【摘要】：目的:慢性疼痛不僅是臨床癥狀,同時(shí)也是一種疾病。疼痛包括痛感覺(jué)和痛情緒兩種成分,前者反映疼痛的性質(zhì)、強(qiáng)度和位置等屬性,后者則包括了因痛而生的焦慮、厭惡、恐懼等不良情緒。隨著人們對(duì)疼痛認(rèn)識(shí)的深入,逐步發(fā)現(xiàn)慢性疼痛時(shí)往往痛情緒帶來(lái)比痛感受本身更為嚴(yán)重的傷害,因此對(duì)痛情緒的研究也日益受到關(guān)注。來(lái)自人類和動(dòng)物的行為學(xué)、電生理學(xué)和腦成像等相關(guān)研究表明,大腦額葉內(nèi)側(cè)前扣帶皮層(anterior cingulate cortex,ACC)作為情緒情感環(huán)路中的重要核團(tuán),其吻側(cè)部(rostral ACC,r ACC)在痛情緒的發(fā)生過(guò)程中起了十分重要的作用。眾所周知,電針可以激活阿片受體產(chǎn)生明顯的鎮(zhèn)痛效應(yīng),但是否同樣可以緩解痛情緒呢?這類資料目前還很少。我們先前的研究和相關(guān)實(shí)驗(yàn)室的資料都證實(shí)了電針也可以緩解痛情緒,而且還可通過(guò)激活μ-阿片受體和抑制NMDA受體對(duì)CFA誘導(dǎo)的條件位置回避(conditioned place avoidance,CPA)產(chǎn)生明顯的緩解作用。阿片受體有多種亞型,其中最受關(guān)注的有μ、δ和κ三種阿片受體亞型。如前所述,我們已經(jīng)證實(shí)μ-阿片受體參與介導(dǎo)了電針緩解痛情緒的作用,但其它受體亞型是否有類似的作用還不清楚。因此本研究將以δ-阿片受體為研究對(duì)象,利用大鼠腳掌注射CFA誘導(dǎo)的CPA(C-CPA)反應(yīng)作為痛情緒行為模型,采用行為檢測(cè)與多通道電生理學(xué)記錄技術(shù)相結(jié)合的手段探索激活r ACC腦區(qū)δ-阿片受體是否可以起到緩解痛情緒的作用?以及r ACC腦區(qū)δ-阿片受體是否介導(dǎo)電針緩解痛情緒的過(guò)程。方法:自制給藥管和帶給藥管的多通道電極,通過(guò)手術(shù)將自制的給藥管和帶給藥管的多通道電極埋置于大鼠r ACC腦區(qū),待術(shù)后一周大鼠完全恢復(fù)之后,給大鼠左側(cè)后腳掌注射完全弗氏佐劑(CFA),誘導(dǎo)產(chǎn)生炎性痛并與環(huán)境相匹配,建立條件性位置回避(conditioned place avoidance,CPA)反應(yīng)模型,r ACC腦區(qū)通過(guò)使用微量進(jìn)樣泵、PE給藥管注射藥物,通過(guò)大鼠腳掌回縮潛伏期(PWL)觀察痛感受行為變化,通過(guò)條件位置回避裝置觀察痛情緒行為變化,同時(shí)利用在體多通道技術(shù)記錄、處理、分析r ACC腦區(qū)神經(jīng)元的信號(hào)變化。使用雙因素進(jìn)行分組,具體模式如下:12組動(dòng)物(n=6-8只/組)=2(CFA,生理鹽水)×6(δ-激動(dòng)劑:5個(gè)劑量,生理鹽水);8組動(dòng)物(n=6-8只/組)=2(CFA,生理鹽水)×2(δ-拮抗劑,生理鹽水)×2(EA,sham EA)。結(jié)果:(1)大鼠建立條件位置回避(CPA)模型1)模型大鼠行為學(xué)觀察腳掌熱縮足反應(yīng)潛伏期比較,CFA+NS組“痛環(huán)境”匹配后明顯縮短,有統(tǒng)計(jì)學(xué)差異(P0.05),NS+NS組“痛環(huán)境”匹配前后沒(méi)有差異(P0.05),大鼠后腳掌左側(cè)注射CFA,產(chǎn)生炎癥性疼痛。NS+NS組“痛環(huán)境”匹配前后在“痛環(huán)境”停留時(shí)間比較沒(méi)有差異(P0.05),CFA+NS組“痛環(huán)境”匹配前后在“痛環(huán)境”停留時(shí)間比較,匹配后明顯縮短,有統(tǒng)計(jì)學(xué)差異(P0.05);CFA+NS組與NS+NS組回避分?jǐn)?shù)比較,前者產(chǎn)生明顯的回避反應(yīng),有統(tǒng)計(jì)學(xué)差異(P0.05)�！巴喘h(huán)境”匹配后在“痛環(huán)境”停留時(shí)間明顯縮短,而在“非痛環(huán)境”停留時(shí)間明顯延長(zhǎng),對(duì)“痛環(huán)境”產(chǎn)生明顯的回避,CFA誘導(dǎo)建立條件位置回避模型(CPA)。2)模型大鼠r ACC腦區(qū)神經(jīng)元?jiǎng)幼麟娢惶卣鞣治鯪S+NS組“痛環(huán)境”和“非痛環(huán)境”神經(jīng)元放電頻率比較,沒(méi)有差異(P0.05)。CFA+NS組“痛環(huán)境”和“非痛環(huán)境”神經(jīng)元放電頻率比較,“痛環(huán)境”明顯增高,有統(tǒng)計(jì)學(xué)差異(P0.05)。CFA+NS組與NS+NS組“痛環(huán)境”匹配之后在“痛環(huán)境”神經(jīng)元放電頻率比較,前者明顯增高,有統(tǒng)計(jì)學(xué)差異(P0.05)。動(dòng)作電位結(jié)果與行為一致。3)模型大鼠r ACC腦區(qū)局部場(chǎng)電位功率譜密度分析NS+NS組“痛環(huán)境”匹配后“痛環(huán)境”和“非痛環(huán)境”δ、θ、α、β、λ五種波的頻率譜密度值比較,均無(wú)差異(P0.05)。CFA+NS組“痛環(huán)境”匹配后“痛環(huán)境”和“非痛環(huán)境”δ、θ、α、β、λ五種波的頻率譜密度值比較,“痛環(huán)境”均明顯增高有統(tǒng)計(jì)學(xué)差異(P0.05)。CFA+NS組與NS+NS組“痛環(huán)境”匹配后在“痛環(huán)境”δ、θ、α、β、λ五種頻率波對(duì)應(yīng)的頻率譜密度值比較,前者均明顯增高,有統(tǒng)計(jì)學(xué)差異(P0.05)。(2)激活大鼠r ACC腦區(qū)δ-阿片受體緩解大鼠痛情緒的行為-電生理學(xué)得觀察1)大鼠r ACC腦區(qū)微量注射δ-阿片受體激動(dòng)劑行為學(xué)觀察CFA+DADLE(0.625/1.25/2.5/5/10μg/ul)組和CFA+NS組“痛環(huán)境”匹配前后在“痛環(huán)境”停留時(shí)間比較,CFA+DADLE(1.25/2.5/5/10μg/ul)組匹配前后均無(wú)差異(P0.05);CFA+DADLE(0.625μg/ul)組和CFA+NS組匹配前后,“痛環(huán)境”停留時(shí)間均明顯縮短,有統(tǒng)計(jì)學(xué)差異(P0.05)。CFA+DADLE(0.625/1.25/2.5/5/10μg/ul)組分別與CFA+NS組回避分?jǐn)?shù)比較,CFA+DADLE(0.625μg/ul)組和CFA+NS組比較沒(méi)有差異(P0.05),CFA+DADLE(1.25/2.5/5/10μg/ul)組均沒(méi)有產(chǎn)生明顯的回避,CFA+DADLE(0.625μg/ul)組和CFA+NS組產(chǎn)生明顯回避,有統(tǒng)計(jì)學(xué)差異(P0.05)。大鼠r ACC腦區(qū)注射不同濃度δ-阿片受體激動(dòng)劑DADLE對(duì)CFA誘導(dǎo)產(chǎn)生的條件位置回避(CPA)產(chǎn)生不同影響,太低濃度DADLE對(duì)CPA沒(méi)有明顯作用,隨著DADLE濃度的增加,對(duì)CPA有抑制作用,但抑制作用沒(méi)有隨DADLE濃度的增加而增強(qiáng);低劑量的δ-阿片受體激動(dòng)劑自身不會(huì)引起條件性獎(jiǎng)賞。2)大鼠r ACC腦區(qū)微量注射δ-阿片受體激動(dòng)劑神經(jīng)元spike放電特征分析CFA+DADLE組“痛環(huán)境”和“非痛環(huán)境”神經(jīng)元放電頻率比較,沒(méi)有差異(P0.05)。CFA+DADLE組與CFA+NS組“痛環(huán)境”匹配之后在“痛環(huán)境”神經(jīng)元放電頻率比較,CFA+NS組放電明顯增多,有統(tǒng)計(jì)差異(P0.05)。NS+DADLE組“痛環(huán)境”和“非痛環(huán)境”神經(jīng)元放電頻率比較,沒(méi)有差異(P0.05)。NS+DADLE組與NS+NS組“痛環(huán)境”匹配之后在“痛環(huán)境”神經(jīng)元放電頻率比較,沒(méi)有差異(P0.05)。3)大鼠r ACC腦區(qū)微量注射δ-阿片受體激動(dòng)劑神經(jīng)元局部場(chǎng)電位變化及功率譜密度分析CFA+DADLE組和NS+DADLE組“痛環(huán)境”匹配后“痛環(huán)境”和“非痛環(huán)境”δ、θ、α、β、λ五種波的頻率譜密度值比較,均無(wú)差異(P0.05)。CFA+DADLE組與CFA+NS組“痛環(huán)境”匹配后在“痛環(huán)境”δ、θ、α、β、λ五種頻率波對(duì)應(yīng)的頻率譜密度值比較,CFA+NS組均明顯增強(qiáng),有統(tǒng)計(jì)學(xué)差異(P0.05)。(3)大鼠r ACC腦區(qū)δ-阿片受體介導(dǎo)電針緩解痛情緒作用的電生理學(xué)研究1)大鼠r ACC腦區(qū)給予不同濃度拮抗劑后,電針刺激大鼠環(huán)跳穴,對(duì)CFA誘導(dǎo)產(chǎn)生的條件位置回避反應(yīng)(CPA)無(wú)明顯作用。我們實(shí)驗(yàn)室已經(jīng)完成,由其他同學(xué)詳細(xì)描述。2)大鼠r ACC腦區(qū)微量注射δ-阿片受體拮抗劑后電針刺激環(huán)跳穴神經(jīng)元spike放電特征分析NS+NS+sham EA組、CFA+NS+EA組、NS+Naltrindole+sham EA組“痛環(huán)境”和“非痛環(huán)境”神經(jīng)元放電頻率比較,沒(méi)有差異(P0.05)。CFA+NS+sham EA組、CFA+Naltrindole+EA組“痛環(huán)境”和“非痛環(huán)境”神經(jīng)元放電頻率比較,“痛環(huán)境”神經(jīng)元放電明顯增強(qiáng),有統(tǒng)計(jì)學(xué)差異(P0.05)。CFA+NS+EA組與CFA+NS+sham EA組“痛環(huán)境”匹配之后在“痛環(huán)境”神經(jīng)元放電頻率比較,CFA+NS+sham EA組明顯增強(qiáng),有統(tǒng)計(jì)學(xué)差異(P0.05)。CFA+Naltrindole+EA組與CFA+NS+EA組“痛環(huán)境”匹配之后在“痛環(huán)境”神經(jīng)元放電頻率比較,CFA+Naltrindole+EA組明顯增強(qiáng),有統(tǒng)計(jì)學(xué)差異(P0.05)。NS+Naltrindole+sham EA組與NS+NS+sham EA組“痛環(huán)境”匹配之后在“痛環(huán)境”神經(jīng)元放電頻率的比較,沒(méi)有差異(P0.05)。3)大鼠r ACC腦區(qū)微量注射δ-阿片受體拮抗劑后電針刺激環(huán)跳穴神經(jīng)元局部場(chǎng)電位變化及功率譜密度分析NS+NS+sham EA組、CFA+NS+EA組、NS+Naltrindole+sham EA組“痛環(huán)境”匹配后“痛環(huán)境”和“非痛環(huán)境”δ、θ、α、β、λ五種波的頻率譜密度值比較,均無(wú)差異(P0.05)。CFA+NS+sham EA組、CFA+Naltrindole+EA組“痛環(huán)境”匹配后“痛環(huán)境”和“非痛環(huán)境”δ、θ、α、β、λ五種波的頻率譜密度值比較,“痛環(huán)境”均明顯增強(qiáng),有統(tǒng)計(jì)學(xué)差異(P0.05)。CFA+NS+sham EA組與CFA+NS+EA組“痛環(huán)境”匹配后在“痛環(huán)境”δ、θ、α、β、λ五種頻率波對(duì)應(yīng)的頻率譜密度值分別比較,CFA+NS+sham EA組均明顯增強(qiáng),有統(tǒng)計(jì)學(xué)差異(P0.05)。CFA+NS+EA組與CFA+Naltrindole+EA組“痛環(huán)境”匹配后在“痛環(huán)境”δ、θ、α、β、λ五種頻率波對(duì)應(yīng)的頻率譜密度值比較,CFA+Naltrindole+EA組均明顯增強(qiáng),有統(tǒng)計(jì)學(xué)差異(P0.05)。NS+NS+sham EA組與NS+Naltrindole+sham EA組“痛環(huán)境”匹配后在“痛環(huán)境”δ、θ、α、β、λ五種頻率波對(duì)應(yīng)的頻率譜密度值比較,均無(wú)差異(P0.05)。結(jié)論:通過(guò)以上結(jié)果可知:1)大鼠r ACC腦區(qū)注射δ-阿片受體激動(dòng)劑DADLE,激活δ-阿片受體,減弱大鼠腳掌注射CFA產(chǎn)生的CPA反應(yīng),抑制CFA誘導(dǎo)的電活動(dòng);2)大鼠r ACC腦區(qū)注射δ-阿片受體拮抗劑,電針對(duì)CFA誘導(dǎo)的電活動(dòng)的抑制作用被阻斷;3)電針可激活大鼠r ACC腦區(qū)的δ-阿片受體,減弱CFA誘導(dǎo)產(chǎn)生的條件位置回避(CPA)反應(yīng),反轉(zhuǎn)CFA誘導(dǎo)的電活動(dòng)。
[Abstract]:Objective: Chronic pain is not only a clinical symptom, but also a disease. Pain includes pain sensation and pain emotion, the former reflects the nature, intensity and location of pain, the latter includes anxiety, aversion, fear and other adverse emotions caused by pain. Pain emotions are often more harmful than pain perception itself, so more and more attention has been paid to the study of pain emotions. Rostral ACC (r ACC) plays an important role in the development of pain. It is well known that electroacupuncture can activate opioid receptors to produce significant analgesic effects, but can it also alleviate pain? This kind of data is still very few. Our previous studies and related laboratory data have confirmed that electroacupuncture is also effective. Opioid receptors have a variety of subtypes, of which three opioid receptor subtypes, mu, Delta and kappa, are the most concerned. As mentioned above, we have confirmed mu-a. Tablet receptors mediate the analgesic effect of Electroacupuncture on pain, but it is not clear whether other receptor subtypes have similar effects. Therefore, this study will take the delta-opioid receptor as the research object, using the CPA (C-CPA) response induced by CFA injection into the paw of rats as a pain behavior model, using behavior detection and multi-channel electrophysiological recording techniques. To explore whether activating the delta-opioid receptor in the brain region of R ACC can alleviate pain and whether the delta-opioid receptor in the brain region of R ACC can mediate the process of alleviating pain by conducting needles. After a week of complete recovery, the rats were injected with CFA into the left hind paw of the rats to induce inflammatory pain and to match the environment. The conditioned place avoidance (CPA) reaction model was established. The changes of pain perception behavior were observed by the paw retraction latency (PWL) of rats, and the changes of pain emotion behavior were observed by the conditioned position avoidance device. At the same time, the signals of neurons in the R ACC brain region were recorded and processed by the in vivo multi-channel technique. The specific patterns were grouped by two factors as follows: 12 groups of animals (n=6-8 rats/group) = 2 (CFA, physiology). Results: (1) Comparing the latency of foot contraction in the CFA + NS group, CFA + NS group significantly contracted after matching the pain environment. There was no significant difference between the NS + NS group and the control group (P Comparing with NS + NS group, CFA + NS group had significant avoidance reaction, and there was significant difference (P 0.05). After matching, the residence time in "pain environment" was significantly shortened, while the residence time in "non-pain environment" was significantly prolonged, and the "pain environment" was significantly produced. To avoid, CFA induced conditioned place avoidance model (CPA). 2) Characteristic analysis of action potential of neurons in the R ACC area of rats in the CFA-induced conditioned place avoidance model (CPA). There was no significant difference in discharge frequencies between "pain environment" and "non-pain environment" neurons in NS+NS group (P 0.05). The discharge frequencies of "pain environment" and "non-pain environment" neurons in CFA+NS group were significantly different. Compared with NS + NS group, the firing frequency of neurons in "pain environment" was significantly higher in CFA + NS group after matching with NS + NS group (P 0.05). Action potential results were consistent with behavior. 3) Power spectral density analysis of R ACC brain area of model rats after matching with NS + NS group "pain environment" There was no significant difference in the frequency spectral densities between "pain environment" and "non-pain environment" (P 0.05). The frequency spectral densities of "pain environment" and "non-pain environment" (P 0.05) were significantly higher in CFA+NS group than in NS+N group (P 0.05). After matching the "pain environment" in group S, the corresponding spectral densities of delta, theta, alpha, beta, and lambda in the "pain environment" were significantly higher than those in the "pain environment" (P The retention time of CFA+DADLE(0.625/1.25/2.5/5/10 ug/ul) group and CFA+NS group before and after the matching of "pain environment" had no significant difference between CFA+DADLE(1.25/2.5/5/10 ug/ul) group and CFA+NS group before and after the matching of "pain environment"(P 0.05); There was no significant difference between CFA+DADLE (0.625/1.25/2.5/5/10 ug/ul) group and CFA+NS group (0.625 ug/ul) in avoidance score, CFA+DADLE (0.625 ug/ul) group and CFA+NS group (P 0.05), CFA+DADLE (1.25/2.5/5/10 ug/ul) group had no significant avoidance, CFA+DADLE (0.625 ug/ul) group and CFA+NS group had significant avoidance score, there was statistical difference. Different concentrations of delta-opioid receptor agonist DADLE had different effects on conditioned position avoidance (CPA) induced by CFA. Too low concentration of DADLE had no obvious effect on CPA. With the increase of DADLE concentration, it had inhibitory effect on CPA, but the inhibitory effect did not increase with the increase of DADLE concentration. The spike discharges of delta-opioid receptor agonist neurons injected into the R ACC area of rats showed no significant difference between the "pain environment" and "non-pain environment" neurons in the CFA+DADLE group (P 0.05). There was no significant difference between the "pain environment" and the "pain environment" in the CFA+DADLE group after matching the "pain environment" in the CFA+DADLE group with the "pain environment" in the CFA+NS group. There was no significant difference in the discharge frequencies of neurons in the "pain environment" and "non-pain environment" between the NS + DADLE group and the NS + NS group (P 0.05). There was no difference in the discharge frequencies of neurons in the "pain environment" between the NS + DADLE group and the NS + NS group (P 0.05). Changes of local field potentials and power spectral density of delta-opioid receptor agonist neurons after microinjection into the R ACC brain area of rats The frequency spectral densities of delta,theta,alpha,beta and lambda in the "pain environment" and "non-pain environment" after matching the "pain environment" of CFA+DADLE group and NS+DADLE group showed no difference (P 0.05). Comparing the spectral densities of delta, theta, alpha, beta and lambda frequencies in the "pain environment" matched with the "environment", the CFA + NS group showed a significant increase (P 0.05). (3) Electrophysiological study on the effect of delta-opioid receptor-mediated conductive acupuncture on pain relief in the R ACC brain region of rats (1) Electroacupuncture stimulation after different concentrations of antagonists were given to the R ACC brain region of rats. Our laboratory has been completed and described in detail by other students. 2) spike discharge characteristics of neurons stimulated by Electroacupuncture at acupoint Huantiao after microinjection of delta-opioid receptor antagonist in the R ACC area of rats. NS+NS+sham EA group, CFA+NS+EA group, NS+Naltrindole+sham EA group, NS+Naltrindole+sham EA group There was no significant difference in discharge frequencies between "pain environment" and "non-pain environment" neurons in EA group (P 0.05). The discharge frequencies of "pain environment" and "pain environment" neurons in CFA+NS+sham EA group, CFA+Naltrindole+EA group and "pain environment" neurons in "pain environment" were significantly higher than those in "pain environment" neurons in "pain environment" and "pain environment" neurons in "pain environment" group (P 0.05). Compared with the control group, the firing frequency of CFA + NS + sham EA group was significantly higher than that of CFA + NS + sham EA group (P 0.05). The firing frequency of CFA + Naltrindole + EA group was significantly higher than that of CFA + NS + EA group (P 0.05). There was no difference in the firing frequency of neurons in the "pain environment" between the rindole + sham EA group and NS + NS + sham EA group (P 0.05). There was no significant difference in the frequency spectral density between the "pain environment" and the "non-pain environment" after matching the "pain environment" in Naltrindole+sham EA group (P 0.05). The frequency spectral density ratio of the "pain environment" and the "non-pain environment" after matching the "pain environment" in CFA+NS+sham EA group and the "pain environment" in CFA+Naltrindole+EA group was higher than that in the "non-pain environment". Comparing with the pain environment of CFA+NS+sham EA group and CFA+NS+EA group, the frequency spectral density values of the five frequencies of delta, theta, alpha, beta and lambda in the pain environment of CFA+NS+sham EA group and CFA+NS+Naltrindole+EA group were significantly increased (P 0.05). Comparing the spectral densities of the five frequencies of the "pain environment" after matching, the CFA + Naltrindole + EA group was significantly higher than that of the NS + Naltrindole + Sham EA group (P 0.05). The frequencies of the five frequencies of the "pain environment" after matching were delta, theta, alpha, beta, and lambda in the "pain environment" of the NS + NS + sham EA group and the NS + Naltrindole + sham EA group. CONCLUSIONS: 1) Injecting DADLE into the R ACC brain area of rats, activating delta-opioid receptor, attenuating the CPA response and inhibiting the electrical activity induced by CFA, and 2) injecting delta-opioid receptor antagonist into the R ACC brain area of rats, electroacupuncture on the electrical activity induced by CFA. 3) EA could activate the delta-opioid receptor in the R ACC region of rats, weaken the conditioned position avoidance (CPA) response induced by CFA, and reverse the electrical activity induced by CFA.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R402

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