| Spinal cord injury (SCI) caused by traffic accident,sports,building and mine accident which become more and more with the development of economy has been a major threat to mankind life and healthy. For the past few years, the incidence is usually high in the youth and the adult which result in permanent disability,and bring about a huge burden to their families and the all society.The traditional therapy of spinal cord injury usually included spine fracture-dislocation stabilization,relive spinal cord compression,symptom therapy and rehabilitative treatment.However,it is a pity that there are no effective and useful methods for SCI patients in clinic. With the development of tissue engineering and the pathology mechanism cognition of SCI,scientists are discovering a new way to cure SCI by transplanting BMSCs,and some satisficated were found during the thereapy results.Bone marrow stromal cells(BMSCs)are not haemopoietic stem cells in hematopoietic system.They are one of an ideal kind of seed cells of tissue engineering for their easy to obtain, self duplication, and multi-directional differentiation. It can not only differentiate into many kinds of mesenchymal tissue under specified inductions, such as bone, cartilage, muscle, but also can differentiate into neural cells. During the process of growth and proliferation,BMSCs can change the microenvironment and secrete some nutrition factors. It is benifical for SCI.So BMSCs has become ideal seeds cells on tissue engineering.With time went on,the neural cells and nutrition factors gradualy decreased. To investigate the influence of different transplantating times on the survival and immigration of BMSCs in injured spinal cord by Subarachnoid administration, and to evaluate the most optimal Subarachnoid administration times for BMSCs, in order to therpy SCI by transplantation of auto-BMSCs in future clinic application.Objective1. we transplanted the labeled-BMSCs in model of SCI by Subarachnoid administration.2. To investigate the influence of different transplantating times on the survival and immigration of the BMSCs in injured spinal cord.3. observed the changes of neural cells and the the expression of Nestin,NF200 GFAP in injured spinal cord and BBB scores in model of SCI rats in the influence of different transplantating times.4. to evaluate the most optimal Subarachnoid administration times for BMSCs and to study possible therapeutic mechanism of SCI.Method1. we applyed density gradient centrifugation and adherence character of BMSCs to isolate BMSCs. The bone marrow of eight adult male rats were used to isolated,cultured, purified and labeled with Hoechst33342 in vitro.2. Rats were induced by improved Allen's method to establish the spinal cord rat models at T9-T10 level. The ninty injured rats were randomly divided into 5 groups, group A, B, C, D andE. The labeled BMSCs with a dose of 1×106 /0.2ml were injected into subarachnoid space of the rats via a catheters under the Subarachnoid space to deliver BMSCs over the lesion site in groupsA,B,CandD. The cells administered only once in group A 1-week after injury. However, they were injected repeatedly in group B 1 and 3 weeks, in group C 1,3 and 5 weeks and in groupD1, 3,5,7 and 9 weeks after injury, respectively. Group E was treated with equal volume phosphate-buffered saline(PBS)via the same ways in the same injected way after injury.3.Neurological functions were evaluated using the Basso-Beattie-Bresnahan (BBB) Scale 1,3,5,7,9and 12 weeks after transplantation. Sections of tissue were analyzed with HE,immunohistochemistry and fluorescence microscopy observe them igration, survival, differentiation and histomorphological changes of BMSCs.Results1. All the BBB scores of rats were less than 4 points afer SCI lweek. At 3 weeks after injury, there were significant difference in theBBB scores between group E and groupsA, B, C, D (P< 0.01). The BBB scores in groupA improved gradually recovery and reached the plateau around at 5 weeks after SCI. At 7,9 and12 weeks, the BBB scores were significantly higher in groups C and D than in groups A and B (P< 0.01), and in group B than ingroup A (P< 0.01). There were no significant differences in the BBB scores between groups C and D (P> 0.05).2. The fluorescence microscopy showed that the transplanted BMSCs survived and grew in the injured region at 3 weeks after injury and as time went on, the planted cells gradually decreased in group A; BMSCs count reached the peak values at 5 in groups B and 7weeks in groups C and D,and then gradually decreased. At 12 weeks, the survival BMSCs were significantly more in groups C and D than in groups A and B(P<0.01).3. HE staining showed that the formation of cavity was observed in each group at 3 weeks after injury and the area of cavity gradually decreased in groups A, B, C and D. At 12 weeks, the area of cavity was the miximal in group C, the moderate in groups A and B, and the maximal in groups E.4. The immunohistochemistry staining indicated that the expression of NF200 was like the trend of changed BMSCs. The expression of NF200 was survived and grew in the injured region at 3 weeks after injury and then cells gradually decreased in group A. NF-positive retain for several weeks in groups B, C and D.By the time of 12 weeks after SCI,the great of NF-positive kept on in groups C.However,a few of NF-positive kept on in groups A.The NF200 expressed more intense in groups C and D than in groups A and B. The expression of NF-positive fibers was more intense through injuried area in group C than in group A. In group E,there was not the expression of NF200 in the SCI area. In the groupE, the expression of Nestin was seen at ependymal cells of central canal and reached the peak value at lweek after SCI,and then dispeared at 3 week after SCI. In the experimental groups, the expression of Nestin reached the peak value in the injured sites and adjacent sites 3 weeks in groupA,5 weeks in groupB and 7 week in group C,D, and followed by gradual decrease.In the groupE,GFAP-postive reached the peak level near the site of injury and the adjacent tissues lweek after the injury in groups E. There was a layer of thick glial scar around the regin of SCI at 7weeks. The expression of GFAP reached the peak value in the injured sites and adjacent sites 3 weeks in groupA,5 weeks in groupB and 7 week in group C,D, and followed by gradual decrease.7 week after SCI,GFAP positive reactive astrocytes delineated the margin of the epicenter of the lesion with their tightly interwoven processes. GFAP-postiv cells were adjust and formed net-like scaffolds which can reconstructan anatomical pathway along which axons can grow.Conclusion1. We applyed density gradient centrifugation and adherence character of BMSCs to isolate BMSCs.This method is simple and useful.Rats were induced by improved Allen's method to establish the spinal cord models that were effected.2. Multiple administration of BMSCs improves the density of BMSCs and change the microenvironment in local SCI. the three BMSCs-treated groups achieved more functional recovery than the other BMSC-treated and control groups.3. The Immunohistochemistry staining indicated that transplantation BMSCs can expressed pre-neuronal marker Nestine and expressed neuronal marker neurofilament200(NF200).The expression density of NF200 and NF-positive fibers was most intense in the three BMSC-treated groups. Furthermore, The expression of NF-positive fibers pentrate the area of SCI.The expression of GFAP interwoven to net-like scaffold after transplatation BMSCs.A few axons could be seen to regrow into the net-like scaffold of layer of glial scar.4. In sum, The study showed that multiple transplantations of BMSCs into subarachnoid space were beneficial, to some extend, to the repair of SCI, and three times for BMSCs transplantation are best. It is effective and economic.It is one of the mechanism of improves the density of BMSCs and create a more supportive environment for survival of neurons and regrowth of axons after injury, down-regulated glial scar and reduce cavity of the injured area of spinal cord.
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