长期低剂量氯胺酮对青春期大脑前额叶的损伤效应与机制研究
发布时间：2018-08-25 11:18
【摘要】：1前言 氯胺酮(ketamine)是使用较为广泛的镇痛麻醉剂,但目前已成为主流娱乐性质的毒品,且滥用现象日趋严重。以英国为例,1999-2003年,娱乐性使用氯胺酮的人群增加了近2倍,且以青少年群体为主。研究提示,使用低于麻醉剂量的氯胺酮可出现难以言喻的愉快体验,如同从地狱跃入天堂。另有研究表明,正常成年人使用亚麻醉剂量的氯胺酮能够产生类似精神分裂症的阳性和阴性症状,包括知觉改变、幻视、幻觉、去人格化和现实感丧失等。滥用氯胺酮引起的个体和群体效应已经引起国际广泛关注。目前,氯胺酮在青少年亚群体中的普遍流行和滥用,原因之一是人们认为它不会导致躯体性依赖；其二,青少年群体具有追求新奇事物和新奇感觉的年龄特征。现氯胺酮在多数国家已成为限制性药物。 氯胺酮是N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMD A)受体的非竞争性拮抗剂,调节谷氨酸和天冬氨酸等兴奋性氨基酸(excitatory amino acids, EAAs)的功能,在突触可塑性和学习记忆中扮演着重要的角色。最近的研究发现,静脉给予成年人短期氯胺酮可损害认知功能和心理健康。但对长期低剂量氯胺酮是否对脑功能产生影响、其生理机制如何却鲜有研究。而长期低剂量使用方式与娱乐场所的氯胺酮滥用状态更为相似。 大多数成瘾药物是通过脑奖赏环路(VTA-NAcc-PFC神经环路)实现成瘾药物的奖赏效应。前额叶皮层(prefrontal cortex, PFC)接受来自腹侧被盖区(ventral tegmental area, VTA)的多巴胺能神经纤维支配,与情感行为、学习与记忆等诸多脑的高级功能密切相关。认知是指人们认识活动的过程,即个体对感觉信号接收、检测、转换、简约、合成、编码、储存、提取、重建、概念形成、判断和问题解决的信息加工处理过程。已经证实PFC是认知过程的关键脑区,构成了认知过程的中枢管理和工作记忆缓冲的基础结构。PFC内部合理的联系为合成不同范围的信息提供了完美的基础结构。有证据表明,PFC的多巴胺能系统对氯胺酮尤为敏感,对啮齿类动物研究发现,持续给予一定量氯胺酮(20mg/kg)可引起中枢神经系统(central nervous system, CNS)特定区域神经元细胞发生程序性细胞死亡,通过线粒体和胞质内质网通路凋亡通路,增加促凋亡因子的表达,加速细胞凋亡。且有证据表明,发育期大脑的这种损伤效应会对成年期的脑功能产生影响。 本研究假设长期娱乐剂量氯胺酮可能通过细胞凋亡途径引起大脑特定脑区,尤其是PFC脑区的细胞毒性作用,造成自发行为、认知功能的损伤。 2目的 2.1观察长期低剂量氯胺酮对青少年期ICR小鼠体重和神经肌肉强度、痛觉末梢和空间学习记忆的影响。 2.2观察长期低剂量氯胺酮对青少年期食蟹猴体重、自发行为的影响。 2.3探讨长期低剂量氯胺酮对青春期脑前额叶神经元凋亡表达的影响。 2.4揭示长期低剂量氯胺酮对青春期脑功能的影响和相关机制。 3材料和方法 3.1ICR小鼠实验动物模型制备 90只小鼠随机分为3组(每组30只),按给药时间长短分为一个月、三个月和六个月组。各组又随机分为对照组(10只)和氯胺酮组(20只),总实验周期为6个月。氯胺酮组每天腹腔注射给予盐酸氯胺酮30mg/kg,对照组给予生理盐水1mg/kg。 3.2ICR小鼠体重测量 实验开始前测量ICR小鼠基础体重,之后每周测量并记录体重变化情况,并随时调整给药剂量,观察长期给予氯胺酮是否会影响ICR小鼠体重增长。 3.3ICR小鼠行为学观察 在造模结束后进行连续3天的行为学测试,内容包括悬挂实验、热板实验和水迷宫实验。 3.4ICR小鼠前额叶神经元凋亡检测 采用核糖核酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, TUNEL)检测PFC中神经元细胞凋亡的情况。取PFC石蜡切片蛋白酶K (proteinase K, pK)孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色,显微镜观察计数神经元凋亡的数量。 3.5ICR小鼠前额叶免疫印迹分析(Western Blotting) 总蛋白经抽提后蛋白定量,按照上样量为100μg的量计算上样体积,经加热变性处理后上样,电泳,5%浓缩胶80V,12%分离胶120V；电转后移至PVDF膜上,5%脱脂奶粉室温封闭1h,一抗4℃过夜,洗膜3次,二抗室温1h,洗膜3次,用ODYSSEY红外成像系统记录显影条带的光密度值。 3.6食蟹猴实验动物模型制备 24只食蟹猴随机分为3组(每组8只),按给药时间的长短分为一个月氯胺酮组,六个月氯胺酮组和六个月对照组。实验组每天静脉注射给予盐酸氯胺酮1mg/kg,对照组给予生理盐水1mg/kg。 3.7食蟹猴体重测量 实验开始前测量食蟹猴基础体重,之后每月测量并记录体重变化情况,观察长期氯胺酮是否会影响食蟹猴体重增长。 3.8食蟹猴行为学观察 在实验的第1、3、7、14、30、31、56、112、183、184、185天给药后录像观察食蟹猴自发行为(行为量表)15min。观察内容包括移动、行走、攀爬、跳跃四种行为。 3.9食蟹猴前额叶神经元凋亡检测 TUNEL染色来检测PFC神经元细胞凋亡情况。取PFC石蜡切片pK孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色,显微镜观察计数神经元凋亡数目。 3.10食蟹猴前额叶免疫印迹分析Western Blotting 总蛋白经抽提后蛋白定量,按照上样量为30μg的量计算上样体积,经加热变性处理后上样,电泳,5%浓缩胶80V,12%分离胶120V；电转后移至PVDF膜上,5%脱脂奶粉室温封闭1h,一抗4℃过夜,洗膜3次,二抗室温1h,洗膜3次,暗室中显影定影,胶片经扫描后用Image J14.0软件分析目的条带的光密度值。 4实验结果 4.1长期低剂量氯胺酮对ICR小鼠体重增长的影响 实验过程中,对照组和氯胺酮组小鼠体重均有不同程度的增加,与初始体重比较均有统计学差异。与对照组相比,氯胺酮组体重增加速度缓慢。不同用药时间的三个组别中氯胺酮组与对照组的体重增长幅度无统计学差异。 4.2长期低剂量氯胺酮对ICR小鼠行为的影响 悬挂实验：一个月和三个月氯胺酮组小鼠掉落软垫所需的延迟时间与其对照组比较,差异无统计学意义,而六个月氯胺酮组在延迟时间上显著少于对照组,具有统计学差异。 热板实验：一个月和三个月氯胺酮组小鼠热板运动总和与其对照组比较,差异无统计学意义,而六个月氯胺酮组的热板运动总和显著少于对照组,具有统计学差异。 水迷宫实验：不同用药时间氯胺酮小鼠寻找逃避平台所需的潜伏期与对照组相比无统计学差异。 4.3长期低剂量氯胺酮对ICR小鼠大脑PFC神经元细胞凋亡的影响 氯胺酮组和对照组TUNEL阳性细胞计数无统计学差异。蛋白印迹分析结果显示,在一个月、三个月和六个月的氯胺酮组与对照组比较,Bax和caspase-3表达增高,Bax/Bcl-2比值增高,Bcl-2表达降低,但差异均无统计学意义。 4.4长期低剂量氯胺酮对青年期食蟹猴体重增加的影响 在6个月的实验过程中,各组食蟹猴体重均增加。氯胺酮组食蟹猴体重增长速度低于对照组,但差异无统计学意义。 4.5长期低剂量氯胺酮对青年期食蟹猴自发活动的影响 与对照组比较,氯胺酮组的自发运动有下降的趋势。给药一个月后,氯胺酮组跳跃显著低于对照组(F=7.439,P0.01)；在其后的153天中,氯胺酮组移动、行走、跳跃和自发活动总量自身比较具有统计学差异(移动：F=4.048,P0.05；行走：F=10.753,P0.01；跳跃：F=4.180,P0.05)。氯胺酮组移动、攀爬和自发行为总量显著少于对照组,差异有统计学意义(移动：F=10.798,P0.001；攀爬：F=4.769,P0.05；自发活动总量：F=5.793,P0.05)。氯胺酮组自发活动总量随用药时间延长而减少(F=12.914,P0.0001),且与药物处理存在交互作用(F=7.342,P0.001)。 4.6长期低剂量氯胺酮对PFC细胞凋亡的影响 与对照组相比,一个月氯胺酮组前额叶TUNEL染色阳性细胞数、促凋亡蛋白(Bax和caspase-3)和Bcl-2表达水平均无显著性差异。六个月氯胺酮组PFC阳性细胞、促凋亡蛋白(Bax和caspase-3)表达水平明显增加,差异有统计学意义。 5结论 5.1长期低剂量氯胺酮可导致青春期小鼠和食蟹猴体重增长速度减慢；小鼠肌肉力量降低和伤害感受受损；抑制食蟹猴自发行为活动。 5.2长期低剂量氯胺酮可诱导食蟹猴前额叶发生明显地细胞凋亡现象,产生神经元细胞毒性作用,进而造成大脑功能受损。 5.3氯胺酮对中枢神经的损伤存在动物种属的差异,与啮齿类动物相比较,非人灵长类动物神经系统对氯胺酮更敏感。这为进一步研究慢性氯胺酮的生理心理机制和制定其临床靶向治疗策略提供了一定的理论基础。
[Abstract]:1 Preface
Ketamine is a widely used analgesic anesthetic, but it has become a mainstream entertainment drug, and the abuse of ketamine is becoming more and more serious. In Britain, for example, in 1999-2003, the number of recreational ketamine users nearly doubled, mainly among young people. Research suggests that the use of ketamine below the anesthetic dose can occur. Unutterable pleasure experiences are like jumping from hell to heaven. Other studies have shown that normal adults using subanesthetic doses of ketamine can produce positive and negative symptoms similar to those of schizophrenia, including changes in perception, hallucinations, hallucinations, depersonalization, and loss of realism. Individual and group effects of ketamine abuse have been reported. At present, ketamine is prevalent and abused in the subgroup of adolescents, one of the reasons is that it does not lead to physical dependence; the other is that the adolescent group has the age characteristics of pursuing novelty and novelty sensation. Now ketamine has become a restrictive drug in most countries.
Ketamine is a non-competitive antagonist of N-methyl-D-aspartate (NMD) receptor, which regulates the function of excitatory amino acids (EAAs), such as glutamate and aspartate, and plays an important role in synaptic plasticity, learning and memory. Recent studies have found that intravenous administration of short-term chlorine to adults plays an important role in synaptic plasticity, learning and memory. Aminone impairs cognitive function and mental health. However, little is known about the physiological mechanism of the effects of long-term low-dose ketamine on brain function.
The prefrontal cortex (PFC) receives dopaminergic nerve fibers from the ventral tegmental area (VTA) and is closely related to many brain functions, such as emotional behavior, learning and memory. Cognition refers to the process of cognition, that is, the process of information processing in which an individual receives, detects, transforms, simplifies, synthesizes, encodes, stores, extracts, reconstructs, conceptualizes, judges and solves problems. PFC has been proved to be the key brain area in cognitive process, constituting the central management of cognitive process and the buffer of working memory. Infrastructure. Reasonable connections within PFC provide the perfect infrastructure for synthesizing information in different ranges. Evidence suggests that the dopaminergic system of PFC is particularly sensitive to ketamine. Studies in rodents have found that sustained administration of a certain amount of ketamine (20mg/kg) can cause specific central nervous system (CNS). Programmed cell death occurs in regional neurons, which increases the expression of pro-apoptotic factors and accelerates apoptosis through the apoptotic pathway of mitochondria and endoplasmic reticulum, and there is evidence that this damage effect in the developing brain may affect brain function in adulthood.
This study hypothesizes that long-term recreational dose ketamine may induce cytotoxicity in specific brain regions, especially PFC, through apoptosis pathway, resulting in spontaneous behavioral and cognitive impairment.
2 purposes
2.1 To observe the effects of long-term low-dose ketamine on body weight, neuromuscular strength, pain terminals and spatial learning and memory in adolescent ICR mice.
2.2 to observe the effects of long-term low dose ketamine on body weight and spontaneous behavior in juvenile cynomolgus monkeys.
2.3 to investigate the effect of long-term low-dose ketamine on the expression of neuronal apoptosis in prefrontal cortex in puberty.
2.4 to reveal the effects of long-term low-dose ketamine on brain function in adolescence and its related mechanisms.
3 materials and methods
Preparation of 3.1ICR mouse experimental animal model
Ninety mice were randomly divided into three groups (30 mice in each group) and divided into three groups according to the duration of administration: one month, three months and six months. Each group was randomly divided into control group (10 mice) and ketamine group (20 mice) with a total experimental period of six months.
Body weight measurement of 3.2ICR mice
The basal body weight of ICR mice was measured before the experiment began, and then the changes of body weight were recorded weekly. The dosage of ketamine was adjusted at any time to observe whether long-term ketamine administration could affect the weight gain of ICR mice.
Behavioral observation of 3.3ICR mice
Behavioral tests were conducted for three consecutive days after modeling, including suspension test, hot-plate test and water maze test.
Detection of apoptosis in prefrontal neurons of 3.4ICR mice
The apoptosis of neurons in PFC was detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). The number of apoptotic neurons was counted.
Western blot analysis of prefrontal lobe in 3.5ICR mice (Western Blotting)
The total protein was extracted and quantified. The sample volume was calculated according to the sample volume of 100 ug. After heating and denaturing, the sample was electrophoresis, 5% concentrated gel 80V, 12% separating gel 120V. After electrophoresis, the protein was transferred to PVDF membrane, 5% skimmed milk powder was sealed at room temperature for 1 hour, the first resistance was overnight at 4 ~C, the second resistance was washed three times at room temperature, and the second resistance was washed three times with ODYSSEY infrared imaging system. The optical density of the developing strip.
Preparation of experimental animal model for 3.6 cynomolgus monkeys
Twenty-four cynomolgus monkeys were randomly divided into three groups (8 in each group) according to the duration of administration: ketamine group for one month, ketamine group for six months and control group for six months.
3.7 body weight measurement of cynomolgus monkeys
The basal body weight of cynomolgus monkeys was measured before the experiment, and then the changes of body weight were recorded monthly to observe whether long-term ketamine could affect the weight gain of cynomolgus monkeys.
Behavioral observation of 3.8 cynomolgus monkeys
The spontaneous behavior of cynomolgus monkeys (Behavior Scale) was recorded for 15 minutes on day 1, 3, 7, 14, 30, 31, 56, 112, 183, 184 and 185 after administration.
3.9 detection of neuronal apoptosis in prefrontal cortex of cynomolgus monkeys
The apoptosis of PFC neurons was detected by TUNEL staining. The number of apoptotic neurons was observed and counted by microscope.
3.10 the prefrontal lobe of cynomolgus monkey by Western blot analysis Western Blotting
After extraction, the total protein was quantified, and the sample volume was calculated according to the amount of 30 ug. After heat denaturation, the sample was loaded with electrophoresis, 5% concentrated gel 80V, 12% separating gel 120V. After electrophoresis, the protein was transferred to PVDF membrane, 5% skimmed milk powder was sealed at room temperature for 1 hour, the first resistance overnight, the second resistance was washed three times at room temperature, the film was washed three times, the film was developed and fixed in darkroom, and the film was scanned. After tracing, the optical density of the target strip was analyzed by Image J14.0 software.
4 experimental results
4.1 long term low-dose ketamine on weight gain in ICR mice
During the experiment, the body weight of the control group and ketamine group increased in varying degrees, which was statistically different from the initial body weight. Compared with the control group, the body weight of ketamine group increased slowly.
4.2 the effect of long-term low-dose ketamine on the behavior of ICR mice
Suspension test: There was no significant difference in the delayed time of falling pad between one month and three months ketamine group and the control group, but the delayed time of six months ketamine group was significantly less than the control group.
Hot-plate test: There was no significant difference in the sum of hot-plate exercise between one month and three months ketamine group and the control group, but the sum of hot-plate exercise in six months ketamine group was significantly less than the control group.
Water maze test: There was no significant difference in the latency of ketamine mice in searching for escape platform between the two groups.
4.3 effects of long-term low-dose ketamine on apoptosis of PFC neurons in ICR mice
There was no significant difference in TUNEL positive cell count between ketamine group and control group. Western blot analysis showed that the expression of Bax and caspase-3 increased, the ratio of Bax to Bcl-2 increased and the expression of Bcl-2 decreased in ketamine group compared with control group at one month, three months and six months, but the difference was not statistically significant.
4.4 the effect of long-term low dose ketamine on body weight gain in young cynomolgus monkeys.
During the 6-month experiment, the weight of cynomolgus monkeys in each group increased. The weight growth rate of cynomolgus monkeys in ketamine group was lower than that of control group, but the difference was not statistically significant.
4.5 the effect of long-term low dose ketamine on spontaneous activity of young cynomolgus monkeys.
Compared with the control group, the spontaneous movement of ketamine group showed a downward trend. One month after administration, the jump of ketamine group was significantly lower than that of the control group (F = 7.439, P 0.01); in the subsequent 153 days, the total amount of movement, walking, jumping and spontaneous activity of ketamine group was statistically different (movement: F = 4.048, P 0.05; walking: F = 10.753, P 0.01). The total amount of movement, climbing and spontaneous behavior in ketamine group was significantly less than that in control group, and the difference was statistically significant (movement: F = 10.798, P 0.001; climbing: F = 4.769, P 0.05; total amount of spontaneous activity: F = 5.793, P 0.05). The total amount of spontaneous activity in ketamine group decreased with the prolongation of treatment (F = 12.914, P 0.0001), and with the drug. Processing interaction exists (F=7.342, P0.001).
4.6 the effect of long term low-dose ketamine on apoptosis of PFC cells
Compared with the control group, there was no significant difference in the number of TUNEL-positive cells, the expression of pro-apoptotic protein (Bax and caspase-3) and Bcl-2 in the prefrontal lobe of ketamine group in one month.
5 Conclusion
5.1 Long-term low-dose ketamine can slow down the weight gain of adolescent mice and cynomolgus monkeys, decrease the muscle strength and impair the sense of injury, and inhibit the spontaneous behavior of cynomolgus monkeys.
5.2 Long-term low-dose ketamine could induce apoptosis in the prefrontal lobe of cynomolgus monkeys, which could induce neurotoxicity and damage the brain function.
5.3 Ketamine is more sensitive to ketamine in non-human primates than in rodents. This provides a theoretical basis for further study of the physiological and psychological mechanism of chronic ketamine and the development of clinical targeted therapy strategies.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.61
[Abstract]:1 Preface
Ketamine is a widely used analgesic anesthetic, but it has become a mainstream entertainment drug, and the abuse of ketamine is becoming more and more serious. In Britain, for example, in 1999-2003, the number of recreational ketamine users nearly doubled, mainly among young people. Research suggests that the use of ketamine below the anesthetic dose can occur. Unutterable pleasure experiences are like jumping from hell to heaven. Other studies have shown that normal adults using subanesthetic doses of ketamine can produce positive and negative symptoms similar to those of schizophrenia, including changes in perception, hallucinations, hallucinations, depersonalization, and loss of realism. Individual and group effects of ketamine abuse have been reported. At present, ketamine is prevalent and abused in the subgroup of adolescents, one of the reasons is that it does not lead to physical dependence; the other is that the adolescent group has the age characteristics of pursuing novelty and novelty sensation. Now ketamine has become a restrictive drug in most countries.
Ketamine is a non-competitive antagonist of N-methyl-D-aspartate (NMD) receptor, which regulates the function of excitatory amino acids (EAAs), such as glutamate and aspartate, and plays an important role in synaptic plasticity, learning and memory. Recent studies have found that intravenous administration of short-term chlorine to adults plays an important role in synaptic plasticity, learning and memory. Aminone impairs cognitive function and mental health. However, little is known about the physiological mechanism of the effects of long-term low-dose ketamine on brain function.
The prefrontal cortex (PFC) receives dopaminergic nerve fibers from the ventral tegmental area (VTA) and is closely related to many brain functions, such as emotional behavior, learning and memory. Cognition refers to the process of cognition, that is, the process of information processing in which an individual receives, detects, transforms, simplifies, synthesizes, encodes, stores, extracts, reconstructs, conceptualizes, judges and solves problems. PFC has been proved to be the key brain area in cognitive process, constituting the central management of cognitive process and the buffer of working memory. Infrastructure. Reasonable connections within PFC provide the perfect infrastructure for synthesizing information in different ranges. Evidence suggests that the dopaminergic system of PFC is particularly sensitive to ketamine. Studies in rodents have found that sustained administration of a certain amount of ketamine (20mg/kg) can cause specific central nervous system (CNS). Programmed cell death occurs in regional neurons, which increases the expression of pro-apoptotic factors and accelerates apoptosis through the apoptotic pathway of mitochondria and endoplasmic reticulum, and there is evidence that this damage effect in the developing brain may affect brain function in adulthood.
This study hypothesizes that long-term recreational dose ketamine may induce cytotoxicity in specific brain regions, especially PFC, through apoptosis pathway, resulting in spontaneous behavioral and cognitive impairment.
2 purposes
2.1 To observe the effects of long-term low-dose ketamine on body weight, neuromuscular strength, pain terminals and spatial learning and memory in adolescent ICR mice.
2.2 to observe the effects of long-term low dose ketamine on body weight and spontaneous behavior in juvenile cynomolgus monkeys.
2.3 to investigate the effect of long-term low-dose ketamine on the expression of neuronal apoptosis in prefrontal cortex in puberty.
2.4 to reveal the effects of long-term low-dose ketamine on brain function in adolescence and its related mechanisms.
3 materials and methods
Preparation of 3.1ICR mouse experimental animal model
Ninety mice were randomly divided into three groups (30 mice in each group) and divided into three groups according to the duration of administration: one month, three months and six months. Each group was randomly divided into control group (10 mice) and ketamine group (20 mice) with a total experimental period of six months.
Body weight measurement of 3.2ICR mice
The basal body weight of ICR mice was measured before the experiment began, and then the changes of body weight were recorded weekly. The dosage of ketamine was adjusted at any time to observe whether long-term ketamine administration could affect the weight gain of ICR mice.
Behavioral observation of 3.3ICR mice
Behavioral tests were conducted for three consecutive days after modeling, including suspension test, hot-plate test and water maze test.
Detection of apoptosis in prefrontal neurons of 3.4ICR mice
The apoptosis of neurons in PFC was detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). The number of apoptotic neurons was counted.
Western blot analysis of prefrontal lobe in 3.5ICR mice (Western Blotting)
The total protein was extracted and quantified. The sample volume was calculated according to the sample volume of 100 ug. After heating and denaturing, the sample was electrophoresis, 5% concentrated gel 80V, 12% separating gel 120V. After electrophoresis, the protein was transferred to PVDF membrane, 5% skimmed milk powder was sealed at room temperature for 1 hour, the first resistance was overnight at 4 ~C, the second resistance was washed three times at room temperature, and the second resistance was washed three times with ODYSSEY infrared imaging system. The optical density of the developing strip.
Preparation of experimental animal model for 3.6 cynomolgus monkeys
Twenty-four cynomolgus monkeys were randomly divided into three groups (8 in each group) according to the duration of administration: ketamine group for one month, ketamine group for six months and control group for six months.
3.7 body weight measurement of cynomolgus monkeys
The basal body weight of cynomolgus monkeys was measured before the experiment, and then the changes of body weight were recorded monthly to observe whether long-term ketamine could affect the weight gain of cynomolgus monkeys.
Behavioral observation of 3.8 cynomolgus monkeys
The spontaneous behavior of cynomolgus monkeys (Behavior Scale) was recorded for 15 minutes on day 1, 3, 7, 14, 30, 31, 56, 112, 183, 184 and 185 after administration.
3.9 detection of neuronal apoptosis in prefrontal cortex of cynomolgus monkeys
The apoptosis of PFC neurons was detected by TUNEL staining. The number of apoptotic neurons was observed and counted by microscope.
3.10 the prefrontal lobe of cynomolgus monkey by Western blot analysis Western Blotting
After extraction, the total protein was quantified, and the sample volume was calculated according to the amount of 30 ug. After heat denaturation, the sample was loaded with electrophoresis, 5% concentrated gel 80V, 12% separating gel 120V. After electrophoresis, the protein was transferred to PVDF membrane, 5% skimmed milk powder was sealed at room temperature for 1 hour, the first resistance overnight, the second resistance was washed three times at room temperature, the film was washed three times, the film was developed and fixed in darkroom, and the film was scanned. After tracing, the optical density of the target strip was analyzed by Image J14.0 software.
4 experimental results
4.1 long term low-dose ketamine on weight gain in ICR mice
During the experiment, the body weight of the control group and ketamine group increased in varying degrees, which was statistically different from the initial body weight. Compared with the control group, the body weight of ketamine group increased slowly.
4.2 the effect of long-term low-dose ketamine on the behavior of ICR mice
Suspension test: There was no significant difference in the delayed time of falling pad between one month and three months ketamine group and the control group, but the delayed time of six months ketamine group was significantly less than the control group.
Hot-plate test: There was no significant difference in the sum of hot-plate exercise between one month and three months ketamine group and the control group, but the sum of hot-plate exercise in six months ketamine group was significantly less than the control group.
Water maze test: There was no significant difference in the latency of ketamine mice in searching for escape platform between the two groups.
4.3 effects of long-term low-dose ketamine on apoptosis of PFC neurons in ICR mice
There was no significant difference in TUNEL positive cell count between ketamine group and control group. Western blot analysis showed that the expression of Bax and caspase-3 increased, the ratio of Bax to Bcl-2 increased and the expression of Bcl-2 decreased in ketamine group compared with control group at one month, three months and six months, but the difference was not statistically significant.
4.4 the effect of long-term low dose ketamine on body weight gain in young cynomolgus monkeys.
During the 6-month experiment, the weight of cynomolgus monkeys in each group increased. The weight growth rate of cynomolgus monkeys in ketamine group was lower than that of control group, but the difference was not statistically significant.
4.5 the effect of long-term low dose ketamine on spontaneous activity of young cynomolgus monkeys.
Compared with the control group, the spontaneous movement of ketamine group showed a downward trend. One month after administration, the jump of ketamine group was significantly lower than that of the control group (F = 7.439, P 0.01); in the subsequent 153 days, the total amount of movement, walking, jumping and spontaneous activity of ketamine group was statistically different (movement: F = 4.048, P 0.05; walking: F = 10.753, P 0.01). The total amount of movement, climbing and spontaneous behavior in ketamine group was significantly less than that in control group, and the difference was statistically significant (movement: F = 10.798, P 0.001; climbing: F = 4.769, P 0.05; total amount of spontaneous activity: F = 5.793, P 0.05). The total amount of spontaneous activity in ketamine group decreased with the prolongation of treatment (F = 12.914, P 0.0001), and with the drug. Processing interaction exists (F=7.342, P0.001).
4.6 the effect of long term low-dose ketamine on apoptosis of PFC cells
Compared with the control group, there was no significant difference in the number of TUNEL-positive cells, the expression of pro-apoptotic protein (Bax and caspase-3) and Bcl-2 in the prefrontal lobe of ketamine group in one month.
5 Conclusion
5.1 Long-term low-dose ketamine can slow down the weight gain of adolescent mice and cynomolgus monkeys, decrease the muscle strength and impair the sense of injury, and inhibit the spontaneous behavior of cynomolgus monkeys.
5.2 Long-term low-dose ketamine could induce apoptosis in the prefrontal lobe of cynomolgus monkeys, which could induce neurotoxicity and damage the brain function.
5.3 Ketamine is more sensitive to ketamine in non-human primates than in rodents. This provides a theoretical basis for further study of the physiological and psychological mechanism of chronic ketamine and the development of clinical targeted therapy strategies.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.61
【参考文献】
相关期刊论文 前3条
1 王玉瑾,刘玲,贾娟,廖林川,王英元;氯胺酮的薄层层析和气相色谱质谱分析[J];中国医院药学杂志;2005年06期
2 贾娟;曹洁;王玉瑾;，
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