發(fā)布時間：2018-09-03 20:57

【摘要】：目的：深部腦刺激（deep brain stimulation，DBS)臨床上已用于帕金森病難治性強(qiáng)迫癥癲癇和抑郁癥等精神疾病的治療。和其他電刺激技術(shù)或腦手術(shù)相比，DBS具有可逆性、微創(chuàng)性和安全性高等優(yōu)點(diǎn)。近幾年，有實(shí)驗研究和臨床的初步觀察提示DBS具有治療藥物依賴及防復(fù)吸潛力。但有關(guān)DBS治療海洛因復(fù)吸的基礎(chǔ)和臨床研究剛起步，治療作用、最佳刺激參數(shù)和合適靶核團(tuán)的選擇以及作用途徑尚待進(jìn)一步系統(tǒng)研究。本課題旨在利用大鼠海洛因自身給藥模型，選用內(nèi)側(cè)前額皮層（medial prefrontal cortex，mPFC）的兩個亞區(qū)（背和腹內(nèi)側(cè)前額皮層）進(jìn)行DBS處理，觀察DBS對大鼠海洛因覓藥行為消退和恢復(fù)的影響，并初步探討其可能的分子生物學(xué)作用機(jī)制。 實(shí)驗一、慢性高頻或低頻刺激背內(nèi)側(cè)前額皮層（dorsal medial prefrontalcortex，dmPFC）對大鼠海洛因覓藥行為消退和恢復(fù)的影響 方法：SD成年雄性大鼠經(jīng)頸靜脈插管和埋置電極手術(shù)后恢復(fù)7天，所有大鼠均進(jìn)行每天4h的海洛因固定比率（FR1）自身給藥訓(xùn)練，連續(xù)訓(xùn)練14h，建立海洛因自身給藥模型。27只大鼠隨機(jī)分為假刺激對照組（Sham組）高頻刺激處理組（H-DBS組）和低頻刺激處理組（L-DBS組）(n=9)，然后進(jìn)行環(huán)境消退訓(xùn)練，每天2h。每天消退訓(xùn)練前，刺激處理組大鼠在dmPFC腦區(qū)給予1h高頻（頻率130HZ，脈寬100μs，電流0.2mA）或低頻（頻率10HZ，脈寬100μs，，電流0.2mA）刺激處理，共連續(xù)刺激10d，然后在最后一次消退訓(xùn)練結(jié)束24h后進(jìn)行線索誘導(dǎo)的海洛因覓藥行為測試。假刺激對照組刺激電流為0mA，其余同刺激處理組。 結(jié)果：已建立穩(wěn)定的海洛因自身給藥大鼠，在隨后的第1-10天的消退訓(xùn)練中，H-DBS組和L-DBS組有效鼻觸數(shù)較Sham對照組有效鼻觸數(shù)均無顯著性差異（P0.05）；在線索誘導(dǎo)的海洛因覓藥行為恢復(fù)測試中，單因素方差分析發(fā)現(xiàn)：H-DBS組有效鼻觸數(shù)顯著低于Sham對照組有效鼻觸數(shù)（F(2,24)=5.304, P 0.05），而L-DBS組有效鼻觸數(shù)與Sham對照組相比無顯著性差異（P0.05）。 實(shí)驗二慢性高頻或低頻刺激腹內(nèi)側(cè)前額皮層(ventral medial prefrontalcortex，vmPFC)對大鼠海洛因覓藥行為消退和恢復(fù)的影響 方法：海洛因自身給藥海洛因自身給藥模型建立、消退訓(xùn)練、DBS電極埋置和刺激參數(shù)等同實(shí)驗一。所有海洛因自身給藥大鼠隨機(jī)分為vmPFC腦區(qū)高頻刺激處理組（H-DBS組）、低頻刺激處理組（L-DBS組）和假刺激對照組（Sham組）(n=9)。 結(jié)果：與Sham對照組相比，消退訓(xùn)練第1-3天，H-DBS組有效鼻觸數(shù)有增高趨勢，但無顯著性差異，隨著消退訓(xùn)練天數(shù)的繼續(xù)，消退訓(xùn)練第4-7天，H-DBS組有效鼻觸數(shù)顯著高于Sham對照組有效鼻觸數(shù)（P0.05），而在消退訓(xùn)練第8-10天中，與Sham對照組相比，H-DBS組有效鼻觸數(shù)均無顯著性差異；而L-DBS組在消退訓(xùn)練第1-10天中，其有效鼻觸數(shù)較Sham對照組相比均無顯著性差異（P0.05）；在線索誘導(dǎo)的海洛因覓藥行為恢復(fù)測試中，單因素方差分析發(fā)現(xiàn)：與Sham對照組相比，H-DBS組有效鼻觸數(shù)顯著增加（F(2,24)=8.489, P 0.05），而L-DBS組有效鼻觸數(shù)無顯著性差異（P0.05） 實(shí)驗三慢性高頻刺激dmPFC對大鼠伏隔核核部（nucleus accumbensshell, NAc shell）和殼部（nucleus accumbens core, NAc core）p-CREBp-ERK和p-Akt表達(dá)的影響 方法：海洛因自身給藥海洛因自身給藥模型建立、消退訓(xùn)練、DBS電極埋置、刺激參數(shù)和線索誘導(dǎo)的覓藥行為恢復(fù)測試等同實(shí)驗一，所有海洛因自身給藥大鼠隨機(jī)分為dmPFC腦區(qū)高頻刺激處理組（H-DBS組）和假刺激對照組（Sham組）(n=9)。線索誘導(dǎo)的覓藥行為測試結(jié)束，立即斷頭取腦：采用Western blot方法檢測NAc shell和NAc core中p-CREBp-ERK和p-AKt的表達(dá)水平變化；運(yùn)用免疫組化方法，觀察高頻刺激dmPFC對NAc shell和NAccore中p-CREB蛋白陽性表達(dá)的影響。 結(jié)果：Western blot檢測發(fā)現(xiàn)：與Sham對照組相比，H-DBS組NAc core中p-CREB表達(dá)顯著升高（t(6)=9.342，P0.01），p-ERK與p-AKt表達(dá)均顯著減少（t(6)=13.347，P0.01；t(6)=11.678，P0.01），但H-DBS組NAc shell中p-CREB和p-ERK均無顯著性變化（P0.05），而p-AKt表達(dá)顯著減少（t(6)=3.863，P0.05）；同時，在p-CREB免疫組化實(shí)驗中發(fā)現(xiàn)，與Sham對照組相比， H-DBS組NAc core中p-CREB陽性細(xì)胞數(shù)顯著增加（t(6)=12.107，P0.05），而NAcshell中p-CREB陽性細(xì)胞數(shù)無顯著變化（P0.05）。 結(jié)論 高頻刺激dmPFC能抑制線索誘導(dǎo)的海洛因覓藥行為恢復(fù)，其作用可能與NAc中磷酸化CREBERK和AKt蛋白的表達(dá)改變有關(guān)。高頻刺激vmPFC可促進(jìn)線索誘導(dǎo)的海洛因覓藥行為的恢復(fù)，并對大鼠覓藥行為消退反應(yīng)有一定的抑制作用；但是，低頻刺激dmPFC或vmPFC對大鼠覓藥行為消退和線索誘導(dǎo)的覓藥行為的恢復(fù)均沒有明顯的干預(yù)作用。
[Abstract]:OBJECTIVE: Deep brain stimulation (DBS) has been used clinically for the treatment of Parkinson's disease, refractory obsessive-compulsive disorder, epilepsy and depression. Compared with other electrical stimulation techniques or brain surgery, DBS has the advantages of reversibility, minimally invasiveness and high safety. However, the basic and clinical studies on DBS in the treatment of heroin relapse have just begun, and the therapeutic effects, the optimal stimulation parameters, the selection of appropriate target nuclei and the pathway of action need to be further systematically studied. Two subareas (dorsal and ventromedial prefrontal cortex) of L prefrontal cortex (mPFC) were treated with DBS to observe the effects of DBS on the regression and recovery of heroin seeking behavior in rats, and to explore the possible molecular biological mechanism.
Experiment 1. Effects of chronic high or low frequency stimulation of dorsal medial prefrontal cortex (dmPFC) on the regression and recovery of heroin seeking behavior in rats
METHODS: SD adult male rats recovered 7 days after jugular vein catheterization and implantation of electrodes. All rats were trained for 4 hours a day for self-administration of heroin (FR1). Heroin self-administration model was established after 14 hours of continuous training. 27 rats were randomly divided into sham group, high-frequency stimulation group (H-DBS group) and low-frequency stimulation group (H-DBS group). Frequency stimulation group (L-DBS group) (n=9), and then environmental regression training, 2 hours a day. Before regression training, stimulation group rats were given 1 hour high frequency (frequency 130HZ, pulse width 100 mus, CURRENT 0.2 mA) or low frequency (frequency 10HZ, pulse width 100 mus, CURRENT 0.2 mA) stimulation in the brain region of dmPFC for 10 days, and then the last regression training session. The cue-induced heroin seeking behavior was tested 24 hours later. The stimulation current of the sham stimulation control group was 0 mA, and the rest of the sham stimulation group was the same as the stimulation treatment group.
RESULTS: Stable heroin self-administered rats were established, and there was no significant difference between H-DBS group and L-DBS group (P 0.05) in effective nasal contact number compared with Sham control group in the following 1-10 days of regression training. The number of effective nasal contacts in L-DBS group was significantly lower than that in Sham control group (F(2,24)=5.304,P 0.05), but there was no significant difference between L-DBS group and Sham control group (P 0.05).
Effect of chronic high or low frequency stimulation of ventral medial prefrontal cortex (vmPFC) on the regression and recovery of heroin seeking behavior in rats
METHODS: Heroin self-administration model was established, regression training, DBS electrode implantation and stimulation parameters were the same as experiment 1. All heroin self-administration rats were randomly divided into three groups: high-frequency stimulation group (H-DBS group), low-frequency stimulation group (L-DBS group) and sham control group (n=9).
Results: Compared with Sham control group, the number of effective nasal contacts in H-DBS group increased from 1 to 3 days of regression training, but there was no significant difference. With the continuation of regression training days, the number of effective nasal contacts in H-DBS group was significantly higher than that in Sham control group on 4-7 days of regression training (P 0.05), but in 8-10 days of regression training, the number of effective nasal contacts in H-DBS group was significantly higher than that in Sham control group. There was no significant difference in the number of effective nasal contacts between DBS group and Sham control group, but there was no significant difference in the number of effective nasal contacts between L-DBS group and Sham control group (P 0.05). (F (2,24) =8.489, P 0.05), but there was no significant difference in the number of effective nasal contacts in the L-DBS group (P0.05).
Effect of chronic high frequency stimulation of dmPFC on the expression of p-CREB? P-ERK and p-Akt in nucleus accumbens (NAc shell) and nucleus accumbens core (NAc core) of rats
METHODS: Heroin self-administration model was established, regression training, DBS electrode implantation, stimulus parameters and cue-induced drug-seeking behavior recovery test were the same as experiment 1. All heroin self-administration rats were randomly divided into dmPFC high-frequency stimulation group (H-DBS group) and sham control group (n=9). The expression of p-CREB?P-ERK and p-AKt in NAc shell and NAc core was detected by Western blot, and the effect of high frequency stimulation of dmPFC on the expression of p-CREB protein in NAc shell and NAc core was observed by immunohistochemistry.
Results: Western blot showed that the expression of p-CREB in NAc core of H-DBS group was significantly higher than that of Sham control group (t (6) = 9.342, P 0.01), and the expression of p-ERK and p-AKt were significantly decreased (t (6) = 13.347, P 0.01; t (6) = 11.678, P 0.01), but the expression of p-CREB and p-ERK in NAc shell of H-DBS group was not significantly changed (P 0.05), but p-AKt (6) = 3.863, P 0.863, P 0.01). At the same time, compared with Sham control group, the number of p-CREB positive cells in NAc core of H-DBS group increased significantly (t(6) = 12.107, P 0.05), while the number of p-CREB positive cells in NAc Shell did not change significantly (P 0.05).
conclusion
High frequency stimulation of dmPFC can inhibit the recovery of clue-induced heroin seeking behavior, which may be related to the changes of phosphorylated CREB?ERK and AKt protein expression in NAc. There was no significant effect of mPFC or vmPFC on the regression of drug-seeking behavior and the recovery of clue-induced drug-seeking behavior in rats.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R749.64

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