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.     

Syntenin‐a promotes spinal cord regeneration following injury in adult zebrafish

In contrast to mammals, adult zebrafish recover locomotor function after spinal cord injury, in part due to the capacity of the central nervous system to repair severed connections. To identify molecular cues that underlie regeneration, we conducted mRNA expression profiling and found that syntenin‐a expression is upregulated in the adult zebrafish spinal cord caudal to the lesion site after injury. Syntenin is a scaffolding protein involved in mammalian cell adhesion and movement, axonal outgrowth, establishment of cell polarity, and protein trafficking. It could thus be expected to be involved in supporting regeneration in fish. Syntenin‐a mRNA and protein are expressed in neurons, glia and newly generated neural cells, and upregulated caudal to the lesion site on days 6 and 11 following spinal cord injury. Treatment of spinal cord‐injured fish with two different antisense morpholinos to knock down syntenin‐a expression resulted in significant inhibition of locomotor recovery at 5 and 6 weeks after injury, when compared to control morpholino‐treated fish. Knock‐down of syntenin‐a reduced regrowth of descending axons from brainstem neurons into the spinal cord caudal to the lesion site. These observations indicate that syntenin‐a is involved in regeneration after traumatic insult to the central nervous system of adult zebrafish, potentially leading to novel insights into the cellular and molecular mechanisms that require activation in the regeneration‐deficient mammalian central nervous system.     

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.     

Bioinformatic identification of key candidate genes and pathways in axon regeneration after spinal cord injury in zebrafish

Zebrafish and human genomes are highly homologous;however,despite this genomic similarity,adult zebrafish can achieve neuronal proliferation,regeneration and functional restoration within 6–8 weeks after spinal cord injury,whereas humans cannot.To analyze differentially expressed zebrafish genes between axon-regenerated neurons and axon-non-regenerated neurons after spinal cord injury,and to explore the key genes and pathways of axonal regeneration after spinal cord injury,microarray GSE56842 was analyzed using the online tool,GEO2R,in the Gene Expression Omnibus database.Gene ontology and protein-protein interaction networks were used to analyze the identified differentially expressed genes.Finally,we screened for genes and pathways that may play a role in spinal cord injury repair in zebrafish and mammals.A total of 636 differentially expressed genes were obtained,including 255 up-regulated and 381 down-regulated differentially expressed genes in axon-regenerated neurons.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were also obtained.A protein-protein interaction network contained 480 node genes and 1976 node connections.We also obtained the 10 hub genes with the highest correlation and the two modules with the highest score.The results showed that spectrin may promote axonal regeneration after spinal cord injury in zebrafish.Transforming growth factor beta signaling may inhibit repair after spinal cord injury in zebrafish.Focal adhesion or tight junctions may play an important role in the migration and proliferation of some cells,such as Schwann cells or neural progenitor cells,after spinal cord injury in zebrafish.Bioinformatic analysis identified key candidate genes and pathways in axonal regeneration after spinal cord injury in zebrafish,providing targets for treatment of spinal cord injury in mammals.     

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.     

RhoA, RhoB, RhoC, Rac1, Cdc42, and Tc10 mRNA levels in spinal cord, sensory ganglia, and corticospinal tract neurons and long-lasting specific changes following spinal cord injury

Inhibition of RhoA has been shown to enhance axonal regeneration following spinal cord injury. Here we mapped mRNA expression patterns of RhoA, B, and C, Rac1, Cdc42, and Tc10 in spinal cord, sensory ganglia, and sensorimotor cortex in uninjured rats, and following spinal cord injury or sham laminectomy. In the intact spinal cord, neurons displayed high levels of Rac1, Cdc42, and Tc10 mRNA hybridization signal. GFAP-immunoreactive astrocytes expressed primarily RhoB and Rac1, while oligodendrocyte-like cells expressed RhoA, Rac1, and Cdc42. Injury caused profound, long-lasting upregulation of RhoA, Rac1, Cdc42, and Tc10 mRNA in the spinal cord, while RhoB was modestly increased and RhoC did not change. GFAP-immunoreactive reactive astrocytes exhibited a dramatic increase of RhoA mRNA expression along with increases of Rac1 and Cdc42. Injury also led to elevation of RhoA, Cdc42, and Tc10 in neurons and modest increases of RhoA, Rac1, and Tc10 in oligodendrocyte-like cells. Laminectomy caused similar, but less pronounced alterations of investigated mRNA species. In dorsal root ganglia neuronal RhoA, Rac1, Cdc42, and Tc10 mRNA levels were increased similarly by spinal cord injury and sham surgery. The CST pyramidal cells expressed Tc10 mRNA and the CST itself was Tc10-immunoreactive. Tc10-immunoreactivity disappeared distal to injury. We conclude that there are gene-specific patterns of expression of the six different Rho-GTPases in normal spinal cord and dorsal root ganglia, and that specific changes of temporal and spatial expression patterns occur in response to spinal cord injury, suggesting different roles of these GTPases in the cellular sequelae of CNS injury.     

RhoA as a target to promote neuronal survival and axon regeneration

Paralysis following spinal cord injury (SCI) is due to failure of axonal regeneration. It is believed that the capacities of neurons to regrow their axons are due partly to their intrinsic characteristics, which in turn are greatly influenced by several types of inhibitory molecules that are present, or even increased in the extracellular environment of the injured spinal cord. Many of these inhibitory molecules have been studied extensively in recent years. It has been suggested that the small GTPase RhoA is an intracellular convergence point for signaling by these extracellular inhibitory molecules, but due to the complexity of the central nervous system (CNS) in mammals, and the limitation of pharmacological tools, the specific roles of RhoA are unclear. By exploiting the anatomical and technical advantages of the lamprey CNS, we recently demonstrated that RhoA knockdown promotes true axon regeneration through the lesion site after SCI. In addition, we found that RhoA knockdown protects the large, identified reticulospinal neurons from apoptosis after their axons were axotomized in spinal cord. Therefore, manipulation of the RhoA signaling pathway may be an important approach in the development of treatments that are both neuroprotective and axon regeneration-promoting, to enhance functional recovery after SCI.     

Differences in the regenerative response of neuronal cell populations and indications for plasticity in intraspinal neurons after spinal cord transection in adult zebrafish

In zebrafish, the capacity to regenerate long axons varies among different populations of axotomized neurons after spinal cord transection. In specific brain nuclei, 84-92% of axotomized neurons upregulate expression of the growth-related genes GAP-43 and L1.1 and 32-51% of these neurons regrow their descending axons. In contrast, 16-31% of spinal neurons with axons ascending to the brainstem upregulate these genes and only 2-4% regrow their axons. Dorsal root ganglion (DRG) neurons were not observed to regrow their ascending axons or to increase expression of GAP-43 mRNA. Expression of L1.1 mRNA is high in unlesioned and axotomized DRG neurons. In the lesioned spinal cord, expression of growth-related molecules is increased in a substantial population of non-axotomized neurons, suggesting morphological plasticity in the spinal-intrinsic circuitry. We propose that locomotor recovery in spinal-transected adult zebrafish is influenced less by recovery of ascending pathways, but more by regrowth of descending tracts and rearrangement of intraspinal circuitry.     

Application of recombinant adenovirus for in vivo gene delivery to spinal cord

One strategy for treating spinal cord injury is to supply damaged neurons with the appropriate neurotrophins either by direct delivery or by transfer of the corresponding genes using viral vectors. Here we report the feasibility of using recombinant adenovirus for in vivo gene transfer in spinal cord. After injection of a recombinant adenovirus carrying a β-galactosidase (β-gal) reporter gene into the mid-thoracic spinal cord of adult rats, transgene expression occurred not only in several types of cells around the injection site but also in neurons whose axons project to this region from rostral or caudal to the injection site. Among labeled neurons were those of the red nucleus, the vestibular nuclei, reticular formation, locus coeruleus, and Clarke's nucleus. A non-specific immune reaction, which could be blocked by immunosuppression with Cyclosporin A, reduced the number of transduced cells surviving at the injection site by 1 month. In neurons away from the injection site, where the immune response was minimal, transgene expression lasted for at least 2 months. These results support the idea that recombinant adenovirus can be used in the spinal cord for in vivo delivery of therapeutic genes important for supporting neuron survival and axon regeneration.     

Role of Exosomes Derived from miR-133b Modified MSCs in an Experimental Rat Model of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) has poor outcomes due to high mortality and morbidity, but until now, the effective treatments remain limited. MicroRNAs (miRNAs) are vital regulators of gene expression and demonstrated to be linked to the pathogenesis of various central nervous system (CNS) diseases. Exosomes are considered as cell-to-cell communication vectors and secreted largely by mesenchymal stromal cells (MSCs). The present study investigated the role of miR-133b delivered by exosomes secreted from MSCs to brain tissues in rats after ICH. An autologous arterial blood ICH model in adult male Sprague–Dawley (SD) rats was used in this study. At 72 h after transfection with miR-133b mimics in MSCs, miR-133b-modified MSC-derived exosomes were collected from medium of MSCs and then injected to rats via tail vein. The levels of miR-133b in secreted exosomes and brain tissues of rats in various groups and the levels of RhoA, phosphorylations of extracellular signal regulating kinase (ERK1/2), and cAMP response element-binding protein (CREB) were detected by real-time PCR and western blot analysis, respectively. The effects of miR-133b on neuronal apoptosis and degeneration were respectively evaluated by TUNEL and fluoro-jade B staining. The miR-133b levels were reduced in brain tissues of rats at 24 h and peaked at 72 h after ICH. At 24 h after miR-133b-modified exosome administration, the level of miR-133b was significantly increased, while the apoptotic and neurodegenerative neurons were obviously reduced in brain tissues after ICH. The results of western blot analysis showed that miR-133b modified exosomes treatment remarkably suppressed RhoA expression and activated ERK1/2/CREB in brain tissues after ICH. Collectively, our investigation suggested that exosomes derived from miR-133b modified MSCs exhibited neuroprotective role for anti-apoptotic effect of miR-133b mediating RhoA and ERK1/2/CREB in rats after ICH.     

Efficacy of chitosan and sodium alginate scaffolds for repair of spinal cord injury in rats

Spinal cord injury results in the loss of motor and sensory pathways and spontaneous regeneration of adult mammalian spinal cord neurons is limited. Chitosan and sodium alginate have good biocompatibility, biodegradability, and are suitable to assist the recovery of damaged tissues, such as skin, bone and nerve. Chitosan scaffolds, sodium alginate scaffolds and chitosan-sodium alginate scaffolds were separately transplanted into rats with spinal cord hemisection. Basso-Beattie-Bresnahan locomotor rating scale scores and electrophysiological results showed that chitosan scaffolds promoted recovery of locomotor capacity and nerve transduction of the experimental rats. Sixty days after surgery, chitosan scaffolds retained the original shape of the spinal cord. Compared with sodium alginate scaffolds-and chitosan-sodium alginate scaffolds-transplanted rats, more neurofilament-H-immunoreactive cells(regenerating nerve fibers) and less glial fibrillary acidic protein-immunoreactive cells(astrocytic scar tissue) were observed at the injury site of experimental rats in chitosan scaffold-transplanted rats. Due to the fast degradation rate of sodium alginate, sodium alginate scaffolds and composite material scaffolds did not have a supporting and bridging effect on the damaged tissue. Above all, compared with sodium alginate and composite material scaffolds, chitosan had better biocompatibility, could promote the regeneration of nerve fibers and prevent the formation of scar tissue, and as such, is more suitable to help the repair of spinal cord injury.     

Leukocyte common antigen-related phosphatase is a functional receptor for chondroitin sulfate proteoglycan axon growth inhibitors

Chondroitin sulfate proteoglycans (CSPGs) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division, and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane leukocyte common antigen-related phosphatase (LAR). CSPGs bind LAR with high affinity in transfected COS-7 cells and coimmunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knock-out mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG-LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.     

Axonal regeneration after spinal cord injury in zebrafish and mammals: differences, similarities, translation

Spinal cord injury (SCI) in mammals results in functional deficits that are mostly permanent due in part to the inability of severed axons to regenerate. Several types of growth-inhibitory molecules expressed at the injury site contribute to this regeneration failure. The responses of axons to these inhibitors vary greatly within and between organisms, reflecting axons’ characteristic intrinsic propensity for regeneration. In the zebrafish (Danio rerio) many but not all axons exhibit successful regeneration after SCI. This review presents and compares the intrinsic and extrinsic determinants of axonal regeneration in the injured spinal cord in mammals and zebrafish. A better understanding of the molecules and molecular pathways underlying the remarkable individualism among neurons in mature zebrafish may support the development of therapies for SCI and their translation to the clinic.     

Axonal guidance molecules and the failure of axonal regeneration in the adult mammalian spinal cord

A wide variety of molecules are involved as attractive or repulsive guidance cues in the developing nervous system. Some of these molecules are also expressed in the CNS of adult mammals where, following injury, they may repel regenerating axons, inhibit axonal regrowth, or control the behaviour of other cells important for the development of the meningeal and glial scars or the immune response to injury. Ephrins, semaphorins, Slits, Netrins, bone morphogenetic proteins (BMPs) and Wnts are among the most likely molecules to be involved in limiting axonal regeneration in the injured spinal cord. The receptors for these molecules are not universally expressed by neurons but there is evidence that ephrins and semaphorins limit regeneration of particular classes of axon into spinal cord lesion sites. It is likely that other repulsive guidance cues will also differentially affect the regeneration of specific tracts within the spinal cord. In addition to direct effects on axonal regeneration, many axonal guidance molecules have effects on glial, meningeal or immune system cells which also modulate the responses of CNS tissue to injury.     

Lesional RhoA+ cell numbers are suppressed by anti-inflammatory, cyclooxygenase-inhibiting treatment following subacute spinal cord injury

Inhibition of the small GTPase RhoA or its downstream target Rho-associated coiled kinase (ROCK) has been shown to promote axon regeneration and to improve functional recovery following spinal cord injury (SCI) in the adult rat. RhoA has also been implicated in delayed secondary injury pathophysiology, such as free radical formation and loss of endothelial integrity leading to edema formation. In the present report, we have analyzed the effect of the central nervous system (CNS) permissive, putatively neuroprotective, anti-inflammatory cyclooxygenase-1/-2 (COX-1/-2) inhibitor indomethacin in CNS effective dosage (2 mg/kg/day) on lesional RhoA expression following subacute spinal cord injury. In control rats receiving vehicle alone, RhoA+ cells accumulate at the lesion site (Th8). At day 3 following SCI, the RhoA+ cellular composition is composed prevailingly of microglia/macrophages and polymononuclear granulocytes, but few reactive astrocytes. In contrast, in the verum group, lesional numbers of RhoA cells were reduced by indomethacin treatment by more than 60% (P < 0.0001). Inflammation-dependent RhoA expression accessible by cyclooxygenase inhibition proposes an immune-related mechanism. Our results identify COX blockers as candidates for a safe, synergistic, adjuvant treatment option in combination with cell-specific approaches to Rho inactivation, effectively minimizing the pool of RhoA+ cells at the lesion site following SCI.     

Olfactory ensheathing cell transplantation and inhibition of NogoA, NgR and RhoA expression in the damaged zone to ameliorate spinal cord injury

BACKGROUND:Olfactory ensheathing cell(OEC)transplantation promotes repair of spinal cord injury. Neural regeneration inhibits binding of the myelin protein Nogo to its receptor(NgR),activates downstream inhibitory signal RhoA, and leads to axonal degeneration.OBJECTIVE: To determine the relationship between OECs transplantation for spinal cord injury and NogoA, NgR, and RhoA protein expression in the damaged zone.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed from September 2006 to May 2007 at the Key Laboratory of Environment and Genes in Xi'an Jiaotong University School of Medicine, China.MATERIALS: OECs were harvested from healthy, adult, male, Sprague Dawley rats aged 6 months. Mouse anti-rat NogoA, NgR, and RhoA monoclonal antibodies were utilized for detection.METHODS: A total of 40 adult Sprague Dawley rats were randomly assigned to four groups: normal, model, OECs, and DF12, with 10 animals in each group. Transverse section spinal cord injury was established in the OECs and DF12 groups, followed by injection of 1 μL OECs suspension(1×108/mL)or equivalent DF12 medium at 1 mm above and below the injury site.MAIN OUTCOME MEASURES: Immunohistochemistry and Western blot were utilized to detect NogoA, NgR, and RhoA expression in the spinal cord injury lesions. Morphological changes were observed by argyrophilia staining, and lower extremity function of the animals was assessed using Basso, Beattie, and Bresnahan scores.RESULTS: Eight weeks following OECs transplantation, a significant increase in new axons was observed in the OECs group, and nerve fibers crossed the injury site to repair spinal cord injury.Qualitative and quantitative results from the OECs group were superior to the model and DF12 groups. At 8 weeks after transplantation, Basso, Beattie, and Bresnahan scores were significantly greater in the OECs group compared with the model and DF12 groups(P< 0.01), but expression of NogoA, NgR, and RhoA protein was significantly decreased compared with the model and DF12 groups(P< 0.05).CONCLUSION: OEC transplantation could inhibit NogoA, NgR, and RhoA expression in spinal cord injury lesions, thereby promoting repair of spinal cord injury.     

Intraspinal transplants

Transplants of embryonic central nervous system tissue have long been used to study axon growth during development and regeneration, and more recently to promote recovery in models of human diseases. Transplants of embryonic substantia nigra correct some of the deficits found in experimental Parkinson's disease, for example, by mechanisms that are thought to include release of neurotransmitter and reinnervation of host targets, as well as by stimulating growth of host axons. Similar mechanisms appear to allow intraspinal transplants of embryonic brainstem to reverse locomotor and autonomic deficits due to experimental spinal cord injuries. Embryonic spinal cord transplants offer an additional strategy for correcting the deficits of spinal cord injury because, by replacing damaged populations of neurons, they may mediate the restoration of connections between host neurons. We have found that spinal cord transplants permit regrowth of adult host axons resulting in reconstitution of synaptic complexes within the transplant that in many respects resemble normal synapses. Transplants of fetal spinal cord may also contribute to behavioral recovery by rescuing axotomized host neurons that otherwise would have died. Electrophysiological and behavioral investigations of functional recovery after intraspinal transplantation are preliminary, and the role of transplants in the treatment of human spinal cord injury is uncertain. Transplants are contributing to our understanding of the mechanisms of recovery, however, and are likely to play a role in the development of rational treatments.     

RhoA/Rho kinase in spinal cord injury

A spinal cord injury refers to an injury to the spinal cord that is caused by a trauma instead of diseases. Spinal cord injury includes a primary mechanical injury and a much more complex secondary injury pro-cess involving inlfammation, oxidation, excitotoxicity, and cell death. During the secondary injury, many signal pathways are activated and play important roles in mediating the pathogenesis of spinal cord injury. Among them, the RhoA/Rho kinase pathway plays a particular role in mediating spinal degeneration and regeneration. In this review, we will discuss the role and mechanism of RhoA/Rho kinase-mediated spinal cord pathogenesis, as well as the potential of targeting RhoA/Rho kinase as a strategy for promoting both neuroprotection and axonal regeneration.     

Identification of key genes involved in recovery from spinal cord injury in adult zebrafish

Zebrafish are an effective vertebrate model to study the mechanisms underlying recovery after spinal cord injury.The subacute phase after spinal cord injury is critical to the recovery of neurological function,which involves tissue bridging and axon regeneration.In this study,we found that zebrafish spontaneously recovered 44%of their swimming ability within the subacute phase(2 weeks)after spinal cord injury.During this period,we identified 7762 differentially expressed genes in spinal cord tissue:2950 were up-regulated and 4812 were down-regulated.These differentially expressed genes were primarily concentrated in the biological processes of the respiratory chain,axon regeneration,and cell-component morphogenesis.The genes were also mostly involved in the regulation of metabolic pathways,the cell cycle,and gene-regulation pathways.We verified the gene expression of two differentially expressed genes,clasp2 up-regulation and h1m down-regulation,in zebrafish spinal cord tissue in vitro.Pathway enrichment analysis revealed that up-regulated clasp2 functions similarly to microtubule-associated protein,which is responsible for axon extension regulated by microtubules.Down-regulated h1m controls endogenous stem cell differentiation after spinal cord injury.This study provides new candidate genes,clasp2 and h1m,as potential therapeutic intervention targets for spinal cord injury repair by neuroregeneration.All experimental procedures and protocols were approved by the Animal Ethics Committee of Tianjin Institute of Medical&Pharmaceutical Sciences(approval No.IMPS-EAEP-Q-2019-02)on September 24,2019.     

Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts

Experiments were carried out to determine if neurons damaged by injury to the spinal cord retain the ability to regenerate their axonal process for a prolonged period of time after the initial response to injury and if peripheral nerve (PN) grafts could support the regrowth of these processes. True blue (TB) was injected into one side of the adult rat lumbar spinal cord to label neurons with axons coursing through this region. Seven days later spinal cord tissue surrounding the injection sites was removed by aspiration to create a hemisection cavity 3-4 mm in length. Four weeks later scar tissue lining the lesion cavity was removed prior to grafting 1 cm segments of autologous tibial nerve to the rostral and the caudal surfaces of the cavity wall. The distal end of each graft was ligated and left unapposed to spinal cord tissue. Four weeks later the distal end of each PN graft was exposed to nuclear yellow (NY) to retrogradely label neurons that had grown an axon into the graft. Neurons containing both TB and NY were deemed capable of axonal regeneration while in a chronically injured state. Double-labeled (TB/NY) neurons were found in the ipsilateral spinal cord in laminae IV through X, excluding IX, and in Laminae VI and VII contralateral to the lesion. Most neurons were located within 10 mm of the lesion, with the majority caudal to the lesion. Nearly 50% (range 24-74%) of lumbar dorsal root ganglion neurons containing TB also were labeled with NY.(ABSTRACT TRUNCATED AT 250 WORDS)