Grafted Dopamine Neurons Research Articles

Additive Effects of Caspase Inhibitor and Lazaroid on the Survival of Transplanted Rat and Human Embryonic Dopamine Neurons

Major practical constraints on neural grafting in Parkinson's disease are the shortage of human donor tissue and the great loss of dopamine neurons during the grafting procedure. The vast majority of implanted embryonic dopamine neurons are believed to die within a few days of transplantation surgery, at least in part through apoptosis. We have previously found that survival of nigral grafts in rodents can be significantly augmented by pretreatment with the caspase inhibitor Ac-YVAD-cmk or by lazaroids (lipid peroxidation inhibitors). We now report that pretreatment with the caspase inhibitor Ac-DEVD-cmk, but not z-VAD-fmk, results in a significantly improved survival of transplanted dopamine neurons of similar magnitude to that achieved in this study using Ac-YVAD-cmk (both 220–230% of control). In addition, we found that treatment of the graft tissue with tirilazad mesylate (a lazaroid allowed for clinical use) almost doubled the survival of grafted dopamine neurons. When Ac-YVAD-cmk and tirilazad mesylate treatments were combined, the number of surviving dopamine neurons increased significantly further to 280% of control. Importantly, the same combination of neuroprotectants enhanced the survival of human dopamine neurons xenotransplanted to immunosuppressed rats (to 240% of control). In conclusion, these results suggest that combining treatments that counteract oxidative stress and caspase activation is a valuable strategy to enhance nigral graft survival that should be considered for clinical application.

Improving the survival of grafted dopaminergic neurons: a review over current approaches.

Neural transplantation is developing into a therapeutic alternative in Parkinson's disease. A major limiting factor is that only 3-20% of grafted dopamine neurons survive the procedure. Recent advances regarding how and when the neurons die indicate that events preceding actual tissue implantation and during the first week thereafter are crucial, and that apoptosis plays a pivotal role. Triggers that may initiate neuronal death in grafts include donor tissue hypoxia and hypoglycemia, mechanical trauma, free radicals, growth factor deprivation, and excessive extracellular concentrations of excitatory amino acids in the host brain. Four distinct phases during grafting that can involve cell death have been identified: retrieval of the embryo; dissection and preparation of the donor tissue; implantation procedure followed by the immediate period after graft injection; and later stages of graft maturation. During these phases, cell death processes involving free radicals and caspase activation (leading to apoptosis) may be triggered, possibly involving an increase in intracellular calcium. We review different approaches that reduce cell death and increase survival of grafted neurons, typically by a factor of 2-4. For example, changes in transplantation procedure such as improved media and implantation technique can be beneficial. Calcium channel antagonists such as nimodipine and flunarizine improve nigral graft survival. Agents that counteract oxidative stress and its consequences, such as superoxide dismutase overexpression, and lazaroids can significantly increase the survival of transplanted dopamine neurons. Also, the inhibition of apoptosis by a caspase inhibitor has marked positive effects. Finally, basic fibroblast growth factor and members of the transforming growth factor-beta superfamily, such as glial cell line-derived neurotrophic factor, significantly improve the outcome of nigral transplants. These recent advances provide hope for improved survival of transplanted neurons in patients with Parkinson's disease, reducing the need for human embryonic donor tissue and increasing the likelihood of a successful outcome.

Chapter 10 Improving the survival of grafted embryonic dopamine neurons in rodent models of Parkinson's disease

Publisher Summary This chapter describes recent animal experiments suggesting that the concept of “one donor–one recipient” may not be impossible to reach within the foreseeable future. Rapid advances have provided more information about how to more accurately estimate graft survival, and when and why grafted dopamine (DA) neurons die. In addition, new data focuses on the cellular mechanisms that may underlie the excessive death of DA neurons in nigral implants. The chapter describes these experiments and highlights the studies examining treatments that result in enhanced survival of grafted DA neurons. Recently, the effect of the antioxidant tirilazad mesylate has been evaluated during the preparation and in the final dissociated mesencephalic cell suspension for grafting. Tirilazad mesylate and Ac-YVADcmk, when present alone, increased the survival of grafted DA neurons to around 190% and 220% of control values, respectively (Hansson et al., 2000). In contrast, the combination of both compounds increased the number of surviving DA neurons significantly more, to around 290% of control values. These results show that the combination of antioxidants and caspase inhibitors represents a valuable strategy to improve the survival of grafted DA neurons.

Dopamine cells in nigral grafts differentiate prior to implantation.

The yield of surviving dopamine cells in nigral grafts is typically low. It is unclear whether the dopamine neurons that do survive are postmitotic at the time of implantation, or are precursor cells that differentiate into dopamine neurons following transplantation in the host brain. We have therefore compared the survival of dopamine neurons in grafts that have been labelled with BrdU at different times prior to or following implantation in order to identify those cells that undergo final cell division at each stage of the procedure. Seven groups of rats were prepared with unilateral nigrostriatal lesions. Three groups received nigral grafts derived from E14 embryos labelled with BrdU in utero on either E12, E13 or E14 days of embryonic age (the E14 injection made 2 h prior to preparation of the graft cell suspension). Three further groups received nigral grafts from untreated E14 embryos, and then dividing cells within the grafts were labelled by injection of BrdU into the host lateral ventricle, 2 h, 1 day or 2 days after implantation (equivalent to E14, E15 and E16 days of embryonic age). The control group received standard (unlabelled) E14 grafts. Five weeks after the transplantation surgery, the host brains were processed using double immunohistochemical techniques to detect tyrosine hydroxylase (TH)-positive neurons which had incorporated BrdU. In the grafts labelled with BrdU prior to implantation, there was an increasing proportion of double-labelled cells (out of the total TH-positive cells surviving in the grafts) with birth dates on E12, E13 and E14 (1%, 12% and 10% per day, respectively). By contrast, grafts labelled following implantation, although containing many dividing neurons, had very few of these BrdU-labelled cells expressing a dopaminergic phenotype; < 1% surviving TH-positive cells were double-labelled from the 2 h post-transplant injection, and < 0.1% from each subsequent injection. This suggests not only that the great majority of TH-positive neurons in nigral grafts were already differentiated at the time of implantation, but also that transplantation of E14 ventral mesencephalic tissue either kills dopaminergic precursors or (more likely in our opinion) prevents their differentiation into a dopaminergic phenotype. Precursor cells that would differentiate into dopaminergic neurons beyond E14 if left in situ in the intact ventral mesencephalon do not readily differentiate into mature dopamine neurons following transplantation. If we are to enhance yields of functional dopamine-rich transplants, then we must identify strategies both to protect predifferentiated dopamine neurons in the grafts and to promote differentiation of a dopaminergic phenotype in precursor cells that continue to divide within the grafts following transplantation into an adult host environment.

Patterns of Cell Death and Dopaminergic Neuron Survival in Intrastriatal Nigral Grafts

Previous studies indicate that 80–95% of grafted dopamine neurons die following implantation of embryonic ventral mesencephalic tissue into the striatum. It is believed that the majority die within the first 1–3 weeks after surgery. The aim of this study was to study when and where the implanted neurons die, using the novel fluorescent stain Fluoro-Jade. Fluoro-Jade has recently been shown to stain cell bodies, dendrites, axons, and terminals of degenerating neurons. We transplanted dissociated ventral mesencephalic tissue from embryonic day 14 rat embryos into intact adult rat striatum. After perfusion and sectioning of the implanted rat brains, the number and distribution of Fluoro-Jade and tyrosine hydroxylase-positive neurons were evaluated at 6, 10, 14, and 42 days posttransplantation. Intensely Fluoro-Jade stained neurons were numerous in the grafts at 6 and 10 days after graft surgery; appeared in reduced numbers at 14 days; and had disappeared by the 42-day time point. The number of surviving tyrosine hydroxylase-positive, dopaminergic neurons in the grafts did not change between 6 and 42 days and the low survival rate confirmed that over 90% of these neurons had died during the first week. Assessment of the distribution of neurons positive for Fluoro-Jade or tyrosine hydroxylase revealed higher numbers of neurons stained for these markers located at the periphery than the center of the grafts, and this pattern did not change over time. This study indicates that transplanted neurons continue to die up to 14 days after grafting. Since the majority of transplanted tyrosine hydroxylase-positive neurons most probably die before 6 days after transplantation, neuroprotective strategies should primarily focus on the transplantation procedure and the first week after implantation.

Diminished Viability, Growth, and Behavioral Efficacy of Fetal Dopamine Neuron Grafts in Aging Rats with Long-Term Dopamine Depletion: An Argument for Neurotrophic Supplementation

We examined the behavioral and morphological correlates of the response to a single intrastriatal dispersed cell graft of fetal rat ventral mesencephalic tissue in male Fischer-344 rats of varying age (4, 17, and 24-26 months old) and history of mesostriatal dopamine (DA) depletion (1 or 14 months). Our goal was to determine the impact of advancing age and duration of DA depletion in the host on DA graft viability and function. The findings can be summarized as follows. (1) Fetal DA neuron grafts that were effective in completely ameliorating amphetamine-induced rotational behavior in young rats with short-term lesions were virtually without effect in aged rats with long-term lesions. Middle-aged rats with long-term lesions responded to these grafts with partial behavioral recovery. (2) Age of the host at the time of transplantation, and not duration of DA depletion, was the primary determinant of response to DA grafts. (3) Diminished efficacy of grafts in lesioned aging rats was related to decreased survival and neurite extension of transplanted DA neurons. (4) Co-grafts of DA neurons with Schwann cells as a source of neurotrophic support improved the behavioral outcome of grafts in aged lesioned rats. These findings support the view that the DA-depleted striatum of aged rats is an impoverished environment for survival, growth, and function of DA grafts. Consistent with this view, local supplementation of the neurotrophic environment of grafted DA neurons with products of co-grafted Schwann cells, a demonstrated source of neurotrophic activity for embryonic DA neurons, improved graft outcome.

Effects of surgical anaesthesia on the viability of nigral grafts in the rat striatum

Only a small proportion of dopamine neurons in nigral grafts typically survive transplantation into the adult striatum. Since many anaesthetics reduce blood flow and disturb a variety of brain metabolites, surgical anaesthesia may be one of the factors that compromise graft survival. Conversely, the lowered core body temperature induced by some anaesthetics might promote the survival of grafted cells by slowing their metabolism. In an initial screen, the widely-used surgical anaesthetic, equithesin, was found to reduce core temperature, mean arterial blood pressure, and to increase the partial pressure of oxygen in arterial blood without producing any significant alteration in arterial pH or the partial pressure of carbon dioxide. In the main experiment, rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle received dopamine-rich embryonic nigral grafts injected into the deafferented neostriatum via previously implanted guide cannulae, which allowed comparison to be made of graft survival after transplantation into awake and in re-anaesthetised animals. There were no significant differences between groups in either the functional effects of the grafts to compensate amphetamine-induced rotation, or in the survival and growth of the grafts as measured in post mortem histology. We therefore conclude that anaesthesia per se is not a major contributory factor in the relatively poor survival of dopamine neurons following transplantation into the rat striatum.

Effects of surgical anaesthesia on the viability of nigral grafts in the rat striatum.

Only a small proportion of dopamine neurons in nigral grafts typically survive transplantation into the adult striatum. Since many anaesthetics reduce blood flow and disturb a variety of brain metabolites, surgical anaesthesia may be one of the factors that compromise graft survival. Conversely, the lowered core body temperature induced by some anaesthetics might promote the survival of grafted cells by slowing their metabolism. In an initial screen, the widely-used surgical anaesthetic, equithesin, was found to reduce core temperature, mean arterial blood pressure, and to increase the partial pressure of oxygen in arterial blood without producing any significant alteration in arterial pH or the partial pressure of carbon dioxide. In the main experiment, rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle received dopamine-rich embryonic nigral grafts injected into the deafferented neostriatum via previously implanted guide cannulae, which allowed comparison to be made of graft survival after transplantation into awake and in re-anaesthetised animals. There were no significant differences between groups in either the functional effects of the grafts to compensate amphetamine-induced rotation, or in the survival and growth of the grafts as measured in post mortem histology. We therefore conclude that anaesthesia per se is not a major contributory factor in the relatively poor survival of dopamine neurons following transplantation into the rat striatum.

Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons

To enhance the current therapeutic benefit of dopamine (DA) neuron grafts in Parkinson's disease, strategies must be developed that increase both DA neuron survival and fiber outgrowth into the denervated striatum. Previous work in our laboratory has demonstrated that dopaminergic neurons grow to greater size when co-grafted with striatal cell suspensions and display extensive tyrosine hydroxylase-positive (TH+) projections, but no conclusion could be reached concerning enhancement of survival of grafted DA neurons. The aim of the present study was to characterize further the potential trophic effects of striatal co-grafts on grafted mesencephalic DA neuron survival. Unilaterally lesioned male Fischer 344 rats were grafted with either a suspension of mesencephalic cells or with both mesencephalic and striatal cell suspensions. Co-grafts were either mixed together or placed separately into the striatum. Lesioned rats receiving no graft served as controls. Rotational behavior was assessed following amphetamine challenge at 2 weeks prior to grafting and at 4 and 8 weeks following grafting. Only rats receiving co-grafts of nigral and striatal suspensions separated by a distance of 1 mm showed significant behavioral recovery from baseline rotational asymmetry. Both mixed and separate striatal co-grafts were associated with a doubling of DA neuron survival compared with solo mesencephalic grafts. In the mixed co-graft experiment, DA neurite branching appeared enhanced and TH-rich patches were observed, whereas with co-grafts that were separated, TH+ innervation of the intervening host striatum was increased significantly. These results provide the first evidence suggesting that nigral-striatal co-grafts, particularly those placed separately and in proximity to each other, increase both DA neuron survival and neurite extension from the mesencephalic component of the grafts.

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.

Intrastriatal Mesencephalic Grafts Affect Neuronal Activity in Basal Ganglia Nuclei and Their Target Structures in a Rat Model of Parkinson’s Disease

Nigrostriatal dopamine (DA) lesions lead to changes of neuronal activity in basal ganglia nuclei such as the globus pallidus (GP, the rodent homolog of lateral globus pallidus), entopeduncular nucleus (EP, the rodent homolog of medial globus pallidus), substantia nigra pars reticulata (SNR), and subthalamic nucleus (STN). We investigated in rats whether embryonic mesencephalic DA neurons grafted in the striatum may affect the lesion-induced alterations of neuronal activity in these structures. Regional neuronal activity was determined by use of quantitative cytochrome oxidase histochemistry. It was also examined in lesioned rats whether the grafts may regulate the expression of c-Fos after systemic administration of apomorphine in the basal ganglia nuclei as well as their target structures, including the ventromedial thalamic nucleus (VM), superior colliculus (SC), and pedunculopontine nucleus (PPN). Lesioned rats exhibited an increased activity of CO in the GP, EP, SNR, and STN ipsilateral to the lesion. Intrastriatal nigral grafts reversed the increases in the CO activity in the EP and SNR, whereas the grafts failed to affect the enzyme activity in the GP or STN. Apomorphine induced an increased expression of c-Fos in the GP, STN, VM, SC, and PPN on the lesioned side. The enhanced expression of this protein in all the structures except for the STN was attenuated by nigral grafts. The present results indicate that intrastriatal DA neuron grafts can normalize the lesion-induced changes of neuronal activity in the output nuclei of the basal ganglia as well as their target structures.

Growth factors improve immediate survival of embryonic dopamine neurons after transplantation into rats

Embryonic dopamine neurons survive poorly after transplant into models of Parkinson's disease, possibly due to programmed cell death (apoptosis). Apoptosis in cultured dopamine neurons can be reduced by growth factors such as glial cell line-derived neurotrophic factor (GDNF) or a combination of insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF). To improve the survival of dopamine neurons in grafts, strands of E15 rat ventral mesencephalon were pretreated with a combination of GDNF, IGF-I, and bFGF and then transplanted into 6-hydroxydopamine-lesioned rats. In control animals, only 32% of dopamine neuron profiles survived the first 24 h after transplant. Growth factor pretreatment increased survival to 49% on day 1. Growth factors reduced the apoptotic rate of transplanted cells, just as they had in the previous in vitro experiments. Apoptotic nuclear morphology was observed in the transplanted dopamine neurons. We conclude that the majority of transplanted dopamine neurons die in grafts within the first 24 h after transplant, most likely by an apoptotic mechanism. Prevention of apoptosis with anti-apoptotic agents may improve the viability of dopamine neurons grafted for Parkinson's disease.

Tirilazad mesylate improves survival of rat and human embryonic mesencephalic neurons in vitro.

The survival rate of embryonic dopamine (DA) neurons after transplantation to the striatum is only 5-20%. Therefore, mesencephalic tissue from several donors needs to be implanted in a parkinsonian patient to induce a therapeutic improvement. Lazaroids are a group of neuroprotective compounds which inhibit lipid peroxidation. Previously, two lazaroids (U-74389G and U-83836F) have been found to improve the survival of both cultured and grafted rat DA neurons. The only lazaroid approved for human use is tirilazad mesylate. The objective of the present study was to explore the effects of tirilazad mesylate on DA neuron survival in cultures of rat ventral mesencephalon and its capacity to promote the in vitro cell viability of embryonic rat and human mesencephalic tissue, treated and dissociated in the same way as in clinical trials. After 7 days in vitro, the number of tyrosine hydroxylase-immunopositive, presumed DA neurons was 140% higher in rat cultures treated with 0.3 microM tirilazad mesylate than that in control cultures. Rat and human cell suspensions supplemented with tirilazad mesylate maintained a high degree of viability for several hours longer than control suspensions. These results indicate that tirilazad mesylate promotes the survival of both rat and human embryonic mesencephalic neurons in vitro. Tirilazad mesylate can be administered clinically and may become a useful tool for increasing survival of grafted DA neurons in patients, thereby reducing the needed quantity of human donor tissue.

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 β-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.

Addition of allogeneic spleen cells causes rejection of intrastriatal embryonic mesencephalic allografts in the rat

To address the importance of antigen-presenting cells for the survival of intracerebral neural allografts, allogeneic spleen cells were added to the graft tissue before transplantation. Dissociated embryonic, dopamine-rich mesencephalic and adult spleen tissues were prepared from either inbred Lewis or Sprague–Dawley rats. A mixture of neural and spleen cells was stereotaxically transplanted into the right striatum of adult Sprague–Dawley rats. Controls were neural allografts without addition of allogeneic spleen cells and syngeneic neural grafts with or without the addition of syngeneic spleen cells. Six weeks after transplantation, brain sections were processed immunocytochemically for tyrosine hydroxylase, specific for grafted dopamine neurons, and a bank of markers for various components in the immune and inflammatory responses. The neural allografts which were mixed with allogeneic spleen cells were rejected. In these rats, there was a high expression of major histocompatibility complex class I and II antigens, intense cellular infiltration including macrophages and activated microglial cells, and a presence of cluster of differentiation 4- and 8-immunoreactive cells in the graft sites. Moreover, there were increased levels of intercellular adhesion molecule-1, tumour necrosis factor-α and interleukin-6 in and around the grafts which were undergoing rejection. In contrast, syngeneic neural grafts survived well regardless of whether they were mixed with syngeneic spleen cells or not, and control neural allografts also exhibited unimpaired survival. No significant difference was observed in the number of grafted dopamine neurons among these three latter groups. The levels of expression of the different markers for inflammation and rejection were generally lower in these grafts than in implants of combined allogeneic neural and spleen cells. In summary, intrastriatal neural allografts, which normally survive well in our animal model, were rejected if allogeneic spleen cells from the same donor were added to the graft tissue. The added spleen cells caused strong host immune and inflammatory responses. The study gave support to the notion that immunological privilege of the brain does not provide absolute protection to immunogenetically histoincompatible neural grafts.

Constructing a New Nigrostriatal Pathway in the Parkinsonian Model with Bridged Neural Transplantation in Substantia Nigra

The physical repair and restoration of a completely damaged pathway in the brain has not been achieved previously. In a previous study, using excitatory amino acid bridging and fetal neural transplantation, we demonstrated that a bridged mesencephalic transplant in the substantia nigra generated an artificial nerve pathway that reinnervated the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats. In the current study, we report that a bridged mesencephalic transplant can anatomically, neurochemically, and functionally reinstate the 6-OHDA-eradicated nigro-striatal pathway. An excitatory amino acid, kainic acid, laid down in a track during the transplant generated a trophic environment that effectively guided the robust growth of transplanted neuronal fibers in a bundle to innervate the distal striatum. Growth occurred at the remarkable speed of approximately 200 microm/d. Two separate and distinct types of dopamine (DA) innervation from the transplant have been achieved for the first time: (1) DA innervation of the striatum, and (2) DA innervation of the pars reticularis of the substantia nigra. In addition, neuronal tracing revealed that reciprocal connections were achieved. The grafted DA neurons in the SNr innervated the host's striatum, whereas the host's striatal neurons, in turn, innervated the graft within 3-8 weeks. Electrochemical volt- ammetry recording revealed the restoration of DA release and clearance in a broad striatal area associated with the DA reinnervation. Furthermore, the amphetamine-induced rotation was attenuated, which indicates that the artificial pathways were motor functional. This study provides additional evidences that our bridged transplantation technique is a potential means for the repair of a completely damaged neuronal pathway.

Early gestational mesencephalon grafts, but not later gestational mesencephalon, cerebellum or sham grafts, increase dopamine in caudate nucleus of MPTP-treated monkeys.

The mechanism of the behavioral improvement observed in parkinsonian primates that receive intrastriatal transplants of fetal dopamine neurons has not been firmly established. Dopamine production by grafted neurons may be the basis of the behavioral recovery. Alternatively, stimulation of the host dopamine system by the transplant procedure itself may be central to the outcome. The present study examined whether dopamine concentration was raised in the caudate nucleus of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated primate following grafting, and if so, whether the elevation was dependent on either (i) the introduction of the implantation cannula (sham), (ii) the brain region that was grafted, or (iii) the gestational age of fetal tissue that was grafted. Transplantation of early gestational age fetal ventral mesencephalon (embryonic days 40-50) was associated with significant elevation of caudate nucleus dopamine concentration to a mean of approximately 20% of control values in the vicinity (within 2 mm) of the graft, compared with more distant sites in the caudate nucleus. With early gestational age fetal ventral mesencephalon, the ratio of homovanillic acid/dopamine concentration near the graft site was normalized compared to the elevated value found in the caudate nucleus distant from the graft site. Grafts of later stage fetal ventral mesencephalon, or fetal cerebellum, or sham implantation did not increase dopamine concentration or lower homovanillic acid/dopamine ratio near the graft site. Biochemical and histochemical evidence suggests that host dopamine neurons terminating in the nucleus accumbens are not the source of the changes. Numerous tyrosine hydroxylase-positive neurons at the graft site were only observed in the MPTP-treated monkeys that received grafts of early gestational age fetal ventral mesencephalon. These data lend strong support to the hypothesis that dopamine derived from grafted dopamine neurons is the major basis for behavioral recovery observed following intrastriatal transplantation in our MPTP-treated monkeys.

The lazaroid U-83836E improves the survival of rat embryonic mesencephalic tissue stored at 4°C and subsequently used for cultures or intracerebral transplantation

We assessed the effects of addition of the lazaroid U-83836E to a preservation medium on the survival of rat dopamine neurons stored before culturing or intracerebral transplantation. Embryonic ventral mesencephalic tissue was preserved at 4°C for 8 days with or without the addition of 0.3 μM of U-83836E to a chemically defined “hibernation” medium. Freshly dissected mesencephalic tissue was used in control groups. For culture experiments, the mesencephalic tissue was dissociated and grown in serum-containing medium. Following 24–48 h in vitro, the number of dopamine neurons in cultures derived from tissue hibernated without the lazaroid was 40% of fresh control, compared with 67% of control in cultures prepared from tissue stored in the presence of U-83836E. When mesencephalic tissue was transplanted to the dopamine-depleted striatum of hemiparkinsonian rats following 8 days storage at 4°C in a medium without U-83836E, the mean number of surviving dopamine neurons in the grafts was significantly reduced to 40% of control. In contrast, grafts of tissue which had been hibernated in U-83836E-containing medium contained as many dopamine neurons as transplants of freshly dissected tissue. High yields of surviving grafted dopamine neurons were correlated to a significantly faster onset of functional recovery of amphetamine-induced motor asymmetry. We conclude that the storage period for rat mesencephalic tissue can be prolonged up to 8 days when using lazaroid-supplemented hibernation medium. As lazaroids have undergone clinical safety testing, the application of lazaroids for tissue storage in clinical transplantation trials can be envisaged.

The detrimental effect of levodopa on behavioral efficacy of fetal dopamine neuron grafts in rats is reversible following prolonged withdrawal of chronic dosing

In previous studies, we observed that chronic levodopa treatment resulted in impaired morphology and function of grafted dopamine neurons in rats. To begin to better understand how levodopa treatment might influence dopamine neurons, we examined whether subsequent discontinuation of chronic levodopa treatment might allow for recovery of graft efficacy. Function of embryonic mesencephalic tissue grafts was assessed by monitoring rotational behavior elicited by amphetamine in lesioned, grafted rats initially treated for 6 weeks with levodopa followed by a 6 week drug-free period. As observed previously, control grafted animals, but not levodopa treated animals, showed behavioral improvement. However, following a 6 week withdrawal period, the levodopa animals demonstrated a significant reduction in amphetamine rotations which was reminiscent of control animals. This suggests that grafted neurons can recover functionally after levodopa treatment is withdrawn, which may be of significance in clinical transplantation trials.

Immunobiology of neural transplants and functional incorporation of grafted dopamine neurons

AbstractIn contrast to the views put forth by Stein &amp; Glasier, we support the use of inbred strains of rodents in studies of the immunobiology of neural transplants. Inbred strains demonstrate homology of the major histocompatibility complex (MHC). Virtually all experimental work in transplantation immunology is performed using inbred strains, yet very few published studies of immune rejection in intracerebral grafts have used inbred animals.