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.     

The role of Nogo-A in axonal plasticity, regrowth and repair

Axonal damage leads to permanent deficits in the adult central nervous system (CNS) not only because of the weak intrinsic ability of adult neurons to activate their growth program but importantly also because of the presence of specific growth inhibitors in the CNS tissue and the environment of the damaged axons. The well-studied myelin-derived protein Nogo-A is involved in various cellular and molecular events contributing to the failure of CNS axons to regrow and reconnect after transection. Recent studies have shown that, by acting in a negative way on the cytoskeleton and on the growth program of axotomized neurons, Nogo-A exerts fast and chronic inhibitory effects on neurite outgrowth. On the other hand, the blockade of Nogo-A results in a marked enhancement of compensatory and regenerative axonal extension in vivo; this enhancement is often paralleled by significant functional recovery, for example, of locomotion or skilled forelimb reaching after spinal cord or stroke lesions in rats and monkeys. Surprisingly, the blockade of Nogo-A or its receptor NgR in the hippocampus has recently been demonstrated to enhance long-term potentiation. A role of Nogo-A in synaptic plasticity/stability might therefore represent an additional, new and important aspect of CNS circuit remodeling. Function-blocking anti-Nogo-A antibodies are currently being tested in a clinical trial for improved outcome after spinal cord injury.     

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.     

Fasciculation and guidance of spinal motor axons in the absence of FGFR2 signaling

During development, fibroblast growth factors (FGF) are essential for early patterning events along the anterior-posterior axis, conferring positional identity to spinal motor neurons by activation of different Hox codes. In the periphery, signaling through one of four fibroblast growth factor receptors supports the development of the skeleton, as well as induction and maintenance of extremities. In previous studies, FGF receptor 2 (FGFR2) was found to interact with axon bound molecules involved in axon fasciculation and extension, thus rendering this receptor an interesting candidate for the promotion of proper peripheral innervation. However, while the involvement of FGFR2 in limb bud induction has been extensively studied, its role during axon elongation and formation of distinct nervous projections has not been addressed so far. We show here that motor neurons in the spinal cord express FGFR2 and other family members during the establishment of motor connections to the forelimb and axial musculature. Employing a conditional genetic approach to selectively ablate FGFR2 from motor neurons we found that the patterning of motor columns and the expression patterns of other FGF receptors and Sema3A in the motor columns of mutant embryos are not altered. In the absence of FGFR2 signaling, pathfinding of motor axons is intact, and also fasciculation, distal advancement of motor nerves and gross morphology and positioning of axonal projections are not altered. Our findings therefore show that FGFR2 is not required cell-autonomously in motor neurons during the formation of initial motor projections towards limb and axial musculature.     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

FGF1 improves functional recovery through inducing PRDX1 to regulate autophagy and anti‐ROS after spinal cord injury

Fibroblast growth factor 1 (FGF1) is thought to exert protective and regenerative effects on neurons following spinal cord injury (SCI), although the mechanism of these effects is not well understood. The use of FGF1 as a therapeutic agent is limited by its lack of physicochemical stability and its limited capacity to cross the blood‐spinal cord barrier. Here, we demonstrated that overexpression of FGF1 in spinal cord following SCI significantly reduced tissue loss, protected neurons in the ventricornu, ameliorated pathological morphology of the lesion, dramatically improved tissue recovery via neuroprotection, and promoted axonal regeneration and remyelination both in vivo and in vivo. In addition, the autophagy and the expression levels of PRDX1 (an antioxidant protein) were induced by AAV‐FGF1 in PC12 cells after H₂O₂ treatment. Furthermore, the autophagy levels were not changed in PRDX1‐suppressing cells that were treated by AAV‐FGF1. Taken together, these results suggest that FGF1 improves functional recovery mainly through inducing PRDX1 expression to increase autophagy and anti‐ROS activity after SCI.     

Molecular targets for therapeutic intervention after spinal cord injury

In an effort to develop therapies for promoting neurological recovery after spinal cord injury, much work has been done to identify the cellular and molecular factors that control axonal regeneration within the injured central nervous system. This review summarizes the current understanding of a number of the elements within the spinal cord environment that inhibit axonal growth and outlines the factors that influence the neuron's ability to regenerate its axon after injury. Recent insights in these areas have identified important molecular pathways that are potential targets for therapeutic intervention, raising hope for victims of spinal cord injury.     

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.     

Aquaporin-4 Mitigates Retrograde Degeneration of Rubrospinal Neurons by Facilitating Edema Clearance and Glial Scar Formation After Spinal Cord Injury in Mice

Atrophy of upper motor neurons hampers axonal regeneration and functional recovery following spinal cord injury (SCI). Apart from the severity of primary injury, a series of secondary pathological damages including spinal cord edema and glial scar formation affect the fate of injured upper motor neurons. The aquaporin-4 (AQP4) water channel plays a critical role in water homeostasis and migration of astrocytes in the central nervous system, probably offering a new therapeutic target for protecting against upper motor neuron degeneration after SCI. To test this hypothesis, we examined the effect of AQP4 deficiency on atrophy of rubrospinal neurons after unilateral rubrospinal tract transection at the fourth cervical level in mice. AQP4 gene knockout (AQP4^?/?) mice exhibited high extent of spinal cord edema at 72 h after lesion compared with wild-type littermates. AQP4^?/? mice showed impairments in astrocyte migration toward the transected site with a greater lesion volume at 1 week after surgery and glial scar formation with a larger cyst volume at 6 weeks. More severe atrophy and loss of axotomized rubrospinal neurons as well as axonal degeneration in the rubrospinal tract rostral to the lesion were observed in AQP4^?/? mice at 6 weeks after SCI. AQP4 expression was downregulated at the lesioned spinal segment at 3 days and 1 week after injury, but upregulated at 6 weeks. These results demonstrated that AQP4 not only mitigates spinal cord damage but also ameliorates retrograde degeneration of rubrospinal neurons by promoting edema clearance and glial scar formation after laceration SCI. This finding supports the notion that AQP4 may be a promising therapeutic target for SCI.     

Peripheral Nerve Injury Modulates Neurotrophin Signaling in the Peripheral and Central Nervous System

Peripheral nerve injury disrupts the normal functions of sensory and motor neurons by damaging the integrity of axons and Schwann cells. In contrast to the central nervous system, the peripheral nervous system possesses a considerable capacity for regrowth, but regeneration is far from complete and functional recovery rarely returns to pre-injury levels. During development, the peripheral nervous system strongly depends upon trophic stimulation for neuronal differentiation, growth and maturation. The perhaps most important group of trophic substances in this context is the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), which signal in a complex spatial and timely manner via the two structurally unrelated p75^NTR and tropomyosin receptor kinase (TrkA, Trk-B and Trk-C) receptors. Damage to the adult peripheral nerves induces cellular mechanisms resembling those active during development, resulting in a rapid and robust increase in the synthesis of neurotrophins in neurons and Schwann cells, guiding and supporting regeneration. Furthermore, the injury induces neurotrophin-mediated changes in the dorsal root ganglia and in the spinal cord, which affect the modulation of afferent sensory signaling and eventually may contribute to the development of neuropathic pain. The focus of this review is on the expression patterns of neurotrophins and their receptors in neurons and glial cells of the peripheral nervous system and the spinal cord. Furthermore, injury-induced changes of expression patterns and the functional consequences in relation to axonal growth and remyelination as well as to neuropathic pain development will be reviewed.     

Transplantation of D15A-Expressing Glial-Restricted-Precursor-Derived Astrocytes Improves Anatomical and Locomotor Recovery after Spinal Cord Injury

The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI). In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs)-derived astrocytes (GDAs) could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs). Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.     

The acute administration of eicosapentaenoic acid is neuroprotective after spinal cord compression injury in rats

The aim of the present study was to investigate the effects of treatment with eicosapentaenoic acid (EPA) after spinal cord compression injury in adult rats. Saline or EPA (250 nmol/kg) was administered intravenously 30 min after compression injury. Locomotor recovery was assessed daily using the BBB open-field locomotor score. One week after injury, animals were sacrificed and the spinal cord tissue containing the compression epicenter, and the adjacent rostral and caudal segments, was immunostained using specific markers for neurons, oligodendrocytes, axonal injury, and macrophages/microglia. Administration of EPA resulted in decreased axonal injury and increased neuronal and oligodendrocyte survival, in the lesion epicenter and adjacent tissue. The behavioural assessment mirrored the neuroprotective effects and showed a significantly improved functional recovery in animals treated with EPA compared to the saline-treated controls over the 7-day period. These observations suggest that EPA has neuroprotective properties when administered after spinal cord trauma.     

The Adhesion Molecule-Characteristic HNK-1 Carbohydrate Contributes to Functional Recovery After Spinal Cord Injury in Adult Zebrafish

The human natural killer cell antigen-1 (HNK-1) is functionally important in development, synaptic activity, and regeneration after injury in the nervous system of several mammalian species. It contains a sulfated glucuronic acid which is carried by neural adhesion molecules and expressed in nonmammalian species, including zebrafish, which, as opposed to mammals, spontaneously regenerate after injury in the adult. To evaluate HNK-1’s role in recovery of function after spinal cord injury (SCI) of adult zebrafish, we assessed the effects of the two HNK-1 synthesizing enzymes, glucuronyl transferase and HNK-1 sulfotransferase. Expression of these two enzymes was increased at the messenger RNA (mRNA) level 11 days after injury in the brainstem nuclei that are capable of regrowth of severed axons, namely, the nucleus of medial longitudinal fascicle and intermediate reticular formation, but not at earlier time points after SCI. mRNA levels of glucuronyl transferase and sulfotransferase were increased in neurons, not only of these nuclei but also in the spinal cord caudal to the injury site at 11 days. Mauthner neurons which are not capable of regeneration did not show increased levels of enzyme mRNAs after injury. Reducing protein levels of the enzymes by application of anti-sense morpholinos resulted in reduction of locomotor recovery for glucuronyl transferase, but not for HNK-1 sulfotransferase. The combined results indicate that HNK-1 is upregulated in expression only in those neurons that are intrinsically capable of regeneration and contributes to regeneration after spinal cord injury in adult zebrafish in the absence of its sulfate moiety.     

Neuronal cytoskeleton in synaptic plasticity and regeneration

During development, dynamic changes in the axonal growth cone and dendrite are necessary for exploratory movements underlying initial axo‐dendritic contact and ultimately the formation of a functional synapse. In the adult central nervous system, an impressive degree of plasticity is retained through morphological and molecular rearrangements in the pre‐ and post‐synaptic compartments that underlie the strengthening or weakening of synaptic pathways. Plasticity is regulated by the interplay of permissive and inhibitory extracellular cues, which signal through receptors at the synapse to regulate the closure of critical periods of developmental plasticity as well as by acute changes in plasticity in response to experience and activity in the adult. The molecular underpinnings of synaptic plasticity are actively studied and it is clear that the cytoskeleton is a key substrate for many cues that affect plasticity. Many of the cues that restrict synaptic plasticity exhibit residual activity in the injured adult CNS and restrict regenerative growth by targeting the cytoskeleton. Here, we review some of the latest insights into how cytoskeletal remodeling affects neuronal plasticity and discuss how the cytoskeleton is being targeted in an effort to promote plasticity and repair following traumatic injury in the central nervous system.     

Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury

Spinal cord injuries (SCIs) in humans and experimental animals are often associated with varying degrees of spontaneous functional recovery during the first months after injury. Such recovery is widely attributed to axons spared from injury that descend from the brain and bypass incomplete lesions, but its mechanisms are uncertain. To investigate the neural basis of spontaneous recovery, we used kinematic, physiological and anatomical analyses to evaluate mice with various combinations of spatially and temporally separated lateral hemisections with or without the excitotoxic ablation of intrinsic spinal cord neurons. We show that propriospinal relay connections that bypass one or more injury sites are able to mediate spontaneous functional recovery and supraspinal control of stepping, even when there has been essentially total and irreversible interruption of long descending supraspinal pathways in mice. Our findings show that pronounced functional recovery can occur after severe SCI without the maintenance or regeneration of direct projections from the brain past the lesion and can be mediated by the reorganization of descending and propriospinal connections. Targeting interventions toward augmenting the remodeling of relay connections may provide new therapeutic strategies to bypass lesions and restore function after SCI and in other conditions such as stroke and multiple sclerosis.     

The Cell Neural Adhesion Molecule Contactin-2 (TAG-1) Is Beneficial for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

The cell neural adhesion molecule contactin-2 plays a key role in axon extension and guidance, fasciculation, and myelination during development. We thus asked, whether contactin-2 is also important in nervous system regeneration after trauma. In this study, we used an adult zebrafish spinal cord transection model to test the functions of contactin-2 in spinal cord regeneration. The expression patterns of contactin-2 at different time points after spinal cord injury were studied at the mRNA level by qPCR and in situ hybridization, and contactin-2 protein levels and immunohistological localization were detected by Western blot and immunofluorescence analyses, respectively. Contactin-2 mRNA and protein levels were increased along the central canal at 6 days and 11 days after spinal cord injury, suggesting a requirement for contactin-2 in spinal cord regeneration. Co-localization of contactin-2 and islet-1 (a motoneuron marker) was observed in spinal cords before and after injury. To further explore the functions of contactin-2 in regeneration, an anti-sense morpholino was used to knock down the expression of contactin-2 protein by application at the time of injury. Motion analysis showed that inhibition of contactin-2 retarded the recovery of swimming functions when compared to standard control morpholino. Anterograde and retrograde tracing at 6 weeks after injury showed that knock down of contactin-2 inhibited axonal regrowth from NMLF neurons beyond lesion site. The combined observations indicate that contactin-2 contributes to locomotor recovery and successful regrowth of axons after spinal cord injury in adult zebrafish.     

Axonal remodeling for motor recovery after traumatic brain injury requires downregulation of ��-aminobutyric acid signaling

Remodeling of the remnant neuronal network after brain injury possibly mediates spontaneous functional recovery; however, the mechanisms inducing axonal remodeling during spontaneous recovery remain unclear. Here, we show that altered γ-aminobutyric acid (GABA) signaling is crucial for axonal remodeling of the contralesional cortex after traumatic brain injury. After injury to the sensorimotor cortex in mice, we found a significant decrease in the expression of GABA_AR-α1 subunits in the intact sensorimotor cortex for 2 weeks. Motor functions, assessed by grid walk and cylinder tests, spontaneously improved in 4 weeks after the injury to the sensorimotor cortex. With motor recovery, corticospinal tract (CST) axons from the contralesional cortex sprouted into the denervated side of the cervical spinal cord at 2 and 4 weeks after the injury. To determine the functional implications of the changes in the expression of GABA_AR-α1 subunits, we infused muscimol, a GABA R agonist, into the contralesional cortex for a week after the injury. Compared with the vehicle-treated mice, we noted significantly inhibited recovery in the muscimol-treated mice. Further, muscimol infusion greatly suppressed the axonal sprouting into the denervated side of the cervical spinal cord. In conclusion, recovery of motor function and axonal remodeling of the CST following cortical injury requires suppressed GABA_AR subunit expression and decreased GABAergic signaling.     

Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.     

A Neonatal Mouse Spinal Cord Injury Model for Assessing Post-Injury Adaptive Plasticity and Human Stem Cell Integration

Despite limited regeneration capacity, partial injuries to the adult mammalian spinal cord can elicit variable degrees of functional recovery, mediated at least in part by reorganization of neuronal circuitry. Underlying mechanisms are believed to include synaptic plasticity and collateral sprouting of spared axons. Because plasticity is higher in young animals, we developed a spinal cord compression (SCC) injury model in the neonatal mouse to gain insight into the potential for reorganization during early life. The model provides a platform for high-throughput assessment of functional synaptic connectivity that is also suitable for testing the functional integration of human stem and progenitor cell-derived neurons being considered for clinical cell replacement strategies. SCC was generated at T9–T11 and functional recovery was assessed using an integrated approach including video kinematics, histology, tract tracing, electrophysiology, and high-throughput optical recording of descending inputs to identified spinal neurons. Dramatic degeneration of axons and synaptic contacts was evident within 24 hours of SCC, and loss of neurons in the injured segment was evident for at least a month thereafter. Initial hindlimb paralysis was paralleled by a loss of descending inputs to lumbar motoneurons. Within 4 days of SCC and progressively thereafter, hindlimb motility began to be restored and descending inputs reappeared, but with examples of atypical synaptic connections indicating a reorganization of circuitry. One to two weeks after SCC, hindlimb motility approached sham control levels, and weight-bearing locomotion was virtually indistinguishable in SCC and sham control mice. Genetically labeled human fetal neural progenitor cells injected into the injured spinal cord survived for at least a month, integrated into the host tissue and began to differentiate morphologically. This integrative neonatal mouse model provides opportunities to explore early adaptive plasticity mechanisms underlying functional recovery as well as the capacity for human stem cell-derived neurons to integrate functionally into spinal circuits.     

A Surgery Protocol for Adult Zebrafish Spinal Cord Injury

Adult zebrafish has a remarkable capability to recover from spinal cord injury,providing an excellent model for studying neuro-regeneration. Here we list equipment and reagents,and give a detailed protocol for complete transection of the adult zebrafish spinal cord. In this protocol,potential problems and their solutions are described so that the zebrafish spinal cord injury model can be more easily and reproducibly performed.In addition,two assessments are introduced to monitor the success of the surgery and functional recovery:one test to assess free swimming capability and the other test to assess extent of neuroregeneration by in vivo anterograde axonal tracing.In the swimming behavior test,successful complete spinal cord transection is monitored by the inability of zebrafish to swim freely for 1 week after spinal cord injury,followed by the gradual reacquisition of full locomotor ability within 6 weeks after injury.As a morphometric correlate,anterograde axonal tracing allows the investigator to monitor the ability of regenerated axons to cross the lesion site and increasingly extend into the gray and white matter with time after injury,confirming functional recovery.This zebrafish model provides a paradigm for recovery from spinal cord injury,enabling the identification of pathways and components of neuroregeneration.