【作者】 姚永兴；

【导师】 姜桢； 赵志奇； 张玉秋；

【作者基本信息】 复旦大学 ， 麻醉学， 2008， 博士

【摘要】 疼痛是一种与组织损伤或潜在的损伤相关的,人类共有而个体差异很大的不愉快的主观感觉和情感体验。疼痛提供机体受到威胁的警报信号,同时也给患者带来痛苦。疼痛按时程可以分为急性疼痛和慢性疼痛。各种原因引起的慢性疼痛是导致劳动力丧失的重要原因。在过去的几十年中,从外周到中枢,对疼痛及其机制的研究取得了长足的进展,发现了一些新的药物作用靶点,并且开发了多种用于治疗的药物。但是,仍有大量病人时刻遭受着疼痛的折磨。前扣带皮层(anteior cingulate cortex,ACC)是边缘系统的重要组成部分。ACC接受来自皮层下多种信息的传入又可恒定地被外周伤害性刺激所激活,成为近年来疼痛研究的热点区域。研究表明ACC不仅参与痛情绪的形成,还对疼痛感觉有调节作用。但是,外周伤害性刺激的持续传入使ACC产生可塑性变化的分子机制,仍然知之甚少。研究认为,中枢突触功能的重塑是疼痛慢性化的重要机制。谷氨酸及其受体系统是介导伤害性信息传递和突触重塑最重要的递质/受体系统。突触后致密物(postsynaptic density,PSD)是新近在谷氨酸能突触后膜中发现的一种特殊结构。PSD中的多种蛋白质,能够在突触水平对谷氨酸受体进行募集整合。Homerlb/c是PSD家族的一员,最近研究发现Homerlb/c与慢性疼痛的发生有关,但未有进一步的研究报道。在慢性疼痛的研究中,完全弗氏佐剂(CFA)致大鼠慢性单关节炎模型(MA)和大鼠坐骨神经慢性缩窄(chronic constriction injury,CCI)模型是两种广泛应用的慢性痛模型,良好地模拟了人类慢性疼痛所表现的自发痛和刺激诱发痛等现象。本研究应用动物行为学、免疫组织化学、免疫沉淀和共沉淀、以及Western-Blot等方法,在大鼠关节炎和坐骨神经慢性缩窄模型上探讨了突触蛋白Homerlb/c在介导慢性疼痛发生发展中的可能作用及其机制,为研究慢性疼痛的发病机理及寻找有效的药物靶位,提供实验依据。本实验分为三个部分:1.在大鼠关节炎和坐骨神经慢性缩窄模型上,应用免疫组织化学、免疫沉淀和共沉淀等方法,检测了脊髓背角以及前扣带皮层(ACC)Homerlb/c的表达以及Homerlb/c与另一种突触蛋白PSD-95的相互作用;2.应用动物疼痛指标测定的方法,观察了鞘内注射Homerlb/c反义寡核苷酸对关节炎大鼠机械痛敏和热痛敏的影响;3.应用免疫组织化学染色,在大鼠鞘内注射Homerlb/c反义寡核苷酸后,观察Homerlb/c在脊髓背角Fos表达和cAMP反应元件结合蛋白(CREB)磷酸化中的作用。结果发现:1.Homerlb/c在脊髓背角的表达特征与其他PSD蛋白的表达方式相似,Homerlb/c阳性细胞主要集中在脊髓背角浅层,在其他层次只看到很浅的免疫反应阳性信号。在高倍镜下观察,Homerlb/c免疫反应阳性物位于细胞膜,细胞呈圆形或椭圆形。免疫印迹结果发现,Homerlb/c在大鼠脊髓背角有较高表达,而脊髓腹角未检测到信号。2.CFA致炎后,大鼠脊髓背角Homerlb/c的表达增加在CFA致炎后,大鼠脊髓背角Homerlb/c的表达呈单侧性增加,大鼠脊髓背角Homerlb/c和PSD-95的共沉淀增加。而PSD-95的表达未见增加。相反,在炎症的基础上,对大鼠踝关节施加强制曲伸的急性刺激(retrival)后,PSD-95的表达减少。3.在CFA致炎后,大鼠ACC Homerlb/c和PSD-95表达以及相互作用发生变化大鼠在CFA致炎后,对侧ACC Homerlb/c和PSD-95表达增加;对侧ACCHomerlb/c和PSD-95的共沉淀增加。该结果说明,在慢性疼痛状态下,突触后膜信号蛋白的相互作用加强。4.Homerlb/c参与神经病理性疼痛的维持与先前研究结果一致,大鼠坐骨神经慢性缩窄性损伤(CCI)后,损伤同侧脊髓背角Homerlb/c表达增加;我们同时发现,损伤对侧ACC部位Homerlb/c和PSD-95的共沉淀增加。说明Homerlb/c同样参与神经病理性疼痛的维持。5.抑制Homerlb/c的表达对MA大鼠的痛敏反应有减轻作用鞘内应用Homerlb/c反义寡核苷酸(10μg/10μl),每日1次,连续4天,抑制大鼠脊髓背角Homerlb/c的表达的同时,减轻了炎症痛大鼠对机械刺激和热刺激的痛敏反应。这种作用具有剂量依赖性。6.鞘内给予Homerlb/c反义寡核苷酸(ODN)对大鼠急性疼痛和运动功能无影响鞘内给予错义寡核苷酸(10μg/10μl)或反义寡核苷酸(10μg/10μl),连续4天,测定机械和热刺激反应阈值。结果发现,与给药前比较,反义寡核苷酸对正常动物的机械缩足反射阈值(PWT)和热缩足反射潜伏期(PWL)无显著影响。对大鼠后肢的回缩,抓握功能以及整体动物的翻正反射评分显示,鞘内给予Homerlb/c反义寡核苷酸(ODN)对大鼠运动功能无影响。7.Homerlb/c介导初级中枢伤害性感受免疫组化结果显示,鞘内应用Homerlb/c反义寡核苷酸,10μg/10μl,每日1次,连续4天,减轻了MA大鼠对伤害性刺激后,脊髓背角Fos蛋白的表达和CREB的磷酸化。该结果表明,Homerlb/c介导慢性疼痛时,初级中枢伤害性感受的信号转导,以及下游的突触功能的重塑。由以上结果得出如下结论:1.脊髓背角和前扣带皮层Homerlb/c参与了慢性炎症性疼痛和神经病理性疼痛的维持;2.慢性疼痛状态下,脊髓Homerlb/c介导初级中枢伤害性感受信号的转导和基因转录;3.前扣带皮层(ACC)PSD蛋白的变化可能是高级中枢参与慢性疼痛调制的分子机制。更多还原

【Abstract】 Chronic pain still a problem that continues to plague us,even after more than a decade of aggressive efforts at improvement.Persuasive epidemiologic evidence, mainly drawn from developed nations,has proven that chronic pain is a widespread public health issue.Despite the heterogeneity of study methods,surveys find that 15%-25%of adults suffer from chronic pain at any given time,a figure that increases to 50%in those older than 65 yr.Chronic pain is linked with a constellation of maladaptive physical,psychologic,family,and social consequences,and can be regarded as a disease entity per se.Inadequately treated pain has major physiological, psychological,economic,and social ramifications for patients,their families,and society.The majority of research into neuropathic pain mechanisms has concentrated on changes in the peripheral nerve or spinal cord after peripheral nerve injury and, therefore,most available evidence relates to changes in these parts of the nervous system.Nevertheless,it is important to recognize that alterations in the brain have also been demonstrated following peripheral nerve injury,but much less is known about the significance of these changes.It is well known that the excitatory amino acid glutamate is the major excitatory neurotransmitter released at the central terminals of primary afferent nociceptive neurones after noxious stimulation.Whilst glutamate acts at a number of post-synaptic receptors.Previous studies have demonstrated that postsynaptic protein, Homer 1b/c,a molecular scaffolding protein that binds and clusters mGluR receptors at neuronal synapses,plays an important role in the signaling process of GluRs. Moreover,a large body of evidence suggests that the ionotropic NMDA sub-type is one of the most intimately involved in both inflammation and nerve injury-induced central sensitization.Previous studies have demonstrated that postsynaptic protein-95(PSD-95),that binds and clusters NMDA receptors,plays an crucial role in the signaling process.Chronic monoarthritis(MA),induced by intraarticular injection of complete Freund’s adjuvant(CFA) into the tibiotarsal joint,provides a localized and stable form of unilateral inflammation with animals displaying hyperalgesia and allodynia and is very suitable for studies of persistent(nociceptive) arthritic pain.Chronic MA is one of the most used animal models to study neuronal plasticity and chronic pain.Besides monoarthritis,chronic constriction inury(CCI) of the sciatic nerve is another one of most used experimental mononeuropathy model in chronic pain research.The current study,utilizing animal behavioral test,immunohistochemistry and co-immunoprecipitation,investigated the possible involvement of Homer 1b/c and PSD-95 in the maintenance of inflammatory pain induced by complete Freund’s adjuvant(CFA),and neuropathic pain induced by chronic constriction inury(CCI) of the sciatic nerve in rats.The results are as follows:1.Exclusive expression of Homer1b/c protein in the spinal cord.Immunohistochemistry found that Homer1b/c immunoreactivity was localized mainly in laminaeⅠ-Ⅱand outer laminaⅢ.Under high magnification,Homer1b/c immunoreactive neuronal cell bodies were observed in the dorsal horn.Western blot confirmed the high level of Homer1b/c expression in the dorsal hom,and the undetectable level in the ventral hom.2.Increased expression of Homer1b/c following inflammatory pain induced by CFA.Injection of CFA into the tibio-tarsal joint of the rat produced a stable monoarthritic model.Injected ankle joint showed redness and swelling at day 1 after CFA injection,and maintained for 14 days or longer.Unilateral intra-articulation of CFA induced significantly increased expression of Homer1b/c in the ipsilateral spinal dorsal hom.While expression of PSD-95 is not affected by CFA-induced inflammatory pain,moreover,pain retrival of the inflammatory ankle significantly decreased expression of PSD-95 in the ipsilateral spinal dorsal horn.3.Increased interaction of PSD protein-95 and Homer1b/c following chronic painCo-immunoprecipitation of PSD protein-95 and Homer1b/c is increased significantly in the spinal dorsal horn and(anteior cingulate cortex,ACC) following CFA-induced inflammatory pain,but acute pain retrival of the inflammatory ankle significantly decreased co-immunoprecipitation of these two proteins in the ipsilateral spinal dorsal horn.4.Homer1b/c mediates neuropathic painUnilateral CCI induced significantly increased expression of Homer1b/c in the Ipsilateral spinal dorsal horn,and co-immunoprecipitation of PSD protein-95 and Homer1b/c is increased significantly in the contralateral ACC following CCI-induced neuropathic pain.This result suggested that Homer1b/c,both in ACC and spinal dorsal hom,plays role in the maintance of neupathic pain induced by chronic constriction inury of the sciatic nerve.5.Homer1b/c ASODN attenuated paw withdrawal responses to mechanical Stimuli Intrathecal injection of the the highest doses(10μg/10μl) of Homer1b/c antisense oligonucleotide(ASODN) every 24 h for 4 days,reduced expression of Homer1b/c protein in the spinal cord.Meanwhile,Homer1b/c ASODN attenuated CFA-induced mechanical hyperalgesia at day 3,4,5 and 6 after ODN injection.The lowest dose(2.5μg/10μl) of Homer1b/c AS ODN and missense ODN(10μg/10μl) had no effect.6.Homer1b/c ASODN attenuated paw withdrawal responses to thermal stimuliIntrathecal injection of a higher dose(5μg and 10μg/10μl) of Homer1b/c AS ODN every 24 h for 4 days,reduced CFA-induced thermal hyperalgesia significantly at day 3,4 after ODN injection.The low dose(2.5μg/10μl) of Homer1b/c AS ODN and Missense ODN(10μg/10μl) had no effect on paw withdrawal responses to thermal stimuli.7.Homer1b/c ASODN has no effect on acute painFollowing the intrathecal injection of antisense ODN(10μg/10μl) every 24 h for 4 days,no significant change of behavioral was found in paw-withdrawal threshold (PWT) to mechanical stimuli with von Frey filaments and paw-withdrawal latencies (PWL) to heat stimuli.Denotes knock down of Homer1b/c in the spinal has no effect on acute pain in intact animals.8.Homer1b/c mediates c-fos expression and CREB phosphorylationExpression of Fos protein and phosphorylated CREB(pCREB) in the spinal dorsal horn following retrival of inflammatory pain induced by CFA injection was reduced by intrathecal injection of Homer1b/c anti-sense ODN.This suggests expression of Fos protein and pCREB following retrival is,at least partly,mediated by Homer1b/c.In conclusion,the present study demonstrated that1.Homer1b/c in the spinal dorsal horn and ACC plays role in the maintance of CFA-induced inflammatory pain and neuropathic pain.2.Interaction of Homer1b/c and PSD-95 may be the possible mechanism underlying chronic pain.3.Homer1b/c mediates c-fos expression and CREB phosphorylation in the spinal dorsal horn following retrival of inflammatory nociception.更多还原