Objective: Adenosine triphosphate (ATP) can promote the repair of spinal cord injury (SCI). To investigate the effect of ATP combined with bone marrow mesenchymal stem cells (BMSCs) transplantation on SCI, and to evaluate the synergistic action of ATP and BMSCs in the repair of SCI and the feasibility of the combined transplantation in the treatment of SCI.
Methods: BMSCs were isolated from the marrow of the tibia and the femur of a male SD rat (weighing 120 g), the 3rd generation BMSCs were labeled with BrdU, then BMSCs suspension of 5.0 x 10(7) cell/mL were prepared. Forty-eight adult female SD rats (weighing 240-260 g) were made SCI models at T2 levels according to the improved Allen's method, and were randomly divided into 4 groups (groups A, B, C, and D, n = 12). In group A, ATP (40 mg/kg) and BMSCs (6 microL) were injected to the central point and the other 2 points which were 1 mm from the each side of head and tail of the injured spinal cord; after blending the BMSCs suspension, the cells amount was about 3.0 x 10(5). In groups B, C, and D, the BMSCs suspension (6 microL), ATP (40 mg/kg), and PBS (40 mg/kg) were injected to the points by the same method as group A, respectively. The general conditions of the rats were observed after operation. The nerve function of low extremities was evaluated using the improved Tarlov scale and the Rivlin inclined plane test at 1, 3, 7, 14, 21, and 28 days after operation. At 28 days after operation, the reparative effect of SCI was observed using histological and immunohistochemical staining.
Results: One rat of group A, 2 of group B, 2 of group C, and 3 of group D died of infection and anorexic, the others survived to the end of the experiment. Paralysis symptom in low extremities occurred in all rats after operation and was improved at 2-3 weeks postoperatively, the improvement of group A was the best, groups B and C were better, group D was the worst. There was no significant difference in the Tarlov scale and the Rivlin inclined plane test among 4 groups at 1 and 3 days after operation and between groups B and C at 7, 14, 21, and 28 days after operation (P > 0.05), but there were significant differences among other groups at 7, 14, 21, and 28 days after operation (P < 0.05). At 28 days after operation, HE staining demonstrated that the injured region in group A was finely restored, without obvious scar tissue and cavity, and there existed clear stem cell differentiation characters; there was small amount of scar tissue and cavity in the injury site of groups B and C; and there was great deal of scar tissue in the injury site of group D, in which there were numerous inflammatory cells and fibroblasts infiltration and bigger cavity. Immunohistochemical staining showed that BrdU-positive BMSCs were seen in groups A and B, and positive cells of group A was significantly more than that of group B (P < 0.05). The expressions of neurofilament protein 200 and glial fibrillary acidic protein in group A were significantly higher than those in groups B, C, and D, and groups B and C were significantly higher than group D (P < 0.05).
Conclusion: ATP has protective effects on injured spinal cord, a combination of ATP and BMSCs can synergistically promote the reparation of SCI.