抑制脊髓络丝蛋白表达对神经性疼痛成年大鼠疼痛行为的影响研究
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摘要
背景络丝蛋白是主要参与神经元发育和成熟大脑突触过程的大分子细胞外基质糖蛋白。在神经元迁移过程终止后,脊髓尤其是脊髓背角浅层的神经元中仍有络丝蛋白的表达。神经性疼痛大鼠脊髓背角中络丝蛋白的表达明显减少,但其在伤害性感受和疼痛中的作用仍不明确。目的探讨鞘内注射络丝蛋白shRNA对成年大鼠疼痛行为的影响。方法 2015年,选取8～10周龄的SPF级雄性SD大鼠119只,置管成功108只,采用随机分组法将其分为正常(Norm)组12只、RNA干扰(RNAi)组12只、假手术(Sham)组12只、Sham+RNAi(SR)组6只、慢性坐骨神经压迫性损伤(CCI)组27只、CCI+RNAi(CR)组27只、CCI+空载体(EV)组12只。CCI组、CR组和EV组制备CCI模型,RNAi组、SR组、CR组鞘内给予络丝蛋白shRNA慢病毒载体,EV组给予空载体。计算干预后第4、7、10、14、21天络丝蛋白表达水平,于干预后第10天进行免疫组织化学观察络丝蛋白定位情况,检测干预前及干预后第1、4、7、10、14、17、21天大鼠足底机械缩足阈值(PWMT)、热辐射刺激潜伏期(PWTL)。结果 RNAi组第10天左、右侧络丝蛋白表达水平均高于Norm组,CR组第10天左侧络丝蛋白表达水平低于Sham组、CCI组,CCI组、CR组、EV组第10天右侧络丝蛋白表达水平均低于Sham组,CR组第10天右侧络丝蛋白表达水平低于CCI组,CCI组第4天右侧络丝蛋白表达水平低于CCI组左侧,CR组第4天右侧络丝蛋白表达水平低于CR组左侧,CCI组右侧、CR组左侧第7、10、14、21天络丝蛋白表达水平均低于CCI组左侧,CR组右侧第7、10、14、21天络丝蛋白表达水平均低于CCI组右侧(P<0.05)。Norm组络丝蛋白弥漫性分布于脊髓背角Ⅰ～Ⅱ、Ⅴ板层和外侧脊髓核(LSN);RNAi组双侧脊髓背角的Ⅰ～Ⅱ板层及LSN中仅有极少量络丝蛋白,Ⅴ板层仍存少量表达;Sham组双侧脊髓背角中络丝蛋白表达则无明显影响;CCI组右侧脊髓背角中络丝蛋白表达水平低于对侧;CR组双侧脊髓背角浅层仅有少量络丝蛋白表达;空载体对EV组络丝蛋白的表达无明显影响。第1、4、7、10、14、17、21天CCI组、CR组、EV组的PWMT、PWTL均低于Sham组,第7、10、14、17、21天CR组的PWMT、PWTL均低于CCI组(P<0.05)。结论在生理状态下,脊髓背角络丝蛋白对痛觉阈值无明显作用,但脊髓背角络丝蛋白表达减少可能参与神经损伤引起的神经性疼痛的发展过程。
Background Reelin is a large secreted extracellular matrix glycoprotein that modulates neuronal development and participates to the functioning of adult central synapses.After migrations have ceased,neurons within the spinal cord especially in the superficial dorsal horn continue to express Reelin.The expression of spinal dorsal horn Reelin downregulates significantly in neuropathic pain rats,while the role of Reelin in nociceptive processing and pain is still unknown.Objective To investigate the effects of intrathecal injection of Reelin shRNA on pain behaviors in adult rats.Methods A total of 119 8-10-week-old healthy male SD rats of SPF grade were selected and treated with intrathecal catheter implantation in 2015.The 108 rats with successful implantation were randomly assigned to normal group(Norm gourp,n=12),RNA interference group(RNAi group,n=12),sham-operation group(Sham group,n=12),sham plus RNA interference group(SR group,n=6),chronic compression injury(CCI) of sciatic nerve group(CCI group,n=27),CCI plus RNA interference group(CR group,n=27),and CCI plus empty vector group(EV group,n=12),respectively.Groups of CCI,CR and EV received a partial sciatic nerve ligation to establish CCI models.RNAi,SR and CR groups were intrathecally treated with lentiviral vector of Reelin shRNA,while EV group received intrathecal injection of an empty lentiviral vector.Western blotting was used to test the expression level of Reelin protein on the 4 th,7 th,10 th,14 th and 21 st days of intervention.Immunohistochemical technique was used to observe the localization of Reelin in the spinal dorsal horn on the 10 th day of intervention.Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) were tested before and on the 1 st,4 th,7 th,10 th,14 th,17 th and 21 st days of intervention,respectively.Results On the 10 th day of intervention,compared with rats in Norm group,the mean expression level of Reelin protein increased significantly on both right and left sides of spinal dorsal horn of rats in RNAi group(P<0.05);the mean expression level of Reelin protein in the left spinal dorsal horn in CR group was significantly lower than that of in sham and CCI groups(P<0.05);the mean expression level of Reelin protein in the right spinal dorsal horn in Sham group was significantly higher than that of CCI,CR and EV groups(P<0.05);the mean expression level of Reelin protein in the right spinal dorsal horn in CR group was significantly lower than that in CCI group(P<0.05). The mean expression level of Reelin protein in the right spinal dorsal horn was significantly lower than that in the left spinal dorsal horn in CCI group on the 4 th day of intervention and the same was true of CR group(P<0.05). The mean expression level of Reelin protein in the left spinal dorsal horn in CCI group was much higher than that of Reelin protein in the right spinal dorsal horn in CCI group and that of Reelin protein in the left spinal dorsal horn in CR group on the 7 th,10 th,14 th,and 21 st days of intervention(P<0.05). The mean expression level of Reelin protein on right spinal dorsal horn in CR group was lower than that of the right spinal dorsal horn in CCI group on the 7 th,10 th,14 th,and 21 st days of intervention(P<0.05). Reelin diffusely localized in laminae Ⅰ,Ⅱ,Ⅴ of spinal dorsal horn and in the lateral spinal nucleus(LSN) of rats in normal group.Only a small amount of filament proteins were found in lamina Ⅰ-Ⅱ and LSN of bilateral spinal dorsal horn,and a small amount of expression was found in lamina Ⅴ of the spinal dorsal horn in RNAi group.Reelin protein on both right and left sides of spinal dorsal horn had no changes in Sham group.Reelin protein in the right spinal dorsal horn was less than the contralateral level in CCI group.Only a very small amount of Reelin protein could be detected on both right and left sides of superficial dorsal horn in CR group.Empty vector had no obvious effects on the expression of spinal Reelin protein in EV group.Compared with rats in Sham group,both mean PWMT and PWTL of rats in CCI,CR and EV group were significantly decreased on the 1 st,4 th,7 th,10 th,14 th,17 th,21 st days of intervention(P<0.05). Both mean PWMT and PWTL in CR group were lower than those of CCI group on the 7 th,10 th,14 th,17 th,21 st days of intervention(P<0.05). Conclusion Spinal Reelin may have no significant effects on pain threshold under physiological condition while spinal Reelin downregulation may contribute to the development of neuropathic pain after peripheral nerve injury.
Background Reelin is a large secreted extracellular matrix glycoprotein that modulates neuronal development and participates to the functioning of adult central synapses.After migrations have ceased,neurons within the spinal cord especially in the superficial dorsal horn continue to express Reelin.The expression of spinal dorsal horn Reelin downregulates significantly in neuropathic pain rats,while the role of Reelin in nociceptive processing and pain is still unknown.Objective To investigate the effects of intrathecal injection of Reelin shRNA on pain behaviors in adult rats.Methods A total of 119 8-10-week-old healthy male SD rats of SPF grade were selected and treated with intrathecal catheter implantation in 2015.The 108 rats with successful implantation were randomly assigned to normal group(Norm gourp,n=12),RNA interference group(RNAi group,n=12),sham-operation group(Sham group,n=12),sham plus RNA interference group(SR group,n=6),chronic compression injury(CCI) of sciatic nerve group(CCI group,n=27),CCI plus RNA interference group(CR group,n=27),and CCI plus empty vector group(EV group,n=12),respectively.Groups of CCI,CR and EV received a partial sciatic nerve ligation to establish CCI models.RNAi,SR and CR groups were intrathecally treated with lentiviral vector of Reelin shRNA,while EV group received intrathecal injection of an empty lentiviral vector.Western blotting was used to test the expression level of Reelin protein on the 4 th,7 th,10 th,14 th and 21 st days of intervention.Immunohistochemical technique was used to observe the localization of Reelin in the spinal dorsal horn on the 10 th day of intervention.Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) were tested before and on the 1 st,4 th,7 th,10 th,14 th,17 th and 21 st days of intervention,respectively.Results On the 10 th day of intervention,compared with rats in Norm group,the mean expression level of Reelin protein increased significantly on both right and left sides of spinal dorsal horn of rats in RNAi group(P<0.05);the mean expression level of Reelin protein in the left spinal dorsal horn in CR group was significantly lower than that of in sham and CCI groups(P<0.05);the mean expression level of Reelin protein in the right spinal dorsal horn in Sham group was significantly higher than that of CCI,CR and EV groups(P<0.05);the mean expression level of Reelin protein in the right spinal dorsal horn in CR group was significantly lower than that in CCI group(P<0.05). The mean expression level of Reelin protein in the right spinal dorsal horn was significantly lower than that in the left spinal dorsal horn in CCI group on the 4 th day of intervention and the same was true of CR group(P<0.05). The mean expression level of Reelin protein in the left spinal dorsal horn in CCI group was much higher than that of Reelin protein in the right spinal dorsal horn in CCI group and that of Reelin protein in the left spinal dorsal horn in CR group on the 7 th,10 th,14 th,and 21 st days of intervention(P<0.05). The mean expression level of Reelin protein on right spinal dorsal horn in CR group was lower than that of the right spinal dorsal horn in CCI group on the 7 th,10 th,14 th,and 21 st days of intervention(P<0.05). Reelin diffusely localized in laminae Ⅰ,Ⅱ,Ⅴ of spinal dorsal horn and in the lateral spinal nucleus(LSN) of rats in normal group.Only a small amount of filament proteins were found in lamina Ⅰ-Ⅱ and LSN of bilateral spinal dorsal horn,and a small amount of expression was found in lamina Ⅴ of the spinal dorsal horn in RNAi group.Reelin protein on both right and left sides of spinal dorsal horn had no changes in Sham group.Reelin protein in the right spinal dorsal horn was less than the contralateral level in CCI group.Only a very small amount of Reelin protein could be detected on both right and left sides of superficial dorsal horn in CR group.Empty vector had no obvious effects on the expression of spinal Reelin protein in EV group.Compared with rats in Sham group,both mean PWMT and PWTL of rats in CCI,CR and EV group were significantly decreased on the 1 st,4 th,7 th,10 th,14 th,17 th,21 st days of intervention(P<0.05). Both mean PWMT and PWTL in CR group were lower than those of CCI group on the 7 th,10 th,14 th,17 th,21 st days of intervention(P<0.05). Conclusion Spinal Reelin may have no significant effects on pain threshold under physiological condition while spinal Reelin downregulation may contribute to the development of neuropathic pain after peripheral nerve injury.
引文
[1]FOLSOM T D,FATEMI S H.The involvement of Reelin in neurodevelopmental disorders[J].Neuropharmacology,2013,68:122-135.DOI:10.1016/j.neuropharm.2012.08.015.
[2]KUBASAK M D,BROOKS R,CHEN S,et al.Developmental distribution of reelin-positive cells and their secreted product in the rodent spinal cord[J].J Comp Neurol,2004,468(2):165-178.DOI:10.1002/cne.10946.
[3]AKOPIANS A L,BABAYAN A H,BEFFERT U,et al.Contribution of the Reelin signaling pathways to nociceptive processing[J].Eur J Neurosci,2008,27(3):523-537.DOI:10.1111/j.1460-9568.2008.06056.x.
[4]阳巧云,李晶金,李欢颜,等.慢性压迫性神经痛大鼠脊髓水平Reelin蛋白表达变化[J].山东大学学报(医学版),2014,52(z1):3-7.YANG Q Y,LI J J,LI H Y,et al.The expression of spinal Reelin following chronic constriction injury of the sciatic nerve in rats[J].Journal of Shandong University(Health Science),2014,52(z1):3-7.
[5]YAKSH T L,RUDY T A.Chronic catheterization of the spinal subarachnoid space[J].Physiol Behav,1976,17(6):1031-1036.
[6]BENNETT G J,XIE Y K.A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[J].Pain,1988,33(1):87-107.
[7]YUAN Y,CHEN H,MA G,et al.Reelin is involved in transforming growth factor-β1-induced cell migration in esophageal carcinoma cells[J].PLoS One,2012,7(2):e31802.DOI:10.1371/journal.pone.0031802.
[8]TAL M,BENNETT G J.Extra-territorial pain in rats with a peripheral mononeuropathy:mechano-hyperalgesia and mechano-allodynia in the territory of an uninjured nerve[J].Pain,1994,57(3):375-382.
[9]HARGREAVES K,DUBNER R,BROWN F,et al.A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia[J].Pain,1988,32(1):77-88.
[10]WANG X,BABAYAN A H,BASBAUM A I,et al.Loss of the Reelin-signaling pathway differentially disrupts heat,mechanical and chemical nociceptive processing[J].Neuroscience,2012,226:441-450.DOI:10.1016/j.neuroscience.2012.09.027.
[11]VILLEDA S A,AKOPIANS A L,BABAYAN A H,et al.Absence of Reelin results in altered nociception and aberrant neuronal positioning in the dorsal spinal cord[J].Neuroscience,2006,139(4):1385-1396.DOI:10.1016/j.neuroscience.2006.01.042.
[12]YVONE G M,ZHAO-FLEMING H H,UDEOCHU J C,et al.Disabled-1 dorsal horn spinal cord neurons co-express Lmx1b and function in nociceptive circuits[J].Eur J Neurosci,2017,45(5):733-747.DOI:10.1111/ejn.13520.
[13]HERZ J,CHEN Y.Reelin,lipoprotein receptors and synaptic plasticity[J].Nat Rev Neurosci,2006,7(11):850-859.DOI:10.1038/nrn2009.
[14]POCKETT S.Spinal cord synaptic plasticity and chronic pain[J].Anesth Analg,1995,80(1):173-179.
[2]KUBASAK M D,BROOKS R,CHEN S,et al.Developmental distribution of reelin-positive cells and their secreted product in the rodent spinal cord[J].J Comp Neurol,2004,468(2):165-178.DOI:10.1002/cne.10946.
[3]AKOPIANS A L,BABAYAN A H,BEFFERT U,et al.Contribution of the Reelin signaling pathways to nociceptive processing[J].Eur J Neurosci,2008,27(3):523-537.DOI:10.1111/j.1460-9568.2008.06056.x.
[4]阳巧云,李晶金,李欢颜,等.慢性压迫性神经痛大鼠脊髓水平Reelin蛋白表达变化[J].山东大学学报(医学版),2014,52(z1):3-7.YANG Q Y,LI J J,LI H Y,et al.The expression of spinal Reelin following chronic constriction injury of the sciatic nerve in rats[J].Journal of Shandong University(Health Science),2014,52(z1):3-7.
[5]YAKSH T L,RUDY T A.Chronic catheterization of the spinal subarachnoid space[J].Physiol Behav,1976,17(6):1031-1036.
[6]BENNETT G J,XIE Y K.A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[J].Pain,1988,33(1):87-107.
[7]YUAN Y,CHEN H,MA G,et al.Reelin is involved in transforming growth factor-β1-induced cell migration in esophageal carcinoma cells[J].PLoS One,2012,7(2):e31802.DOI:10.1371/journal.pone.0031802.
[8]TAL M,BENNETT G J.Extra-territorial pain in rats with a peripheral mononeuropathy:mechano-hyperalgesia and mechano-allodynia in the territory of an uninjured nerve[J].Pain,1994,57(3):375-382.
[9]HARGREAVES K,DUBNER R,BROWN F,et al.A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia[J].Pain,1988,32(1):77-88.
[10]WANG X,BABAYAN A H,BASBAUM A I,et al.Loss of the Reelin-signaling pathway differentially disrupts heat,mechanical and chemical nociceptive processing[J].Neuroscience,2012,226:441-450.DOI:10.1016/j.neuroscience.2012.09.027.
[11]VILLEDA S A,AKOPIANS A L,BABAYAN A H,et al.Absence of Reelin results in altered nociception and aberrant neuronal positioning in the dorsal spinal cord[J].Neuroscience,2006,139(4):1385-1396.DOI:10.1016/j.neuroscience.2006.01.042.
[12]YVONE G M,ZHAO-FLEMING H H,UDEOCHU J C,et al.Disabled-1 dorsal horn spinal cord neurons co-express Lmx1b and function in nociceptive circuits[J].Eur J Neurosci,2017,45(5):733-747.DOI:10.1111/ejn.13520.
[13]HERZ J,CHEN Y.Reelin,lipoprotein receptors and synaptic plasticity[J].Nat Rev Neurosci,2006,7(11):850-859.DOI:10.1038/nrn2009.
[14]POCKETT S.Spinal cord synaptic plasticity and chronic pain[J].Anesth Analg,1995,80(1):173-179.