Schwann Cells and Fetal Tectal Tissue Cografted to the Midbrain of Newborn Rats: Fate of Schwann Cells and Their Influence on Host Retinal Innervation of Grafts

Schwann cells transplanted into the adult central nervous system (CNS) can exert powerful growth-promoting effects on damaged axons. An important issue is whether central axons induced to regrow by Schwann cells retain the capacity to recognize and selectively innervate their appropriate target cells. To examine how Schwann cells may influence the specificity of neuron-neuron interactions in CNS neuropil, we cultured neonatal rat Schwann cells and mixed them with dissociated fetal tectal cells. In some instances, Schwann cells were prelabeled with Hoechst dye 33342. Schwann cells comprised between 2.5 and 15% of the combined cell population. After reaggregation, cografts were injected onto the midbrain of newborn rats. One to 6 months later, grafts were examined for the presence of Schwann cells and the pattern and density of host retinal innervation of the cografts was assessed. Immunohistochemical studies showed that areas of the transplants containing large numbers of surviving Hoechst-labeled Schwann cells were strongly immunoreactive for the low-affinity nerve growth factor receptor (p75), S-100, GFAP, and laminin. Very little peripheral (P_o positive) myelin was seen. As in pure fetal tectal grafts, host retinal axons were sometimes observed to innervate superficial, localized areas in the cografts known to be homologous to the retinorecipient layers of the superior colliculus. Unlike pure tectal grafts, however, optic axons were not confined to these regions and fibers were often dispersed within the cograft neuropil. Dense growth was seen in association with Hoechst-labeled Schwann cells and, in some cases, optic axons were observed to grow toward Schwann cells and away from nearby target areas. These observations suggest that, under certain circumstances, Schwann cells can stimulate retinal axons to grow into inappropriate (nontarget) regions in the CNS, presumably by producing growth promoting factors which mask or compete with signals released from the target neurons themselves.     

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.     

Regeneration of axons from adult rat retinal ganglion cells on cultured Schwann cells is not dependent on basal lamina.

The ability of sciatic nerve grafts to support in vivo regeneration of retinal ganglion cell axons in the adult rat raises the question of which peripheral nerve constituents may be required to promote this unexpected central regenerative response. Prime candidates for this role include the surface of the Schwann cell and components of extracellular matrix present in peripheral nerve trunks. To determine the relative importance of Schwann cells and their basal lamina in promoting retinal ganglion cell axon regeneration in the mammalian visual system, we have used an in vitro model. This approach allowed analysis of the abilities of defined peripheral nerve constituents to promote in vitro outgrowth of neurites from explants of adult rat retina harvested 7 to 10 days after in vivo optic nerve crush. Neurite outgrowth was assessed by neurofilament immunofluorescence after 3 to 20 days in vitro. Culture substrata, consisting of isolated Schwann cells (SC), Schwann cells with their assembled extracellular matrix (SC + ECM), or isolated extracellular matrix from which the Schwann cells had been removed (ECM), were prepared by first co-culturing rat Schwann cells with embryonic dorsal root ganglion neurites on a layer of type I collagen, and then manipulating the cultures to produce the desired substrata. Type I collagen alone did not support neurite growth from adult rat retina. SC and SC + ECM supported regeneration of axons from retinal explants at average growth rates of 18 and 30 microns/h, respectively. Isolated ECM was a poor substrate for retinal neurite growth; the few neurites that gained access to this material grew at rates averaging less than 3 microns/h. These observations suggest that regeneration of adult mammalian retinal ganglion cell axons through peripheral nerve grafts (in vivo) is primarily dependent on neurite-promoting factors present on the surface of Schwann cells and does not require organized extracellular matrix.     

Specific Reinnervation of Lesioned Mouse Striatum by Grafted Mesencephalic Dopaminergic Neurons

Selective lesions of the dopaminergic nigrostriatal system and embryonic neuron grafts were used to study the mechanism by which exogenous neurons can restore transmitter function and to examine CNS development and plasticity. C57BL mice treated with acetaldehyde/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine show irreversible loss of substantia nigra dopaminergic neurons. Implants of embryonic mesencephalic dopaminergic neurons functionally reinnervate the striatum and form a dense network of fibres; approximately 20% of the implanted dopaminergic cells survive for several months. However, dopaminergic fibre outgrowth and mesencephalic graft development appear lower in control, non-lesioned, animals. Moreover, implants of embryonic hypothalamic dopaminergic neurons show little or no survival. These results indicate that interactions between embryonic and adult neurons are selective. We suggest that this specificity may be sustained by the action of still unknown trophic and/or tropic factors, possibly produced by the lesioned striatum and by putative inhibitory mechanisms of cell migration and neuritic outgrowth.     

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.     

Membrane-bound CSPG mediates growth cone outgrowth and substrate specificity by Schwann cell contact with the DRG neuron cell body and not via growth cone contact

The central nervous system and peripheral nervous system (CNS/PNS) contain factors that inhibit axon regeneration, including myelin-associated glycoprotein (MAG), the Nogo protein, and chondroitin sulfate proteoglycan (CSPG). They also contain factors that promote axon regeneration, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Axon regeneration into and within the CNS fails because the balance of factor favors inhibiting regeneration, while in the PNS, the balance of factor favors promoting regeneration. The balance of influences in the CNS can be shifted toward promoting axon regeneration by eliminating the regeneration-inhibiting factors, overwhelming them with regeneration-promoting factors, or making axon growth cones non-receptive to regeneration-inhibiting factors. The present in vitro experiments, using adult rat dorsal root ganglion (DRG) neurons, were designed to determine whether the regeneration-inhibiting influences of Schwann cell CSPG are mediated via Schwann cell membrane contact with the DRG neuron cell body or their growth cones. The average longest neurite of neurons in cell body contact with Schwann cells was 7.4-fold shorter than those of neurons without Schwann cell-neuron cell body contact (naked neurons), and the neurites showed substrate specificity, growing only on the Schwann cell membranes and not extending onto the laminin substrate. The neurites of naked neurons showed no substrate specificity and extended over the laminin substrate, as well as onto and off the Schwann cells. After digesting the Schwann cell CSPG with the enzyme C-ABC, neurons in cell body contact with Schwann cells extended neurites the same length as those of naked neurons, and their neurites showed no substrate selectivity. Further, the neurites of naked neurons were not longer than those of naked neurons not exposed to C-ABC. These data indicate that the extent of neurite outgrowth from adult rat DRG neurons and substrate specificity of their growth cone is mediated via contact between the Schwann cell membrane-bound CSPG and the DRG neuron cell body and not with their growth cones. Further, there was no apparent influence of diffusible or substrate-bound CSPG on neurite outgrowth. These results show that eliminating the CSPG of Schwann cells in contact with the cell body of DRG neurons eliminates the sensitivity of their growth cones to the CSPG-induced outgrowth inhibition. This may in turn allow the axons of these neurons to regenerate through the dorsal roots and into the spinal cord.     

Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts

The aim of this study was to investigate the effects of vascular endothelial growth factor (VEGF) on regeneration of the rat sciatic nerve in vivo. To that end we used 10-mm long cell-free nerve grafts to bridge a gap in the sciatic nerve. The grafts were pretreated with either VEGF (50, 100 or 250 ng/ml), nerve growth factor (NGF, 100 ng/ml) or laminin (100 ng/ml) before implantation. Outgrowth of axons, Schwann cells, blood vessels and macrophages were studied 10 days post-implantation by the use of immunocytochemistry and histochemistry. Grafts pretreated with VEGF stimulated the outgrowth of Schwann cells and blood vessels but not axons. In such grafts, the Schwann cells also exhibited a dramatic change in morphology and became filled with large lipid-containing vacuoles. These cells also showed an intense immunoreactivity for the VEGF receptor flk-1. Neither pretreatment with laminin nor NGF affected the outgrowth of Schwann cells. However, NGF treatment increased the number of axons in the graft but was not able to counteract injury-induced downregulation of substance P in the dorsal root ganglia. The results show that local application of VEGF promotes at least two events, invasion of Schwann cells and neovascularization, which are important during nerve regeneration. The findings suggest that the effects of the pretreatment by the growth factors is local and limited to the graft, whereas central events like neuropeptide synthesis is not affected.     

Effects of acetylcholinesterase and butyrylcholinesterase on cell survival, neurite outgrowth, and voltage-dependent calcium currents of embryonic ventral mesencephalic neurons

The aim of this study was to investigate the effect of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) on cell survival, neurite outgrowth and voltage-dependent calcium currents in developing rat ventral mesencephalic (VM) neurons. Both BuChE and AChE have been shown to promote neurite outgrowth in postnatnal preparations. However, the effect of these substances has never been investigated on rat embryonic VM cells, which are used in animal models of foetal transplantation as a treatment for Parkinson's disease. The effects of incubation with BuChE and tetrameric (G(4))- or monomeric (G(1))-AChE on cell survival and neurite outgrowth were characterised over a 7-day period on dopaminergic cells within embryonic VM cultures. The acute effects of these treatments on voltage-dependent calcium currents from embryonic VM cells were then investigated using whole-cell voltage-clamp recordings. The chronic effect of modulating voltage-dependent calcium channels was subsequently explored using the selective calcium channel antagonists omega-agatoxin IVA, omega-conotoxin GVIA, and nifedipine. The results presented here demonstrate firstly trophic effects of BuChE and G(4)- and G(1)-AChE upon dopaminergic neurite outgrowth, secondly that BuChE and G(4)- and G(1)-AChE have an inhibitory effect on voltage-dependent calcium currents, and finally that selective voltage-dependent calcium channel inhibitors also have trophic effects upon dopaminergic neurite outgrowth.     

Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination

Glial cell line-derived neurotrophic factor (GDNF) is the prototypical member of a growth factor family that signals via the cognate receptors ret and GDNF-receptor alpha-1. The latter receptors are expressed on a variety of neurons that project into the spinal cord, including supraspinal neurons, dorsal root ganglia, and local neurons. Although effects of GDNF on neuronal survival in the brain have previously been reported, GDNF effects on injured axons of the adult spinal cord have not been investigated. Using an ex vivo gene delivery approach that provides both trophic support and a cellular substrate for axonal growth, we implanted primary fibroblasts genetically modified to secrete GDNF into complete and partial mid-thoracic spinal cord transection sites. Compared to recipients of control grafts expressing a reporter gene, GDNF-expressing grafts promoted significant regeneration of several spinal systems, including dorsal column sensory, regionally projecting propriospinal, and local motor axons. Local GDNF expression also induced Schwann cell migration to the lesion site, leading to remyelination of regenerating axons. Thus, GDNF exerts tropic effects on adult spinal axons and Schwann cells that contribute to axon growth after injury.     

Growth of embryonic retinal neurites elicited by contact with Schwann cell surfaces is blocked by antibodies to L1

Explants from embryonic rat retina plated on Schwann cell monolayers were used to examine the mechanisms by which these central neurons interact with Schwann cell surfaces. Embryonic retinal explants extend neurites reliably on Schwann cell surfaces (Kleitman et al., 1988, J. Neurosci. 8: 653). Antibodies to molecules thought to be present on Schwann cell surfaces (laminin and the 217C antigen), on retinal neurite surfaces (Thy-1.1), or on both surfaces (L1) were tested for their ability to influence this neurite growth. Of these, only antibodies to L1 were effective in blocking retinal neurite extension on Schwann cells. Inhibition of neurite growth by anti-L1 was shown to be specific to growth on Schwann cell surfaces because neurite growth on air-dried collagen (a substratum known to support retinal neurite outgrowth) was not affected. This blockage was dose-dependent. At a low titer of anti-L1 Fab fragments defasciculation of neurites was prominent; at high titers 95% of neurite outgrowth was inhibited. This virtual elimination of the ability of Schwann cell surfaces to support embryonic retinal neurite growth in the presence of antibodies to L1 indicates that binding of the L1 molecule is a critical component of the mechanism by which Schwann cells foster the growth of these neurites. The present experiments concur with the growing body of evidence that L1 plays an important role in supporting neurite growth on cell surfaces and raise the possibility that L1 may also mediate the striking ability of adult retinal axons to regenerate in a peripheral nerve environment.     

Role of N-cadherin in Schwann cell precursors of growing nerves

In the present paper, we determine the localization and developmental regulation of N-cadherin in embryonic rat nerves and examine the role of N-cadherin in this system. We also identify a major transition in the architecture of embryonic nerves and relating it to N-cadherin expression. We find that in early embryonic nerves, N-cadherin is primarily expressed in Schwann cell precursors. Pronounced expression is seen at distal nerve fronts where these cells associate with growth cones, and the proximal nerve ends, in boundary cap cells. Unexpectedly, N-cadherin is downregulated as precursors generate Schwann cells, coinciding with the time at which most axons make target connections. Therefore, glial N-cadherin expression is essentially restricted to the period of axon outgrowth. We also provide evidence that N-cadherin supports the formation of contacts between Schwann cell precursors and show that these cells are a favorable substrate for axon growth, unlike N-cadherin-negative Schwann cells. Induction of N-cadherin expression in Schwann cells by neuregulin-1 restores their ability to form contacts and support axon growth. Finally, we show that the loss of glial N-cadherin during embryonic nerve development is accompanied by a transformation of nerve architecture, involving the appearance of endoneurial connective tissue space, fibroblasts, Schwann cell basal lamina, and blood vessels. Because N-cadherin is likely to promote the extensive glial contacts typical of the compact embryonic nerve, we suggest that N-cadherin loss at the time of Schwann cell generation allows endoneurial space to appear between the glial cells, a development that eventually permits the extensive interactions between connective tissue and individual axon-Schwann cell units necessary for myelination.     

Immature optic nerve glia of rat do not promote axonal regeneration when transplanted into a peripheral nerve

The ability of immature central nervous system (CNS) glia to promote axonal regeneration was studied by grafting segments of embryonic and neonatal rat optic nerves into the sciatic nerves of adult rats. Unexpectedly, very few axons regenerated through these grafts. The majority of the axons bypassed the grafts and were associated with Schwann cells. These results were similar to those obtained with grafts of adult rat optic nerves. The failure of immature CNS glia to promote axonal regeneration under these conditions suggests that they may be less effective than Schwann cells in promoting the regeneration and growth of axons.     

Strands of embryonic mesencephalic tissue show greater dopamine neuron survival and better behavioral improvement than cell suspensions after transplantation in parkinsonian rats

The 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.     

Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.     

ROCK inhibition promotes adult retinal ganglion cell neurite outgrowth only in the presence of growth promoting factors

Lesioned central nervous system (CNS) axons fail to regenerate because of limited availability of neurotrophic factors (NTF) to promote neuron survival and drive axon regeneration through an environment rich in multiple myelin- and non myelin-derived axon growth inhibitory ligands that initiate growth cone collapse through the Rho/Rho kinase (ROCK) signalling pathway. However, pharmacological inhibition of Rho and ROCK promotes neurite outgrowth in PC12, Ntera-2 cells and embryonic/early postnatal neurons in culture. We have used our well-characterised CNS myelin-inhibited adult rat retinal culture model to show that Y27632 only promotes disinhibited neurite outgrowth if RGC are co-stimulated with ciliary neurotrophic factor (CNTF). Y27632 in CNTF-stimulated retinal cultures promotes optimal RGC neurite outgrowth at 10 μM concentrations, while higher concentrations negatively correlate with RGC neurite outgrowth and survival. Raising the levels of cAMP in Y27632-treated retinal cultures also promotes significant RGC neurite outgrowth, an effect that is potentiated by the further inclusion of CNTF. Our results suggest that Y27632-induced ROCK inhibition promotes robust disinhibited axon regeneration of adult neurons only when growth promoting factors are added and/or cAMP levels are raised.     

Delivery of sonic hedgehog or glial derived neurotrophic factor to dopamine-rich grafts in a rat model of Parkinson's disease using adenoviral vectors Increased yield of dopamine cells is dependent on embryonic donor age

The poor survival of dopamine grafts in Parkinson's disease is one of the main obstacles to the widespread application of this therapy. One hypothesis is that implanted neurons, once removed from the embryonic environment, lack the differentiation factors needed to develop the dopaminergic phenotype. In an effort to improve the numbers of dopamine neurons surviving in the grafts, we have investigated the potential of adenoviral vectors to deliver the differentiation factor sonic hedgehog or the glial cell line-derived neurotrophic factor GDNF to dopamine-rich grafts in a rat model of Parkinson's disease. Adenoviral vectors containing sonic hedgehog, GDNF, or the marker gene LacZ were injected into the dopamine depleted striatum of hemiparkinsonian rats. Two weeks later, ventral mesencephalic cell suspensions were prepared from embryos of donor ages E12, E13, E14 or E15 and implanted into the vector-transduced striatum. Pre-treatment with the sonic hedgehog vector produced a three-fold increase in the numbers of tyrosine hydroxylase-positive (presumed dopaminergic) cells in grafts derived from E12 donors, but had no effect on E13-E15 grafts. By contrast, pre-treatment with the GDNF vector increased yields of dopamine cells in grafts derived from E14 and E15 donors but had no effect on grafts from younger donors. The results indicate that provision of both trophic and differentiation factors can enhance the yields of dopamine neurons in ventral mesencephalic grafts, but that the two factors differ in the age and stage of embryonic development at which they have maximal effects.     

Long distance selective fiber outgrowth of transplanted hNT neurons in white matter tracts of the adult rat brain

Terminally differentiated neurons derived from a human teratocarcinoma cell line (NT2N or hNT neurons) are promising as a cell source for transplantation, as they have been shown to be safe for transplantation in humans. We have shown previously that hNT neurons can express a catecholaminergic phenotype in a rat Parkinson model. In this study, we investigated the long-term survival and ability of hNT neurons to express tyrosine hydroxylase and reconstruct the dopamine-denervated nigrostriatal pathway. Hemiparkinsonian rats received grafts of 400,000 viable hNT neurons into each of the denervated striatum and substantia nigra. Robust hNT grafts were detected up to 24 weeks posttransplantation, although few cells expressed tyrosine hydroxylase. Many hNT fibers were often associated with ipsilateral and contralateral white matter tracts--corpus callosum, rostral migratory stream, optic tract, and external capsule. Fewer fibers were associated with the superior cerebellar peduncle, medial lemniscus, and nigrostriatal pathway. Axons also projected into the frontal cortex and extended parallel to the surface of the brain in the superficial cortical layers. These pathways were seen in all grafted animals, suggesting that specific guidance cues exist in the adult brain governing hNT fiber outgrowth. Injured adult axons and transplanted embryonic neuronal axons rarely extend for such distances in the adult nervous system. We propose that elucidating the factors promoting and guiding hNT axonal outgrowth could provide important clues to enhancing regeneration and target reinnervation in the adult brain, two factors of critical importance for cell restoration strategies aimed at brain repair.     

Ephrins stimulate or inhibit neurite outgrowth and survival as a function of neuronal cell type

The Eph family of tyrosine kinase receptors and ligands play key roles in cell segregation and axon targeting in the developing nervous system. Interactions between the ligands and receptors cause repulsion or degeneration of receptor-positive axons from several brain regions including the retina, hippocampus, thalamus, and midbrain dopaminergic system. We extend these previous observations by showing that three A-ephrins also negatively regulate the growth of neurites from striatal and olfactory neurons. In addition to negative effects, however, we also report a trophic activity of the A-ephrins: Ephrin-A2 and A5 promote survival and neurite outgrowth of sympathetic neurons. These observations provide support to the notion that ephrins may function as either negative or positive signals in the developing nervous system.     

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.     

The neurotrophin NT4/5, but not NT3, enhances the efficacy of nigral grafts in a rat model of Parkinson''s disease

The neurotrophins NT4/5 and NT3 have previously been shown to improve the survival and fibre outgrowth of embryonic dopaminergic neurons in vitro. In the present study we attempted to augment the efficacy of embryonic nigral grafts in vivo. This was done by directly infusing the neurotrophins intraparenchymally in close proximity to transplanted nigral tissue placed in the dopamine depleted striatum of 6-hydroxydopamine lesioned rats. Our results indicated that NT4/5, but not NT3, stimulated fibre growth from embryonic nigral cells and enhanced functional efficacy of the grafts as assessed by metamphetamine-induced rotation.