脊髓小胶质细胞活化状态与前列腺的炎性疼痛

目的:观察化学性刺激大鼠前列腺后相应脊髓节段小胶质细胞活化的状况。方法:实验于2004-05/2005-11在第三军医大学神经生物学教研室实验室完成。①分组:健康雄性SD大鼠24只,随机分为4组:对照组6只;完全弗氏佐剂刺激3d组6只;完全弗氏佐剂刺激10d组6只;完全弗氏佐剂刺激28d组6只。②模型制备:对照组:取下腹部正中直切口,于前列腺腹侧叶注入生理盐水0.1mL,可吸收线缝合伤口;完全弗氏佐剂刺激组:取下腹部正中直切口,于前列腺腹侧叶注入完全弗氏佐剂0.1mL,可吸收线缝合伤口。③用免疫组织化学技术对不同时间段大鼠相应脊髓节段(L6和S1)的小胶质细胞活化进行检测。结果:24只大鼠均进入结果分析。完全弗氏佐剂刺激大鼠前列腺后3,10,28d,L6～S1脊髓背角浅层和深层小胶质细胞的活化与对照组相比均显著增加,其中3d时已显著增强,10d时增强最显著,28d时较10d时活化有所减少,但仍显著高于对照组。结论:完全弗氏佐剂刺激大鼠前列腺引起前列腺炎性疼痛,导致了相应节段L6～S1脊髓背角小胶质细胞的激活,提示小胶质细胞活化在前列腺炎性疼痛的神经病理过程可能发挥了重要作用。     

右美托咪定对慢性炎症痛大鼠疼痛及脊髓高迁移率族蛋白B1表达的影响

目的 采用完全弗氏佐剂诱导慢性炎症痛(chronic inflammatory pain, CIP)大鼠模型,探讨右美托咪定(dexmedetomidine, Dex)对CIP大鼠疼痛及脊髓高迁移率族蛋白B1(high mobility group protein B1, HMGB1)表达的影响。方法 48只大鼠随机分为对照组(不干预)、假手术组(左后足趾皮下注射生理盐水)、CIP组(左后足趾皮下注射完全弗氏佐剂)、Dex组(左后足趾皮下注射完全弗氏佐剂+鞘内注射Dex)各12只。造模后观察各组大鼠行为学表现,造模前、鞘内注射前1 h及注射后2、24 h检测各组大鼠左后肢热缩足反射潜伏期(paw withdrawal thermal latency, PWTL)及机械性缩足反射阈值(mechanical withdrawal threshold, MWT),注射后2、24 h采用ELISA法检测脊髓TNF-α、IL-1β水平,注射后24 h采用实时荧光定量PCR法检测脊髓HMGB1、Toll样受体4(Toll-like receptor 4, TLR4)、核因子-κB(nuclear ...     

脊髓小胶质细胞活化状态与前列腺的炎性疼痛

目的：观察化学性刺激大鼠前列腺后相应脊髓节段小胶质细胞活化的状况。 方法：实验于2004-05／2005—11在第三军医大学神经生物学教研室实验室完成。①分组：健康雄性SD大鼠24只，随机分为4组：对照组6只；完全弗氏佐剂刺激3d组6只；完全弗氏佐剂刺激10d组6只；完全弗氏佐剂刺激28d组6只。②模型制备：对照组：取下腹部正中直切口，于前列腺腹侧叶注入生理盐水0．1mL，可吸收线缝合伤口；完全弗氏佐剂刺激组：取下腹部正中直切口，于前列腺腹侧叶注入完全弗氏佐剂0．1mL，可吸收线缝合伤口。③用免疫组织化学技术对不同时间段大鼠相应脊髓节段（L6和S1）的小胶质细胞活化进行检测。 结果：24只大鼠均进入结果分析。完全弗氏佐剂刺激大鼠前列腺后3，10，28d，L6～S1脊髓背角浅层和深层小胶质细胞的活化与对照组相比均显著增加，其中3d时已显著增强，10d时增强最显著，28d时较10d时活化有所减少，但仍显著高于对照组。 结论：完全弗氏佐剂刺激大鼠前列腺引起前列腺炎性疼痛，导致了相应节段L6-S1脊髓背角小胶质细胞的激活，提示小胶质细胞活化在前列腺炎性疼痛的神经病理过程可能发挥了重要作用。     

A型肉毒毒素对关节炎疼痛大鼠脊髓小胶质细胞活化及肿瘤坏死因子-α表达的影响

目的 观察A型肉毒毒素（BTX-A）注射对佐剂性关节炎大鼠疼痛行为及脊髓小胶质细胞激活、肿瘤坏死因子-α(TNF-α)表达的影响,并探讨BTX-A治疗佐剂性关节炎疼痛的可能机制。 方法 采用随机数字表法将60只清洁级雄性SD大鼠分为假手术组、完全弗氏佐剂组（模型组）和BTX-A组,每组20只大鼠。除假手术组外,其余2组均于左后肢踝关节腔注射50 μl CFA建立佐剂性关节炎疼痛模型,假手术组大鼠关节腔内注射50 μl生理盐水作为对照。造模成功后假手术组和模型组大鼠左后肢踝关节腔注射20 μl生理盐水,BTX-A组大鼠注射20 μl 5 U BTX-A。于造模前1天、造模后第1,3,7,14,21天及给药后第1,3,7,14天对各组大鼠机械痛阈和热痛阈进行测定;并于测试后取出相应时间点脊髓组织,采用免疫蛋白印迹法、免疫荧光染色法检测离子钙接头蛋白(IBA-1)表达及IBA-1免疫阳性细胞(IBA-1-IR)数量;另采用ELISA法、实时荧光定量逆转录-聚合酶链反应（RT-PCR）检测脊髓水平TNF-α蛋白及TNF-α mRNA表达情况。 结果 与模型组比较,BTX-A组踝关节腔注射BTX-A后第3天时其机械痛撤足阈值及热痛阈潜伏期均明显增加,直到注射后第14天时差异均具有统计学意义(P<0.01);脊髓水平IBA-1蛋白表达显著降低,IBA-1免疫阳性细胞数量明显下调（P<0.01）;TNF-α蛋白及TNF-α mRNA表达均明显降低(P<0.05)。 结论 BTX-A可减轻 CFA诱导的关节炎疼痛,其镇痛机制可能与抑制脊髓小胶质细胞活化及TNF-α释放有关。     

慢性前列腺炎模型大鼠脊髓背角降钙素基因相关肽表达的测定

目的:检测慢性前列腺炎模型大鼠脊髓背角降钙素基因相关肽(Calcitonin Gene-Related Peptide,CGRP)的表达.方法:雄性SD大鼠16只,随机分为正常对照组和实验组,实验组在大鼠前列腺侧叶注射完全弗氏佐剂制备慢性前列腺炎模型,Taqman荧光PCR相对定量法检测各组大鼠脊髓背角中CGRP的表达.结果:慢性前列腺炎模型大鼠脊髓背角中CGRP mRNA的表达水平为1.12±0.01,比正常对照组明显增加(P<0.01).结论:CGRP在慢性前列腺炎模型大鼠脊髓背角中表达显著增加,可能参与慢性前列腺炎疼痛的形成.     

疼痛应对策略测量工具的研究概况

摘要 目的：探讨缝隙连接蛋白Cx43在炎性镜像痛大鼠脊髓背角的表达。方法：1、行为学研究：鞘内置管成功的雄性SD大鼠24只,随机分为3组（n＝8）。CFA模型组：于大鼠左后肢踝关节外侧皮下注射完全弗氏佐剂（CFA）50μl建立佐剂性关节炎性痛模型,鞘内注射NS 10μl;CBX处理组：皮下注射CFA50μl,鞘内注射缝隙连接阻滞剂甘珀酸（CBX）100μg（10μl）;NS对照组：皮下注射NS 50μl,鞘内注射NS 10μl。分别于CFA致炎前1d(基础值)和致炎后0.5h、1d、3d、5d、7d、10d、14d每次鞘内注射后2h测定双侧的热缩足反射潜伏期（PWTL）。2、形态学研究：雄性SD大鼠20只,随机分为4组（n＝5）：NS生理盐水组、C1d组、C3d组和C7d组。分别在CFA致炎后相应时间点取腰段脊髓行Cx43免疫组织化学染色分析,测定双侧脊髓背角积分光密度值。结果：1、单侧踝关节外侧皮下注射CFA可以在致炎后2h～7d诱导出双侧的热痛觉过敏,呈现明显的镜像痛和域外痛现象(P<0.01或0.05),鞘内注射CBX可以在炎性痛的不同阶段逆转上述改变。2、单侧CFA致炎后,大鼠脊髓双侧Cx43的表达与对照组相比均显著增加(P<0.01或0.05),主要集中于Ⅰ～Ⅱ层和Ⅹ层,其时程变化与行为学变化基本一致。结论：大鼠单侧CFA致炎诱导的佐剂性关节炎性痛模型,具有显著的镜像痛特征。脊髓双侧背角Cx43表达增加,鞘内应用缝隙连接阻滞剂可以逆转镜像痛现象,提示脊髓缝隙连接可能在疼痛中枢敏感化中扮演重要角色。 关键词 佐剂性关节炎;镜像痛;缝隙连接;连接蛋白43;脊髓背角;甘珀酸     

缝隙连接蛋白CX43在炎性镜像痛大鼠脊髓背角的表达

目的:探讨缝隙连接蛋白Cx43在炎性镜像痛大鼠脊髓背角的表达.方法:(1)行为学研究:鞘内置管成功的雄性SD大鼠24只,随机分为3组(n=8).CFA模型组:于大鼠左后肢踝关节外侧皮下注射完全弗氏佐剂(CFA)50μl建立关节炎性痛模型,鞘内注射NS 10μl;CBX处理组:皮下注射CFA 50μa,鞘内注射缝隙连接阻滞剂甘珀酸(CBX)100μg(10μl);NS对照组:皮下注射NS50μl,鞘内注射NS 10μl.分别于CFA致炎前1d(基础值)和致炎后0.5h、1d、3d、5d、7d、10d、14d每次鞘内注射后2h测定双侧的热缩足反射潜伏期(PWTL).(2)形态学研究:雄性SD大鼠20只,随机分为4组(n=5):NS生理盐水组、C1d组、C3d组和C7d组.分别在CFA致炎后相应时间点取腰段脊髓行Cx43免疫组织化学染色分析,测定双侧脊髓背角积分光密度值.结果:(1)单侧踝关节外侧皮下注射CFA可以在致炎后2h～7d诱导出双侧的热痛觉过敏,呈现明显的镜像痛和域外痛现象(P＜0.01或0.05),鞘内注射CBX可以在炎性痛的不同阶段逆转上述改变.(2)单侧CFA致炎后,大鼠脊髓双侧Cx43的表达与对照组相比均显著增加(P＜0.01或0.05),主要集中于Ⅰ～Ⅱ层和Ⅹ层,其时程变化与行为学变化基本一致.结论:大鼠单侧CFA致炎诱导的关节炎性痛模型,具有显著的镜像痛特征.脊髓双侧背角Cx43表达增加,鞘内应用缝隙连接阻滞剂可以逆转镜像痛现象,提示脊髓缝隙连接可能在疼痛中枢敏感化中起重要作用.     

单关节炎诱导胶质细胞在大鼠颈、胸、腰、骶段脊髓背角的活化

目的:探讨小胶质细胞和星形胶质细胞在单关节炎大鼠颈、胸、腰、骶段脊髓背角的活化.方法:16只SD雄性大鼠随机均分为正常对照组和单关节炎组(n=8).左侧踝关节腔注射完全弗氏佐剂(complete Freund' s adjuvant,CFA)建立单关节炎模型.分别采用Hargreaves’法和von Frey纤毛测定单关节炎大鼠双侧后爪致炎前及致炎后3d的热刺激缩爪反应潜伏期(paw withdrawal latency,PWL)、机械刺激抬腿反应阈值(paw withdrawal threshold,PWT).行为学测定结束后,每组取4只大鼠,灌注后取脊髓颈、胸、腰、骶段,采用免疫荧光组织化学法检测Iba-1(小胶质细胞标记物)和GFAP(星形胶质细胞标记物)在大鼠脊髓背角的表达情况.结果:单关节炎组大鼠致炎侧后爪的PWL和PWT值在致炎3d后均显著低于对照组(P＜0.01),对侧后爪差异无显著性(P＜0.05).单关节炎组大鼠致炎3d后,Iba-1和GFAP的荧光强度在颈、胸、腰段脊髓的双侧背角相较于对照组均明显增高,差异有显著性(P＜ 0.05或P＜0.01),而在骶段无统计学差异(P＜0.05),但有明显增加趋势.致炎侧和对侧脊髓背角Iba-1和GFAP的荧光强度在颈、胸、腰、骶各节段差异均无统计学意义(P＜0.05).结论:单关节致炎导致大鼠患侧后爪出现触诱发痛和热痛觉过敏,并诱导小胶质细胞和星形胶质细胞在大鼠颈、胸、腰段双侧脊髓背角的活化.     

鞘内注射银杏内酯B对神经病理痛大鼠痛阈及脊髓c-fos表达的影响

目的:观察鞘内注射银杏内酯B(BN52021)对SNI神经病理痛大鼠痛阈及脊髓c-fos表达的影响,探讨脊髓血小板活化因子及其受体参与痛觉信号调节的可能机制.方法:鞘内置管后的SD大鼠24只随机等分为4组:假手术组(sham组),SNI组,SNI+DMSO对照组和SNI+BN52021组,建立SNI疼痛模型,手术后1,3,5,7,10和14d鞘内给药并测痛阈,第14d取大鼠腰段脊髓,冰冻切片,免疫组织化学染色检测Fos免疫阳性细胞.结果:SNI神经损伤大鼠机械缩爪阈明显降低(P<0.05),同侧脊髓背角浅层内Fos阳性神经元明显增多(P<0.05);鞘内应用银杏内酯B明显减少脊髓背角神经元c-fos的表达,同时伴有大鼠机械异常痛敏的减轻,各组大鼠辐射热缩爪潜伏期无明显差异.结论:鞘内注射银杏内酯B可减轻SNI大鼠机械异常痛敏,抑制神经损伤后脊髓背角c-fos的表达.     

硫辛酸对炎性痛小鼠脊髓背角c-Fos和Iba-1的影响

目的:观察硫辛酸(α-lipoic acid, ALA)对炎性痛小鼠疼痛行为学及脊髓背角Iba-1和c-Fos表达的影响。方法 :成年SPF级雄性昆明小鼠40只,随机分为4组(n=10):对照组(Control组)、炎性痛组[CFA组:足底注射30μl完全弗氏佐剂(complete Freund's adjuvant, CFA)]、硫辛酸组(ALA组:腹腔注射ALA 100 mg/kg)和炎性痛+硫辛酸组(CFA+ALA组:足底注射30μl CFA3 d后腹腔注射ALA 100 mg/kg)。术后3 d,腹腔注射ALA后分别检测热缩足潜伏期(paw withdrawal latency, PWL)和足趾肿胀程度并用免疫组织化学法检测脊髓背角Iba-1 (Ionized calcium binding adaptor molecule-1)和c-Fos表达。结果:ALA明显抑制炎性痛引起的热缩足潜伏期降低,与此同时,给予ALA能明显降低炎性痛小鼠脊髓背角Iba-1和c-Fos的表达。结论:硫辛酸明显减少炎性痛小鼠脊髓背角Iba-1和c-Fos的表达,对炎性痛产生明显的抑制作用。     

Inhibition of ERK phosphorylation decreases nociceptive behaviour in monoarthritic rats

In this study we investigated the role of the activation of the extracellular signal-regulated kinases 1 and 2 (ERK) in chronic inflammatory articular nociception. Monoarthritis was induced in the left ankle of Wistar rats by injection of complete Freund's adjuvant (CFA). Movement of the inflamed joint increased ERK phosphorylation in neurones of the superficial and deep ipislateral dorsal horn laminae of L3-L5 spinal cord segments. Spinal immunoreactivity to phosphoERK was more intense in animals in which the inflammation lasted longer, 7 days or more, than in rats with less time of inflammation. PhosphoERK levels were transient, since 2h after ankle stimulation spinal immunoreaction had almost disappeared. PhosphoERK immunoreactivity was not induced by movement of ankles from non-arthritic control animals, neither in monoarthritic rats in which the inflamed ankle was not stimulated. Intrathecal administration of PD 98059, an inhibitor of ERK phosphorylation, reduced nociceptive behaviour induced by the ankle bend test in monoarthritic rats. The anti-nociceptive effect of PD 98059 was more prominent and in animals with short lasting (4 days) than in animals with longer (14 days) monoarthritis. Taken together, these findings suggest that ERK phosphorylation in spinal cord neurones plays an important role in chronic inflammatory articular pain and that its inhibition may provide significant anti-nociception.     

Spinal cord protein interacting with C kinase 1 is required for the maintenance of complete Freund’s adjuvant-induced inflammatory pain but not for incision-induced post-operative pain

Protein interacting with C kinase 1 (PICK1) is a PDZ-containing protein that binds to AMPA receptor (AMPAR) GluR2 subunit and protein kinase Cα (PKCα) in the central neurons. It functions as a targeting and transport protein, presents the activated form of PKCα to synaptic GluR2, and participates in synaptic AMPAR trafficking in the nervous system. Thus, PICK1 might be involved in many physiological and pathological processes triggered via the activation of AMPARs. We report herein that PICK1 knockout mice display impaired mechanical and thermal pain hypersensitivities during complete Freund’s adjuvant (CFA)-induced inflammatory pain maintenance. Acute transient knockdown of spinal cord PICK1 through intrathecal injection of PICK1 antisense oligodeoxynucleotide had a similar effect. In contrast, knockout and knockdown of spinal cord PICK1 did not affect incision-induced guarding pain behaviors or mechanical or thermal pain hypersensitivities. We also found that PICK1 is highly expressed in dorsal horn, where it interacts with GluR2 and PKCα. Injection of CFA into a hind paw, but not a hind paw incision, increased PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn. These increases were absent when spinal cord PICK1 was deficient. Given that dorsal horn PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization contribute to the maintenance of CFA-induced inflammatory pain, our findings suggest that spinal PICK1 may participate in the maintenance of persistent inflammatory pain, but not in incision-induced post-operative pain, through promoting PKCα-mediated GluR2 phosphorylation and internalization in dorsal horn neurons.     

Role of spinal microglia in rat models of peripheral nerve injury and inflammation.

Mounting evidence supports the hypothesis that spinal microglia modulate the development and maintenance of some chronic pain states. Here we examined the role of spinal microglia following both peripheral inflammatory insult and peripheral nerve injury. We observed significant ipsilateral dorsal horn microglia activation 2 weeks after injury and bilateral activation 50 days following nerve injury as well as 24 h following intraplantar zymosan but not intraplantar complete Freund's adjuvant (CFA). Ipsilateral but not contralateral microglia activation was associated with hind paw mechanical hyperalgesia. Spinal injection of the glial metabolic inactivator fluorocitrate attenuated ipsilateral hyperalgesia and bilateral spinal microglia activation after peripheral nerve injury. Intrathecal fluorocitrate reversed hyperalgesia after intraplantar zymosan and produced no reversal of CFA-induced hyperalgesia. These data suggest a role for spinal glia in the persistence of mechanical hyperalgesia following peripheral nerve injury. However, activation of spinal microglia contralaterally did not correlate to nociception. Furthermore, it would appear that the time course of microglia activation and their contribution to inflammatory pain is dependent on the inflammatory stimulus administered.     

The CGRP receptor antagonist BIBN4096BS peripherally alleviates inflammatory pain in rats

Calcitonin gene–related peptide (CGRP) is known to play a major role in the pathogenesis of pain syndromes, in particular migraine pain. Here we focus on its implication in a rat pain model of inflammation, induced by injection of complete Freund adjuvant (CFA). The nonpeptide CGRP receptor antagonist BIBN4096BS reduces migraine pain and trigeminal neuronal activity. Here we demonstrate that the compound reduces inflammatory pain and spinal neuronal activity. Behavioural experiments reveal a reversal of the CFA-induced mechanical hypersensitivity and monoiodoacetate (MIA)-induced weight-bearing deficit in rats after systemic drug administration. To further investigate the mechanism of action of the CGRP antagonist in inflammatory pain, in vivo electrophysiological studies were performed in CFA-injected rats. Recordings from wide dynamic range neurons in deep dorsal horn layers of the lumbar spinal cord confirmed a reduction of neuronal activity after systemic drug application. The same amount of reduction occurred after topical administration onto the paw, with resulting systemic plasma concentrations in the low nanomolar range. However, spinal administration of BIBN4096BS did not modify the neuronal activity in the CFA model. Peripheral blockade of CGRP receptors by BIBN4096BS significantly alleviates inflammatory pain.     

Lindernde Wirkung des CGRP-Antagonismus auf Entzündungsschmerz

The neuropeptide calcitonin gene-related peptide (CGRP) is known to play a major role in the pathogenesis of pain syndromes, in particular migraine pain; however, its implication in inflammatory processes is not well known. The CGRP receptor antagonist BIBN4096BS was shown to reduce migraine pain and trigeminal neuronal activity. An analgesic action of this compound can also be found in rats with induced acute inflammation by injection of complete Freund’s adjuvant (CFA) in one hindpaw. In this model the compound reduced inflammatory pain and spinal neuronal activity. Behavioral experiments (Randall-Selitto test) revealed a reversal of the CFA-induced mechanical hyperalgesia in rats after systemic drug administration. In vivo electrophysiological studies performed in rats injected with CFA using recordings of wide dynamic range neurons in deep dorsal horn layers of the lumbar spinal cord, confirmed a reduction of neuronal activity after systemic drug administration. The same considerable amount of reduction occurred after topical administration onto the paw with resulting systemic plasma concentrations in the low nanomolar range. Spinal administration of BIBN4096BS did not modify the neuronal activity in the CFA model which suggests that peripheral blockade of CGRP receptors by BIBN4096BS significantly alleviates inflammatory pain.     

Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain

Previous studies from our laboratory have demonstrated that both chronic inflammatory pain, induced by intraplantar injection of complete Freund's adjuvant (CFA), and prolonged (48 h) stimulation of mu-opioid receptors (muOR) by systemic administration of a variety of selective agonists, resulted in enhanced plasma membrane targeting of delta-opioid receptors (deltaOR) in neurons of the dorsal spinal cord. To determine whether deltaOR trafficking induced by chronic inflammation was dependent on the activation of muOR, we investigated by immunogold cytochemistry the effects of intraplantar CFA injection on the plasma membrane density of deltaOR in muOR knockout (KO) mice. In untreated wild-type (WT) mice, only a small proportion of deltaOR was associated with neuronal plasma membranes in the dorsal horn of the spinal cord. The CFA-induced inflammation produced a significantly higher ratio of plasma membrane to intracellular receptors, as well as a 75% increase in the membrane density of immunoreactive deltaOR, in dendrites of the ipsilateral dorsal horn as compared to untreated mice. This increase in the membrane density of deltaOR was likely due to a recruitment of receptors from intracellular stores since no difference in the overall deltaOR immunolabeling density was evident between CFA-treated and untreated mice. Most importantly, the CFA-induced changes in deltaOR plasma membrane insertion seen in WT animals were not present in the spinal cord of muOR KO mice. These results demonstrate that the integrity of muOR is necessary for CFA-induced changes in deltaOR trafficking to occur and suggest that these changes could be elicited by stimulation of muOR by endogenous opioids released in response to chronic inflammatory pain.     

Peripheral therapeutic ultrasound stimulation alters the distribution of spinal C-fos immunoreactivity induced by early or late phase of inflammation

The purpose of this investigation was to examine the central modulated effects of therapeutic ultrasound (US) on neuronal activity in the spinal cord on early and late phases of inflammation. In this study, induction of c-Fos protein, which reflects neuronal activation (particularly inflammatory nociception), was investigated in the lumbar spinal cord with immunohistochemistry. Inflammatory monoarthritis was induced in 20 male Wistar rats (weighing 250-300 g) via intra-articular injection of complete Freund's adjuvant (CFA) into the tibiotarsal joint. Two phases of arthritis, early phase (18 h after adjuvant injection) and late phase (7 d after adjuvant injection), were studied in the rats. Pulsed-mode US (1 MHz, the spatial average temporal average intensity [I(SATA)] = 0.5 W/cm(2), 50% duty cycle) was applied for 5 min. The effects of US and sham treatments against these phases of arthritis were demonstrated by spinal c-Fos-like immunoreactivity (c-Fos-LI). All data were evaluated statistically with the paired t-test or analysis of variance with Bonferroni corrections. c-Fos-LI neurons were abundant (average 264.2 +/- 11.9) in the L3 and L4 neurons of the spinal cord in areas ipsilateral to the CFA-induced arthritic leg in the early phase, but few were present (average 40.4 +/- 4.5) in the late phase in sham-treated animals. Bonferroni corrections to the alpha level were used to check the group differences in spinal c-Fos expression, and significance was reached when p < 0.025. In the early inflammatory phase, US treatment significantly suppressed the increased number of c-Fos-LI neurons associated with CFA-induced arthritis in superficial laminae, nucleus proprius, deep laminae and ventral horn of the spinal cord. However, during the late inflammatory phase, US significantly triggered c-Fos expression in most laminae, particularly in the nucleus proprius, deep laminae and ventral horn of the spinal cord. The results of our study suggest that administration of US causes a reduction of early nociceptive inflammatory processing, as shown by a decrease in CFA-induced c-Fos-LI neurons at the level of the spinal cord. In contrast, the US did not suppress, but rather enhanced, the number of c-Fos-LI neurons during the late inflammatory phase. The peripheral influences of US on the central modulation of the spinal nociceptive processing system is suggested and may reflect the work being done through the neuroplasticity of spinal cord in response to peripheral stimulation of US. Therefore, we propose a difference in spinal expression of c-Fos-LI neurons between effects of peripheral US stimulation in arthritic models that underlie early and late inflammatory pain. (E-mail: sherrie@sunrise.hk.edu.tw).     

Effect of genetic knockout or pharmacologic inhibition of neuronal nitric oxide synthase on complete Freund's adjuvant-induced persistent pain

Nitric oxide (NO) acts as a neurotransmitter or neuromodulator involving in the modulation of thermal and/or inflammatory hyperalgesia. The neuronal nitric oxide synthase (nNOS) is a key enzyme for NO production in normal neuronal tissues, but its functional role in chronic pain remains unclear. The present study combined a genetic strategy with a pharmacologic approach to address the role of nNOS in the central mechanism of complete Freund's adjuvant (CFA)-induced chronic inflammatory pain. Targeted disruption of the nNOS gene significantly reduced CFA-induced mechanical pain hypersensitivity during the maintenance (but not the development) of inflammatory pain, while it failed to attenuate either development or maintenance of CFA-induced thermal pain hypersensitivity. Intraperitoneal administration of L-N(G)-nitro-arginine methyl ester (L-NAME), a non-specific NOS inhibitor, blocked CFA-evoked thermal and mechanical pain hypersensitivity at both development (2h) and maintenance (24h) phase in wild type mice, but had no effect in the knockout mice. Furthermore, intrathecal injection of either L-NAME or 7-nitroindazole, a selective nNOS inhibitor, markedly attenuated mechanical pain hypersensitivity at both 2 and 24h after CFA injection. Finally, spinal cord nNOS (but not endothelial NOS or inducible NOS) expression was up-regulated at 24h after CFA injection, occurring mainly in the ipsilateral superficial dorsal horn. Together, these data indicate that spinal cord nNOS may be essential for the maintenance of mechanical pain hypersensitivity and that it may also be sufficient for the development of mechanical pain hypersensitivity and for the development and maintenance of thermal pain hypersensitivity after chronic inflammation. Our findings suggest that spinal cord nNOS might play a critical role in central mechanisms of the development and/or maintenance of chronic inflammatory pain.     

Chronic secondary hypersensitivity of dorsal horn neurones following inflammation of the knee joint

Intra-articular injection of Freund's complete adjuvant (FCA) into the rat knee joint produces a swelling of the joint and long lasting hypersensitivity. In this study we have used this model and in vivo electrophysiology to investigate the time course of spinal changes underlying chronic secondary hypersensitivity, by stimulating the ankle joint (an area outside the site of primary hypersensitivity), and have compared the results with behavioural data from the same population of animals at 4-8, 13-17 and 55-59 days following FCA injection. The magnitude of responses and the proportion of dorsal horn neurones receiving inputs from A beta- A delta- and C-fibre afferents were monitored. At all time points, there was a significant increase in the ongoing activity of deep dorsal horn neurones when compared to nai ve rats, correlating well with the behavioural hypersensitivity. Both the magnitude of neuronal responses, and the proportion of neurones responding to electrical or mechanical stimulation in an area of secondary hypersensitivity, were significantly increased 4-8 and 13-17 days following FCA injection. However, while there was still behavioural hypersensitivity at 55-59 days there was a substantial decline in the responses to mechanical stimulation and A-fibre responses to electrical stimulation, although the proportion of neurones responding in the C-fibre latency remained elevated. These results suggest that the behavioural hypersensitivity is due to hyperexcitability at the level of the dorsal horn reflected as an increase of both C-fibre responses and spontaneous activity.     

Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund’s adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats

The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum- and glucocorticoid-inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1)/Rab4-dependent GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund’s adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4-5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)-GRASP-1, GRASP-1-Rab4, and Rab4-GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA-enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP-1, Rab4, and GluR1 by immunofluorescence. GSK-650394 (an SGK1 activation antagonist, 1, 10, and 30 μM, 10 μL/rat, intrathecally) dose-dependently prevented CFA-induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein-protein interactions at 1 day after CFA administration. Intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPAR antagonist, 1, 3, and 10 μM, 10 μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA-induced hyperalgesia and the associated GluR1 trafficking and Rab4-GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP-1/Rab4 cascade, plays a pivotal role in CFA-induced inflammatory pain by regulating GluR1-containing AMPAR recycling in the dorsal horn.