NB‐3 signaling mediates the cross‐talk between post‐traumatic spinal axons and scar‐forming cells

Little is known about the molecules mediating the cross‐talk between post‐traumatic axons and scar‐forming cells after spinal cord injury. We found that a sustained NB‐3 induction was simultaneously present in the terminations of post‐traumatic corticospinal axons and scar‐forming cells at the spinal lesion site, where they were in direct contact when axons tried to penetrate the glial scar. The regrowth of corticospinal axons was enhanced in vivo with NB‐3 deficiency or interruption of NB‐3 trans‐homophilic interactions. Biochemical, in vitro and in vivo evidence demonstrated that NB‐3 homophilically interacted in trans to initiate a growth inhibitory signal transduction from scar‐forming cells to neurons by modulating mTOR activity via CHL1 and PTPσ. NB‐3 deficiency promoted BMS scores, electrophysiological transmission, and synapse reformation between regenerative axons and neurons. Our findings demonstrate that NB‐3 trans‐homophilic interactions mediate the cross‐talk between post‐traumatic axons and scar‐forming cells and impair the intrinsic growth ability of injured axons.     

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.     

Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin‐associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1‐day‐old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1‐to 22‐day‐old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1‐ and 15‐day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15‐day cord overwhelmed permissive or growth promoting molecules in membranes from 1‐day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 393–406, 1999     

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.     

Generation of an OMgp allelic series in mice

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin‐derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type‐specific roles of OMgp in CNS injury‐induced axonal plasticity. genesis 47:751–756, 2009. © 2009 Wiley‐Liss, Inc.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Phosphorylation of Dpsyl2 (CRMP2) and Dpsyl3 (CRMP4) is required for positioning of caudal primary motor neurons in the zebrafish spinal cord

Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co‐injection of phosphorylation‐mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 911–920, 2013     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Metformin ameliorates BSCB disruption by inhibiting neutrophil infiltration and MMP‐9 expression but not direct TJ proteins expression regulation

Blood‐spinal cord barrier (BSCB) disruption is a major process for the secondary injury of spinal cord injury (SCI) and is considered to be a therapeutic target for SCI. Previously, we demonstrated that metformin could improve functional recovery after SCI; however, the effect of metformin on BSCB is still unknown. In this study, we found that metformin could prevent the loss of tight junction (TJ) proteins at day 3 after SCI in vivo, but in vitro there was no significant difference of these proteins between control and metformin treatment in endothelial cells. This indicated that metformin‐induced BSCB protection might not be mediated by up‐regulating TJ proteins directly, but by inhibiting TJ proteins degradation. Thus, we investigated the role of metformin on MMP‐9 and neutrophils infiltration. Neutrophils infiltration is the major source of the enhanced MMP‐9 in SCI. Our results showed that metformin decreased MMP‐9 production and blocked neutrophils infiltration at day 1 after injury, which might be related to ICAM‐1 down‐regulation. Also, our in vitro study showed that metformin inhibited TNF‐α‐induced MMP‐9 up‐regulation in neutrophils, which might be mediated via an AMPK‐dependent pathway. Together, it illustrated that metformin prevented the breakdown of BSCB by inhibiting neutrophils infiltration and MMP‐9 production, but not by up‐regulating TJ proteins expression. Our study may help to better understand the working mechanism of metformin on SCI.     

Failure of Schwann cells as supporting cells for adult neural progenitor cell grafts in the acutely injured spinal cord

Adult neural progenitor cells (NPC) co-grafted with fibroblasts replace cystic lesion defects and promote cell-contact-mediated axonal regeneration in the acutely injured spinal cord. Fibroblasts are required as a platform to maintain NPC within the lesion; however, they are suspected to create an inhospitable milieu for regenerating central nervous system (CNS) axons. Therefore, we thought to replace fibroblasts by primary Schwann cells, which might serve as a superior scaffold to maintain NPC within the lesion and might further enhance axon regrowth and remyelination following spinal cord injury. Adult rats underwent a cervical dorsal column transection immediately followed by transplantation of either NPC/Schwann cell or NPC/Schwann cell/fibroblast co-grafts. Animals receiving Schwann cell or fibroblast grafts alone, or Schwann cell/fibroblast co-grafts served as controls. At 3 weeks after injury/transplantation, histological analysis revealed that only fibroblast-containing grafts were able to replace the cystic lesion defect. In both co-cultures and co-grafts, Schwann cells and NPC were segregated. Almost all NPC migrated out of the graft into the adjacent host spinal cord. As a consequence, only peripheral-type myelin, but no CNS-type myelin, was detected within co-grafts containing NPC/Schwann cells. Corticospinal axon regeneration into Schwann-cell-containing co-grafts was reduced. Taken together, Schwann cells within NPC grafts contribute to remyelination. However, Schwann cells fail as a supporting platform to maintain NPC within the graft and impair CNS axon regeneration; this makes them an unfavorable candidate to support/augment NPC grafts following spinal cord injury.This work was supported by the Institute International de Recherche en Paraplégie Geneva, on behalf of an anonymous donation, and ReForM-Program, University of Regensburg, School of Medicine.     

The C‐terminal domain of tetanus toxin protects motoneurons against acute excitotoxic damage on spinal cord organotypic cultures

The C‐terminal domain of tetanus toxin (Hc‐TeTx) has been suggested to act as a neuroprotective agent by activating signaling pathways related to neurotrophins and also to exert anti‐apoptotic effects. Here, we show the beneficial properties of the recombinant protein Hc‐TeTx to protect spinal motoneurons against excitotoxic damage. In vitro spinal cord organotypic cultures were used to assess acute glutamate excitotoxic damage. Our results indicate that Hc‐TeTx treatment improves motoneuron survival within a short therapeutical window (the first 2 h post‐injury). Within this interval, we found that p44/p42 MAP kinase (ERK1/2) and glycogen synthase kinase‐3 (GSK3β) signaling pathways play a crucial role in the neuroprotective effect. Moreover, we demonstrated that Hc–TeTx treatment initiate autophagy which is ERK1/2‐ and GSK3β‐dependent. These findings suggest a possible therapeutical tool to improve motoneuron survival immediately after excitotoxic insults or during the secondary injury phase that occurs after spinal cord trauma.     

Monoamine and Tissue Fluid Levels in Contused Spinal Cord of Sheep

Methods are described for the determination of sheep spinal cord tissue fluid content and norepinephrine, serotonin, and dopamine concentrations after experimental injury. The amount of tissue fluid varied in different regions of sheep spinal cord following injury. Norepinephrine and serotonin wet weight concentrations were corrected for this variation in tissue fluid. Corrected norepinephrine wet weight cord concentrations did not change up to 3 hr after injury. Levels of serotonin at 60 min after injury were similar to controls. Dopamine was not detected in sheep spinal cord. α-Methyl tyrosine significantly reduced fluid in the spinal cord at 75 min after injury. Hinwood B. G. et al. Monoamine and tissue fluid levels in contused spinal cord of sheep. J. Neurochem. 35, 786–791 (1980).     

Stem‐Cell‐Driven Growth and Regrowth of the Adult Spinal Cord in Teleost Fish

The spinal cord of the teleost fish Apteronotus leptorhynchus continues to grow during adulthood, in concert with the overall body growth. Immunohistological studies, combined with mathematical modeling, suggest that this growth is driven by proliferative activity of Sox2‐expressing stem/progenitor cells (SPCs) and by cell drift due to population pressure. The SPCs exhibit high volumetric density in the caudal filament and the ependymal layer. Nevertheless, the majority of these cells are found in the parenchyma throughout the rostro‐caudal axis of the spinal cord, albeit at much lower volumetric densities than in the ependymal layer. The SPCs give rise, via transit‐amplifying cells, to neurons and glia. The relative number of neurons and glia is primarily regulated through apoptosis of supernumerary neurons. Quantitative analysis has demonstrated that the continued cell proliferation results in additive neurogenesis. This addition includes adult‐born spinal electromotoneurons, thereby resulting in a continuous increase in the amplitude of the fish’s electric organ discharge during adult life. Amputation of the caudal part of the spinal cord induces initially a degenerative response, dominated by massive apoptotic cell death in spinal cord tissue immediately rostral to the injury site, and distinguished by a partial loss of the electric organ discharge amplitude. This phase is followed by a regenerative response, characterized by absence of gliosis and by rapid stem‐cell‐driven tissue regrowth. Although the quality of the regenerated tissue is variable among individuals, the structural repair has led in every fish examined thus far to full recovery of the electric organ discharge amplitude.     

Zebrafish Spinal Cord Repair Is Accompanied by Transient Tissue Stiffening

Severe injury to the mammalian spinal cord results in permanent loss of function due to the formation of a glial-fibrotic scar. Both the chemical composition and the mechanical properties of the scar tissue have been implicated to inhibit neuronal regrowth and functional recovery. By contrast, adult zebrafish are able to repair spinal cord tissue and restore motor function after complete spinal cord transection owing to a complex cellular response that includes axon regrowth and is accompanied by neurogenesis. The mechanical mechanisms contributing to successful spinal cord repair in adult zebrafish are, however, currently unknown. Here, we employ atomic force microscopy-enabled nanoindentation to determine the spatial distributions of apparent elastic moduli of living spinal cord tissue sections obtained from uninjured zebrafish and at distinct time points after complete spinal cord transection. In uninjured specimens, spinal gray matter regions were stiffer than white matter regions. During regeneration after transection, the spinal cord tissues displayed a significant increase of the respective apparent elastic moduli that transiently obliterated the mechanical difference between the two types of matter before returning to baseline values after the completion of repair. Tissue stiffness correlated variably with cell number density, oligodendrocyte interconnectivity, axonal orientation, and vascularization. This work constitutes the first quantitative mapping of the spatiotemporal changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides the tissue mechanical basis for future studies into the role of mechanosensing in spinal cord repair.     

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.Download video file.^{(224M, mp4)}     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti‐inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV‐2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS‐induced M1 BV‐2 microglia, also the inflammatory secretion phenotype of M1 BV‐2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK‐/mTOR‐mediated autophagic flux stimulation.