| Brachial plexus roots avulsion is a devasteting injury which is characterized by the interruption of rootlets from cervical cord and apoptosis or necrosis of spinal cord neurons and always lead to an flaccid arm.Till now, brachial plexus roots avulsion has been treated by neurotization or nerve transposition with some exciting clinical outcomes.But the limitation of donor nerves make those procedures unpredictable.To explore better alternatives, intraspinal reimplantation of avulsed nerve roots has been tried experimentally with some encouraging results.Carlstedt et al performed reimplantation procedure clinically to treat traumatic adult brachial plexus root avulsion and obstetric brachial plexus palsy with some certain return of related muscles strenghth.Brachial plexus root avulsion,which always lead to intraspinal neurons apoptosis or necrosis and failed axonal outgrowth with in the non-permisive environment,is considered to be a problem of the central nervous system(CNS).Because of the above affairs,the evental functional recovery is limited if treated only by nerve root reimplantation.We have investigated the pathological and biochemical events within spinal cord after brachial plexus root avulsion and intend to combine reimplantation and neural stem cells(a type of premitive neuroepithelial stem cells)transplantation to reconstruct the continuity between peripheral nerves and spinal cord fot the treatment of brachial plexus root avulsion.NSCs(Neural stem cells),which derive from embry or premitive nervous tissues,have the capacity of self-renewal and multiple potential.It is the above capacity that provides NSCs with the possible application for the treatment of central nervous system injury or disease.NT-3,a member of neurotrophin family,can prevent motoneurons apoptosis,maintain neurons survival and promote neural differentiation and axonal outgrowth.It is our present strategy that NSCs are used as a gene segement carrier to express NT-3 within injured spinal cord region and the functional differentiation,such as motoneurons or oligodendrocytes,In the present study,PGET-1 TA plasmid and pLEGFP-C1 plasmid were employed to carry the amplified NT-3 gene segment into NSCs,and the transfected NSCs were transplanted into the corresponding spinal cord segment after brachial plexus root avulsion and the survival,migration,differentiation and motoneuron protection effect were observed.1.Establishment of experimental rat model of intraspinal reimplantation after brachial plexus root avulsionReimplantation of avulsed brachial plexus root was performed through posterior subscapular approach within Wistar rats and the structure of rat brachial plexus and musculocutaneous nerve,localization maker of cervical nerve roots,microanatomy of transvers cervical spinal cord section,and selection of implantation point were investigated.Conclusions:1.The nerve fibers of rat musculocutaneous nerve are mainly from C5～C7 nerve roots and no fibers from C8 take part in the formation of musculocutaneous nerve.2.Rat model of reimplantation of avulsed root after brachial plexus avulsion can be successfully established through posterior subscapular approach.3.Concerning the avulsion procedure of nerve roots, intradural manipulation is regarded as an efficient and safe alternative with less bleeding.4.In addition to the bigger C2,T1 spinal processes,nutrition vessel holes on the C3 vertebral lamina and the C2～C3 ligamentum flavum are also reliable markers for the nerve roots identification and localization.5.We take the midpoint between anterolateral sulcus and lateral midline of spinal cord as implantation point and the depth of intraspinal implantation is 0.5 mm beneath the dura matter.2.Isolation,cultivation and identification of neural stem cells derived from embryonic rat brain and its differentiation under Velvet Antler Polypepitides.Neural stem cells were isolated from embryonic 14 days rat brain and cultivated by serum-free culture techniques within DMEM-F12 medium including B27,20 ug/l of EGF and 20 ug/l of bFGF.While self-renewal capacity of the isolated cells was identified by clonal analysis,anti-Musashi and anti-Nestin immunocytochemistry staining were employed to identified the neural stem cells specially.After withdraw of EGF and bFGF from the medium, isolated neural stem cells were induced into differentiation within medium including 10%of fetal bovine serum.Anti-NSE and anti-GFAP immunocytochemistry staining were employed to identify the newly born neurons and astrocytes respectively.Also,the biological effect of the VAP on neural stem cells was studied.Conclusions:1.We successfully isolated neural stem cells from embryonic rat brain utilizing serum-free culture techniques.2.The isolated neural stem cells proved to have capacity of self-renewal and multiple potential.3.Embryonic 13～14 days rat brains are better source of neural stem cells.4.During the isolation procedure,mechanical trituration should be the better choice than enzymic digestion which always lead to cell damage.5.The VAP can induce neural stem cells into neurons and the rate of new born neurons reached to 75% under 50μg/L of VAP.3.Construction of pLEGFP-C1-NT-3 recombinant plasmid and transfection of neural stem cells.Tatal RNA was extracted from newborn rat brain followed by amplification of NT-3 gene by RT-PCR.PGET-1 TA plasmid and pLEGFP-C1 plasmid were employed to transfer the amplified NT-3 gene into neural stem cells.It was documented that NT-3 protein was efficiently expressed within the transfected neural stem cells.Conclusions:1.We successfully constructed pLEGFP-C1-NT-3 recombinant plasmid.2.Neural stem cells were efficiently transfected by pLEGFP-C1-NT-3 recombinant plasmid after packaging of those plasmids within PA317 cells.3.NT-3 protein was efficiently expressed within the transfected neural stem cells.4.Transplantation of NT-3 modified neural stem cells combined with reimplantation of avulsed nerve root.The rats were divided into 4 groups:A group(only nerve root reimplantation),B group(nerve root reimplantation+neural stem cells transplantation),C group(nerve root reimplantation+pLEGFP-C1 modified neural stem cells transplantation)and D group(nerve root reimplantation+ NT-3 modified neural stem cells transplantation).The function of involved extremity,Latency and CAMP of related musculocutaneous nerves,pathological changes and motoneurons score and migration,survival and differentiation of transplanted neural stem cells were observed 1 month,2 months,4 months and 6 months postoperatively.Conclusions:1.There was no signicicant difference upon function of involved extremity between the above groups.2.Compared with the control groups, Latency and CAMP of musculocutaneous nerves of rats treated with NT-3 modified neural stem cells transplantation improved significantly.3.Compared with the control groups,transplanted NT-3 modified neural stem cells can protect injured motoneurons with significant effect.4.The transplanted NT-3 modified neural stem cells can survive for a long time span(even for 6 months) and express NT-3 protein within transplantation site.5.The transplanted NT-3 modified neural stem cells can migrate out of the transplantation region and differentiated into neurons.
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