慢性吗啡处理及戒断后大鼠海马CA1区钙结合蛋白Calbindin的表达变化

目的:观察慢性吗啡处理及戒断后大鼠海马CA1区中钙结合蛋白Calbindin(CB)的表达变化,为其功能研究提供形态学依据.方法:36只雄性SD大鼠随机分为吗啡依赖组和生理盐水对照组.吗啡依赖组大鼠腹膜腔注射吗啡,2次/d,起始剂量为5mg/kg,逐日递增5mg/kg,至第10d为50mg/kg;对照组注射同体积的生理盐水.于末次注射后动物分别存活3h、3d、14d和30d.免疫组化方法和相对平均灰度值检测海马CAl区CB的表达变化.结果:在生理盐水处理组各存活时间点海马CAI区CB的表达相同.和生理盐水对照组相比,吗啡依赖组3h时海马CA1区CB的表达明显增加(P<0.05),3 d时进一步增加,至第14 d时CA1区CB的表达开始恢复.结论:慢性吗啡处理及戒断后海马CA1区CB的表达上调可能对戒断早期海马神经元有保护作用.     

鞘内注射M受体和GDNF反义寡脱氧核苷酸抑制吗啡戒断大鼠蓝斑c-Fos表达

应用免疫组化方法观察鞘内注射毒蕈碱型乙酰胆碱(muscarinic acetylcholine receptor,M) 受体和胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)反义寡脱氧核苷酸对吗啡戒断大鼠蓝斑(locus coeruleus,LC)区内Fos表达的影响。结果显示,鞘内注射M_2受体和GDNF反义寡脱氧核苷酸明显减少大鼠吗啡戒断症状评分值(n=6,P<0.05)。正常大鼠LC区神经元Fos基础表达较低,吗啡依赖大鼠LC区神经元Fos表达增加,吗啡依赖大鼠纳酪酮(4mg/Kg,ip)催促戒断后,Fos表达进一步增加;鞘内注射M_2受体和GDNF反义寡脱氧核苷酸处理后均减少吗啡戒断大鼠LC区神经元Fos表达(n=5,P<0.05)。而鞘内注射M_1受体反义寡脱氧核苷酸处理组LC 区神经元Fos表达较吗啡戒断组没有显著差异(n=5,P>O.05)。结果提示:脊髓M_2受体调节吗啡戒断时LC区的神经元激活,而这种神经上行性激活涉及神经元与胶质细胞之间的适应性调节。     

脊髓水平iNOS在吗啡依赖大鼠戒断反应中的作用

目的:探讨脊髓水平诱导型一氧化氮合酶在吗啡依赖大鼠戒断反应中的作用。方法:健康雄性SD大鼠72只,体重200~250 g,吗啡剂量每次10 mg/kg,每日2次,隔日每次增加10 mg/kg,至第6天末次注射50 mg/kg,大鼠腹腔注射纳洛酮4 mg/kg建立吗啡依赖及戒断模型,在纳洛酮激发戒断前30 min鞘内注射iNOS特异性抑制剂氨基胍(AG)150μg。分为正常对照组、吗啡依赖组、吗啡戒断组、AG组。采用行为学(n=8)、免疫组织化学(n=6)和Western blot(n=4)方法观察鞘内应用iNOS特异性抑制剂氨基胍对吗啡依赖大鼠纳洛酮催促戒断反应和脊髓神经元iNOS表达的影响。结果:AG组戒断症状评分和戒断组促诱发痛评分均低于戒断组(P<0.05)。免疫组织化学和Western blot显示戒断组大鼠脊髓iNOS阳性神经元的数目和蛋白的表达增高,而AG组大鼠脊髓iNOS阳性神经元的数目和iNOS蛋白的表达低于戒断组(P<0.05)。结论:脊髓水平iNOS表达上调可能参与介导吗啡戒断反应。     

脊髓水平细胞外信号调节激酶激活参与吗啡依赖的形成及戒断反应的表达

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察吗啡依赖及戒断大鼠脊髓神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal-regulated kinase,pERK）表达的变化,及鞘内注射促分裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase,MEK）抑制剂U0126或ERK反义寡核苷酸对吗啡依赖大鼠纳洛酮催促戒断反应、触诱发痛及脊髓神经元pERK表达的影响,探讨脊髓水平pERK在介导吗啡依赖和戒断过程中的作用。结果显示：（1）在吗啡依赖形成过程中,大鼠脊髓胞浆与胞核非磷酸化ERK表达没有改变,但pERK表达逐渐增加,纳洛酮催促戒断后,仍有进一步增加的趋势,戒断1h后,其表达量明显下降,但仍高于对照组。（2）鞘内预先注射MEK抑制剂U0126或ERK反义寡核苷酸能明显抑制吗啡戒断反应和戒断引起的痛觉异常;与行为学结果一致,脊髓背角pERK阳性神经元表达与脊髓胞浆和胞核pERK表达也明显降低。上述结果提示,脊髓水平ERK激活和核转位参与吗啡依赖的形成及戒断反应的表达。     

慢性吗啡依赖大鼠条件性位置厌恶模型杏仁中央核蛋白激酶A表达变化

目的:分析慢性吗啡依赖大鼠纳洛酮催瘾戒断条件性位置厌恶(CPA)建立前后、消退后及重建后,与成瘾密切相关的脑区杏仁中央核(CeA)内蛋白激酶A(PKA)蛋白表达的适应性变化,探讨阿片依赖戒断后厌恶动机形成的生物学基础。方法:①将雄性SD大鼠分为实验组(慢性吗啡注射+纳洛酮催瘾组,MN),对照组(慢性吗啡注射+生理盐水催瘾组,MS),慢性生理盐水注射+纳洛酮催瘾组,SN),每组24只。采用慢性吗啡注射(10 mg/kg,BID,ip)后给予一次纳洛酮(0.3 mg/kg)催瘾注射(同时与条件性位置训练箱搭配)建立大鼠CPA模型。②在CPA建立前后、消退后及重建后,采用免疫组织化学方法检测CeA内PKA蛋白表达情况。结果:MN组在CPA建立前后、消退后及重建后CeA内PKA的蛋白表达出现适应性变化(P<0.05),建立后(Day7,134.43±4.481,P<0.05),消退后(Day13,141.01±3.360,P<0.01)及重建后(Day14,137.18±40.330,P<0.05)PKA蛋白表达水平均低于建立前(Day5,124.48±6.722)。而MS组(P>0.05)和SN组(P>0.05)在CPA的各个时间点PKA的蛋白表达变化差异均无显著性。结论:①CeA内PKA蛋白的低表达导致的厌恶的中枢状态,可能是CPA建立的关键的神经机制。②CeA内PKA的适应性变化可能是物质依赖戒断后CPA相关神经可塑性变化的重要分子基础。     

NO参与介导吗啡戒断大鼠脊髓神经元敏感化

运用Fos免疫组织化学、NADPH－d组织化学、F/NADPH－d双标、鞘内注射和反义寡核苷酸技术,观察吗啡戒断大鼠脊髓神经元活动变化及NO在其中的作用,结果发现：非吗啡依赖大鼠急性应用纳洛酮和吗啡依赖大鼠脊髓水平Fos-LI和NADPH－d阳性神经元表达与对照组相比无明显变化,二者也无Fos／NADPH－d双标神经元表达;吗啡依赖纳洛酮催促戒断大鼠脊髓Fos-LI、NADPH－d阳性神经元、纤维和终末表达明显增加,且出现Fos／NADPH－d双标神经元表达。Fos-LI和Fos／NADPH－d双标神经元呈现双侧脊髓全层分布,NADPH－d阳性神经元、纤维和终末主要位于双侧脊髓背角浅层。鞘内注射NOS抑制剂L－NA和nNOS反义寡核苷酸均明显降低吗啡依赖大鼠纳洛酮催促戒断症状评分,减少吗啡戒断大鼠脊髓Fos-LI表达。上述结果提示：NO参与介导吗啡戒断大鼠脊髓神经元敏感化。     

慢性吗啡给予、戒断及再给药过程中大鼠海马感觉门控的动态变化

药物成瘾被认为是药物长期作用于脑而产生的一种慢性复吸性脑疾病,长期反复的药物（如吗啡）滥用会导致一系列严重后果,如药物依赖、药物耐受、强迫性药物寻求等。本实验利用条件化位置偏好（conditioned place preference,CPP）模型来检测大鼠对吗啡依赖和心理渴求等过程;采用双声刺激听觉诱发电位来研究大鼠在慢性吗啡给予、戒断以及再给药过程中海马感觉门控（N40）的动态变化。吗啡组大鼠注射吗啡（10mg／kg,i．p．）12d,经历第一次戒断12d,再次注射吗啡（2．5mg／kg,i．P．）1d,之后经历第二次戒断2d;对照组大鼠注射同体积生理盐水,其余实验条件与吗啡组相同。CPP实验表明,这种药物给予方法促使大鼠对吗啡产生药物依赖和心理渴求。双声刺激诱发电位实验表明,吗啡组大鼠在吗啡给予期间海马感觉门控受到损伤;第一次戒断期的第1～2天海马感觉门控能力减弱,第3天增强,第4～12天逐渐恢复到正常水平;再次给予吗啡后海马感觉门控能力与对照组相比显著降低,并且随后2d的戒断期内海马感觉门控能力也一直保持较低水平,表明再次给药使大鼠海马感觉门控对吗啡更加敏感化。结果提示,长期反复的吗啡给予及再给药干扰了海马的感觉门控能力,吗啡成瘾对大脑可能产生长期影响。     

M受体对吗啡戒断大鼠脊髓和脑干NMDA受体基因表达及中脑导水管周围灰质中谷氨酸释放的调节

应用鞘内注射反义寡脱氧核苷酸技术和RT—PCR反应,观察毒蕈碱型乙酰胆碱受体(muscarinic acetylcholine receptor,M)对吗啡依赖大鼠脊髓和脑干NMDA受体NR1A和NR2A mRNA表达和中脑导水管周围灰质区(periaqueductal grey,PAG)中谷氨酸释放的影响。结果显示,吗啡依赖大鼠脊髓NR1A和NR2A mRNA表达明显升高,而脑干中NR1A和NR2A mRNA表达没有显著变化;注射纳洛酮后1h,吗啡戒断大鼠脊髓和脑干中NR1A和NR2A表达显著高于依赖组,经NMDA受体拮抗剂MK801(0．125mg／kg,i．p．)、M受体拮抗剂东莨菪碱(0．5mg／kg,i．p．)、M1受体拮抗剂呱伦西平(10mg/kg,i．p．)和NOS抑制剂L-NAME(10mg／kg,i．p．)处理后,脊髓和脑干中NR1A和NR2A基因表达都较戒断组明显减少。在纳洛酮激发前24h鞘内注射NR1A和M2受体的反义寡脱氧核苷酸(4μg／只),戒断症状评分值及脊髓和脑干的NR1A mRNA的表达均较对照组明显减少。吗啡依赖大鼠在纳洛酮注射前24h鞘内注射M2受体反义寡脱氧核苷酸(4μg/只),可以明显减少PAG内透析液中谷氨酸含量。上述结果提示：NMDA受体的基因表达和谷氨酸释放参与吗啡戒断过程,而这种表达受到M受体的调节。     

鞘内注射U0126对吗啡戒断大鼠脊髓神经元磷酸化CREB表达的影响

目的：研究鞘内注射细胞外信号调节激酶（ERK）抑制剂U0126对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓磷酸化cAMP反应元件结合蛋白（p-CREB）表达的影响。方法：建立大鼠吗啡依赖和戒断模型,分为正常对照组、吗啡依赖组、吗啡戒断组、U0126组、溶媒（DMSO）组,采用行为学（n=8）、免疫组织化学（n=6）和Western blot（n=4）方法观察鞘内应用U0126对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓p-CREB表达的影响。结果：①鞘内注射u0126可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28．6±4．89,U0126组为22．5±4．09（P〈0．05）;戒断组促诱发痛评分（TEAscore）为13．5±2．55,U0126组为10．0±2．76（P〈0．05）。②鞘内注射U0126可明显减少胸腰段脊髓背角p-CREB阳性神经元的数目,U0126组为287±54,低于戒断组（380±71,P〈0．05）。 ③westem blot结果显示：鞘内注射U0126明显抑制吗啡戒断期间脊髓p-CREB表达的增加。结论：鞘内注射U0126能明显抑制吗啡戒断大鼠脊髓神经元p-CREB的表达。     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

Co-administration of dextromethorphan during pregnancy and throughout lactation significantly decreases the adverse effects associated with chronic morphine administration in rat offspring.

In this study, we have focused our investigation of the facts whether co-administration of a NMDA antagonist dextromethorphan (DM) with morphine during pregnancy and throughout lactation could prevent the adverse effects associated with chronic morphine administration in rat offspring. Adult female Sprague-Dawley rats were randomly separated into four groups and were received subcutaneous injection of either saline, morphine, morphine + dextromethorphan or dextromethorphan twice a day and progressively increased 1 mg/kg at 7-day intervals from a beginning dose of 2 mg/kg for both morphine and dextromethorphan. The rats were mated between days 7 and 8. Administration of drugs was continued during pregnancy. After rat offspring were born, the doses of morphine or dextromethorphan injected into the maternal rats were increased by 1 mg/kg every two weeks till the offspring were 30 day old. The results showed that mortality of morphine group is much higher than control group. The offspring of morphine group weighed significantly less than control group on postnatal day 14 (p14), p30 or p60. The antinociceptive effect of morphine on p14 rats was reduced in the morphine group and indicated the development of morphine tolerance. The hippocampal NMDA receptor densities have been shown decreased on p14 rats. The precipitated withdrawal symptoms were assessed on p7 rats. Rats in morphine group showed greater frequency of abdominal stretch and wet dog shake in 2 hr than control group. On the other hand, co-administration of DM with morphine effectively prevented all these adverse effects of morphine to the offspring rats. DM co-administered with morphine also partially prevented the development of morphine tolerance in maternal rats. If this effect of dextromethorphan is applied to clinical pregnant patients with morphine addiction or chronic pain, it will have a great value for the benefit of their children.     

Effect of morphine withdrawal on food and water intake, urine output and electrolyte excretion in the rat: participation of the renin-aldosterone-system in renal excretory changes

Male Wistar rats received two i.p. injections of morphine-HCl, 2.5 mg/kg at 8.00 a.m. and 2.00 p.m. on the 1st day: the dose was doubled every other day to reach a total daily dose of 40 mg/kg on the 4th day. This schedule was maintained for 12 days. On day 16 the animals received the last injection of morphine, 20 mg/kg. One hour later (9.00 a.m.) six rats were decapitated and PRA, PAC and ACTH were measured by radioimmunoassay. Groups of six rats were killed at 9.00 a.m. on the 1st, 2nd, 5th and the 8th day after morphine withdrawal. Control data for PRA, PAC and ACTH were obtained from eighteen saline-injected rats. Nine out of morphine-treated animals were kept in metabolism cages to investigate simultaneously food and water intake. and renal excretion. Morphine withdrawal after chronic morphine treatment in the rat resulted in antidiuresis and a reduction of electrolyte excretion which were not due to a reduction in water and food intake. The simultaneous increase of PRA and PAC associated with decreased electrolyte excretion indicates that, in addition to antidiuretic hormone, also the renin-aldosterone-system probably play a relevant role in the renal excretory changes after morphine withdrawal.     

慢性吗啡耐受大鼠脑内孤啡肽生成与释放增加

本文彩放射免疫分析法测定了慢性吗啡耐受过程中大鼠脑室灌流液、中脑导水管周围灰质（ＰＡＧ）及杏仁核中孤啡肽（ＯＦＱ）免疫活性的动态变化。结果观察到：（１）大鼠连续５ｄ皮下注射递增剂量的盐酸吗中民慢性吗啡耐受,其脑室灌流中ＯＦＱ－ｉｒ随吗啡注射剂量和注射次数的增加逐渐上升,第５ｄ注射后较对照组升高了５２％;（２）皮下注射吗啡１ｄ、３ｄ、５ｄ的大鼠ＰＡＧ中ＯＦＱ－ｉｒ比对照组分别升高了１７％、４８％和８     

The effect of pentobarbital on the antinociceptive action of morphine in morphine-tolerant and non-tolerant rats

The interaction of sodium pentobarbital with morphine sulfate in both morphine-tolerant and non-tolerant rats was investigated using the tail-compression test for analgesia. Male Sprague-Dawley rats (300–350 g) were given pentobarbital (4, 8, or 16 mg/kg) 5 min before morphine (2, 4, 6, or 8 mg/kg). Control animals received two saline injections, or pentobarbital plus saline, or saline plus morphine. All injections were subcutaneous. Prior to the first injection, a baseline nociceptive threshold was determined for each rat by applying a modified micrometer to its tail and increasing the pressure until a squeak was elicited. Test readings were taken every half-hour for 2 hr beginning 30 min after the second injection. For the chronic studies, animals were first made tolerant to morphine by the administration of the narcotic twice a day for 3 days, increasing the dose from 10 to 50 mg/kg/injection. Identical testing procedures were then followed with these rats except that the test dose of morphine given on day 4 was in the range 8–128 mg/kg. It was found that Na pentobarbital, in the subanesthetic doses used, had neither antinociceptive nor hyperalgesic properties. Furthermore, the barbiturate had no effect on the antinociceptive action of morphine in either morphine-tolerant or non-tolerant rats.     

Dorsal noradrenergic bundle lesions fail to alter opiate withdrawal or suppression of opiate withdrawal by clonidine

Previous work has shown that clonidine effectively supresses many of the signs of opiate withdrawal. The present study was designed to test the hypothesis that the supression of opiate withdrawal by clonidine is mediated by forebrain noradrenergic projections of the locus coeruleus. Two groups of 24 rats each were subjected to either a 6-hydroxydopamine lesion of the dorsal noradrenergic bundle (Lesion group) or a sham, vehicle injection (Sham group). All rats were made dependent on morphine by subcutaneous implantation of one 75 mg silastic morphine pellet for three days followed by 3 more days with two additional 75 mg pellets. Following removal of the morphine pellet, withdrawal was precipitated in all rats by subcutaneous injection of 4 mg/kg of naloxone. Pretreatment 10 min. before withdrawal with clonidine (0.1 or 0.2 mg/kg) produced a significant attenuation of withdrawal signs as compared to saline injected rats; this effect was equally significant in both sham and lesion groups. Lesions of the locus coeruleus had no effect on withdrawal, nor did they affect the ameliorating action of clonidine. These results substantiate the observation that clonidine can effectively attenuate signs of opiate withdrawal in the rat, but fail to support the hypothesis that these effects are mediated by the forebrain projections of the locus coeruleus.