Ventral Mesencephalic Tissue Research Articles

Regulation of neuropeptide mRNA expression in the basal ganglia by intrastriatal and intranigral transplants in the rat Parkinson model

Previous studies have shown that intrastriatal transplants of dopamine (DA)-rich fetal ventral mesencephalic (VM) tissue can correct denervation-induced changes in the cellular expression of neuropeptide and receptor mRNAs in the rat Parkinson model. However, with the standard transplantation approach normalization of all cellular parameters has not been obtained. This may be due either to the incomplete striatal reinnervation achieved by these transplants, or to the ectopic placement of the grafts. In the present study we have used a microtransplantation approach to obtain a more complete reinnervation of the denervated striatum (20 micrograft deposits spread over the entire structure). Neurons were also implanted directly into the substantia nigra. In rats with multiple intrastriatal VM transplants the lesion-induced upregulation of mRNAs encoding for preproenkephalin (PPE), the D 2-type DA-receptor, and the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD 67) was normalized throughout the striatum, whereas the lesion-induced downregulation of preprotachykinin mRNA was unaffected. Intranigral grafts of either fetal DA-rich VM tissue or GABA-rich striatal tissue did not induce any changes in striatal neuropeptide and D 2-receptor mRNA expression despite significant behavioral improvement. Comparison of the behavioral data with levels of neuropeptide expression showed that in rats with intrastriatal VM transplants a complete normalization of striatal PPE and GAD 67 mRNA expression did not translate into a complete recovery of spontaneous motor behaviors. The results show that extensive DA reinnervation of the host striatum by multiple VM microtransplants is insufficient to obtain full recovery of all lesion-induced changes at both the cellular and the behavioral level. A full reconstruction of the nigrostriatal pathway or, alternatively, modulation of basal ganglia function by grafting in non-striatal regions may be required to further improve the functional outcome in the DA-denervated brain.

Ultrastructural characterization of dissociated embryonic ventral mesencephalic tissue treated with neuroprotectants.

Poor survival and differentiation of grafted dopamine neurons limits the application of clinical transplantation in Parkinson's disease. The survival of grafted dopamine neurons is only improved by a factor of 2-3 by adding neuroprotectants during tissue preparation. We used dye exclusion cell viability and electron microscopy to investigate the effects of the caspase inhibitor ac-YVAD-cmk and the lazaroid tirilazad mesylate on ultrastructural changes in dissociated embryonic mesencephalic cells. In addition, we examined whether the neuroprotectants selectively counteracted specific signs of neurodegeneration. Cell viability decreased significantly over time in both control and treated cell suspensions, but the number of viable cells remaining was significantly higher in tirilazad mesylate-treated cell suspensions. In control samples, the proportion of cells with an ultrastructure consistent with healthy cells decreased from 70%, immediately after dissociation, to 30% after 8 h of incubation. Similar changes were also observed in cell suspensions treated with neuroprotectants. Thus, the neuroprotectants examined did not block the development of specific morphological signs of neurodegeneration. However, when also taking into account that dead cells lysed and disappeared from each cell suspension with time, we found that the total number of remaining viable cells with healthy nuclear chromatin or intact membrane integrity was significantly higher in the tirilazad mesylate-treated group. The results indicate that tirilazad mesylate protects only a small subpopulation of embryonic mesencephalic cells from degeneration induced by mechanical trauma during tissue dissection and dissociation.

Behavioral and subthalamic effects of combining a fetal ventral mesencephalic transplant in striatum with an electrolytic lesion of the entopeduncular nucleus in the rat with a unilateral 6-OHDA lesion of substantia nigra.

Behavioral and electrophysiological methods were used to determine whether a transplant of dopamine-rich fetal tissue in striatum combined with an electrolytic lesion of the entopeduncular nucleus have additive effects in the unilaterally lesioned rat model for Parkinson's disease. The subjects were rats with the left substantia nigra lesioned with 6-hydroxydopamine (6-OHDA) and responding to systemic amphetamine with rotation toward the side of the lesion (ipsilateral rotation). The motor response to amphetamine was fractionated into six aspects, half reflecting the unilateral deafferentation in striatum and half representing those aspects of the response evoked in normal rats. After collection of baseline values, 25 rotators received a transplant of fetal ventral mesencephalic tissue in the left striatum and 20 received a transplant and, at the same time, an electrolytic lesion of the left entopeduncular nucleus. Testing for the motor response to amphetamine resumed after 4 weeks of recovery and continued at weekly intervals for 5 weeks. Upon completion of these tests, each rotator was implanted with multiple electrodes in the subthalamic nucleus. After recovery, multiunit responses to amphetamine and apomorphine were recorded from several electrodes in parallel during the motor response to the drugs. In rotators with transplant only, treatment with amphetamine evoked oral stereotypy and an attenuated ipsilateral rotation response. In rotators with combined transplant and entopeduncular lesion, ipsilateral rotation did not change or increased. Subthalamic responses to amphetamine and apomorphine were larger in rotators with combined transplant and entopeduncular lesion than in rotators with transplant alone. These findings indicate that the combination of transplant and pallidotomy in the 6-OHDA rat model for parkinsonism does not lead to additive benefits, an effect that may have been due to the nonselectivity of the electrolytic damage and/or of the lesion extending beyond the entopeduncular nucleus into the lateral hypothalamus.

Characterisation of cell damage and death in embryonic mesencephalic tissue: a study on ultrastructure, vital stains and protease activity

Dissociated embryonic ventral mesencephalic tissue is a source of dopaminergic neurones in both cell culture and neural transplantation studies. Around 90% of grafted dopaminergic neurones die within 1 week after transplantation. Little is known about when the cell death is triggered and what forms of cell death predominate. Using electron microscopy, we characterised ultrastructural changes in dissected embryonic day 14 rat mesencephalic tissue before and after tissue dissociation. In addition, cell viability was evaluated using Trypan Blue and Hoechst/Ethidium Homodimer. Several cells exhibited leaky outer membranes (permitting entry of vital stains) and ultrastructural degeneration already immediately after the mesencephalon was dissected, and before it was mechanically disrupted. After 2 h at room temperature, 90% of the remaining cells had intact outer membranes. However, when estimating cells lost acutely in the tissue dissociation, in addition to cells exhibiting condensed chromatin and organellar changes, we suggest that only around 14% of the cells initially dissected in the mesencephalic tissue pieces remained healthy after 2 h. There was a peak in calpain activity (specific cleavage of fodrin) immediately following tissue dissociation, and it subsided during the next few hours. Caspase-3 activity was initially low, but increased almost 20-fold 4 h after tissue disruption. Interestingly, extensive degradation of caspase-3 occurred already directly after dissection and was at least partly calpain-dependent. Our data suggest that, in addition to cells undergoing primary necrosis, some cells undergo apoptotic or related changes soon after tissue harvesting, and eventually undergo a secondary necrosis. In summary, embryonic mesencephalic cells exhibit multiple degenerative changes very early on in the neural transplant/tissue culture preparation protocol.

Rat nigral xenografts survive in the brain of MHC class II-, but not class I-deficient mice

We have examined the role of the indirect pathway of antigen recognition and T cells in neural xenografts rejection by using major histocompatibility complex (MHC) class II-deficient mice as xenograft recipients. Dissociated embryonic ventral mesencephalic tissue from Sprague–Dawley rats was stereotaxically injected as a cell suspension into the striatum of MHC class II-deficient adult mice as well as MHC class I-deficient and wild-type mice as controls. All of the MHC class II-deficient mice had surviving grafts in the striatum 4 weeks post-grafting. In contrast, only a few of the MHC class I-deficient mice exhibited very small grafts and none of the wild-type mice had any surviving grafts. The mean number of surviving transplanted dopamine neurons in the MHC class II-deficient group was significantly larger than that observed in the other two groups. Moderate levels of MHC class I antigen expression were seen in the transplantation sites of some animals in the MHC class II-deficient group. No helper or cytotoxic T cells were observed infiltrating into the graft sites of this group. However, there were markedly increased levels of expression of MHC class I and class II antigens, and a number of T cells infiltrating in the graft sites in both the MHC class I-deficient and wild-type groups. These results show that rat embryonic nigral tissue can survive transplantation in the brain of the MHC class II-deficient mice for at least 4 weeks without any overt signs of rejection, suggesting that the indirect pathway of foreign antigen recognition mediated by host MHC class II molecules and helper T cells plays an important role in the rejection responses to intracerebral xenografts.

The release of excitatory amino acids, dopamine, and potassium following transplantation of embryonic mesencephalic dopaminergic grafts to the rat striatum, and their effects on dopaminergic neuronal survival in vitro.

A major limitation to the effectiveness of grafts of fetal ventral mesencephalic tissue for parkinsonism is that about 90-95% of grafted dopaminergic neurones die. In rats, many of the cells are dead within 1 day and most cell death is complete within 1 week. Our previous results suggest that a major cause of this cell death is the release of toxins from the injured CNS tissue surrounding the graft, and that many of these toxins have dissipated within 1 h of inserting the grafting cannula. In the present experiments we measured the change over time in the concentration of several potential toxins around an acutely implanted grafting cannula. We also measured the additional effect of injecting suspensions of embryonic mesencephalon, latex microspheres, or vehicle on these concentrations. Measurements of glutamate, aspartate, and dopamine by microdialysis showed elevated levels during the first 20-60 min, which then declined to baseline. In the first 20 min glutamate levels were 10.7 times, aspartate levels 5 times, and dopamine levels 24.3 times baseline. Potassium levels increased to a peak of 33 +/- 10.6 mM 4-5 min after cannula insertion, returning to baseline of <5 mM by 30 min. Injection of cell suspension, latex microspheres, or vehicle had no significant effect on these levels. We then assayed the effect of high concentrations of glutamate, aspartate, dopamine, and potassium on dopaminergic neuronal survival in E14 ventral mesencephalic cultures. In monolayer cultures only dopamine at 200 microM showed toxicity. In three-dimensional cultures only the combination of raised potassium, dopamine, glutamate, and aspartate together decreased dopaminergic neuronal survival. We conclude that toxins other than the ones measured are the main cause of dopaminergic cell death after transplantation, or the effects of the toxins measured are enhanced by anoxia and metabolic challenges affecting newly inserted grafts.

Lipid-Mediated Glial Cell Line-Derived Neurotrophic Factor Gene Transfer to Cultured Porcine Ventral Mesencephalic Tissue

Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM tissue grafts to PD patients indicate that one reason for the poor outcome of neural transplantation is the low survival and differentiation of grafted dopaminergic neurons. To improve dopaminergic cell survival through a gene-therapeutic approach we have established and report here results of lipid-mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly increased transfection yields. Expression of human GDNF via an episomal vector could be detected by in situ hybridization and by the measuring of GDNF protein secreted into the culture medium. When compared to mock-transfected controls, VM tissue expressing recombinant GDNF contained significantly higher numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo- and xenotransplantation treatment in Parkinson'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.

A multiple target neural transplantation strategy for Parkinson's disease.

Intracerebral transplantation of embryonic ventral mesencephalic tissue is a potential treatment for patients with Parkinson's disease for whom medical management is unsatisfactory. Neural transplantation for parkinsonism has been studied experimentally in animal models of Parkinson's disease for more than two decades. These animal studies have shown significant graft survival, synapse formation, graft induced-dopamine release, and behavioural recovery in transplanted animals. Encouraged by these results, clinical programs have been initiated over the past 15 years; more than 250 patients worldwide have undergone neural transplantation. Both animal and clinical studies indicate that neural transplantation has the potential to become a valuable treatment option for Parkinson's disease. However, while many transplant recipients obtain clinically useful symptom relief, in all cases functional recovery is incomplete. Certain symptoms do not respond well to transplant therapy, and those symptoms that do typically do not resolve completely. This has spurred efforts to optimize the transplant procedure. One important approach is exploring novel methods such as multiple site transplantation. This transplantation strategy results in a more complete reinnervation of the dopaminergic circuitry that is affected in Parkinson's disease. In principle, multiple site transplantation should provide a more satisfactory resolution of symptoms. Here we review the progress made in multiple site neural transplantation for Parkinson's disease. The effects of intrastriatal, intranigral, intrasubthalamic nucleus, and intrapallidal grafts in animal models of Parkinson's disease are analysed. The current data suggest that intrastriatal grafts alone are inadequate to promote complete functional recovery. A multiple target strategy may restore dopaminergic input to affected basal ganglia nuclei and improve outcomes of neural transplantation in Parkinson's disease.

Generation of tyrosine hydroxylase-immunoreactive neurons in ventral mesencephalic tissue of Nurr1 deficient mice

Nurr1 is an orphan nuclear receptor belonging to the family of evolutionary conserved steroid/thyroid hormone receptors. It has been shown that Nurr1 is required for development of ventral mesencephalic dopaminergic cells in vivo and that Nurr1 regulates the tyrosine hydroxylase (TH) gene. The aim of this study was to investigate the possibility of finding ventral mesencephalic TH-positive neurons in Nurr1 deficient tissue when developed in the presence of wild type (WT) striatum. Therefore, fetal ventral mesencephalic tissue from embryonic day (E) 9.5–10.5 fetuses from Nurr1 mutant mice was co-cultured with lateral ganglionic eminence (LGE) from WT fetuses using the ‘roller-drum’ culture technique. TH-immunohistochemistry revealed similar number of positive neurons in WT, heterozygous, and Nurr1 deficient tissue, respectively. When ventral mesencephalon, dissected from E10.5 fetuses, was cultured alone without the presence of LGE, significantly more TH-immunoreactive neurons were found in WT and Nurr1 +/− than that seen in Nurr1 −/− cultures. In single ventral mesencephalic cultures dissected from E15.5, TH-positive neurons were found in all tissue cultures derived from knockout animals. Interestingly, the formation of TH-positive nerve fiber bundles was obvious in WT cultures while not observed in cultures of knockout tissue. When ventral mesencephalon was cultured alone in serum-free medium, almost no TH-positive neurons were found in cultures of knockout tissue. The addition of the growth factors epidermal growth factor and fibroblast growth factor-8 did not induce TH-immunoreactivity in serum-free Nurr1 −/− tissue cultures. In conclusion, TH-positive neurons may be generated in ventral mesencephalic tissue of Nurr1 deficient mice, suggesting that Nurr1 is not required for TH gene expression in ventral midbrain in vitro.

Porcine Neural Xenografts in Rats and Mice: Donor Tissue Development and Characteristics of Rejection

Embryonic ventral mesencephalic tissue from the pig is a potential alternative donor tissue for neural transplantation to Parkinson's disease patients. For stable graft survival, the host immune response has to be prevented. This study was performed in order to analyze the mechanisms and dynamics of neural xenograft rejection, as well as neurobiological properties of the donor tissue. Adult normal mice and rats, and cyclosporin A-treated rats, received intrastriatal transplants of dissociated embryonic ventral mesencephalic pig tissue that was 27 or 29 embryonic days of age (E27 and E29). The animals were perfused at 2, 4, 6, and 12 weeks after grafting and the brains were processed for immunohistochemistry of dopaminergic (tyrosine hydroxylase positive) neurons, CD4+ and CD8+ lymphocytes, natural killer cells, macrophages, microglia, and astrocytes. Thirty-five rats received daily injections of BrdU for 5 consecutive days at different time points after transplantation and were perfused at 6 weeks. These animals were analyzed for proliferation of cells in the donor tissue, both in healthy and in rejecting grafts. No tyrosine hydroxylase-positive cells proliferated after grafting. Our results demonstrated that E27 was superior to E29 donor tissue for neurobiological reasons. Cyclosporin A immunosuppression was protective only during the first weeks and failed to protect the grafts in a long-term perspective. Grafts in mice were invariably rejected between 2 and 4 weeks after transplantation, while occasional grafts in untreated rats survived up to 12 weeks without signs of an ongoing rejection process. CD8+ lymphocytes and microglia cells are most likely important effector cells in the late, cyclosporin A-resistant rejection process.

Astrocytes from Cerebral Cortex or Striatum Attract Adult Host Serotoninergic Axons into Intrastriatal Ventral Mesencephalic Co-Grafts

The identification of axon growth inhibitory molecules offers new hopes for repair of the injured CNS. However, the navigational ability of adult CNS axons and the guidance cues they can recognize are still essentially unknown. Astrocytes may express guidance molecules and are known to have different regional phenotypes. To evaluate their influence on the affinity of adult serotoninergic (5-HT) axons for a projection target, we co-implanted astrocytes from the neonatal striatum, cortex, or ventral mesencephalon together with fetal ventral mesencephalic tissue into the striatum of adult rats. Two months after surgery, quantification after in vitro 5-[1,2-(3)H]serotonin ([(3)H]5-HT) uptake and autoradiography showed that ventral mesencephalic grafts with co-grafted cortical or striatal astrocytes were four times and three times, respectively, more densely innervated by host 5-HT axons than control ventral mesencephalic grafts with or without co-grafted ventral mesencephalic astrocytes. Immunohistochemistry for glial fibrillary acidic protein, vimentin, or chondroitin-sulfate proteoglycans revealed no qualitative or quantitative differences in host astroglial scar or production of inhibitory molecules that could explain these differences in 5-HT innervation. These results demonstrate that astrocytes grown in culture from different brain regions have the potential to influence the growth and maintenance of adult 5-HT axons in a graft of neural tissue from another brain region. It should now be feasible to identify the molecules expressed by cultured cortical or striatal, but not by ventral mesencephalic, astrocytes that have these tropic actions on 5-HT axons of the neostriatum.

Neuroprotection of Grafted Neurons with a GDNF/Caspase Inhibitor Cocktail

Transplantation of fetal ventral mesencephalic (VM) tissue shows great promise as an experimental therapy for patients with Parkinson's disease. However, cell survival in brain tissue grafts is poor, with survival rates of only 5–15%. We have utilized a combination of the caspase inhibitor bocaspartyl (OMe)-fluoromethylketone (BOC-ASP-CH2F) and glial cell line-derived neurotrophic factor (GDNF) to enhance survival of grafted dopamine neurons. The VM tissue was dissected from embryonic day 13–15 rat fetuses, incubated in different doses of BOC-ASP-CH2F and GDNF, and transplanted to the anterior chamber of the eye of adult rats. Growth of the tissue was assessed through the translucent cornea. Doses of 50 and 100 micromolar of the general caspase inhibitor appeared to have detrimental effects on mesencephalic tissue, while 20 micromolar had beneficial effects on overall transplant growth. A combination of the caspase inhibitor and GDNF appeared to have more prominent effects on cell survival as well as dopaminergic fiber density than either agent by itself. The transplants doubled in size when they were treated with a combination of BOC-ASP-CH2F and GDNF, and cell death markers were significantly reduced at both 48 h and 4–6 days postgrafting. This is, to our knowledge, the first combined approach using apoptotic blockers with trophic factors, and demonstrates a viable strategy for protection of developing neurons, since several different aspects of graft function may be addressed simultaneously.

The development of dopamine overflow from foetal nigral grafts in the intact rat striatum and their influence on contralateral striatal dopamine overflow

In this study, cell suspensions of foetal rat ventral mesencephalic dopaminergic tissue were grafted to the intact (non-lesioned) striatum of adult rats. Differential pulse voltammetry at carbon-fibre micro electrodes (12 μm diameter) was employed to first, monitor the development of dopamine overflow over a 20 week period within the grafts and secondly, their influence on contralateral striatal dopamine overflow. At 8 and 20 weeks, animals were pre-treated with pargyline and both striata were monitored for dopamine overflow for 90 min following d-amphetamine administration. Amphetamine led to a significant increase in dopamine overflow in both the grafted striatum and the contralateral striatum. The time course of dopamine overflow in both the grafted striatum and the striatum contralateral to the graft was similar in all groups of animals. Although the actual concentration of dopamine measured in 20 week old grafts was more (≈21%) than that measured in 8 week old grafts, there was no significant difference between the two time points. The concentration of dopamine measured in the striatum contralateral to 8 week old grafts was significantly lower (≈43%) than that measured in the striatum of a normal control rats. There was no significant difference between the concentration of dopamine measured in the striatum contralateral to 20 week old grafts and normal control rats. In conclusion, dopamine overflow from a ventral mesencephalic graft does not change significantly between 8 and 20 weeks following grafting. However, the grafted tissue causes a decrease of d-amphetamine-induced dopamine overflow in the contralateral side 8 weeks following grafting, which is restored 12 weeks later.

Evidence for Target-Specific Nerve Fiber Outgrowth from Subpopulations of Grafted Dopaminergic Neurons: A Retrograde Tracing Study Using in Oculo and Intracranial Grafting

Efforts have been made to counteract the symptoms of Parkinson's disease by substituting the loss of dopaminergic neurons with fetal ventral mesencephalic grafts. One of the postulated limiting factors in this treatment is the relatively poor cell survival and limited graft-derived fiber outgrowth. Recent results documenting enhanced survival of grafted dopaminergic neurons showed no positive correlation to enhanced innervation of the striatal target. Therefore this study was undertaken to investigate whether all surviving grafted dopaminergic neurons projected to the striatal target. Hence, fetal ventral mesencephalic tissue was implanted adjacent to mature versus immature striatal tissue using in oculo and intraventricular grafting techniques. In in oculo grafting, fetal ventral mesencephalon was implanted simultaneously with fetal lateral ganglionic eminence (immature striatal target) or to already matured striatal in oculo grafts (mature striatal target). Furthermore, fetal ventral mesencephalon was implanted into the lateral ventricle adjacent to mature dopamine-depleted striatum. The retrograde tracer fluorogold was injected into the striatal portion of the in oculo cografts and into reinnervated areas of the adult brain. Immunohistochemistry revealed that a significantly larger proportion of tyrosine hydroxylase-positive neurons in the ventral mesencephalic graft was innervating in oculo immature striatal tissue, and hence was fluorogold-positive, in comparison with the number of tyrosine hydroxylase-positive neurons innervating mature striatal tissue. Moreover, intracranial transplantations showed that tyrosine hydroxylase-positive neurons were distributed within the grafts in dense clusters of cells. In most clusters tyrosine hydroxylase-positive cells were fluorogold-negative but calbindin-positive. In a few tyrosine hydroxylase-positive cell clusters, neurons were coexpressing fluorogold but were calbindin-negative. In conclusion, significantly more dopamine neurons projected to immature than to mature striatal tissue and thus, a subpopulation of grafted dopaminergic neurons was not projecting into adult striatum. Thus, the results from this study show that further attempts to enhance survival of grafted dopamine neurons in purpose to enhance graft-derived fiber outgrowth and efficacy should also consider different subtypes of dopamine neurons.

Effects of immunosuppressive treatment on host responses against intracerebral porcine neural tissue xenografts in rats.

Embryonic xenogeneic neural tissue is an alternative for transplantation in Parkinson's disease, but immune responses limit the application. The aims of this study were to enhance the in vitro viability rates by donor tissue pretreatment; to compare the efficacy of cyclosporine A (CsA) and tacrolimus (FK) in inhibiting xenograft rejection in rats; to evaluate additional inductive therapy with prednisolone (PRE) or mycophenolate mofetil (MMF). Tirilazad (a lipid peroxidase inhibitor) or FK and acYVAD-cmk (a caspase inhibitor), were added to embryonic porcine ventral mesencephalic tissue and viability was assessed in vitro. Tirilazad-treated tissue was grafted to the striatum of rats that were either left untreated or immunosuppressed with FK (1 mg/kg) or CsA (15 mg/kg) alone or in combination with a 2-week PRE (20 mg/kg) or MMF (40 mg/kg) induction course. Xenograft survival and host responses were determined using immunohistochemistry. Pretreatment with tirilazad enhanced tissue survival in vitro. After transplantation into untreated controls, there was no graft survival at twelve weeks. Neural cell counts were significantly improved in immunosuppressed recipients, but there were no differences between the treatment groups. Additional inductive treatment reduced the infiltration with CD4+ and CD8+ cells, and macrophage infiltration was reduced compared with animals given CsA or FK alone. Pretreatment of the donor tissue with free-radical scavengers reduces cell loss caused by tissue trauma. Porcine neural tissue xenografts survive significantly better in animals immunosuppressed with either FK or CsA. Additional inductive treatment with PRE or MMF reduced the infiltration of host cells into the xenografts.

Inhibitors of p38 MAP kinase increase the survival of transplanted dopamine neurons

Fetal cell transplantation therapies are being developed for the treatment of a number of neurodegenerative disorders including Parkinson’s disease [10–12,21,22,24,36,43]. Massive apoptotic cell death is a major limiting factor for the success of neurotransplantation. We have explored a novel protein kinase pathway for its role in apoptosis of dopamine neurons. We have discovered that inhibitors of p38 MAP kinase (the pyridinyl imidazole compounds: PD169316, SB203580, and SB202190) improve survival of rat dopamine neurons in vitro and after transplantation into hemiparkinsonian rats. In embryonic rat ventral mesencephalic cultures, serum withdrawal led to 80% loss of dopamine neurons due to increased apoptosis. Incubation of the cultures with p38 MAP kinase inhibitors at the time of serum withdrawal prevented dopaminergic cell death by inhibiting apoptosis. In the hemiparkinsonian rat, preincubation of ventral mesencephalic tissue with PD169316 prior to transplantation accelerated behavioral recovery and doubled the survival of transplanted dopamine neurons. We conclude that inhibitors of stress-activated protein kinases improve the outcome of cell transplantation by preventing apoptosis of neurons after grafting.

Fetal intra-nigral ventral mesencephalon and kidney tissue bridge transplantation restores the nigrostriatal dopamine pathway in hemi-parkinsonian rats

We have previously demonstrated that intranigral transplantation of fetal ventral mesencephalic (VM) tissue and nigrostriatal administration of glial cell line-derived neurotrophic factor (GDNF) restores striatal dopamine input in hemiparkinsonian rats. Since it has been found that GDNF is highly expressed in fetal kidney, we examined the possibility that fetal kidney tissue may provide trophic support, similar to GDNF, to an intranigral dopamine (DA) transplant and restore the nigrostriatal pathway. Adult Sprague–Dawley rats were anesthetized and unilaterally injected with 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. Completeness of the lesion was evaluated by measuring amphetamine-induced rotation. One month after 6-OHDA lesioning, fetal VM cells were grafted into the lesioned nigral area followed by transplantation of fetal kidney tissue or vehicle along a pathway from nigra to striatum. Animals receiving these transplants showed a significant decrease both in amphetamine-induced rotation and in postural asymmetry 1 to 3 months after grafting. Immunocytochemical studies demonstrated tyrosine hydroxylase (TH) positive fiber tracts in the lesioned striatum. Control animals that received vehicle injection after the intranigral graft or no transplantation showed no alterations in amphetamine-induced turning and no TH-positive fibers in the lesioned striatum. These results indicate that combinations of fetal nigral and kidney transplants may restore the nigrostriatal DA pathway in Parkinsonian rats. As fetal kidney contains a variety of trophic proteins, it may provide a synergistic admixture to optimally promote DA fiber outgrowth.

Cell Transplantation for the Treatment of Parkinson's Disease

Cell transplantation is an experimental therapy for Parkinson's disease (PD) and other movement disorders. Several open-label research trials have shown clinically meaningful improvement in parkinsonian signs and symptoms after striatal transplantation of allogeneic fetal ventral mesencephalic (FVM) tissue. However, ethical concerns, variability in surgical techniques, and reports of unusual late complications in a few patients in a clinical trial have limited the use of allogeneic FVM tissue to a few research centers. Research into alternative cell sources such as porcine FVM and allogeneic retinal pigment epithelial cells has shown promising results in preclinical trials, and they are currently being tested in clinical trials. Novel strategies to improve cell survival and to avoid immune rejection of transplants show promising results in preclinical trials. This article focuses on these recent advances and compares the potential clinical utility of these emerging cell therapies for the treatment of advanced PD.

Nonviral Glial Cell-Derived Neurotrophic Factor Gene Transfer Enhances Survival of Cultured Dopaminergic Neurons and Improves Their Function after Transplantation in a Rat Model of Parkinson’s Disease

Transplantation of dopaminergic fetal mesencephalic tissue into the striatum is currently being developed for treatment of patients with advanced Parkinson's disease. Ethical concerns regarding the use of human fetal tissue, and the limited availability as well as poor survival and differentiation of dopaminergic neurons after transplantation have reduced the extent and outcome of this approach so far. With the purpose of finding means to increase the yield of dopaminergic neurons in transplants, and to reduce the amount of fetal tissue needed for each transplanted patient, we transfected rat fetal ventral mesencephalic (VM) tissue grown as organotypic free-floating roller tube (FFRT) cultures with a vector encoding human glial cell-derived neurotrophic factor (hGDNF). For transfer of an episomal expression vector (pRep7-GDNF8) a nonviral, nonliposomal cationic transfection technique was applied and optimized. Recombinant hGDNF expression resulted in a higher number of TH-positive neurons in the cultures as measured 6 days after transfection. Ventral mesencephalic cultures expressing hGDNF were then grafted into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Grafting of genetically modified VM cultures resulted in earlier functional recovery compared with grafting nontransfected cultures. We conclude that organotypic free-floating roller tube cultures can be successfully transfected to produce hGDNF with effects on TH-expressing neurons in vitro and functional effects after grafting in a rat Parkinson's disease model.