Experimental Study Of FK506 In Combination With Neural Stem Cells On Nerve Regeneration After Nerve Allograft

Partâ… The study on isolation and culture of neural stem cells from rat brain invitroObjective To investigate culturing neural stem cells(NSCs) that derived from ratembryos in vitro and observe their growth and differentiation.Methods NSCs wereisolated from hippocampus of SD rat embryos by mechanical and chemicalmethods.Cells were cultured in DMEM/F12 medium.The process of cell proliferationwere observed by microscope.The cell types were identified byimmuno-cytochemical analyses .Nestin,NSE and GFAP were used separately toidentify NSCs,neuron and astrocyte after cell differentiation.Results NSCs grewwell in the serum-free conditional medium with nestin positive cell express.Afterdifferentiation,the cells demonstrated NSE and GFAP immunoreactive.ConclusionNSCs could be cultured in serum-free conditional medium and they could be inducedtodifferentiate into neurons and astrocytes.Partâ…¡Experimental study of FK506 in combination with neural stem cellsimprove the regeneration of nerve allograftsObjective To study the effect of FK506 and neural stem cells on the regeneration innerve allografts.Methods A rat sciatic nerve model was successfully established anda total of 50 rats were used in our study [Lewis(L),n=40;Brown-Norway (BN),n=10].Sciatic nerve (1cm) deficits were created in the Lewis rats and sciatic nerves wereharvested from BN rats to repair the deficits of the Lewis rats.The animals wererandomly selected tomake four treatment groups.Group A received notreatment.Group B received neural stem cells.Group B received neural stem cells. Group D received FK506.Group C received both neural stem cells and FK506.Nerveregeneration was evaluated at 6 and 12 weeks by standardized pin-prick andtoe-spread tests.Nerve samples were harvested at 6 and 12 weeks Axonalregeneration was assessed by electrophysiological test.The MCV was measured foreach nerve allograft.Muscle denervation atrophy was evaluated by gastrocnemiusweights.Two step IHC detection reagent Brdu and GFAP were used to evaluate thechange of protein.NGF and GAP-43 ISH detection kit to test the change ofRNA.Results Rats of group C showed less motion,malnutrition and failed to gainweight during the first week.After 6 weeks,morphometric analysis confirmed thatthe number of myelinated fiber,myelin width and axon diameter were maximallyincreased in the C group.Muscle weight of group C&D were larger than that of groupA&B.MNCV and conductive time in group C are better than other groups.Improvedfunctional and histomorphometric outcomes were observed in animals treated withneural stem cells and FK506 after nerve allograft transplantation when compared toanimals receiving no treatment and FK506 or neural stem cells alone.ConclusionThe combination of neural stem cells and FK506 could afford nerve regeneration.Itmay provides an expanded source of nerve tissue to alleviate the morbidity ofharvesting peripheral nerves from multiple sites for those afflicted with extensiveperipheral nerve injuries.