長期低劑量氯胺酮對青春期大腦前額葉的損傷效應(yīng)與機制研究
發(fā)布時間：2018-08-25 11:18
【摘要】：1前言 氯胺酮(ketamine)是使用較為廣泛的鎮(zhèn)痛麻醉劑,但目前已成為主流娛樂性質(zhì)的毒品,且濫用現(xiàn)象日趨嚴(yán)重。以英國為例,1999-2003年,娛樂性使用氯胺酮的人群增加了近2倍,且以青少年群體為主。研究提示,使用低于麻醉劑量的氯胺酮可出現(xiàn)難以言喻的愉快體驗,如同從地獄躍入天堂。另有研究表明,正常成年人使用亞麻醉劑量的氯胺酮能夠產(chǎn)生類似精神分裂癥的陽性和陰性癥狀,包括知覺改變、幻視、幻覺、去人格化和現(xiàn)實感喪失等。濫用氯胺酮引起的個體和群體效應(yīng)已經(jīng)引起國際廣泛關(guān)注。目前,氯胺酮在青少年亞群體中的普遍流行和濫用,原因之一是人們認(rèn)為它不會導(dǎo)致軀體性依賴；其二,青少年群體具有追求新奇事物和新奇感覺的年齡特征�，F(xiàn)氯胺酮在多數(shù)國家已成為限制性藥物。 氯胺酮是N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMD A)受體的非競爭性拮抗劑,調(diào)節(jié)谷氨酸和天冬氨酸等興奮性氨基酸(excitatory amino acids, EAAs)的功能,在突觸可塑性和學(xué)習(xí)記憶中扮演著重要的角色。最近的研究發(fā)現(xiàn),靜脈給予成年人短期氯胺酮可損害認(rèn)知功能和心理健康。但對長期低劑量氯胺酮是否對腦功能產(chǎn)生影響、其生理機制如何卻鮮有研究。而長期低劑量使用方式與娛樂場所的氯胺酮濫用狀態(tài)更為相似。 大多數(shù)成癮藥物是通過腦獎賞環(huán)路(VTA-NAcc-PFC神經(jīng)環(huán)路)實現(xiàn)成癮藥物的獎賞效應(yīng)。前額葉皮層(prefrontal cortex, PFC)接受來自腹側(cè)被蓋區(qū)(ventral tegmental area, VTA)的多巴胺能神經(jīng)纖維支配,與情感行為、學(xué)習(xí)與記憶等諸多腦的高級功能密切相關(guān)。認(rèn)知是指人們認(rèn)識活動的過程,即個體對感覺信號接收、檢測、轉(zhuǎn)換、簡約、合成、編碼、儲存、提取、重建、概念形成、判斷和問題解決的信息加工處理過程。已經(jīng)證實PFC是認(rèn)知過程的關(guān)鍵腦區(qū),構(gòu)成了認(rèn)知過程的中樞管理和工作記憶緩沖的基礎(chǔ)結(jié)構(gòu)。PFC內(nèi)部合理的聯(lián)系為合成不同范圍的信息提供了完美的基礎(chǔ)結(jié)構(gòu)。有證據(jù)表明,PFC的多巴胺能系統(tǒng)對氯胺酮尤為敏感,對嚙齒類動物研究發(fā)現(xiàn),持續(xù)給予一定量氯胺酮(20mg/kg)可引起中樞神經(jīng)系統(tǒng)(central nervous system, CNS)特定區(qū)域神經(jīng)元細胞發(fā)生程序性細胞死亡,通過線粒體和胞質(zhì)內(nèi)質(zhì)網(wǎng)通路凋亡通路,增加促凋亡因子的表達,加速細胞凋亡。且有證據(jù)表明,發(fā)育期大腦的這種損傷效應(yīng)會對成年期的腦功能產(chǎn)生影響。 本研究假設(shè)長期娛樂劑量氯胺酮可能通過細胞凋亡途徑引起大腦特定腦區(qū),尤其是PFC腦區(qū)的細胞毒性作用,造成自發(fā)行為、認(rèn)知功能的損傷。 2目的 2.1觀察長期低劑量氯胺酮對青少年期ICR小鼠體重和神經(jīng)肌肉強度、痛覺末梢和空間學(xué)習(xí)記憶的影響。 2.2觀察長期低劑量氯胺酮對青少年期食蟹猴體重、自發(fā)行為的影響。 2.3探討長期低劑量氯胺酮對青春期腦前額葉神經(jīng)元凋亡表達的影響。 2.4揭示長期低劑量氯胺酮對青春期腦功能的影響和相關(guān)機制。 3材料和方法 3.1ICR小鼠實驗動物模型制備 90只小鼠隨機分為3組(每組30只),按給藥時間長短分為一個月、三個月和六個月組。各組又隨機分為對照組(10只)和氯胺酮組(20只),總實驗周期為6個月。氯胺酮組每天腹腔注射給予鹽酸氯胺酮30mg/kg,對照組給予生理鹽水1mg/kg。 3.2ICR小鼠體重測量 實驗開始前測量ICR小鼠基礎(chǔ)體重,之后每周測量并記錄體重變化情況,并隨時調(diào)整給藥劑量,觀察長期給予氯胺酮是否會影響ICR小鼠體重增長。 3.3ICR小鼠行為學(xué)觀察 在造模結(jié)束后進行連續(xù)3天的行為學(xué)測試,內(nèi)容包括懸掛實驗、熱板實驗和水迷宮實驗。 3.4ICR小鼠前額葉神經(jīng)元凋亡檢測 采用核糖核酸末端轉(zhuǎn)移酶介導(dǎo)的缺口末端標(biāo)記法(terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, TUNEL)檢測PFC中神經(jīng)元細胞凋亡的情況。取PFC石蠟切片蛋白酶K (proteinase K, pK)孵育,平衡液處理,TUNEL反應(yīng)混合液孵育,DAB顯色,顯微鏡觀察計數(shù)神經(jīng)元凋亡的數(shù)量。 3.5ICR小鼠前額葉免疫印跡分析(Western Blotting) 總蛋白經(jīng)抽提后蛋白定量,按照上樣量為100μg的量計算上樣體積,經(jīng)加熱變性處理后上樣,電泳,5%濃縮膠80V,12%分離膠120V；電轉(zhuǎn)后移至PVDF膜上,5%脫脂奶粉室溫封閉1h,一抗4℃過夜,洗膜3次,二抗室溫1h,洗膜3次,用ODYSSEY紅外成像系統(tǒng)記錄顯影條帶的光密度值。 3.6食蟹猴實驗動物模型制備 24只食蟹猴隨機分為3組(每組8只),按給藥時間的長短分為一個月氯胺酮組,六個月氯胺酮組和六個月對照組。實驗組每天靜脈注射給予鹽酸氯胺酮1mg/kg,對照組給予生理鹽水1mg/kg。 3.7食蟹猴體重測量 實驗開始前測量食蟹猴基礎(chǔ)體重,之后每月測量并記錄體重變化情況,觀察長期氯胺酮是否會影響食蟹猴體重增長。 3.8食蟹猴行為學(xué)觀察 在實驗的第1、3、7、14、30、31、56、112、183、184、185天給藥后錄像觀察食蟹猴自發(fā)行為(行為量表)15min。觀察內(nèi)容包括移動、行走、攀爬、跳躍四種行為。 3.9食蟹猴前額葉神經(jīng)元凋亡檢測 TUNEL染色來檢測PFC神經(jīng)元細胞凋亡情況。取PFC石蠟切片pK孵育,平衡液處理,TUNEL反應(yīng)混合液孵育,DAB顯色,顯微鏡觀察計數(shù)神經(jīng)元凋亡數(shù)目。 3.10食蟹猴前額葉免疫印跡分析Western Blotting 總蛋白經(jīng)抽提后蛋白定量,按照上樣量為30μg的量計算上樣體積,經(jīng)加熱變性處理后上樣,電泳,5%濃縮膠80V,12%分離膠120V；電轉(zhuǎn)后移至PVDF膜上,5%脫脂奶粉室溫封閉1h,一抗4℃過夜,洗膜3次,二抗室溫1h,洗膜3次,暗室中顯影定影,膠片經(jīng)掃描后用Image J14.0軟件分析目的條帶的光密度值。 4實驗結(jié)果 4.1長期低劑量氯胺酮對ICR小鼠體重增長的影響 實驗過程中,對照組和氯胺酮組小鼠體重均有不同程度的增加,與初始體重比較均有統(tǒng)計學(xué)差異。與對照組相比,氯胺酮組體重增加速度緩慢。不同用藥時間的三個組別中氯胺酮組與對照組的體重增長幅度無統(tǒng)計學(xué)差異。 4.2長期低劑量氯胺酮對ICR小鼠行為的影響 懸掛實驗：一個月和三個月氯胺酮組小鼠掉落軟墊所需的延遲時間與其對照組比較,差異無統(tǒng)計學(xué)意義,而六個月氯胺酮組在延遲時間上顯著少于對照組,具有統(tǒng)計學(xué)差異。 熱板實驗：一個月和三個月氯胺酮組小鼠熱板運動總和與其對照組比較,差異無統(tǒng)計學(xué)意義,而六個月氯胺酮組的熱板運動總和顯著少于對照組,具有統(tǒng)計學(xué)差異。 水迷宮實驗：不同用藥時間氯胺酮小鼠尋找逃避平臺所需的潛伏期與對照組相比無統(tǒng)計學(xué)差異。 4.3長期低劑量氯胺酮對ICR小鼠大腦PFC神經(jīng)元細胞凋亡的影響 氯胺酮組和對照組TUNEL陽性細胞計數(shù)無統(tǒng)計學(xué)差異。蛋白印跡分析結(jié)果顯示,在一個月、三個月和六個月的氯胺酮組與對照組比較,Bax和caspase-3表達增高,Bax/Bcl-2比值增高,Bcl-2表達降低,但差異均無統(tǒng)計學(xué)意義。 4.4長期低劑量氯胺酮對青年期食蟹猴體重增加的影響 在6個月的實驗過程中,各組食蟹猴體重均增加。氯胺酮組食蟹猴體重增長速度低于對照組,但差異無統(tǒng)計學(xué)意義。 4.5長期低劑量氯胺酮對青年期食蟹猴自發(fā)活動的影響 與對照組比較,氯胺酮組的自發(fā)運動有下降的趨勢。給藥一個月后,氯胺酮組跳躍顯著低于對照組(F=7.439,P0.01)；在其后的153天中,氯胺酮組移動、行走、跳躍和自發(fā)活動總量自身比較具有統(tǒng)計學(xué)差異(移動：F=4.048,P0.05；行走：F=10.753,P0.01；跳躍：F=4.180,P0.05)。氯胺酮組移動、攀爬和自發(fā)行為總量顯著少于對照組,差異有統(tǒng)計學(xué)意義(移動：F=10.798,P0.001；攀爬：F=4.769,P0.05；自發(fā)活動總量：F=5.793,P0.05)。氯胺酮組自發(fā)活動總量隨用藥時間延長而減少(F=12.914,P0.0001),且與藥物處理存在交互作用(F=7.342,P0.001)。 4.6長期低劑量氯胺酮對PFC細胞凋亡的影響 與對照組相比,一個月氯胺酮組前額葉TUNEL染色陽性細胞數(shù)、促凋亡蛋白(Bax和caspase-3)和Bcl-2表達水平均無顯著性差異。六個月氯胺酮組PFC陽性細胞、促凋亡蛋白(Bax和caspase-3)表達水平明顯增加,差異有統(tǒng)計學(xué)意義。 5結(jié)論 5.1長期低劑量氯胺酮可導(dǎo)致青春期小鼠和食蟹猴體重增長速度減慢；小鼠肌肉力量降低和傷害感受受損；抑制食蟹猴自發(fā)行為活動。 5.2長期低劑量氯胺酮可誘導(dǎo)食蟹猴前額葉發(fā)生明顯地細胞凋亡現(xiàn)象,產(chǎn)生神經(jīng)元細胞毒性作用,進而造成大腦功能受損。 5.3氯胺酮對中樞神經(jīng)的損傷存在動物種屬的差異,與嚙齒類動物相比較,非人靈長類動物神經(jīng)系統(tǒng)對氯胺酮更敏感。這為進一步研究慢性氯胺酮的生理心理機制和制定其臨床靶向治療策略提供了一定的理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R749.61
【參考文獻】
相關(guān)期刊論文 前3條
1 王玉瑾,劉玲,賈娟,廖林川,王英元;氯胺酮的薄層層析和氣相色譜質(zhì)譜分析[J];中國醫(yī)院藥學(xué)雜志;2005年06期
2 賈娟;曹潔;王玉瑾;，
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