The Effect Of Buyang-huanwu Decoction Combined With Neural Stem Cells On The Repair Of Injured Spinal Cord In Rats

Objective:To investigate the effect of Buyang-Huanwu Decoction(BYHWD) on survival, proliferation, migration and differentiation of neural stem cells transplanted into rats after SCI. In addition, we investigated whether there was a synergy between BYHWD and NSCs to recovery of neurological function and remyelination.Methods:Embryonic neural tube-derived neural stem cells were cultured according to the methods established by our laboratory. NSCs suspensions of second generation were collected, then neurospheres were dispersed into single cell suspension and transferred to a culture flask. DMEM/F12complete culture solution was added into the culture flask with BrdU at the final concentration of10μmmol/L. Cell suspensions were dispersed into single cell suspension48hours after BrdU labeling. Single cell suspensions were adjusted to a final density of1×106cells/ml. Female Sprague-Dawley (SD) rats, weighing220-250g, were randomly divided into4groups:1.the spinal cord injury(SCI) group;2.the Buyang Huanwu Decoction treatment (BYHWD) group;3.the NSCs transplantation (NSCs) group; and4. the NSCs transplantation combined with Buyang Huanwu Decoction (BYHWD+NSCs) group. Each group had three time points:7days,14days and28days. Rats used in the following experiments received the guiding principles of care and consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Anesthesia was acheived by4%chloral hydrate (0.1ml/kg). Laminectomy was used in the rats in the sham-control group. The spinal cords of rats in the remaining four groups were transected completely at thoracic cord10. During the surgery, gelatin sponges containing NSCs labeled with BrdU were transplanted into the lesion of rats in the NSCs group and the BYHWD+NSCs group. According to the conversion factor of the dosage between humans and rats, rat dose (mg/kg)=W×human dose (mg/kg)(W:the conversion factor6.25), the dose in rats is about14.8g/(kg day). So the rats in the BYHWD group and the BYHWD+NSCs group were given Buyang Huanwu Decoction at this dose by introgastric infusion, while same volume of saline was given to the rats in the NSCs group. Postoperative care was performed according to the NIH guide.Results:1. The cells derived from the posterior segment of neural tube of E11.5d rat were cultured with good proliferation to get a large number of cells, expressing the primitive neurofilament nestin, which was deemed as specific antigen and the mark of NSCs. After labeled by BrdU, nuclei in neurospheres appeared bright green under confocal laser scanning microscopy.2. Difference between the BYHWD+NSCs group and other groups respective were obviously observed by methods of BBB score of hind limb motor function over time (P<0.05). The recovery of animal limb function was significant.3. Transmission Electron Microscope (TEM) showed:In the BYHWD+NSCs group, the extent of myelin loose reduced distinctly, regenerated fiber increased, the structure of myelin was clear and neat and the arrangement orderly, the swelling of mitochondria became lightened, vacuolar degeneration of microtubules got less, a lot of synapses were found, thin myelin sheath regeneration increased significantly.4. After transplantation, the grafted cells, NSCs, could easily be identified mainly in the lesion site in the NSCs group and the BYHWD+NSCs group and integrated well with the host tissue. There was no BrdU-positive cell in Sham-operated group, SCI group and the BYHWD group. The number of BrdU-positive cells at each time point in the BYHWD+NSCs group was greater than that in the NSCs group (p<0.05). The number of BrdU-positive cells at28d was the most among three time points of the BYHWD group (p<0.05).5. A double-label immunofluorescence study showed that the number of glial fibrillary acidic protein (GFAP) and BrdU double positive cells in the BYHWD+NSCs group was significantly less than that in NSCs group (P<0.05). There were myelin basic protein (MBP) and BrdU double positive cells and neuron specific enolase (NSE) and BrdU double positive cells in the BYHWD combined with NSCs group (BYHWD+NSCs), whereas corresponding double positive cells were rare in the NSCs group. Conclusion:The transplanted NSCs can survive, proliferate and migrate after SCI. BYHWD could improve the survival and proliferation of transplantated NSCs after SCI. The immigrating distance of transplanted NSCs toward head and caudal tissue in the BYHWD+NSCs group was longer than that in NSCs group. BYHWD may enhance the differentiation of NSCs to neurons and oligodendrocytes reduce the differentiation to astrocytes in injured spinal cord. There was a synergy to recovery of neurological function and remyelination between BYHWD and NSCs.