Major vault protein promotes locomotor recovery and regeneration after spinal cord injury in adult zebrafish

In contrast to mammals, adult zebrafish recover locomotor functions after spinal cord injury (SCI), in part due to axonal regrowth and regeneration permissivity of the central nervous system. Upregulation of major vault protein (MVP) expression after spinal cord injury in the brainstem of the adult zebrafish prompted us to probe for its contribution to recovery after SCI. MVP is a multifunctional protein expressed not only in many types of tumours but also in the nervous system, where its importance for regeneration is, however, unclear. Using an established zebrafish SCI model, we found that MVP mRNA and protein expression levels were increased in ependymal cells in the spinal cord caudal to the lesion site at 6 and 11 days after SCI. Double immunolabelling showed that MVP was co‐localised with Islet‐1 or tyrosine hydroxylase around the central canal of the spinal cord in sham‐injured control fish and injured fish 11 days after surgery. MVP co‐localised with the neural stem cell marker nestin in ependymal cells after injury. By using an in vivo morpholino‐based knock‐down approach, we found that the distance moved by MVP morpholino‐treated fish was reduced at 4, 5 and 6 weeks after SCI when compared to fish treated with standard control morpholino. Knock‐down of MVP resulted in reduced regrowth of axons from brainstem neurons into the spinal cord caudal to the lesion site. These results indicate that MVP supports locomotor recovery and axonal regrowth after SCI in adult zebrafish.     

Expression of protein zero is increased in lesioned axon pathways in the central nervous system of adult zebrafish

The immunoglobulin superfamily molecule protein zero (P0) is important for myelin formation and may also play a role in adult axon regeneration, since it promotes neurite outgrowth in vitro. Moreover, it is expressed in the regenerating central nervous system (CNS) of fish, but not in the nonregenerating CNS of mammals. We identified a P0 homolog in zebrafish. Cell type-specific expression of P0 begins in the ventromedial hindbrain and the optic chiasm at 3-5 days of development. Later (at 4 weeks) expression has spread throughout the optic system and spinal cord. This is consistent with a role for P0 in CNS myelination during development. In the adult CNS, glial cells constitutively express P0 mRNA. After an optic nerve crush, expression is increased within 2 days in the entire optic pathway. Expression peaks at 1 to 2 months and remains elevated for at least 6 months postlesion. After enucleation, P0 mRNA expression is also upregulated but fails to reach the high levels observed in crush-lesioned animals at 4 weeks postlesion. Spinal cord transection leads to increased expression of P0 mRNA in the spinal cord caudal to the lesion site. The glial upregulation of P0 mRNA expression after a lesion of the adult zebrafish CNS suggests roles for P0 in promoting axon regeneration and remyelination after injury.     

Cysteine- and glycine-rich protein 1a is involved in spinal cord regeneration in adult zebrafish

In contrast to mammals, adult zebrafish have the ability to regrow descending axons and gain locomotor recovery after spinal cord injury (SCI). In zebrafish, a decisive factor for successful spinal cord regeneration is the inherent ability of some neurons to regrow their axons via (re)expressing growth-associated genes during the regeneration period. The nucleus of the medial longitudinal fascicle (NMLF) is one of the nuclei capable of regenerative response after SCI. Using microarray analysis with laser capture microdissected NMLF, we show that cysteine- and glycine-rich protein (CRP)1a (encoded by the csrp1a gene in zebrafish), the function of which is largely unknown in the nervous system, was upregulated after SCI. In situ hybridization confirmed the upregulation of csrp1a expression in neurons during the axon growth phase after SCI, not only in the NMLF, but also in other nuclei capable of regeneration, such as the intermediate reticular formation and superior reticular formation. The upregulation of csrp1a expression in regenerating nuclei started at 3 days after SCI and continued to 21 days post-injury, the longest time point studied. In vivo knockdown of CRP1a expression using two different antisense morpholino oligonucleotides impaired axon regeneration and locomotor recovery when compared with a control morpholino, demonstrating that CRP1a upregulation is an important part of the innate regeneration capability in injured neurons of adult zebrafish. This study is the first to demonstrate the requirement of CRP1a for zebrafish spinal cord regeneration.     

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR-133b during zebrafish spinal cord regeneration and show upregulation of miR-133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR-133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR-133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR-133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth-related molecules. Our results indicate that miR-133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR-133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.     

Serotonergic innervation of the caudal spinal stump in rats after complete spinal transection: Effect of olfactory ensheathing glia

Spinal cord injury studies use the presence of serotonin (5‐HT)‐immunoreactive axons caudal to the injury site as evidence of axonal regeneration. As olfactory ensheathing glia (OEG) transplantation improves hindlimb locomotion in adult rats with complete spinal cord transection, we hypothesized that more 5‐HT‐positive axons would be found in the caudal stump of OEG‐ than media‐injected rats. Previously we found 5‐HT‐immunolabeled axons that spanned the transection site only in OEG‐injected rats but detected labeled axons just caudal to the lesion in both media‐ and OEG‐injected rats. Now we report that many 5‐HT‐labeled axons are present throughout the caudal stump of both media‐ and OEG‐injected rats. We found occasional 5‐HT‐positive interneurons that are one likely source of 5‐HT‐labeled axons. These results imply that the presence of 5‐HT‐labeled fibers in the caudal stump is not a reliable indicator of regeneration. We then asked if 5‐HT‐positive axons appose cholinergic neurons associated with motor functions: central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more 5‐HT‐positive varicosities in lamina X adjacent to central canal cluster cells in lumbar and sacral segments of OEG‐ than media‐injected rats. SMNs and partition cells are less frequently apposed. As nonsynaptic release of 5‐HT is common in the spinal cord, an increase in 5‐HT‐positive varicosities along motor‐associated cholinergic neurons may contribute to the locomotor improvement observed in OEG‐injected spinal rats. Furthermore, serotonin located within the caudal stump may activate lumbosacral locomotor networks. J. Comp. Neurol. 515:664–676, 2009. © 2009 Wiley‐Liss, Inc.     

Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish

We analyzed pathway choices of regenerating, mostly supraspinal, descending axons in the spinal cord of adult zebrafish and the cellular changes in the spinal cord caudal to a lesion site after complete spinal transection. Anterograde tracing (by application of the tracer rostral to the spinal lesion site) showed that significantly more descending axons (74%) regenerated in the spinal gray matter of the caudal spinal cord than would be expected from random growth. Retrograde tracing (by application of the tracer caudal to the spinal lesion site) showed that, rostral to the lesion, most of these axons (80%) extended into the major white matter tracts. Thus, ventral descending tracts often were devoid of labeled axons caudal to a spinal lesion but contained many axons rostral to the lesion in the same animals, indicating a pathway switch of descending axons from the white matter to the gray matter. Ascending axons of spinal neurons were not observed regrowing to the rostral tracer application site; therefore, they most likely did not contribute to the axonal populations analyzed. A macrophage/microglia response within 2 days of spinal cord transection, along with phagocytosis of myelin, was observed caudal to the transection by immunohistochemistry and electron microscopy. Nevertheless, caudal to the lesion, descending tracts in the white matter were filled with myelin debris during the time of axonal regrowth, at least up to 6 weeks postlesion. We suggest that the spontaneous regeneration of axons of supraspinal origin after spinal cord transection in adult zebrafish may be due in part to the axons' ability to negotiate novel pathways in the spinal cord gray matter.     

Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish

Monoaminergic innervation of the spinal cord has important modulatory functions for locomotion. Here we performed a quantitative study to determine the plastic changes of tyrosine hydroxylase-positive (TH1(+); mainly dopaminergic), and serotonergic (5-HT(+)) terminals and cells during successful spinal cord regeneration in adult zebrafish. TH1(+) innervation in the spinal cord is derived from the brain. After spinal cord transection, TH1(+) immunoreactivity is completely lost from the caudal spinal cord. Terminal varicosities increase in density rostral to the lesion site compared with unlesioned controls and are re-established in the caudal spinal cord at 6 weeks post lesion. Interestingly, axons mostly fail to re-innervate more caudal levels of the spinal cord even after prolonged survival times. However, densities of terminal varicosities correlate with recovery of swimming behavior, which is completely lost again after re-lesion of the spinal cord. Similar observations were made for terminals derived from descending 5-HT(+) axons from the brain. In addition, spinal 5-HT(+) neurons were newly generated after a lesion and transiently increased in number up to fivefold, which depended in part on hedgehog signaling. Overall, TH1(+) and 5-HT(+) innervation is massively altered in the successfully regenerated spinal cord of adult zebrafish. Despite these changes in TH and 5-HT systems, a remarkable recovery of swimming capability is achieved, suggesting significant plasticity of the adult spinal network during regeneration.     

Impact of neural cell adhesion molecule deletion on regeneration after mouse spinal cord injury

The neural cell adhesion molecule (NCAM) plays important functional roles in development of the nervous system. We investigated the influence of a constitutive ablation of NCAM on the outcome of spinal cord injury. Transgenic mice lacking NCAM (NCAM?/?) were subjected to severe compression injury of the lower thoracic spinal cord using wild‐type (NCAM+/+) littermates as controls. According to the single‐frame motion analysis, the NCAM?/? mice showed reduced locomotor recovery in comparison to control mice at 3 and 6 weeks after injury, indicating an overall positive impact of NCAM on recovery after injury. Also the Basso Mouse Scale score was lower in NCAM?/? mice at 3 weeks after injury, whereas at 6 weeks after injury the difference between genotypes was not statistically significant. Worse locomotor function was associated with decreased monoaminergic and cholinergic innervation of the spinal cord caudal to the injury site and decreased axonal regrowth/sprouting at the site of injury. Astrocytic scar formation at the injury site, as assessed by immunohistology for glial fibrillary acidic protein at and around the lesion site was increased in NCAM?/? compared with NCAM+/+ mice. Migration of cultured monolayer astrocytes from NCAM?/? mice was reduced as assayed by scratch wounding. Numbers of Iba‐1 immunopositive microglia were not different between genotypes. We conclude that constitutive NCAM deletion in young adult mice reduces recovery after spinal cord injury, validating the hypothesized beneficial role of this molecule in recovery after injury.     

Lesion-induced generation of interneuron cell types in specific dorsoventral domains in the spinal cord of adult zebrafish

In contrast to mammals, adult zebrafish regenerate neurons in the lesioned spinal cord. For example, motor neurons are generated from an olig2‐expressing population of pMN‐like ependymoradial glial cells in a ventrolateral position at the central canal. However, the extent of neuronal regeneration is unclear. Here we show, using a transgenic fish in which V2 interneurons are labeled by green fluorescent protein (GFP) under the control of the vsx1 promoter, that after a complete spinal cord transection, large numbers of V2 interneurons are generated in the vicinity of the lesion site. Tg(vsx1:GFP)⁺ cells are not present in the unlesioned spinal cord and label with the proliferation marker bromodeoxyuridine (BrdU) after a lesion. Some mediolaterally elongated Tg(vsx1:GFP)⁺ cells contact the central canal in a medial position. These cells likely arise from a p2‐like domain of ependymoradial glial progenitor cells, indicated by coexpression of Pax6 and Nkx6.1, but not DsRed driven by the olig2 promoter in these cells. We also present evidence that Pax2⁺ interneurons are newly generated after a spinal lesion, whereas the generation rate for a dorsal population of parvalbuminergic interneurons is comparatively low. Our results identify the regenerative potential of different interneuron types for the first time and support a model in which different progenitor cell domains in distinct dorsoventral positions around the central canal are activated by a lesion to give rise to diverse neuronal cell types in the adult zebrafish spinal cord. J. Comp. Neurol. 520:3604–3616, 2012. © 2012 Wiley Periodicals, Inc.     

Adult neural progenitor cells provide a permissive guiding substrate for corticospinal axon growth following spinal cord injury

Adult neural progenitor cells (NPC) are an attractive source for cell transplantation and neural tissue replacement after central nervous system (CNS) injury. Following transplantation of NPC cell suspensions into the acutely injured rat spinal cord, NPC survive; however, they migrate away from the lesion site and are unable to replace the injury-induced lesion cavity. In the present study we examined (i) whether NPC can be retained within the lesion site after co-transplantation with primary fibroblasts, and (ii) whether NPC promote axonal regeneration following spinal cord injury. Co-cultivation of NPC with fibroblasts demonstrated that NPC adhere to fibroblasts and the extracellular matrix produced by fibroblasts. In the presence of fibroblasts, the differentiation pattern of co-cultivated NPC was shifted towards glial differentiation. Three weeks after transplantation of adult spinal-cord-derived NPC with primary fibroblasts as mixed cell suspensions into the acutely injured cervical spinal cord in adult rats, the lesion cavity was completely replaced. NPC survived throughout the graft and differentiated exclusively into glial cells. Quantification of neurofilament-labeled axons and anterogradely labeled corticospinal axons indicated that NPC co-grafted with fibroblasts significantly enhanced axonal regeneration. Both neurofilament-labeled axons and corticospinal axons aligned longitudinally along GFAP-expressing NPC-derived cells, which displayed a bipolar morphology reminiscent of immature astroglia. Thus, grafted astroglial differentiated NPC promote axon regrowth following spinal cord injury by means of cellular guidance.     

Contrasting neuropathology and functional recovery after spinal cord injury in developing and adult rats

Conflicting findings exist regarding the link between functional recovery and the regrowth of spinal tracts across the lesion leading to the restoration of functional contacts. In the present study, we investigated whether functional locomotor recovery was attributable to anatomical regeneration at postnatal day 1 (PN1), PN7, PN14 and in adult rats two months after transection injury at the tenth thoracic segment of the spinal cord. The Basso, Beattie, and Bresnahan scores showed that transection led to a failure of hindlimb locomotor function in PN14 and adult rats. However, PN1 and PN7 rats showed a significant level of stepping function after complete spinal cord transection. Unexpectedly, unlike the transected PN14 and adult rats in which the spinal cord underwent limited secondary degeneration and showed a scar at the lesion site, the rats transected at PN1 and PN7 showed massive secondary degeneration both anterograde and retrograde, leaving a >5-mm gap between the two stumps. Furthermore, retrograde tracing with fluorogold (FG) also showed that FG did not cross the transection site in PN1 and PN7 rats as in PN14 and adult rats, and re-transection of the cord caused no apparent loss in locomotor performance in the rats transected at PN1. Thus, these three lines of evidence strongly indicated that the functional recovery after transection in neonatal rats is independent of regrowth of spinal tracts across the lesion site. Our results support the notion that the recovery of locomotor function in developing rats may be due to intrinsic adaptations in the spinal circuitry below the lesion that control hindlimb locomotor activity rather than the regrowth of spinal tracts across the lesion. The difference in secondary degeneration between neonatal and adult rats remains to be explored.     

Collagen IV deposits do not prevent regrowing axons from penetrating the lesion site in spinal cord injury

Scarring is suggested to impede axon regrowth across the lesion site in the injured adult mammalian central nervous system. Collagen Type IV, as a major component of the scar formed after injury, is an impediment for successful axonal regeneration and a decrease in its amount is a prerequisite for regrowing axons to cross the lesion in the postcommissural fornix in the injured adult rat (Stichel et al. [1999] Neurosci. 93:321-333). The aim of the present study was to analyze the relationship between collagen IV deposits and regrowing axons at various times after dorsal hemi-section of the adult rat spinal cord. Immunohistochemical double staining revealed that penetrating neurofilament-positive axons and collagen IV deposits were co-localized in the lesion site in the initial stages of axonal sprouting (between 7 and 14 days post-operatively) and were still present 1 and 2 months post-operatively. Interestingly, collagen IV-immunoreactive areas located around cystic cavities formed at the site of injury 1 month post-operatively, were devoid of axons. In conclusion, our observations indicate that collagen IV deposits after spinal cord injury do not prevent neurofilament-positive regrowing axons from penetrating the lesion site.     

Delayed treatment with Rho-kinase inhibitor does not enhance axonal regeneration or functional recovery after spinal cord injury in rats

Axonal regeneration in the central nervous system is blocked by many different growth inhibitory factors. Some of these inhibitors act on neurons by activating RhoA and Rho-kinase, an effector of RhoA. Several studies have shown that Rho-kinase inhibition immediately after spinal cord injury enhances axonal sprouting and functional recovery. In this study, we ask whether delayed treatment with Rho-kinase inhibitor is effective in promoting regeneration and functional recovery. We administered Fasudil, a Rho-kinase inhibitor, locally to the injury site 4 weeks or immediately after contusion of the thoracic spinal cord in rats. Although the immediate treatment significantly stimulated axonal sprouting and recovery of hindlimb function, treatment started 4 weeks after surgery had no effect on fiber sprouting or locomotor recovery. Our findings suggest that RhoA/Rho-kinase alone may not account for the irreversible arrest of axon outgrowth in the chronic stage of injury in the central nervous system.     

Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord injury in rats

Nogo-66 receptor (NgR), a common receptor for the three known myelin-associated inhibitors, i.e., Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), plays a key role in the failure of axonal regeneration in the adult mammalian central nervous system (CNS). Here we report a novel vaccine approach that stimulates the production of anti-NgR antibody to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). We showed that adult rats immunized with recombinant NgR produced high titers of the anti-NgR antibody and that antisera obtained from the immunized rats promoted neurite outgrowth of rat cerebellar neurons on the inhibitory MAG substrate in vitro. In a spinal cord dorsal hemisection model, NgR immunization promoted regeneration of lesioned corticospinal tract (CST) axons, anterogradely labeled with biotin dextran amine (BDA), beyond the lesion site. In a contusive SCI model, NgR immunization markedly reduced the total lesion volume and improved Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and grid walking performance. Thus, the NgR vaccine approach may represent a promising repair strategy to promote structural and functional recovery following SCI.     

Low‐dose fractionated irradiation promotes axonal regeneration beyond reactive gliosis and facilitates locomotor function recovery after spinal cord injury in beagle dogs

Injury to the adult central nervous system (CNS) results in the formation of glial scar tissues. Glial scar‐induced failure of regenerative axon pathfinding may limit axon regrowth beyond the lesion site and cause incorrect reinnervation and dystrophic appearance of stalled growth after CNS trauma. Glial scars also upregulate chondroitin sulphate proteoglycans (CSPGs) and expression of proinflammatory factor(s) that form a barrier to axonal regeneration. Therefore, interventions for glial scarring are an attractive strategy for augmenting axonal sprouting and regeneration and overcoming the physical and molecular barriers impeding functional repair. The glial reaction occurs shortly after spinal cord injury (SCI) and can persist for days or weeks with upregulation of cell cycle proteins. In this study, we utilised Beagle dogs to establish a preclinical SCI model and examine the efficacy of low‐dose fractionated irradiation (LDI) treatment, which was performed once a day for 14 days (2 Gy per dose, 28 Gy in total). Low‐dose fractionated irradiation is a stable method for suppressing cell activation and proliferation through interference in the cell cycle. Our results demonstrated that LDI could reduce astrocyte and microglia activation/proliferation and attenuate CSPGs and IL‐1β expression. Low‐dose fractionated irradiation also promoted and provided a pathway for long‐distance axon regeneration beyond the lesion site, induced reinnervation of axonal targets and restored locomotor function after SCI in Beagle dogs. Taken together, our findings suggest that LDI would be a promising therapeutic strategy for targeting glial scarring, promoting axon regeneration and facilitating reconstruction of functional circuits after SCI.     

From cortex to cord: motor circuit plasticity after spinal cord injury

Spinal cord injury is associated with chronic sensorimotor deficits due to the interruption of ascending and descending tracts between the brain and spinal cord. Functional recovery after anatomically complete spinal cord injury is limited due to the lack of long-distance axonal regeneration of severed fibers in the adult central nervous system. Most spinal cord injuries in humans, however, are anatomically incomplete.Although restorative treatment options for spinal cord injury remain currently limited, research from experimental models of spinal cord injury have revealed a tremendous capability for both spontaneous and treatment-induced plasticity of the corticospinal system that supports functional recovery. We review recent advances in the understanding of corticospinal circuit plasticity after spinal cord injury and concentrate mainly on the hindlimb motor cortex, its corticospinal projections, and the role of spinal mechanisms that support locomotor recovery. First, we discuss plasticity that occurs at the level of motor cortex and the reorganization of cortical movement representations. Next, we explore downstream plasticity in corticospinal projections. We then review the role of spinal mechanisms in locomotor recovery. We conclude with a perspective on harnessing neuroplasticity with therapeutic interventions to promote functional recovery.     

Basal lamina formation at the site of spinal cord transection

The pia-glial basal lamina (BL) at the site of spinal cord injury could be an important physical impediment to central nervous system regeneration. We used an epithelial BL-specific immunohistochemical stain to determine the location of the pia-glial BL after spinal cord transection. Small segments of BL were found at the margin of the lesion 5 days after transection. After 10 days, longer and more numerous segments were seen. At 20 days, the entire transected end of the spinal cord was capped by a layer of BL.     

Self‐assembling peptide amphiphile promotes plasticity of serotonergic fibers following spinal cord injury

Injection into the injured spinal cord of peptide amphiphile (PA) molecules that self‐assemble and display the laminin epitope IKVAV at high density improved functional recovery after spinal cord injury (SCI) in two different species, rat and mouse, and in two different injury models, contusion and compression. The improvement required the IKVAV epitope and was not observed with the injection of an amphiphile displaying a nonbioactive sequence. To explore the mechanisms underlying these improvements, the number of serotonergic fibers in the lesioned spinal cord was compared in animals receiving the IKVAV‐PA, a nonbioactive PA (PA control), or sham injection. Serotonergic fibers were distributed equally in all three groups rostral to the injury but showed a significantly higher density caudal to the injury site in the IKVAV PA‐injected group. Furthermore, this difference was not present in the subacute phase following injury but appeared in the chronically injured cord. The IKVAV PA‐injected groups also trended higher both in the total number neurons adjacent to the lesion and in the number of long propriospinal tract connections from the thoracic to the lumbar cord. IKVAV PA injection did not alter myelin thickness, total axon number caudal to the lesion, axon size distribution, or total axon area. Serotonin can promote stepping even in complete transection models, so the improved function produced by the IKVAV PA treatment may reflect the increased serotonergic innervation caudal to the lesion in addition to the previously demonstrated regeneration of motor and sensory axons through the lesion. © 2010 Wiley‐Liss, Inc..