Synergistic effects of Buyang Huanwu decoction and embryonic neural stem cell transplantation on the recovery of neurological function in a rat model of spinal cord injury

Abstract

The aim of the present study was to investigate the therapeutic effect of a combined treatment of Buyang Huanwu decoction (BYHWD), a well-known formula of traditional Chinese medicine, and neural stem cells (NSCs) on spinal cord injury (SCI) and the associated underlying mechanisms. A SCI model was established by surgery via a complete transection of the T10 vertebra of female Sprague-Dawley rats. Gelatin sponges were used to absorb NSCs labeled with the thymidine analog, 5-bromo-2-deoxyuridine (BrdU), and were transferred into the transected spinal cords. BYHWD was administered once a day by introgastric infusion. Motor functions of the hind limbs were evaluated using the 21-point locomotor rating scale developed by Basso, Beattie and Bresnahan (BBB). The fate of the transplanted NSCs under the various conditions was examined by double immunofluorescence staining, using markers for neurons, astrocytes and oligodendrocytes, with BrdU. Ultrastructural changes of the SCI site following the various treatments were examined under a transmission electron microscope. The number of double positive cells for glial fibrillary acidic protein and BrdU in the BYHWD + NSC group was significantly decreased when compared with that in the NSC group (P<0.05). However, the number of cells that were labeled double positive for myelin basic protein and BrdU, as well as neuron specific enolase and BrdU, was greater in the BYHWD + NSC group when compared with the NSC group. Electron microscopy demonstrated that treatment with BYHWD combined with NSCs significantly alleviated demyelination. Results from the BBB motor function test exhibited a significant improvement in the BYHWD + NSC group when compared with the SCI, BYHWD and NSC only groups. In conclusion, the results demonstrated that the traditional Chinese medicine formula, BYHWD, exerted an effect on the differentiation and migration of NSCs. Combining the administration of BYHWD with NSCs was shown to have a synergistic effect on the recovery of neurological function, mitigating the progress of demyelination or ameliorating the recovery of myelination.

Keywords: Buyang Huanwu decoction; neural stem cells; spinal cord injury; traditional Chinese medicine; transplantation.

Figure 1

Characterization and differentiation of the cultured isolated NSCs from the rat embryo neural tube. (A) Cultured NSCs grew into neurospheres in the culture dish, expressing the neural stem cell marker, nestin. (B) NSCs continued to proliferate in the in vitro culture, as shown by the incorporation of BrdU (green). (C) Transplanted NSCs in the injured spinal cord differentiated into neurons, labeled with the neuron marker, NSE (red) and BrdU (green). (D) Transplanted NSCs differentiated into oligodendrocytes, expressing MBP and BrdU, and incorporated into neural networks. (E and F) BYHWD treatment exerted a suppressive effect on astrogliosis in the spinal cord injury (SCI) site, as compared with NSC treatment alone. (G) BYHWD treatment was shown to promote NSC survival at the SCI site at the three examined time points (^*P<0.05; n=36). (H) BYHWD treatment was shown to inhibit astrogliogenesis in the surrounding SCI site at the two examined time points (^*P<0.05; n=36). BYHWD, Buyang Huanwu decoction; NSC, neural stem cells; BrdU, 5-bromo-2-deoxyuridine; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; NSE, neuron specific enolase. Scale bars, 50 μm (A and C), 30 μm (B) and 100 μm (D, E and F).

Figure 2

(A-F) BYHWD promotes NSC migration in the spinal cord injury site in rostral and caudal directions. NSCs were shown to migrate into the white matter and gray matter, closely integrating with the nerve tract and neural networks. At the same level of the spinal cord, there were more fluorescein immunofluorescence-labelled Brdu incorporated NSCs (white arrows) in the BYHWD-treated group (D, E and F) compared with the NSC only group (A, B and C) at the corresponding time points. (G) BYHWD promotes the migration of NSCs in the spinal cord (^*P<0.05; n=36). BYHWD, Buyang Huanwu decoction; NSC, neural stem cell. Scale bar, 100 μm.

Figure 3

Ultrastructure of the spinal cord at the SCI site and the effect of NSCs and BYHWD treatment on SCI rehabilitation, as measured by the BBB locomotor rating scale. Ultrastructure examination revealed the effect of the different treatments on the SCI after 28 days. (A and B) No treatment resulted in onion-like demyelination (white arrow). (C and D) Treatment with BYHWD only mitigated the demyelination (black arrow). (E and F) Treatment with NSCs only further promoted the mitigation of demyelination when compared with the BYHWD and control group (black arrows). (G and H) Combination treatment with BYHWD and NSCs notably alleviated the demyelination, and the myelination wrapping the axons was shown to recover to relative normality. B, D, F and H are higher magnification compared with A, C, E and G, respectively. (I) Motor rehabilitation, as measured using the BBB locomotor rating scale. Treatment with BYHWD + NSCs demonstrated a synergistic effect on locomotion recovery. BYHWD, Buyang Huanwu decoction; NSC, neural stem cells; SCI, spinal cord injury; BBB, Basso, Beattie and Brasnahan. Scale bar, 2 μm.