针刺对创伤性脑损伤大鼠神经损伤的影响
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摘要
目的:通过观察小胶质细胞表达状态探讨针刺对创伤性颅脑损伤大鼠神经损伤的影响机制。方法:54只SD雄性大鼠按随机数字表分成正常组、模型组和针刺组,每组各18只。参照Feeney自由落体撞击法制备创伤性颅脑损伤大鼠模型。针刺组给予针刺"百会""水沟""风府"透"哑门"、双侧"合谷"穴,每天1次。治疗第3、7、14天后,分别进行改良大鼠神经功能缺损评分(mNSS),HE染色和免疫荧光标记法检测脑损伤的程度和小胶质细胞的表达量,采用酶联免疫法(ELISA)检测血清神经元特异性烯醇化酶(NSE)和勿动蛋白A(Nogo-A)含量的变化。结果:与正常组比较,模型组第1、3、7、14天mNSS显著升高(P<0.01);与模型组比较,针刺组第3、7、14天mNSS显著降低(P<0.05,P<0.01)。HE染色和免疫荧光标记结果可见,与正常组比较,模型组3个时点的组织结构持续破坏,神经元坏死,小胶质细胞大量活化(P<0.05);与模型组比较,针刺组3个时点组织结构破坏程度减轻,小胶质细胞活化减少(P<0.05)。与正常组比较,模型组3个时点血清NSE、Nogo-A含量显著升高(P<0.01);与模型组比较,针刺组3个时点血清NSE含量显著降低(P<0.01),第7、14天血清Nogo-A含量显著降低(P<0.01)。结论:针刺可能通过抑制小胶质细胞的表达及活化,继而抑制继发神经损害,促进神经功能恢复。
Objective To observe the effect of acupuncture on activities of microglia in traumatic brain injury(TBI)rats.Methods Fifty-four male SD rats were randomly and equally divided into normal control,model and acupuncture groups according to the random number table(n=18 rats in each group).The TBI model was established by using a free fall brain injury striking device after exposing the local cranial bone(to induce the left parietal cerebral contusion).Acupoints"Baihui"(GV20),"Shuigou"(GV26),"Fengfu"(GV16),"Yamen"(GV15)and bilateral"Hegu"(LII4)were stimulated intensively by twirling the filiform needles with force at a range of>360°and a frequency of 160-180 cycles/min for 10 sec in every acupoint,once every5 min during the 15 minutes' needle retaining.The treatment was given once every day for successive 14 days.The rats of the normal and model groups were grabbed and fixed with the same procedure.The behavioral changes were tested using modified neurological severity score(mNSS).The histopathological changes of the injured cerebral cortex tissues were observed by using hematoxylin-eosin(H.E.)staining,and the fluorescence intensity of Iba-1(marker of microglia)positive products in the surrounding tissue of the cerebral focus was displayed by immunofluorescence staining,and the contents of neuron specific enolate(NSE)and neurite outgrowth inhibitor-A(Nogo-A)in serum(indicating a secondary nerve damage)were assayed by ELISA.Results The mNSS scores were significantly increased on day 1,3,7 and 14 in the model group in comparison with the normal group(P<0.01)and considerably decreased at the 4 time-points after acupuncture intervention relevant to the model group(P<0.05,P<0.01).H.E.staining showed that modeling induced pathological changes such as the excursion of cell nucleus,cellular swelling,vacuole-like change,neuron death,karyopyknosis dissolution,and proliferation of fibrous tissue were relatively milder in the acupuncture group.The average fluorescence intensity values of Iba-1-positive products,serum NSE and Nogo-A contents on day3,7 and 14 were significantly higher in the model group than in the normal group(P<0.05,P<0.01),and notably down-regulated in the acupuncture group than in the model group(P<0.05,P<0.01,except Nogo-A on day 3).Conclusion Acupuncture intervention may accelerate neurological function recovery in TBI rats,which is closely related to its effects in inhibiting the activation of microglia and secondary nerve damage.
Objective To observe the effect of acupuncture on activities of microglia in traumatic brain injury(TBI)rats.Methods Fifty-four male SD rats were randomly and equally divided into normal control,model and acupuncture groups according to the random number table(n=18 rats in each group).The TBI model was established by using a free fall brain injury striking device after exposing the local cranial bone(to induce the left parietal cerebral contusion).Acupoints"Baihui"(GV20),"Shuigou"(GV26),"Fengfu"(GV16),"Yamen"(GV15)and bilateral"Hegu"(LII4)were stimulated intensively by twirling the filiform needles with force at a range of>360°and a frequency of 160-180 cycles/min for 10 sec in every acupoint,once every5 min during the 15 minutes' needle retaining.The treatment was given once every day for successive 14 days.The rats of the normal and model groups were grabbed and fixed with the same procedure.The behavioral changes were tested using modified neurological severity score(mNSS).The histopathological changes of the injured cerebral cortex tissues were observed by using hematoxylin-eosin(H.E.)staining,and the fluorescence intensity of Iba-1(marker of microglia)positive products in the surrounding tissue of the cerebral focus was displayed by immunofluorescence staining,and the contents of neuron specific enolate(NSE)and neurite outgrowth inhibitor-A(Nogo-A)in serum(indicating a secondary nerve damage)were assayed by ELISA.Results The mNSS scores were significantly increased on day 1,3,7 and 14 in the model group in comparison with the normal group(P<0.01)and considerably decreased at the 4 time-points after acupuncture intervention relevant to the model group(P<0.05,P<0.01).H.E.staining showed that modeling induced pathological changes such as the excursion of cell nucleus,cellular swelling,vacuole-like change,neuron death,karyopyknosis dissolution,and proliferation of fibrous tissue were relatively milder in the acupuncture group.The average fluorescence intensity values of Iba-1-positive products,serum NSE and Nogo-A contents on day3,7 and 14 were significantly higher in the model group than in the normal group(P<0.05,P<0.01),and notably down-regulated in the acupuncture group than in the model group(P<0.05,P<0.01,except Nogo-A on day 3).Conclusion Acupuncture intervention may accelerate neurological function recovery in TBI rats,which is closely related to its effects in inhibiting the activation of microglia and secondary nerve damage.
引文
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[2]娄梅,罗云华.针刺联合高压氧对重度颅脑损伤昏迷患者的促醒作用[J].当代医学,2013,19(1):159-160.
[3]蔡文栋,杨洁,赵春燕,等.针刺为主综合治疗颅脑外伤后综合征临床研究[J].河北医药,2013,35(2):304-305.
[4]李丹,周兆祥,刘志坚,等.针刺智三针和四神聪穴对脑外伤患者认知障碍的临床观察[J].湖南中医药大学学报,2013,33(5):99-102.
[5]张毅敏,唐纯志,程少冰,等.针刺对脑挫伤大鼠脑组织坏死面积及脑细胞凋亡的影响[J].中医杂志,2010,51(6):528-530.
[6]张毅敏,陈爱连,唐纯志,等.电针对重型颅脑损伤昏迷患者促醒作用的临床观察[J].针刺研究,2013,38(2):158-162.
[7]JIANG S T,CHEN W H,ZHANG Y M,et al.Acupuncture induces the proliferation and differentiation of endogenous neural stem cells in rats with traumatic brain injury[J].Evid Based Complement Alternat Med,2016,2016:2047412.
[8]贺晓生.重视对创伤性脑损伤炎症反应反应的研究[J].第四军医大学学报,2004,25(9):769-771.
[9]DAISUKE I,YOSHINORI I,KEIKO O,et al.Microglia specific localisation of a novel calcium binding protein,Iba-1[J].Mol Brain Res,1998,57(1):1-9.
[10]ITO D,TANAKA K,SUZUKI S,et al.Enhanced expression of Iba-1,ionized calcium-binding adapter molecule 1,after transient focal cerebral ischemia in rat brain[J].Stroke,2001,32(5):1208-1215.
[11]邓春雷.实验针灸学[M].北京:人民卫生出版社,1998.
[12]LU M,CHEN J,LU D,et al.Global test statistics for treatment effect of stroke and traumatic brain injury in rats with administration of bone marrow stromal cells[J].J Neurosci Methods,2003,128(1-2):183-190.
[13]MORGANTI-KOSSMANN M C,RANCAN M,STAHEL PF,et al.Inflammatory response in acute traumatic brain injury:a double-edged sword[J].Curr Opin Crit Care,2002,8(2):101-105.
[14]ALLAN S M,ROTHWELL N J.Cytokines and acute neurodegeneration[J].Nat Rev Neurosci,2001,2(10):734-744.
[15]TSUIKI K,TAKADA A,NAGAHIRO S,et al.Synthesis of serotonin in traumatized rat brain[J].J Neurochem,1995,64(3):1319-1325.
[16]董晓巧,刘伟国,杨小锋.纳洛酮对颅脑损伤后细胞凋亡及自由基影响的实验研究[J].浙江临床医学,2005,7(12):1235-1236.
[17]BEAUMONT A,MARMAROU A,FATOUROS P,et al.Secondary insults worsen blood brain barrier dysfunction assessed by MRI in cerebral contusion[J].Acta Neurochir Suppl,2002,81:217-219.
[18]KUMAR A,LOANE D J.Neuroinflammation after traumatic brain injury:opportunities for therapeutic intervention[J].Brain Behav Immun,2012,26(8):1191-1201.
[19]SAVID S,KRONER A.Repertoire of microglial and macrophage responses after spinal cord injury[J].Nat Rev Neurosci,2011,12(7):388-399.
[20]MA L,NAGAI J,UEDA H.Microglial activation mediates de novo lysophosphatidic acid production in a model of neuropathic pain[J].J Neurochem,2010,115(3):643-653.
[21]FUJITA R,MA Y,UEDA H.Lysophosphatidic acid-induced membrane ruffling and brain-derived neurotrophic factor gene expression are mediated by ATP release in primary microglia[J].J Neurochem,2008,107(1):152-160.
[22]靳国伟,韩世波,陈跃平.神经元特异性烯醇化酶的临床应用研究进展[J].中国临床神经外科杂志,2013,18(3):189-191.
[23]李怀斌,吴锋,熊克仁,等.针药结合对局灶性脑缺血大鼠海马CA1和CA3区神经干细胞增殖与分化相关蛋白表达的影响[J].针刺研究,2015,40(6):455-460.
[24]PLEINES U E,MORGANTI-KOSSMANN M C,RANCANM,et al.S-100beta reflects the extent of injury and outcome,whereas neuronal specific enolase is a better indicator of neuroinflammation in patients with severe traumatic brain injury[J].J Neurotrauma,2001,18(5):491-498.
[25]韩永升,徐银,韩咏竹,等.电针对局灶性脑缺血再灌注大鼠神经血管单元的保护作用[J].针刺研究,2013,38(3):173-180.
[26]钟琳,凌杰.急性颅脑损伤患者血清神经元特异性烯醇化酶、Nogo-A蛋白和髓鞘碱性蛋白水平的变化[J].中国医药导报,2013,10(30):120-122.
[27]赵永烈,史海霞.近年来针灸治疗颅脑损伤的研究概况[J].甘肃中医学院学报,2002,19(3):47-50.
[28]林志诚,杨珊莉,薛偕华,等.针刺百合穴改善脑卒中患者记忆力的中枢机制[J].中国康复理论与实践,2005,21(2):184-188.
[2]娄梅,罗云华.针刺联合高压氧对重度颅脑损伤昏迷患者的促醒作用[J].当代医学,2013,19(1):159-160.
[3]蔡文栋,杨洁,赵春燕,等.针刺为主综合治疗颅脑外伤后综合征临床研究[J].河北医药,2013,35(2):304-305.
[4]李丹,周兆祥,刘志坚,等.针刺智三针和四神聪穴对脑外伤患者认知障碍的临床观察[J].湖南中医药大学学报,2013,33(5):99-102.
[5]张毅敏,唐纯志,程少冰,等.针刺对脑挫伤大鼠脑组织坏死面积及脑细胞凋亡的影响[J].中医杂志,2010,51(6):528-530.
[6]张毅敏,陈爱连,唐纯志,等.电针对重型颅脑损伤昏迷患者促醒作用的临床观察[J].针刺研究,2013,38(2):158-162.
[7]JIANG S T,CHEN W H,ZHANG Y M,et al.Acupuncture induces the proliferation and differentiation of endogenous neural stem cells in rats with traumatic brain injury[J].Evid Based Complement Alternat Med,2016,2016:2047412.
[8]贺晓生.重视对创伤性脑损伤炎症反应反应的研究[J].第四军医大学学报,2004,25(9):769-771.
[9]DAISUKE I,YOSHINORI I,KEIKO O,et al.Microglia specific localisation of a novel calcium binding protein,Iba-1[J].Mol Brain Res,1998,57(1):1-9.
[10]ITO D,TANAKA K,SUZUKI S,et al.Enhanced expression of Iba-1,ionized calcium-binding adapter molecule 1,after transient focal cerebral ischemia in rat brain[J].Stroke,2001,32(5):1208-1215.
[11]邓春雷.实验针灸学[M].北京:人民卫生出版社,1998.
[12]LU M,CHEN J,LU D,et al.Global test statistics for treatment effect of stroke and traumatic brain injury in rats with administration of bone marrow stromal cells[J].J Neurosci Methods,2003,128(1-2):183-190.
[13]MORGANTI-KOSSMANN M C,RANCAN M,STAHEL PF,et al.Inflammatory response in acute traumatic brain injury:a double-edged sword[J].Curr Opin Crit Care,2002,8(2):101-105.
[14]ALLAN S M,ROTHWELL N J.Cytokines and acute neurodegeneration[J].Nat Rev Neurosci,2001,2(10):734-744.
[15]TSUIKI K,TAKADA A,NAGAHIRO S,et al.Synthesis of serotonin in traumatized rat brain[J].J Neurochem,1995,64(3):1319-1325.
[16]董晓巧,刘伟国,杨小锋.纳洛酮对颅脑损伤后细胞凋亡及自由基影响的实验研究[J].浙江临床医学,2005,7(12):1235-1236.
[17]BEAUMONT A,MARMAROU A,FATOUROS P,et al.Secondary insults worsen blood brain barrier dysfunction assessed by MRI in cerebral contusion[J].Acta Neurochir Suppl,2002,81:217-219.
[18]KUMAR A,LOANE D J.Neuroinflammation after traumatic brain injury:opportunities for therapeutic intervention[J].Brain Behav Immun,2012,26(8):1191-1201.
[19]SAVID S,KRONER A.Repertoire of microglial and macrophage responses after spinal cord injury[J].Nat Rev Neurosci,2011,12(7):388-399.
[20]MA L,NAGAI J,UEDA H.Microglial activation mediates de novo lysophosphatidic acid production in a model of neuropathic pain[J].J Neurochem,2010,115(3):643-653.
[21]FUJITA R,MA Y,UEDA H.Lysophosphatidic acid-induced membrane ruffling and brain-derived neurotrophic factor gene expression are mediated by ATP release in primary microglia[J].J Neurochem,2008,107(1):152-160.
[22]靳国伟,韩世波,陈跃平.神经元特异性烯醇化酶的临床应用研究进展[J].中国临床神经外科杂志,2013,18(3):189-191.
[23]李怀斌,吴锋,熊克仁,等.针药结合对局灶性脑缺血大鼠海马CA1和CA3区神经干细胞增殖与分化相关蛋白表达的影响[J].针刺研究,2015,40(6):455-460.
[24]PLEINES U E,MORGANTI-KOSSMANN M C,RANCANM,et al.S-100beta reflects the extent of injury and outcome,whereas neuronal specific enolase is a better indicator of neuroinflammation in patients with severe traumatic brain injury[J].J Neurotrauma,2001,18(5):491-498.
[25]韩永升,徐银,韩咏竹,等.电针对局灶性脑缺血再灌注大鼠神经血管单元的保护作用[J].针刺研究,2013,38(3):173-180.
[26]钟琳,凌杰.急性颅脑损伤患者血清神经元特异性烯醇化酶、Nogo-A蛋白和髓鞘碱性蛋白水平的变化[J].中国医药导报,2013,10(30):120-122.
[27]赵永烈,史海霞.近年来针灸治疗颅脑损伤的研究概况[J].甘肃中医学院学报,2002,19(3):47-50.
[28]林志诚,杨珊莉,薛偕华,等.针刺百合穴改善脑卒中患者记忆力的中枢机制[J].中国康复理论与实践,2005,21(2):184-188.