Erythropoietin facilitates the recruitment of bone marrow mesenchymal stem cells to sites of spinal cord injury

Abstract

Despite the successes of bone marrow mesenchymal stem cell (BMSC) transplantation for the treatment of spinal cord injuries, only a small fraction of grafted cells migrate to the target areas. Therefore, there remains a need for more efficient strategies of BMSC delivery. The present study was designed to explore this. Rat models of spinal cord injury (SCI) were established and exposed to phosphate buffered saline (control), BMSCs or BMSCs + erythropoietin (EPO). Basso, Beattie and Bresnahan (BBB) locomotor scale and grid walk tests were then utilized to estimate neurological rehabilitation. Additionally, the following assays were performed: Immunofluorescence localization of BMSCs to the site of SCI; the transwell migration assay to detect in vitro cellular migration; the terminal deoxynucleotidyl transferase dUTP nick end labeling assay to determine the apoptotic index of the lesion; and western blotting analysis to evaluate the expression of vascular endothelial growth factor (VEGF) and brain derived neurotrophic factor (BDNF) at the site of SCI. The BBB scores of the BMSC + EPO treated group were significantly increased compared with the BMSC treatment group (P<0.05). For example, BMSC + EPO treated rats had a significantly decreased number of hind limb slips compared with the BMSC treatment group (P<0.05). Furthermore, EPO significantly increased the migration capacity of BMSCs compared with the control group (P<0.001). In addition, the apoptotic index of the BMSC + EPO group was significantly decreased compared with the BMSC group (P<0.05). Green fluorescent protein-labeled BMSCs were detected at the site of SCI in the BMSC and BMSCs + EPO groups, with the signal being notably stronger in the latter. Moreover, the expression of VEGF and BDNF in the BMSCs + EPO group was significantly increased compared with the BMSC group (P<0.05). In conclusion, the results of the present study indicate that EPO can facilitate the recruitment of BMSCs to sites of SCI, increase expression of BDNF and VEGF, and accelerate recovery of neurological function following SCI.

Keywords: bone marrow; erythropoietin; mesenchymal stem cells; spinal cord injury.

Figure 1.

Locomotor function recovery after SCI in rat models treated with BMSCs or BMSCs + EPO. (A) BBB scale scores, which represent the restoration of hind limb function post-SCI. (B) Grid walk test of hind limbs post-SCI to evaluate motor sensitivity and capacity for precise control of the hind limbs. Results are presented as the mean ± standard deviation. *P<0.05 vs. control group; ^ҰP<0.05 vs. BMSCs group; ^#P<0.01 vs. control group. SCI, spinal cord injury; BMSCs, bone marrow mesenchymal stem cells; EPO, erythropoietin; BBB, Basso, Beattie and Bresnahan locomotor scale.

Figure 2.

Effect of EPO on improving the migration capacity of BMSCs in vitro. (A) Images of the BMSCs under a light microscope, magnification ×400. The BMSCs were dyed with crystal violet and the cells transplanted were violet. (B) Transwell migration assay results. Cells were measured three times, then the average was taken. Results are presented as the mean ± standard deviation. *P<0.001 vs. BMSCs alone group. EPO, erythropoietin; BMSCs, bone marrow mesenchymal stem cells.

Figure 3.

Effect of EPO on the apoptotic index of SCI sites. Images showing apoptosis in the sites of SCI 7 days post-SCI at ×400 magnification in (A) the control, (B) the BMSC treatment group and (C) the BMSCs + EPO group. Apoptotic cells were stained dark brown, normal nuclei were blue. (D) Apoptotic index (percentage of apoptotic cells) in each group following SCI. The number of apoptotic cells was decreased in the BMSCs + EPO group compared with the control and BMSC treatment groups. Results are presented as the mean ± standard deviation. *P<0.01 vs. control group, ^¥P<0.05 vs. BMSC group, ^#P<0.05 vs. control group. SCI, spinal cord injury; BMSC, bone marrow mesenchymal stem cells; EPO, erythropoietin.

Figure 4.

Effect of erythropoietin (EPO) on the localization of bone marrow mesenchymal stem cells (BMSCs) transplanted into spinal cord injury (SCI) sites 28 days following SCI in (A) the control group, (B) the BMSC treatment group and (C) the BMSC + EPO group. GFP-labeled BMSCs were observed at the site of SCI in the BMSC and BMSC + EPO groups.

Figure 5.

Expression of VEGF and BDNF 28 days following spinal cord injury. Protein expression levels were determined through (A) western blotting followed by (B) analysis. Expression levels were normalized to GAPDH. Results are presented as the mean ± standard deviation. *P<0.05 vs. control; ^†P<0.05 vs. Control, ^¥P<0.05 vs. BMSC group. VEGF, vascular endothelial growth factor; BDNF, brain derived neurotrophic factor; BMSC, bone marrow mesenchymal stem cell; EPO, erythropoietin.