any morphological parameter of the graft (whole graft volume, P-zone-volume). DISCUSSION

Behavioural responses in lesioned animals Amphetamine-induced rotation. Consistent with previous reports animals with a unilateral striatal 3-NP lesion exhibited ipsiversive rotations in response to amphetamine. 32 The number of net ipsiversive turns in response to amphetamine appears to be higher in rats with 3-NP compared to QA-induced striatal lesions

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Fig. 2.

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589

Fig. 3. (A) Photomicrograph showing a coronal section of an animal receiving a mesencephalic and striatal co-graft. Immunostaining with GFAP antibody reveals intense glial activation on the graft-host border (see arrowheads). 3-NP-induced striatal degeneration and dilatation of the lateral ventricle (V) can be observed (magni®cation £ 200). (B) Photomicrograph of an OX-18 immunostained coronal section of a co-grafted animal showing demarcation of the implant (see arrowheads; magni®cation £ 200). V, lateral ventricle.

re¯ecting the additional loss of dopaminergic neurons in SNc. 32 Apomorphine-induced rotation. Animals with unilateral 3-NP striatal lesions also rotated ipsiversively in response to apomorphine. In contrast, apomorphine induces contraversive rotation in the 6-OHDA rat model, re¯ecting dopamine receptor supersensitivity within the denervated striatum. 44 Therefore, it is likely that the observed apomorphine-induced ipsi- rather than contraversive rotation in 3-NP lesioned rats was mainly due to the destruction of the striatum, and that the partial reduction of dopaminergic neurons in the SNc did not signi®cantly affect turning behaviour. 32 Behavioural responses in grafted animals Amphetamine-induced rotation. Amphetamine exerts its effect on rotational behaviour by enhancement of

dopamine release from the nigrostriatal terminals. In 6OHDA-lesioned rats complete reversal of ipsiversive amphetamine-induced circling occurs within ®ve to 10 weeks after transplantation of fetal mesencephalic tissue. 3,13 Reduction of amphetamine-induced rotation has been shown to require dopaminergic ®bre outgrowth from the graft into adult recipient striatum. 17,38 Previous studies have shown that embryonic striatal grafts placed into the excitotoxically lesioned striatum can reduce the ipsiversive rotation score. 15 This effect requires a functional innervation of the graft tissue by dopaminergic neurons of host origin. Anatomical observations of ingrowth and synaptic connectivity are compatible with host derived innervation of embryonic striatal tissue grafted into ibotenic-lesioned recipient striatum. 8,35,53 In addition formation of efferent connectivity has been shown to mediate reversal of amphetamine-induced rotation behaviour in rat models of HD. 16 Tracer studies demonstrated that efferent projections from striatal grafts

Fig. 2. (A) Photomicrograph showing a representative coronal section immunohistochemically stained with DARPP-32 antibody of an animal receiving a mesencephalic and striatal co-graft. Mesencephalic and striatal cell suspensions had been grafted separately but merged to form a single mass. P-zones (indicated by arrows) and NP-zones can be clearly distinguished within the graft. Injection of 500 nmol 3-NP resulted in a severe striatal atrophy and dilatation of the lateral ventricle (V) on the operated side (magni®cation £ 200). (B) Photomicrograph showing an AChE-stained coronal section of the same co-grafted animal. Again P-zones (indicated by arrows) and NP-zones are clearly distinguishable (magni®cation £ 200). V, lateral ventricle. (C) Photomicrograph showing a coronal section of a co-grafted animal immunostained with TH-antibody. Surviving TH-positive neurons and cell clusters (indicated by arrows) are visible (magni®cation £ 400). V, lateral ventricle.

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innervate host globus pallidus. 52 Ultra-structural studies showed that graft-derived ®bres form morphologically normal synaptic contacts onto host pallidal neurons. 50,51 In the unilateral 3-NP-lesion model both striatal lesions as well as loss of nigrostriatal terminals are likely to determine amphetamine-induced rotation scores. In our present study, transplantation of embryonic dopaminergic neurons failed to reverse amphetamine-induced rotational behaviour in a novel unilateral 3-NP rat model of advanced SND. Graft-derived dopamine release may not have been suf®cient to reverse amphetamine-induced rotational behaviour. In addition, no outgrowth of THimmunoreactive ®bres from the graft through the glial scar into the surrounding brain tissue could be detected. Finally, there was marked destruction of adult dopamine receptor bearing striatum. Embryonic mesencephalic cells survived in all animals of the co-graft as well as the pure mesencephalic graft group. The number of surviving dopaminergic neurons was variable, however, a signi®cantly greater number of surviving dopaminergic neurons was observed in the co-graft compared to the pure mesencephalic graft group. Several in vitro studies have shown that embryonic striatal neurons exert a trophic in¯uence on the development of embryonic dopaminergic neurons. Both growth and development of grafted embryonic mesencephalic cells were enhanced when they were co-cultured with striatal primordial cells, 12 striatal membranes 36 or soluble striatal extracts. 43 Separate mesencephalic and striatal co-grafts have been reported to induce signi®cant recovery of amphetamineinduced rotation behaviour compared to rats with pure mesencephalic or mixed co-grafts. 7,9,10,54 This observation may be due to increased number of dopaminergic cells. 11 However, in vivo studies in the rat Parkinson model report discrepancies regarding the effects of striatal cells on dopaminergic cell survival. Increased numbers of surviving embryonic mesencephalic neurons were found in mixed 10,11,41 as well as separate co-grafts. 41 Other studies have failed to demonstrate increased survival of TH-positive cells. 7,9,49 Despite a higher survival rate of TH-positive embryonic neurons in the co-graft group, no signi®cant difference between the pure mesencephalic and the co-graft group in the reduction of amphetamine-induced ipsiversive rotations was observed. This ®nding might be explained by preferential interaction of embryonic mesencephalic ®bres with embryonic striatal cells that was observed using double labelling immunocytochemistry (data not shown). Furthermore, dopamine release of a restricted number of surviving mesencephalic cells might compensate the partial dopamine de®cit and lead to saturation of all dopamine binding sites in the spared striatum. A threshold effect of dopaminergic grafts has also been reported in the 6OHDA-rat model. 38 Apomorphine-induced rotation. Apomorphine is a mixed dopamine D1 and D2 receptor agonist that acts by direct stimulation of dopamine receptors. In 6-OHDA lesioned rats mesencephalic grafts produce a partial reduction of contraversive apomorphine-induced rotation behaviour. 14 Characteristic contraversive rotation pattern

is caused by dopamine receptor supersensitivity due to lack of nigrostriatal afferents. Released dopamine from surviving embryonic neurons leads to a partial reversal of postsynaptic dopamine receptor supersensitivity. In the HD rat model striatal grafts partially reduce the apomorphine-induced ipsiversive rotation score. 15,16 Such compensation of ipsiversive apomorphine-induced rotation suggests that striatal grafts express functional dopamine receptors, consistent with autoradiographic observations of spiroperidol binding to dopamine D2 receptors in striatal grafts. 24,37 Furthermore, dopaminergic activation of grafted striatal neurons is able to in¯uence efferent targets by virtue of projections of grafted neurons to the globus pallidus or the substantia nigra pars reticulata.35 In the present study, there was no reduction of apomorphine induced rotation asymmetry consistent with major destruction of the host striatopallidal projection. 15 Reversal of apomorphine-induced rotation behaviour by grafted embryonic striatal neurons in HD animal models is critically dependent on regeneration of striatopallidal circuitry and expression of functionally active dopamine receptors. 15 In the present MSA±P rat model P-zones, indicating surviving embryonic striatal cells, were found in six of eight (75%) co-grafted rats and seven of eight (83.3%) rats in the striatal graft group. These ®ndings suggest that apomorphine binding to dopamine receptors on embryonic striatal neurons did not exert any functional effect due to lack of efferent projections to host targets. As an increase of apomorphine-induced rotation throughout the post-grafting period was found in all groups, including sham animals, non-speci®c effects of transplantation surgery on prelesioned adult host striatum may partly account for this functional change. Paw reaching Embryonic striatal grafts have been demonstrated to ameliorate several behavioural de®cits induced by excitotoxic striatal lesions in HD animal models. 5 These behavioural de®cits include complex motor disturbances like impaired skilled forelimb use. 15,25,30 Since accurate task performance normally is dependent on the integrity of the nigrostriatal dopamine pathway as well as on the integrity of the neostriatum itself, 15 effects of the striatal grafts on paw use require a functional dopaminergic afferent input. In contrast, nigral grafts fail to improve contralateral paw reaching impairments induced by unilateral 6-OHDA lesions of the nigrostriatal pathway. 30 The signi®cant recovery of skilled paw reaching de®cits that is observed in the striatal graft model compared to the nigral graft model can be partly attributed to the placement of the grafts. Due to their homotopic implantation site, striatal grafts are able to reconnect with host targets in the adjacent globus pallidus. 35 These connections may provide the basis for the reformation of a functional circuitry. 15 By contrast, the nigral grafts are placed ectopically into the denervated neostriatum where they remain disconnected from host afferent neural systems. Nigral grafts in the rat PD model provide a tonic reinnervation of the deafferented striatum which may be insuf®cient to restore

Neurotransplantation in a model of advanced MSA

de®cits of complex motor control. 14,18 In the 3-NP model animals of all groups increased the number of pellets taken and eaten by the ipsilateral good paw compared to the ®rst test performed. This ipsilateral improvement is unlikely to be graft-mediated but rather related to further practise. 30 In contrast, rats failed to develop a learning curve on the contralateral side. It is known that manipulations of the neostriatum cause an impairment in tasks requiring a memory component for their execution. 39 Graft-derived reversal of complex motor de®cits due to striatal lesions requires restoration of nigro-striato-pallidal circuits. 15 Results of drug-induced rotation tests suggest that grafts in the advanced SND model were poorly integrated into the host neuronal circuitry. The lack of neuroanatomical reconstruction is consistent with absent recovery of skilled paw use de®cits. In order to assess the degree of integration of embryonic grafts into striatopallidal and striatonigral circuits further experiments like retrograde tracer studies are necessary. CONCLUSION

In the unilateral 3-NP rat model of SND (MSA±P)

591

drug-induced rotation asymmetries and contralateral paw reaching de®cits remained unchanged following transplantation of dopamine rich embryonic ventral mesencephalon. The lack of behavioural effects may be explained by severe atrophy of the adult striatum, additional nigral lesions as well as glial demarcation of embryonic grafts preventing dopaminergic re-innervation. The inef®cacy of embryonic neuronal grafts suggests that neurotransplantation may not be a useful strategy for improving motor function in patients with advanced MSA±P. However, several approaches may increase graft-derived bene®ts in the 3-NP model including induction of mild or moderate striatal lesions by low dose 3-NP injections, thus mimicking earlier stages of MSA±P, as well as grafting at an earlier time-point following lesion placement.

AcknowledgementsÐThis study was supported by the Austrian Science Foundation (P11748-MED). Technical assistance of Bernd Weiler, Harald Granbichler and Christian TrawoÈger is gratefully acknowledged.

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