Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

Abstract

Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10(6) cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10(6) cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

Figures

Figure 1. Basso, Beattie and Bresnahan (BBB) locomotor rating scale scores after treatment with mononuclear cells 1 h (Panel A) and 9 days (Panel B) after rat spinal cord injury. Data are reported as means ± SD for 8-14 rats in each group. BBB scale, scores range from 0 (complete paralysis) to 21 (normal gait). *P ≤ 0.05 for the control and sham groups compared to the injured and treated groups. **P ≤ 0.05 for the injured group compared to the treated group (ANOVA followed by the Mann-Whitney test).

Figure 2. Volume of the thoracic spinal cord lesions of the group treated 1 h (Panel A) or 9 days (Panel B) after spinal cord injury. Data are reported as means ± SD for 5 rats in each group. *P ≤ 0.05, difference between the control groups and the spinal cord injured group (Mann-Whitney test). #P ≤ 0.05, difference between the control groups and treated group (Duncan test).

Figure 3. Identification of transplanted cells expressing human-specific anti-nucleus and mitotic cell antigen (NUMA). Mononuclear cells from human umbilical cord (arrowheads) present at the lesion site, at the T9 level, administered into the injury site 1 h (A) and into the cistern magna 9 days (B) after the lesion.

Figure 4. Immunofluorescence staining for antinucleus and mitotic cell antigen (NUMA) (A, D, G), glial fibrillary acidic protein (GFAP) (B), synaptophysin (E), and chondroitin sulfate proteoglycan (NG2) (H) in sections of spinal cord at the T9 level of animals transplanted with mononuclear cells from umbilical cord blood 1 h after the lesion. Overlapping of the antibodies is represented as: C, NUMA (green) and GFAP (red); F, NUMA (green) and synaptophysin (red); I, NUMA (green) and chondroitin sulfate proteoglycan (NG2) (red). Arrowheads indicate: NUMA-positive cells (A, D, G); positive cells when labeled with GFAP (B), synaptophysin (E), and NG2 (H); doublelabeling with NUMA and GFAP (C), synaptophysin (F), and NG2 (I).

Figure 5. Immunofluorescence staining for nucleus and mitotic cell antigen (NUMA) (A, D, G), glial fibrillary acidic protein (GFAP) (B), synaptophysin (E) and chondroitin sulfate proteoglican (NG2) (H) in sections of spinal cord at the T9 level of animals transplanted with mononuclear cells from umbilical cord blood 9 days after the lesion. Overlapping of the antibodies is represented as: C, NUMA (green) and GFAP (red); F, NUMA (green) and synaptophysin (red); I, NUMA (green) and NG2 (red). Arrowheads indicate: NUMA-positive cells (A, D, G); positive cells when labeled with GFAP (B), synaptophysin (E), and NG2 (H); double-labeling with NUMA and GFAP (C), synaptophysin (F), and NG2 (I).