电针对神经病理性疼痛大鼠痛觉过敏及脊髓背角钾-氯离子协同转运体2表达的影响
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摘要
目的:观察患侧电针和健侧电针足三里穴、阳陵泉穴对神经病理性疼痛大鼠行为学表现以及脊髓背角钾-氯离子协同转运体2(KCC2)表达的影响,探讨患侧选穴和健侧选穴两种针刺方案的镇痛效应,以及KCC2在电针镇痛效应中的作用。方法:将40只雄性SD大鼠随机分为4组:假模组、模型组、患侧电针组、健侧电针组,每组10只。建立大鼠坐骨神经慢性压迫性损伤(CCI)模型,治疗组于术后1周开始电针治疗,每天1次,连续7天。各组于术前(0天)及术后3、5、7、10、12、14天分别测量大鼠患侧足机械足反射阈值(MWT)和热缩足反射潜伏期(TWL)。术后14天处死大鼠,取脊髓组织行HE染色观察组织病理学改变,并采用免疫组化方法检测脊髓背角KCC2蛋白的表达。结果:与术前比较,CCI各组大鼠痛阈明显降低,出现痛觉过敏(P<0.001),电针治疗后大鼠MWT和TWL均有不同程度的持续升高,且患侧与健侧电针相比无明显差异。HE染色结果提示,患侧电针组与健侧电针组脊髓背角组织及神经元病变程度较模型组减轻。术后14天患侧与健侧电针组较模型组脊髓背角患侧KCC2表达显著增加(P<0.05),并且患侧电针组与健侧电针组无明显差异。结论:患侧电针和健侧电针能产生类似的镇痛效果,均可减轻外周神经损伤后引起的痛觉过敏。患侧电针与健侧电针治疗均可抑制KCC2蛋白的表达下调,电针的镇痛作用可能是通过增加脊髓背角中KCC2的表达实现的。
Objective: To investigate the effect of electro-acupuncture(EA) on the behaviors and the expression of KCC2 in spinal cord dorsal horn by applying Zusanli acupoint(ST-36) and Yanglingquan acupoint(GB-34) in chronic constriction injury(CCI) rats.Method: Forty male SD rats were randomly divided into 4 groups: sham CCI group, CCI group, ipsilateral EA group and contralateral EA group, with 10 cases in each group. EA groups received EA treatment for 7days. The mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) were tested on the day before operation and at 3,5,7,10,12,14 days after CCI operation. In addition, the pathological changes of spinal cord dorsal horn were observed by HE stain and the expression of KCC2 was detected by immunohistochemistry.Result: The MWT and TWL values were decreased after CCI operation when compared with pre-operation(P<0.001). After EA treatment for 7 days, the MWT and TWL values for two EA groups were markedly increased(P<0.05). EA treatment significantly attenuated pathological changes of spinal cord dorsal horn. Furthermore, compared with the CCI group, the KCC2 expression was upregulated in the ipsilateral EA group and contralateral EA group after EA treatment(P<0.05), and there was no significant difference between the ipsilateral EA group and contralateral EA group.Conclusion: EA could reduce CCI-induced hyperalgesia. The analgesic effect of EA may be achieved by inhibiting the expression of KCC2 in spinal cord dorsal horn.
Objective: To investigate the effect of electro-acupuncture(EA) on the behaviors and the expression of KCC2 in spinal cord dorsal horn by applying Zusanli acupoint(ST-36) and Yanglingquan acupoint(GB-34) in chronic constriction injury(CCI) rats.Method: Forty male SD rats were randomly divided into 4 groups: sham CCI group, CCI group, ipsilateral EA group and contralateral EA group, with 10 cases in each group. EA groups received EA treatment for 7days. The mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) were tested on the day before operation and at 3,5,7,10,12,14 days after CCI operation. In addition, the pathological changes of spinal cord dorsal horn were observed by HE stain and the expression of KCC2 was detected by immunohistochemistry.Result: The MWT and TWL values were decreased after CCI operation when compared with pre-operation(P<0.001). After EA treatment for 7 days, the MWT and TWL values for two EA groups were markedly increased(P<0.05). EA treatment significantly attenuated pathological changes of spinal cord dorsal horn. Furthermore, compared with the CCI group, the KCC2 expression was upregulated in the ipsilateral EA group and contralateral EA group after EA treatment(P<0.05), and there was no significant difference between the ipsilateral EA group and contralateral EA group.Conclusion: EA could reduce CCI-induced hyperalgesia. The analgesic effect of EA may be achieved by inhibiting the expression of KCC2 in spinal cord dorsal horn.
引文
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[22]Modol L,Cobianchi S,Navarro X.Prevention of NKCC1phosphorylation avoids downregulation of KCC2 in central sensory pathways and reduces neuropathic pain after peripheral nerve injury[J].Pain,2014,155(8):1577—1590.
[23]Miletic G,Miletic V.Loose ligation of the sciatic nerve is associated with Trk B receptor-dependent decreases in KCC2 protein levels in the ipsilateral spinal dorsal horn[J].Pain,2008,137(3):532—539.
[2]Coull JA,Boudreau D,Bachand K,et al.Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain[J].Nature,2003,424(6951):938—942.
[3]Aguado F,Carmona MA,Pozas E,et al.BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl-cotransporter KCC2[J].Development,2003,130(7):1267—1280.
[4]Zhao B,Wong AY,Murshid A,et al.Identification of a novel di-leucine motif mediating K+/Cl-cotransporter KCC2 constitutive endocytosis[J].Cell Signal,2008,20(10):1769—1779.
[5]Zhang Z,Wang X,Wang W,et al.Brain-derived neurotrophic factor-mediated downregulation of brainstem K+-Cl-cotransporter and cell-type-specific GABA impairment for activation of descending pain facilitation[J].Mol Pharmacol,2013,84(4):511—520.
[6]Kim SK,Park JH,Bae SJ,et al.Effects of electroacupuncture on cold allodynia in a rat model of neuropathic pain:mediation by spinal adrenergic and serotonergic receptors[J].Exp Neurol,2005,195(2):430—436.
[7]Bennett GJ,Xie YK.A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[J].Pain,1988,33(1):87—107.
[8]华兴邦,周浩良.大鼠穴位图谱的研制[J].实验动物与动物实验,1991,(1):1—5.
[9]Xing GG,Liu FY,Qu XX,et al.Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain[J].Exp Neurol,2007,208(2):323—332.
[10]Jensen TS,Baron R,Haanpaa M,et al.A new definition of neuropathic pain[J].Pain,2011,152(10):2204—2205.
[11]Silva JR,Silva ML,Prado WA.Analgesia induced by 2-or100-Hz electroacupuncture in the rat tail-flick test depends on the activation of different descending pain inhibitory mechanisms[J].J Pain,2011,12(1):51—60.
[12]娄必丹,龚萍,章薇,等.针刺“对应穴”治疗痛证的理论探讨[J].中国针灸,2015,35(1):77—79.
[13]Millan MJ.The induction of pain:an integrative review[J].Prog Neurobiol,1999,57(1):1—164.
[14]Millan MJ.Descending control of pain[J].Prog Neurobiol,2002,66(6):355—474.
[15]阚宇.慢性痛大鼠海马NO/c GMP/PKG信号通路在针刺累积镇痛效应中的作用分析[D].中国中医科学院,2013.
[16]陈波,李明月,丁沙沙,等.针刺调节神经-内分泌-免疫网络研究进展(英文)[J].World Journal of Acupuncture-moxibustion,2014,24(4):49—53.
[17]Zhang W,Liu LY,Xu TL.Reduced potassium-chloride cotransporter expression in spinal cord dorsal horn neurons contributes to inflammatory pain hypersensitivity in rats[J].Neuroscience,2008,152(2):502—510.
[18]Rivera C,Voipio J,Thomas-Crusells J,et al.Mechanism of activity-dependent downregulation of the neuron-specific KCl cotransporter KCC2[J].J Neurosci,2004,24(19):4683—4691.
[19]Payne JA,Stevenson TJ,Donaldson LF.Molecular characterization of a putative K-Cl cotransporter in rat brain.A neuronal-specific isoform[J].J Biol Chem,1996,271(27):16245—16252.
[20]Price TJ,Cervero F,Gold MS,et al.Chloride regulation in the pain pathway[J].Brain Res Rev,2009,60(1):149—170.
[21]Morales-Aza BM,Chillingworth NL,Payne JA,et al.Inflammation alters cation chloride cotransporter expression in sensory neurons[J].Neurobiol Dis,2004,17(1):62—69.
[22]Modol L,Cobianchi S,Navarro X.Prevention of NKCC1phosphorylation avoids downregulation of KCC2 in central sensory pathways and reduces neuropathic pain after peripheral nerve injury[J].Pain,2014,155(8):1577—1590.
[23]Miletic G,Miletic V.Loose ligation of the sciatic nerve is associated with Trk B receptor-dependent decreases in KCC2 protein levels in the ipsilateral spinal dorsal horn[J].Pain,2008,137(3):532—539.