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神经元细胞骨架蛋白在推拿治疗坐骨神经损伤中的作用及机理探讨
【作者】 高玉峰；
【导师】 于天源；
【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2014， 博士
【摘要】 [目的]研究推拿干预对坐骨神经夹持损伤大鼠行为学、形态学、微管相关蛋白2(MAP-2)、神经丝蛋白M(NF-M)、髓鞘相关糖蛋白(MAG)、Rho激酶Ⅱ(ROCKII)的影响,从神经元细胞骨架蛋白角度,揭示推拿在周围神经损伤修复过程中的作用机制。[方法]本实验以坐骨神经损伤为例研究周围神经损伤,以SD大鼠为实验动物,将大鼠随机分为正常组、假手术组、模型组、模型对照组和推拿组。模型组、模型对照组和推拿组大鼠制备坐骨神经夹持损伤模型,模型对照组大鼠给予布袋束缚,推拿组在造模后7天开始选取患侧“殷门”、“承山”、“阳陵泉”穴,采用推拿手法模拟仪模拟点法、拨法、揉法进行干预,每日1次,每10次休息1天,共治疗20次。行为学主要采用斜板实验、光热耐痛阈实验的检测,考察坐骨神经损伤大鼠经推拿手法干预后运动和感觉功能恢复情况；形态学主要采用大鼠脊髓、坐骨神经HE染色,考察推拿手法干预后神经再生修复程度；通过大鼠腓肠肌湿重的测量和腓肠肌恢复率的计算,评价推拿干预后靶组织恢复情况；通过检测坐骨神经和脊髓中MAP-2、NF-M、MAG、ROCKII的变化,探究推拿对周围神经损伤再生修复作用机制。[结果]行为学结果显示,①斜板实验：造模7天后模型组斜板实验评分低于正常组和假手术组有显著性差异(P<0.05),说明坐骨神经损伤后,大鼠失神经支配区肌力降低,模型成功。推拿手法干预10次后,推拿组斜板试验评分高于模型组、模型对照组,有显著性差异(P<0.05),推拿手法干预20次后,推拿组斜板试验评分接近正常组水平,与正常组相比无显著性差异(P>0.05),与模型组、模型对照组比较有显著性差异(P<0.05),说明推拿能改善神经损伤后大鼠下肢肌力的恢复。②光热耐痛阈实验：造模7天后模型组光热耐痛阈值高于正常组和假手术组有显著性差异(P<0.05),说明坐骨神经损伤后,神经纤维的连续性受到破坏,靶组织的痛觉传导出现障碍。推拿手法干预10次、20次后,推拿组光热耐痛阈值低于模型组、模型对照组有显著性差异(P<0.05),说明推拿可促进神经损伤后感觉功能的恢复。形态学观察,①坐骨神经HE染色：正常组雪旺细胞排列整齐,结构完整；假手术组与正常组相似；模型组神经纤维散乱,轴索崩解,髓鞘与轴索形成卵圆体,雪旺细胞增殖；模型对照组与模型组相似；推拿组神经纤维排列较整齐,轴索清晰,雪旺细胞基本正常。②脊髓HE染色：正常组脊髓腹角神经元细胞核居中,核仁清晰,神经细胞排列整齐,尼氏体分布均匀；假手术组与正常组相似；模型组细胞出现肿胀、变性,神经元数量减少,排列不规则,尼氏体溶解消失；模型对照组与模型组具有相似的损伤表现；推拿组大部分神经元呈现正常形态,排列较规则,神经元变性、损伤程度较轻。③腓肠肌湿重结果：造模7天后,模型组大鼠患侧腓肠肌湿重和恢复率低于正常组和假手术组(P<0.05),说明坐骨神经损伤后神经纤维与腓肠肌的联系遭到破坏,腓肠肌发生退变、萎缩。推拿干预10次后,推拿组大鼠患侧腓肠肌湿重和恢复率高于模型组、模型对照组有显著性差异(P<0.05),与正常组相比有显著性差异(P<0.05)。推拿干预20次后,推拿组大鼠患侧腓肠肌湿重和恢复率仍高于模型组、模型对照组有显著性差异(P<0.05),说明推拿促进损伤神经再生,在一定程度上阻止或逆转腓肠肌萎缩。机理研究,①MAP-2免疫组化结果：造模7天后,模型组大鼠坐骨神经、脊髓中MAP-2积分光密度高于正常组和假手术组(P<0.05),说明大鼠坐骨神经损伤后机体自然修复过程中MAP-2可应激性的表达。正常组与假手术组之间无明显差异(P>0.05),说明坐骨神经未损伤时,其神经元结构和功能未受到破坏,不会引起MAP-2的变化。推拿干预10次及20次后,推拿组坐骨神经、脊髓中MAP-2积分光密度明显高于模型组和模型对照组(P<0.05),表明推拿可促进促MAP-2在坐骨神经、脊髓中表达；模型对照组与模型组之间无明显差异(P>0.05),说明布袋束缚不会引起机体MAP-2表达的变化。ONF-M免疫组化结果：造模7天后,模型组大鼠坐骨神经、脊髓中NF-M积分光密度高于正常组和假手术组(P<0.05),正常组与假手术组之间无差异(P>0.05),推拿干预10次及20次后,推拿组坐骨神经、脊髓中NF-M积分光密度明显高于模型组和模型对照组(P<0.05),表明推拿可促进促NF-M在坐骨神经、脊髓中表达；模型组与模型对照组之间无明显差异(P>0.05)。③MAG免疫组化结果：造模7天后,模型组脊髓中MAG积分光密度高于正常组和假手术组(P<0.05),正常组与假手术组之间无差异(P>0.05)。推拿干预10次及20次后,推拿组脊髓中MAG积分光密度明显低于模型组和模型对照组(P<0.05),表明推拿可抑制MAG在脊髓中表达,模型组与模型对照组之间无差异(P>0.05)。④ROCKII免疫组化结果：造模7天后,模型组脊髓中ROCKII积分光密度高于正常组和假手术组(P<0.05),正常组与假手术组之间无差异(P>0.05)。推拿干预10次及20次后,推拿组脊髓中ROCKII积分光密度明显低于模型组和模型对照组(P<0.05),表明推拿可抑制ROCKII在脊髓中表达；模型对照组与模型组之间无明显差异(P>0.05),说明布袋束缚不会对坐骨神经损伤大鼠ROCKII的表达产生影响。[结论]1推拿可促进坐骨神经损伤大鼠神经损伤后的修复,重新与靶组织建立联系,提高腓肠肌肌力和恢复率,并且提升痛觉敏感度,从而促进神经损伤大鼠感觉和运动功能的恢复。2推拿能够提高坐骨神经损伤大鼠脊髓和坐骨神经中神经元细胞骨架蛋白MAP-2、NF-M的表达,维持神经元细胞骨架完整性,促进受损神经元内部结构和功能的恢复,从而保护神经元,起到促进轴突再生的作用。3推拿能够降低坐骨神经损伤大鼠脊髓中轴突生长抑制因子MAG、ROCKII的表达,抑制微管与神经丝解聚,维护神经元细胞骨架结构稳定,促进神经元的存活,从而起到促进神经再生修复的作用。4推拿治疗周围神经损伤机理之一是通过调控轴突生长抑制因子,促进神经元细胞骨架蛋白的合成,从而起到保护神经元,促进受损神经再生修复。
【Abstract】 [Objects]This thesis studies the influence of massage intervention on the sciatic nerve clamping damage behavior of rats, morphology, MAP-2, NF-M, MAG and ROCKII, and further reveals the mechanism of massage action during the repair of peripheral nerve injury.[Methods]In this experimental studies, the SD rats were randomly divided into normal group, sham operation group, model group, model control group and massage group, and further investigated the peripheral nerve injury around nervi ischiadicus. The sciatic nerve clipping injury model were prepared for the rats of model group, model control group and the massage group. The cloth binding was used for the model control group, and acupuncture points of ’yinmen’,’chengshan’ and ’yanglingquan’ were selected to intervene the massage group through the pointing, shifting and kneading methods,1times a day with each of the10times and1days of rest (a total of20therapy). In the behavior analysis, we investigated the recovery of the kinematic and sensory functions of the rats through oblique board test and thermal pain tolerance test,while in the morphology analysis, the degree of nerve regeneration, repair and recovery after massage intervention were investigated based on the HE coloration of the rat’s spinal cord and nervus ischiadicus, the recover degree of target tissue were investigated based on the gastrocnemius wet weight and the recovery rate of the gastrocnemius wet weight. Finally, we explored the regeneration mechanism of massage on peripheral nerve injury through detections of changes of MAP-2, NF-M. MAG. ROCKII in the sciatic nerve and medulla spinalis.[Results]The behavioral results show that,①oblique board test:after modeling of7days, oblique board test score of the model group is lower than that of the normal group and sham operation group with significant differences (P<0.05), illustrating that muscle of the innervation area was decreased, and the model was succeed. Oblique board test score of the massage group is higher than that of the model group and model control group with significant differences (P<0.05) after10times of massage intervention. After20times of massage intervention, oblique board test score approaches the level of normal group with insignificant differences (P>0.05), but shows a significant differences (P<0.05) in the comparison of model group and model control group, illustrating that massage has the ability to recover the muscle strength of lower limbs for the rats with neurologic damage.②Thermal pain tolerance test:thermal pain tolerance threshold of the model group is higher than that of normal group and sham operation group (P<0.05) after7days of modeling, illustrating that continuity of nerve fiber is damaged after sciatic nerve injury, and then obstructing pain transmission of the target tissue. However, thermal pain tolerance threshold of the massage group becomes lower than that of model group and model control group with significant differences (P<0.05) after10,20times of massage intervention, illustrating that massage can enhances the recovery of sensory function from neurologic damage.Morphological observation,①Sciatic nerve HE staining:the schwann cells of the normal group are regulated in good orders and integrated. The sham operation group is similar to the normal group. The Nerve fiber distribution of the model group is scattered, the axon becomes disintegrated, the myelin sheath and the axon cohere to form the egg-round body, and the schwann cells increase. The model control group is similar to the model group. The Nerve fiber of the massage group are regulated in good orders, the axon is clear and the schwann cells are normal.②Medulla spinalis HE staining:the spinal cord ventral horn neurons in nucleus are centrally located with clear nucleolus, nerve cells are arranged regularly, and nissl body is distributed uniformly; there is no significant difference between sham operation group and normal group, cell swelling and degeneration appear in the model group with the decreasing number of neuron, in irregular arrangement and with dissolved tigroid body, and the performance of model control group is similar to the model group in terms of injury. In the massage group, the most part of the neurons appear in normal shape and regular arrangement, and with lesser degenerated and injured neuron.③the gastrocnemius wet weight results:The ipsilateral gastrocnemius muscle wet weight and its recovery rate of the model group are lower than those of normal group and sham operation group (P<0.05), illustrating that the contact of the nerve fibers and gastrocnemius were damaged, and lead to the regression and shrivel of gastrocnemius. After10times of massage intervention, the ipsilateral gastrocnemius muscle wet weight and recovery rate of the massage group are changed to become higher than those of model group and model control group with significant difference (P<0.05), but has a difference when compared to the normal group (P<0.05). After20times, the ipsilateral gastrocnemius muscle wet weight and its recovery rate of the massage group still higher than the model group and model control group with significant difference (P<0.05), showing massage can promote the regeneration of injury nerve, and then prevent or even reverse, in a certain degree, the atrophy of gastrocnemius.Mechanism:①MAP-2immunohistochemistry results:after7days of modelling, the MAP-2Integral optical density in the sciatic nerve and spinal cord for the massage group are higher than that of the normal group and sham operation group (P<0.05),showing the MAP-2Stress-induced-expression during the natural recovering process from the injury of nervi ischiadicus of rats. The insignificant difference (P>0.05) between normal group and sham operation group shows that neuronal structure and function have not been damaged for sciatic nerve without injury, leading to invariance of MAP-2. After10,20times of massage intervention, MAP-2integral optical density in the sciatic nerve and spinal cord for massage group are significantly higher than the model group and model control group (P<0.05), showing that massage could be able to promote the MAP-2expression in the sciatic nerve and spinal cord; there are no significant difference between the model group and model control group (P>0.05), indicating that cloth binding have not triggered the change of MAP-2expression.②NF-M immunohistochemistry results:after7days of modeling, the NF-M integral optical density in the sciatic nerve and spinal cord for the model group are higher than that of the normal group and sham operation group (P<0.05), and there is non-obvious difference between the latter two groups(P>0.05). After10,20times of massage intervention, NF-M integral optical density in the sciatic nerve and spinal cord for massage group are significantly higher than the model group and model control group (P<0.05), showing that massage could be able to promote the NF-M expression in the sciatic nerve and spinal cord; there are no significant difference between the model group and model control group (P>0.05).③MAG immunohistochemistry results:after7days of modeling, the MAG integral optical density in the spinal cord for the model group are higher than that of the normal group and sham operation group (P<0.05), and there is non-obvious difference between the latter two groups (P>0.05). After10,20times of massage intervention, MAG integral optical density in the spinal cord for massage group are significantly lower than the model group and model control group (P<0.05), showing that massage could be able to prevent the MAG expression in the spinal cord; there are no significant difference between the model group and model control group (P>0.05).④ROCKII immunohistochemistry results:after7days of modeling, the ROCKII integral optical density in the spinal cord for the model group are higher than that of the normal group and sham operation group (P<0.05), and there is non-obvious difference between the latter two groups (P>0.05). After10,20times of massage intervention, ROCKII integral optical density in the spinal cord for massage group becomes significantly lower than the model group and model control group (P<0.05), showing that massage could be able to restrain the ROCKII expression in the spinal cord; there are no significant difference between the model group and model control group (P>0.05), showing cloth binding have no effect on the ROCKII expression of rats with sciatic nerve injury.[Conclusions]1. Massage could promote the repair of injured nervi ischiadicus of SD rats, rebuild the connection with the target tissue, increase the gastrocnemius muscle strength and its recovery rate, increase pain sensitivity, and thus promote the recovery of sensory and motor function of the rats with nerve injury.2. Massage increases the MAP-2, NF-M expression of the neuronal cytoskeleton protein in the medulla spinalis and the ischiadic nerve for the rats with injury of sciatic nerve, maintains the integrity of the neuronal cytoskeleton, restores the damaged neurons of the internal structure and function, protects the neurons, and then plays part in the axonal regeneration.3. Massage could be able to decrease the MAG, ROCKII expression in the inhibitor of axon growth of spinal cord for the rats with injured sciatic nerve, to inhibit the microtubule depolymerization and neurofilament, to maintain the neuronal cytoskeleton structure stability, to promote neuronal survival, and thereby to enhance the regeneration and repair of nerve function.4. One of the massage mechanisms for therapy of the peripheral nerve injury is based on the regulation of axon growth inhibitory factor and promotion of the synthesis of neuronal cytoskeletal proteins, leading to the protection of neurons, promote the regeneration and repair of injured nerve.