Effects Of ChABC,NEP1–40 Combined With Neural Stem Cells Transplantation On The Recovery Of Neurological Function In Rats With Spinal Cord Injurey

Objective: To investigate the effects of combined transplantation of chondroitinase ABC(ChABC),Nogo-66(1-40)antagonist peptide(NEP1–40)and rat embryonic neural stem cells(NSCs)on axonal regeneration,growth of neuron and the recovery of neurological function in rat with spinal cord injury.Methods:(1)The recovery of NSCs cryopreserved in our preliminary experiment have been performed before isolation and cultivation.The NSCs were cultured and induced by ATRA(the concentration of 1.0 umol/L)in vitro.The NSCs induced by ATRA(the concentration of 1.0 umol/L)were marked by BrdU before transplantation.(2)There are 60 adult Sprague-Daw female rats were randomly divided into six groups,namely,the sham operation group(group A,n=10),the injury control group(group B,n=10),the NSCs treatment group(group C,n=10),the NSCs combined with ChABC treatment group(group D,n=10),the NSCs combined with NEP1-40 treatment group(group E,n=10),and NSCs combined with ChABC and NEP1-40 treatment group(group F,n=10).Complete spinal cord injury models of rat were performed by totally transecting at T9-T11 segment except the sham group before transplantation.Group A(sham operation group)and group B(model control group)received an equivalent transplantation withphosphate buffer saline(n=10)only.Group C received transplantation with NSCs(n=10).Combined transplantation of NSCs with ChABC was performed in group D(n=10).Group E received transplantation with NSCs and NEP1-40(n=10).NSCs in combination with ChABC and NEP1-40 transplantation was performed in group F(n=10).(3)Basso,Beattie,Bresnahan(BBB)score and Somatosensory evoked potentials(SEP)test were used to evaluate the functional changes preoperatively,postoperatively and 2 weeks,5 weeks,as well as 8 weeks after transplantation,respectively.The injured spinal cords was sampled for observing 8 weeks after transplantation and HE staining was performed to observe the morphological alteration in the injured spinal cord.The number,distribution and differentiation of transplanted NSCs were examined by double Immunofluorescence staining,using microtubule-associated protein 2(MAP-2)and BrdU.Data were analyzed with the software SPSS 17.0,One-way ANOVA was used to analyze one-variable experiments with six groups andtwo groups were compared by Student’s t tests.Data are reported as means ± SEMs,and p < 0.05 was considered significant.Result:(1)2 weeks after operation,recovery was observed in SCI groups,and the recovery of all transplanted treatment groups was better than that of SCI model group,the outcome of neurological functional recovery in group F significantly surpassed that of group C,D and E among all observing time point of post-transplantation,At any observing point of post-transplantation,the BBB score showed significant difference in all transplanted treatment groups(P<0.05): Group B had the lowest BBB scores when compared with that of group C,D,E and F(P<0.05),and group F had the highest BBB score when compared with group C,D and E(P<0.05).(2)At each time point,the improving of SEP in group B was not dramatically,the SEP of all transplanted treatment groups had the shorter SEP than it of SCI model group,there was an obvious difference in all transplanted treatment groups(P<0.05): the SEP was significantly longer in group B than it of group C,D,E and F(P<0.05),and the group F had shortest SEP when compared with group C,D and E(P<0.05).(3)8 weeks after transplantation,HE staining in group A showed complete morphological structure and regular arrangement of cells;while the morphological structure was seriously damaged in group B,where irregular arrangement of cells as well as a large number of scar and cysts could be seen;and there were obvious cell and capillary hyperplasia and a few small cysts in group C,D,E,and group F in particularly.(4)The viable positive cells labeled by BrdU could only be observed in group C,D,E and F,the body of BrdU positive cells was stained to be orange and it mainly distributed into the surrounding of white and gray.The difference of the number of Brdu-positive cells was enormous in all transplanted treatment groups(P<0.05): The group F had the most numbers when compared with group C,D and E(P<0.05).(5)Using Fluorescence staining,the positive cells marked by MAP-2 and BrdU could be detected in group C,D,E and F,the nuclei was stained to be dark red and the body and axon was stained to be green.The axonal regeneration and growth was significantly promoted and the number and surface area of positive cells by labeled MAP-2 had significant difference(P<0.05): group F had the most maximum cells and the largest area when compared with group A.B.C,D and E(P<0.05).Conclusion:(1)The transplantation of NSCs induced by ATRA,ChABC combined with NSCs and NEP1-40 combined with NSCs could strongly improve the functional recovery of the SCI in rats.(2)Combined transplantation of NSCs induced by ATRA,chondroitinase ABC and NEP1-40 was more effective than ChABC alone or combined with NEP1-40 on enhancing the functional recovery after spinal cord injury(3)ChABC and NEP1–40 had a synergistic effect on the recovery of neurological function in rats with spinal cord injury.