Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into parkinsonian mice.

Clinical trials have provided proof of principle that new dopamine neurons isolated from the developing ventral midbrain and transplanted into the denervated striatum can functionally integrate and alleviate symptoms in Parkinson's disease patients. However, extensive variability across patients has been observed, ranging from long-term motor improvement to the absence of symptomatic relief and development of dyskinesias. Heterogeneity of the donor tissue is likely to be a contributing factor in the variable outcomes. Dissections of ventral midbrain used for transplantation will variously contain progenitors for different dopamine neuron subtypes as well as different neurotransmitter phenotypes. The overall impact of the resulting graft will be determined by the functional contribution from these different cell types. The A9 substantia nigra pars compacta dopamine neurons, for example, are known to be particularly important for motor recovery in animal models. Serotonergic neurons, on the other hand, have been implicated in unwanted dyskinesias. Currently little knowledge exists on how variables such as donor age, which have not been controlled for in clinical trials, will impact on the final neuronal composition of fetal grafts. Here we performed a birth dating study to identify the time-course of neurogenesis within the various ventral midbrain dopamine subpopulations in an effort to identify A9-enriched donor tissue for transplantation. The results show that A9 neurons precede the birth of A10 ventral tegmental area dopamine neurons. Subsequent grafting of younger ventral midbrain donor tissue revealed significantly larger grafts containing more mitotic dopamine neuroblasts compared to older donor grafts. These grafts were enriched with A9 neurons and showed significantly greater innervation of the target dorso-lateral striatum and DA release. Younger donor grafts also contained significantly less serotonergic neurons. These findings demonstrate the importance of standardized methods to improve cell therapy for Parkinson's disease and have significant implications for the generation and selectivity of dopamine neurons from stem cell based sources.

Directing dopaminergic fiber growth along a preformed molecular pathway from embryonic ventral mesencephalon transplants in the rat brain.

To identify guidance molecules to promote long-distance growth of dopaminergic axons from transplanted embryonic ventral mesencephalon (VM) tissue, three pathways were created by expressing green fluorescent protein (GFP), glial cell line-derived neurotrophic factor (GDNF), or a combination of GDNF/GDNF receptor α1 (GFRα1) along the corpus callosum. To generate the guidance pathway, adenovirus encoding these transcripts was injected at four positions along the corpus callosum. In all groups, GDNF adenovirus was also injected on the right side 2.5 mm from the midline at the desired transplant site. Four days later, a piece of VM tissue from embryonic day 14 rats was injected at the transplant site. All rats also received daily subcutaneous injections of N-acetyl-L-cysteinamide (NACA; 100 µg per rat) as well as chondroitinase ABC at transplant site (10 U/ml, 2 µl). Two weeks after transplantation, the rats were perfused and the brains dissected out. Coronal sections were cut and immunostained with antibody to tyrosine hydroxylase (TH) to identify and count dopaminergic fibers in the corpus callosum. In GFP-expressing pathways, TH(+) fibers grew out of the transplants for a short distance in the corpus callosum. Very few TH(+) fibers grew across the midline. However, pathways expressing GDNF supported more TH(+) fiber growth across the midline into the contralateral hemisphere. Significantly greater numbers of TH(+) fibers grew across the midline in animals expressing a combination of GDNF and GFRα1 in the corpus callosum. These data suggest that expression of GDNF or a combination of GDNF and GFRα1 can support the long-distance dopaminergic fiber growth from a VM transplant, with the combination having a superior effect.

Selected changes in spinal cord morphology after T4 transection and olfactory ensheathing cell transplantation.

Spinal cord transection at T4 results in severe damage of the nervous tissue, with impairment of motor, sensory and autonomic functions. Transplantation of olfactory ensheathing cells (OECs) has the potential to improve these functions through a number of mechanisms, which include facilitation of regeneration and neuroprotection. For cardiovascular functions, we have previously shown that OECs reduce the duration of autonomic dysreflexia, without evidence of regeneration. To further understand the mechanisms underpinning this improvement, we have studied changes in selected morphological features (cavitation, non-cavity tissue loss, morphology of sympathetic preganglionic neurons and primary afferent fibre density) in the T4-transected rat spinal cord over 9 weeks, both in control and OEC-transplanted animals. T4 transection led to a number of structural changes: gradual formation of cavities, non-cavity tissue loss, a long-term increase in soma size of sympathetic preganglionic neurons and a temporary increase in the extent of their dendritic arbours, and an increase in the density of primary afferent fibres caudal to the lesion. OECs decreased the cavitation and normalised soma size of the sympathetic preganglionic neurons below the lesion, while increasing the extent of dendritic arbours in the preganglionic neurons above the lesion. Thus the OECs may contribute to the normalisation of the dysreflexic hypertension through tissue preservation and normalisation of the morphology of the preganglionic neurons caudal to the lesion, while enhancing the input on the rostral preganglionic neurons, whose vasomotor control remains intact. We hypothesise that these changes are mediated through secretion of soluble trophic factors by the transplanted OECs.

Histological findings on fetal striatal grafts in a Huntington's disease patient early after transplantation.

Cell transplantation is a promising therapeutic approach that has the potential to replace damaged host striatal neurons and, thereby, slow down or even reverse clinical signs and symptoms during the otherwise fatal course of Huntington's disease (HD). Open-labeled clinical trials with fetal neural transplantation for HD have demonstrated long-term clinical benefits for HD patients. Here we report a postmortem analysis of an individual with HD 6 months after cell transplantation and demonstrate that cells derived from grafted fetal striatal tissue had developed into graft-derived neurons expressing dopamine-receptor related phosphoprotein (32 kDa) (DARPP-32), neuronal nuclear antigen (NeuN), calretinin and somatostatin. However, a fully mature phenotype, considered by the expression of developmental markers, is not reached by engrafted neurons and not all types of interneurons are being replaced at 6 months, which is the earliest time point human fetal tissue being implanted in a human brain became available for histological analysis. Host-derived tyrosine hydroxylase (TH) fibers had already heavily innervated the transplants and formed synaptic contacts with graft-derived DARPP-32 positive striatal neurons. In parallel, the transplants contained a considerable number of immature neuroepithelial cells (doublecortin+, Sox2+, Prox-1+, ss3-tubulin+) that exhibited a pronounced migration into the surrounding host striatal tissue and considerable mitotic activity. Graft-derived astrocytes could also be found. Interestingly, the immunological host response in the grafted area showed localized increase of immunocompetent host cells within perivascular spaces without deleterious effects on engrafted cells under continuous triple immunosuppressive medication. Thus this study provides for a better understanding of the developmental processes of grafted human fetal striatal neurons in HD and, in addition, has implications for stem cell-based transplantation approaches in the CNS.

A patient with Huntington's disease and long-surviving fetal neural transplants that developed mass lesions.

Transplantation of human fetal neural tissue into adult neostriatum is an experimental therapy for Huntington's disease (HD). Here we describe a patient with HD who received ten intrastriatal human fetal neural transplants and, at one site, an autologous sural nerve co-graft. Although initially clinically stable, she developed worsening asymmetric upper motor neuron symptoms in addition to progression of HD, and ultimately died 121 months post transplantation. Eight neural transplants, up to 2.9 cm, and three ependymal cysts, up to 2.0 cm, were identified. The autologous sural nerve co-graft was found adjacent to the largest mass lesion, which, along with the ependymal cyst, exhibited pronounced mass effect on the internal capsules bilaterally. Grafts were composed of neurons and glia embedded in disorganized neuropil; robust Y chromosome labeling was present in a subset of grafts and cysts. The graft-host border was discrete, and there was no evidence of graft rejection or HD pathologic changes within donor neurons. This report, for the first time, highlights the potential for graft overgrowth in a patient receiving fetal neural transplantation.

Dopamine neurons implanted into people with Parkinson's disease survive without pathology for 14 years.

Postmortem analysis of five subjects with Parkinson's disease 9-14 years after transplantation of fetal midbrain cell suspensions revealed surviving grafts that included dopamine and serotonin neurons without pathology. These findings are important for the understanding of the etiopathogenesis of midbrain dopamine neuron degeneration and future use of cell replacement therapies.

Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease.

Fourteen years after transplantation into the striatum of an individual with Parkinson's disease, grafted nigral neurons were found to have Lewy body-like inclusions that stained positively for alpha-synuclein and ubiquitin and to have reduced immunostaining for dopamine transporter. These pathological changes suggest that Parkinson's disease is an ongoing process that can affect grafted cells in the striatum in a manner similar to host dopamine neurons in the substantia nigra. These findings have implications for cell-based therapies and for understanding the cause of Parkinson's disease.

Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation.

Two subjects with Parkinson's disease who had long-term survival of transplanted fetal mesencephalic dopaminergic neurons (11-16 years) developed alpha-synuclein-positive Lewy bodies in grafted neurons. Our observation has key implications for understanding Parkinson's pathogenesis by providing the first evidence, to our knowledge, that the disease can propagate from host to graft cells. However, available data suggest that the majority of grafted cells are functionally unimpaired after a decade, and recipients can still experience long-term symptomatic relief.

Progressive reparative gliosis in aged hosts and interferences with neural grafts in an animal model of Huntington's disease.

1. Neural transplantation in Huntington's diseased patients is currently the only approach in the treatment of this neurodegenerative disorder. The clinical trial, unfortunately, includes only a small number of patients until now, since many important questions have not been answered yet. One of them is only mild to moderate improvement of the state in most of grafted patients. 2. We examined the morphological correlates in the response to intrastriatal grafting of fragments of foetal rat ventral mesencephalic tissue 1 month after transplantation in male Wistar rats within varying durations (from 2 to 38 weeks) of experimentally induced neurodegenerative process of the striatum (used as a model of Huntington's disease). Our goal was to determine the impact of advanced striatal damage and gliosis on the graft viability and host-graft integration. 3. The findings can be summarized as follows: The progressive reactive gliosis, which is not able to compensate continual reduction of the grey matter leading to an extensive atrophy of the striatum in a long-term lesions, results in formation of the compact glial network. This tissue cannot be considered the suitable terrain for successful graft development and formation of host-graft interconnections. 4. The progression of irreversible morphological changes in long-lasting neurodegenerative process within the striatum can be supposed one of the important factors, which may decrease our prospect of distinct improvement after neural grafting in patients in advanced stage of Huntington's disease, who still remain the leading group in clinical trials.

Functional improvement with low-dose dopaminergic grafts in hemiparkinsonian rats.

OBJECTIVE: The beneficial functional effects of neural transplantation in Parkinson's disease are often directly attributed to the number of surviving dopaminergic cells within a graft. However, recent clinical trials of fetal neural transplantation suggest that a high number of dopaminergic cells may induce serious side effects. In this study, we explored the ability of low-dose dopaminergic grafts to produce functional benefits in the 6-hydroxydopamine rodent model of Parkinson's disease over a long period of observation. METHODS: Twelve rats received either 50,000 or 400,000 fetal ventral mesencephalic cells implanted into the striatum. Rotational behavior was assessed after the lesion and at 3, 6, 9, and 12 weeks after transplantation. Twelve weeks after transplantation, animals were perfused, and microtome sections were stained for tyrosine hydroxylase, glial fibrillary acidic protein, heat-shock protein 27, and vimentin. RESULTS: The low-dose group had a three-fold increase in tyrosine hydroxylase-positive cell survival rate compared with the high-dose group rate. The low-dose group also had a mean cell diameter significantly higher than the high-dose group. There was no significant difference between groups in fiber density; however, a higher percentage of longer fibers was encountered in the low-dose group. The low-dose group had a lower degree of trauma in the striatum, as assessed by optical density scores from glial fibrillary acidic protein, heat-shock protein 27, and vimentin staining. There was significant improvement in rotational behavior in the high-dose group at 3 weeks after transplantation, whereas the rotational behavior normalized in the low-dose group at 6 weeks after grafting. There was no significant difference in rotational behavior scores between groups at 6 weeks after grafting. CONCLUSION: This study demonstrates that over time, a low-dose dopaminergic graft has the capability of eliciting the same functional effect as a high-dose graft. Furthermore, low-dose grafts may increase graft survival, fiber outgrowth, and dopamine production and decrease trauma to the brain.

The future of cell-based transplantation therapies for neurodegenerative disorders.

Parkinson's disease is a common neurodegenerative disease with a lifetime incidence of 2.5% and a prevalence of at least 2% in individuals over 70 years old. Patients can be effectively treated with drugs that target the dopaminergic nigro-striatal pathway, but over time the efficacy of these medications is limited by the development of profound motor fluctuations and dyskinesias. This has prompted the search for alternative treatments, including the use of cell replacement therapies. Over the last decade, human fetal nigral transplants have demonstrated that dopaminergic neurons can survive and provide clinical benefit for patients with Parkinson's disease. However, there are clearly ethical concerns and a limit to the supply of this tissue as well as more recently anxieties over side effects. As a result, alternative sources of tissue have been investigated, and one such source are stem cells, which provide an attractive renewable tissue supply. In this review, we will discuss the current state-of-the-art and the characteristics of Parkinson's disease that increase its attraction as a target of stem cell therapy against results of current clinical trials using fetal neural grafts. Then we will discuss the various types and sources of stem cells, and some early transplantation results in animal models of Parkinson's disease. Finally we will discuss the prospect of using stem cells to deliver drugs and neurotrophic factors involved in neuroprotective and neuroreparative strategies in Parkinson's disease and other neurodegenerative conditions.

Motor impairment in normal aging, clinically possible Parkinson's disease, and clinically probable Parkinson's disease: longitudinal evaluation of a cohort of prospective brain donors.

This study presents data on the antemortem evaluations of a cohort of individuals registered in a brain donation program. Clinical evaluation determined that many individuals were unaware they had clinical signs of Parkinson's disease (PD) (rest tremor, bradykinesia, rigidity). Quantitative motor testing (timed tapping test and Purdue pegboard test) revealed a graded reduction in performance in those clinically found to have clinically possible and clinically probable PD. Longitudinal examinations over 4 years revealed some individuals progressed from control to clinically possible PD and clinically possible PD to clinically probable PD. This study underscores the importance of longitudinal antemortem testing of prospective brain donors as well as the potential value of quantitative motor testing.

Chronic gliosis triggers Alzheimer's disease-like processing of amyloid precursor protein.

Alzheimer's disease is a progressively dementing illness characterized by the extracellular accumulation and deposition of beta-amyloid. Early onset Alzheimer's disease is linked to mutations in three genes, all of which lead to increased beta-amyloid production. Inflammatory changes and gliosis may also play a role in the disease process, but the importance of these reactive events remains unclear. We recently reported that chronic cortical gliosis in heterotopic fetal rat cortical transplants is associated with significant changes in the levels of some of the proteins implicated in the pathogenesis of Alzheimer's disease. Because rodent beta-amyloid does not form extracellular amyloid deposits, we have now extended this model of chronic cortical gliosis to transgenic mice expressing the Swedish mutant form of human amyloid precursor protein. In addition, apolipoprotein E knockout mice were used to elucidate the role of this protein in reactive gliosis. The expression of mutant and murine proteins was assayed 6 or 10 months after transplantation using immunohistochemical and western blot methods. Heterotopic transplantation of fetal cortex onto the midbrain of neonatal mice consistently resulted in reactive gliosis, independent of apolipoprotein E status. In contrast, in homotopic cortex-to-cortex grafts there was little alteration in glial reactivity, a result similar to that obtained previously in rats. By 10 months post-transplantation the level of presenilin-1 expression was lower in heterotopic grafts than in host cortex and there was increased expression of transgenic amyloid precursor protein, but only in the gliotic cortex-to-midbrain grafts. Most importantly, increased levels of beta-amyloid, and particularly its precursor, C-99, were selectively found in these heterotopic transplants. Our results show that chronic gliosis is associated with altered processing of the amyloid precursor protein in vivo and thus may initiate or exacerbate pathological changes associated with Alzheimer's disease.

Simultaneous inhibition of B7 and LFA-1 signaling prevents rejection of discordant neural xenografts in mice lacking CD40L.

Transplantation of embryonic human neural tissue can restore dopamine neurotransmission and improve neurological function in patients with Parkinson's disease. Logistical and ethical factors limit the availability of human embryonic allogeneic tissue. Embryonic xenogeneic neural tissue from porcine donors is an alternative form of donor tissue, but effective immunomodulatory techniques are warranted for neural xenotransplantation to become clinically feasible. We transplanted embryonic porcine ventral mesencephalic tissue into the brains of adult untreated C57BL/6 mice, untreated CD40L-/-mice and CD40L-/-mice that received injections of anti-LFA-1, CTLA41g or both compounds. Double-treated CD40L-/-mice had large grafts with high numbers of dopaminergic neurons 4 wk after transplantation. The grafts were completely devoid of lymphocytes, macrophages and activated microglia. Untreated C57BL/6 mice had rejected their grafts. Untreated CD40L-/-mice and CD40L-/-mice treated with monotherapy of anti-LFA-1 or CTLA41g had smaller grafts and more microglial and lymphocytic infiltration than double-treated CD40L-/-mice. We conclude that immunomodulation with concomitant inhibition of LFA-1 and B7 signaling in the perioperative period in CD40L-/-mice prevented the rejection of discordant neural xenografts. The treatment most likely reduced antigen presenting capacity and interfered with the costimulatory signaling needed for T cell activation to occur.

Xenotransplantation for brain repair: reduction of porcine donor tissue immunogenicity by treatment with anti-Gal antibodies and complement.

BACKGROUND: Transplantation of embryonic neural tissue is a potential treatment for Parkinson's disease. Because human donor material is in short supply, porcine xenografts are considered a useful alternative. Current immunosuppressive therapies fail, however, to protect intracerebral neural xenografts from host CD4 T lymphocytes. To reduce the immunogenicity of porcine donor tissue, we attempted to remove microglial cells with antibodies against the alpha-galactosyl epitope (Galalpha1,3Galbeta1,4GlcNAc-R), or anti-Gal, and complement, and studied whether this pretreatment can reduce direct and indirect T-cell responses to the tissue. METHODS: Brain tissue from 27-day-old pig embryos was dissociated and treated with human anti-Gal and rabbit complement. The microglial content was analyzed by flow cytometry. [3H]thymidine incorporation in cocultures of the brain cells and purified human CD4 T cells was used to determine direct T-cell responses. Indirect T-cell responses were studied by grafting pretreated and control-pretreated (no anti-Gal) nigral tissue into the lesioned striatum of immunocompetent rats with 6-hydroxydopamine-induced hemiparkinsonism. Amphetamine-induced circling behavior was used to measure graft function. RESULTS: Anti-Gal and complement reduced the microglial content to 11-24% of control and abolished the ability of the brain cells to induce human CD4 T-cell proliferation. Pretreated nigral tissue reduced hemiparkinsonism by more than 50% in five of eight rats at some point during the 10-week follow-up. Rats receiving control-pretreated nigral tissue did not display this degree of improvement. CONCLUSIONS: Pretreatment with anti-Gal and complement can reduce the immunogenicity of porcine neural tissue, and might, therefore, be a valuable alternative or supplement to immunosuppression in neural xenotransplantation.

The effects of dexamethasone therapy on permeability, blood flow and iNOS expression in experimental glioma.

Most studies of dexamethasone (DXN) effects on experimental glioma have used doses 10-500 higher (on mg/kg basis) than those used for patients with brain tumour. The relevance of findings to patients with glioma are therefore uncertain. In order to evaluate the effects of clinical doses of DXN (0.22 mg kg(-1)day(-1)) on the pathophysiology of an experimental glioma we have treated rodents with established C6 gliomas for 3 days. The effects of therapy on local cerebral blood flow (LCBF), tumour blood flow (TBF), tumour capillary permeability (TCP), and inducible nit ric oxide synthase (iNOS) mRNA expression were evaluated. DXN caused a significant reduction in TCP (21 +/- 1.9 to 7.7 +/- 2.2 ml.gm(-1)min(-1)10(-3)) and iNOS mRNA production within and around tumour, but no significant change in either TBF or LCBF. The reduction in TCP was identical to that reported after higher doses of DXN and is probably mediated by glucocorticoid receptors. Further in vivo stud ies using either behavioural or neuropathological paradigms in rodents with established cerebral glioma should be treated with similar doses of DXN to optimise clinical relevance.

Delta opioid peptide augments functional effects and intrastriatal graft survival of rat fetal ventral mesencephalic cells.

Delta enkephalin analogue [D-Ala(2),D-Leu(5)]enkephalin (DADLE) has been shown to protect dopamine transporters from methamphetamine-induced neurotoxicity. In the present study, we demonstrate that exposure of embryonic ventral mesencephalic cells to DADLE (0.01 g/ml), prior to intrastriatal transplantation, enhanced functional recovery and graft survival in 6-hydroxydopamine-induced hemiparkinsonian rats. At 6 and 8 weeks posttransplantation, animals that received DADLE-treated cell grafts exhibited significantly higher (near normal) spontaneous locomotor behaviors, as well as trends of greater reversal of motor asymmetrical behaviors compared with animals that received nontreated cell grafts. Histological examination revealed that animals transplanted with DADLE-treated cell grafts exhibited about twice the number of surviving tyrosine hydroxylase-immunoreactive grafted neurons compared with those animals that received nontreated cell grafts. These results suggest that DADLE should be considered as an adjunctive agent for neural transplantation therapy in Parkinson's disease.

Prolonged postlesion transplantation delay adversely influences survival of both homotopic and heterotopic fetal hippocampal cell grafts in Kainate-lesioned CA3 region of adult hippocampus.

Fetal hippocampal CA3 cell grafts exhibit dramatically enhanced survival when transplanted at an early postlesion delay of 4 days into the lesioned CA3 region of adult hippocampus. However, survival of these homotopic grafts following placement at late postlesion time points when the host milieu is considerably less receptive to grafts is unknown. We hypothesize that an extended postlesion delay at the time of grafting will lead to significant diminution in cell survival of both homotopic and heterotopic fetal transplants grafted to lesioned adult CNS. We quantitatively investigated absolute cell survival of 5'-bromodeoxyuridine-labeled fetal hippocampal CA3 and CA1 cell grafts, following transplantation into the lesioned CA3 region of adult rat hippocampus, at a delay of 45 days after a unilateral intracerebroventricular administration of kainic acid (KA). Survival of these grafts was also analyzed in intact CA3 of the hippocampus contralateral to KA administration for comparison. In lesioned CA3 region, CA3 (homotopic) and CA1 (heterotopic) grafts exhibited comparable but only moderate survival, with a recovery of only 21-31% of injected cells. Cell survival in these grafts into lesioned hippocampus was similar to survival of grafts placed into the contralateral intact CA3 region. These results are in sharp contrast to increased graft survival measured following transplants performed at 4 days postlesion. In such grafts placed early, there was both a significantly higher cell survival than grafts placed into the intact CA3 region and also a characteristic differential survival based on graft cell specificity to the lesioned CA3 region (Zaman et al., Exp. Neurol., 161:535-561, 2000). Thus, the enhanced conduciveness of lesioned CA3 region for survival of homotopic CA3 cell grafts observed at 4 days postlesion wanes by 45 days postlesion to that of intact CA3 region, in spite of residual loss of CA3 neurons with the lesion. Strategies that considerably augment graft cell survival may therefore be critical for optimal integration of fetal grafts into the adult CNS at late postlesion time points.

Grafting of neural tissue in chronically injured spinal cord: influence of the donor tissue on regenerative activity.

BACKGROUND: To determine the influence of different nervous tissue grafts on the regenerative activity of chronically injured spinal cord, an experimental study examining the expression of the proliferating cell nuclear antigen (PCNA) in chronically injured spinal cord subjected to neural grafting was performed. METHODS: Three months after induced spinal cord injury, paraplegic Wistar rats were subjected to grafting of neural tissue. Grafts consisted of fetal brain cortex, fetal spinal cord, crushed adult peripheral nerve tissue, or fetal brain cortex combined with crushed adult peripheral nerve tissue. Four months later, the spinal cord was removed and the grafted zone was studied by means of immunohistochemical demonstration of PCNA. RESULTS: Different patterns of PCNA expression were recorded in the different experimental groups. PCNA-immunostained cells in injured spinal cord tissue, mainly ependymal cells and astrocytes, increased when co-transplantation of fetal brain cortex and crushed adult peripheral nerve tissue was used, in comparison to other neural donor tissues. In the grafted tissue, proliferative activity was greater when fetal brain cortex, alone or with peripheral nerve, was used, in comparison to the use of fetal spinal cord or adult peripheral nerve tissue. Nevertheless, the number of PCNA-positive cells does not seem to be influenced by the presence of peripheral nerve tissue in the donor tissue. CONCLUSIONS: Our present findings suggest the effectiveness of co-transplantation of peripheral nerve tissue and fetal brain tissue in attempts at spinal cord reconstruction after injury.