Experimental Investigation of Grafting Pathways of Bone Marrow Stromal Cells into Spinal Cord Injury in Rats.
|School||Dalian Medical University|
|Keywords||Bone marrow mesenchymal stem cells Spinal cord injury Cell transplantation|
Objective: To compare effects of Bone marrow stromal cells(BMSCs)transplantation on the recovery of neurological function in rats with spinal cord injury by direct injection into the injury,lumbar punctur(eLP),femoral venous delivery(IV),and select proper pathway for clinical application.Methods:1. Providing healthy rats of 6 weeks old and 120-140g weight,separating and culturing BMSCs by means of whole marrow method,amplifying with generations,performing observation on cell morphology under the inverted phase contrast microscope,and 5th-generation BMSCs can be identified using a flow cytometry. BMSCs were labeled with bromodeoxyuridine(Brdu)at 48 hours before use.2. 48 rats were employed to establish the T10 spinal cord injury(SCI)models by modified Allen’s Weight Drop.Following 3 days,rats were randomly assigned into 4 groups,Group A:model control,Group B:directly into the injury,Group C:femoral venous delivery,Group D:lumbar puncture delivery. Rats in the model control group did not undergo cell transplantation. Nerve regeneration and functional recovery were observed in 3,7,14,21days after cells transplantation.3. Recovery of hindlimbs locomotor function was assessed in open field according to BBB score.4. The rats were killed after 3、7、14、21 days to observe whether the BMSCs can move to injury location and pathology change. Brdu+ cells , Brdu+GFAP and Brdu+NF-200 were identified by immunohistochemistry. Use image analysis system to calculate Mean Optical Density(MOD)of NF-200 and GFAP in each group.5. Statistical Analyses:All data were expressed as mean±standard deviation. The data were evaluated using SPSS16.0 software package for single-factor analysis. Differences were deemed statistically significant at p<0.05 level.Results: 1. The 5th-generation cells which had unitary fusiform shape and grew in vortex and in parallel with each other. The isolated cells were BMSCs by using flow cytometry.2. Locomotor function of Group A、B、C and D gradually recovered after spinal cord injury. From the beginning of 3 days,BBB scores were significantly lower in Group A than in Group B、C and D. Recovery of hindlimbs locomotor function of Group B was significantly better than C and D. And BBB scores of Group D were higher than Group C. The difference was statistically significant.3. Hematoxylin-eosin staining demonstrated many cavities and plenty of vacuole in spinal cord tissues in the model control group. No avities,small and few vacuoles and slightly interstitial edema were detected in the local injection group. No cavities,small and few vacuoles and slightly interstitial edema were detected in the other groups. 4. Brdu positive cells had been observed on 3、7、14、21day after transplantation. The number of Brdu+ cells of Group B was the most,and cells of Group D were more than Group C. The number of Brdu positive cells were gradually reduced with time(P<0.05). Brdu+GFAP double positive cells were observed in the spinal cord slice on 14 day,at the same time,Brdu+NF-200 double positive cells were observed on 21day,and the quantity of double positive cells is scarce. The mean optical density values of GFAP and NF-200 in Group B、C、D were significantly higher than Group A(P<0.05).Compare with Group C and D,MOD of Group B was the highest. And D group is better than C group.Conclusions:1. BMSCs were obtained by means of the whole marrow method.2. Transplanted BMSCs via direct injection,the femoral vein injection and lumbar puncture can survive and differentiate at injured area of the spinal cord and can improve neurological function.3. Comprehensive comparison and optimization of BMSCs transplantation pathways for treatment of spinal cord injury in rats,LP is an ideal minimally invasive technique. And it is superior to IV delivery and direct injection delivery,together with the potential for autologous transplantation, lends itself for clinical application.