| BackgroundSpinal cord injury (SCI) is a central nervous system trauma that causes severe damage for which there is currently no effective treatment. SCI causes partly permanent disability; thus, preventing SCI-induced neuronal apoptosis, replacing dead neurons, inhibiting scar formation, and creating a microenvironment conducive to neural regeneration are critical and challenging aspects in the treatment of SCI. The efficacy of electroacupuncture (EA) along the Du Meridian in the treatment of SCI has been well established in the medical community. This study aims to research the effect of EA on tissue repair in rats with acute SCI. Adult male Sprague-Dawley (SD) rats were used to construct the animal model of SCI. The experimental groups were assigned as follows: Group A was the control group, Group B was the sham group, Group C was the SCI group, Group D was the sham+EA group, and Group E was the SCI+EA group. The repair effect of EA on rats with SCI was determined at 1,3,7,14,21,28, and 56 d after establishing the model by Basso, Beattie, and Bresnahan (BBB) behavioral scoring, hematoxylin and eosin (HE) staining, immunohistochemistry, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). In addition, molecules in the notch signaling pathway were analyzed by real time PCR (qRT-PCR) and western blot (WB) to investigate the effect of notch signaling pathway in the repair process. The results show that EA treatment of SCI is mediated by inhibiting the notch signaling pathway, thus stimulating the proliferation and differentiation of endogenous neural stem cells, promoting tissue repair after SCI.Chapter 1. Establishment and electroacupuntucre therapy of acute thoracic spinal cord injury model with clip compression in Sprague-Dawley ratsObjectiveClip compression for spinal cord injury model has been widely used in the research of experimental spinal cord injury rat. Clamping force can influence on the back and ventral sides of spinal cord, and it has the same mechanism with human spinal cord injury, it is closed to human injury type. Electroacupuncture have merits of Chinese acupuncture point stimulation and physical stimulation, electroacupuncture can repair the spinal cord injury. In this experiment, we evaluated the spinal cord injury and electroacupuncture models using many methods, including survival curve, body weight, BBB scores, HE staining and pathological morphology at 1,3,7,14,21,28,56 d.Methods1. In this experiment,210 SD rats were randomly divided into five groups:control group, sham group, SCI group, sham+EA group, SCI+EA group, and every group have 42 rats. SCI group have spinal cord injury by aneurysm clip; control group without any treatment; sham group exposed spinal cord but not injury; SCI+EA group with spinal cord injury and the EA treatment in next day; sham+EA group exposed spinal cord and treatment with EA in next day.2. Observation of rat survival in every group and construction of survival curve is intuitive way to evaluate spinal cord injury. Record the death situation of rats and draw the survival curve with the time as x axis and the survival ratio as y axis.3. Weighing weight of rats is also a very direct method to evaluate spinal cord injury. Draw the weight change curve with the time as x axis and the weight as y as axis.4. BBB score has a very good correlation of ethology after spinal cord injury. Assessing the recovery of neural function of rats through BBB score in every group at 1, 3,7,14,21,28 and 56d; Score range from 0 to 21, the normal rat has the highest BBB score 21.5. The HE staining of spinal cord in every group at at 1,3,7,14,21,28 and 56d, and observation of white matter and gray matter of the spinal cord injury, and the necrosis and bleeding holes.6. All data were statistical anylsis with one-way ANOVA statistic by SPSS13.0 and Graphpad prism 5.0 software, α=0.05 as test standard.Results1. The death rate of the SCI model group was significantly higher than that of the SCI+ EA group, and this difference was statistically significant (P<0.05), demonstrating that EA can repair tissue after SCI. The control group, sham group, and sham+EA group rats were not significantly different in weight, whereas the weight of the SCI group and SCI+EA group rats had decreased 1 d after surgery; however, the SCI group decreased more than the SCI+EA group. After 21 d, the body weight stopped decreasing and began increasing. The body weights of the SCI group decreased from 1 to 21 d, became constant after 21 d, and started increasing after 56 d.2. The BBB scoring after establishment of the SCI, control, sham, and sham+EA groups all scored 21 points at different time points. The SCI and SCI+EA groups scored 0 points at 1 d post-surgery, indicating successful establishment of the model, although the SCI group resulted in paralysis of the hind limbs of the rats. Over time, the SCI group and SCI+EA group rats gradually increased in BBB scores, and the BBB scores of the SCI+EA group after 1,3,7,14,21,28, and 56 d increased significantly compared to that of the SCI group, and the difference was statistically significant (P< 0.05). However, these scores were significantly lower compared to that of the normal control, sham, and sham+EA groups.3. In the SCI group after 1 d, there were structural disruptions in the central canal, edema in the white matter, and traces of hemorrhage. In the SCI group after 3 d, there was edema in the gray matter and white matter at the central damaged area. There was structural disruption of the gray and white matter with substantial hemorrhage of the white matter at the affected region. The structure of the spinal cord neuron was disrupted, and empty vacuoles were visible between the gray and white matter at the damaged region; in addition, the neurons were swollen, pyknosis of part of the neuronal nuclei was observed, and the boundary for the gray matter in the spinal cord was blurred. After 7 d, the structure of the gray and white matter was disorganized with unclear boundaries, the grey matter lamina was lost, and empty vacuoles remained visible between the grey and white matter at the damaged region. After 14 d, the gray and white matter boundary was still blurred with no hemorrhage, and there were visible empty vacuoles between the gray and white matter. After 21 d, a subtle gray and white matter boundary was visible, and neuronal edema was alleviated; however, the empty vacuoles remained visible at the site of SCI. After 28 d, abnormalities of the white matters were gradually restored, and a clear gray and white matter boundary appeared with empty vacuoles present. After 56 d, swelling and small vacuoles were slightly visible with a clear gray and white matter boundary.ConclusionIn the present study, we established rat model of actue clip-type spinal cord injury via medical cerebral aneurysm clip, examined the effects of electroacupuncture (EA) through acupoints (GV14 and GV4) on actue clip-type spinal cord injury by the detection method of ethology (BBB scores) and pathology (HE staining). The results suggested that SCI+EA treated group displays faster and better hindlimb motor function compared with SCI group, and the difference is more significant with the extension time using EA treatment. While the hindlimb motor function is possible to recover to a certain extent of SCI group in rats, and the recovery of SCI+EA treated group was significantly better than SCI group, indicating that the EA treatment can promote the recovery of spinal cord injury. On biopsy, HE staining also showed that the spinal cord injury models is successfully constructed and demonstrated that the EA treatment can alleviate and repair the spinal cord injury in the paraffin sections of spinal cord. In our experimental setup, there are five groups, including normal group, sham-operation group, SCI group, sham-operation+EA group and SCI+EA group. Seven time points (1d,3d,7d,14d,21d,28d and 56d) were set up in each group, which provide the powerful experimental basis for the subdivision of the repair and treatment of spinal cord injury. The establishment of rat model of actue clip-type spinal cord injury could reflect the pathological changes of spinal cord injury, had the advantage of operability, easy to repeat, simple and stable, and provided a solid foundation for the pathophysiology of spinal cord injury in the future study.Chapter 2. Effect of electroacupuncture on inflammatory factor in adult rats with spinal cord injuryObjectiveSpinal cord injury can induce some secondary injury, break of blood-spinal cord barrier after spinal cord injury can increase many inflammatory factors concentration, and inhibit the repair of spinal cord injury.This experiment will research the effect of electroacupuncture on inflammatory factor in adult rats with spinal cord injury, and investigated the mechanism of repair spinal cord injury.Methods1. The rats were randomly divided into 5 groups:control group, sham group, SCI group, sham+EA group, SCI+EA group. We collected blood samples by femoral vein in every group at 1,3,7,14,21,28,56d.2. Collecting surm after centrifugation, test the concentration of TNF-a, IL-1a, IL-6 and IL-10 using ELSIA.3. All data were statistical anylsis with one-way ANOVA statistic by SPSS 13.0 and Graphpad prism 5.0 software, α=0.05 as test standard.Results1. Compared between different groups in same point, the inflammatory factor of control group, sham group and sham+SCI group have not change; inflammatory factors have the highest concentration in SCI group than other group; moreover, the inflammatory factors have the lower concentration in SCI+EA group than the SCI group, but still higher than control group.2. In SCI group, the inflammatory factors have the highest expression at 3d and have a decline trend after 7d; the SCI+EA group also have the same trend as the SCI group.Conclusion1. The spinal cord injury can induce inflammatory reactions.2. Electroacupuncture can inhibit the inflammatory reaction induced by spinal cord injury.Chapter 3. Effect of electroacupuncture on endogenous neural stem cell in adult rats with spinal cord injuryObjectiveAfter spinal cord injury, the endogenous neural stem cells (ENSCs) can differentiated into astrocyte and oligodendrocyte which formed keloids and suppressed the repair of spinal cord injury; but the proliferation of endogenous neural stem cells can promote the repair of spinal cord injury. Nestin, GFAP, GALC proteins are respectively the marker of neural stem cell, astrocyte and oligodendrocyte. In this chapter, we will research the effect of electroacupuncture on the expression of Nestin, GFAP and GALC, further investigate the-effect of electroacupuncture on the proliferation and differentiation of eNSCs.Methods1. The rats were randomly divided into 5 groups:control group, sham group, SCI group, sham+EA group, SCI+EA group. We collected the spinal cord samples in every group at 1,3,7,14,21,28,56d.2. Testing the expression of Nestin, GFAP and GALC by the real time PCR assay in mRNA level.3. Testing the expression of Nestin, GFAP and GALC by the western blot assay in protein level.4. Testing the expression of Nestin, GFAP and GALC by immunohistochemistry and immunofluorescence assay.5. All data were statistical anylsis with one-way ANOVA statistic by SPSS 13.0 and Graphpad prism 5.0 software, α=0.05 as test standard.Results1. The results of QPCR showed that compared between different groups in same point, the expression of Nestin, GFAP, GALC in control group, sham group and sham+SCI group have not change; but in SCI group, the expression of GFAP and GALC were increased with time and decreased after 14d, the expression of Nestin was decreased with time and stabilized after 14d; The GFAP, GALC and Nestin in SCI+EA group have the same trend with SCI group, but compared with SCI group, the EA treatment can inhibit the expression of GFAP and GALC, and promote the expression of Nestin.2. The results of western blot showed the similar change with QPCR assay.3. The results of IHC showed that the number of GFAP, GALC positive cells in SCI group and SCI+EA group were more higher than control group, sham group and sham+EA group, and were highest at 14d; Nestin was the exact opposite. However, compared with the SCI group, after the EA treatment, the number of GFAP, GALC positive cells were obviously decreased and the number of Nestin positive cell were obviously increased in SCI+EA group.4. The results of IF for GFAP and Nestin have the similar trend with IHC results.Conclusion1. The spinal cord injury can suppress the proliferation of ENSCs, and promote the ENSCs differentiated into the astrocyte and oligodendrocyte.2. Electroacupuncture can promote the proliferation of ENSCs, and inhibit the ENSCs differentiated into the astrocyte and oligodendrocyte, thereby inhibiting the formation of glial scar to repair the spinal cord injury.Chapter 4. Effect of electroacupuncture on Notch signaling pathway in rats with spinal cord injuryObjectiveEndogenous neural stem cells (ENSCs) have an important role in repair of spinal cord injury, and it was regulated by many signaling pathways. Notch signal pathway is one of the major signal pathways for regulating ENSCs, what changes of Notch signaling pathway after spinal cord injury? What role the electroacupuncture in Notch signaling pathway? Above questions will be researched in this chapter. In this chapter, we investigated the effect of electroacupuncture on Notch signaling pathway at aspect of Notch signal pathway relative proteins (Notch 1, Notch3, Notch4, ps1 and Hes1).Methods1. The rats were randomly divided into 5 groups:control group, sham group, SCI group, sham+EA group, SCI+EA group. We collected the spinal cord samples in every group at 1,3,7,14,21,28,56d.2. Testing the expression of Notch1, Notch3, Notch4, ps1 and Hes1 by the real time PCR assay in mRNA level.3. Testing the expression of Notch1, Notch3, Notch4, ps1 and Hes1 by the western blot assay in protein level.4. All data were statistical anylsis with one-way ANOVA statistic by SPSS 13.0 and Graphpad prism 5.0 software, α=0.05 as test standard.Results1. The results of QPCR showed that compared between different groups in same point, the expression of Notch1, Notch3, Notch4, ps1 and Hes1 in control groupp,sham group and sham+SCI group have not change; but in SCI group, the expression of Notchl, Notch3, Notch4, psl and Hes1 were increased with time and highest at 14d, were decreased after 14d; The Notch1, Notch3, Notch4, psl and Hes1 in SCI+EA group have the same trend with SCI group, but compared with SCI group, the EA treatment decreased the expression of Notch1, Notch3, Notch4, ps1 and Hes1.2. The results of western blot showed the similar change with QPCR assay.Conclusion1. The spinal cord injury activiated the relative proteins of Notch signaling pathway.2. Electroacupuncture can inhibit the Notch signaling pathway induced by spinal cord injury.3. Electroacupuncture might promote spinal cord injury repair through inhibiting the activation of Notch signaling pathway. |
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