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In contrast to humans, lampreys spontaneously recover their swimming capacity after a complete spinal cord injury (SCI). This recovery process involves the regeneration of descending axons. Spontaneous axon regeneration in lampreys has been mainly studied in giant descending neurons. However, the regeneration of neurochemically distinct descending neuronal populations with small-caliber axons, as those found in mammals, has been less studied. Cholecystokinin (CCK) is a regulatory neuropeptide found in the brain and spinal cord that modulates several processes such as satiety, or locomotion. CCK shows high evolutionary conservation and is present in all vertebrate species. Work in lampreys has shown that all CCKergic spinal cord axons originate in a single neuronal population located in the caudal rhombencephalon. Here, we investigate the spontaneous regeneration of CCKergic descending axons in larval lampreys following a complete SCI. Using anti-CCK-8 immunofluorescence, confocal microscopy and lightning adaptive deconvolution, we demonstrate the partial regeneration of CCKergic axons (81% of the number of axonal profiles seen in controls) 10 weeks after the injury. Our data also revealed a preference for regeneration of CCKergic axons in lateral spinal cord regions. Regenerated CCKergic axons exhibit colocalization with synaptic vesicle marker SV2, indicative of functional synaptic connections. We also extracted swimming dynamics in injured animals by using DeepLabCut. Interestingly, the degree of CCKergic reinnervation correlated with improved swimming performance in injured animals, suggesting a potential role in locomotor recovery. These findings open avenues for further exploration into the role of specific neuropeptidergic systems in post-SCI spinal locomotor networks.

Abstract Spinal cord injury (SCI) represents a profound central nervous system affliction, resulting in irreversibly compromised daily activities and disabilities. SCI involves excessive inflammatory responses, which are characterized by the existence of high levels of proinflammatory M1 macrophages, and neuronal mitochondrial energy deficit, exacerbating secondary damage and impeding axon regeneration. This study delves into the mechanistic intricacies of SCI, offering insights from the perspectives of neuroimmune regulation and mitochondrial function, leading to a pro-fibrotic macrophage phenotype and energy-supplying deficit. To address these challenges, we developed a smart scaffold incorporating enzyme mimicry nanoparticle-ceriumoxide (COPs) into nanofibers (NS@COP), which aims to pioneer a targeted neuroimmune repair strategy, rescuing CGRP receptor on macrophage and concurrently remodeling mitochondrial function. Our findings indicate that the integrated COPs restore the responsiveness of pro-inflammatory macrophages to calcitonin gene-related peptide (CGRP) signal by up-regulating receptor activity modifying protein 1 (RAMP1), a vital component of the CGRP receptor. This promotes macrophage fate commitment to an anti-inflammatory pro-resolution M2 phenotype, then alleviating glial scar formation. In addition, NS@COP implantation also protected neuronal mitochondrial function. Collectively, our results suggest that the strategy of integrating nanozyme COP nanoparticles into a nanofiber scaffold provides a promising therapeutic candidate for spinal cord trauma via rational regulation of neuroimmune communication and mitochondrial function.

Spinal cord injury has the characteristics of high mortality and disability rates, and the treatment of spinal cord injury has been a worldwide problem. With the deepening of spinal cord injury research and the advancement of treatment methods, the survival rate of patients has improved, but the clinical efficacy is still not ideal. New research has found that the immune response is involved in the entire process of pathological changes and regeneration and repair after spinal cord injury. Among them, the complement system plays an important role in the occurrence and development of spinal cord injury. This article mainly reviews the role of the complement system in secondary spinal cord injury and regeneration and repair after spinal cord injury, including the doubleedged sword effect of the complement system, the impact of the complement system on axonal regeneration and synaptic maintenance, and possible signaling pathways, with a view to improving the treatment of spinal cord injury, providing new treatment ideas.

Abstract Background The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear. Methods We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs). Results The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites. Conclusion These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration.

IntroductionAnesthetic conditioning has been shown to provide neuroprotection in several neurological disorders. Whether anesthetic conditioning provides protection against peripheral nerve injuries remains unknown. The aim of our current study is to investigate the impact of isoflurane conditioning on the functional outcomes after peripheral nerve injury (PNI) in a rodent sciatic nerve injury model.MethodsAdult male Lewis rats underwent sciatic nerve cut and repair and exposed to none (Group 1, sham), single isoflurane exposure (Group 2), three-time isoflurane exposure (Group 3), and six-time isoflurane exposure (Group 4). Isoflurane conditioning was established by administration of 2% isoflurane for 1 hour, beginning 1-hour post sciatic nerve cut and repair. Groups 3 and 4 were exposed to isoflurane for 1 hour, 3 and 6 consecutive days respectively. Functional outcomes assessed included compound muscle action potential (CMAP), evoked muscle force (tetanic and specific tetanic force), wet muscle mass, and axonal counting.ResultsWe observed an increase in axons, myelin width and a decrease in G-ratio in the isoflurane conditioning groups (3- and 6-days). This correlated with a significant improvement in tetanic and specific tetanic forces, observed in both groups 3 and 4.DiscussionIsoflurane conditioning (3- and 6-day groups) resulted in improvement in functional outcomes at 12 weeks post peripheral nerve injury and repair in a murine model. Future experiments should be focused on identifying the therapeutic window of isoflurane conditioning and exploring the underlying molecular mechanisms responsible for isoflurane conditioning induced neuroprotection in PNI.

Peripheral nerve damage often leads to the onset of neuropathic pain (NeuP). This condition afflicts millions of people, significantly burdening healthcare systems and putting strain on families’ financial well-being. Here, we will focus on the role of peripheral sensory neurons, specifically the Dorsal Root Ganglia neurons (DRG neurons) in the development of NeuP. After axotomy, DRG neurons activate regenerative signals of axons-soma communication to promote a gene program that activates an axonal branching and elongation processes. The results of a neuronal morphological cytoskeleton change are not always associated with functional recovery. Moreover, any axonal miss-targeting may contribute to NeuP development. In this review, we will explore the epidemiology of NeuP and its molecular causes at the level of the peripheral nervous system and the target organs, with major focus on the neuronal cross-talk between intrinsic and extrinsic factors. Specifically, we will describe how failures in the neuronal regenerative program can exacerbate NeuP.

Peripheral nerve injuries (PNIs) are common and devastating. The current standard of care relies on the slow and inefficient process of nerve regeneration after surgical intervention. Electrical stimulation (ES) has been shown to both experimentally and clinically result in improved regeneration and functional recovery after PNI for motor and sensory neurons; however, its effects on sympathetic regeneration have never been studied. Sympathetic neurons are responsible for a myriad of homeostatic processes that include, but are not limited to, blood pressure, immune response, sweating, and the structural integrity of the neuromuscular junction. Almost one quarter of the axons in the sciatic nerve are from sympathetic neurons, and their importance in bodily homeostasis and the pathogenesis of neuropathic pain should not be underestimated. Therefore, as ES continues to make its way into patient care, it is not only important to understand its impact on all neuron subtypes, but also to ensure that potential adverse effects are minimized. This piece gives an overview of the effects of ES in animals models and in humans while offering a perspective on the potential effects of ES on sympathetic axon regeneration.

Abstract Spinal cord injury (SCI) has no effective treatment modalities. It faces a significant global therapeutical challenge, given its features of poor axon regeneration, progressive local inflammation, and inefficient systemic drug delivery due to the blood–spinal cord barrier (BSCB). To address these challenges, a new nano complex that achieves targeted drug delivery to the damaged spinal cord is proposed, which contains a mesoporous silica nanoparticle core loaded with microRNA and a cloaking layer of human umbilical cord mesenchymal stem cell membrane modified with rabies virus glycoprotein (RVG). The nano complex more readily crosses the damaged BSCB with its exosome‐resembling properties, including appropriate size and a low‐immunogenic cell membrane disguise and accumulates in the injury center because of RVG, where it releases abundant microRNAs to elicit axon sprouting and rehabilitate the inflammatory microenvironment. Culturing with nano complexes promotes axonal growth in neurons and M2 polarization in microglia. Furthermore, it showed that SCI mice treated with this nano complex by tail vein injection display significant improvement in axon regrowth, microenvironment regulation, and functional restoration. The efficacy and biocompatibility of the targeted delivery of microRNA by nano complexes demonstrate their immense potential as a noninvasive treatment for SCI.

Valeriana jatamansi Jones (VJJ), renowned for its extensive history in traditional Chinese medicine and ethnomedicine within China, is prevalently utilized to alleviate ailments such as epigastric distension and pain, gastrointestinal disturbances including food accumulation, diarrhea, and dysentery, as well as insomnia and other diseases. Moreover, the Iridoid-rich fraction derived from Valeriana jatamansi Jones (IRFV) has demonstrated efficacy in facilitating the recuperation of motor functions after spinal cord injury (SCI). This study is aimed to investigate the therapeutic effect of IRFV on SCI and its underlying mechanism. Initially, a rat model of SCI was developed to assess the impact of IRFV on axonal regeneration. Subsequently, employing the PC12 cell model of oxidative damage, the role and mechanism of IRFV in enhancing axonal regeneration were explored using the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway inhibitor LY294002. Ultimately, the same inhibitor was administered to SCI rats to confirm the molecular mechanism through which IRFV promotes axonal regeneration by activating the PI3K/Akt signaling pathway. The results showed that IRFV significantly enhanced motor function recovery, reduced pathological injury, and facilitated axonal regeneration in SCI rats. In vitro experiments revealed that IRFV improved PC12 cell viability, augmented axonal regeneration, and activated the PI3K/Akt signaling pathway. Notably, the inhibition of this pathway negated the therapeutic benefits of IRFV in SCI rats. In conclusion, IRFV promote promotes axonal regeneration and recovery of motor function after SCI through activation of the PI3K/Akt signaling pathway.

Rab5 is a GTPase protein that is involved in intracellular membrane trafficking. It functions by binding to various effector proteins and regulating cellular responses, including the formation of transport vesicles and their fusion with the cellular membrane. Rab5 has been reported to play an important role in the development of the zebrafish embryo; however, its role in axonal regeneration in the central nervous system remains unclear. In this study, we established a zebrafish Mauthner cell model of axonal injury using single-cell electroporation and two-photon axotomy techniques. We found that overexpression of Rab5 in single Mauthner cells promoted marked axonal regeneration and increased the number of intra-axonal transport vesicles. In contrast, treatment of zebrafish larvae with the Rab kinase inhibitor CID-1067700 markedly inhibited axonal regeneration in Mauthner cells. We also found that Rab5 activated phosphatidylinositol 3-kinase (PI3K) during axonal repair of Mauthner cells and promoted the recovery of zebrafish locomotor function. Additionally, rapamycin, an inhibitor of the mechanistic target of rapamycin downstream of PI3K, markedly hindered axonal regeneration. These findings suggest that Rab5 promotes the axonal regeneration of injured zebrafish Mauthner cells by activating the PI3K signaling pathway.

Objective Herein, we investigated whether mesenchymal stem cells (MSCs) transplantation combined with electroacupuncture (EA) treatment could decrease the proportion of proinflammatory microglia/macrophages and neurotoxic A1 reactive astrocytes and inhibit glial scar formation to enhance axonal regeneration after spinal cord injury (SCI). Methods Adult rats were divided into 5 groups after complete transection of the spinal cord at the T10 level: a control group, a nonacupoint EA (NA-EA) group, an EA group, an MSC group, and an MSCs+EA group. Immunofluorescence labeling, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blots were performed. Results The results showed that MSCs+EA treatment reduced the proportion of proinflammatory M1 subtype microglia/macrophages, but increased the differentiation of anti-inflammatory M2 phenotype cells, thereby suppressing the mRNA and protein expression of proinflammatory cytokines (tumor necrosis factor-α and IL-1β) and increasing the expression of an anti-inflammatory cytokine (interleukin [IL]-10) on days 7 and 14 after SCI. The changes in expression correlated with the attenuated neurotoxic A1 reactive astrocytes and glial scar, which in turn facilitated the axonal regeneration of the injured spinal cord. In vitro, the proinflammatory cytokines increased the level of proliferation of astrocytes and increased the expression levels of C3, glial fibrillary acidic protein, and chondroitin sulfate proteoglycan. These effects were blocked by administering inhibitors of ErbB1 and signal transducer and activator of transcription 3 (STAT3) (AG1478 and AG490) and IL-10. Conclusion These findings showed that MSCs+EA treatment synergistically regulated the microglia/macrophage subpopulation to reduce inflammation, the formation of neurotoxic A1 astrocytes, and glial scars. This was achieved by downregulating the ErbB1-STAT3 signal pathway, thereby providing a favorable microenvironment conducive to axonal regeneration after SCI.

Following spinal cord injury (SCI), the regenerative capacity of the central nervous system (CNS) is severely limited by the failure of axonal regeneration. The regeneration of CNS axons has been shown to occur by grafting predegenerated peripheral nerves (PPNs) and to be promoted by the transplantation of neural precursor cells (NPCs). The introduction of a combinatorial treatment of PPNs and NPCs after SCI has to address the additional problem of glial scar formation, which prevents regenerating axons from leaving the implant and making functional connections. Previously, we discovered that the synthetic sulfoglycolipid Tol-51 inhibits astrogliosis. The objective was to evaluate axonal regeneration and locomotor function improvement after SCI in rats treated with a combination of PPN, NPC, and Tol-51. One month after SCI, the scar tissue was removed and replaced with segments of PPN or PPN+Tol-51; PPN+NPC+Tol-51. The transplantation of a PPN segment favors regenerative axonal growth; in combination with Tol-51 and NPC, 30% of the labeled descending corticospinal axons were able to grow through the PPN and penetrate the caudal spinal cord. The animals treated with PPN showed significantly better motor function. Our data demonstrate that PPN implants plus NPC and Tol-51 allow successful axonal regeneration in the CNS.

Providing cellular support and modifying the glial scar around the lesion are two key strategies for promoting axonal regeneration after spinal cord injury. We showed previously that over-expressing polysialic acid (PSA) on Schwann cells (SCs) by lentiviral vector (LV)-mediated expression of polysialyltransferase (PST) facilitated their integration and migration in the injured spinal cord. We also showed that PSA over-expression in the injured spinal cord modified the glial scar and promoted the growth of ascending sensory axons. In this study, we combined the PST/SC transplantation with LV/PST injection in spinal cords after dorsal column transection and found the combined treatments led to faster and more profound locomotor functional recovery compared with animals receiving combined GFP/SC transplantation with LV/GFP injection. Histological examination showed significantly more injured corticospinal axons growing close to the lesion/transplant borders and into the caudal spinal cord in the PST group than in the GFP group. We also found over -expressing PSA around the lesion site did not cause allodynia and hyperalgesia in our injury model. These results demonstrate the promising therapeutic benefit of over-expressing PSA in transplanted SCs and spinal cord in promoting axonal growth and restoring motor function.

Abstract Background Preinjury of peripheral nerves triggers dorsal root ganglia (DRG) axon regeneration, a biological change that is more pronounced in young mice than in old mice, but the complex mechanism has not been clearly explained. Here, we aim to gain insight into the mechanisms of axon regeneration after conditioning lesion in different age groups of mice, thereby providing effective therapeutic targets for central nervous system (CNS) injury. Methods The microarray GSE58982 and GSE96051 were downloaded and analyzed to identify differentially expressed genes (DEGs). The protein–protein interaction (PPI) network, the miRNA-TF-target gene network, and the drug-hub gene network of conditioning lesion were constructed. The L4 and L5 DRGs, which were previously axotomized by the sciatic nerve conditioning lesions, were harvested for qRT-PCR. Furthermore, histological and behavioral tests were performed to assess the therapeutic effects of the candidate drug telmisartan in spinal cord injury (SCI). Results A total of 693 and 885 DEGs were screened in the old and young mice, respectively. Functional enrichment indicates that shared DEGs are involved in the inflammatory response, innate immune response, and ion transport. QRT-PCR results showed that in DRGs with preinjury of peripheral nerve, Timp1, P2ry6, Nckap1l, Csf1, Ccl9, Anxa1, and C3 were upregulated, while Agtr1a was downregulated. Based on the bioinformatics analysis of DRG after conditioning lesion, Agtr1a was selected as a potential therapeutic target for the SCI treatment. In vivo experiments showed that telmisartan promoted axonal regeneration after SCI by downregulating AGTR1 expression. Conclusion This study provides a comprehensive map of transcriptional changes that discriminate between young and old DRGs in response to injury. The hub genes and their related drugs that may affect the axonal regeneration program after conditioning lesion were identified. These findings revealed the speculative pathogenic mechanism involved in conditioning-dependent regenerative growth and may have translational significance for the development of CNS injury treatment in the future.

ObjectiveThis study analyzed the current research hotspots and future development trends of the therapeutic effects of microRNA on PNI axonal regeneration through bibliometric methods. Moreover, the current advantages and disadvantages of this field as well as future development prospects are discussed in depth.MethodsCiteSpace V and VOSviewer were used as bibliometric tools to complete the analysis of the research focus and direction of the published articles. To supplement, sort out, and summarize, we analyzed the research status of the study on the application of microRNAs for axonal regeneration after peripheral nerve injury from 2013 to 2023.ResultsA total of 207 publications were retrieved from the Web of Science database. After exclusion and screening, a final selection of 174 articles that met the research criteria. These 174 articles were authored by a total of 846 individuals, representing 24 countries and 199 institutions. Additionally, this study presents information on the annual publication output, country distribution, top 5 contributing authors, top 5 most cited articles, and top 10 contributing institutions.ConclusionAs one of the hottest topics today, microRNAs have become the current research hotspot in neural inflammation, neural cell repair and regeneration, neural protection, and functional recovery. With more investment in research in this field, more high-quality articles will be published in both domestic and international outstanding journals, which will bring a new era for the treatment of peripheral nerve injury.

IntroductionMenstrual blood-derived stem cells (MenSCs) are vital in treating many degenerative and traumatic disorders. However, the underlying molecular mechanisms remain obscure in MenSCs-treating spinal cord injury (SCI) rats.MethodsMenSCs were adopted into the injured sites of rat spinal cords at day 7 post surgery and the tissues were harvested for total RNA sequencing analysis at day 21 after surgery to investigate the expression patterns of RNAs. The differentially expressed genes (DEGs) were analyzed with volcano and heatmap plot. DEGs were sequentially analyzed by weighted gene co-expression network, functional enrichment, and competitive endogenous RNAs (ceRNA) network analysis. Next, expression of selected miRNAs, lncRNAs, circRNAs and mRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics packages and extra databases were enrolled to scoop the genes functions and their interaction relationships.ResultsA total of 89 lncRNAs, 65 circRNAs, 120 miRNAs and 422 mRNAs were significantly upregulated and 65 lncRNAs, 72 circRNAs, 74 miRNAs, and 190 mRNAs were significantly downregulated in the MenSCs treated rats compared to SCI ones. Current investigation revealed that MenSCs treatment improve the recovery of the injured rats and the most significantly involved pathways in SCI regeneration were cell adhesion molecules, nature killer cell mediated cytotoxicity, primary immunodeficiency, chemokine signaling pathway, T cell receptor signaling pathway and B cell receptor signaling pathway. Moreover, the lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA network of SCI was constructed. Finally, the protein-protein interaction (PPI) network was constructed using the top 100 DE mRNAs. The constructed PPI network included 47 nodes and 70 edges.DiscussionIn summary, the above results revealed the expression profile and potential functions of differentially expressed (DE) RNAs in the injured spinal cords of rats in the MenSCs-treated and SCI groups, and this study may provide new clues to understand the mechanisms of MenSCs in treating SCI.

BackgroundSpinal cord injury (SCI) is a devastating disease that results in permanent paralysis. Currently, there is no effective treatment for SCI, and it is important to identify factors that can provide therapeutic intervention during the course of the disease. Zinc, an essential trace element, has attracted attention as a regulator of inflammatory responses. In this study, we investigated the effect of zinc status on the SCI pathology and whether or not zinc could be a potential therapeutic target.MethodsWe created experimental mouse models with three different serum zinc concentration by changing the zinc content of the diet. After inducing contusion injury to the spinal cord of three mouse models, we assessed inflammation, apoptosis, demyelination, axonal regeneration, and the number of nuclear translocations of NF-κB in macrophages by using qPCR and immunostaining. In addition, macrophages in the injured spinal cord of these mouse models were isolated by flow cytometry, and their intracellular zinc concentration level and gene expression were examined. Functional recovery was assessed using the open field motor score, a foot print analysis, and a grid walk test. Statistical analysis was performed using Wilcoxon rank-sum test and ANOVA with the Tukey-Kramer test.ResultsIn macrophages after SCI, zinc deficiency promoted nuclear translocation of NF-κB, polarization to pro-inflammatory like phenotype and expression of pro-inflammatory cytokines. The inflammatory response exacerbated by zinc deficiency led to worsening motor function by inducing more apoptosis of oligodendrocytes and demyelination and inhibiting axonal regeneration in the lesion site compared to the normal zinc condition. Furthermore, zinc supplementation after SCI attenuated these zinc-deficiency-induced series of responses and improved motor function.ConclusionWe demonstrated that zinc affected axonal regeneration and motor functional recovery after SCI by negatively regulating NF-κB activity and the subsequent inflammatory response in macrophages. Our findings suggest that zinc supplementation after SCI may be a novel therapeutic strategy for SCI.

[INLINE:1] Vision depends on accurate signal conduction from the retina to the brain through the optic nerve, an important part of the central nervous system that consists of bundles of axons originating from retinal ganglion cells. The mammalian optic nerve, an important part of the central nervous system, cannot regenerate once it is injured, leading to permanent vision loss. To date, there is no clinical treatment that can regenerate the optic nerve and restore vision. Our previous study found that the mobile zinc (Zn2+) level increased rapidly after optic nerve injury in the retina, specifically in the vesicles of the inner plexiform layer. Furthermore, chelating Zn2+ significantly promoted axonal regeneration with a long-term effect. In this study, we conditionally knocked out zinc transporter 3 (ZnT3) in amacrine cells or retinal ganglion cells to construct two transgenic mouse lines (VGATCreZnT3fl/fl and VGLUT2CreZnT3fl/fl, respectively). We obtained direct evidence that the rapidly increased mobile Zn2+ in response to injury was from amacrine cells. We also found that selective deletion of ZnT3 in amacrine cells promoted retinal ganglion cell survival and axonal regeneration after optic nerve crush injury, improved retinal ganglion cell function, and promoted vision recovery. Sequencing analysis of reginal ganglion cells revealed that inhibiting the release of presynaptic Zn2+ affected the transcription of key genes related to the survival of retinal ganglion cells in postsynaptic neurons, regulated the synaptic connection between amacrine cells and retinal ganglion cells, and affected the fate of retinal ganglion cells. These results suggest that amacrine cells release Zn2+ to trigger transcriptomic changes related to neuronal growth and survival in reginal ganglion cells, thereby influencing the synaptic plasticity of retinal networks. These results make the theory of zinc-dependent retinal ganglion cell death more accurate and complete and provide new insights into the complex interactions between retinal cell networks.

Stromal cell-derived factor-1 and its receptor C-X-C chemokine receptor 4 (CXCR4) have been shown to regulate neural regeneration after stroke. However, whether stromal cell-derived factor-1 receptor CXCR7, which is widely distributed in the developing and adult central nervous system, participates in neural regeneration remains poorly understood. In this study, we established rat models of focal cerebral ischemia by injecting endothelin-1 into the cerebral cortex and striatum. Starting on day 7 after injury, CXCR7-neutralizing antibody was injected into the lateral ventricle using a micro drug delivery system for 6 consecutive days. Our results showed that CXCR7-neutralizing antibody increased the total length and number of sprouting corticospinal tract fibers in rats with cerebral ischemia, increased the expression of vesicular glutamate transporter 1 and growth-related protein 43, markers of the denervated spinal cord synapses, and promoted the differentiation and maturation of oligodendrocyte progenitor cells in the striatum. In addition, CXCR7 antibody increased the expression of CXCR4 in the striatum, increased the protein expression of RAS and ERK1/2 associated with the RAS/ERK signaling pathway, and improved rat motor function. These findings suggest that CXCR7 improved neural functional recovery after ischemic stroke by promoting axonal regeneration, synaptogenesis, and myelin regeneration, which may be achieved by activation of CXCR4 and the RAS/ERK1/2 signaling pathway.

Treadmill exercise and mesenchymal stem cell transplantation are both practical and effective methods for the treatment of cerebral ischemia. However, whether there is a synergistic effect between the two remains unclear. In this study, we established rat models of ischemia/reperfusion injury by occlusion of the middle cerebral artery for 2 hours and reperfusion for 24 hours. Rat models were perfused with bone marrow mesenchymal stem cell-derived exosomes (MSC-exos) via the tail vein and underwent 14 successive days of treadmill exercise. Neurological assessment, histopathology, and immunohistochemistry results revealed decreased neuronal apoptosis and cerebral infarct volume, evident synaptic formation and axonal regeneration, and remarkably recovered neurological function in rats subjected to treadmill exercise and MSC-exos treatment. These effects were superior to those in rats subjected to treadmill exercise or MSC-exos treatment alone. Mechanistically, further investigation revealed that the activation of JNK1/c-Jun signaling pathways regulated neuronal apoptosis and synaptic-axonal remodeling. These findings suggest that treadmill exercise may exhibit a synergistic effect with MSC-exos treatment, which may be related to activation of the JNK1/c-Jun signaling pathway. This study provides novel theoretical evidence for the clinical application of treadmill exercise combined with MSC-exos treatment for ischemic cerebrovascular disease.

Vimentin is a major type III intermediate filament protein that plays important roles in several basic cellular functions including cell migration, proliferation, and division. Although vimentin is a cytoplasmic protein, it also exists in the extracellular matrix and at the cell surface. Previous studies have shown that vimentin may exert multiple physiological effects in different nervous system injuries and diseases. For example, the studies of vimentin in spinal cord injury and stroke mainly focus on the formation of reactive astrocytes. Reduced glial scar, increased axonal regeneration, and improved motor function have been noted after spinal cord injury in vimentin and glial fibrillary acidic protein knockout (GFAP–/–VIM–/–) mice. However, attenuated glial scar formation in post-stroke in GFAP–/– VIM–/– mice resulted in abnormal neuronal network restoration and worse neurological recovery. These opposite results have been attributed to the multiple roles of glial scar in different temporal and spatial conditions. In addition, extracellular vimentin may be a neurotrophic factor that promotes axonal extension by interaction with the insulin-like growth factor 1 receptor. In the pathogenesis of bacterial meningitis, cell surface vimentin is a meningitis facilitator, acting as a receptor of multiple pathogenic bacteria, including E. coli K1, Listeria monocytogenes, and group B streptococcus. Compared with wild type mice, VIM–/– mice are less susceptible to bacterial infection and exhibit a reduced inflammatory response, suggesting that vimentin is necessary to induce the pathogenesis of meningitis. Recently published literature showed that vimentin serves as a double-edged sword in the nervous system, regulating axonal regrowth, myelination, apoptosis, and neuroinflammation. This review aims to provide an overview of vimentin in spinal cord injury, stroke, bacterial meningitis, gliomas, and peripheral nerve injury and to discuss the potential therapeutic methods involving vimentin manipulation in improving axonal regeneration, alleviating infection, inhibiting brain tumor progression, and enhancing nerve myelination.

Tanshinone IIA serves as a coenzyme for certain biochemical reactions, exhibiting various pharmacological effects in the treatment of neurological diseases including spinal cord injury (SCI), however, its working mechanism in the treatment of SCI is not clear. Based on previous research, we believe that tanshinone IIA promotes the survival and repair of nerves after spinal cord injury through its pharmacological effects such as anti-inflammatory, antioxidant, and prevention of cellular apoptosis in the spinal cord.

Abstract Background Bu-Shen-Huo-Xue (BSHX) decoction has been used in the postoperative rehabilitation of patients with spinal cord injury in China. In the present study, we aim to reveal the bioactive compounds in BSHX decoction and comprehensively explore the effects of BSHX decoction and the underlying mechanism in spinal cord injury recovery. Methods The main chemical constituents in BSHX decoction were determined by UPLC–MS/MS. SCI mice were induced by a pneumatic impact device at T9–T10 level of the vertebra, and treated with BSHX decoction. Basso–Beattie–Bresnahan (BBB) score, footprint analysis, hematoxylin–eosin (H&E) staining, Nissl staining and a series of immunofluorescence staining were performed to investigate the functional recovery, glial scar formation and axon regeneration after BSHX treatment. Immunofluorescent staining of bromodeoxyuridine (BrdU), neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) was performed to evaluate the effect of BSHX decoction on neural stem cells (NSCs) proliferation and differentiation. Results We found that the main compounds in BSHX decoction were Gallic acid, 3,4-Dihydroxybenzaldehyde, (+)-Catechin, Paeoniflorin, Rosmarinic acid, and Diosmetin. BSHX decoction improved the pathological findings in SCI mice through invigorating blood circulation and cleaning blood stasis in the lesion site. In addition, it reduced tissue damage and neuron loss by inhibiting astrocytes activation, and promoting the polarization of microglia towards M2 phenotype. The functional recovery test revealed that BSHX treatment improved the motor function recovery post SCI. Conclusions Our study provided evidence that BSHX treatment could improve the microenvironment of the injured spinal cord to promote axonal regeneration and functional recovery in SCI mice.

Unlike mammals, adult zebrafish are able to fully regenerate axons and functionally recover from neuronal damage in the mature central nervous system (CNS). Decades of research have tried to identify the mechanisms behind their spontaneous regenerative capacity, but the exact underlying pathways and molecular drivers remain to be fully elucidated. By studying optic nerve injury-induced axonal regrowth of adult zebrafish retinal ganglion cells (RGCs), we previously reported transient dendritic shrinkage and changes in the distribution and morphology of mitochondria in the different neuronal compartments throughout the regenerative process. These data suggest that dendrite remodeling and temporary changes in mitochondrial dynamics contribute to effective axonal and dendritic repair upon optic nerve injury. To further elucidate these interactions, we here present a novel adult zebrafish microfluidic model in which we can demonstrate compartment-specific alterations in resource allocation in real-time at single neuron level. First, we developed a pioneering method that enables to isolate and culture adult zebrafish retinal neurons in a microfluidic setup. Notably, with this protocol, we report on a long-term adult primary neuronal culture with a high number of surviving and spontaneously outgrowing mature neurons, which was thus far only very limitedly described in literature. By performing time-lapse live cell imaging and kymographic analyses in this setup, we can explore changes in dendritic remodeling and mitochondrial motility during spontaneous axonal regeneration. This innovative model system will enable to discover how redirecting intraneuronal energy resources supports successful regeneration in the adult zebrafish CNS, and might facilitate the discovery of new therapeutic targets to promote neuronal repair in humans.

Traumatic optic neuropathy (TON) is a condition that causes massive loss of retinal ganglion cells (RGCs) and their axonal fibers, leading to visual insufficiency. Several intrinsic and external factors can limit the regenerative ability of RGC after TON, subsequently resulting in RGC death. Hence, it is important to investigate a potential drug that can protect RGC after TON and enhance its regenerative capacity. Herein, we investigated whether Huperzine A (HupA), extracted from a Chinese herb, has neuroprotective effects and may enhance neuronal regeneration following the optic nerve crush (ONC) model. We compared the three modes of drug delivery and found that intravitreal injection of HupA could promote RGC survival and axonal regeneration after ONC. Mechanistically, HupA exerted its neuroprotective and axonal regenerative effects through the mTOR pathway; these effects could be blocked by rapamycin. To sum up, our findings suggest a promising application of HupA in the clinical treatment of traumatic optic nerve.

Axonal regeneration and functional recovery are poor after spinal cord injury (SCI), typified by the formation of an injury scar. While this scar was traditionally believed to be primarily responsible for axonal regeneration failure, current knowledge takes a more holistic approach that considers the intrinsic growth capacity of axons. Targeting the SCI scar has also not reproducibly yielded nearly the same efficacy in animal models compared to these neuron-directed approaches. These results suggest that the major reason behind central nervous system (CNS) regeneration failure is not the injury scar but a failure to stimulate axon growth adequately. These findings raise questions about whether targeting neuroinflammation and glial scarring still constitute viable translational avenues. We provide a comprehensive review of the dual role of neuroinflammation and scarring after SCI and how future research can produce therapeutic strategies targeting the hurdles to axonal regeneration posed by these processes without compromising neuroprotection.

Oscillating field stimulation (OFS) is a potential method for treating spinal cord injury. Although it has been used in spinal cord injury (SCI) therapy in basic and clinical studies, its underlying mechanism and the correlation between its duration and nerve injury repair remain poorly understood. In this study, we established rat models of spinal cord contusion at T10 and then administered 12 weeks of OFS. The results revealed that effectively promotes the recovery of motor function required continuous OFS for more than 6 weeks. The underlying mechanism may be related to the effects of OFS on promoting axon regeneration, inhibiting astrocyte proliferation, and improving the linear arrangement of astrocytes. This study was approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (supplemental approval No. AEEI-2021-204) on July 26, 2021.

Graphene and graphene-based materials have the ability to induce stem cells to differentiate into neurons, which is necessary to overcome the current problems faced in the clinical treatment of spinal cord injury. This review summarizes the advantages of graphene and graphene-based materials (in particular, composite materials) in axonal repair after spinal cord injury. These materials have good histocompatibility, and mechanical and adsorption properties that can be targeted to improve the environment of axonal regeneration. They also have good conductivity, which allows them to make full use of electrical nerve signal stimulation in spinal cord tissue to promote axonal regeneration. Furthermore, they can be used as carriers of seed cells, trophic factors, and drugs in nerve tissue engineering scaffolds to provide a basis for constructing a local microenvironment after spinal cord injury. However, to achieve clinical adoption of graphene and graphene-based materials for the repair of spinal cord injury, further research is needed to reduce their toxicity.

Spinal cord injury (SCI) is a serious and disabling disease with a high mortality rate. It often leads to complete or partial sensory and motor dysfunction and is accompanied by a series of secondary outcomes, such as pressure sores, pulmonary infections, deep vein thrombosis in the lower extremities, urinary tract infections, and autonomic dysfunction. Currently, the main treatments for SCI include surgical decompression, drug therapy, and postoperative rehabilitation. Studies have shown that cell therapy plays a beneficial role in the treatment of SCI. Nonetheless, there is controversy regarding the therapeutic effect of cell transplantation in SCI models. Meanwhile exosomes, as a new therapeutic medium for regenerative medicine, possess the advantages of small size, low immunogenicity, and the ability to cross the blood-spinal cord barrier. Certain studies have shown that stem cell-derived exosomes have anti-inflammatory effects and can play an irreplaceable role in the treatment of SCI. In this case, it is difficult for a single treatment method to play an effective role in the repair of neural tissue after SCI. The combination of biomaterial scaffolds and exosomes can better transfer and fix exosomes to the injury site and improve their survival rate. This paper first reviews the current research status of stem cell-derived exosomes and biomaterial scaffolds in the treatment of SCI respectively, and then describes the application of exosomes combined with biomaterial scaffolds in the treatment of SCI, as well as the challenges and prospects.

Conductive scaffolds have been shown to exert a therapeutic effect on patients suffering from peripheral nerve injuries (PNIs). However, conventional conductive conduits are made of rigid structures and have limited applications for impaired diabetic patients due to their mechanical mismatch with neural tissues and poor plasticity. We propose the development of biocompatible electroconductive hydrogels (ECHs) that are identical to a surgical dressing in this study. Based on excellent adhesive and self-healing properties, the thin film-like dressing can be easily attached to the injured nerve fibers, automatically warps a tubular structure without requiring any invasive techniques. The ECH offers an intimate and stable electrical bridge coupling with the electrogenic nerve tissues. The in vitro experiments indicated that the ECH promoted the migration and adhesion of the Schwann cells. Furthermore, the ECH facilitated axonal regeneration and remyelination in vitro and in vivo through the MEK/ERK pathway, thus preventing muscle denervation atrophy while retaining functional recovery. The results of this study are likely to facilitate the development of non-invasive treatment techniques for PNIs in diabetic patients utilizing electroconductive hydrogels.

Spinal cord injury (SCI) induces neurological deficits associated with long-term functional impairments. Since the current treatments remain ineffective, novel therapeutic options are needed. Besides its effect on bipolar mood disorder, lithium was reported to have neuroprotective activity in different neurodegenerative conditions, including SCI. In SCI, the effects of lithium on long-term neurological recovery and neuroplasticity have not been assessed. We herein investigated the effects of intraperitoneally administered lithium chloride (LiCl) on motor coordination recovery, electromyography (EMG) responses, histopathological injury and remodeling, and axonal plasticity in mice exposed to spinal cord transection. At a dose of 0.2, but not 2.0 mmol/kg, LiCl enhanced motor coordination and locomotor activity starting at 28 days post-injury (dpi), as assessed by a set of behavioral tests. Following electrical stimulation proximal to the hemitransection, LiCl at 0.2 mmol/kg decreased the latency and increased the amplitude of EMG responses in the denervated hindlimb at 56 dpi. Functional recovery was associated with reduced gray and white matter atrophy rostral and caudal to the hemitransection, increased neuronal survival and reduced astrogliosis in the dorsal and ventral horns caudal to the hemitransection, and increased regeneration of long-distance axons proximal and distal to the lesion site in mice receiving 0.2 mmol/kg, but not 2 mmol/kg LiCl, as assessed by histochemical and immunohistochemical studies combined with anterograde tract tracing. Our results indicate that LiCl induces long-term neurological recovery and neuroplasticity following SCI.

Glaucoma is an optic neuropathy and is currently one of the most common diseases that leads to irreversible blindness. The axonal degeneration that occurs before retinal ganglion neuronal loss is suggested to be involved in the pathogenesis of glaucoma. G protein-coupled receptor 3 (GPR3) belongs to the class A rhodopsin-type GPCR family and is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate the Gαs protein without a ligand, which elevates the basal intracellular cAMP level. Our earlier reports suggested that GPR3 enhances both neurite outgrowth and neuronal survival. However, the potential role of GPR3 in axonal regeneration after neuronal injury has not been elucidated. Herein, we investigated retinal GPR3 expression and its possible involvement in axonal regeneration after retinal injury in mice. GPR3 was relatively highly expressed in retinal ganglion cells (RGCs). Surprisingly, RGCs in GPR3 knockout mice were vulnerable to neural death during aging without affecting high intraocular pressure (IOP) and under ischemic conditions. Primary cultured neurons from the retina showed that GPR3 expression was correlated with neurite outgrowth and neuronal survival. Evaluation of the effect of GPR3 on axonal regeneration using GPR3 knockout mice revealed that GPR3 in RGCs participates in axonal regeneration after optic nerve crush (ONC) under zymosan stimulation. In addition, regenerating axons were further stimulated when GPR3 was upregulated in RGCs, and the effect was further augmented when combined with zymosan treatment. These results suggest that GPR3 expression in RGCs helps maintain neuronal survival and accelerates axonal regeneration after ONC in mice.

Abstract Background Traumatic spinal cord injury (SCI) is a severely disabling disease that leads to loss of sensation, motor, and autonomic function. As exosomes have great potential in diagnosis, prognosis, and treatment of SCI because of their ability to easily cross the blood–brain barrier, the function of Schwann cell-derived exosomes (SCDEs) is still largely unknown. Methods A T10 spinal cord contusion was established in adult female mice. SCDEs were injected into the tail veins of mice three times a week for 4 weeks after the induction of SCI, and the control group was injected with PBS. High-resolution transmission electron microscope and western blot were used to characterize the SCDEs. Toll-like receptor 2 (TLR2) expression on astrocytes, chondroitin sulfate proteoglycans (CSPGs) deposition and neurological function recovery were measured in the spinal cord tissues of each group by immunofluorescence staining of TLR2, GFAP, CS56, 5-HT, and β-III-tublin, respectively. TLR2f/f mice were crossed to the GFAP-Cre strain to generate astrocyte specific TLR2 knockout mice (TLR2−/−). Finally, western blot analysis was used to determine the expression of signaling proteins and IKKβ inhibitor SC-514 was used to validate the involved signaling pathway. Results Here, we found that TLR2 increased significantly on astrocytes post-SCI. SCDEs treatment can promote functional recovery and induce the expression of TLR2 on astrocytes accompanied with decreased CSPGs deposition. The specific knockout of TLR2 on astrocytes abolished the decreasing CSPGs deposition and neurological functional recovery post-SCI. In addition, the signaling pathway of NF-κB/PI3K involved in the TLR2 activation was validated by western blot. Furthermore, IKKβ inhibitor SC-514 was also used to validate this signaling pathway. Conclusion Thus, our results uncovered that SCDEs can promote functional recovery of mice post-SCI by decreasing the CSPGs deposition via increasing the TLR2 expression on astrocytes through NF-κB/PI3K signaling pathway.

Functional recovery after peripheral nerve injuries is critically dependent on axonal regeneration. Several autonomous and non-cell autonomous processes regulate axonal regeneration, including the activation of a growth-associated transcriptional program in neurons and the reprogramming of differentiated Schwann cells (dSCs) into repair SCs (rSCs), triggering the secretion of neurotrophic factors and the activation of an inflammatory response. Repair Schwann cells also release pro-regenerative extracellular vesicles (EVs), but is still unknown whether EV secretion is regulated non-cell autonomously by the regenerating neuron. Interestingly, it has been described that nerve activity enhances axonal regeneration by increasing the secretion of neurotrophic factors by rSC, but whether this activity modulates pro-regenerative EV secretion by rSC has not yet been explored. Here, we demonstrate that neuronal activity enhances the release of rSC-derived EVs and their transfer to neurons. This effect is mediated by activation of P2Y receptors in SCs after activity-dependent ATP release from sensory neurons. Importantly, activation of P2Y in rSCs also increases the amount of miRNA-21 present in rSC-EVs. Taken together, our results demonstrate that neuron to glia communication by ATP-P2Y signaling regulates the content of SC-derived EVs and their transfer to axons, modulating axonal elongation in a non-cell autonomous manner.

Neuronal plasma membrane proteins are essential for integrating cell extrinsic and cell intrinsic signals to orchestrate neuronal differentiation, growth and plasticity in the developing and adult nervous system. Here, we shed light on the family of plasma membrane proteins phospholipid phosphatase-related proteins (PLPPRs) (alternative name, PRGs; plasticity-related genes) that fine-tune neuronal growth and synaptic transmission in the central nervous system. Several studies uncovered essential functions of PLPPRs in filopodia formation, axon guidance and branching during nervous system development and regeneration, as well as in the control of dendritic spine number and excitability. Loss of PLPPR expression in knockout mice increases susceptibility to seizures, and results in defects in sensory information processing, development of psychiatric disorders, stress-related behaviors and abnormal social interaction. However, the exact function of PLPPRs in the context of neurological diseases is largely unclear. Although initially described as active lysophosphatidic acid (LPA) ecto-phosphatases that regulate the levels of this extracellular bioactive lipid, PLPPRs lack catalytic activity against LPA. Nevertheless, they emerge as atypical LPA modulators, by regulating LPA mediated signaling processes. In this review, we summarize the effects of this protein family on cellular morphology, generation and maintenance of cellular protrusions as well as highlight their known neuronal functions and phenotypes of KO mice. We discuss the molecular mechanisms of PLPPRs including the deployment of phospholipids, actin-cytoskeleton and small GTPase signaling pathways, with a focus on identifying gaps in our knowledge to stimulate interest in this understudied protein family.

Abstract Background Exosomes derived from the bone marrow mesenchymal stem cell (MSC) have shown great potential in spinal cord injury (SCI) treatment. This research was designed to investigate the therapeutic effects of miR-26a-modified MSC-derived exosomes (Exos-26a) following SCI. Methods Bioinformatics and data mining were performed to explore the role of miR-26a in SCI. Exosomes were isolated from miR-26a-modified MSC culture medium by ultracentrifugation. A series of experiments, including assessment of Basso, Beattie and Bresnahan scale, histological evaluation, motor-evoked potential recording, diffusion tensor imaging, and western blotting, were performed to determine the therapeutic influence and the underlying molecular mechanisms of Exos-26a in SCI rats. Results Exos-26a was shown to promote axonal regeneration. Furthermore, we found that exosomes derived from miR-26a-modified MSC could improve neurogenesis and attenuate glial scarring through PTEN/AKT/mTOR signaling cascades. Conclusions Exosomes derived from miR-26a-modified MSC could activate the PTEN-AKT-mTOR pathway to promote axonal regeneration and neurogenesis and attenuate glia scarring in SCI and thus present great potential for SCI treatment. Graphical abstract

Abstract Introduction Stem cell therapy using neural progenitor cells (NPCs) shows promise in mitigating the debilitating effects of spinal cord injury (SCI). Notably, myelin stimulates axonal regeneration from mammalian NPCs. This led us to hypothesize that myelin-associated proteins may contribute to axonal regeneration from NPCs. Methods We conducted an R-based bioinformatics analysis to identify key gene(s) that may participate in myelin-associated axonal regeneration from murine NPCs, which identified the serine protease myelin basic protein (Mbp). We employed E12 murine NPCs, E14 rat NPCs, and human iPSC-derived Day 1 NPCs (D1 hNPCs) with or without CRISPR/Cas9-mediated Mbp knockout in combination with rescue L1-70 overexpression, constitutively-active VP16-PPARγ2, or the PPARγ agonist ciglitazone. A murine dorsal column crush model of SCI utilizing porous collagen-based scaffolding (PCS)-seeded murine NPCs with or without stable Mbp overexpression was used to assess locomotive recovery and axonal regeneration in vivo. Results Myelin promotes axonal outgrowth from NPCs in an Mbp-dependent manner and that Mbp’s stimulatory effects on NPC neurite outgrowth are mediated by Mbp’s production of L1-70. Furthermore, we determined that Mbp/L1-70’s stimulatory effects on NPC neurite outgrowth are mediated by PPARγ-based repression of neuron differentiation-associated gene expression and PPARγ-based Erk1/2 activation. In vivo, PCS-seeded murine NPCs stably overexpressing Mbp significantly enhanced locomotive recovery and axonal regeneration in post-SCI mice. Conclusions We discovered that Mbp supports axonal regeneration from mammalian NPCs through the novel Mbp/L1cam/Pparγ signaling pathway. This study suggests that bioengineered, NPC-based interventions can promote axonal regeneration and functional recovery post-SCI.

Alterations in environmentally sensitive epigenetic mechanisms (e.g., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.

Nervous system disorders are prevalent health issues that will only continue to increase in frequency as the population ages. Dying-back axonopathy is a hallmark of many neurologic diseases and leads to axonal disconnection from their targets, which in turn leads to functional impairment. During the course of many of neurologic diseases, axons can regenerate or sprout in an attempt to reconnect with the target and restore synapse function. In amyotrophic lateral sclerosis (ALS), distal motor axons retract from neuromuscular junctions early in the disease-course before significant motor neuron death. There is evidence of compensatory motor axon sprouting and reinnervation of neuromuscular junctions in ALS that is usually quickly overtaken by the disease course. Potential drugs that enhance compensatory sprouting and encourage reinnervation may slow symptom progression and retain muscle function for a longer period of time in ALS and in other diseases that exhibit dying-back axonopathy. There remain many outstanding questions as to the impact of distinct disease-causing mutations on axonal outgrowth and regeneration, especially in regards to motor neurons derived from patient induced pluripotent stem cells. Compartmentalized microfluidic chambers are powerful tools for studying the distal axons of human induced pluripotent stem cells-derived motor neurons, and have recently been used to demonstrate striking regeneration defects in human motor neurons harboring ALS disease-causing mutations. Modeling the human neuromuscular circuit with human induced pluripotent stem cells-derived motor neurons will be critical for developing drugs that enhance axonal regeneration, sprouting, and reinnervation of neuromuscular junctions. In this review we will discuss compensatory axonal sprouting as a potential therapeutic target for ALS, and the use of compartmentalized microfluidic devices to find drugs that enhance regeneration and axonal sprouting of motor axons.

Peripheral nerve injuries occur as the result of sudden trauma and lead to reduced quality of life. The peripheral nervous system has an inherent capability to regenerate axons. However, peripheral nerve regeneration following injury is generally slow and incomplete that results in poor functional outcomes such as muscle atrophy. Although conventional surgical procedures for peripheral nerve injuries present many benefits, there are still several limitations including scarring, difficult accessibility to donor nerve, neuroma formation and a need to sacrifice the autologous nerve. For many years, other therapeutic approaches for peripheral nerve injuries have been explored, the most notable being the replacement of Schwann cells, the glial cells responsible for clearing out debris from the site of injury. Introducing cultured Schwann cells to the injured sites showed great benefits in promoting axonal regeneration and functional recovery. However, there are limited sources of Schwann cells for extraction and difficulties in culturing Schwann cells in vitro. Therefore, novel therapeutic avenues that offer maximum benefits for the treatment of peripheral nerve injuries should be investigated. This review focused on strategies using mesenchymal stem cells to promote peripheral nerve regeneration including exosomes of mesenchymal stem cells, nerve engineering using the nerve guidance conduits containing mesenchymal stem cells, and genetically engineered mesenchymal stem cells. We present the current progress of mesenchymal stem cell treatment of peripheral nerve injuries.

Axon regeneration and remyelination of the damaged region is the most common repair strategy for spinal cord injury. However, achieving good outcome remains difficult. Our previous study showed that porcine decellularized optic nerve better mimics the extracellular matrix of the embryonic porcine optic nerve and promotes the directional growth of dorsal root ganglion neurites. However, it has not been reported whether this material promotes axonal regeneration in vivo. In the present study, a porcine decellularized optic nerve was seeded with neurotrophin-3-overexpressing Schwann cells. This functional scaffold promoted the directional growth and remyelination of regenerating axons. In vitro, the porcine decellularized optic nerve contained many straight, longitudinal channels with a uniform distribution, and microscopic pores were present in the channel wall. The spatial micro topological structure and extracellular matrix were conducive to the adhesion, survival and migration of neural stem cells. The scaffold promoted the directional growth of dorsal root ganglion neurites, and showed strong potential for myelin regeneration. Furthermore, we transplanted the porcine decellularized optic nerve containing neurotrophin-3-overexpressing Schwann cells in a rat model of T10 spinal cord defect in vivo. Four weeks later, the regenerating axons grew straight, the myelin sheath in the injured/transplanted area recovered its structure, and simultaneously, the number of inflammatory cells and the expression of chondroitin sulfate proteoglycans were reduced. Together, these findings suggest that porcine decellularized optic nerve loaded with Schwann cells overexpressing neurotrophin-3 promotes the directional growth of regenerating spinal cord axons as well as myelin regeneration. All procedures involving animals were conducted in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Sun Yat-sen University (approval No. SYSU-IACUC-2019-B034) on February 28, 2019.

Inhibitor of DNA binding 2 (Id2) can promote axonal regeneration after injury of the central nervous system. However, whether Id2 can promote axonal regeneration and functional recovery after peripheral nerve injury is currently unknown. In this study, we established a mouse model of bilateral sciatic nerve crush injury. Two weeks before injury, AAV9-Id2-3×Flag-GFP was injected stereotaxically into the bilateral ventral horn of lumbar spinal cord. Our results showed that Id2 was successfully delivered into spinal cord motor neurons projecting to the sciatic nerve, and the number of regenerated motor axons in the sciatic nerve distal to the crush site was increased at 2 weeks after injury, arriving at the tibial nerve and reinnervating a few endplates in the gastrocnemius muscle. By 1 month after injury, extensive neuromuscular reinnervation occurred. In addition, the amplitude of compound muscle action potentials of the gastrocnemius muscle was markedly recovered, and their latency was shortened. These findings suggest that Id2 can accelerate axonal regeneration, promote neuromuscular reinnervation, and enhance functional improvement following sciatic nerve injury. Therefore, elevating the level of Id2 in adult neurons may present a promising strategy for peripheral nerve repair following injury. The study was approved by the Experimental Animal Ethics Committee of Jinan University (approval No. 20160302003) on March 2, 2016.

Millions of people worldwide are affected by traumatic spinal cord injury, which usually results in permanent sensorimotor disability. Damage to the spinal cord leads to a series of detrimental events including ischaemia, haemorrhage and neuroinflammation, which over time result in further neural tissue loss. Eventually, at chronic stages of traumatic spinal cord injury, the formation of a glial scar, cystic cavitation and the presence of numerous inhibitory molecules act as physical and chemical barriers to axonal regrowth. This is further hindered by a lack of intrinsic regrowth ability of adult neurons in the central nervous system. The intracellular signalling molecule, cyclic adenosine 3′,5′-monophosphate (cAMP), is known to play many important roles in the central nervous system, and elevating its levels as shown to improve axonal regeneration outcomes following traumatic spinal cord injury in animal models. However, therapies directly targeting cAMP have not found their way into the clinic, as cAMP is ubiquitously present in all cell types and its manipulation may have additional deleterious effects. A downstream effector of cAMP, exchange protein directly activated by cAMP 2 (Epac2), is mainly expressed in the adult central nervous system, and its activation has been shown to mediate the positive effects of cAMP on axonal guidance and regeneration. Recently, using ex vivo modelling of traumatic spinal cord injury, Epac2 activation was found to profoundly modulate the post-lesion environment, such as decreasing the activation of astrocytes and microglia. Pilot data with Epac2 activation also suggested functional improvement assessed by in vivo models of traumatic spinal cord injury. Therefore, targeting Epac2 in traumatic spinal cord injury could represent a novel strategy in traumatic spinal cord injury repair, and future work is needed to fully establish its therapeutic potential.

Axonal regeneration plays an important role in functional recovery after nervous system damage. However, after axonal injury in mammals, regeneration is often poor. The deletion of Krüppel-like factor-4 (Klf4) has been shown to promote axonal regeneration in retinal ganglion cells. However, the effects of Klf4 deletion on the corticospinal tract and peripheral nervous system are unknown. In this study, using a mouse model of sciatic nerve injury, we show that the expression of Klf4 in dorsal root ganglion sensory neurons was significantly reduced after peripheral axotomy, suggesting that the regeneration of the sciatic nerve is associated with Klf4. In vitro, dorsal root ganglion sensory neurons with Klf4 knockout exhibited significantly enhanced axonal regeneration. Furthermore, the regeneration of the sciatic nerve was enhanced in vivo following Klf4 knockout. Finally, AAV-Cre virus was used to knockout the Klf4 gene in the cortex. The deletion of Klf4 enhanced regeneration of the corticospinal tract in mice with spinal cord injury. Together, our findings suggest that regulating KLF4 activity in neurons is a potential strategy for promoting axonal regeneration and functional recovery after nervous system injury. This study was approved by the Animal Ethics Committee at Soochow University, China (approval No. SUDA20200316A01).

Biomaterial bridging provides physical substrates to guide axonal growth across the lesion. To achieve efficient directional guidance, combinatory strategies using permissive matrix, cells and trophic factors are necessary. In the present study, we evaluated permissive effect of poly (acrylonitrile-co-vinyl chloride) guidance channels filled by different densities of laminin-precoated unidirectional polypropylene filaments combined with Schwann cells, and glial cell line-derived neurotrophic factor for axonal regeneration through a T10 hemisected spinal cord gap in adult rats. We found that channels with filaments significantly reduced the lesion cavity, astrocytic gliosis, and inflammatory responses at the graft-host boundaries. The laminin coated low density filament provided the most favorable directional guidance for axonal regeneration which was enhanced by co-grafting of Schwann cells and glial cell line-derived neurotrophic factor. These results demonstrate that the combinatorial strategy of filament-filled guiding scaffold, adhesive molecular laminin, Schwann cells, and glial cell line-derived neurotrophic factor, provides optimal topographical cues in stimulating directional axonal regeneration following spinal cord injury. This study was approved by Indiana University Institutional Animal Care and Use Committees (IACUC #:11011) on October 29, 2015.

Human amniotic membrane (hAM) and collagen nerve wraps are biomaterials that have been investigated as therapies for improving outcomes of peripheral nerve regeneration; however, their efficacy has not been compared. The purpose of this study is to compare the efficacy of collagen and human amniotic membrane nerve wraps in a rodent sciatic nerve reverse autograft model. Lewis rats (n = 29) underwent sciatic nerve injury and repair in which a 10-mm gap was bridged with reverse autograft combined with either no nerve wrap (control), collagen nerve wrap or hAM nerve wrap. Behavioral analyses were performed at baseline and 4, 8 and 12 weeks. Electrophysiological studies were conducted at 8, 10 and 12 weeks. Additional outcomes assessed included gastrocnemius muscle weights, nerve adhesions, axonal regeneration and scarring at 12 weeks. Application of both collagen and hAM nerve wraps resulted in improvement of functional and histologic outcomes when compared with controls, with a greater magnitude of improvement for the experimental group treated with hAM nerve wraps. hAM-treated animals had significantly higher numbers of axons compared to control animals (p

Abstract Electroconductive hydrogels are very attractive candidates for accelerated spinal cord injury (SCI) repair because they match the electrical and mechanical properties of neural tissue. However, electroconductive hydrogel implantation can potentially aggravate inflammation, and hinder its repair efficacy. Bone marrow stem cell‐derived exosomes (BMSC‐exosomes) have shown immunomodulatory and tissue regeneration effects, therefore, neural tissue‐like electroconductive hydrogels loaded with BMSC‐exosomes are developed for the synergistic treatment of SCI. These exosomes‐loaded electroconductive hydrogels modulate microglial M2 polarization via the NF‐κB pathway, and synergistically enhance neuronal and oligodendrocyte differentiation of neural stem cells (NSCs) while inhibiting astrocyte differentiation, and also increase axon outgrowth via the PTEN/PI3K/AKT/mTOR pathway. Furthermore, exosomes combined electroconductive hydrogels significantly decrease the number of CD68‐positive microglia, enhance local NSCs recruitment, and promote neuronal and axonal regeneration, resulting in significant functional recovery at the early stage in an SCI mouse model. Hence, the findings of this study demonstrate that the combination of electroconductive hydrogels and BMSC‐exosomes is a promising therapeutic strategy for SCI repair.