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.     

Induction of axon growth into Schwann cell implants grafted into lesioned adult rat spinal cord.

Polymerized collagen rolls enclosing Schwann cells (SCs) raised in culture were grafted into cystic cavities formed after lesioning the thoracic spinal cord of adult rats. Axons were already present within the graft by 14 days after implantation and both ensheathed and myelinated axons were numerous by 28 days. This axonal ingrowth was maintained over longer survival periods. The axons within the graft always appeared related to Schwann cells. Acellular collagen rolls did not show axonal ingrowth. These Schwann cell-collagen implants resemble peripheral nerve grafts in their ability to induce axonal regeneration into the graft.     

Nerve growth factor promotes CNS cholinergic axonal regeneration into acellular peripheral nerve grafts

Peripheral nerve grafts promote vigorous regeneration of adult mammalian CNS axons. Elimination of nerve-associated cells by freeze-thawing abolishes this promoting quality, possibly by creating inhibitory cellular debris and/or destroying the production of stimulatory factors by living Schwann or other cells. Here, debris-free acellular peripheral nerve segments placed between the disconnected septum and the hippocampal formation acquired almost no cholinergic axons after 1 month. However, such acellular nerve grafts treated before implantation with purified beta-nerve growth factor (NGF) contained nearly as many longitudinally oriented cholinergic axons as did fresh cellular nerve grafts. These results suggest that (i) NGF is required for the regeneration of adult CNS cholinergic axons into nerve grafts and (ii) an important function of living cells within peripheral nerve may be the production of neuronotrophic factors such as NGF.     

Regrowth of axons into the distal spinal cord through a Schwann-cell-seeded mini-channel implanted into hemisected adult rat spinal cord

Schwann cells (SCs) have been shown to be a key element in promoting axonal regeneration after being grafted into the central nervous system (CNS). In the present study, SC-supported axonal regrowth was tested in an adult rat spinal cord implantation model. This model is characterized by a right spinal cord hemisection at the eighth thoracic segment, implantation of a SC-containing mini-channel and restoration of cerebrospinal fluid circulation by suturing the dura. We demonstrate that a tissue cable containing grafted SCs formed an effective bridge between the two stumps of the hemicord 1 month after transplantation. Approximately 10 000 myelinated and unmyelinated axons (1 : 9) per cable were found at its midpoint. In addition to propriospinal axons and axons of peripheral nervous system (PNS) origin, axons from as many as 19 brainstem regions also grew into the graft without additional treatments. Most significantly, some regenerating axons in the SC grafts were able to penetrate through the distal graft-host interface to re-enter the host environment, as demonstrated by anterograde axonal labelling. These axons coursed toward, and then entered the grey matter where terminal bouton-like structures were observed. In channels containing no SCs, limited axonal growth was seen within the graft and no axons penetrated the distal interface. These findings further support the notion that SCs are strong promotors of axonal regeneration and that the mini-channel model may be appropriate for further investigation of axonal re-entry, synaptic reconnection and functional recovery following spinal cord injury.     

Adenoviral vector-mediated expression of a foreign gene in peripheral nerve tissue bridges implanted in the injured peripheral and central nervous system

Axons of the CNS do normally not regenerate after injury, in contrast to axons of the PNS. This is due to a different microenvironment at the site of the lesion as well as a particular intrinsic program of axonal regrowth. Although transplantation of peripheral nerve tissue bridges is perhaps the most successful approach to promoting regeneration in the CNS, ingrowth of CNS nerve fibers with such transplants is limited. Genetic modification of peripheral nerve bridges to overexpress outgrowth-promoting proteins should, in principle, improve the permissive properties of peripheral nerve transplants. The present study shows that pieces of peripheral intercostal nerve, subjected to ex vivo adenoviral vector-mediated gene transfer and implanted as nerve bridges in transected sciatic nerve, avulsed ventral root, hemi-sected spinal cord and intact brain, are capable of expressing a foreign gene. In vitro studies showed expression of the reporter gene LacZ up to 30 days in Schwann cells. After implantation, LacZ expression could be detected at 7 days postimplantation, but had virtually disappeared at 14 days. Schwann cells of the transduced nerve bridges retained the capacity of guiding regenerative peripheral and central nerve fiber ingrowth. Transduction of intercostal nerve pieces prior to implantation should, in principle, enable enhanced local production of neurotrophic factors within the transplant and has the potential to improve the regeneration of injured axons into the graft.     

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.     

Nerve growth factor infusion into the denervated adult rat hippocampal formation promotes its cholinergic reinnervation

The well-documented but little-understood failure of lengthy axonal regeneration after injury of the adult mammalian CNS may be caused by an insufficient availability of local growth-promoting factors. If so, identifying and supplying the missing factors may result in better central axonal regeneration. This hypothesis was tested in an adult rat CNS model in which peripheral nerve grafts were placed into a lesion cavity between the septum and hippocampal formation. Continuous infusion of nerve growth factor (NGF) into the dorsal hippocampal tissue dramatically enhanced and accelerated the regrowth and penetration of cholinergic axons into the hippocampal formation. Thus, NGF can overcome the apparent resistance of the hippocampal CNS tissue to cholinergic reinnervation.     

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.     

Expression of insulin-like growth factors and corresponding binding proteins (IGFBP 1–6) in rat spinal cord and peripheral nerve after axonal injuries

Insulin-like growth factors (IGFs) exert trophic effects on several different cell types in the nervous system, including spinal motoneurons. After peripheral nerve injury, the increased expression of IGFs in the damaged nerve has been suggested to facilitate axonal regeneration. Here we have examined the expression pattern of mRNAs encoding IGF-1 and and -2, IGF binding proteins (IGFBPs) 1–6 in the rat spinal cord and peripheral nerve in three lesion models affecting lumbar motoneurons, i.e., sciatic nerve transection, ventral root avulsion, and a cut lesion in the ventral funiculus of the spinal cord. The expression was also studied in enriched Schwann cell and astrocyte cultures. The injured sciatic nerve expressed IGF-1 and IGF-2 as well as IGFBP-4 and IGFBP-5, whereas central nervous system (CNS) scar tissue expressed IGF-1, IGFBP-2, and IGFBP-5. IGFBP-6 mRNA was strongly upregulated in spinal motoneurons after all three types of lesions. IGFBP-6-like immunoreactivity was present in motoneuron cell bodies, dendrites in the ventral horn, and axons in the sciatic nerve. In line with the in vivo findings, cultured Schwann cells expressed IGF-1, IGF-2, IGFBP-4, and IGFBP-5 mRNAs, whereas cultured astrocytes expressed IGF-1, IGFBP-2, and IGFBP-5 mRNAs. These findings show that IGF-1 is available for lesioned motoneurons both after peripheral and central axonal lesions, whereas there are clear differences in the expression patterns for IGF-2 and some of the binding proteins in CNS and peripheral nervous system (PNS) scar tissue. The robust upregulation of IGFBP-6 mRNA in lesioned motoneurons suggests that this binding protein may be of special relevance for the severed cells. J. Comp. Neurol. 400:57–72, 1998. © 1998 Wiley-Liss, Inc.     

The expression of nerve growth factor receptor on Schwann cells and the effect of these cells on the regeneration of axons in traumatically injured human spinal cord

To investigate the effects of Schwann cells and nerve growth factor receptor (NGFR) on the regeneration of axons, autopsy specimens of spinal cord from 21 patients with a survival time of 2 h to 54 years after spinal cord trauma were studied using immunohistochemistry and electron microscopy. Regenerating sprouts of axons could be observed as early as 4 days after trauma. At 4.5 months after trauma, many regenerating nests of axons appeared in the injured spinal cord. The regeneration nests contained directionally arranged axons and Schwann cells. Some axons were myelinated. In injured levels of the spinal cord, the Schwann cells exhibited an increased expression of NGFR within spinal roots. These results show that an active regeneration process occurs in traumatically injured human spinal cord. The NGFR expressed on Schwann cells could mediate NGF to support and induce the axon regeneration in the central nervous system. Received: 20 June 1995 / Revised, accepted: 18 September 1995     

Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: ultrastructural location, suppression by axonal contact, and binding properties

Axotomy of sciatic nerve fibers in adult rats induces expression of NGF receptor in the entire population of Schwann cells located distal to the injury (Taniuchi et al., 1986b). In the present study we have used immunocytochemistry, with a monoclonal antibody directed against the rat NGF receptor, to examine axotomized peripheral nerves by light and electron microscopy. We have found that (1) the NGF receptor molecules were localized to the cell surface of Schwann cells forming bands of Bungner; (2) axonal regeneration into the distal portion of sciatic nerve coincided temporally and spatially with a decrease in Schwann cell expression of NGF receptor; (3) Schwann cell NGF receptor could be induced by axotomy of NGF-independent neurons, such as motoneurons and parasympathetic neurons; and (4) the presence of axon-Schwann cell contact was inversely related to expression of Schwann cell NGF receptor. Using biochemical assays we have found that, in striking contrast to peripheral nerves, there was no detectable induction of NGF receptor in the spinal cord and brain after axotomy of NGF receptor-bearing fibers. Filtration assays of 125I-NGF binding to the induced NGF receptors of Schwann cells measured a Kd of 1.5 nM and a fast dissociation rate, both characteristics of class II receptor sites. We conclude that Wallerian degeneration induces Schwann cells, but not central neuroglia, to produce and position upon their plasmalemmal surface the class II NGF receptor molecules. The induction is ubiquitous among Schwann cells, irrespective of the type of axon they originally ensheathed. Expression of Schwann cell NGF receptor is negatively regulated by axonal contact, being induced when axons degenerate and suppressed when regenerating axons grow out along the Schwann cell surface. We propose that the induced NGF receptors function to bind NGF molecules upon the Schwann cell surface and thereby provide a substratum laden with trophic support and chemotactic guidance for regenerating sensory and sympathetic neurons.     

A combination of insulin-like growth factor-I and platelet-derived growth factor enhances myelination but diminishes axonal regeneration into Schwann cell grafts in the adult rat spinal cord

Insulin-like growth factor-I (IGF-I) promotes axonal regeneration in the peripheral nervous system and this effect is enhanced by platelet-derived growth factor (PDGF). We decided, therefore, to study the effects of these factors on axonal regeneration in the adult rat spinal cord. Semipermeable polymer tubes, closed at the distal end, containing Matrigel mixed with cultured rat Schwann cells and IGF-I/PDGF, were placed at the proximal stump of the spinal cord after removal of the thoracic T9-11 segments. Control animals received implants of only Matrigel and Schwann cells or only Matrigel and IGF-I/PDGF. Four weeks after implantation, electron microscopic analysis showed that the addition of IGF-I/PDGF resulted in an increase in the myelinated:unmyelinated fiber ratio from 1:7 to 1:3 at 3 mm in the Schwann cell graft, and that myelin sheath thickness was increased 2-fold. The reduced number of unmyelinated axons was striking in electron micrographs. These results suggested that IGF-I/PDGF enhanced myelin formation of regenerated axons in Schwann cell implants, but there was a 36% decrease in the total number of myelinated axons at the 3 mm level of the graft. This finding and the altered myelinated:unmyelinated fiber ratio revealed that the overall fiber regeneration into Schwann cell implants was diminished up to 63% by IGF-I/PDGF. Histological evaluation revealed that there were more larger cavities in tissue at the proximal spinal cord-graft interface in animals receiving a Schwann cell implant with IGF-I/PDGF. Such cavitation might have contributed to the reduction in axonal ingrowth. In sum, the results indicate that whereas the combination of IGF-I and PDGF enhances myelination of regenerating spinal cord axons entering implants of Matrigel and Schwann cells after midthoracic transection, the overall regeneration of axons into such Schwann cell grafts is diminished. GLIA 19:247–258, 1997. © 1997 Wiley-Liss, Inc.     

Microtubule stabilization promoted axonal regeneration and functional recovery after spinal root avulsion

A spinal root avulsion injury disconnects spinal roots with the spinal cord. The rampant motoneuron death, inhibitory CNS/PNS transitional zone (TZ) for axonal regrowth and limited regeneration speed together lead to motor dysfunction. Microtubules rearrange to assemble a new growth cone and disorganized microtubules underline regeneration failure. It has been shown that microtubule‐stabilizing drug, Epothilone B, enhanced axonal regeneration and attenuated fibrotic scaring after spinal cord injury. Here, we are reporting that after spinal root avulsion+ re‐implantation in adult rats, EpoB treatment improved motor functional recovery and potentiated electrical responses of motor units. It facilitated axons to cross the TZ and promoted more and bigger axons in the peripheral nerve. Neuromuscular junctions were reformed with better preserved postsynaptic structure, and muscle atrophy was prevented by EpoB administration. Our study showed that EpoB was a promising therapy for promoting axonal regeneration after peripheral nerve injury.     

Nerve growth factor signaling of p75 induces differentiation and ceramide-mediated apoptosis in Schwann cells cultured from degenerating nerves.

In peripheral nerve regeneration or remyelination, immature Schwann cells expressing p75(NTR) play cardinal roles in the support and regeneration of axons (Griffin JW, Hoffman PN. Peripheral Neuropathy 361-376, 1993). Only one of four to six Schwann cells participate in remyelination of damaged or regenerating axons. The rest of the cells, or supernumerary Schwann cells, show severe atrophy and gradually decrease in number, reestablishing a 1:1 axon-Schwann cell relationship (Said G, Duckett S. Acta Neuropathol (Berl) 53:173-179, 1981). Recent reports demonstrated that severely atrophied supernumerary Schwann cells are eliminated by apoptosis during axonal regeneration or remyelination (Hirata H, Hibasami H. Apoptosis 3:353-360, 1998; Berciano MT, Calle E. Acta Neuropathol (Berl) 95:269-279, 1998). The mechanism to induce selective death of supernumerary Schwann cells without causing any damage to axon-associated Schwann cells or axons remains to be determined. In this article, we report that p75(NTR), the low-affinity receptor for all members of neurotrophins, signals both cell differentiation and apoptosis through intracellular ceramide elevation. The final response is dependent on the intracellular ceramide level and Schwann cells modulate their response by changing expression level of p75(NTR). This effect was selective for nerve growth factor (NGF). Taken together, the present study suggests that NGF contributes both to phenotypic regulation and to elimination of the dedifferentiated Schwann cells, while supporting survival or regeneration of certain types of axons during peripheral nerve repair or regeneration.     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

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.     

BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury

Bone marrow stromal cells (MSCs) constitute a heterogeneous cell layer in the bone marrow, supporting the growth and differentiation of hematopoietic stem cells. Recently, it has been reported that MSCs harbor pluripotent stem cells capable of neural differentiation and that simple treatment of MSCs with chemical inducing agents leads to their rapid transdifferentiation into neural cells. We examined whether native or neurally induced MSCs would reconstitute an axonal growth-promoting milieu after cervical spinal cord injury (SCI), and whether such cells could act as vehicles of growth factor gene delivery to further augment axonal growth. One month after grafting to cystic sites of SCI, native MSCs supported modest growth of host sensory and motor axons. Cells "neurally" induced in vitro did not sustain a neural phenotype in vivo and supported host axonal growth to a degree equal to native MSCs. Transduction of MSCs to overexpress brain-derived neurotrophic factor (BDNF) resulted in a significant increase in the extent and diversity of host axonal growth, enhancing the growth of host serotonergic, coerulospinal, and dorsal column sensory axons. Measurement of neurotrophin production from implanted cells in the lesion site revealed that the grafts naturally contain nerve growth factor (NGF) and neurotrophin-3 (NT-3), and that transduction with BDNF markedly raises levels of BDNF production. Despite the extensive nature of host axonal penetration into the lesion site, functional recovery was not observed on a tape removal or rope-walking task. Thus, MSCs can support host axonal growth after spinal cord injury and are suitable cell types for ex vivo gene delivery. Combination therapy with other experimental approaches will likely be required to achieve axonal growth beyond the lesion site and functional recovery.     

Peripheral nerve grafts after cervical spinal cord injury in adult cats

Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with chondroitinase ABC 2 days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4-20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model.     

Axonal regeneration induced by blockade of glial inhibitors coupled with activation of intrinsic neuronal growth pathways

Several pharmacological approaches to promote neural repair and recovery after CNS injury have been identified. Blockade of either astrocyte-derived chondroitin sulfate proteoglycans (CSPGs) or oligodendrocyte-derived NogoReceptor (NgR1) ligands reduces extrinsic inhibition of axonal growth, though combined blockade of these distinct pathways has not been tested. The intrinsic growth potential of adult mammalian neurons can be promoted by several pathways, including pre-conditioning injury for dorsal root ganglion (DRG) neurons and macrophage activation for retinal ganglion cells (RGCs). Singly, pharmacological interventions have restricted efficacy without foreign cells, mechanical scaffolds or viral gene therapy. Here, we examined combinations of pharmacological approaches and assessed the degree of axonal regeneration. After mouse optic nerve crush injury, NgR1-/- neurons regenerate RGC axons as extensively as do zymosan-injected, macrophage-activated WT mice. Synergistic enhancement of regeneration is achieved by combining these interventions in zymosan-injected NgR1-/- mice. In rats with a spinal dorsal column crush injury, a preconditioning peripheral sciatic nerve axotomy, or NgR1(310)ecto-Fc decoy protein treatment or ChondroitinaseABC (ChABC) treatment independently support similar degrees of regeneration by ascending primary afferent fibers into the vicinity of the injury site. Treatment with two of these three interventions does not significantly enhance the degree of axonal regeneration. In contrast, triple therapy combining NgR1 decoy, ChABC and preconditioning, allows axons to regenerate millimeters past the spinal cord injury site. The benefit of a pre-conditioning injury is most robust, but a peripheral nerve injury coincident with, or 3days after, spinal cord injury also synergizes with NgR1 decoy and ChABC. Thus, maximal axonal regeneration and neural repair are achieved by combining independently effective pharmacological approaches.