Presence of tau pathology within foetal neural allografts in patients with Huntington's and Parkinson's disease.

Cell replacement has been explored as a therapeutic strategy to repair the brain in patients with Huntington's and Parkinson's disease. Post-mortem evaluations of healthy grafted tissue in such cases have revealed the development of Huntington- or Parkinson-like pathology including mutant huntingtin aggregates and Lewy bodies. An outstanding question remains if tau pathology can also be seen in patients with Huntington's and Parkinson's disease who had received foetal neural allografts. This was addressed by immunohistochemical/immunofluorescent stainings performed on grafted tissue of two Huntington's disease patients, who came to autopsy 9 and 12 years post-transplantation, and two patients with Parkinson's disease who came to autopsy 18 months and 16 years post-transplantation. We show that grafts also contain tau pathology in both types of transplanted patients. In two patients with Huntington's disease, the grafted tissue showed the presence of hyperphosphorylated tau [both AT8 (phospho-tau Ser202 and Thr205) and CP13 (pSer202) immunohistochemical stainings] pathological inclusions, neurofibrillary tangles and neuropil threads. In patients with Parkinson's disease, the grafted tissue was characterized by hyperphosphorylated tau (AT8; immunofluorescent staining) pathological inclusions, neurofibrillary tangles and neuropil threads but only in the patient who came to autopsy 16 years post-transplantation. Abundant tau-related pathology was observed in the cortex and striatum of all cases studied. While the striatum of the grafted Huntington's disease patient revealed an equal amount of 3-repeat and 4-repeat isoforms of tau, the grafted tissue showed elevated 4-repeat isoforms by western blot. This suggests that transplants may have acquired tau pathology from the host brain, although another possibility is that this was due to acceleration of ageing. This finding not only adds to the recent reports that tau pathology is a feature of these neurodegenerative diseases, but also that tau pathology can manifest in healthy neural tissue transplanted into the brains of patients with two distinct neurodegenerative disorders.

Mutant huntingtin is present in neuronal grafts in Huntington disease patients.

OBJECTIVE: Huntington disease (HD) is caused by a genetically encoded pathological protein (mutant huntingtin [mHtt]), which is thought to exert its effects in a cell-autonomous manner. Here, we tested the hypothesis that mHtt is capable of spreading within cerebral tissue by examining genetically unrelated fetal neural allografts within the brains of patients with advancing HD. METHODS: The presence of mHtt aggregates within the grafted tissue was confirmed using 3 different types of microscopy (bright-field, fluorescence, and electron), 2 additional techniques consisting of Western immunoblotting and infrared spectroscopy, and 4 distinct antibodies targeting different epitopes of mHtt aggregates. RESULTS: We describe the presence of mHtt aggregates within intracerebral allografts of striatal tissue in 3 HD patients who received their transplants approximately 1 decade earlier and then died secondary to the progression of their disease. The mHtt(+) aggregates were observed in the extracellular matrix of the transplanted tissue, whereas in the host brain they were seen in neurons, neuropil, extracellular matrix, and blood vessels. INTERPRETATION: This is the first demonstration of the presence of mHtt in genetically normal and unrelated allografted neural tissue transplanted into the brain of affected HD patients. These observations raise questions on protein spread in monogenic neurodegenerative disorders of the central nervous system characterized by the formation of mutant protein oligomers/aggregates.

Transplantation of rat neural stem cells reduces stereotypic behaviors in rats after intrastriatal microinfusion of Tourette syndrome sera.

Tourette syndrome (TS) is a heterogenous neuropsychiatric disorder. In most cases, tics are self-limited or can be treated by behavioral or pharmacological therapy. However, for some individuals, tics can cause lifelong impairment and life-threatening symptoms, which are intractable to traditional treatment. Neural stem cell (NSC) is a potential tool to treat certain neurological diseases. In this study, we proposed to use neural stem cell transplantation as a novel therapy to treat TS and discussed its efficacy. Wistar rats were microinfused with TS sera into the striatum followed by the transplantation of NSCs or vehicle at the infusion site. The sera of the TS patients were identified to have enriched antineural antibodies. Prior to grafting, rat embryonic NSCs were co-cultured with 5-bromodeoxyuridine (Brdu) for 24 h. Stereotypic behaviors were counted at 1, 7, 14 and 21 days after transplantation of NSCs. Morphological analyses revealed that NSCs survived and differentiated into neurons and astrocytes in the striatum 3 weeks after grafting. To sum it up, rat embryonic neural stem cell grafts survived and differentiated in the striatum of TS rat may help relieve stereotypic behaviors of the host. Our results suggest that transplantation of NSCs intrastriatum may have therapeutic potential for TS.

Human fetal striatal transplantation in huntington's disease: a refinement of the stereotactic procedure.

BACKGROUND: Human fetal striatal transplantation (HFST) is an experimental stereotactic intervention in the treatment of Huntington's disease (HD). This procedure has proved feasible, safe, well tolerated and it offers a potential strategy for brain repair in HD patients. Target areas are the nucleus caudatus caput (NCc) and the precommissural and postcommissural putamen (Pu). A suboptimal spatial distribution of grafts was frequently reported, especially for the postcommissural Pu, because of striatal atrophy and the concurrent ventricular frontal horn enlargement. An improvement of the stereotactic procedure aimed to optimize the intrastriatal placement of grafts is therefore considered a timely issue. METHODS: Eight consecutive HD patients underwent bilateral HFST. For the first 6 procedures (first group) we performed both caudate and putaminal tracks through a single frontal entry point. For the following 10 procedures (second group), we adopted two completely distinct routes, with two separate entry points, for NCc and Pu tracks. The average number of stereotactic tracks and the average infused volume of tissue suspension were compared between the two groups. RESULTS: The average number of putaminal tracks and the average infused volume of suspension were significantly higher in the second group. CONCLUSION: Adopting two separate routes for caudate and putaminal trajectories allowed us to achieve a larger amount of fetal tissue deposits and a better spatial distribution of grafts.

The corridor task: striatal lesion effects and graft-mediated recovery in a model of Huntington's disease.

Experimental validation of cell replacement therapy as a treatment of neurodegenerative diseases requires the demonstration of graft-mediated behavioural recovery. The Corridor task proved to be simple and efficient to conduct with a robust ipsilateral retrieval bias in our rodent Huntington's disease model. The Corridor task is a viable behavioural option, particularly to non-specialised laboratories, for the evaluation of lateralised striatal damage and the probing of alternative therapeutic strategies, including transplantation.

Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat.

Repeated systemic administration of low doses of kainic acid (KA) induces spontaneous convulsive seizures [Hellier JL, Patrylo PR, Buckmaster PS, Dudek FE. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res 1998;31:73-84]. In this study, male Sprague-Dawley animals received intranigral transplants of a control cell line M213-2O, or a cell line transfected with human GAD67 cDNA (M213-2O CL4) [Conejero-Goldberg C, Tornatore C, Abi-Saab W, Monaco MC, Dillon-Carter O, Vawter M, et al. Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content. Exp Neurol 2000;161:453-61], or no transplant. Eight weeks after transplantation surgery, KA was administered (5 mg/kg/h) until animals reached stage V seizures as described by Racine [Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281-94]. The group transplanted with CL4 required a larger dose of KA and a longer latency to reach a stage V seizure. In addition, this group exhibited significantly fewer stage III and IV seizures. These results indicate that intranigral transplants of a GABA-producing cell line can decrease the number of kainic acid-induced seizures.

Simultaneous intrastriatal and intranigral dopaminergic grafts in the parkinsonian rat model: role of the intranigral graft.

The current transplantation strategy in experimental and clinical Parkinson's disease (PD) has been to place nigral dopaminergic grafts not in their ontogenic site (substantia nigra) but in their target area (striatum). Although intrastriatal dopaminergic grafts are capable of reinnervating the striatum, they fail to reinnervate the nigra, which may be an important factor limiting the efficacy of fetal tissue transplantation in parkinsonian patients. We have previously shown that simultaneous intrastriatal and intranigral dopaminergic grafts (double grafts) may provide a more complete restoration of the nigrostriatal circuitry (Mendez et al. [1996] J Neurosci 16:7216-7227; Mendez and Hong [1997] Brain Res 778:194-205). In the present study, we investigated the contribution of the intranigral graft to functional recovery in double-grafted hemiparkinsonian rats. Twenty Wistar rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway were divided into two groups and received either double grafts (n = 10) or intrastriatal grafts alone (n = 10). Following transplantation, both intrastriatally and double-grafted animals had a significant decrease in rotational behavior. However, only animals with double grafts exhibited a significant increase in contralateral adjusting step performance. The intranigral graft was subsequently lesioned by a second 6-OHDA injection. Following the second lesion, animals with double grafts exhibited a significant reversal of rotational behavior and a 51% reduction in contralateral adjusting step performance. The reversal in functional recovery correlated with a significant loss of intranigral grafted neurons. These results suggest that the intranigral graft has an important role in the functional recovery of double-grafted animals. Restoration of dopaminergic innervation to both the nigra and the striatum may be crucial for optimizing graft efficacy and may be a superior strategy in neural transplantation for PD.

Rationale for intrastriatal grafting of striatal neuroblasts in patients with Huntington's disease.

Huntington's disease is a genetic disease, autosomal and dominant, that induces motor disorders, an inexorable deterioration of higher brain functions and psychiatric disturbances. At present, there are no known therapeutics against Huntington's disease. The Network of European CNS Transplantation and Restoration (NECTAR) has begun a program aimed at defining the conditions under which intrastriatal transplantation of fetal striatal cells could be attempted as an experimental treatment for Huntington's disease. This review presents the reasons why our group is considering participating in these trials. The validity of this therapeutic approach is supported by three main series of data: (i) neuropathological, clinical and imaging data indicate that Huntington's disease is, above all, a localized affection of a specific neuronal population ("medium-spiny" neurons) in the striatum; (ii) a large body of experimental results, obtained in rats and non-human primates, demonstrates that transplanted fetal striatal cells are able to integrate the host brain and to substitute for previously lesioned host striatal neurons; (iii) expertise in clinical neural transplantation has now been acquired from the treatment of patients with Parkinson's disease. These different sets of data are presented and discussed in this review. There are a number of problems which do not yet appear to be entirely resolved, nor are they likely to be using the experimental models currently available. These problems are identified and explicitly presented as working hypotheses. (1) Anatomo-functional results obtained in rodents and non-human primates with excitotoxic striatal lesions can serve as a basis for the extrapolation of what can be obtained from patients with Huntington's disease. (2). Huntington's disease can be efficiently fought by substituting degenerated striatal neurons alone. (3) Huntington's disease is due to a genetic defect which either hits the neurons that carry it directly or hits them indirectly only after several decades. Transplanted neurons, because they do not carry the gene or because they are of fetal origin, will not be rapidly affected by the ongoing disease process. Given the current state of knowledge, intracerebral transplantation appears to be the most serious opportunity (if not the only one that has been experimentally validated) for clinical improvement to be obtained in patients with Huntington's disease. The purpose of this review is to open a scientific discussion on its experimental bases before actual clinical trials start.

Descriptive morphology of developing fetal neostriatal allografts in the rhesus monkey: a correlated light and electron microscopic Golgi study.

Primate fetal striatal neurons were transplanted into the ibotenic acid lesioned rhesus monkey striatum. Ten weeks after transplantation the monkeys were transcardially perfused and graft tissue was histologically stained. Golgi impregnated, and processed for electron microscopy. The monkeys received magnetic resonance imaging (MRI) scans before lesioning, after lesioning, and ten weeks after transplantation to noninvasively study the striatal grafts. The study demonstrated that fetal striatal grafts, measuring up to 0.4 x 0.8 cm, can survive for extended periods of time in the non-human primate. Hematoxylin-eosin stained sections of the transplant demonstrated that neuronal, glial, vascular, and lymphocytic cells were present in the graft. The majority of the neurons had somatic diameters between 8 and 20 microns and were characterized by nuclei containing multiple nucleoli. A few neurons within the graft had somatic diameters up to 40 microns. These larger neurons exhibited more mature cytoplasm containing a moderate amount of Nissl substance. Some of the blood vessels within the graft were surrounded by a large number of plasma cells, but there was no evidence of hemorrhage or necrosis. Bielschowsky staining and Golgi impregnation of the transplanted tissue demonstrated that there were neurons at various degrees of differentiation. Some of the neurons had varicose dendrites, growth cones, and filopodia, which are all characteristics of immature neurons, while others had a much more mature appearance, including a moderate number of dendritic spines. Some of these neurons had an appearance typical of differentiating "medium spiny" neurons of the normal striatum. Electron microscopic analysis of the transplanted tissue and individual Golgi-impregnated neurons within the transplant confirmed that there were developing neurons within the graft. These neurons had an increased nuclear-to-cytoplasmic ratio and had nuclei containing multiple nucleoli. The neuropil surrounding these neurons was loosely organized and contained large areas of extracellular space. The neuropil exhibited developing dendrites, numerous growth cones, and mature synapses. In summary, the study demonstrated that fetal striatal allografts can survive for up to three months in the rhesus monkey and undergo normal differentiation as assessed by Golgi impregnation and electron microscopy.

DARPP-32-rich zones in grafts of lateral ganglionic eminence govern the extent of functional recovery in skilled paw reaching in an animal model of Huntington's disease.

Grafts of striatal tissue comprise two different types of tissue: regions with (P-zones) and without (NP-zones) neurons that express markers characteristic of the striatum, such as dopamine- and cyclic AMP-regulated phosphoprotein with a mol. wt of 32,000 (DARPP-32). It remains unclear whether P-zones alone play a crucial role in functional effects of striatal grafts in an animal model of Huntington's disease. The present study has been performed to determine: (i) the yield of DARPP-32-positive neurons in grafts of lateral ganglionic eminence; (ii) whether treatment of graft tissue with the spin-trapping agent alpha-phenyl-tert-butyl nitrone enhances the survival of implanted DARPP-32-positive neurons; and (iii) the relationship between the number of DARPP-32-positive neurons in the grafts and functional effects of the grafts on paw-reaching ability in rats with unilateral quinolinic acid lesions of the striatum. Dissociated tissue derived from the lateral ganglionic eminence of rat embryos (embryonic day 14), with or without addition of alpha-phenyl-tert-butyl nitrone (3 mM), was implanted into the quinolinic acid-lesioned striatum. Compared to unlesioned normal animals, rats with striatal lesions showed substantial impairment in paw-reaching ability, particularly on the side contralateral to the lesion, as judged from the number of pellets retrieved by each paw. Intrastriatal grafts gave rise to a significant improvement in paw-reaching ability. The mean total number of surviving DARPP-32-positive cells in grafts without alpha-phenyl-tert-butyl nitrone treatment was estimated at 115 x 10(3), which did not significantly differ from that in alpha-phenyl-tert-butyl nitrone-treated grafts. The paw-reaching scores were significantly correlated with the volumes of P-zones and the number of DARPP-32-positive neurons, but with neither the volumes of NP-zones nor the total graft volume. The results suggest that P-zones in striatal grafts mediate graft-derived functional recovery in a complex task such as skilled forelimb use. Although the antioxidant treatment with alpha-phenyl-tert-butyl nitrone failed to promote graft survival, the positive correlation between the yield of DARPP-32-positive cells in the graft and the extent of the functional recovery highly warrants further attempts to increase the yield of the striatal component in the graft.

Intrastriatal grafts of embryonic mesencephalic rat neurons genetically modified using an adenovirus encoding human Cu/Zn superoxide dismutase.

Intrastriatal grafting of embryonic dopamine-containing neurons is a promising approach for treating clinical and experimental Parkinson's disease. However, neuropathological analyses of grafted patients and transplanted rats have demonstrated that the survival of grafted dopamine neurons is relatively poor. In the present study, we pursued a strategy of transferring a potentially neuroprotective gene into rat embryonic mesencephalic rat cells in vitro, before grafting them into the denervated striatum of 6-hydroxydopamine-lesioned rats. We performed intrastriatal grafts of embryonic day 14 mesencephalic cells infected with replication-defective adenoviruses bearing either the human copper-zinc superoxide dismutase gene or, as a control, the E. coli lac Z marker gene. The transgenes were expressed in the grafts four days after transplantation and the expression persisted for at least five weeks thereafter. After five weeks postgrafting, there was more extensive functional recovery in the superoxide dismutase group as compared to the control (uninfected cells) and beta-galactosidase groups. The functional recovery was significantly correlated with the number of tyrosine hydroxylase-positive cells in the grafts, although the clear trend to increased survival of the dopamine neurons in the superoxide dismutase grafts did not reach statistical significance. Only a moderate inflammatory reaction was revealed by OX-42 immunostaining in all groups, suggesting that ex vivo gene transfer using adenoviral vectors is a promising method for delivering functional proteins into brain grafts.

Immune reactions following systemic immunization prior or subsequent to intrastriatal transplantation of allogeneic mesencephalic tissue in adult rats.

We have previously found that dissociated mesencephalic tissue, which differs from the host at both major histocompatibility complex and non-major histocompatibility complex gene loci, can survive stereotaxic transplantation to the striatum of adult rats. We have now studied the outcome of intrastriatal neural allografts in rats that were systemically immunized by an orthotopic skin allograft either prior or subsequent to intracerebral implantation surgery. Dissociated mesencephalic tissue from Lewis rat embryos was stereotaxically injected into the dopamine-depleted striatum of hemi-parkinsonian Sprague-Dawley rats. One group was immunized by an orthotopic allogeneic skin graft of the same genetic origin as the neural graft, six weeks before the neural transplantation (the pre-immunized group). Another group was post-immunized by an orthotopic skin allograft, six weeks after the neural transplantation (the post-immunized group). A control group of rats was not challenged by a skin allograft. Marked behavioural recovery was observed in six of seven rats in the control group, in six of eight rats in the post-immunized group, and in none of the pre-immunized rats. Tyrosine hydroxylase-immunopositive cells were found in rats from the two behaviourally compensated groups, but not in the pre-immunized group. The immune responses were evaluated by OX-18 (monoclonal antibody against major histocompatibility complex class I antigen), OX-6 (major histocompatibility complex class II antigen), OX-42 (microglia and macrophages), glial fibrillary acidic protein (astrocytes), OX-8 (cytotoxic T-lymphocytes) and W3/25 (helper T-lymphocytes) immunocytochemistry. All the neural allografts in the pre-immunized group were rejected, leaving scars only. There were more intense immune responses to the allografts in the post-immunized group than the control group, in terms of immunocytochemically higher expression of major histocompatibility complex class I and II antigens and more intense cellular reactions consisting of macrophages, activated microglia and astrocytes, in addition to CD8- and CD4-positive lymphocytes. In summary, the results show the following: (i) systemic pre-immunization leads to complete rejection of intrastriatal neural allografts, implying that the status of the host immune system before transplantation determines the outcome for intrastriatal neural allografts; (ii) established intrastriatal neural allografts can survive for at least six weeks after systemic immunization, in spite of increased host immune responses in and around the allografts; (iii) there are no marked immune reactions against intrastriatal neural allografts 13 weeks after implantation in rats which have not been systemically immunized by a skin allograft; (iv) pre-immunized rats may provide a very useful animal model to investigate the role of inflammatory lymphokines in immune rejection and to test alternative immunosuppressive drugs.

Changes in striatal immediate early gene expression following neonatal dopaminergic lesion and effects of intrastriatal dopaminergic transplants.

To evaluate the functional integration of neonatal dopaminergic transplants within host brain we studied the postsynaptic effects induced by their stimulation by following the expression of immediate early genes c-fos, c-jun and egr-1. This study was conducted nine months after the intrastriatal implantation of embryonic mesencephalic neurons to rat pups having sustained a unilateral lesion of the nigrostriatal dopaminergic system. We examined whether, when challenged with d-amphetamine: (1) dopaminergic grafts transplanted into the previously denervated neonatal neostriatum lead to a normal activation of postsynaptic striatal neurons in term of immediate early genes activation; and (2) whether this activation is related to the action of the dopamine released from the grafts using a dopaminergic D1 antagonist. Following a mild stress-injection of saline-c-fos expression was high in the lesioned neostriatum when compared with control animals. This effect was only partially counteracted by a pre-treatment with the D1 antagonist SCH 23390, but was abolished by the graft. Administration of d-amphetamine increased c-fos expression in the neostriatum and the globus pallidus of the control group. This activation was partially blocked by the lesion. The transplant reversed the effect of the lesion and, moreover, led to a c-fos over-expression in the dorsolateral neostriatum and the globus pallidus. These overcompensations positively correlated with the abnormal rotation induced by d-amphetamine in the same animals. Pre-treatment with SCH 23390 blocked the effect of d-amphetamine on c-fos expression in control and grafted animals. Similar results were found for egr-1 but not c-jun expression. It is concluded that the neonatal lesion of the nigrostriatal dopaminergic pathway, in contrast to the adult-stage lesion, modifies the reactivity of c-fos in the neostriatum to stress, presumably in relation with compensatory reorganizations occurring following the neonatal lesion. Grafts made into neonates, when challenged with amphetamine, induce an abnormal c-fos expression which can predict the degree of overshoot observed for rotation activity. This over-expression, which depends upon the stimulation of D1 receptors, indicate an abnormal activation of postsynaptic target cells by the grafts.

Intrathalamic striatal grafts survive and affect circling behaviour in adult rats with excitotoxically lesioned striatum.

Current models of basal ganglia disorders suggest that choreoathetosis is the end result of reduced GABAergic inhibition of the motor thalamus. Graft-derived release of GABA from intrastriatal striatal grafts has also been reported. In the present work, cell suspension grafts from embryonic day 14-15 rat striatal primordia were implanted close to the ventromedial thalamic nucleus to investigate whether they can develop and survive in this ectopic location, and whether they induce changes in the circling behaviour of the host. The grafts were implanted either in normal rats or in rats whose striatum had been lesioned with ibotenic acid. These grafts were implanted either ipsilateral or contralateral to the lesioned striatum. Additionally, some rats received intrastriatal grafts, and lesioned but non-grafted rats and lesioned rats that had received injections of saline or of cell suspensions from fetal spinal cord in the thalamus were used as control. Four to eight months after transplantation, circling behaviour after amphetamine or apomorphine injection was evaluated. Serial sections were stained with Cresyl Violet and studied immunohistochemically with antibodies against DARPP-32 (dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein, as striatal marker), Fos protein, glutamate decarboxylase (67,000 mol. wt), glutamate decarboxylase (65,000 mol. wt) and GABA. Cresyl Violet sections showed that the intrathalamic striatal grafts developed into tissue masses resembling those observed in intrastriatal striatal grafts. DARPP-32 immunohistochemistry revealed that the grafts were composed of DARPP-32 immunoreactive (striatum-like) and DARPP-32-negative patches. The intrathalamic grafts of rats which had received a low dose of apomorphine (0.25 mg/kg) 2 h before perfusion showed clusters of intensely Fos-immunoreactive nuclei throughout the transplant, indicating that these cells had developed dopamine receptors and supersensitivity to dopamine agonists. Double Fos and DARPP-32 immunohistochemistry revealed that the Fos-positive nuclei were located in the striatum-like areas. Finally, the intrathalamic grafts also contained neurons immunoreactive to GABA and glutamate decarboxylase (65,000 and 67,000 mol. wt). Rats that had received intrathalamic grafts contralateral to the lesioned striatum (i.e. contralateral to the lesion-induced turning direction) showed a significant reduction of circling both after amphetamine (78% reduction) or apomorphine (77% reduction) injection. Rats that had received grafts ipsilateral to the lesioned striatum showed a 75% decrease in amphetamine-induced circling, but no significant change in apomorphine-induced circling. No significant drug-induced circling was observed in non-lesioned and grafted rats. Sham grafting (saline) or grafting of weakly GABAergic tissue (fetal spinal cord) had no significant effects on lesion-induced circling behaviour.(ABSTRACT TRUNCATED AT 400 WORDS)

Glial cell line-derived neurotrophic factor improves intrastriatal graft survival of stored dopaminergic cells.

Glial cell line-derived neurotrophic factor, the newest member of the transforming growth factor-beta superfamily, has been shown to promote the survival and differentiation of dopaminergic neurons in the ventral mesencephalon. Glial cell line-derived neurotrophic factor has been implicated in both the in vitro and in vivo recovery of mesencephalic dopaminergic cells challenged with the neurotoxins 1-methyl-4-phenylpyridinium and 6-hydroxydopamine. Previous studies have shown increased survival of intrastriatally transplanted dopaminergic cells when followed by infusion of neurotrophic factors such as basic fibroblast growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. However, the effects of glial cell line-derived neurotrophic factor co-administered with dopaminergic cells prior to implantation in the host striatum have not been studied. In the present study, the hypothesis was that treating fetal ventral mesencephalic tissue containing the dopaminergic substantia nigra with glial cell line-derived neurotrophic factor either during storage or at the time of transplantation, would enhance grafted dopaminergic cell survival and functional reinnervation of the host striatum in the unilaterally 6-hydroxydopamine-lesioned rat. To test this hypothesis, two experiments were performed. In the first experimental group (n = 7), fetal ventral mesencephalons from embryonic day 14 rats were maintained in hibernation medium containing glial cell line-derived neurotrophic factor (1 migrogram/ml) at 4 degrees C for six days prior to dissociation and stereotactic implantation into the host striatum: the control group (n = 5) received tissue hibernated without glial cell line-derived neurotrophic factor. The second experimental group (n = 8) received fresh fetal ventral mesencephalic tissue treated with glial cell line-derived neurotrophic factor (0.2 microgram/microliter) while the control group (n = 5) received the fresh graft with no glial cell line-derived neurotrophic factor. Transplantation success was assessed by behavioural analysis (rotometry) and tyrosine hydroxylase immunohistochemistry. Cell counts of tyrosine hydoxylase-stained sections revealed a statistically significant increase in tyrosine hydroxylase-positive neurons in grafts exposed to glial cell line-derived neurotrophic factor during hibernation as compared to control grafts. In addition, there was a statistically significant enhancement of fibre density in the glial cell line-derived neurotrophic factor hibernation graft group as compared to the glial cell line-derived neurotrophic factor fresh graft group. Behavioural analysis three weeks post-grafting exhibited a statistically significant decrease in amphetamine-induced rotations in animals transplanted with glial cell line-derived neurotrophic factor grafts as compared to control grafts. These findings suggest that storing dopaminergic cells in a glial cell line-derived neurotrophic factor-containing medium prior to transplantation increases graft survival, graft derived fibre outgrowth, and behavioural recovery in the adult host. This observation has potential implications for enhancing the efficacy of neural transplantation in the treatment of Parkinson's disease.

Dopamine-rich grafts in the neostriatum and/or nucleus accumbens: effects on drug-induced behaviours and skilled paw-reaching.

This study compares the behavioural efficiency of dopaminergic mesencephalic neurons implanted into the rat neostriatum and/or the nucleus accumbens. The dopaminergic mesotelencephalic pathway was unilaterally destroyed by injection of 6-hydroxydopamine into the medial forebrain bundle at the level of the lateral hypothalamus. Three weeks later, embryonic dopaminergic mesencephalic neurons were implanted into the denervated neostriatum, or the nucleus accumbens or into both locations (double grafts). All animals were tested over a four month period for amphetamine- and apomorphine-induced rotation, apomorphine-induced locomotor activity, and on a skilled paw reaching task. The characteristic ipsilateral rotation induced by amphetamine observed in lesioned animals was significantly reduced by neostriatal and double grafts, but persisted in animals with grafts in the nucleus accumbens alone. Four months after grafting, an overcompensation of rotation was observed for the neostriatal and double grafted animals, which now rotated contralaterally, i.e. away from the grafted side. The rotation induced by apomorphine in lesioned rats was decreased by neostriatal and double grafts and to a lesser extent by grafts implanted into the nucleus accumbens. Apomorphine-induced locomotor hyperactivity in lesioned animals was ameliorated by the nucleus accumbens and by double grafts. In the paw-reaching task, lesioned animals showed severe impairment in the use of the contralateral limb, which none of the grafts alleviated. Pretreatment with amphetamine had variable effects on the paw-reaching task which persisted in subsequent drug-free trials, suggesting that a conditioning mechanism may be involved. These findings suggest that the simultaneous reinnervation of the neostriatum and the nucleus accumbens by dopaminergic transplants is not sufficient to re-establish normal function in more complex behavioural tasks.

Striatal tissue transplantation in non-human primates.

The caudate nucleus and putamen form part of a complex but topographically connected circuitry that links the cortex, the basal ganglia and the thalamus. Within this complex system lie a series of functionally and anatomically segregated loops that allow the concurrent processing of a wide range of cognitive and motor information (Alexander et al., 1986; Alexander and Crutcher, 1990). As a constituent of these loops it has been shown that the striatum is involved in movement initiation, response selection and attentional processes (Robbins and Brown, 1990; Alexander, 1994; Lawrence et al., 1998). Although it is the medium spiny GABAergic projection neurones that are primarily lost in HD, it is not sufficient merely to replace the GABA. Instead it is crucial for striatal tissue transplants to integrate with the host tissue in such a way that the cortico-striatal-thalamic circuitry is restored and is functional. Rodent studies have progressed a long way in establishing the principle that striatal grafts can, at least partially, restore function and integrate appropriately with the host (Dunnett and Svendsen, 1993; Björklund et al., 1994; Sanberg et al., 1998) but the limited behavioural repertoire and the undifferentiated striatum meant that it was inevitable that studies should progress into primate models. Anatomical tracing studies have demonstrated that motor, premotor and somatosensory cortical areas send corticostriatal projections primarily to the putamen region in primates, whereas the head and body of the caudate nucleus mostly receive efferent input from associative cortical areas (Kemp and Powell, 1970; Kunzle, 1975, 1977, 1978; Selemon and Goldman-Rakic, 1985). Based on such anatomical, and functional, studies Alexander and colleagues have proposed the existence of at least five cortico-striatal-thalamic loops including a motor, a dorsolateral-prefrontal and an orbito-frontal loop (Alexander et al., 1986). The concentration of motor inputs to the putamen region suggests a particular involvement of this structure in the motor loop. Indeed, unilateral lesions of the putamen disrupt motor performance in the marmoset and generate apomorphine-induced dyskinesias in larger primates (Burns et al., 1995; Kendall et al., 2000). The implantation of striatal grafts into marmosets that had previously received unilateral putamen lesions ameliorated some of the motor impairments, which suggested at least partial restoration of the motor loop. In support of this we found direct evidence of host-graft cortico-striatal connectivity using an anterograde tracer injected in the primary motor cortical region (Kendall et al., 1998a). In larger primates, with lesions of the caudate and putamen, striatal [figure: see text] allografts and xenografts have been shown to reduce apomorphine-induced dyskinesias (Isacson et al., 1989; Hantraye et al., 1992; Palfi et al., 1998). The mechanism by which dyskinesias are elicited is not fully understood but alterations in firing patterns within both segments of the globus pallidus have been identified during dyskinetic movements (Matsumura et al., 1995). It seems likely that it would actually require re-establishment of afferent connections between the implanted putamen and the globus pallidus as well as of functioning dopamine receptors within the graft for the reduction in the dyskinetic profile to be observed. Certainly there is evidence, from rodent studies and the marmoset study described here, that close proximity of the graft to the globus pallidus yields better functional recovery (Isacson et al., 1986). In addition, anatomical tracing studies in rats have demonstrated connections between the implanted tissue and the host globus pallidus (Wictorin et al., 1989b, 1990) However, the relationship between graft placement and functional recovery remains to be fully substantiated.

Restoration of function by neural transplantation in the ischemic brain.

Stroke remains a major brain disorder that often renders patients severely impaired and permanently disabled. There is no available treatment for reversing these deficits. Hippocampal, striatal and cortical grafting studies demonstrate that fetal cells/tissues, immortalized cells, and engineered cell lines can survive grafting into the ischemic adult brain, correct neurotransmitter release, establish both afferent and efferent connections with the host brain, and restore functional and cognitive deficits in specific models of stroke. The success of neural transplantation depends on several factors: the stroke model (location, extent, and degree of infarction), the donor cell viability and survival at pre- and post-transplantation, and the surgical technique, among others. Further exploitation of knowledge of neural transplantation therapy already available from our experience in treating Parkinson's disease needs to be critically considered for stroke therapy. While the consensus is to create a functional neuronal circuitry in the damaged host brain, there is growing evidence that trophic action of the grafts and host, as well as exogenous application of trophic factors may facilitate functional recovery in stroke. Current treatment modules, specifically that of rehabilitative medicine, should also be explored with neural transplantation therapy. However, validation of neural transplantation and any other treatment for stroke should be critically assessed in laboratory experiments and limited clinical trials. No direct treatment is recognized as safe and effective for reversing the stroke-induced brain damage and functional/cognitive deficits. The first clinical trial of neural transplantation in stroke patients is a mile-stone in stroke therapy, but subsequent large-scale trials should be approached with caution.

Lazaroid-enhanced survival of grafted dopamine neurons does not increase target innervation.

The lazaroid U-74006F enhances survival of grafted ventral mesencephalic neurons. In this study the intraocular grafting model was used and survival and outgrowth from fetal ventral mesencephalic grafts treated with U-74006F was evaluated in nigrostriatal co-grafts. Fetal lateral ganglionic eminence was implanted into the anterior eye chamber and left to mature. Fetal ventral mesencephalon was then implanted and the eyes were treated with U-74006F. The lazaroid treatment enhanced survival of tyrosine hydroxylase (TH)-positive neurons, but did not enhance TH-positive nerve fiber growth into the striatal portions of the co-grafts. However, a marked increase in nerve fiber formation was found within the ventral mesencephalic grafts. In conclusion, increased cell survival enhanced nerve fiber formation within the ventral mesencephalic portion of the co-graft and not, as expected, in the striatal part.

Development of the human striatum: implications for fetal striatal transplantation in the treatment of Huntington's disease.

Fetal neural transplantation has recently been demonstrated to ameliorate motor and other behavioral deficits in animals models of Huntington's disease, and reconstruct many of the damaged striatal circuits. However, there has been significant variability in the histological appearance of these grafts, most likely related to differences of the regions of dissection of the donor tissue. Selective dissection and transplantation of the lateral ventricular eminence in rodents has resulted in grafts consisting of primarily striatal-like tissue. This data, combined with data from our own and other laboratories has led to a description of the development of human striatum, with a particular emphasis on the relevance of human striatal development to the field of fetal tissue transplantation for the treatment of Huntington's disease. If the goal of transplantation is to graft GABAergic striatal projection neurons, it is our impression that optimal grafting results will occur when transplants are derived from the lateral ventricular eminence and the lateral aspect of the body of the ventricular eminence anterior to the foramen of Monro. Optimal results are likely to occur when donor ages range from Stage 19 to 23, with possible graft success when donor age extends to as late as postovulatory week 22.