The feasibility and the mechanism study about valproic acid combination with bone marrow mesenchymal stem cells to repair the acute spinal cord injury in rats

Abstract

Objective To investigate the feasibility of valproic acid (VPA) combined with bone marrow mesenchymal stem cells (BMSCs) to promote restoration of the rat spinal cord injury (SCI) and its mechanism of action. Methods BMSCs from Sprague-Dawley (SD) rats were cultured in vitro. The third generation BMSCs were detected by flow cytometry and collected for use. Spinal cord injury model was Made by modified Allen's techniqtechnique. According to the random number table method sixty adult male SD rats were divided into five groups: control group, SCI model group, VPA treated group, BMSCs transplantation group and VPA combined with MBSCs group. At BMSCs transplantation group and VPA combined with MBSCs group, 1 mL BMSCS, it's concentration is 1×106 /mL, were injected into the spinal cord of injured Segment; while control group, SCI model group and VPA treated group received the same dose of normal saline at the same time point. VPA(300 mg/kg) was administrated in rats through hypodermic injection immediately after injury, then repeated per 12 h until killing; while control group, SCI model group and BMSCs transplantation group received the same dose of normal saline at the same time point. The recovery of the locomotor function of each group was evaluated with basso Basso-beattieBeattie-bresnahan Bresnahan (BBB) scale at 7 d, and 14 d after injury, then the rats were killed. The sections were stained with hematoxylin and eosin (HE) for Syringomyelia area.The expression of Caspase-3 protein was detected with immunohistochemistry. Then the neuronal apoptosis was observed by TUNEL staining. Results (1) By the flow cytometry instrument detection third-generation cultured BMSCs can be stable expressed CD34 and CD44. (2)The motor function of the control group was not affected. The BBB score of the three treatment groups was significantly higher than that of the SCI model group(all P values<0.05). The score of VPA combined with MBSCs group was significantly higher than that of VPA treated group and D on the 14th day after SCI(all P values<0.05). (3)HE staining results showed the area of the lesion area of the four groups were respectively (4.57±0.26), (3. 34±0.21), (3.51±0.18) and (2.43±0.35), respectively. VPA combined with MBSCs group was significantly lower than the other three group(all P values<0.05). (4)Immunohistochemical staining showed that a small amount of Caspase-3 expression was found in control group. The expression of Caspase-3 in three treatment groups was significantly lower than that in SCI model group(P< 0.05). While the expression of Caspase-3 in VPA combined with MBSCs group was significantly lower than that in VPA treated group and D(all P values<0.05). (5)A small number of apoptosis-positive cells were found in control group at 14 days after injury. Compared with SCI model group, the apoptosis index of the three treatment groups was lower(all P values<0.05). The apoptosis index of VPA combined with MBSCs group was significantly lower than that of VPA treated group and BMSCs transplantation group(all P values< 0.05). Conclusions Hypodermic injection of VPA for BMSCs transplantation promote SCI repair in rats is feasible, especially in the lower expression of Caspase-3, inhibition of neural cell apoptosis and promote motor nerve functional recovery effect is remarkable. Key words: Spinal cord injury; Valproic acid; Bone marrow mesenchymal stem cells; Injury repair; Experimental study