| Spinal cord injury (SCI) is a common injury during traffic, working and sports accident with high rate of disability and mortality. SCI contains primary injury and secondary injury. Primary injury is inconvertible. Pathological changes of secondary injury contain axonotmesis, myelin disintegration, neurons necrosis and apoptosis. The main clinical treatments includes reconstruct spinal stability, solve spinal compression symptom and control secondary injury. In recent years, domestic and foreign scholars research on the treatment of spinal cord injury in a wide range, elaborate a series of views on its own renewable capacity, the growth of the environment as well as the factors which promote regeneration. To sum up, including: Applicant drugs and neurotrophic factors to control secondary injury; (2) transplant neural stem cells, olfactory ensheathing cells, spinal stromal cells, embryonic tissue or gene cells of regulation functions, and bridge connection damage site; (3) study cell signal transduction mecha- nisms, stimulate or regulate signaling pathways of cell growth cone; (4) eliminate inhibition in spinal cord regeneration in a variety of factors, such as the degradation of glial mark, the elimination of inhibition of axon regeneration in a variety of factors; (5) demyelizate the axons re- myelination. And how to bridge connection spinal cord stump and to promote the reconstruction of nerve conduction pathway is an important research direction. Activated Macrophages Transplantation (AMT) is in rich sources, based on simple materials, easy isolation and culture. Local micro- environment can be improved to reduce secondary spinal cord injury by gathered a large number of AMT. We transferred AMT to rats'site of spinal cord injury to observe the function of recovery. In recent years, people's awareness of macrophages is macrophages as a transplantation material to promote regeneration of axons. Experimental studies have proved that the AMT have a certain role in the recovery of the spinal cord sensory and motor nerve damage. AMT is survived in the host spinal cord and promoted functional recovery of spinal cord, and its possible mechanisms: (1) protect and restore some of the organizations injury; (2) removal partial damage of the cell and tissue necrosis of the wreckage, in particular, inhibit axon regeneration material– sphingomyelin derived renewable inhibitory factor; (3) stimulate and secret nutrient requirements to cells regeneration, and regulate cells substrate and protease requirements to axon regeneration; (4) reduce stump after spinal cord injury in the formation of glial scar effectively.Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells in the adult animal or human bone marrow with the characteristics of multi-potent stem cells can differentiate into multiple types of cells. Experimental research has shown that, MSCs have differentiated into neural cells induced by the potential, and the same as neural stem cells to migration and integration in the central nervous system (brain and spinal cord), can secrete neurotrophic factors, cytokines and other biologically active factor, and demonstrate a new and ideal method for the treatment of spinal cord injury. MSCs transplanted into the rat spinal cord injury site, showing MSCs can survive, and well integrated into the host tissue, and also can promote the growth of axons in the absence of gene-modified. Compared to other transplanted cells, MSCs have a wide range of sources, based on simple materials, have always been to maintain its multi- directional differentiation potential in long-term culture in vitro process, have weak response in vivo transplantation, overcome ethics and the problem of immune rejection, etc., so explore the MSCs transplantation in the treatment of SCI have important clinical significance. Some studies had shown that lipid peroxidation mediated by oxygen free radical played a major role on the secondary spinal cord injury. The extent of lipid peroxidation also affects the fate of engrafted cells in great part. According to the theory of traditional Chinese medicine, the para- plegia caused by SCI belongs to the field of body laziness and paralysis. Its treatment is mainly depended on activating blood circulation, removing blood stasis, deoppilating du channel, dredging meridian, tonifying pneuma, invigorating kidney and replenishing essence. The kidney in charge of bones produces essence depending on the incessant supplying of spleen and stomach, which is the foundation of acquiring nutriments and the source of metaplasia between pneuma and blood. Renal injury can make essence lost. Blood and essence may transform each other. Nourishing piyin remedy (NPR) has the function of tonifying pneuma, activating blood, invigorating kidney, replenishing essence, engorging spleen and stomach and so on. It can protect neurocyte by enhancing antioxygenic activity of the mitoc- hondria, accommodating phospholipid metabolism, inhibiting responsive gliosis and inflammatory reaction. The present study investigated the protective role and mechanism of serum nourishing piyin remedy on MSC slipid peroxidation injury.Part 1 Experimental Study of Function Recovery of Rat Spinal Cord Injuries Using Activated Macrophages TransplantationObjective To investigate the promoting effects of activated macro- phages transplantation on function recovery of rat spinal cord injuries (SCI).Methods The spinal cord of adult rats was injured with modified Allen method, and the rats were randomly divided into 2 groups. In one group (A group), slight saline solution (20μl) was injected into the spinal cord injury site, while in the other group (B group) activated macrophages (20μl) were transplanted instead. The inclined plane test and Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) were used to observe the recovery of rats'behavioral function on 7, 14 and 28 days after the transplantation, while spinal evoked potential tests were used to observe the recovery of spinal cord nervousness function, hematoxylin- eosin (HE) staining to reflect the change of cavity volume, and immunohistochemical method to observe the survival of activated macro- phages transplantation and regeneration of damaged nerve fibers.Results On the 28th day after transplantation, inclined plane critical point was significantly different between group A and B, which were 44.96°±5.70°and 52.72°±6.51°respectively (p<0.05). On the 28th day after transplantation, BBB scale had significant difference between group A and group B, which were 6.8±1.2 and 10.2±4.1 (p<0.05). On the 28th day after transplantation, MEP and SEP latencies had significant differences. The MEP latencies of group A and group B were 4.69±0.47 (ms) and 3.62±1.29, respectively (p<0.05) and the SEP latencies of group A and group B were 4.19±1.97 and 2.51±0.76 respectively (p<0.05). On the 28th day after transplantation, nerves axons count (strip/mm2) was significantly different between group A and group B, which were 32.8±6.1 and 40.8±9.0 respe- ctively (p<0.05). In B group, macrophages survived in the injury area, cavity volume of the injured site was significantly reduced and there was significant regeneration of nerve fiber.Conclusions Activated macrophages can survive in the injury site to relieve spinal cord injuries, reduce cavity volume and promote axonal regeneration and improve functional recovery.Part 2 The effects of mesenchymal stem cells transplantation on the function recovery of rat with spinal cord injuriesObjective To investigate the promoting effects of mesenchymal stem cells (MSCs) transplantation on function recovery of rat spinal cord injuries (SCI).Methods The spinal cord of adult rats was injured with modified Allen method, and the rats were randomly divided into 2 groups:the control group(group A) on the 9th day after SCI, were injected into the spinal cord injury site with slight saline solution (5μl), while the experimental group (group B) were injected into the spinal cord injury site with mesenchymal stem cells (MSCs) (5μl). The inclined plane test and Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) were used to observe the recovery of rats'behavioral function on 7th, 14th and 28th days after the transplantation, spinal evoked potential tests were used to observe the recovery of spinal cord nervousness function, hematoxylin-eosin (HE) staining to reflect the change of cavity volume, and immunohistochemical method to observe the survival of mesenchymal stem cells ( MSCs ) transplantation and regeneration of damaged nerve fibers.Results On the 28th day after transplantation,inclined plane critical point was significantly different between group A and B, which were 44.96°±5.70°and 53.19°±6.51°respectively (p<0.05). On the 28th day after transplantation, BBB scale had significant difference between group A and group B, which were 6.8±1.2 and 10.1±3.5 (p<0.05). On the 28th day after transplantation, MEP and SEP latencies had significant differences. The MEP latencies of group A and group B were 4.69±0.47 (ms) and 3.97±0.83, respectively (p<0.05) and the SEP latencies of group A and group B were 4.19±1.97 and 2.60±0.92 respectively (p<0.05). On the 28th day after transplantation, nerves axons count (strip/mm2) was significantly different between group A and group B, which were 32.8±6.1 and 39.0±4.6. respectively (p<0.05). In B group, there was significant regeneration astrocytes and nerve fiber,cavity volume of the injured site was significantly reduced.Conclusions Mesenchymal stem cells(MSCs) can differentiate into neurons and astrocytes, reduce cavity volume and promote axonal regeneration and improve functional recovery.Part 3 Experimental Study of Nourishing Piyin Remedy (NPR) to Protect Mesenchymal Stem Cells (MSCs) Transplantation in the Treatment of Rats with Spinal Cord InjuryObjective To investigate the promoting effects of mesenchymal stem cells (MSCs) transplantationon function recovery of rat spinal cord injuries (SCI), and the protective effectiveness of serum nourishing piyin remedy on the survival, migration and differentiation of transplanted cells in vivo.Method Ninety adult female SD rats were randomly divided into fore groups as Group A (control group), Group B (MSCs transplantationon group), Group C (MSCs engrafted group combined with blank serum) and Group D (MSCs nourishing piyin remedy serum). Injury models were established by self-designed electronic impact device. The inclined plane test and Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) were used to observe the recovery of rats'behavioral function on 7, 14 and 28 days after the transplantation, while spinal evoked potential tests were used to observe the recovery of spinal cord nervousness function, hematoxylin-eosin (HE) staining to reflect the change of cavity volume, and immunohistochemical method to observe the survival and differentiation of MSCs and regeneration of damaged nerve fibers. Results On the 28th day after transplantation, inclined plane critical point was significantly different among group A, B, C and D, which were 44.96°±5.69°, 48.11°±5.47°, 49.59°±5.76°and 55.75°±4.14°respectively (p<0.05); BBB scale had significant difference among groups, which were 6.9±1.3, 8.5±1.1 10.7±0.9 and 12.3±10.6 respectively (p<0.05). At the same time, MEP latencies had significant differences among group A, B, C and D, which were 4.57±0.33 (ms), 4.49±0.47 (ms), 4.39±0.65 (ms) and 3.77±0.76 (ms) respectively (p<0.05), SEP latencies had significant differences among group A, B, C and D, which were 4.19±0.93 (ms), 3.71±1.15 (ms), 3.63±0.77 (ms) and 2.52±0.57 (ms) respectively (p<0.05). Nerves axons count (strip/mm2) was significantly different among group A, B, C and D, which were 32.8±6.21, 34.5±5.5, 36.2±5.4 and 40.9±3.1 respectively (p<0.05). On the 28th day after transplantation, most cavity volume of the injured site was filled; tastrocytes and nerve fiber here were regenerated in group D.Conclusion The MSCs can survive, migrate and differentiate into mature phenotypes after transplantation, improve repair and regeneration of rat nerve fibers after SCI. The serum NPR can benefit the survival, migration and differentiation of transplanted MSCs by ameliorating the microenvironment of injured spinal cord. Transplantation with MSCs could facilitate recovery and reorganization of nerve fiber in moderate SCI models, and reveal synergistic effect when used combined with serum NPR.To sum up, through nourishing piyin remedy (NPR) to protect mesenchymal stem cells (MSCs) transplantation in the treatment of rats with spinal cord injury researched by this experiment, obtain the following results:1. Activated Macrophages Transplantation (AMT) is in rich sources, based on simple materials, easy isolation and culture. Local micro-environment can be improved to reduce secondary spinal cord injury by gathered a large number of AMT.2. The MSCs could retain survival, migrate and differentiate into mature phenotypes-neuron, astrocyte and oligodentrocyte, after trans- plantation into the moderate contused spinal cord, thus, facilitate the recovery and regeneration of nerve fiber.3. The serum nourishing piyin remedy could improve the SCI microenvironment, decrease the lipid peroxidation injury, benefit the survival, migration and differentiation of transplanted MSCs, and demo- nstrate synergistic effect when used in combination.4. How to enhance the antioxygenic activity of nourishing piyin remedy, still need to be further investigated. There are many prospective aspects for the transplantation of MSCs combined with tissue engineering scaffold and gene transfection technique.
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