【作者】 高永静；

【导师】 赵志奇；

【作者基本信息】 复旦大学 ， 神经生物学， 2004， 博士

【摘要】 疼痛是一种与组织损伤或潜在损伤相关的不愉快的主观感觉和情绪体验。该定义赋予疼痛两重含义:感觉分辨和情绪反应。近四十年来,对疼痛感觉分辨机制的研究已取得了很大进展。然而,由于缺乏痛情绪研究的行为学指标和相应的动物模型,极大地限制了对痛情绪机制的研究。调查表明,临床上慢性疼痛患者遭受的恶性情绪体验,如焦虑、恐惧、孤独、甚至厌世等给病人造成的身心伤害远比疼痛本身更为严重。因此,阐明痛情绪反应的机制对于我们全面、深入地理解疼痛的本质以及治疗和缓解慢性顽固性疼痛病人的不良情绪反应具有重要意义。前扣带皮层(Anterior Cingulate Cortex, ACC)是边缘系统的重要结构。大量证据表明,ACC神经元的激活与情绪、记忆和疼痛密切相关。电生理研究显示ACC部分神经元对伤害性刺激或预示伤害性刺激的环境信息产生反应。PET研究表明,伤害性刺激和疼痛引起的不愉快可以激活ACC。动物行为学研究显示,ACC参与介导热板刺激、福尔马林诱导的条件性位置回避以及激光痛刺激等引起的情绪样反应。我们最近的研究显示,对福尔马林诱导的条件性位置回避进行“唤醒”后,ACC内的Fos表达显著增加。临床上,切除包括ACC在内的皮层组织可以减轻病人的痛情绪,但不影响对痛的刺激强度和部位的判断。因此,这些研究表明,ACC参与痛感觉和痛情绪的信息加工。cAMP反应元件结合蛋白(CREB)是细胞核内的重要转录因子,它参与长时记忆和持续性疼痛诱导的脊髓背角神经元中枢敏化的形成。细胞外信号调节激酶(ERK)是 有丝分裂原激活的蛋白激酶(MAPK)家族的成员之一,它的磷酸化可引起CREB激活。到目前为止,CREB是否是痛相关情绪反应中的关键因子,以及MAPK/ERK信号通路介导的CREB的激活是否参与痛相关的情绪反应还不得而知。 <WP=7>本研究探讨了ACC参与痛厌恶情绪的神经生物学机制,主要结果如下:1.采用荧光金(FG)逆行束路追踪技术研究ACC的传入投射。将3%的荧光金(0.2μl)注入到大鼠吻侧ACC(rACC)或尾侧ACC(cACC)。7天以后灌注取材,将40μm厚的切片贴于载玻片于荧光显微镜下观察。结果显示:FG注射在rACC或cACC,阳性神经元的分布区域没有显著不同,且FG阳性细胞主要分布在注射区的同侧。ACC接受皮层和皮层下的许多结构的纤维投射,包括:丘脑中线核群和板内核群、杏仁核、次级视皮层、次级听皮层、外嗅皮层和嗅周皮层等。表明ACC不但接受来自丘脑的伤害性信息传入,也接受来自视、听、嗅皮层等的环境信息的传入,为ACC参与痛的感觉分辨和情绪反应提供了形态学证据。2.采用疼痛刺激(大鼠后爪注射福尔马林)或非疼痛性恐惧刺激(0.5mA电击)与条件位置装置相结合的方法,建立了四天训练模式的福尔马林诱导的条件性位置回避(F-CPA)模型和电击诱导的条件性位置回避(S-CPA)模型。F-CPA模型可以反映动物由于受到伤害性刺激而产生的不良情绪状态(痛厌恶);S-CPA反应大鼠的恐惧厌恶情绪。在条件训练中,足底注射福尔马林以及电击都可以使大鼠产生条件性位置回避。微量注射兴奋性毒素损毁大鼠的ACC或杏仁核, F-CPA程度明显降低。但S-CPA程度的降低只见于杏仁核损毁大鼠,ACC损毁对S-CPA没有显著影响。ACC或杏仁核损毁后,福尔马林引起的急性的痛行为反应未见明显改变。提示杏仁核参与痛和恐惧引起的厌恶情绪;大鼠ACC特异性参与疼痛刺激引起的厌恶反应。3.在F-CPA训练的第二天,足底注射福尔马林前10分钟,双侧ACC内微量注射MAPK激酶的抑制剂PD98059,部分阻断大鼠痛厌恶反应;但单侧注射PD98059或双侧注射溶剂,不能阻断F-CPA的形成。双侧ACC内注射PD98059不影响福尔马林引起的大鼠急性伤害性行为。4.免疫细胞化学和Western Blot研究显示F-CPA的“唤醒” 能够诱导CREB和ERK在ACC的激活,与非“唤醒”组大鼠相比,ACC内pCREB(磷酸化的CREB)和pERK(磷酸化的ERK)的激活明显升高。F-CPA诱导的pERK和pCREB的激活高峰分别在痛经历“唤醒”后的5 min和30 min。对照组(假<WP=8>训练和假唤醒)的大鼠ACC内的pERK和pCREB的激活明显低于F-CPA组。同时,福尔马林注射前,双侧ACC给予PD98059的大鼠,“唤醒”后pERK和pCREB的激活也降低。以上各不同处理组的大鼠ACC内非磷酸化的CREB和ERK没有显著变化。5.单独皮下注射福尔马林诱导ACC内CREB和ERK的磷酸化。pERK和pCREB的激活高峰也分别在福尔马林刺激后的5 min和30 min。非磷酸化的CREB和ERK没有显著变化。综上所述, 大鼠ACC参与痛厌恶情绪的产生;ACC内的ERK-CREB信号通路在福尔马林引起的痛厌恶情绪的形成和“唤醒”中有重要作用。更多还原

【Abstract】 Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, which includes a sensory-discriminative and an affective-motivational component. During past decades, a great progress has been made on the study of the sensory-discriminative component of pain. However, the data on the pain-related affective aspect are quite limited, due to the lack of a behavioral index or available animal model relating to the affective component. A line of investigation indicated the patients with chronic pain in clinic suffer from much more the emotional disturbance, such as anxiety, fear, pessimism, than pain itself. Thus, clarify the mechanism of pain-related affective dimension is very important not only for deepening and expanding our understanding of pain but also for the cure and remission of chronic intractable negative emotion induced by pain.Anterior cingulate cortex is an important part of the limbic system. A great body of evidence indicates that ACC is related to emotion, memory and pain. Electrophysiological studies showed that some neurons within the ACC responded to either noxious stimulus per se or environmental cues that predict a painful stimulus. Positron emission tomography (PET) studies revealed that both noxious stimuli and pain-induced unpleasantness activated the ACC. Animal behavioral studies indicated that the ACC mediated the affective-like responses of tonic pain in hot-plate, formalin-induced conditioned place avoidance and laser-pain conditioning test. Our recent findings showed that following retrieval of formalin-induced conditioned place avoidance, Fos expression in the ACC was significantly increased. Clinically, surgical ablation of the ACC and surrounding cortical tissue obviously <WP=10>decreased pain-related unpleasantness/dysphoria without affecting the patient’s ability to discriminate the intensity or localization of the noxious stimulus. Taken together, these studies suggest that the ACC is involved in processing both of pain sensation and pain emotion. The cAMP-responsive element binding protein (CREB) is a transcription factor that is involved in the formation of long-term memory and the persistent pain-induced central sensitization of the spinal dorsal horn neurons. Extracellular signal-regulated kinase (ERK) is a member of mitogen-activated protein kinase (MAPK) family that may be linked to mediate the activation of CREB. It is unknown whether CREB is a critical factor for pain-related affective states and whether MAPK/ERK signaling cascade mediate the activation of CREB during the induction of pain-related emotion.The present study was mainly to address the neurobiological mechanisms underlying the roles of ACC in pain aversive affection.1. Using fluorogold (FG) retrograde tract-tracing technique, the afferent projection of the ACC was traced. FG (3%, 0.2 μl) was injected into unilateral rACC or cACC. Seven days later, the rats were perfused. Forty-micrometer-thick sections were mounted on slides and observed under microscopy. The results showed that the distribution of the FG-labeled cells had no significant difference between rACC injection or cACC injection. FG-labeled cells were mainly distributed in the ipsilateral injection site. The ACC received afferent axons from many cortical and subcortical areas, including the midline and intralaminar thalamic nuclei, amygdala, secondary visual cortex, secondary auditory cortex, ectorhinal and perirhinal cortex. It is indicated that the ACC receives not only the nociceptive information from the thalamus but also the environmental information from visual, auditory, olfactory cortex. The results provided morphological evidence for an involvement of ACC in pain sensation and emotion.2. Combination of painful stimulus (injection of formalin into the rat’s hindpaw) <WP=11>or unpainful stimulus (0.5 mA electric shock) with a place conditioning apparatus, we developed formalin-induced conditioned place avoidance (F-CPA) and electric foot-shock-induced conditioned pla更多还原