Experimental Study On The Repair Of Spinal Cord Injuried Mice By Transplantation Of Electric Fields-induced Engineered Neural Tissue

Objective:EGFP transgenic mouse neural stem cells(EGFP-NSCs)were used as seed cells to construct three-dimensional(3D)engineered neural tissues(ENT)in vitro by applying physiological electric fields(EFs).The constructed EGFP-ENT was transplanted into mice with spinal cord injury,and the migration and differentiation of EGFP-ENT in host spinal cord tissue and the recovery of motor function in mice were examined at the histological and functional levels to explore the feasibility of EFs-induced ENT transplantation for repairing spinal cord injury.Methods:EGFP-NSCs were successfully isolated from EGFPTg/+ mouse embryos,expanded in vitro by suspension culture method,and the differentiation and growth of NSCs were regulated by 150 m V/mm EFs in 3D Matrigel as the scaffold material.Immunofluorescence staining was used to detect the cellular composition and tissue structure of EGFP-ENT.A mouse spinal cord injury model was constructed,and ENT was transplanted into mice with spinal cord injury.The experiment was divided into normal mice control group,the sham surgery group,the SCI group,the EGFP-NSCs group and the EGFP-ENT group.The BMS scores of motor function of the experimental animals were performed continuously after transplantation.The survival and differentiation of ENT in mice and its integration with mouse spinal cord were analyzed by immunofluorescence histochemical staining technique and histological detection after sampling at different time points.Results:(1)EGFP-NSCs were cultured in suspension to form neurospheres,and immunofluorescent chemical staining demonstrated that these Neurospheres express Nestin and Musashi1,specific markers of NSCs;under differentiation conditions,EGFP-NSCs have the potential for multidirectional differentiation into neurons and astrocytes.(2)When the EGFP-ENT constructed in vitro was observed by laser confocal microscopy,MAP2 positive neurons were evenly distributed and synaptophysin expression could be observed throughout the tissue,indicating that 3D ENT with a certain proportion of neurons forming a well-developed neural network was successfully constructed in vitro.(3)EGFP-ENT was transplanted into spinal cord-injured mice,and immunofluorescence staining showed that EGFP+/Nestin+ double positive NSCs were detected at the central canal of the host spinal cord,indicating that EGFP-ENT-derived NSCs could migrate to the central canal of the spinal cord as reserve NSCs for the host after EGFP-ENT was transplanted into the spinal cord of mice with spinal cord injury.(4)EGFP+/ MAP2+ double positive neurons were detected in the host spinal cord gray matter,indicating that EGFP-ENT survived after transplantation into the spinal cord of spinal cord-injured mice and migrated into the gray matter and differentiated to form mature neurons.(5)EGFP+/SYP+ double positive neurons were detected in the host spinal cord gray matter,indicating that EGFP-ENT-derived transplanted cells formed functional synaptic connections with different cells in the host spinal cord.(6)The results of BMS score in mice showed that the recovery of motor function was significantly better in the EGFP-ENT group than in the SCI and the EGFP-NSCs groups,indicating that ENT transplantation could promote the recovery of motor function in mice with spinal cord injury.Conclusion:(1)EGFP-ENT with a certain proportion of neurons and well-developed neural networks was constructed in vitro by applying EFs stimulation;(2)After transplantation of in vitro-constructed EGFP-ENT into the spinal cord of spinal cord-injured mice,the NSCs in EGFP-ENT survived,migrated and differentiated in the host spinal cord,and exhibited homing behavior;(3)The transplanted EGFP-ENT formed corresponding functional connections with the host spinal cord tissue,which could effectively promote the recovery situation of motor function in spinal cord-injured mice.