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.

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.

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.

Electroacupuncture Facilitates the Integration of Neural Stem Cell-Derived Neural Network with Transected Rat Spinal Cord.

The hostile environment of an injured spinal cord makes it challenging to achieve higher viability in a grafted tissue-engineered neural network used to reconstruct the spinal cord circuit. Here, we investigate whether cell survival and synaptic transmission within an NT-3 and TRKC gene-overexpressing neural stem cell-derived neural network scaffold (NN) transplanted into transected spinal cord could be promoted by electroacupuncture (EA) through improving the microenvironment. Our results showed that EA facilitated the cell survival, neuronal differentiation, and synapse formation of a transplanted NN. Pseudorabies virus tracing demonstrated that EA strengthened synaptic integration of the transplanted NN with the host neural circuit. The combination therapy also promoted axonal regeneration, spinal conductivity, and functional recovery. The findings highlight EA as a potential and safe supplementary therapeutic strategy to reinforce the survival and synaptogenesis of a transplanted NN as a neuronal relay to bridge the two severed ends of an injured spinal cord.

Electro-acupuncture promotes the survival and differentiation of transplanted bone marrow mesenchymal stem cells pre-induced with neurotrophin-3 and retinoic acid in gelatin sponge scaffold after rat spinal cord transection.

In the past decades, mesenchymal stem cells (MSCs) as a promising cell candidate have received the most attention in the treatment of spinal cord injury (SCI). However, due to the low survival rate and low neural differentiation rate, the grafted MSCs do not perform well as one would have expected. In the present study, we tested a combinational therapy to improve on this situation. MSCs were loaded into three-dimensional gelatin sponge (GS) scaffold. After 7 days of induction with neurotrophin-3 (NT-3) and retinoic acid (RA) in vitro, we observed a significant increase in TrkC mRNA transcription by Real-time PCR and this was confirmed by in situ hybridization. The expression of TrkC was also confirmed by Western blot and immunohistochemistry. Differentiation potential of MSCs in vitro into neuron-like cells or oligodendrocyte-like cells was further demonstrated by using immunofluorescence staining. The pre-induced MSCs seeding in GS scaffolds were then grafted into the transected rat spinal cord. One day after grafting, Governor Vessel electro-acupuncture (GV-EA) treatment was applied to rats in the NR-MSCs + EA group. At 30 days after GV-EA treatment, it found that the grafted MSCs have better survival rate and neuron-like cell differentiation compared with those without GV-EA treatment. The sustained TrkC expression in the grafted MSCs as well as increased NT-3 content in the injury/graft site by GV-EA suggests that NT-3/TrkC signaling pathway may be involved in the promoting effect. This study demonstrates that GV-EA and pre-induction with NT-3 and RA together may promote the survival and differentiation of grafted MSCs in GS scaffold in rat SCI.