摘要
第一部分脊神经结扎大鼠脊髓星形胶质细胞ToⅡ样受体(TLR3)的表达
目的评价脊神经结扎疼痛模型大鼠脊髓星形胶质细胞TLR3的表达以及在慢性神经病理性痛中的作用。
方法雄性SD大鼠66只，体重180～250g，随机分为两组：SHAM组(假手术组，n=30)，分离右侧L_5脊神经但不结扎。SNL组(n=36)，结扎右侧L_5脊神经。每组大鼠均于术前1d、术后1、3、5、7、10、14、21、28d行行为学实验；SNL组于结扎后7d随机取6只大鼠利用双重免疫荧光标记法观察脊髓背角GFAP和TLR3的表达；每组于术后3、7、14、21、28d各随机取6只大鼠断头处死，取L_(4-5)脊髓节段采用RT-PCR法测定TLR3 mRNA的表达。
结果与SHAM组比较，SNL组PWPT和PWL降低(P＜0．05)；SNL组大鼠术后7d脊髓背角(术侧)GFAP阳性表达的细胞，TLR3也呈阳性表达。与基础值比较，SNL组脊髓背角TLR3 mRNA表达上调(P＜0．05)。
结论脊髓背角星形胶质细胞TLR3表达上调可能参与了脊神经结扎大鼠慢性神经病理性痛的发生与发展。
第二部分脊髓星形胶质细胞TLR3在大鼠神经病理性痛中的作用
实验一鞘内注射聚肌苷-多聚胞嘧啶核苷酸(poly(I:C))诱发神经病理性痛的实验研究
目的评价脊髓星形胶质细胞TLR3在大鼠神经病理性痛中的作用。
方法雄性SD大鼠，体重180～250g，126只鞘内置管成功的大鼠，随机分为3组，正常对照组(C组，n=42)；生理盐水组(NS组，n=42)鞘内注射NS 0．5 ml/kg，1次/d，连续7 d；神经病理性痛组(NP组，n=42)腹腔注射米诺环素40 mg/kg+鞘内注射poly(I:C)0．5 mg/kg，1次/d，连续7 d。各组大鼠于鞘内给药前1 d和鞘内给药后1、3、5、7、10、14、21、28 d测定机械痛阈和热痛阈；各组于鞘内给药后7 d各取6只大鼠，取L_(4-5)脊髓节段，采用免疫组化法测定脊髓背角胶质纤维酸性蛋白(GFAP)的表达；各组于鞘内给药前1 d和后1、7、14、21、28 d各取处死6只大鼠，取L_(4-5)脊髓节段，采用RT-PCR法测定TLR3 mRNA的表达变化趋势，以及术后7 d IL-6、TNF-a、IFN-β和IP-10 mRNA的表达，并使用ELISA法测定IL-6和IP-10蛋白变化。
结果与C组和NS组比较，NP组PWPT降低并持续至术后28天(P＜0．05)；NP组脊髓背角GFAP免疫反应阳性产物表达相对面积分别增加93．1％(P＜0．01)和90．9％(P＜0．01)；与基础值比较，NP组脊髓背角TLR3 mRNA表达上调(P＜0．05)。与NS组比较，NP组鞘内注射poly(I:C)后7 d天脊髓IL-6、TNF-a、IFN-β和IP-10 mRNA表达均上调(P＜0．05)，脊髓IL-6(9．5ng/g)和IP-10(12．3ng/g)蛋白表达显著上调(P＜0．05)。
结论脊髓背角星形胶质细胞TLR3可能参与了大鼠神经病理性痛的形成与维持。
实验二鞘内注射TLR3反义寡聚核苷酸对脊神经结扎大鼠的镇痛作用
目的探讨脊髓星形胶质细胞TLR3在神经病理性疼痛中的作用。
方法雄性SD大鼠，体重180～250 g。实验Ⅰ108只鞘内置管大鼠随机分为：生理盐水(NS)20μl+脊神经结扎(SNL)(A组，n=36)，错义寡聚核苷酸(MM-ODN)20μg/d+SNL(B组，n=36)，反义寡聚核苷酸(AS-ODN)20μg/d+SNL(C组，n=36)。实验Ⅱ108只SNL后7d大鼠随机分为：SNL+NS 20μl(D组，n=36)，SNL+MM-ODN 20μg/d(E组，n=36)，SNL+AS-ODN 20μg/d(F组，n=36)。实验Ⅰ和实验Ⅱ分别于SNL前1d和后7d开始鞘注，每天一次，共7d；于术后-1、1、3、5、7、10、14d和-1、7、10、12、14 d行行为学实验。实验Ⅰ和实验Ⅱ分别于术后7d和14d每组各随机取6只大鼠利用免疫组织化学法观察脊髓背角GFAP的表达，并于术后-1、3、7、14d和术后-1、7、10、14d每组各随机取6只大鼠断头处死，取L_(4-5)脊髓节段采用RT-PCR法测定TLR3和IL-6mRNA的表达，以及于术后7 d和术后14 d使用ELISA法测定IL-6和IP-10蛋白变化。
结果实验Ⅰ与A组比较，C组PWPT升高并持续至术后14d(P＜0．05)，C组各时点PWL无明显变化(P＞0．05)；与A组和B组比较，C组脊髓背角GFAP免疫反应阳性产物表达相对面积分别下降46．4％(P＜0．01)和45．7％(P＜0．01)；与A组和B组比较，C组鞘内注射AS-ODN抑制了TLR3和IL-6 mRNA以及IL-6和IP-10蛋白的表达(P＜0．05)。实验ⅡD组、E组和F组在PWPT、PWL、GFAP表达、TLR3和IL-6 mRNA以及IL-6和IP-10蛋白表达方面的差异无统计学意义(P＞0．05)。
结论脊髓背角星形胶质细胞TLR3可能参与了神经病理性疼痛的发生与发展。
第三部分脊髓背角c-Jun氨基末端激酶(JNK)活化在TLR3介导的神经病理性痛中的作用
目的观察大鼠鞘内注射poly(I:C)及TLR3反义寡聚核苷酸后脊髓背角c-Jun氨基末端激酶(JNK)活化水平的变化，探讨JNK在TLR3介导的神经病理性痛中作用。
方法雄性SD大鼠24只，体重180～250g，随机分为4组：A组(n=6)，鞘内注射poly(I:C)0．5mg/kg；B组(n=6)，鞘内注射SP600125 50nmol+poly(I:C)0．5mg/kg，A和B组每天鞘内注射一次，共7 d；C组(n=6)，鞘内注射MM-ODN20μg/d+脊神经结扎(SNL)；D组(n=6)，鞘内注射AS-ODN 20μg/d+SNL，C和D组于SNL前一天开始鞘内注射；每天一次，共7 d。A、B组和C、D组分别于鞘内注射和SNL后-1、1、3、5、7d行行为学实验，并分别于鞘内注射和SNL后7d每组取6只大鼠利用免疫荧光组织化学法观察脊髓背角pJNK的表达。
结果与A组比较，B组PWPT下降被SP600125阻止(P＜0．05)，PWL无明显变化(P＞0．05)；与C组比较，D组AS-ODN阻止了PWPT下降(P＜0．05)，PWL无明显变化(P＞0．05)。与A组比较，B组大鼠脊髓背角pJNK表达明显减少，阳性细胞数减至20±3(P＜0．05)；与C组比较，D组大鼠脊髓背角pJNK表达亦明显减少，阳性细胞数为27±5(P＜0．05)。
结论脊髓背角JNK活化水平增加，JNK通路激活，可能参与了TLR3介导的神经病理性痛的形成。
Part 1 TLR3 expression in spinal astrocytes in rats following spinal nerveligation
Objective To investigate the role of changes in TLR3 expression in spinalastrocytes induced by spinal nerve ligation(SNL).
Methods Sixty-six male SD rats weighing 180～250 g were randomly divided into 2groups: SHAM group(n=30) and SNL group(n=36). The animals were anesthetizedwith intraperitoneal 10% Chloral Hydrate 0.3～0.35ml/100g. Neuropathic pain wasinduced by L_5 spinal nerve ligation. The right spinal nerve was exposed and ligatedwith 4-0 silk thread. In sham operation group the right spinal nerve was exposed butnot ligated. Pain threshold was estimated by measuring paw withdrawal pressurethreshold and latency with Von Fery filament stimulation at 1 d before operation(baseline) and 1, 3, 5, 7, 10, 14, 21, 28 day after SNL. The animals were terminatedon the 7th day after SNL and the lumbar segment of the spinal cord was removed and the expression of TLR3 and GFAP positive cells in the spinal dorsal horn weredetermined by immunofluorescence double-staining.The rats were terminated at 3, 7,14, 21, 28d after operation in each group and the L_(4～5) segment of the spinal cord wasremoved for determination of expression of TLR3 mRNA by RT-PCR.
Result In SNL group PWPT and PWL was significantly decreased compared withthose in SHAM group (P＜0.05). Immunofluoresence double-staining indicated thatTLR3 and GFAP were colocalizated in the spinal astrocytes in SNL group. Theexpression of TLR3 mRNA in the ipsilateral spinal dorsal horn was significantlyincreased in SNL group compared with SHAM group (P＜0.05).
Conclusion Up-regulation of TLR3 gene expression in the spinal dorsal horn maybe involved in the mechanism of SNL-induced neuropathic pain.
Part 2 The role of TLR3 of spinal astrocytes in neuropathic pain
Study 1 Neuropathic pain induced by intrathecal injection of poly(I:C)
Objective To investigate the role of TLR3 of spinal astrocytes in neuropathic paininduced by intrathecal(i.t.) poly(I:C).
Methods: 126 male SD rats weighing 180～250 g were randomly divided into 3groups: group C (n=42),control group; group NS(n=42), NS (0.5ml/kg,i.t.), once aday for 7 consecutive days; group NP(n=42), minocycline(40mg/kg,i.p.)+ Poly(I:C)(0.5mg/kg, i.t.), once a day for 7 consecutive days. Pain threshold was estimated bymeasuring paw withdrawal pressure threshold and latency with Von Fery filamentstimulation at -1 (1 d before operation), 1, 3, 5, 7, 10, 14, 21, 28 day after injection.The animals were killed on the 7th day after injection and the lumbar segment of thespinal cord was removed and the expression of GFAP positive cells in the spinaldorsal horn were determined by immunohistochemistry ABC method. The rats were killed at -1, 1, 7, 14, 21, 28d after injection and the L_(4-5) segment of the spinal cordwas removed for determination of expression of IL-6, TNF-a, IFN-β, IP-10 and TLR3mRNA by RT-PCR. To test whether the upregulation of the IL-6 and IP-10 in protein,we measured IL-6 and IP-10 protein levels in the media by ELISA.
Result In group NP, PWPT was significantly decreased compared with group C andNS (P＜0.05). Immunohistochemistry indicated that staining of GFAP immunoreactivecells in group NP much more than group C and NS(P＜0.05).The expressionof TLR3 mRNA in the spinal dorsal hom was significantly increased in group NPcompared with group C and NS (P＜0.05). Significantly higher spinal expression ofmRNA for IL-6, TNF-a, IFN-βand IP-10 was observed at day 7 after injection ofpoly(I:C) compared with saline (P＜0.05). IL-6 (9.5ng/g) and IP-10 (12.3ng/g) proteinlevels in poly(I:C)-stimulated rats were significantly elevated at day 7 (P＜0.05) afterintrathecal injection compared with saline control animals(P＜0.05).
Conclusion TLR3 of spinal astrocytes may be involved in the mechanism ofpoly(I:C)-induced neuropathic pain.
Study 2 Analgesic effect of intrathecal antisence oligodeoxynucleotide of TLR3on rats following spinal nerve ligation
Objective To investigate the analgesic effect of TLR3 expression of spinalastrocytes on neuropathic pain induced by spinal nerve ligation(SNL).
Methods Male SD rats weighing 180～250 g were included in this experiment.Catheter was placed intrathecally with a tip located at lumbo-sacral segment of spinalcord for intrathecal(i.t.) drug administration in all animals. Neuropathic pain wasinduced by ligation of right L_5 spinal nerve with 4-0 silk thread. In PartⅠ, 108 animalswere randomly and equally divided into 3 groups: group A (n=36), normal saline(NS)(20μl,i.t.)+SNL; group B (n=36), mismatch oligodeoxynucleotide (MM-ODN) (20μg/d,i.t.) +SNL; group C (n=36), antisense oligodeoxy- nucleotide (AS-ODN)(20μg/d,i.t.)+SNL, once a day for 7 consecutive days. In PartⅡ,on the 7~(th) day afterSNL another 108 rats were randomly and equally divided into 3 groups: group D(n=36), SNL + NS(20μl,i.t.); group E (n=36), SNL + MM-ODN(20μg/d,i.t.); group F(n=36), SNL +AS-ODN (20μg/d,i.t.), once a day for 7 consecutive days. Painthreshold was estimated by measuring paw withdrawal pressure threshold(PWPT) andlatency(PWL) with Von Fery filament stimulation at -1 (1 d before operation), 1, 3, 5,7, 10, 14 day in PartⅠand -1, 7, 10, 12, 14 day in PartⅡafter SNL, respectively. Theanimals were killed on the 7~(th)(PartⅠ) or 14~(th)(PartⅡ) day after SNL and lumbarsegments of spinal cord were removed and immunohisto-chemistry ABC method wasused to determine the expression of GFAP positive cells in the spinal dorsal horn. Therats were killed at -1, 3, 7, 14 day in PartⅠand -1,7, 10, 14 day in PartⅡafteroperation in each group and the L_(4-5) segment of the spinal cord was removed fordetermination of expression of TLR3 and IL-6 mRNA by RT-PCR. To test thevariance of the IL-6 and IP-10 in protein, we measured IL-6 and IP-10 protein levelsin the media by ELISA.
Results PartⅠ: In group C, PWPT was significantly increased compared with group A(P＜0.05), PWL has no significant difference at different time (P＞0.05). Immunohisto-chemistryindicated that the relative area of staining of GFAP immuno-reactive cellsin group C decreased 46.4% and 45.7% (P＜0.05) respectively, when compared withthat in group A and B. The expression of TLR3 and IL-6 mRNA, IL-6 and IP-10protein was significantly inhibited in group C (P＜0.05). PartⅡ: There was nosignificant difference in the above-mentioned markers among group D, group E andgroup F (P＞0.05).
Conclusion TLR3 of spinal astrocytes in the spinal dorsal horn may be involved inthe development of SNL-induced neuropathic pain.
Part 3 Activation of JNK in spinal cord dorsal horn following neuropathic painmediated by TLR3
Objective To investigate whether the intracellular signal transduction pathway isinvolved in the mechanisms of neuropathic pain, we examine the activation of c-JunN-terminal kinase (JNK) in the dorsal horn of the spinal cord in a rat model afterintrathecal(i.t.) poly(I:C) or antisense oligodeoxynucleotide of TLR3.
Methods Male SD rats weighing 180～250 g were used in this experiment. In allanimals catheter was placed intrathecally with the tip located at lumbo-sacral segmentof the spinal cord for intrathecal drug administration. Neuropathic pain was inducedby right L_5 spinal nerve ligation with 4-0 silk thread. 24 animals were randomlydivided into 4 groups (n=6 each): group A, Poly(I:C) (0.5ml/kg,i.t.); group B,SP600125 50nmol + Poly(I:C) (0.5ml/kg,i.t.); group C, mismatch oligodeoxynucleotide(MM-ODN) (20μg/d,i.t.) +SNL; group D, antisense oligodeoxynucleotide (ASODN)(20μg/d,i.t.) +SNL, once a day for 7 consecutive days. Pain threshold wasestimated by measuring paw withdrawal pressure threshold(PWPT) and latency(PWL)with Von Fery filament stimulation at -1(1 d before operation), 1, 3, 5, 7 day in groupA and B after i.t. or in group C and D after SNL. The animals were killed on the7~(th)day in group A and B after i.t. or in group C and D after SNL and the lumbarsegment of the spinal cord was removed and the expression of pJNK positive cells inthe spinal dorsal horn were determined by immuno fluorescence histochemistry.
Results Poly(I:C) induced dramatic mechanical allodynia from day 1 to day 7 afteri.t., An increase in the phosphorylation of JNK in the dorsal horn of the spinal cordwas also observed after i.t.; Intrathecal infusion of a JNK peptide inhibitor, SP600125,did not affect normal pain responses but potently prevented poly(I:C)-inducedmechanical allodynia and the activation of JNK. Intrathecal infusion of antisense oligodeoxynucl-eotide of TLR3 also potently prevented SNL-induced mechanicalallodynia and the activation of JNK.
Conclusion Activation of JNK in spinal cord dorsal horn may be involved in themechanism of TLR3-mediated neuropathic pain in rats.
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1. Milligan ED, Twining C, Chacur M, et al. Spinal gila and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J Neurosci, 2003, 23: 1026- 1040.
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