Tail Nerve Electrical Stimulation and Electro-Acupuncture Can Protect Spinal Motor Neurons and Alleviate Muscle Atrophy after Spinal Cord Transection in Rats.

Spinal cord injury (SCI) often results in death of spinal neurons and atrophy of muscles which they govern. Thus, following SCI, reorganizing the lumbar spinal sensorimotor pathways is crucial to alleviate muscle atrophy. Tail nerve electrical stimulation (TANES) has been shown to activate the central pattern generator (CPG) and improve the locomotion recovery of spinal contused rats. Electroacupuncture (EA) is a traditional Chinese medical practice which has been proven to have a neural protective effect. Here, we examined the effects of TANES and EA on lumbar motor neurons and hindlimb muscle in spinal transected rats, respectively. From the third day postsurgery, rats in the TANES group were treated 5 times a week and those in the EA group were treated once every other day. Four weeks later, both TANES and EA showed a significant impact in promoting survival of lumbar motor neurons and expression of choline acetyltransferase (ChAT) and ameliorating atrophy of hindlimb muscle after SCI. Meanwhile, the expression of neurotrophin-3 (NT-3) in the same spinal cord segment was significantly increased. These findings suggest that TANES and EA can augment the expression of NT-3 in the lumbar spinal cord that appears to protect the motor neurons as well as alleviate muscle atrophy.

Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells.

Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.

Effects of tail nerve electrical stimulation on the activation and plasticity of the lumbar locomotor circuits and the prevention of skeletal muscle atrophy after spinal cord transection in rats.

INTRODUCTION: Severe spinal cord injury results in the loss of neurons in the relatively intact spinal cord below the injury area and skeletal muscle atrophy in the paralyzed limbs. These pathological processes are significant obstacles for motor function reconstruction. OBJECTIVE: We performed tail nerve electrical stimulation (TNES) to activate the motor neural circuits below the injury site of the spinal cord to elucidate the regulatory mechanisms of the excitatory afferent neurons in promoting the reconstruction of locomotor function. METHODS: Eight days after T10 spinal cord transection in rats, TNES was performed for 7 weeks. Behavioral scores were assessed weekly. Electrophysiological tests and double retrograde tracings were performed at week 8. RESULTS: After 7 weeks of TNES treatment, there was restoration in innervation, the number of stem cells, and mitochondrial metabolism in the rats' hindlimb muscles. Double retrograde tracings of the tail nerve and sciatic nerve further confirmed the presence of synaptic connections between the tail nerve and central pattern generator (CPG) neurons in the lumbar spinal cord, as well as motor neurons innervating the hindlimb muscles. CONCLUSION: The mechanisms of TNES induced by the stimulation of primary afferent nerve fibers involves efficient activation of the motor neural circuits in the lumbosacral segment, alterations of synaptic plasticity, and the improvement of muscle and nerve regeneration, which provides the structural and functional foundation for the future use of cutting-edge biological treatment strategies to restore voluntary movement of paralyzed hindlimbs.

Mesenchymal Stem Cells Combined With Electroacupuncture Treatment Regulate the Subpopulation of Macrophages and Astrocytes to Facilitate Axonal Regeneration in Transected Spinal Cord.

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-1ß) 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.

The therapeutic mechanism of transcranial iTBS on nerve regeneration and functional recovery in rats with complete spinal cord transection.

Background: After spinal cord transection injury, the inflammatory microenvironment formed at the injury site, and the cascade of effects generated by secondary injury, results in limited regeneration of injured axons and the apoptosis of neurons in the sensorimotor cortex (SMC). It is crucial to reverse these adverse processes for the recovery of voluntary movement. The mechanism of transcranial intermittent theta-burst stimulation (iTBS) as a new non-invasive neural regulation paradigm in promoting axonal regeneration and motor function repair was explored by means of a severe spinal cord transection. Methods: Rats underwent spinal cord transection and 2 mm resection of spinal cord at T10 level. Four groups were studied: Normal (no lesion), Control (lesion with no treatment), sham iTBS (lesion and no functional treatment) and experimental, exposed to transcranial iTBS, 72 h after spinal lesion. Each rat received treatment once a day for 5 days a week; behavioral tests were administered one a week. Inflammation, neuronal apoptosis, neuroprotective effects, regeneration and synaptic plasticity after spinal cord injury (SCI) were determined by immunofluorescence staining, western blotting and mRNA sequencing. For each rat, anterograde tracings were acquired from the SMC or the long descending propriospinal neurons and tested for cortical motor evoked potentials (CMEPs). Regeneration of the corticospinal tract (CST) and 5-hydroxytryptamine (5-HT) nerve fibers were analyzed 10 weeks after SCI. Results: When compared to the Control group, the iTBS group showed a reduced inflammatory response and reduced levels of neuronal apoptosis in the SMC when tested 2 weeks after treatment. Four weeks after SCI, the neuroimmune microenvironment at the injury site had improved in the iTBS group, and neuroprotective effects were evident, including the promotion of axonal regeneration and synaptic plasticity. After 8 weeks of iTBS treatment, there was a significant increase in CST regeneration in the region rostral to the site of injury. Furthermore, there was a significant increase in the number of 5-HT nerve fibers at the center of the injury site and the long descending propriospinal tract (LDPT) fibers in the region caudal to the site of injury. Moreover, CMEPs and hindlimb motor function were significantly improved. Conclusion: Neuronal activation and neural tracing further verified that iTBS had the potential to provide neuroprotective effects during the early stages of SCI and induce regeneration effects related to the descending motor pathways (CST, 5-HT and LDPT). Furthermore, our results revealed key relationships between neural pathway activation, neuroimmune regulation, neuroprotection and axonal regeneration, as well as the interaction network of key genes.

A biocompatible gelatin sponge scaffold confers robust tissue remodeling after spinal cord injury in a non-human primate model.

We previously constructed a three-dimensional gelatin sponge (3D-GS) scaffold as a delivery vehicle for therapeutic cells and trophic factors in the treatment of spinal cord injury (SCI), and this study aimed to assess the biosafety and efficacy of the scaffold in a non-human primate SCI model. However, because it has only been tested in rodent and canine models, the biosafety and efficacy of the scaffold should ideally be assessed in a non-human primate SCI model before its use in the clinic. No adverse reactions were observed over 8 weeks following 3D-GS scaffold implantation into in a Macaca fascicularis with hemisected SCI. Scaffold implantation also did not add to neuroinflammatory or astroglial responses already present at the injured site, suggesting good biocompatibility. Notably, there was a significant reduction in α-smooth muscle actin (αSMA)-positive cells at the injury/implantation interface, leading to alleviation of fibrotic compression of the residual spinal cord tissue. The regenerating tissue in the scaffold showed numerous cells migrating into the implant secreting abundant extracellular matrix, resulting in a pro-regenerative microenvironment. Consequently, nerve fiber regeneration, myelination, vascularization, neurogenesis, and electrophysiological improvements were achieved. These results indicated that the 3D-GS scaffold had good histocompatibility and effectiveness in the structural repair of injured spinal cord tissue in a non-human primate and is suitable for use in the treatment of patients with SCI.

Tail nerve electrical stimulation promoted the efficiency of transplanted spinal cord-like tissue as a neuronal relay to repair the motor function of rats with transected spinal cord injury.

Following transected spinal cord injury (SCI), there is a critical need to restore nerve conduction at the injury site and activate the silent neural circuits caudal to the injury to promote the recovery of voluntary movement. In this study, we generated a rat model of SCI, constructed neural stem cell (NSC)-derived spinal cord-like tissue (SCLT), and evaluated its ability to replace injured spinal cord and repair nerve conduction in the spinal cord as a neuronal relay. The lumbosacral spinal cord was further activated with tail nerve electrical stimulation (TNES) as a synergistic electrical stimulation to better receive the neural information transmitted by the SCLT. Next, we investigated the neuromodulatory mechanism underlying the action of TNES and its synergism with SCLT in SCI repair. TNES promoted the regeneration and remyelination of axons and increased the proportion of glutamatergic neurons in SCLT to transmit brain-derived neural information more efficiently to the caudal spinal cord. TNES also increased the innervation of motor neurons to hindlimb muscle and improved the microenvironment of muscle tissue, resulting in effective prevention of hindlimb muscle atrophy and enhanced muscle mitochondrial energy metabolism. Tracing of the neural circuits of the sciatic nerve and tail nerve identified the mechanisms responsible for the synergistic effects of SCLT transplantation and TNES in activating central pattern generator (CPG) neural circuits and promoting voluntary motor function recovery in rats. The combination of SCLT and TNES is expected to provide a new breakthrough for patients with SCI to restore voluntary movement and control their muscles.

Neurotrophin-3 gene-modified Schwann cells promote TrkC gene-modified mesenchymal stem cells to differentiate into neuron-like cells in poly(lactic-acid-co-glycolic acid) multiple-channel conduit.

Rapid progress in the field of nerve tissue engineering has opened up the way for new therapeutic strategies for spinal cord injury (SCI). Bone marrow-derived mesenchymal stem cells (MSCs) could be differentiated into neural lineages, which can be used as a potential cell source for nerve repair. Schwann cells (SCs) have been reported to support structural and functional recovery of SCI. In this study, we co-cultured neurotrophin-3 (NT-3) gene-modified SCs and NT-3 receptor tyrosine protein kinase C (TrkC) gene-modified MSCs in a three-dimensional porous poly(lactic-acid-co-glycolic acid) (PLGA) conduit with multiple channels in vitro for 14 days. Our results showed that more than 50% of the grafted MSCs were MAP2- and ß-III-tubulin-positive cells, and the MSCs expressed a high level of ß-III-tubulin detected by Western blotting, indicating a high rate of neuronal differentiation. Furthermore, immunostaining of PSD95 revealed the formation of a synapse-like structure, which was confirmed under electron microscopy. In conclusion, co-culture of NT-3 gene-modified SCs and TrkC gene-modified MSCs in the PLGA multiple-channeled conduit can promote MSCs' differentiation into neuron-like cells with synaptogenesis potential. Our study provides a biological basis for future application of this artificial MSCs/SCs/PLGA complex in the SCI treatment.

Electro-acupuncture and its combination with adult stem cell transplantation for spinal cord injury treatment: A summary of current laboratory findings and a review of literature.

The incidence and disability rate of spinal cord injury (SCI) worldwide are high, imposing a heavy burden on patients. Considerable research efforts have been directed toward identifying new strategies to effectively treat SCI. Governor Vessel electro-acupuncture (GV-EA), used in traditional Chinese medicine, combines acupuncture with modern electrical stimulation. It has been shown to improve the microenvironment of injured spinal cord (SC) by increasing levels of endogenous neurotrophic factors and reducing inflammation, thereby protecting injured neurons and promoting myelination. In addition, axons extending from transplanted stem cell-derived neurons can potentially bridge the two severed ends of tissues in a transected SC to rebuild neuronal circuits and restore motor and sensory functions. However, every single treatment approach to severe SCI has proven unsatisfactory. Combining different treatments-for example, electro-acupuncture (EA) with adult stem cell transplantation-appears to be a more promising strategy. In this review, we have summarized the recent progress over the past two decades by our team especially in the use of GV-EA for the repair of SCI. By this strategy, we have shown that EA can stimulate the nerve endings of the meningeal branch. This would elicit the dorsal root ganglion neurons to secrete excess amounts of calcitonin gene-related peptide centrally in the SC. The neuropeptide then activates the local cells to secrete neurotrophin-3 (NT-3), which mediates the survival and differentiation of donor stem cells overexpressing the NT-3 receptor, at the injury/graft site of the SC. Increased local production of NT-3 facilitates reconstruction of host neural tissue such as nerve fiber regeneration and myelination. All this events in sequence would ultimately strengthen the cortical motor-evoked potentials and restore the motor function of paralyzed limbs. The information presented herein provides a basis for future studies on the clinical application of GV-EA and adult stem cell transplantation for the treatment of SCI.

Transcription Profiling of a Revealed the Potential Molecular Mechanism of Governor Vessel Electroacupuncture for Spinal Cord Injury in Rats.

OBJECTIVE: This study aimed to identify differentially expressed genes (DEGs) by transcriptome analysis to elucidate a potential mechanism by which governor vessel electroacupuncture (GV-EA) promotes neuronal survival, axonal regeneration, and functional recovery after complete transection spinal cord injury (SCI). METHODS: Sham, control, or GV-EA group adult female Sprague Dawley rats underwent a complete transection SCI protocol. SCI area RNA-seq investigated the DEGs of coding and noncoding RNAs 7 days post-SCI. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were used to classify DEGs functions, to explain a possible molecular mechanism. Immunofluorescence and BBB (Basso, Beattie, and Bresnahan) score were used to verify a GV-EA treatment effect following SCI. RESULTS: GV-EA treatment could regulate the expression of 173 mRNA, 260 lncRNA, and 153 circRNA genes among these DEGs resulted by SCI. GO enrichment analysis showed that the DEGs were most enriched in membrane, actin binding, and regulation of Toll-like receptor signaling pathway. KEGG pathway analysis showed enriched pathways (e.g. , Toll-like receptors, MAPK, Hippo signaling). According to the ceRNA network, miR-144-3p played a regulatory role by interacting with lncRNA and circRNA. GV-EA also promoted the injured spinal cord neuron survival, axonal regeneration, and functional improvement of hind limb locomotion. CONCLUSION: Results of our RNA-seq suggest that post-SCI GV-EA may regulate characteristic changes in transcriptome gene expression, potential critical genes, and signaling pathways, providing clear directions for further investigation into the mechanism of GV-EA in subacute SCI treatment. Moreover, we found that GV-EA promotes neuronal survival, nerve fiber extension, and motor function recovery in subacute SCI.

Construction of a niche-specific spinal white matter-like tissue to promote directional axon regeneration and myelination for rat spinal cord injury repair.

Directional axon regeneration and remyelination are crucial for repair of spinal cord injury (SCI), but existing treatments do not effectively promote those processes. Here, we propose a strategy for construction of niche-specific spinal white matter-like tissue (WMLT) using decellularized optic nerve (DON) loaded with neurotrophin-3 (NT-3)-overexpressing oligodendrocyte precursor cells. A rat model with a white matter defect in the dorsal spinal cord of the T10 segment was used. The WMLT transplantation group showed significant improvement in coordinated motor functions compared with the control groups. WMLT transplants integrated well with host spinal cord white matter, effectively addressing several barriers to directional axonal regeneration and myelination during SCI repair. In WMLT, laminin was found to promote development of oligodendroglial lineage (OL) cells by binding to laminin receptors. Interestingly, laminin could also guide linear axon regeneration via interactions with specific integrins on the axon surface. The WMLT developed here utilizes the unique microstructure and bioactive matrix of DON to create a niche rich in laminin, NT-3 and OL cells to achieve significant structural repair of SCI. Our protocol can help to promote research on repair of nerve injury and construction of neural tissues and organoids that form specific cell niches.

Stem cell-derived neuronal relay strategies and functional electrical stimulation for treatment of spinal cord injury.

The inability of adult mammals to recover function lost after severe spinal cord injury (SCI) has been known for millennia and is mainly attributed to a failure of brain-derived nerve fiber regeneration across the lesion. Potential approaches to re-establishing locomotor function rely on neuronal relays to reconnect the segregated neural networks of the spinal cord. Intense research over the past 30 years has focused on endogenous and exogenous neuronal relays, but progress has been slow and the results often controversial. Treatments with stem cell-derived neuronal relays alone or together with functional electrical stimulation offer the possibility of improved repair of neuronal networks. In this review, we focus on approaches to recovery of motor function in paralyzed patients after severe SCI based on novel therapies such as implantation of stem cell-derived neuronal relays and functional electrical stimulation. Recent research progress offers hope that SCI patients will one day be able to recover motor function and sensory perception.

Developing a mechanically matched decellularized spinal cord scaffold for the in situ matrix-based neural repair of spinal cord injury.

Tissue engineering is a promising strategy to repair spinal cord injury (SCI). However, a bioscaffold with mechanical properties that match those of the pathological spinal cord tissue and a pro-regenerative matrix that allows robust neurogenesis for overcoming post-SCI scar formation has yet to be developed. Here, we report that a mechanically enhanced decellularized spinal cord (DSC) scaffold with a thin poly (lactic-co-glycolic acid) (PLGA) outer shell may fulfill the requirements for effective in situ neuroengineering after SCI. Using chemical extraction and electrospinning methods, we successfully constructed PLGA thin shell-ensheathed DSC scaffolds (PLGA-DSC scaffolds) in a way that removed major inhibitory components while preserving the permissive matrix. The DSCs exhibited good cytocompatibility with neural stem cells (NSCs) and significantly enhanced their differentiation toward neurons in vitro. Due to the mechanical reinforcement, the implanted PLGA-DSC scaffolds showed markedly increased resilience to infiltration by myofibroblasts and the deposition of dense collagen matrix, thereby creating a neurogenic niche favorable for the targeted migration, residence and neuronal differentiation of endogenous NSCs after SCI. Furthermore, PLGA-DSC presented a mild immunogenic property but prominent ability to polarize macrophages from the M1 phenotype to the M2 phenotype, leading to significant tissue regeneration and functional restoration after SCI. Taken together, the results demonstrate that the mechanically matched PLGA-DSC scaffolds show promise for effective tissue repair after SCI.

Decellularized optic nerve functional scaffold transplant facilitates directional axon regeneration and remyelination in the injured white matter of the rat spinal cord.

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.

Governor Vessel Electro-Acupuncture Promotes the Intrinsic Growth Ability of Spinal Neurons through Activating Calcitonin Gene-Related Peptide/α-Calcium/Calmodulin-Dependent Protein Kinase/Neurotrophin-3 Pathway after Spinal Cord Injury.

Spinal cord injury (SCI) invariably results in neuronal death and failure of axonal regeneration. This is attributed mainly to the hostile microenvironment and the poor intrinsic regrowth capacity of the injured spinal neurons. We have reported previously that electro-acupuncture on Governor Vessel acupoints (GV-EA) can promote neuronal survival and axonal regeneration of injured spinal cord. However, the underlying mechanism for this has remained uncertain. The present study aimed to explore the neural afferent pathway of GV-EA stimulation and the possible mechanism by which GV-EA can activate the intrinsic growth ability of injured spinal neurons. By cholera toxin B (CTB) retrograde labeling, immunostaining, and enzyme-linked immunosorbent assay (ELISA), we showed here that GV-EA could stimulate the spinal nerve branches of the dorsal root ganglion cells. This would then increase the release of calcitonin gene-related peptide (CGRP) from the afferent terminals in the spinal cord. It is of note that the effect was abrogated after dorsal rhizotomy. Additionally, both in vivo and in vitro results showed that CGRP would act on the post-synaptic spinal cord neurons and triggered the synthesis and secretion of neurotrophin-3 (NT-3) by activating the calcitonin gene-related peptide (CGRP)/ receptor activity-modifying protein (RAMP)1/calcium/calmodulin-dependent protein kinase (αCaMKII) pathway. Remarkably, the observed effect was prevented by the dorsal rhizotomy and the blockers of the CGRP/RAMP1/αCaMKII pathway. More importantly, increase in NT-3 promoted the survival, axonal regrowth, and synaptic maintenance of spinal cord neurons in the injured spinal cord. Therefore, it is concluded that increase in NT-3 production is one of the mechanisms by which GV-EA can activate the intrinsic growth ability of spinal neurons after SCI. The experimental results have reinforced the theoretical basis of GV-EA for its clinical efficacy in patients with SCI.

Electroacupuncture facilitates the integration of a grafted TrkC-modified mesenchymal stem cell-derived neural network into transected spinal cord in rats via increasing neurotrophin-3.

AIMS: This study was aimed to investigate whether electroacupuncture (EA) would increase the secretion of neurotrophin-3 (NT-3) from injured spinal cord tissue, and, if so, whether the increased NT-3 would promote the survival, differentiation, and migration of grafted tyrosine kinase C (TrkC)-modified mesenchymal stem cell (MSC)-derived neural network cells. We next sought to determine if the latter would integrate with the host spinal cord neural circuit to improve the neurological function of injured spinal cord. METHODS: After NT-3-modified Schwann cells (SCs) and TrkC-modified MSCs were co-cultured in a gelatin sponge scaffold for 14 days, the MSCs differentiated into neuron-like cells that formed a MSC-derived neural network (MN) implant. On this basis, we combined the MN implantation with EA in a rat model of spinal cord injury (SCI) and performed immunohistochemical staining, neural tracing, electrophysiology, and behavioral testing after 8 weeks. RESULTS: Electroacupuncture application enhanced the production of endogenous NT-3 in damaged spinal cord tissues. The increase in local NT-3 production promoted the survival, migration, and maintenance of the grafted MN, which expressed NT-3 high-affinity TrkC. The combination of MN implantation and EA application improved cortical motor-evoked potential relay and facilitated the locomotor performance of the paralyzed hindlimb compared with those of controls. These results suggest that the MN was better integrated into the host spinal cord neural network after EA treatment compared with control treatment. CONCLUSIONS: Electroacupuncture as an adjuvant therapy for TrkC-modified MSC-derived MN, acted by increasing the local production of NT-3, which accelerated neural network reconstruction and restoration of spinal cord function following SCI.

An NT-3-releasing bioscaffold supports the formation of TrkC-modified neural stem cell-derived neural network tissue with efficacy in repairing spinal cord injury.

The mechanism underlying neurogenesis during embryonic spinal cord development involves a specific ligand/receptor interaction, which may be help guide neuroengineering to boost stem cell-based neural regeneration for the structural and functional repair of spinal cord injury. Herein, we hypothesized that supplying spinal cord defects with an exogenous neural network in the NT-3/fibroin-coated gelatin sponge (NF-GS) scaffold might improve tissue repair efficacy. To test this, we engineered tropomyosin receptor kinase C (TrkC)-modified neural stem cell (NSC)-derived neural network tissue with robust viability within an NF-GS scaffold. When NSCs were genetically modified to overexpress TrkC, the NT-3 receptor, a functional neuronal population dominated the neural network tissue. The pro-regenerative niche allowed the long-term survival and phenotypic maintenance of the donor neural network tissue for up to 8 weeks in the injured spinal cord. Additionally, host nerve fibers regenerated into the graft, making synaptic connections with the donor neurons. Accordingly, motor function recovery was significantly improved in rats with spinal cord injury (SCI) that received TrkC-modified NSC-derived neural network tissue transplantation. Together, the results suggested that transplantation of the neural network tissue formed in the 3D bioactive scaffold may represent a valuable approach to study and develop therapies for SCI.

Decellularization optimizes the inhibitory microenvironment of the optic nerve to support neurite growth.

Allogeneic or homologous tissue transplantation is an effective strategy to repair tissue injury. However, the central nervous tissues like the brain, spinal cord, and optic nerve are not ideal materials for nervous tissue regeneration due to the excessive axonal inhibitor cues in their microenvironments. In the present study, we found that decellularization optimizes the function of the adult optic nerve in supporting the oriented outgrowth of dorsal root ganglion (DRG) neurites. The neurites growing on the decellularized optic nerve (DON) showed longer extension distances than those growing on the normal optic nerve (ON). Neurite branching was also significantly increased on the DON compared to on the ON. Decellularization selectively removed some axon-inhibitory molecules such as myelin-associated glycoprotein (basically not detected in DON) and chondroitin sulfate proteoglycans (detected in DON at a level less than 0.3 fold that in ON) and preserved some axon-promoted extracellular matrix (ECM) proteins, including collagen IV and laminin (detected at levels 6.0-fold higher in DON than in ON). Furthermore, collagen IV and laminin were shown to be preserved in DON, and their binding activities with integrin α1 were retained to promote the extension of DRG neurites. Together, the findings provide a feasible way to optimize the axon-inhibited microenvironment of central nervous tissues and establish a theoretical basis for the application of DON scaffolds in repairing central nervous injury.

Electro-acupuncture promotes survival, differentiation of the bone marrow mesenchymal stem cells as well as functional recovery in the spinal cord-transected rats.

BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) are one of the potential tools for treatment of the spinal cord injury; however, the survival and differentiation of MSCs in an injured spinal cord still need to be improved. In the present study, we investigated whether Governor Vessel electro-acupuncture (EA) could efficiently promote bone marrow mesenchymal stem cells (MSCs) survival and differentiation, axonal regeneration and finally, functional recovery in the transected spinal cord. RESULTS: The spinal cords of adult Sprague-Dawley (SD) rats were completely transected at T10, five experimental groups were performed: 1. sham operated control (Sham-control); 2. operated control (Op-control); 3. electro-acupuncture treatment (EA); 4. MSCs transplantation (MSCs); and 5. MSCs transplantation combined with electro-acupuncture (MSCs+EA). After 2-8 weeks of MSCs transplantation plus EA treatment, we found that the neurotrophin-3 (NT-3), cAMP level, the differentiation of MSCs, the 5-HT positive and CGRP positive nerve fibers in the lesion site and nearby tissue of injured spinal cord were significantly increased in the MSCs+EA group as compared to the group of the MSCs transplantation or the EA treated alone. Furthermore, behavioral test and spinal cord evoked potentials detection demonstrated a significantly functional recovery in the MSCs +EA group. CONCLUSION: These results suggest that EA treatment may promote grafted MSCs survival and differentiation; MSCs transplantation combined with EA treatment could promote axonal regeneration and partial locomotor functional recovery in the transected spinal cord in rats and indicate a promising avenue of treatment of spinal cord injury.

Pretreatment with Rhodiola rosea extract reduces cognitive impairment induced by intracerebroventricular streptozotocin in rats: implication of anti-oxidative and neuroprotective effects.

OBJECTIVE: To investigate the pretreatment effects of Rhodiola rosea (R. rosea) extract on cognitive dysfunction, oxidative stress in hippocampus and hippocampal neuron injury in a rat model of Alzheimer's disease (AD). METHODS: Male Sprague-Dawley rats were pretreated with R. rosea extract at doses of 1.5, 3.0, and 6.0 g/kg for 3 weeks, followed by bilateral intracerebroventricular injection with streptozotocin (1.5 mg/kg) on days 1 and 3. Behavioral alterations were monitored after 2 weeks from the lesion using Morris water maze task. Three weeks after the lesion, the rats were sacrificed for measuring the malondialdehyde (MDA), glutathione reductase (GR) and reduced glutathione (GSH) levels in hippocampus and histopathology of hippocampal neurons. RESULTS: The MDA level was significantly increased while the GR and GSH levels were significantly decreased with striking impairments in spatial learning and memory and severe damage to hippocampal neurons in the model rat induced by intracerebroventricular injection of streptozotocin. These abnormalities were significantly improved by pretreatment with R. rosea extract (3.0 g/kg). CONCLUSION: R. rosea extract can protect rats against cognitive deficits, neuronal injury and oxidative stress induced by intracerebroventricular injection of streptozotocin, and may be used as a potential agent in treatment of neurodegenerative diseases such as AD.