| 1 ObjectiveSpinal cord injury(SCI) is one of the higher disability disease, it not only bring suffering to patients in the physiological and psychological, but also bring heavy economic burden to patients and their families. At present, there are no effective treatment for SCI. So, it is still the extremely difficult problem and hot research topic which should be strong demand to capture by the medical profession in the world. The pathological mechanism of SCI is very complicated. Neurotrophic factor (NTFs) and its receptor changes in central nervous system after injury is an important pathological reaction, and NTFs deficiency affects nerve repair in late SCI. Electro acupuncture as a traditional Chinese medicine treatment, has attracted more and more researchers attention. In this study, neurotrophic factors 3(NT-3) which is closely related to the repair of spinal cord injured nerve as a starting point, Du meridian electroacupuncture therapy as an intervention for SCI, using behavioral science, morphology and molecular biology methods, to investigate the effect of Du meridian electroacupuncture to related protein of different NT-3 receptor mediated signal pathway in rats’spinal cord tissue with SCI, to evaluate the effect of Du meridian electroacupuncture treatment on SCI, which provides experimental data support for the clinical treatment of Du meridian electroacupuncture for SCI and other nervous system injury diseases.2 MethodsThis research was carried out by the form of animal experiments, SD rats of clean grade were selected as the research object. They were randomly divided into five group:blank group(B group), control group(C group), sham-operating group(S group), model group(M group) and Du meridian electroacupuncture group(EA group). Modified Allen’s methods was used to duplicate SCI model. The rats were sacrificed in day1, day3, day7 post-SCI, after receiving Du meridian electroacupuncture treatment. BBB test was used to detect motor function of rats’ hind limbs among groups, in order to evaluate the effect of Du meridian electroacupuncture treatment for the recovery of motor function after SCI; HE staining and Nissl staining were used to observe the pathological changes of injured spinal cord tissue and the condition of neurons, in order to evaluate the effect of Du meridian electroacupuncture treatment for the histomorphology after SCI; TUNEL was used to detect the situation of cell apoptosis; immunohistochemical method and western blot were used to assay the expression of related protein in the signal pathway mediated by NT-3 different receptors in the injured spinal tissue, to discuss the effect of Du meridian electroacupuncture treatment for SCI from macro to micro different levels.3 Results3.1 BBB results:(1) at dayl post-SCI:compared with B group, C group and S group, the BBB score of M group significantly decreased(P<0.01), while there was no statistical differences between EA group and M group(P>0.05); (2) at day3 post-SCI:compared with B group, C group and S group, the BBB score of M group significantly decreased(.P<0.01), compared with M group, the BBB score of EA group increased, but without statistical differences(P>0.05); (3) at day7 post-SCI:compared with B group, C group and S group, the BBB score of M group significantly decreased(P<0.01), compared with M group, the BBB score of EA group increased with statistical differences(P<0.01).3.2 HE staining results:(1) at day 1 post-SCI:Group M showed that spinal cord tissue structure was destroyed, gray, white matter boundary was not clear, gray matter within the patchy hemorrhage, a large number of red blood cell aggregation, motor neurons in gray matter regions of the cell swelling, interstitial edema, nuclear pyknosis, white matter nerve fiber swelling thickening, arranged in disorder, the pathological changes of EA group was similar to the M group;(2) at day 3 post-SCI:The M group showed that white matter regions continued to appear the situation of swelling and thickening of the nerve fibers, the organizational structure appear disordered and sparse, even a cavity formed, and infiltration of microglia, motor neuron in gray matter of larger scope swelling, and even the emergence of lysis, necrosis, leukocytes infiltration, the central tube structure disordered, EA group showed that bleeding relatively reduced, the other pathological changes began to appear improvement; (3) at day 7 post-SCI:The M group showed that less bleeding, white matter and gray matter boundary was still obscure, the amount of gray matter neurons was rare, a large number of small glial cell proliferation, white matter nerve fiber structure obviously swelling, bleeding of EA group decreased, neurons began to appear clear edge, microglial cell hyperplasia also appeared, but relatively less than group M.3.3 Nissl staining results:(1) at dayl post-SCI:The number of neurons in M group showed reduced, neuronal cells remained appeared different degrees of swelling, Nissl body staining cells reduced, or even disappeared, staining fade, the site in the central nucleus was most obvious, the improvement of EA group tissue morphology changes was not obvious, neurons also had different degrees of reduction, Nissl staining was light; (2) at day 3 post-SCI:M group showed that motor neuron in gray matter region appeared obvious atrophy, part of neurons dissolved, the quantity of Nissl body significantly reduced, or even disappeared, group EA showed that the phenomenon of neuronal atrophy and reduced still existed, but the quantity of neurons reduced more less than that of group M, and the Nissl body were faintly visible; (3) at day 7 post-SCI:M group showed that a small amount of Nissl body appeared again, EA group showed that the Nissl body of surviving neurons appeared again, and significantly more than that of M group.3.4 TUNEL results:(1) at day1 post-SCI:The spinal cord sections of M group and EA group showed more positive particles, which mainly distributed in the injured region and edge, there was significant difference between the two groups; (2) at day 3 post-SCI:Group M showed that positive cells increased obviously, and the distribution scope enlarged, the emergence of positive cells appeared in the gray matter and white matter, the size of apoptosis cell were irregular, EA group decreased more than that of M group, there was statistical differences between two groups; (3) at day 7 post-SCI:Apoptotic cells in group M reduced than before, the number of positive cells in EA group decreased more significantly than that in M group, there was significant difference between groups.3.5 Effects of Du meridian electroacupuncture on ERK signal pathway mediated by NT-3 specific receptor TrkC:(1) Immunohistochemical method(IHC):in day1, day3, day7 post-SCI, compared with blank group, control group and sham-operating group, the expression of NT-3, TrkC, MEK2 and p-ERK1 in injured spinal cord were decreased in model group, while Du meridian electroacupuncture could up-regulate their expression;(2) Western Blot results were in accordance with that of IHC.3.6 Effects of Du meridian electroacupuncture on JNK signal pathway mediated by NT-3 receptor p75NIR:(1) Immunohistochemical method(IHC):in day1, day3, day7 post-SCI, compared with blank group, control group and sham-operating group, the expression of p75NTR, p-JNK and p-c-Jun in injured spinal cord were increased in model group, while Du meridian electroacupuncture could down-regulate their expression;(2) Western Blot results were in accordance with that of IHC.4 Conclusions4.1 SCI model duplicated by Allen’s weight drop method could simulate the pathological features and clinical manifestation of SCI well. Du meridian electroacupuncture treatment could improve motor dysfunction of SCI rats’hind limbs, and promote the recovery of neurological function.4.2 Du meridian electroacupuncture treatment could improve the damaged neurons’situation of SCI rats, promote survival of neuron, and has protective effect to neurons.4.3 Du meridian electroacupuncture treatment could reduce the occurring of cell apoptosis of SCI rats, promote survival of neuron, and has certain protective effects to the repairment of injured nerve.4.4 Du meridian electroacupuncture treatment could promote endogenous NT-3 and its specific receptor TrkC synthesis and secretion, and activate of ERK signal transduction pathway, increase the expression of MEK2 and p-ERKl, then to protect neurons, promote the survival of neurons, inhibit cell apoptosis.4.5 Du meridian electroacupuncture therapy can reduce the activation of p75NTR receptors, inhibit the activation of JNK pathway, reduced JNK, c-Jun phosphorylation and expression, to achieve the purpose of inhibiting cell apoptosis.
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