The Effect Of Transplantation Of Peripheral Nerve With Neural Stem Cell Plus Lithium Chloride Injection In The Treatment Of Rat Spinal Cord Injury

Objectives: To obtain neural stem cells (NSC) from rat embryonic spinal cord,culture in vitro and identify cells.Methods: The spinal cord was dissected out from green fluorescent protein (GFP)transgenic E14rat embryo, and single cells were dissociated using enzymaticdigestion and mechanical separation, then cultured in serum free medium. Theexpression of nestin, BrdU, NeuN, and GFAP of cultured cells were detected usingimmunofluorescence technique to identify the properties of these cells.Results: Neural cells derived from the spinal cord of GFP transgenic rat embryogrowth well in vitro, expressing GFP protein. The expression of nestin was observedin most of these cultured cells. When fetal bovine serum was added into culturemedium, the expression of NeuN and GFAP were detected among these cells.Conclusions: Neural cells derived from GFP transgenic rat embryo expressed specificprotein of NSC, and had the properties of self-renewal and multiple differentiationpotency, which confirmed that the cultured cells were neural stem cells. Objectives: To investigate the effect of lithium chloride on the proliferation and differentiation of cultured NSC.Methods: Lithium chloride was added into culture media in different concentrationsto observe the growth of cultured NSC. The expressions of BrdU, NeuN, and GFAPwere investigated when1mM lithium chloride were add into culture media.Results: Lithium chloride at1mM did not affect the growth of cultured NSCs, whileLiCl at3mM significant suppressed the growth of cultured NSCs, and addition of6mM of LiCl resulted in a lot of NSC death. Lithium chloride at1mM significantlypromoted the proliferation of NSCs with or without the present of EGF and bFGF.Compared with the control group, the addition of lithium chloride at1mM resulted ina higher percentage of NeuN positive cells, and a lower percentage of GFAP positivecells among cultured neural stem cells.Conclusions: Lithium chloride at1mM is good for the growth of cultured NSCs.Lithium chloride promoted the proliferation and neural differentiation of NSCs, whileinhibited the glial differentiation of cultured stem cells. Objectives: To investigate the effect of lithium chloride on the survival anddifferentiation of NSC after transplanted into rat tibial nerve, and the effect oftransplanted NSC with lithium treatment on host tibial nerve.Methods: The right tibial nerve of adult rat was ligated, and dissociated NSCs weretransplanted into distal dump of tibial nerve, followed intraperitoneal injection ofeither lithium or normal saline for one week. After that, the expression of nestin,NeuN, ChAT, GFAP in transplanted NSCs, the expression of NF200, ED1, andBDNF in host tibial nerve were investigated.Results: One week after transplantation, transplanted NSCs survived well, migratedfrom injection site to surrounding tissue through axon interspace. There was noobvious expression of nestin, NeuN, and ChAT was found in transplanted NSCs.Most of the NSCs expressed GFAP protein. After tibial nerve ligation, most of the axons degenerated. Compared to control group without NSC transplantation, animalreceived NSC transplantation had more NF200positive residual axons; and lithiuminjection result in more residual axons. Lithium also reduced ED1expression in tibialnerve after NSC transplantation. The expression of BDNF increased in tibial nervewith NSC transplantation, or lithium injection without NSC transplantation. Thecombination of NSC transplantation and lithium injection up regulated more BDNFexpression than other groups.Conclusions: Transplanted NSCs survived well in tibial nerve, and most of the NSCsdifferentiated into GFAP positive glia. Lithium injection reduced the GFAPdifferentiation of transplanted NSCs and inhibited macrophage infiltration after NSCtransplantation. NSC transplantation plus lithium treatment reduced host axonsdegeneration, and improve BDNF expression, Objectives: To investigate the effect of transplantation of NSCs in tibial nerve withlithium chloride injection in the treatment of spinal cord injury.Methods: One week after transplanted NSCs into tibial nerve, the rat right spinal cordhemisection injury was created and a portion of spinal cord was removed. Tibial nervewith or without implanted NSCs was then transplanted into the removed area of spinalcord, followed with intraperitoneal injection of lithium chloride or natural saline for4weeks. The locomotor function of right hind limb was evaluated at2and4weeksafter surgery. The survival and differentiation of transplanted NSCs, the number ofhost motoneurons, the expression of NF200in regenerated axons, the glial scarformation and macrophage infiltration were investigated4weeks after surgery.Results: Two and four weeks after surgery, tibial nerve transplantation withimplanted NSC or tibial nerve alone with lithium injection had better functionalrecovery compared to tibial nerve alone, while tibial nerve with implanted NSCs pluslithium injection had best functional recovery. Transplanted tibial nerve well integrated into host spinal cord. NSCs inside tibial nerve survived well after secondtransplantation. Lithium promoted transplanted NSC extent neurites into host spinalcord, and promoted NSCs differentiated into NF200positive neurons. Compared totibial nerve alone, transplantation of NSCs and lithium injection resulted in more hostmotoneurons survived in the injured side of spinal cord, more regenerated axons intibial nerve, reduced glial scar area and ED1expression. Combination of NSCtransplantation and lithium injection had better results than other groups in all aspectsmentioned above.Conclusions: Tibial nerve transplantation with implanted NSC or lithium treatmentalone significantly improved functional recovery after spinal cord hemi-section injury,reduced host neuron death, decreased glial scar formation and macrophage infiltration.Combination of NSC transplantation and lithium injection had better effect in thetreatment of spinal cord injury.