Spontaneous and augmented growth of axons in the primate spinal cord: effects of local injury and nerve growth factor-secreting cell grafts

Little is known about molecular and cellular responses to spinal cord injury in primates. In this study, the normal milieu of the primate spinal cord was disturbed by multiple needle penetrations and cell injections in the mid-thoracic spinal cord; subsequent effects on local axons and expression of extracellular matrix (ECM) molecules were examined, together with effects of cellular delivery of nerve growth factor (NGF) to the injured region. Four adult rhesus monkeys each received injections of two grafts of autologous fibroblasts genetically modified to secrete human NGF, and, in control injection sites, two separate grafts of autologous fibroblasts transduced to express the reporter gene, beta-galactosidase. Three months later, Schwann cells extensively infiltrated the region of localized injury and penetrated both NGF and control fibroblast grafts. Marked upregulation of several ECM molecules occurred, including chondroitin and heparan sulfate proteoglycans and type IV collagen, in or adjacent to all injection sites. Schwann cells were an apparent source of some ECM expression. Spinal cord sensory axons and putative coerulospinal axons extended into both graft types, but they penetrated NGF grafts to a significantly greater extent. Many of these axons expressed the cell adhesion molecule L1. Thus, extensive cellular and molecular changes occur at sites of localized primate spinal cord injury and grafting, attributable in part to migrating Schwann cells, and are accompanied by spontaneous axonal plasticity. These molecular and cellular events closely resemble those observed in the rodent spinal cord after injury. Furthermore, as in rodent studies, cellular delivery of a trophic factor significantly augments axonal plasticity in the primate spinal cord.     

GDNF‐enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons

We previously demonstrated that coadministration of glial cell line‐derived neurotrophic factor (GDNF) with grafts of Schwann cells (SCs) enhanced axonal regeneration and remyelination following spinal cord injury (SCI). However, the cellular target through which GDNF mediates such actions was unclear. Here, we report that GDNF enhanced both the number and caliber of regenerated axons in vivo and increased neurite outgrowth of dorsal root ganglion neurons (DRGN) in vitro, suggesting that GDNF has a direct effect on neurons. In SC‐DRGN coculture, GDNF significantly increased the number of myelin sheaths produced by SCs. GDNF treatment had no effect on the proliferation of isolated SCs but enhanced the proliferation of SCs already in contact with axons. GDNF increased the expression of the 140 kDa neural cell adhesion molecule (NCAM) in isolated SCs but not their expression of the adhesion molecule L1 or the secretion of the neurotrophins NGF, NT3, or BDNF. Overall, these results support the hypothesis that GDNF‐enhanced axonal regeneration and SC myelination is mediated mainly through a direct effect of GDNF on neurons. They also suggest that the combination of GDNF administration and SC transplantation may represent an effective strategy to promote axonal regeneration and myelin formation after injury in the spinal cord. © 2009 Wiley‐Liss, Inc.     

Neurotrophism without neurotropism: BDNF promotes survival but not growth of lesioned corticospinal neurons.

Neurotrophic factors exert many effects on the intact and lesioned adult central nervous system (CNS). Among these effects are prevention of neuronal death (neurotrophism) and promotion of axonal growth (neurotropism) after injury. To date, however, it has not been established whether survival and axonal growth functions of neurotrophins can be independently modulated in injured adult neurons in vivo. To address this question, the ability of brain-derived neurotrophic factor (BDNF) to influence corticospinal motor neuronal survival and axonal growth was examined in two injury paradigms. In the first paradigm, a survival assay, adult Fischer 344 rats underwent subcortical lesions followed by grafts to the lesion cavity of syngenic fibroblasts genetically modified to secrete high amounts BDNF or, in control subjects, the reporter gene green fluorescent protein. In control subjects, only 36.2 +/- 7.0% of the retrogradely labeled corticospinal neurons survived the lesion, whereas 89.8 +/- 5.9% (P < 0.001) of the corticospinal neurons survived in animals that received BDNF-secreting grafts. However, in an axonal growth assay, BDNF-secreting cell grafts that were placed into either subcortical lesion sites or sites of thoracic spinal cord injury failed to elicit corticospinal axonal growth. Despite this lack of a neurotropic effect on lesioned corticospinal axons, BDNF-secreting cell grafts placed in the injured spinal cord significantly augmented the growth of other types of axons, including local motor, sensory, and coerulospinal axons. Immunolabeling for tyrosine kinase B (trkB) demonstrated that BDNF receptors were present on corticospinal neuronal somata and apical dendrites but were not detected on their projecting axons. Thus, single classes of neurons in the adult CNS appear to exhibit disparate survival and growth sensitivity to neurotrophic factors, potentially attributable at least in part to differential trafficking of neurotrophin receptors. The possibility of tropic/trophic divergence must be considered when designing strategies to promote CNS recovery from injury.     

Glial cell line-derived neurotrophic factor-enriched bridging transplants promote propriospinal axonal regeneration and enhance myelination after spinal cord injury

Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-beta (TGF-beta) family, is widely expressed in the developing and adult central nervous system (CNS). At present, limited information is available regarding the effects of GDNF in the repair of spinal cord injury (SCI). In the present study, mini-guidance channels containing either: (1) Matrigel (MG, a basement membrane component), (2) Schwann cells (SCs, 120 x 10(6)/ml) in MG (SC-MG), (3) recombinant human GDNF (rhGDNF, 3 microg/microl) in MG (GDNF-MG), and (4) a combination of all three components (GDNF-SC-MG) were grafted into a T9 hemisection-gap lesion in adult rats to examine the effects of GDNF on axonal regeneration and myelination following SCI. Thirty days post-transplantation, limited axonal growth was observed within guidance channels containing MG-alone (MG). When SCs were added to the channels (SC-MG group), consistent axonal ingrowth containing both myelinated and unmyelinated axons was observed, confirming our previous findings. The addition of GDNF-alone without SCs (GDNF-MG) resulted in substantial ingrowth of unmyelinated axons, suggesting that GDNF has a direct neurite-growth promoting effect on these axons. Implantation of channels containing both GDNF and SCs (GDNF-SC-MG) produced a significant and synergistic increase in axonal regeneration and myelination. In addition, GDNF reduced the extent of reactive gliosis, infiltration of activated macrophages/microglia, and cystic cavitation at the graft-host interfaces. Retrograde tracing revealed that grafts of SC-seeded channels containing GDNF promoted a significant increase in the number of propriospinal neurons which had regenerated their axons into the grafts, as compared to SC-MG-seeded channels. These results indicate that GDNF may play a novel therapeutic role in promoting propriospinal axonal regeneration, enhancing myelin formation, and improving graft-host interfaces after SCI.     

Intrathecal injection of GDNF and BDNF induces immediate early gene expression in rat spinal dorsal horn

Glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are potent trophic factors for dorsal root ganglion cells. In addition, these factors are produced in subsets of dorsal root ganglion cells and transported anterogradely to their terminals in the superficial dorsal horn of the spinal cord, where they constitute the only source of GDNF and BDNF. We investigated the effect of 10 mug GDNF and BDNF injected by lumbar puncture on the expression of the immediate early gene (IEG) products c-Fos, c-Jun, and Krox-24 in the adult rat dorsal horn. In the dorsal horn of S1 spinal segments, GDNF and BDNF induced a strong increase in IEG expression, which was most pronounced in laminae I and II (2.9- to 4.5-fold). More distal from the injection site, in the dorsal horn of L1/L2 spinal segments, the increase in IEG expression was less pronounced, suggesting a concentration-dependent effect. In order to explain the effects of intrathecally injected GDNF, we investigated whether lumbo-sacral dorsal horn neurons expressed RET protein, the signal-transducing element of the receptor complex for GDNF. It was found that several of these neurons contained RET immunoreactivity and that some of the RET-labeled neurons had the appearance of nociceptive-specific cells, confirming their presumed role in pain transmission. Additionally, using double-labeling immunofluorescence combined with confocal microscopy, it was found that after intrathecal GDNF injection 35% of c-Fos-labeled cells were also labeled for RET. These results demonstrate that intrathecally administered GDNF and BDNF induce IEG expression in dorsal horn neurons in the adult rat, supposedly by way of their cognate receptors, which are present on these neurons. We further suggest that the endogenous release of GDNF and BDNF, triggered by nociceptive stimuli, is involved in the induction of changes in spinal nociceptive transmission as in various pain states.     

Differential Regulation of mRNAs for GDNF and its Receptors Ret and GDNFRα After Sciatic Nerve Lesion in the Mouse

Glial cell line-derived neurotrophic factor (GDNF), first characterized for its effect on dopamine uptake in central dopaminergic neurons, appears to be a powerful neurotrophic factor for motor neurons. GDNF has recently been shown to signal through a multisubunit receptor. This receptor is composed of a ligand-binding subunit, called GDNF receptor α (GDNFRα), and a signalling tyrosine kinase subunit, Ret. To gain further insight into GDNF function, we investigated the expression of GDNF and its receptors after nerve lesion in adult mice. Analysis of expression in muscle, nerve and spinal cord by RNase protection assay and in situ hydridization revealed that, in adult non-lesioned mice, GDNF mRNA was expressed in the nerve and GDNFRα mRNA in the nerve and the spinal cord, while the expression of Ret was restricted to spinal cord motor neurons. After a sciatic nerve crush a rapid increase in GDNF mRNA was observed in the distal part of the nerve and a delayed elevation in the muscle, while GDNFRα mRNA was up-regulated in the distal part of the sciatic nerve but not in proximal nerve or spinal cord. The lesion also induced a rapid increase in Ret mRNA expression, but the increase was observed only in spinal cord motor neurons and in dorsal root ganglion neurons. A pattern of expression of GDNF and its receptors similar to that seen after lesion in the adult was detected during embryonic development. Administration of GDNF enhanced sciatic nerve regeneration measured by the nerve pinch test. Taken together, these results suggest that GDNF has an important role during regeneration after nerve damage in the adult.     

Robust Growth of Chronically Injured Spinal Cord Axons Induced by Grafts of Genetically Modified NGF-Secreting Cells

Little spontaneous regeneration of axons occurs after acute and chronic injury to the CNS. Previously we have shown that the continuous local delivery of neurotrophic factors to the acutely injured spinal cord induces robust growth of spinal and supraspinal axons. In the present study we examined whetherchronicallyinjured axons also demonstrate significant neurotrophin responsiveness. Adult rats underwent bilateral dorsal hemisection lesions that axotomize descending supraspinal pathways, including the corticospinal, rubrospinal, and cerulospinal tracts, and ascending dorsal spinal sensory projections. One to three months later, injured rats received grafts of syngenic fibroblasts genetically modified to produce nerve growth factor (NGF). Control subjects received unmodified cell grafts or cells transduced to express the reporter gene β-galactosidase. Three to five months after grafting, animals that received NGF-secreting grafts showed dense growth of putative cerulospinal axons and primary sensory axons of the dorsolateral fasciculus into the grafted lesion site. Growth from corticospinal, raphaespinal, and local motor axons was not detected. Thus, robust growth of defined populations of supraspinal and spinal axons can be elicited in chronic stages after spinal cord injury by localized, continuous transgenic delivery of neurotrophic factors.     

IGF-I gene delivery promotes corticospinal neuronal survival but not regeneration after adult CNS injury

An unmet challenge of spinal cord injury research is the identification of mechanisms that promote regeneration of corticospinal motor axons. Recently it was reported that IGF-I promotes corticospinal axon growth during nervous system development. We therefore investigated whether IGF-I also promotes regeneration or survival of adult lesioned corticospinal neurons. Adult Fischer 344 rats underwent C3 dorsal column transections followed by grafts of IGF-I-secreting marrow stromal cell grafts into the lesion cavity. IGF-I secreting cell grafts promoted growth of raphespinal and cerulospinal axons, but not corticospinal axons, into the lesion/graft site. We then examined whether IGF-I-secreting cell grafts promote corticospinal motor neuron survival or axon growth in a subcortical axotomy model. IGF-I expression coupled with infusion of the IGF binding protein inhibitor NBI-31772 significantly prevented corticospinal motor neuron death (93% cell survival compared to 49% in controls, P < 0.05), but did not promote corticospinal axon regeneration. Coincident with observed effects of IGF-I on corticospinal survival but not growth, expression of IGF-I receptors was restricted to the somal compartment and not the axon of adult corticospinal motor neurons. Thus, whereas IGF-I influences corticospinal axonal growth during development, its application to sites of adult spinal cord injury or subcortical axotomy fails to promote corticospinal axonal regeneration under conditions that are sufficient to prevent corticospinal cell death and promote the growth of other supraspinal axons. We conclude that developmental patterns of growth factor responsiveness are not simply recapitulated after adult injury, potentially due to post-natal shifts in patterns of IGF-I receptor expression.     

GDNF‐treated acellular nerve graft promotes motoneuron axon regeneration after implantation into cervical root avulsed spinal cord

T‐H. Chu, L. Wang, A. Guo, V. W‐K. Chan, C. W‐M. Wong and W. Wu (2012) Neuropathology and Applied Neurobiology 38, 681–695 GDNF‐treated acellular nerve graft promotes motoneuron axon regeneration after implantation into cervical root avulsed spinal cord It is well known that glial cell line‐derived neurotrophic factor (GDNF) is a potent neurotrophic factor for motoneurons. We have previously shown that it greatly enhanced motoneuron survival and axon regeneration after implantation of peripheral nerve graft following spinal root avulsion. Aims: In the current study, we explore whether injection of GDNF promotes axon regeneration in decellularized nerve induced by repeated freeze‐thaw cycles. Methods: We injected saline or GDNF into the decellularized nerve after root avulsion in adult Sprague–Dawley rats and assessed motoneuron axon regeneration and Schwann cell migration by retrograde labelling and immunohistochemistry. Results: We found that no axons were present in saline‐treated acellular nerve whereas Schwann cells migrated into GDNF‐treated acellular nerve grafts. We also found that Schwann cells migrated into the nerve grafts as early as 4 days after implantation, coinciding with the first appearance of regenerating axons in the grafts. Application of GDNF outside the graft did not induce Schwann cell infiltration nor axon regeneration into the graft. Application of pleiotrophin, a trophic factor which promotes axon regeneration but not Schwann cell migration, did not promote axon infiltration into acellular nerve graft. Conclusions: We conclude that GDNF induced Schwann cell migration and axon regeneration into the acellular nerve graft. Our findings can be of potential clinical value to develop acellular nerve grafting for use in spinal root avulsion injuries.     

Glial cell line-derived neurotrophic factor increases calcitonin gene-related peptide immunoreactivity in sensory and motoneurons in vivo

Calcitonin gene-related peptide (CGRP) is expressed at high levels in roughly 50% of spinal sensory neurons and plays a role in peripheral vasodilation as well as nociceptive signalling in the spinal cord. Spinal motoneurons express low levels of CGRP; motoneuronal CGRP is thought to be involved in end-plate plasticity and to have trophic effects on target muscle cells. As both sensory and motoneurons express receptors for glial cell line-derived neurotrophic factor (GDNF) we sought to determine whether CGRP was regulated by GDNF. Rats were treated intrathecally for 1-3 weeks with recombinant human GDNF or nerve growth factor (NGF) (12 microg/day) and dorsal root ganglia and spinal cords were stained for CGRP. The GDNF treatment not only increased CGRP immunoreactivity in both sensory and motoneurons but also resulted in hypertrophy of both populations. By combined in situ hybridization and immunohistochemistry we found that, in the dorsal root ganglia, CGRP was up-regulated specifically in neurons expressing GDNF but not NGF receptors following GDNF treatment. Despite the increase in CGRP in GDNF-treated rats, there was no increase in thermal or mechanical pain sensitivity, while NGF-treated animals showed significant decreases in pain thresholds. In motoneurons, GDNF increased the overall intensity of CGRP immunoreactivity but did not increase the number of immunopositive cells. As GDNF has been shown to promote the regeneration of both sensory and motor axons, and as CGRP appears to be involved in motoneuronal plasticity, we reason that at least some of the regenerative effects of GDNF are mediated through CGRP up-regulation.     

GDNF, RET and GFRalpha-1-3 mRNA expression in the developing human spinal cord and ganglia.

The identification of endogenous neurotrophic factors and their receptors in human spinal cord is important not only to understand development, but also in the consideration of possible future therapies for neurodegenerative disorders and trauma. Using in situ hybridization, the expression of glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), persephin (PSP), GFRalpha-1, GFRalpha-2, GFRalpha-3 and RET mRNA in human fetal spinal cord was studied. Strong GDNF mRNA hybridization signal, presumably restricted to Clarke's nucleus, was detected in the thoracic spinal cord. mRNA encoding GFRalpha-1 was expressed in the entire spinal cord gray matter with particularly high expression in the ventral horn. GFRbeta-1 was also expressed more weakly in dorsal root ganglia. NTN and persephin mRNA were not detected in either the fetal spinal cord or the dorsal root ganglia. mRNA coding for GFRalpha-2, however, was found in most cells of the spinal cord gray matter. A strong expression of GFRalpha-3 mRNA was detected in dorsal root ganglia cells and Schwann cells. The transducing receptor RET was expressed strongly in motorneurons and dorsal root ganglion neurons. We conclude that basic features concerning the role of the GDNF family of ligands and their receptors revealed in rodents applies to humans.     

Oxidized galectin-1 stimulates the migration of Schwann cells from both proximal and distal stumps of transected nerves and promotes axonal regeneration after peripheral nerve injury

Oxidized galectin-1 has recently been identified as a key factor that plays important roles in initial axonal growth in injured peripheral nerves. The aim of this study was to investigate the effects of oxidized galectin-1 on regeneration of rat spinal nerves using acellular autografts (containing no viable cells) and allografts (containing no cell membranes) with special attention to the relationship between axonal regeneration and Schwann cell migration. Immunohistochemically, endogenous galectin-1 was expressed in dorsal root ganglion (DRG) neurons, spinal cord motoneurons, and axons and Schwann cells in normal sciatic nerves. Administration of oxidized recombinant human galectin-1 (rh-gal-lox, 5 ng/ml) in autograft model promoted axonal regeneration from motoneurons as well as from DRG neurons; this was confirmed by a fluorogold tracer study (p < 0.05). Anti-rh-gal-1 antibody (30 microg/ml) strongly inhibited axonal regrowth (p < 0.05). Pretreatment of allografts with rh-gal-lox stimulated the migration of Schwann cells not only from proximal stumps but also from distal stumps into the grafts, resulting in accelerated axonal regeneration (p < 0.05). Moreover, Schwann cell migration preceded the axonal growth in the presence of exogenous rh-gal-lox in the grafts. These results strongly suggest that local administration of exogenous rh-gal-lox promotes the migration of Schwann cells followed by axonal regeneration from both motor and sensory neurons, resulting in acceleration of neuronal repair. This technique may also be of value in the repair of human nerves.     

Expression of receptors for glial cell line-derived neurotrophic factor family ligands in sacral spinal cord reveals separate targets of pelvic afferent fibers

Nerve growth factor has been proposed to mediate many structural and chemical changes in bladder sensory neurons after injury or inflammation. We have examined the expression of receptors for the glial cell line-derived neurotrophic factor (GDNF) family within sensory terminals located in the sacral spinal cord and in bladder-projecting sacral dorsal root ganglion neurons of adult female Sprague-Dawley rats. Nerve fibers immunolabelled for GFRalpha1 (GDNF receptor), GFRalpha2 (neurturin receptor), or GFRalpha3 (artemin receptor) showed distinct distribution patterns in the spinal cord, suggesting separate populations of sensory fibers with different functions: GFRalpha1-labeled fibers were in outer lamina II and the lateral-collateral pathway and associated with autonomic interneurons and preganglionic neurons; GFRalpha2-labeled fibers were only in inner lamina II; GFRalpha3-labeled fibers were in lamina I, the lateral-collateral pathway, and areas surrounding dorsal groups of preganglionic neurons and associated interneurons. Immunofluorescence studies of retrogradely labelled bladder-projecting neurons in sacral dorsal root ganglia showed that approximately 25% expressed GFRalpha1 or GFRalpha3 immunoreactivity, the preferred receptors for GDNF and artemin, respectively. After cyclophosphamide-induced bladder inflammation, fluorescence intensity of GFRalpha1-positive fibers increased within the dorsal horn, but there was no change in the GFRalpha2- or GFRalpha3-positive fibers. These studies have shown that GDNF and artemin may target bladder sensory neurons and potentially mediate plasticity of sacral visceral afferent neurons following inflammation. Our results have also revealed three distinct subpopulations of sensory fibers within the sacral spinal cord, which have not been identified previously using other markers.     

Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1.

Schwann cells contribute to efficient axonal regeneration after peripheral nerve injury and, when grafted to the central nervous system (CNS), also support a modest degree of central axonal regeneration. This study examined (1) whether Schwann cells grafted to the CNS exhibit normal patterns of differentiation and association with spinal axons and what signals putatively modulate these interactions, and (2) whether Schwann cells overexpressing neurotrophic factors enhance axonal regeneration. Thus, primary Schwann cells were transduced to hypersecrete human nerve growth factor (NGF) and were grafted to spinal cord injury sites in adult rats. Comparisons were made to nontransfected Schwann cells. From 3 days to 6 months later, grafted Schwann cells exhibited a phenotypic and temporal course of differentiation that matched patterns normally observed after peripheral nerve injury. Schwann cells spontaneously aligned into regular spatial arrays within the cord, appropriately remyelinated coerulospinal axons that regenerated into grafts, and appropriately ensheathed but did not myelinate sensory axons extending into grafts. Coordinate expression of the cell adhesion molecule L1 on Schwann cells and axons correlated with establishment of appropriate patterns of axon-Schwann cell ensheathment. Transduction of Schwann cells to overexpress NGF robustly increased axonal growth but did not otherwise alter the nature of interactions with growing axons. These findings suggest that signals expressed on Schwann cells that modulate peripheral axonal regeneration and myelination are also recognized in the CNS and that the modification of Schwann cells to overexpress growth factors significantly augments their capacity to support extensive axonal growth in models of CNS injury.     

Axonal responses to cellularly delivered NT-4/5 after spinal cord injury

Neurotrophic factors delivered to the injured spinal cord have been shown to enhance axonal growth, prevent neuronal degeneration and partially improve sensorimotor function. The present study examined the effects of NT-4/5 on growth of spinal and supraspinal axons, glia, and functional outcome after spinal cord injury. Adult Fischer 344 rats received spinal cord dorsal hemisections or complete transections at the midthoracic level. Fibroblasts modified to secrete NT-4/5 or green fluorescent protein as controls were immediately grafted to the lesion site. Axonal growth responses were determined between 3 and 6 months postinjury by retrograde and anterograde tracing and immunohistochemistry. Motor axons, coerulospinal, reticulospinal, and propriospinal axons responded to NT-4/5 delivery after thoracic spinal cord injury with significantly increased axonal penetration into NT-4/5 secreting grafts compared to GFP-expressing control grafts. Axonal growth beyond NT-4/5-producing grafts and functional recovery were not observed. Numerous Schwann cells, but not oligodendrocytes, were present within NT-4/5-secreting grafts and remyelinated axons inside the graft. Thus, NT-4/5 and BDNF appear to be interchangeable to elicit substantial axonal growth in the injured spinal cord.     

The expression of nuclear 3,5,3' triiodothyronine receptors is induced in Schwann cells by nerve transection.

The effects of thyroid hormones on the nervous system are mediated by the presence of nuclear T3 receptors (NT3R). In this study, the expression of NT3R was investigated in spinal cord, dorsal root ganglia (DRG), or sciatic nerve of adult rats after immunostaining with a 2B3-NT3R monoclonal antibody which recognizes both alpha and beta types of NT3R. The specificity of this monoclonal antibody was confirmed by Western blots. The 2B3-NT3R monoclonal antibody recognized one band corresponding to a molecular weight of 57 kDa in extract of spinal cord or DRG. No staining was observed on immunoblot of intact sciatic nerve. In the spinal cord, the nuclei of the neurons and glial cells including both astrocytes and oligodendrocytes exhibited 2B3-NT3R immunoreactivity. While all the nuclei of the DRG sensory neurons expressed the NT3R, all the nuclei of the satellite and Schwann cells were devoid of any immunoreaction. In the sciatic nerve, the nuclei of the Schwann cells also lacked 2B3-NT3R-immunoreactivity. After sciatic nerve transection in vivo, Schwann cell nuclei, which never expressed NT3R in intact nerves of adult rats, displayed a clear 2B3-NT3R immunoreaction in proximal and distal stumps adjacent to the section. Double immunostaining with antibodies raised to 3-sulfogalactosylceramide or S100 confirmed that most of the NT3R containing nuclei belong to Schwann cells. In dissociated cell cultures grown in vitro from sciatic nerves, Schwann cells exhibited 2B3-NT3R immunoreactivity. These data suggest that the inhibition of NT3R expression in Schwann cells ensheathing axons in intact nerve is reversed when the axons are degenerating or lacking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury

Neural stem cells (NSCs) offer the potential to replace lost tissue after nervous system injury. This study investigated whether grafts of NSCs (mouse clone C17.2) could also specifically support host axonal regeneration after spinal cord injury and sought to identify mechanisms underlying such growth. In vitro, prior to grafting, C17.2 NSCs were found for the first time to naturally constitutively secrete significant quantities of several neurotrophic factors by specific ELISA, including nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. When grafted to cystic dorsal column lesions in the cervical spinal cord of adult rats, C17.2 NSCs supported extensive growth of host axons of known sensitivity to these growth factors when examined 2 weeks later. Quantitative real-time RT-PCR confirmed that grafted stem cells expressed neurotrophic factor genes in vivo. In addition, NSCs were genetically modified to produce neurotrophin-3, which significantly expanded NSC effects on host axons. Notably, overexpression of one growth factor had a reciprocal effect on expression of another factor. Thus, stem cells can promote host neural repair in part by secreting growth factors, and their regeneration-promoting activities can be modified by gene delivery.     

A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell line SCTM41 enhances survival and fiber outgrowth from embryonic nigral neurons grafted to the striatum and to the lesioned substantia nigra.

We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson's disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.     

Peripheral nerve autografts to the rat spinal cord: Studies with axonal tracing methods

In young adult female rats, autologous sciatic nerve segments were transplanted to the thoracic region of the spinal cord. The grafts became well innervated but led to no obvious functional improvement. The origin and termination of axons in the grafts was studied by retrograde neuronal labeling with horseradish peroxidase (HRP) and radioautographic axonal tracing. Studies with HRP indicated that some axons in the grafts originated from intrinsic CNS neurons with their cell bodies in nearby segments of the spinal cord and that others arose from dorsal root ganglia at the level of the grafts and at least 7 segments distal to them. After tritiated amino acids were injected into lumbar dorsal root ganglia, labeled axons could be followed into the grafts but not into the rostral spinal cord stumps. Together with other experimental observations, these results demonstrate a correlation between success or failure of elongation of dorsal root fibers and peripheral or central ensheathment at the axonal tip. The corticospinal tract was studied both with radioautography and retrograde axonal transport of HRP but no extension of its axons into peripheral nerve grafts was detected under these experimental conditions. The findings implicate both neuroglial and axonal factors in the feeble regenerative response usually seen after injury to the spinal cord.