小鼠岛叶皮质5型代谢型谷氨酸受体对神经病理性疼痛的调控作用
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摘要
目的:通过保留性神经损伤(SNI)模型,探索神经病理性疼痛发生后,岛叶皮质(IC)中FOS蛋白和5型代谢型谷氨酸受体(m Glu R5)及其下游分子的表达变化,并观察对岛叶皮质局部应用m Glu R5激动剂DHPG和拮抗剂MPEP对动物疼痛行为的影响。方法:将健康成年的雄性C57BL/6小鼠随机分为SNI组和假手术组(Sham组),SNI组采用SNI模型制造神经病理性疼痛模型。造模后通过机械痛行为学检测造模是否成功,通过免疫荧光染色检测岛叶皮质FOS蛋白的表达变化,通过Western Blot检测小鼠IC中m Glu R5和下游分子p-ERK与PI3K的表达变化;通过埋管注射的方式向实验组与假手术组IC内给予m Glu R5拮抗剂MPEP与激动剂DHPG,观察两种药物对小鼠机械性痛行为的影响。结果:SNI术后,小鼠的机械性痛的痛阈出现明显下降,并可维持至术后4周。免疫荧光组织化学染色结果显示:与假手术组相比,SNI组小鼠在术后1、2周IC中FOS蛋白表达增加(P<0.05)。Western Blot检测岛叶皮质m Glu R5及其下游分子p-ERK、PI3K的表达结果显示:术后SNI组m Glu R5表达水平相比假手术组增高(P<0.05),SNI组p-ERK表达水平相比假手术组增高(P<0.05),而PI3K仅在第2周时出现升高(P<0.05)。IC内置管给予MPEP后假手术组小鼠痛阈出现下降(P<0.05),而给予DHPG后SNI组的痛阈出现升高(P<0.05)。结论:小鼠IC中m Glu R5参与神经病理性疼痛的发生或调控,并且IC中m Glu R5表现出抑制性作用。在SNI后m Glu R5出现保护性增高,而m Glu R5激动剂DHPG可起到镇痛作用。
Objective:To investigate the change of FOS protein and metabotropic glutamate receptor 5(m Glu R5) and its downstream molecules expression in insular cortex(IC) after spared nerve injury(SNI) induced neuropathic pain.And to observe the influence of application of mGluR5 antagonist MPEP or agonist DHPG into IC to pain behavior.Methods:Healthy adult male C57 BL/6 mice were randomly divided into two groups:SNI group and Sham group.SNI group mice received a spared nerve injury operation.A mechanical pain behavior test was carried out to investigate whether the surgery was succeeded or not.FOS expression in IC was quantified through immunofluorescent staining.And the expression level of mGluR5 and its downstream molecules p-ERK and PI3 K were quantified through Western Blot.Through cannulas inserted in bilateral IC,DHPG or MPEP was injected into IC.Mechanical pain test was then performed to observe the influence of DHPG or MPEP to pain threshold.Results:After SNI,model animal showed a significant decrease in the pain threshold.Immunofluorescent staining showed that FOS-ir neurons in IC were increased after SNI operation(P < 0.05).Western Blot showed that,compared with Sham group,SNI group IC mGluR5 expression increased after surgery(P < 0.05) and p-ERK expression also increased after surgery(P < 0.05).However,PI3 K expression only increased in the 2 nd week after SNI surgery(P < 0.05).Through cannulas inserted in bilateral IC,DHPG or MPEP was injected into IC.The pain threshold of Sham group decreased after injection of MPEP(P < 0.05),while the pain threshold of SNI group increased after injection of DHPG(P < 0.05).Conclusion:Mice mGluR5 in IC is involved in the occurring or regulation of neuropathic pain and showed an inhibitory effect.There was a protective increase in mGluR5 after SNI,while mGluR5 agonist DHPG showed an analgesic effect.
Objective:To investigate the change of FOS protein and metabotropic glutamate receptor 5(m Glu R5) and its downstream molecules expression in insular cortex(IC) after spared nerve injury(SNI) induced neuropathic pain.And to observe the influence of application of mGluR5 antagonist MPEP or agonist DHPG into IC to pain behavior.Methods:Healthy adult male C57 BL/6 mice were randomly divided into two groups:SNI group and Sham group.SNI group mice received a spared nerve injury operation.A mechanical pain behavior test was carried out to investigate whether the surgery was succeeded or not.FOS expression in IC was quantified through immunofluorescent staining.And the expression level of mGluR5 and its downstream molecules p-ERK and PI3 K were quantified through Western Blot.Through cannulas inserted in bilateral IC,DHPG or MPEP was injected into IC.Mechanical pain test was then performed to observe the influence of DHPG or MPEP to pain threshold.Results:After SNI,model animal showed a significant decrease in the pain threshold.Immunofluorescent staining showed that FOS-ir neurons in IC were increased after SNI operation(P < 0.05).Western Blot showed that,compared with Sham group,SNI group IC mGluR5 expression increased after surgery(P < 0.05) and p-ERK expression also increased after surgery(P < 0.05).However,PI3 K expression only increased in the 2 nd week after SNI surgery(P < 0.05).Through cannulas inserted in bilateral IC,DHPG or MPEP was injected into IC.The pain threshold of Sham group decreased after injection of MPEP(P < 0.05),while the pain threshold of SNI group increased after injection of DHPG(P < 0.05).Conclusion:Mice mGluR5 in IC is involved in the occurring or regulation of neuropathic pain and showed an inhibitory effect.There was a protective increase in mGluR5 after SNI,while mGluR5 agonist DHPG showed an analgesic effect.
引文
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[18]Craig AD.How do you feel-now?The anterior insula and human awareness[J].Nat Rev Neurosci,2009,10:59-70.
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[25]Palazzo E,Marabese I,de Novellis V,et al.Supraspinal metabotropic glutamate receptors:a target for pain relief and beyond[J].Eur J Neurosci,2014,39:444-454.
[26]Li W,Neugebauer V.Differential changes of group II and group III m Glu R function in central amygdala neurons in a model of arthritic pain[J].J Neurophysiol,2006,96:1803-1815.
[27]Attal N,Cruccu G,Baron R,et al.EFNS guidelines on the pharmacological treatment of neuropathic pain:2010 revision[J].Eur J Neurol,2010,17:1113-1188.
[28]Dworkin RH,O’Connor AB,Audette J,et al.Recommendations for the pharmacological management of neuropathic pain:an overview and literature update[J].Mayo Clin Proc,2010,85:S3-S14.
[29]Siddall PJ,Cousins MJ,Otte A,et al.Pregabalin in central neuropathic pain associated with spinal cord injury:a placebo-controlled trial[J].Neurology,2006,67:1792-1800.
[30]Norrbrink C,Lundeberg T.Tramadol in neuropathic pain after spinal cord injury:a randomized,double-blind,placebo-controlled trial[J].Clin J Pain,2009,25:177-184.
[2]于生元.神经病理性疼痛[J].中国现代神经疾病杂志,2013,13:741-743.
[3]Stanwell P,Siddall P,Keshava N,et al.Neuro magnetic resonance spectroscopy using wavelet decomposition and statistical testing identifies biochemical changes in people with spinal cord injury and pain[J].Neuroimage,2010,53:544-552.
[4]王舰.神经病理性痛刺激导致岛叶-杏仁基底外侧核通路发生的可塑性变化及机制研究[D].第四军医大学,2016.
[5]张明.岛叶吻侧无颗粒细胞层大麻素受体1抑制神经病理性痛小鼠机械性触诱发痛行为及其机制[D].第四军医大学,2016.
[6]Dietrich D,Beck H,Kral T,et al.Metabotropic glutamate receptors modulate synaptic transmission in the perforant path:pharmacology and localization of two distinct receptors[J].Brain Res,1997,767:220-227.
[7]Chung G,Young KC,Yun YC,et al.Upregulation of prefrontal metabotropic glutamate receptor 5 mediates neuropathic pain and negative mood symptoms after spinal nerve injury in rats[J].Sci Rep,2017,7:9743.
[8]Peterson CD,Kitto KF,Akgun E,et al.Bivalent ligand that activates mu opioid receptor and antagonizes m Glu R5 receptor reduces neuropathic pain in mice[J].Pain,2017,158:2431-2441.
[9]Vincent K,Cornea VM,Jong YJ,et al.Intracellular m Glu R5 plays a critical role in neuropathic pain[J].Nat Commun,2016,7:10604.
[10]Chaplan SR,Bach FW,Pogrel JW,et al.Quantitative assessment of tactile allodynia in the rat paw[J].J Neurosci Methods,1994,53:55-63.
[11]Dixon WJ.The up-and-down method for small samples[J].J Am Stat Assoc,1965,60:967-978.
[12]Liu MG,Zhuo M.Loss of long-term depression in the insular cortex after tail amputation in adult mice[J].Mol Pain,2014,10:1.
[13]Liu MG,Koga K,Guo YY,et al.Long-term depression of synaptic transmission in the adult mouse insular cortex in vitro[J].Eur J Neurosci,2013,38:3128-3145.
[14]Mao L.The scaffold protein homer1b/c links metabotropic glutamate receptor 5 to extracellular signal-regulated protein kinase cascades in neurons[J].J Neurosci,2005,25:2741-2752.
[15]Karim F,Wang CC,Gereau RT.Metabotropic glutamate receptor subtypes 1 and 5 are activators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice[J].J Neurosci,2001,21:3771-3779.
[16]Cozzoli DK,Goulding SP,Zhang PW,et al.Binge drinking upregulates accumbens m Glu R5-Homer2-PI3K signaling:functional implications for alcoholism[J].J Neurosci,2009,29:8655-8668.
[17]Li XY,Ko HG,Chen T,et al.Alleviating neuropathic pain hypersensitivity by inhibiting PKMzeta in the anterior cingulate cortex[J].Science,2010,330:1400-1404.
[18]Craig AD.How do you feel-now?The anterior insula and human awareness[J].Nat Rev Neurosci,2009,10:59-70.
[19]Jasmin L,Burkey AR,Granato A,et al.Rostral agranular insular cortex and pain areas of the central nervous system:a tract-tracing study in the rat[J].J Comp Neurol,2004,468:425-440.
[20]Frot M,Mauguiere F.Dual representation of pain in the operculoinsular cortex in humans[J].Brain,2003,126:438-450.
[21]Lamm C,Decety J,Singer T.Meta-analytic evidence for common and distinct neural networks associated with directly experienced pain and empathy for pain[J].Neuroimage,2011,54:2492-2502.
[22]Inui K,Tsuji T,Kakigi R.Temporal analysis of cortical mechanisms for pain relief by tactile stimuli in humans[J].Cereb Cortex,2006,16:355-365.
[23]Ferraguti F,Shigemoto R.Metabotropic glutamate receptors[J].Cell Tissue Res,2006,326:483-504.
[24]Chiechio S,Nicoletti F.Metabotropic glutamate receptors and the control of chronic pain[J].Curr Opin Pharmacol,2012,12:28-34.
[25]Palazzo E,Marabese I,de Novellis V,et al.Supraspinal metabotropic glutamate receptors:a target for pain relief and beyond[J].Eur J Neurosci,2014,39:444-454.
[26]Li W,Neugebauer V.Differential changes of group II and group III m Glu R function in central amygdala neurons in a model of arthritic pain[J].J Neurophysiol,2006,96:1803-1815.
[27]Attal N,Cruccu G,Baron R,et al.EFNS guidelines on the pharmacological treatment of neuropathic pain:2010 revision[J].Eur J Neurol,2010,17:1113-1188.
[28]Dworkin RH,O’Connor AB,Audette J,et al.Recommendations for the pharmacological management of neuropathic pain:an overview and literature update[J].Mayo Clin Proc,2010,85:S3-S14.
[29]Siddall PJ,Cousins MJ,Otte A,et al.Pregabalin in central neuropathic pain associated with spinal cord injury:a placebo-controlled trial[J].Neurology,2006,67:1792-1800.
[30]Norrbrink C,Lundeberg T.Tramadol in neuropathic pain after spinal cord injury:a randomized,double-blind,placebo-controlled trial[J].Clin J Pain,2009,25:177-184.