A comparison of dopaminergic cells from the human NTera2/D1 cell line transplanted into the hemiparkinsonian rat.

Human NT cells derived from the NTera2/D1 cell line express a dopaminergic phenotype making them an attractive vehicle to supply dopamine to the depleted striatum of the Parkinsonian patient. In vitro, hNT neurons express tyrosine hydroxylase (TH), depending on the length of time they are exposed to retinoic acid. This study compared two populations of hNT neurons that exhibit a high yield of TH+ cells, MI-hNT and DA-hNT. The MI-hNT and DA-hNT neurons were intrastriatally transplanted into the 6-OHDA hemiparkinsonian rat. Amelioration in rotational behavior was measured and immunohistochemistry was performed to identify surviving hNT and TH+ hNT neurons. Results indicated that both MI-hNT and DA-hNT neurons can survive in the striatum, however, neither maintained their dopaminergic phenotype in vivo. Other strategies used in conjunction with hNT cell replacement are likely needed to enhance and maintain the dopamine expression in the grafted cells.

Transplantation of human fetal striatal tissue in Huntington's disease: rationale for clinical studies.

Huntington's disease is a fatal neurological disorder characterized by chorea and deterioration in cognitive and neuropsychiatric function. Primary pathological changes are found in the striatum, where GABAergic neurons undergo degenerative changes. Local interneurons are relatively spared. Here, we describe the rationale for clinical trials of fetal striatal tissue transplantation for the treatment of Huntington's disease. Specifically, the reasons for utilizing tissue derived from the far lateral aspect of the lateral ventricular eminence as a source of striatal tissue will be discussed.

Brain transplants enhance rather than reduce the impairment of spatial memory and olfaction in bulbectomized rats.

The possibility to compensate the loss of olfactory and non-olfactory functions due to removal of the olfactory bulb by embryonal brain grafts was investigated in adult rats. Spatial working memory was examined in an 8-arm radial water maze task 6 weeks after bulbectomy. During 15 daily trials, performance gradually improved in bulbectomized controls (n = 10) and in rats with olfactory bulb transplants (n = 9), but did not attain that of intact controls (n = 10). No improvement was observed in the rats with substantia nigra grafts (n = 8). Eleven weeks after bulbectomy, the same rats were tested in the water tank navigation task. The performance improved during ten 12-trial sessions in bulbectomized rats less than in intact controls, but more than in the transplanted rats. The olfactory food retrieval test performed 14 weeks after bulbectomy revealed almost full recovery of smell in bulbectomized rats, but not in the transplanted animals. It is concluded that the spatial memory deficit is probably due to bulbectomy-induced interference with septohippocampal function which is not alleviated, but rather enhanced by transplantation. The results suggest that the effect of brain grafting is not always beneficial.

Transplantation of dorsal root ganglion into the olfactory bulb of neonatal rats: a histochemical study.

We have transplanted encapsulated dorsal root ganglia (DRG) from adult Wistar albino rats unilaterally into partially bulbectomized (n = 20) neonatal (P3-5) rats of the same strain. Three months postoperatively the animals were perfused and their brains processed by direct thiocholine method for cholinesterases (Ch), specific acetylcholinesterase (AChE) and nonspecific butyrylcholinesterase (BuChE) or stained by Cresyl violet. Selected sections were immunohistochemically stained for olfactory marker protein (OMP). In 17 cases we found surviving transplanted DRG. Fifteen transplants were well integrated with the spared portion of the olfactory bulb (OB) as clearly demonstrated by AChE and BuChE histochemistry, while two did not integrate. Regenerated OMP positive olfactory axons originating from neuroepithelium and AChE positive fibres from OB remnant penetrated into the transplants. In one case, fibers connected with BuChE positive Schwann cells grew from the transplanted DRG into the host OB. Individual sensory neurons of the transplants revealed variable intensity of the AChE staining, thus resembling the pattern of AChE activity in normal DRG. BuChE activity was mostly localized on the surface of sensory neurons in the ring of satellite cells. Some BuChE positive blood vessels penetrated into the DRG, and were observed around sensory neurons. The results showed a considerable viability and adaptability of the sensory neurons in the new environment after a long-term transplantation.

Migration patterns of neonatal subventricular zone progenitor cells transplanted into the neonatal striatum.

Our previous studies have shown that the progeny of the neuronal progenitor cells localized in a discrete region of the anterior part of the neonatal subventricular zone, referred to as the SVZa, migrate tangentially along a stereotypical and extended pathway to the olfactory bulb, and then turn radially into one of the overlying cellular layers. In this study we have examined whether the SVZa cells retain their ability to migrate and disperse when heterotopically transplanted into the striatum. SVZa cells from P0-P2 rat pups were microdissected, dissociated, labeled with the lipophilic, fluorescent dye PKH26 or the cell proliferation marker BrdU, and then transplanted into the neonatal (P0-P2) striatum. Examination of the striatum a few days after transplantation revealed aggregates of heavily labeled BrdU-positive, SVZa cells in the striatum, often situated near blood vessels. Two to four weeks after transplantation, however, the labeled SVZa cells had disseminated from their site of implantation and showed three patterns of distribution. In none of the cases was the implantation site detectable in the striatum, signifying that the cells had become incorporated in the host brain. Of the 12 brains analyzed for cell distribution, transplanted SVZa cells were confined to the striatum in 4 cases. The cells were present as individual cells or in small groups of usually two to four cells. When PKH26 was used, we found that many of the transplanted cells extended processes into the striatum. In 3 out of the 12 animals, the labeled SVZa cells were distributed along the dorsal and lateral aspects of the striatal boundary. In the remaining five animals, labeled SVZa cells appeared in both locations: within the striatum as well as along the striatal boundary. The dispersion of the transplanted cells within the striatum and the presence of the transplanted SVZa cells all along the striatal boundary, a region corresponding to the lateral cortical stream of migration of the developing forebrain, demonstrates that the isochronically transplanted SVZa cells retained their capacity to migrate.

The effects of taurine on hNT neurons transplanted in adult rat striatum.

Taurine acts as an antioxidant able to protect neurons from free radical-mediated cellular damage. Moreover, it modulates the immune response of astrocytes that participate in neurodegenerative processes. The objective of this study was to examine whether taurine can prevent or attenuate the host inflammatory response induced by the xenotransplantation of neurons derived from the human teratocarcinoma cell line (hNT neurons). Male Sprague-Dawley rats were treated IP with either saline or taurine. Animals from both groups were perfused on the 4th or 11th day and the saline or taurine was administered from the start of the study until the day prior to sacrifice. The brains were processed immunohistochemically using antibodies against glial fibrillary acidic protein (GFAP), microglia (OX42), and human nuclear matrix antigen (NuMA). In the saline group, NuMA labeling revealed small grafts on the 4th day and no surviving cells on the 11th day. However, in the group that received taurine there were surviving grafts at both time points. Strong immunoreactivity for GFAP and OX42 was detected in the saline group surrounding the transplant. These effects were reduced in animals receiving taurine. Taken together, these results demonstrated that taurine was able to facilitate graft survival and attenuate the immune response generated by the xenograft.

Neuronal progenitor cells of the neonatal subventricular zone differentiate and disperse following transplantation into the adult rat striatum.

We 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.

The X-gal caution in neural transplantation studies.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Olfactory bulb transplantation into the olfactory bulb of neonatal rats: an autoradiographic study.

Tritiated thymidine prelabeled presumptive olfactory bulbs (E15-E17, and E19) were homotopically transplanted in unilaterally partially or totally bulbectomized neonatal rats (P1-P5). [3H]thymidine was injected to pregnant rats at the time when the large neurons of the bulb were undergoing cellular division. After postoperative survival times from 20 days to 7 months, the animals were sacrificed and processed for histological, immunohistochemical and autoradiographic observations. The nuclear autoradiographic label allowed easy recognition of the transplanted tissue in totally bulbectomized animals after short survival and in partially bulbectomized animals after long survival. The autoradiographic label was strictly confined to the transplanted tissue and intermingling of host and donor neurons was never observed. The reliability of the autoradiographic technique in our study will enable us to mark those neurons whose axons can be demonstrated, by retrograde tracing methods, to establish connections with the host brain.

Olfactory bulb transplantation into the olfactory bulb of neonatal rats: a WGA-HRP study.

After unilateral bulbectomy in neonatal (P1-P5) rats, autoradiographically prelabeled presumptive olfactory bulbs from E15 and E17 embryos were transplanted in place of the removed tissue. After 2-7 months, the animals received injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the piriform cortex. Nine of the twenty animals revealed WGA-HRP-positive neurons among neurons autoradiographically labeled, providing thus evidence that the axons of the output neurons from the homotopically transplanted olfactory bulb reconnect with the host piriform cortex.

Olfactory bulb transplantation into the olfactory bulb of neonatal rats.

Embryonic olfactory bulbs (E14-E18) were homotopically transplanted into neonatal rats (P1-P5) after partial or total bulb removal. After different survival times, the animals were sacrificed and their brains processed for histological and immunohistochemical observations. The growing sensory axons randomly formed glomeruli within the transplanted tissue which often acquired a typical laminar organization. Myelinated fibers connecting the transplant with the telencephalon were clearly identified in one animal. Thus, the present study has shown that olfactory bulb transplants were able to differentiate, mature and establish relationship with both the periphery and the brain.

Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone.

Earlier studies in our laboratory have demonstrated that a discrete region of the anterior part of the neonatal subventricular zone (SVZa) contains exclusively neuronal progenitor cells. The descendants of the SVZa progenitor cells are destined for the granule cell and glomerular layers of the olfactory bulb, where they differentiate into granule and periglomerular cells, the interneurons of the olfactory bulb, respectively. In the present set of experiments we examined the neurotransmitter phenotype of the SVZa-derived cells. In order to label SVZa-derived cells, the cell proliferation marker bromodeoxyuridine (BrdU) was injected into the SVZa of postnatal day 2 (P2) rats. After 3 weeks, by which time most of the SVZa-derived cells have migrated to their final destination in the bulb, the animals were perfused and their brains processed for immunohistochemistry. To identify the neurotransmitter phenotype of the SVZa-derived cells, sagittal sections of the forebrain, including the olfactory bulb, were double-labeled with an antibody to BrdU in conjunction with an antibody to gamma-amino-butyric acid (GABA) or tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. Using simultaneous indirect immunofluorescence to detect the presence of single- and double-labeled cells, we found that 59% and 51% of the BrdU-positive cells were immunoreactive for GABA in the granule cell and glomerular layers, respectively. In addition, 10% of the BrdU-positive periglomerular cells were immunoreactive for TH. The presence of double-labeled (BrdU-positive/GABA-positive and BrdU-positive/TH-positive) cells in the olfactory bulb, demonstrates that the SVZa is a source of the GABAergic and dopaminergic interneurons of the olfactory bulb during postnatal development.

Dopaminergic phenotype of hNT cells in vitro.

We investigated the catecholaminergic nature of cultured hNT neurons previously treated either for 4 or 5 weeks with retinoic acid (RA). There were significantly more tyrosine hydroxylase (TH)-positive neurons (60%) in cultures treated for 4 weeks with RA compared to 5 week-treated cultures (

Apoptosis in cultured hNT neurons.

Programmed cell death (apoptosis) is an important mechanism shaping the size of different cell populations within the developing nervous system. In our study we used the NT2/D1 clone originally established from the Ntera 2 cell line to investigate the baseline levels of apoptosis in cultured postmitotic hNT (NT2-N) neurons previously treated for 3, 4 or 5 weeks with retinoic acid (RA) and compared it with apoptosis in NT2 precursors unexposed to RA. First, we examined whether different lengths of exposure to RA might affect baseline apoptotic rate in differentiating hNT neurons. Second, we investigated whether cultured hNT neurons, previously shown to possess dopaminergic characteristics, would be preferentially affected by apoptosis. Using the terminal deoxynucleotidyl transferase (tdt)-labeling technique we found that the postmitotic hNT neuronal cells exposed to RA demonstrated significantly higher numbers of apoptotic cells (12.5-15.8%) in comparison to rapidly dividing NT2 precursor cell line (3.6-4.4%) at both studied (1 and 5 days in vitro, DIV) time points. Similar apoptotic nuclear morphology, including a variable extent of nuclear fragmentation was observed in all examined hNT cultures. On the other hand, the incidence of apoptotic nuclei was rare in cultures of NT2 precursors not subjected to RA treatment. Combined immunocytochemistry for tyrosine hydroxylase (TH) and Hoechst staining revealed dopaminergic hNT neurons destined to die. Our double-labeling studies have demonstrated that only a subset of TH-positive hNT cells had condensed chromatin after 1 (approx. 15%) and 5 (approx. 20%) DIV. NT2 precursors were not TH-positive. Collectively, our results demonstrated that exposure to differentiating agent RA triggers an apoptotic commitment in a subset of postmitotic hNT neurons. These results suggest that this cell line may serve as a model of neuronal development to test various pathogenic factors implicated in the etiology of Parkinson's disease (PD), as well as to screen numerous pharmacological treatments that may slow or prevent dopaminergic deterioration.

In vitro induction and in vivo expression of bcl-2 in the hNT neurons.

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.

In vitro and in vivo characterization of hNT neuron neurotransmitter phenotypes.

The hNT neuron exhibits many characteristics of neuroepithelial precursor cells, making them an excellent model to study neuronal plasticity in vitro and in vivo. These cells express a number of neurotransmitters in vitro, including dopamine, gamma-aminobutyric acid and acetylcholine. However, there have been few reports of the neurotransmitters that hNT neurons express in vivo. The present study examined whether hNT neurons express the same neurotransmitters in vivo as they do in vitro. First, the expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT) and the human specific nuclear marker NuMA by hNT neurons was confirmed. Nineteen normal animals were then transplanted with 80,000 hNT neurons aimed at the striatum, hippocampus or cerebral cortex. Five additional animals received injections of medium. All animals received daily intraperitoneal injections of cyclosporine (10 mg/kg) and survived 30 days. Sections through the transplants were examined for NuMA-positive hNT neurons, and for the presence of the three neurotransmitter markers: TH, GAD and ChAT. The hNT neurons were found in the striatum and cortex. Of the hNT neurons found within the rat striatum, 33% were ChAT-positive. In the cortex, only 4% of the neurons expressed ChAT. No GAD-positive hNT neurons were detected at either site. No NuMA-positive neurons were found in the hippocampus. The implanted hNT neurons did not induce activation of astrocytes as determined by immunocytochemistry for glial fibrillary acidic protein (GFAP). Moreover, no hNT neuron was found to express GFAP in vivo. Together, these data suggest that the hNT neurons engraft in the new host tissue, maintain their neuronal identity and may be guided in differentiation according to local environmental cues.

Neural transplantation in Parkinson's disease.

In conclusion, proof of the principle exists that neural grafts can survive transplantation in PD and that this graft survival is related to preliminary evidence of clinical benefit and improvement on FD-PET. Two prospective, randomized, surgical placebo-controlled trials of fetal tissue transplantation for the treatment of PD will be published in the near future, as will results of a placebo-controlled xenograft trial. Lifelong survival of human fetal nigral grafts is likely. The striatum is comparatively simple to target surgically in comparison to other sites such as the subthalamic nucleus. Several new sources of dopamine cells are being developed for transplantation purposes. Long-term monitoring for toxicity, such as the development of dyskinesias, will be needed, and dose-escalation trials should be performed slowly due to the irreversible nature of transplants. There are numerous ways to improve current techniques of neural transplantation.

Neovascularization of the dorsal root ganglia transplanted into the olfactory bulb of neonatal rats.

Dorsal root ganglia (DRG), whose vessels are permeable to blood-born proteins, were syngenically transplanted into 2-5 days old Wistar albino rats after partial unilateral bulbectomy. Our aim was to follow survival and vascularization of the DRG, transplanted into a new environment, the developing olfactory bulb (OB). Three months after grafting the DRG graft was found fused with the spared portion of the OB. Only a subpopulation of the transplanted neurons survived the transplantation. Cholinesterase histochemistry showed BuChE positive vessels of host origin around the surviving DRG neurons. The majority of the vessels was impermeable to i.v. applied fluorescent dyes (Evans blue and lucifer yellow) and only few (1-3 vessel profiles/section) of them were labeled at the graft surface. By lanthanum nitrate tracing at the ultrastructural level, tight junctions were seen in the majority of the blood vessels of the graft. Our study shows that during the neovascularization the transplanted DRG is invaded by the host-derived blood vessels which possess blood brain barrier properties-they are impermeable to applied micro-and macromolecules. The newly formed/reestablished circulation appeared to be sufficient for maintaining a subpopulation of the transplanted sensory neurons.

Distribution of growth associated protein (B-50/GAP-43) and glial fibrillary acidic protein (GFAP) immunoreactivity in rat homotopic olfactory bulb transplants.

Recent studies on olfactory bulb homotopic transplantation after partial or subtotal bulb ablation have shown that regenerated olfactory axon are able to form glomeruli-like structures either in the transplant or in the spared olfactory bulb. To investigate the maturation of olfactory axons and their terminal connections in the transplant and remnant olfactory bulb we performed immunohistochemistry utilizing as markers B-50/GAP43 for neurite outgrowth and glial fibrillary acidic protein (GFAP) for the glial response to the surgery. Radioactively prelabeled olfactory bulbs (E 18) were homotopically transplanted in unilaterally bulbectomized neonatal rats (P6). Two to four months after transplantation in the partially bulbectomized rats the laminar organization of the olfactory bulb remnant depended on the extent of lesion. The transplant was disorganized showing irregularly distributed glomeruli. In a few cases pseudo-laminar organization was observed resembling that of the normal olfactory bulb. In 2 month-old transplants, outgrowing axons had a newly formed glomeruli displayed prominent B-50/GAP43 immunoreactivity. Four months after the operation B-50/GAP43 immunoreactivity was still present in the regrowing axons and some glomeruli in the transplant were B-50/GAP43 positive, while other glomeruli revealed a patchy pattern. The same B-50/GAP43 immunostained patchy structure were present in remnants of the lesioned olfactory bulb. Distinct increase of GFAP activity was observed along the olfactory axons and in the glomeruli-like structures. The persisting B-50/GAP43 immunoreactivity in the glomeruli of the transplant and in the remnants of lesioned olfactory bulb suggests that maturation of the newly formed glomeruli was delayed in comparison to the intact control olfactory bulb. Furthermore, the decrease of B-50/GAP43 immunoreactivity and the patchy distribution may indicate that synaptogenesis occurred in the glomeruli of the transplant, in spite of the altered topography. Together with previous findings on reinnervation of the piriform cortex from projection neurons situated in olfactory bulb transplants, our immunohistochemical data support the notion that olfactory bulb transplantation may reestablish to some degree the neuronal circuits affected by the experimental surgery.

[Neurotransplantation, critical analysis and perspectives]. / Neurotransplantácie, kritická analýza a perspektívy.

Basic morphological and functional properties of basal ganglia are described and their role in the development of Parkinson's disease is discussed in detail. Based on recent experimental data, the problem of human autologous adrenal medulla transplants as well as human dopaminergic neurons is analyzed and prospective approaches to successful therapeutic interventions are considered mainly from the molecular and immunobiological point of view. The relative limitations of the regenerative capacity and transplantability of the nervous tissue are assessed and compared with the critical developmental periods of human neurogenesis (Fig. 6, Ref. 47.).