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吗啡在临床上是效果良好的止痛药,用于治疗慢性疼痛,但是反复使用会导致成瘾,这极大地限制了它在临床上的应用[1]。此外,全球因使用吗啡导致成瘾的人越来越多,严重危害了个人身体健康和公共卫生安全[2]。目前,国内外研究吗啡成瘾的方法有很多,包括条件性位置偏爱模型[3-4]、自身给药模型[5-6]及行为敏化模型等[7-9]。条件性位置偏爱(CPP)模型具有周期短、操作简单的特点,被广泛用于研究药物成瘾,是研究药物滥用的经典动物模型。运用巴普洛夫反射原理,通过小鼠在伴药箱停留的时间多少,来反映小鼠对滥用药物的渴求程度,也是一种经典的筛选抗成瘾药物的动物模型[10]。CPP模型的建立有很多影响因素,如给药方法(增量法、恒量法)、恒量法中的吗啡浓度(5、10、20 mg·kg−1)[11-13]、吗啡注射次数(3、4、6次)[14-15]、给药方式(皮下注射,腹腔注射)[16-17]、给药时程间(6、8、10 d)、训练时间(30、40、50 min)的选择;CPP箱体个数的差异(三箱式、两箱式);CPP箱体环境的差异(箱壁颜色、箱底纹理)及动物种类(小鼠种类、大鼠种类)[18]等都会影响CPP模型建立的效果。在恒量吗啡给药中,不同研究存在吗啡给药时间间隔上的差异[19-20],主要有24 、48 h,目前这两种方法间的差异尚未见报道。本研究针对不同给药时间间隔对吗啡诱导小鼠CPP模型的影响,比较建立模型前后小鼠在各箱的停留时间和CPP模型的消退情况,对吗啡诱导CPP模型进行综合评价,为后续抗吗啡成瘾药物的筛选提供整体动物模型。
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CPP造模前后小鼠在伴药箱停留时间见表1,结果表明,造模前各组小鼠在伴药箱停留时间无明显差异,经多次皮下注射吗啡后,与生理盐水组(24 h)相比,吗啡组(24 h)小鼠在伴药箱的时间极显著增加(P < 0.001);与生理盐水组(48 h)相比,吗啡组(48 h)小鼠在伴药箱的时间也极显著增加(P < 0.001)。计算CPP得分,结果见图1。通过单因素方差分析,与生理盐水组相比,吗啡组24 h和48 h组的CPP得分均明显增加,但二者之间无显著性差异,两种方法都能成功建立CPP模型。
表 1 不同给药时间间隔对CPP模型建立的影响
Table 1. The effect of intervals of drug administration on establishment of conditioned place preference (CPP) model
给药时间间隔
Interval of drug
administration/h组别
Treatment
group造模前伴药箱时间/s
Time spent in drug paired compartment
prior to model establishment造模后伴药箱时间/s
Time spent in drug paired compartment
after model establishmentCPP 得分
CPP score24 生理盐水组 274.26 ± 17.01 287.35 ± 25.03 13.09 ± 16.63 吗啡组 260.45 ± 14.05 452.29 ± 22.52*** 191.85 ± 23.08*** 48 生理盐水组 278.87 ± 20.70 292.48 ± 17.17 13.61 ± 8.18 吗啡组 285.49 ± 12.04 462.47 ± 21.51### 176.98 ± 17.89### 注:***代表与生理盐水组(24 h)相比较具有极显著差异,P< 0.001;###代表与生理盐水组(48 h)相比较具有极显著差异,P< 0.001。
Note:*** indicates highly significant difference at P < 0.001 as against the saline group (24 h); ### indicates highly significant difference at P < 0.001 as compared to the saline group (48 h). -
CPP表达期以后第2天进行消退训练,比较2种给药时间间隔建立CPP模型后的维持情况。每天进行1次15 min的测试。结果(图2)表明,前3 d内,两组小鼠在伴药箱的停留时间差别不大,说明短期(3 d)内时间间隔为24 h和48 h时建立的CPP模型无差别;从第4 天开始,吗啡组(24 h)小鼠在伴药箱停留的时间开始变小,出现消退情况(伴药箱时间与基础值的差值为基础值的20%以内),6~10 d时,消退情况较多,但消退情况不稳定(前天消退,后天又恢复)到10 d时大部分小鼠(8/12,约66%)消退且稳定,而吗啡组(48 h)小鼠在伴药箱的时间一直维持在440 s左右,10 d时,只有少部分(3/12,约25%)小鼠的CPP恢复至基础值左右,在6 d和10 d时,伴药箱的时间显著高于吗啡组(24 h)的时间(F(9,220) = 7.23;6 d,t=3.000,P<0.05;10 d,t=2.947,P< 0.05),这表明给药间隔时间为24 h建立的CPP模型维持时间短、易被消退;对吗啡组(48 h)小鼠继续测试7 d,结果见图3。从图3可见,吗啡组(48 h)在11~13 d时伴药箱内停留依然保持较高水平,与基础值相比具有极显著差异(P< 0.001),在第14天时出现消退情况,到第16天后部分(5/12,42%)小鼠被消退,所以吗啡给药间隔48 h建立的CPP模型可以维持15 d。
Effect of Intervals of Drug Administration on Morphine-induced Conditioned Place Preference Models in Mice
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摘要: 为比较不同给药时间间隔下,同等剂量的吗啡诱导条件性位置偏爱(CPP)模型的差异,为后续抗吗啡依赖药物的筛选奠定基础,本研究建立了不同时间间隔的吗啡诱导CPP模型,(1)吗啡组(24 h):连续4 d皮下注射,上午注射吗啡(5 mg·kg−1),下午注射生理盐水,吗啡给药间隔24 h,建立CPP模型;(2)吗啡组(48 h):连续8 d皮下注射,1、3、5、7 d注射吗啡(5 mg·kg−1),2、4、6、8 d注射生理盐水,吗啡给药间隔48 h,建立CPP模型。测定造模前后小鼠在伴药箱的停留时间\造模后CPP得分及CPP模型维持时间。结果表明,吗啡给药时间间隔24 h和48 h均能显著增加小鼠在伴药箱内的停留时间,造模后CPP得分明显增加,吗啡组(24 h)小鼠CPP模型能持续5 d,而吗啡组(48 h)小鼠CPP模型能持续15 d。短期内给药时间间隔不影响CPP模型的形成,与吗啡组(24 h)进行长期对比,给药时间间隔为48 h时,CPP模型维持时间更长、不易消退。
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关键词:
- 给药时间间隔 /
- 吗啡 /
- 小鼠条件性位置偏爱模型 /
- 维持时间
Abstract: To compare the difference of the morphine-induced conditioned place preference (CPP) models with the same dose of morphine under different time intervals of drug administration, morphine morphine-induced CPP models were established to provide a reference for screening anti-morphine addiction drug. Morphine groups (24 s) and (48 h) and saline groups (24 h) and (48 h) were designed in this experiment. The morphine group (24 h) received a subcutaneous injection for 4 consecutive days, and the mice were injected with morphine at the dose of 5 mg·kg−1 in the morning, and with saline in the afternoon. The interval of morphine administration in this group was 24 hours. In the morphine group (48 h) subcutaneous injections were given to mice for 8 consecutive days, and the mice were injected with morphine at 5 mg·kg−1 at days 1, 3, 5 and 7, and with saline at days 2, 4, 6 and 8. Morphine was administered at an interval of 48 hours in this group. The saline groups (24 h) and (48 h) were given subcutaneous injection but only with saline. CPP models for the morphine groups were then established. The time spent in drug-paired compartments was recorded to explore the establishment and maintenance of the CPP models for the morphine groups, and CPP scores were also calculated. The results showed that morphine significantly increased the time spent in the drug-paired compartments and the CPP scores when administered to the mice at respective intervals of 24 hours and 48 hours. The mouse CPP model of the mice lasted for 5 days in the morphine group (24 h) and for 15 days in the morphine group (48 h). It is concluded that the time interval of drug administration did not affect the establishment of the CPP model in a short term and that the mouse CPP model lasted longer with a delay in the extinction of the mouse CPP in the morphine group (48 h) than in the morphine group (24 h) in a long term. -
图 3 11~17 d内吗啡组(48 h)的消退情况
***代表与基础值相比较具有极显著性差异,且*=P < 0.05,**= P < 0.01,***=P < 0.001。
Fig. 3 The extinction of mouse preference behavior in the morphine group (48 h) in 11-17 days
*** indicates highly significant difference as against the preconditioned preference baseline: * at P < 0.05, ** P < 0.01 and *** P< 0.001.
表 1 不同给药时间间隔对CPP模型建立的影响
Table 1 The effect of intervals of drug administration on establishment of conditioned place preference (CPP) model
给药时间间隔
Interval of drug
administration/h组别
Treatment
group造模前伴药箱时间/s
Time spent in drug paired compartment
prior to model establishment造模后伴药箱时间/s
Time spent in drug paired compartment
after model establishmentCPP 得分
CPP score24 生理盐水组 274.26 ± 17.01 287.35 ± 25.03 13.09 ± 16.63 吗啡组 260.45 ± 14.05 452.29 ± 22.52*** 191.85 ± 23.08*** 48 生理盐水组 278.87 ± 20.70 292.48 ± 17.17 13.61 ± 8.18 吗啡组 285.49 ± 12.04 462.47 ± 21.51### 176.98 ± 17.89### 注:***代表与生理盐水组(24 h)相比较具有极显著差异,P< 0.001;###代表与生理盐水组(48 h)相比较具有极显著差异,P< 0.001。
Note:*** indicates highly significant difference at P < 0.001 as against the saline group (24 h); ### indicates highly significant difference at P < 0.001 as compared to the saline group (48 h). -
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