Regular ArticleA Comparison of Solid Intraventricular and Dissociated Intraparenchymal Fetal Substantia Nigra Grafts in a Rat Model of Parkinson's Disease: Impaired Graft Survival Is Associated with High Baseline Rotational Behavior
Abstract
It has been established that substantia nigra (SN) grafts can produce a behavioral effect in animal models of Parkinson's disease when transplanted either into the lateral ventricles as solid tissue fragments or directly into the striatum as dissociated cells. These two transplantation methods (solid-intraventricular and dissociated-intraparenchymal) were directly compared in the present study. Adult male rats received unilateral SN lesions by stereotaxic injection of 6-hydroxydopamine. Fetal ventral mesencephalon, containing the SN, was harvested from 15-day gestational rat fetuses. Equal weights of tissue were transplanted, either as solid-intraventricular or dissociated-intraparenchymal grafts. In the animals with surviving grafts, similar decreases in apomorphine-induced rotational behavior were seen in both dissociated (40.2% at 6 weeks, 29.6% at 12 weeks, N = 12) and solid (31.8% at 6 weeks, 51.0% at 12 weeks, N = 11) graft groups. Control animals receiving tectal grafts showed smaller decreases (dissociated: 12.6% at 6 weeks, 18.8% at 12 weeks, N = 20; solid: 4.1% at 6 weeks, 8.0% at 12 weeks, N = 20). There were no significant overall differences between the effects of the solid and dissociated grafts; however, in the subgroup of animals with high baseline levels of rotational behavior, the solid-intraventricular grafts produced a significantly larger effect then did the dissociated-intraparenchymal SN grafts. There was also a tendency for the effect of the solid-intraventricular grafts to increase gradually over the 12-week testing period, whereas the effects of the dissociated-intraparenchymal grafts were maximal by 6 weeks after transplantation. For both SN graft groups, there were significant correlations between decreases in rotation and numbers of surviving tyrosine hydroxylase-immunore-active neurons in the grafts. It was also found that animals with no surviving transplanted tyrosine hydroxylase-positive neurons had higher baseline levels of rotational behavior. Therefore, in the present model, solid-intraventricular and dissociated-intraparenchymal SN grafts have similar effects on apomorphine-induced rotational behavior, except that the solid-intra-ventricular grafts tended to produce a larger effect in animals with high baseline levels of rotational behavior.
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Apoptosis in primary cultures of E14 rat ventral mesencephala: Time course of dopaminergic cell death and implications for neural transplantation
1999, Experimental NeurologyTransplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum. However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery. A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear. Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival. Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media. After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically. Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h. Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons. There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h. We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.
Strands of embryonic mesencephalic tissue show greater dopamine neuron survival and better behavioral improvement than cell suspensions after transplantation in parkinsonian rats
1998, Brain ResearchThe success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons. To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands. We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery. We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day 15 rat ventral mesencephalon. Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. To evaluate the effects of striatal cografts and growth factors on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells. Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic factor (GDNF, 100 ng/ml), insulin-like growth factor-I (IGF-I, 1500 ng/ml), and basic fibroblast growth factor (bFGF, 150 ng/ml). Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior. Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry. Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with growth factors.
Neuronal progenitor cells of the neonatal subventricular zone differentiate and disperse following transplantation into the adult rat striatum
1998, Cell TransplantationWe have investigated the suitability of a recently identified and characterized population of neuronal progenitor cells for their potential use in the replacement of degenerating or damaged neurons in the mammalian brain. The unique population of neuronal progenitor cells is situated in a well-delineated region of the anterior part of the neonatal subventricular zone (referred to as SVZa). This region can be separated from the remaining proliferative, gliogenic, subventricular zone encircling the lateral ventricles of the forebrain. Because the neurons arising from the highly enriched neurogenic progenitor cell population of the SVZa ordinarily migrate considerable distances and ultimately express the neurotransmitters GABA and dopamine, we have examined whether they could serve as an alternative source of tissue for neural transplantation. SVZa cells from postnatal day 0–2 rats, prelabeled by intraperitoneal injections of the cell proliferation marker BrdU, were implanted into the striatum of adult rats approximately 1 mo after unilateral denervation by 6-OHDA. To examine the spatio-temporal distribution and phenotype of the transplanted SVZa cells, the experimental recipients were perfused at short (less than 1 wk), intermediate (2–3 wk) and long (5 mo) postimplantation times. The host brains were sectioned and stained with an antibody to BrdU and one of several cell-type specific markers to determine the phenotypic characteristics of the transplanted SVZa cells. To identify neurons we used the neuron-specific antibody TuJ1, or antimembrane-associated protein 2 (MAP-2), and anti-GFAP was used to identify astrocytic glia. At all studied intervals the majority of the surviving SVZa cells exhibited a neuronal phenotype. Moreover, morphologically they could be distinguished from the cells of the host striatum because they resembled the intrinsic granule cells of the olfactory bulb, their usual fate. At longer times, a greater number of the transplanted SVZa cells had migrated from their site of implantation, often towards an outlying blood vessel, and the density of cells within the core of the transplant was reduced. Furthermore, there were rarely signs of transplant rejection or a glial scar surrounding the transplant. In the core of the transplant there were low numbers of GFAP-positive cells, indicating that the transplanted SVZa cells, predominantly TuJ1-positive/MAP2-positive, express a neuronal phenotype. Collectively, the propensity of the SVZa cells to express a neuronal phenotype and to survive and integrate in the striatal environment suggest that they may be useful in the reconstruction of the brain following CNS injury or disease.
A comparative study of preparation techniques for improving the viability of nigral grafts using vital stains, in vitro cultures, and in vivo grafts
1995, Cell TransplantationThe intracerebral transplantation of embryonic dopaminergic nigral neurons, although relatively successful, leads to a fairly low yield of surviving cells. Many factors may influence the viability of dopaminergic grafts and one of these is the preparation of the tissue prior to transplantation. We have investigated the effects of different steps during the preparation and storage of embryonic rat nigral cell suspensions on their subsequent survival at a variety of different time points using a combination of techniques and studies. For studies concerned with the first 24 h we employed vital stains, in the period covering the next 7 days we used in vitro cultures, and in the long term experiment we used in vivo grafts. The results suggest that nigral cell suspensions may remain sufficiently viable for grafting for much longer periods than previously reported. In addition a number of parameters which affect cell survival have been characterised, including the age of the embryonic donor tissue, the use of proteolytic enzymes and the trituration procedure used during the preparation of the suspension. The optimal preparation technique, therefore, uses E13–E14 embryos with the dissected ventral mesencephalon being incubated in purified 0.1% trypsin solutions for 60 min and triturated using a flame polished Pasteur pipette. This may have important implication in improving intracerebral transplantation for Parkinson's disease.
The influence of donor age on the survival of solid and suspension intraparenchymal human embryonic nigral grafts
1995, Cell TransplantationIn many species, graft survival and graft-derived behavioral recovery are affected by the embryonic donor age. We compared the ability of solid and suspension grafts of human embryonic mesencephalic dopaminergic (DA) neurons at different embryonic stages to survive intraparenchymal transplantation into 6-OHDA lesioned immunosuppressed rats. Suspension grafts survived best when donor age was between postconception (PC) days 34 and 56. Transplants displayed numerous healthy tyrosine hydroxylase immunoreactive (TH-IR) neurons which sent extensive neuritic processes into the host striatum. Suspension grafts survived poorly when donor age was greater than 65 days. Solid implants displayed comparable viability of TH-IR neurons when donor age was between 44 and 65 days. No solid grafts contained TH-IR cells when donor tissue was older than 72 days. The suspension and solid methods of transplantation resulted in comparable survival of robust grafts, but solid grafts resulted in more intergraft variability than suspension grafts, particularly among the more marginal implants. Our results demonstrate that the upper limit for survival of human embryonic DA suspension grafts correlates well with the period of development of the human nigrostriatal pathway. The “window” for donor age of solid human embryonic DA grafts appears to be extended by about 9 days in comparison to suspension grafts. These data suggest that the upper age limit for grafting human mesencephalic DA neurons should be PC day 56 for suspension grafts, and PC day 65 for solid implants. Older donors are likely to produce grafts with fewer surviving DA neurons.
Development of human fetal ventral mesencephalic grafts in rats with 6-OHDA lesions of the nigrostriatal pathway
1995, Neuroscience ResearchNeuronal transplantation is an approach that can be exploited to study the development of the human central nervous system as well as being used in attempts to restore neurological function. In the present study, we have examined cellular events that appear to precede the development of dopamine nerve fiber extension by neurons from the human fetal ventral mesencephalon. These cellular events were examined using neuronal cell suspensions from human fetal ventral mesencephalic tissue (gestational ages 7–10 weeks) transplanted into the striatum of unilaterally lesioned 6-hydroxydopamine (6-OHDA) rats. Animals were sacrificed for immunohistochemistry 9–10 weeks after the transplantation prior to the manifestation of behavioral recovery. Histological analysis revealed tyrosine hydroxylase (TH) immunoreactive neurons in the grafts. The majority of these neurons had very short TH positive processes (60–70 um), indicating that the maturation of grafted dopaminergic neurons was still incomplete. Immunostaining for the human specific intermediate neurofilament (hNF, clone: BF-10) showed dense neuronal fibers in the grafts. These fibers extended deeper into the host brain than the TH positive neuronal processes. The whole striatum, particularly the medial part of the striatum, exhibited long NF positive processes. Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed fine astrocytic processes inside the grafts, which were clearly different from host reactive glial cells surrounding the grafts. These graft-derived glial processes tended to extend into the host brain deeper than the TH positive neuronal processes from the grafts. These early histological findings of the grafted human fetal ventral mesencephalon suggest that the graft-derived NF positive neuronal processes, as well as the glial processes, radiate from the grafted tissue and extend into the host brain prior to the extension of TH positive processes. These results further suggest that human-to-rat xenografts can be used to study the neural development of human fetal brain tissue.