Stimulating regeneration in the damaged spinal cord.

Great progress has been made in recent years in experimental strategies for spinal cord repair. In this review we describe two of these strategies, namely the use of neurotrophic factors to promote functional regeneration across the dorsal root entry zone (DREZ), and the use of synthetic fibronectin conduits to support directed axonal growth. The junction between the peripheral nervous system (PNS) and central nervous system (CNS) is marked by a specialized region, the DREZ, where sensory axons enter the spinal cord from the dorsal roots. After injury to dorsal roots, axons will regenerate as far as the DREZ but no further. However, recent studies have shown that this barrier can be overcome and function restored. In animals treated with neurotrophic factors, regenerating axons cross the DREZ and establish functional connections with dorsal horn cells. For example, intrathecal delivery of neurotrophin 3 (NT3) supports ingrowth of A fibres into the dorsal horn. This ingrowth is revealed using a transganglionic anatomical tracer (cholera toxin subunit B) and analysis at light and electron microscopic level. In addition to promoting axonal growth, spinal cord repair is likely to require strategies for supporting long-distance regeneration. Synthetic fibronectin conduits may be useful for this purpose. Experimental studies indicate that fibronectin mats implanted into the spinal cord will integrate with the host tissue and support extensive and directional axonal growth. Growth of both PNS and CNS axons is supported by the fibronectin, and axons become myelinated by Schwann cells. Ongoing studies are aimed at developing composite conduits and promoting axonal growth from the fibronectin back into the spinal cord.     

Actions of neurotrophic factors and their signaling pathways in neuronal survival and axonal regeneration

Adult axons in the mammalian central nervous system do not elicit spontaneous regeneration after injury, although many affected neurons have survived the neurotrauma. However, axonal regeneration does occur under certain conditions. These conditions include: (a) modification of regrowth environment, such as supply of peripheral nerve bridges and transplantation of Schwann cells or olfactory ensheathing glia to the injury site; (b) application of neurotrophic factors at the cell soma and axon tips; (c) blockade of growth-inhibitory molecules such as Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein; (d) prevention of chondroitin-sulfate-proteoglycans-related scar tissue formation at the injury site using chondroitinase ABC; and (e) elevation of intrinsic growth potential of injured neurons via increasing intra-cellular cyclic adenosine monophosphate level. A large body of evidence suggests that these conditions achieve enhanced neuronal survival and axonal regeneration through sometimes over-lapping and sometimes distinct signal transduction mechanisms, depending on the targeted neuronal populations and intervention circumstances. This article reviews the available information on signal transduction pathways underlying neurotrophic-factor-mediated neuronal survival and neurite outgrowth/axonal regeneration. Better understanding of signaling transduction is important in helping us develop practical therapeutic approaches for encouraging neuronal survival and axonal regeneration after traumatic injury in clinical context.     

Extracellular regulators of axonal growth in the adult central nervous system

Robust axonal growth is required during development to establish neuronal connectivity. However, stable fibre patterns are necessary to maintain adult mammalian central nervous system (CNS) function. After adult CNS injury, factors that maintain axonal stability limit the recovery of function. Extracellular molecules play an important role in preserving the stability of the adult CNS axons and in restricting recovery from pathological damage. Adult axonal growth inhibitors include a group of proteins on the oligodendrocyte, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein and ephrin-B3, which interact with axonal receptors, such as NgR1 and EphA4. Extracellular proteoglycans containing chondroitin sulphates also inhibit axonal sprouting in the adult CNS, particularly at the sites of astroglial scar formation. Therapeutic perturbations of these extracellular axonal growth inhibitors and their receptors or signalling mechanisms provide a degree of axonal sprouting and regeneration in the adult CNS. After CNS injury, such interventions support a partial return of neurological function.     

Central nervous system growth and differentiation factors: Clinical horizons—Truth or dare?

Growth factors are potent and effective regulators of nerve-cell differentiation and survival. In the past year, several compelling studies have suggested that two proteins, glial derived neurotrophic factor and ciliary neurotrophic factor, may be useful in clinical approaches to treating injury or diseases of the nervous system. In addition, delivery of such factors to the central nervous system may be facilitated by a number of recently reported technologies: growth factor-antibody conjugates, polymerencapsulation and adenovirus vectors. These recent developments are part of new and innovative approaches towards brain repair.     

Dichotomy of the glial cell response to axonal injury and regeneration

Neurons in the mammalian central nervous system (CNS) have a poor capacity for regenerating their axons after injury. In contrast, neurons in the CNS of lower vertebrates and in the peripheral nervous system (PNS) of mammals are endowed with a high posttraumatic capacity to regenerate. The differences in regenerative capacity have been attributed to the different compositions of the respective cellular environments and to different responses to injury the nonneuronal cells display, which range from supportive and permissive to nonsupportive and hostile for regeneration. The same cell type may support or inhibit regeneration, depending on its state of maturity or differentiation. Astrocytes and oligodendrocytes are examples of cells in which such a dichotomy is manifested. In developing and in spontaneously regenerating nerves, these cells support (astrocytes) and permit (oligodendrocytes) growth. However, in nonregenerating adult mammalian nerves, astrocytes form the nonsupportive scar tissue; and the mature oligodendrocytes inhibit axonal growth. Maturation of these cells may be regulated differently during development than after injury. Among the putative regulators are factors derived from astrocytes, resident microglia; or cytokines produced by macrophages. During development, regulation leads to a temporal separation between axonal growth and maturation of the cellular environment, which might not occur spontaneously after injury in a nonregenerating CNS without intervention at the appropriate time. Data suggest that temporal intervention aimed at the glial cells might enhance the poor regenerative capacity of the mammalian CNS. Possible regulation of the nonneuronal cell response to injury via involvement of protooncogenes is proposed.     

Neurotrophin treatment to promote regeneration after traumatic CNS injury

Neurotrophins are a family of growth factors that have been found to be central for the development and functional maintenance of the nervous system, participating in neurogenesis, neuronal survival, axonal growth, synaptogenesis and activity-dependent forms of synaptic plasticity. Trauma in the adult nervous system can disrupt the functional circuitry of neurons and result in severe functional deficits. The limitation of intrinsic growth capacity of adult nervous system and the presence of an inhospitable environment are the major hurdles for axonal regeneration of lesioned adult neurons. Neurotrophic factors have been shown to be excellent candidates in mediating neuronal repair and establishing functional circuitry via activating several growth signaling mechanisms including neuron-intrinsic regenerative programs. Here, we will review the effects of various neurotrophins in mediating recovery after injury to the adult spinal cord.     

Neurotrophic factors in regeneration.

Nerve growth factor, fibroblast growth factor, and ciliary neurotrophic factor can protect selected populations of neurons from some of the degenerative changes that otherwise follow axonal injury or other insults. The function of diffusible neurotrophic factors in axonal regeneration is still unclear, however. Knowledge of the nerve growth factor congeners, brain-derived neurotrophic factor and neurotrophin-3, is advancing rapidly as is the identification of neurotrophin receptors, several of which are membrane-bound tyrosine kinases.     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a “regeneration cocktail.” In this review, we describe the ability of peptides derived from tenascin-C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

Regeneration in the spinal cord

Important advances have been made in our understanding of conditions that influence the intrinsic capacity of mature CNS neurons to initiate and maintain a regrowth response. The combination of exogenous neurotrophic support with strategies to alter the terrain at the injury site itself suggests that there are important interactions between them that lead to increased axonal regeneration. The ability of chronically injured neurons to initiate a regeneration response is unexpected. Our view of the role that inhibitors play in restricting axonal growth has also expanded. The findings indicate that the windows of opportunity for enhancing growth after spinal cord injury may be more numerous than previously thought.     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors: strategies to improve neuronal regeneration

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a "regeneration cocktail." In this review, we describe the ability of peptides derived from tenascin- C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

Integrins and the extracellular matrix: key mediators of development and regeneration of the sensory nervous system

The somatosensory nervous system is responsible for the transmission of a multitude of sensory information from specialized receptors in the periphery to the central nervous system. Sensory afferents can potentially be damaged at several sites: in the peripheral nerve; the dorsal root; or the dorsal columns of the spinal cord; and the success of regeneration depends on the site of injury. The regeneration of peripheral nerve branches following injury is relatively successful compared to central branches. This is largely attributed to the presence of neurotrophic factors and a Schwann cell basement membrane rich in permissive extracellular matrix (ECM) components which promote axonal regeneration in the peripheral nerve. Modulation of the ECM environment and/or neuronal integrins may enhance regenerative potential of sensory neurons following peripheral or central nerve injury or disease. This review describes the interactions between integrins and ECM molecules (particularly the growth supportive ligands, laminin, and fibronectin; and the growth inhibitory chondroitin sulfate proteoglycans (CSPGs)) during development and regeneration of sensory neurons following physical injury or neuropathy.     

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA-Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.     

Insulin-Like Growth Factor-1 Enhances Motoneuron Survival and Inhibits Neuroinflammation After Spinal Cord Transection in Zebrafish

Cellular and Molecular Neurobiology - Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor produced locally in the central nervous system which can promote axonal regeneration, protect...     

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.     

Masseteric Nerve Injury Increases Expression of Brain-Derived Neurotrophic Factor in Microglia Within the Rat Mesencephalic Trigeminal Tract Nucleus

The distribution of brain-derived neurotrophic factor was examined in the rat mesencephalic trigeminal tract nucleus after transection and crush of the masseteric nerve. In the intact mesencephalic trigeminal tract nucleus, brain-derived neurotrophic factor was detected in small cells with fine processes. These cells and processes were occasionally located adjacent to tyrosine kinase B receptor-immunoreactive sensory neurons. The transection and crush of the masseteric nerve increased expression of brain-derived neurotrophic factor in the nucleus. The number and size of brain-derived neurotrophic factor-immunoreactive cells and processes were dramatically elevated by the nerve injury. As a result, the density of brain-derived neurotrophic factor-immunoreactive profiles in the mesencephalic trigeminal tract nucleus at 7 days after the injury was significantly higher compared with the intact nucleus. Double immunofluorescence method also revealed that brain-derived neurotrophic factor-immunoreactive cells were mostly immunoreactive for OX-42 but not glial fibrillary acidic protein. In addition, the retrograde tracing method demonstrated that brain-derived neurotrophic factor-immunoreactive cells and processes surrounded retrogradely labeled neurons which showed tyrosine kinase B receptor-immunoreactivity. These findings indicate that the nerve injury increases expression of brain-derived neurotrophic factor in microglia within the mesencephalic trigeminal tract nucleus. The glial neurotrophic factor may be associated with axonal regeneration of the injured primary proprioceptor in the trigeminal nervous system.     

Regeneration-based therapies for spinal cord injuries

Although it has been long believed that the damaged central nervous system does not regenerate upon injury, there is an emerging hope for regeneration-based therapy of the damaged central nervous system (CNS) due to the progress of developmental biology and regenerative medicine including stem cell biology. In this review, we have summarized recent studies aimed at the development of regeneration-based therapeutic approaches for spinal cord injuries, including therapy with anti-inflammatory cytokines, transplantation of neural stem/precursor cells and induction of axonal regeneration.     

Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.     

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Axons in the adult mammalian central nervous system (CNS) exhibit little regeneration after injury. It has been suggested that several axonal growth inhibitors prevent CNS axonal regeneration. Recent research has demonstrated that semaphorin3A (Sema3A) is one of the major inhibitors of axonal regeneration. We identified a strong and selective inhibitor of Sema3A, SM-216289, from the fermentation broth of a fungal strain. To examine the effect of SM-216289 in vivo, we transected the spinal cord of adult rats and administered SM-216289 into the lesion site for 4 weeks. Rats treated with SM-216289 showed substantially enhanced regeneration and/or preservation of injured axons, robust Schwann cell-mediated myelination and axonal regeneration in the lesion site, appreciable decreases in apoptotic cell number and marked enhancement of angiogenesis, resulting in considerably better functional recovery. Thus, Sema3A is essential for the inhibition of axonal regeneration and other regenerative responses after spinal cord injury (SCI). These results support the possibility of using Sema3A inhibitors in the treatment of human SCI.     

Nerve injury signaling

Although neurons within the peripheral nervous system (PNS) have a remarkable ability to repair themselves after injury, neurons within the central nervous system (CNS) do not spontaneously regenerate. This problem has remained recalcitrant despite a century of research on the reaction of axons to injury. The balance between inhibitory cues present in the environment and the intrinsic growth capacity of the injured neuron determines the extent of axonal regeneration following injury. The cell body of an injured neuron must receive accurate and timely information about the site and extent of axonal damage in order to increase its intrinsic growth capacity and successfully regenerate. One of the mechanisms contributing to this process is retrograde transport of injury signals. For example, molecules activated at the injury site convey information to the cell body leading to the expression of regeneration-associated genes and increased growth capacity of the neuron. Here we discuss recent studies that have begun to dissect the injury-signaling pathways involved in stimulating the intrinsic growth capacity of injured neurons.