前扣带皮层神经元μ阿片受体介导电针刺激缓解痛情绪的研究

发布时间：2018-07-05 15:12

本文选题：痛情绪 + 条件性位置回避(CPA)　；参考：《山西医科大学》2015年硕士论文

【摘要】：目的:疼痛包含两种成分,即痛的感觉分辨(sensory-discriminative)与痛的情绪反应(emotional-affective dimensions)[1]。痛的感觉分辨感受痛刺激的性质、强度和位置等属性;痛的情绪体验包括焦虑、厌恶、恐惧等不良情绪。大量证据表明,电针刺激可以缓解疼痛的感觉分辨,且电针是通过激活体内的阿片受体来发挥缓解疼痛的作用[2-6]。一些临床研究和动物实验表明,电针刺激也可以缓解人和动物的痛情绪反应,但作用机制尚不清楚[7,8]。人类和动物研究发现,ACC(anterior cingulate cortex,ACC)的吻侧部(r ACC)在痛情绪中有着重要的作用[9-12]。有实验证明,痛情绪引起的条件性位置回避(conditioned place avoidance,CPA)反应可通过切除大鼠双侧r ACC得到缓解[13-16]。因此,我们推测伤害性刺激引起的痛情绪反应,可能会通过激活r ACC神经元的阿片受体得到缓解。前期实验中,我们在大鼠r ACC内给予μ-阿片受体激动剂DAMGO,结果发现其可缓解由完全弗氏佐剂(complete Freund’s adjuvant,CFA)诱导的条件性位置回避,但不影响大鼠的热缩足反射潜伏期(paw withdrawal latency,PWL)[17]。由此得出结论,大鼠r ACC内μ-阿片受体的活化缓解了大鼠的痛情绪反应,且此过程不依赖于大鼠的疼痛感觉分辨而实现。基于前期实验结果,我们进一步推测,电针刺激缓解痛情绪反应也可能通过r ACC内μ-阿片受体介导。实验过程中,我们先观察电针刺激是否可以缓解CFA诱导的CPA反应,然后预先在大鼠r ACC内注射μ-阿片受体拮抗剂CTOP后再给予电针刺激,观察r ACC内μ-阿片受体的拮抗剂是否可反转电针缓解痛情绪的作用,并试图对其机制进行探讨。方法:1.建立由CFA诱导的持续性疼痛模型取250-270g成年雄性SD(Sprague Dawley)大鼠,左侧足底皮下注射0.08ml完全弗氏佐剂(Complete Freund’s adjuvant,CFA),建立炎症性疼痛模型。对照组为左侧足底皮下注射0.08ml生理盐水(NS)。2.建立由CFA诱导的条件性位置回避(CPA)模型将250-270g成年雄性SD大鼠,随机分为2组:(1)足底注射NS(0.08ml)组;(2)足底注射CFA(0.08ml)组。比较大鼠脚掌注射CFA前后大鼠在痛环境中停留的时间,并计算回避分数(CPA Score),判断大鼠是否产生CPA反应。3.电针刺激取成年雄性SD大鼠250-270g,随机分为真电针组(electroacupuncture EA)和假电针组(sham EA)。在脚掌注射NS/CFA后,将针灸针插入大鼠双侧环跳穴(GB30),用胶带固定于大腿。通过导线将针灸针与电针仪相连,通电后开始电针刺激。4.检测热缩足反射潜伏期(Paw Withdrawal Latency,PWL)在CPA的第一天和第三天分别检测各组大鼠的PWL,观察CFA对大鼠的热痛行为反应的影响,以及给予电针刺激、r ACC注射不同剂量CTOP后大鼠PWL数值的变化。5.实验分组根据左侧足底皮下注射、r ACC区给药和双侧环跳穴刺激将大鼠分为以下8组。(1)足底注射NS,r ACC注射NS,EA;(2)足底注射NS,r ACC注射NS,sham EA;(3)足底注射CFA,r ACC注射NS,EA;(4)足底注射CFA,r ACC注射NS,sham EA;(5)足底注射NS,r ACC分别注射2/10/20(nmol/μl)CTOP,EA;(6)足底注射NS,r ACC分别注射2/10/20(nmol/μl)CTOP,sham EA;(7)足底注射CFA,r ACC分别注射2/10/20(nmol/μl)CTOP,EA;(8)足底注射CFA,r ACC分别注射2/10/20(nmol/μl)CTOP,sham EA。结果:1.足底注射CFA可诱导大鼠产生条件性位置回避(CPA)反应。足底注射CFA与足底注射NS后相比,大鼠的热缩足反射潜伏期(PWL)明显缩短(9.1±1.0 s vs 13.1±1.9 s,P0.05,n=5),足底注射CFA可引起大鼠产生炎症性疼痛。足底注射NS组,(Post 352.1±113.8 s vs Pre 317.2±67.8 s,P0.05,n=5),大鼠在痛环境停留时间第三天和第一天相比没有显著性差异。足底注射CFA组,(Post151.4±58.38 s vs Pre 323.5±105.5 s,P0.05,n=6),大鼠在痛环境停留时间第三天明显小于第一天,有统计学差异。足底注射CFA组和足底注射NS组回避分数之间有显著性差别(-172.1±97.3 s vs 12.6±81.2 s,P0.05,n1=5,n2=6),足底注射CFA组回避分数比较大,对痛环境产生厌恶反应。2.电针刺激可缓解CFA诱导的大鼠条件性位置回避(CPA)反应。大鼠左侧足底注射CFA后,在双侧环跳穴分别给予真电针刺激(EA)或者假电针刺激(sham EA)。炎性痛大鼠模型中,假电针刺激后,大鼠在痛环境停留的时间第三天和第一天相比缩短,有显著性差异,(Post 151.4±23.8 s vs Pre 323.5±43.1 s,P0.05,n=6);真电针刺激后,大鼠在痛环境中停留时间第三天与第一天相比没有统计学差异,(Post 248.9±144.1 s vs Pre 234.9±54.57 s,P0.05,n=5)。真电针组(EA)回避分数明显小于假电针组(sham EA),两组有统计学差异(13.9±47.2 s vs-172.1±39.7 s,P0.05,EA组n=5,sham EA组n=6)。大鼠左侧足底注射NS后,在双侧环跳穴分别给予真电针刺激(EA)或者假电针刺激(sham EA)。两组大鼠在痛环境中停留时间第三天与第一天相比均没有统计学差异,假电针组(Post 352.1±50.9 s vs Pre 317.2±30.3 s,P0.05,n=5);真电针组(Post250.2±112.4 s vs Pre 305.6±45.9 s,P0.05,n=5)。两组回避分数相比没有统计学差异(-64.49±52.9 s vs 12.6±36.3 s,P0.05,NS+sham EA组n=5,NS+EA组n=9)。电针刺激正常大鼠不会引起大鼠的厌恶或者奖赏反应。大鼠左侧足底注射CFA后,在双侧环跳穴分别给予真电针刺激(EA)或假电针刺激(sham EA),在第二天测量大鼠的热痛反应,PWL之间没有统计学差异(12.0±1.4s vs 9.1±0.4 s,P0.05,n=5),电针刺激双侧环跳穴不影响刺激后第二天由CFA引起的热痛行为。3.r ACC内注射CTOP反转了电针刺激缓解痛情绪的作用。足底注射CFA组,r ACC内注射μ-阿片受体拮抗剂CTOP后进行电针刺激。足底注射CFA组,在r ACC内注射NS后进行电针刺激,作为对照组。大鼠在痛环境中停留时间第三天与第一天相比:CTOP组(Post 210.5±59.8 s vs Pre 307.8±47.0 s,P0.05,n=5);NS组(Post 248.9±64.5 s vs Pre 234.9±24.4 s,P0.05,n=5)。CTOP组回避分数明显大于NS组,有显著性差异(-127.5±24.1 s vs 13.9±47.2 s,P0.05,n=5)。r ACC内注射CTOP反转了电针刺激缓解痛情绪的作用。足底注射CFA组,r ACC内注射μ-阿片受体的拮抗剂CTOP或NS,不进行电针刺激。大鼠在痛环境中停留时间第三天与第一天相比:CTOP组(Post 255.1±71.8 s vs Pre 403.5±46.2 s,P0.05,n=5);NS组(Post 151.4±23.8 s vs Pre 323.5±43.1 s,P0.05,n=6);CTOP组和NS组回避分数没有统计学差异(-148.4±50.5 s vs-172.1±39.7 s,P0.05,n=5)。r ACC内注射CTOP本身不会影响大鼠的痛情绪反应。r ACC内注射2/10/20(nmol/μl)的μ-阿片受体拮抗剂CTOP,反转电针刺激缓解痛情绪的作用效果不同。大鼠在痛环境中停留时间第三天与第一天相比:2 nmol/μl CTOP组与NS组之间没有统计学差异(-2.0±18.8 s vs 13.9±47.2 s,P0.05,n=7),10 nmol/μl CTOP组与NS组之间有统计学差异(-97.3±30.3 s vs 13.9±47.2 s,P0.05,n=7),20 nmol/μl CTOP组与NS组之间有统计学差异(-127.5±24.1 s vs 13.9±47.2 s,P0.05,n=7);10nmol/μl CTOP组与2 nmol/μl CTOP组之间有统计学差异(-97.3±30.3s vs-2.0±18.8 s,P0.05,n=7),20nmol/μl CTOP组与2 nmol/μl CTOP组之间有统计学差异(-127.5±24.1 s vs-2.0±18.8 s,P0.05,n=7),10nmol/μl CTOP组与20 nmol/μl CTOP组之间没有统计学差异(-97.3±30.3 s vs-127.5±24.1 s,P0.05,n=7)。预先在r ACC内注射2 nmol/μl CTOP未观察到反转电针缓解痛情绪的作用,预先在r ACC内给予10nmol/μl CTOP则反转了电针缓解痛情绪的作用,预先在r ACC内给予20nmol/μl CTOP反转电针缓解痛情绪的作用更强,但与10nmol/μl CTOP的作用相比没有统计学差异。大鼠左侧足底注射CFA,r ACC注射2/10/20(nmol/μl)CTOP的各组大鼠PWL值与r ACC注射NS相比,都没有统计学差异(P0.05)。各组PWL值为:CFA+NS组10.9±0.9 s,n=6;CFA+2nmol/μl CTOP组7.2±0.6 s,n=5;CFA+10nmol/μl CTOP组11.0±1.3 s,n=5;CFA+20nmol/μl CTOP组8.1±2.1 s,n=5。r ACC内注射CTOP不改变CFA引起的热痛行为。结论:1.电针刺激可缓解CFA诱导的大鼠CPA反应(痛情绪)。2.电针刺激缓解CFA诱导的痛情绪的作用是通过激活r ACC内μ-阿片受体实现。
[Abstract]:Objective: the pain consists of two components: the properties, intensity and location of sensory discrimination of [1]. pain in the pain of sensory discrimination (sensory-discriminative) and painful emotional response (emotional-affective dimensions); the emotional experience of pain includes anxiety, dislikes, and fear. The sensory discrimination of pain is alleviated and the electroacupuncture is activated by activating the opioid receptor in the body to play a role in alleviating pain. [2-6]. some clinical studies and animal experiments have shown that electroacupuncture can also relieve the pain and emotional responses of humans and animals, but the mechanism of action is not yet clear in [7,8]. human and animal studies, ACC (anterior cingulate cortex, A) CC (R ACC) plays an important role in the pain mood. [9-12]. has proved that the conditioned place avoidance (conditioned place avoidance, CPA) reaction caused by pain emotion can be relieved by the removal of bilateral R ACC in rats. We speculate that the pain emotional response caused by nociceptive stimuli may be activated by activating R God. The opioid receptor of the trans element was relieved. In the previous experiment, we gave the micron opioid receptor agonist DAMGO in the rat R ACC, and found that it could alleviate the conditional position avoidance induced by the complete Freund's adjuvant (complete Freund 'adjuvant, CFA), but did not affect the latent period of the rat's heat contraction reflex (paw withdrawal latency, PWL). It is concluded that the activation of micron opioid receptor in R ACC in rats relieves the pain response of rats, and this process is not dependent on the pain sensation resolution of rats. Based on the previous experimental results, we further speculate that the electroacupuncture stimulation may also be mediated by the R ACC opioid receptor. Whether the electroacupuncture stimulation can relieve the CPA response induced by CFA, and then injecting the micron opioid receptor antagonist CTOP in the rat R ACC to the electroacupuncture stimulation in advance, observe whether the antagonist of the R opioid receptor in R ACC can reverse the effect of the electroacupuncture on the pain emotion, and tries to explore the mechanism of it. Method: 1. the establishment of CFA induced by CFA The model of 250-270g adult male SD (Sprague Dawley) rats was taken from the persistent pain model, and the left foot was injected subcutaneously with 0.08ml complete Freund's adjuvant (Complete Freund 's adjuvant, CFA), and the inflammatory pain model was established. The control group was subcutaneously injected with 0.08ml physiological saline (NS) to establish a conditioned location avoidance model. The adult male SD rats of 250-270g were randomly divided into 2 groups: (1) the plantar injection of NS (0.08ml) group; (2) the plantar injection of CFA (0.08ml) group. The time of the rats' paws before and after the injection of CFA in the pain environment was compared and the avoidance fraction (CPA Score) was calculated to determine whether the rat generated CPA reaction.3. electroacupuncture stimulation to take adult male SD rats, random. It is divided into the true electroacupuncture group (electroacupuncture EA) and the false electroacupuncture group (sham EA). After the injection of NS/CFA in the foot, the acupuncture needle is inserted into the bilateral ring jump point (GB30) in the rat, and the needle is attached to the thigh with the adhesive tape. The acupuncture needle is connected with the electroacupuncture instrument through the wire. The electroacupuncture stimulates.4. to detect the latent period of the reflex (Paw Withdrawal Latency, PWL) in the CPA. The PWL of rats in each group was detected on the first and third days, and the effect of CFA on the heat pain behavior of rats was observed and the stimulation of electroacupuncture was given. The PWL value of R ACC was given after CTOP in different doses of CTOP. The.5. experiment group was subdivided into 8 groups according to the left foot, and the R ACC district administration and bilateral ring hopping stimulation were divided into the following groups. (1) foot Injection of NS, R ACC injection NS, EA; (2) foot injection NS, R ACC injection NS, sham EA; (3) foot injection CFA. Do not injecting 2/10/20 (nmol/ L) CTOP, EA; (8) CFA in the foot, R ACC injection 2/10/20 (nmol/ Mu L) CTOP, which can induce conditioned place avoidance response in rats. .05, n=5), the plantar injection of CFA could cause inflammatory pain in rats. NS group was injected into the plantar group (Post 352.1 + 113.8 s vs Pre 317.2 + 67.8 s, P0.05, n=5). There was no significant difference between the rats in the pain environment for third days and the first day. There was a significant difference between the third days of the residence time of third days. There were significant differences between the foot injection group and the NS group (-172.1 + 97.3 s vs 12.6 + 81.2 s, P0.05, n1=5, n2=6). The avoidance fraction of the foot injection CFA group was larger, and the negative reaction to the pain ring was relieved by CFA induction. Rat conditioned place avoidance (CPA) reaction. After CFA was injected into the left foot of the rat, real electroacupuncture stimulation (EA) or pseudo electroacupuncture stimulation (sham EA) were given at the bilateral ring jump points. In the model of inflammatory pain rats, the time of the rats in the pain environment was shortened third days compared to the first day, and there was a significant difference (Post 151.4 + 23.8 s VS). Pre 323.5 + 43.1 s, P0.05, n=6); after real electroacupuncture stimulation, there was no statistical difference between the third days in the pain environment and the first day (Post 248.9 + 144.1 s vs Pre 234.9 + 54.57 s, P0.05, n=5). The true electroacupuncture group (EA) avoidance score was significantly smaller than that of the false electroacupuncture group, and the two groups had statistical differences (13.9 + 47.2 S, P0.05, group EA, n=5, sham EA n=6). After injection of NS in the left foot of the rat, the third days of the two groups of rats in the pain environment were not significantly different from those in the first day (sham EA). True electroacupuncture group (Post250.2 + 112.4 s vs Pre 305.6 + 45.9 s, P0.05, n=5). There was no statistical difference between the two groups (-64.49 + 52.9 s vs 12.6 + 36.3 s). No real electroacupuncture stimulation (EA) or pseudo electroacupuncture stimulation (sham EA) was used to measure the heat pain response of rats in second days. There was no statistical difference between PWL (12 + 1.4s vs 9.1 + 0.4 s, P0.05, n=5). Electroacupuncture stimulation of bilateral ring hops did not affect the heat pain caused by CFA second days after stimulation. An injection of CFA in group CFA, R ACC injection of CTOP opioid receptor antagonist CTOP for electroacupuncture stimulation. The plantar injection of CFA group, the injection of NS in R ACC and electroacupuncture stimulation, as the control group. The duration of the stay in the pain environment was third days compared to the first day: CTOP group (Post 210.5 + 59.8 s vs 307.8 + 47 47 The avoidance score of OST 248.9 + 64.5 s vs Pre 234.9 + 24.4 s, P0.05, n=5) was significantly greater than that of the NS group, and there was a significant difference (-127.5 + 24.1 s vs 13.9 + 47.2). Rats in the pain environment were stopped for third days compared with the first day: group CTOP (Post 255.1 + 71.8 s vs Pre 403.5 + 46.2 s, P0.05, n=5), NS group (Post 151.4 + 23.8 s vs 323.5 + 43.1). The body did not affect the pain emotion response of the rats to the.R ACC injection of 2/10/20 (nmol/ Mu L) micron opioid receptor antagonist CTOP, and the effect of the reverse electroacupuncture stimulation on the pain emotion was different. The duration of the stay in the pain environment was third days compared with the first day: there was no statistical difference between the 2 nmol/ Mu L CTOP group and the NS group (-2.0 + 18.8 s 13.9 + 47.2) S, P0.05, n=7), there are statistical differences between the 10 nmol/ L CTOP group and the NS group (-97.3 + 30.3 s vs 13.9 + 47.2 s, P0.05, and P0.05). N=7), there were statistical differences between the 20nmol/ L CTOP group and the 2 nmol/ L CTOP group (-127.5 + 24.1 s vs-2.0 + 18.8 s, P0.05, 24.1). The effect of giving 10nmol/ Mu L CTOP in advance in R ACC reverses the effect of Electroacupuncture on relieving pain emotion, and the effect of giving 20nmol/ u l CTOP reverse electroacupuncture in R ACC to relieve pain emotion is stronger, but there is no statistical difference compared with 10nmol/ micron L. The PWL values of rats were not statistically different from that of R ACC injection NS (P0.05). The PWL values in group CFA+NS were 10.9 + 0.9 s, n=6 and CFA+2nmol/ u l CTOP group 7.2 + 0.6. The effect of CFA induced CPA response (pain).2. electroacupuncture on alleviating CFA induced pain was achieved by activating the R opioid receptor in R ACC.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R402

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