孕中期大鼠母体氨胺酮麻醉致子鼠海马发育异常及情绪障碍

发布时间：2018-06-20 20:49

本文选题：氯胺酮 + 脑发育　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景：近年来,大量实验研究均证实了常用全麻药比如氯胺酮、七氟醚、异氟醚等对啮齿类和灵长类动物发育期的大脑具有神经毒性,可致一系列病理形态学及神经电生理学的改变：(1)可致神经细胞内钙稳态失衡,线粒体功能障碍,多脑区神经细胞凋亡；(2)抑制神经细胞增殖；(3)损伤神经元骨架结构,阻碍神经元骨架结构形成,影响突触的发育,致突触形成障碍；(4)阻碍胶质细胞生长,影响其成熟；(5)影响海马的长时程增强(LTP)等。且与形态学及神经电生理学损害相一致的结果是,全麻药可导致远期的认知功能障碍。临床回顾性研究也表明在四岁之前接受过麻醉和手术的儿童,出现远期读写及计算能力障碍的风险性增加,人们越来越关注常用的全麻药对发育期大脑的安全性。目前大多数关于全麻药神经毒性损害所致的远期神经行为学异常的研究,主要集中于认知功能损害,比如记忆力、读写及计算能力的损害,而忽略了行为学异常的另外一个重要的方面,即情绪障碍。关于情绪与认知关系的研究,近年来引起了学者们极大的关注,一般认为情绪障碍与认知障碍的关系非常复杂,二者既可以共存也可以单独存在,且相互影响,相互作用。对于情绪障碍与认知障碍的病因病理机制及二者发生发展的顺序尚不明确,认知障碍(如记忆力损害)可能只是情绪障碍的一个症状或者说是病情发展到一定程度的结果而已。此外,关于全麻药对发育期大脑神经毒性的研究多集中在出生后发育期的(近似于人类新生儿期、儿童期)动物大脑,而孕期母体麻醉对宫内胎儿神经发育影响的研究则比较少。既往孕期全麻药毒性的研究主要是孕早期全麻药的致畸作用和分娩期母体麻醉用药对新生儿器官功能状态的影响,而孕中期全麻药对胎儿神经发育影响的研究很少。由于孕早期手术麻醉有发生致畸、流产的可能,大多数观点认为母体孕中期手术麻醉是一个相对安全时期。事实上,人类神经元增殖及迁移在孕中期开始加速并在孕中期后达到高峰,突触形成期即“易损期”始于孕中期一直持续至出生后的几年。临床使用的全麻药大多为脂溶性的,易透过胎盘进入胎儿血液循环,再通过血液循环系统作用于胎儿大脑,可能会对其生长发育产生各种不良影响。目前每年大约有0.75%-2%的孕妇在怀孕期间需要行外科手术治疗(比如阑尾和胆囊急性炎症、卵巢肿瘤、外伤等),这些手术多为不可避免的手术。而且,随着外科的发展,腹腔镜手术的日渐增多及胎儿手术的逐渐开展使孕期手术适应症不断增加,这个数字将会越来越大。因此母体孕中期麻醉对胎儿大脑发育造成的危害尤其值得关注。氯胺酮是一种临床常用的全身麻醉药物,经典的NMDA受体拮抗剂。尽管它在发达国家已经不再使用,但在发展中国家的临床麻醉、镇痛镇静中仍发挥着很大的作用。而且近年在亚太地区尤其是中国和印度,氯胺酮娱乐场所滥用情况急剧增加。其复杂的药理药效及毒副作用,如致精神分裂、致幻和认知功能损害等也引起人们的关注。本研究模拟孕中期产妇非产科手术麻醉,给予孕中期大鼠氯胺酮麻醉,兼顾母体、胎盘、孕周期、手术刺激等诸多因素,观察孕中期氯胺酮麻醉对子代鼠成年后认知功能和情绪的影响以及对出生后海马发育的影响,研究其毒性可能的分子机制。对指导孕中期相对安全的全麻药物选择、麻醉方案及防治措施,具有重大意义。实验目的：通过一系列行为学,即糖水偏好实验(SPT)、旷场实验(OFT)测试、水迷宫实验(WMT)、强迫游泳实验(FST),观察孕中期大鼠母体氯胺酮静脉麻醉对子鼠认知功能和情绪的影响；通过Western Blot检测子鼠海马区门冬氨酸(NMDA)受体亚基NRl、NR2A、NR2B及其下游效应因子脑源性营养因子(BDNF).突触后密度蛋白-95(PSD-95)的表达：应用溴脱氧嘧啶(BrdU)标记技术标记成年子代鼠海马齿状回(DG)和室管膜下区(SVZ)神经元以观察神经元增殖情况；应用高尔基银浸染色技术和尼氏染色观察子鼠海马区神经元的形态学改变,研究和探讨母体氯胺酮麻醉对子代鼠神经毒性的相关机制。实验方法： 12只成年雌性SD处鼠,体重约180-220g,适应性饲养一周后,晚上7：00和有性经验的雄性SD大鼠进行交配,次日早晨行阴道图片检查(镜下见到精子记为孕0天,即GO)。将孕鼠饲养至G14随机等分为2组：对照组(C,n=6)和氯胺酮组(K,n=6)。C组孕鼠不做任何处理,K组孕鼠经右侧大腿后部肌群肌肉注射氯胺酮40mg/kg,遂用24G套管针从尾静脉置管,接微量注射泵,以40～60mg/kg.h的速度持续静脉泵注氯胺酮(生理盐水稀释至10mg/ml)2h.随时调整输注速度使孕鼠处于深度镇静至轻度外科水平麻醉的状态,即孕鼠无自主运动,肌肉松弛,对疼痛刺激存在反应,眼睑反射消失。术中使用电加热板使母鼠体温维持在36.5-37.5℃,同时观察呼吸幅度、频率及口唇粘膜颜色。麻醉结束,待孕鼠翻正反射恢复后送回鼠笼继续饲养。至G22,各组随机选取3只孕鼠腹腔注射溴脱氧嘧啶(50mg/kg),2h后剖腹取子鼠大脑行免疫组化检测DG和SVZ区BrdU阳性细胞数目,余孕鼠等待自然分娩(记为P0),出生后约6h随机取各母鼠的2只子鼠大脑行Nissl染色,其余子鼠饲养至21天断奶,继续饲养至P25天。至P25,将各组子鼠随机分为两批(n=11/10,3-4只／母鼠)进行连续3天的行为学测试,一批行水迷宫(WMT)测试和糖水偏好实验(SPT),一批行旷场实验(OFT)和强迫游泳(FST)。行为学结束后,随机取6只子鼠(2只／母鼠)断头处死后取大脑制备石蜡切片行Nissl染色检测海马CA1及CA3区神经元密度；随机取5只子鼠(1-2只／母鼠)腹腔注射溴脱氧嘧啶(50mg/kg),连续3天,末次注射12h后断头处死制备冰冻切片,行免疫组化检测DG和SVZ区BrdU阳性细胞数目；随机取3只子鼠(1只／母鼠)断头处死后,快速取大脑行高尔基银浸染色分别观察海马CA1、CA3锥体细胞树突分支数目、总树突长度、树突空间分布情况、及突触棘密度；随机取3只子鼠(1只／母鼠)断头处死后,在冰面上快速取海马组织行Western Blot检测NMDA受体亚基NR1、NR2A、NR2B,及其下游效应因子BDNF、PSD-95蛋白表达情况。此外,在另一组实验中,取3只G14雌鼠和年龄相当的3只未孕雌鼠,行相同的氯胺酮静脉麻醉处理,麻醉完毕立即经左心室取动脉血行血气分析,此组母鼠不在参与后续的实验。实验结果： 1.所有氯胺酮静脉麻醉的雌鼠麻醉及苏醒过程平稳,无呼吸抑制等异常情况出现,血气分析显示各项生理指标均在正常范围内,且两组子鼠各项指标差异无统计学意义(P0.05)。P28时,氯胺酮组子鼠体重显著低于对照组子鼠(P0.01)。氯胺酮组两只体重偏低的(分别为36g、44g)子鼠不再参与后继的实验。 2.P25-P30时,行为学结果表明氯胺酮组子鼠呈现出抑郁和焦虑样情绪障碍,且伴有学习记忆力的损害。具体表现为：在SPT中,两组子鼠总液体饮用量的差异无统计学意义(P0.05),但是和对照组子鼠相比,氯胺酮组子鼠糖水饮用率显著下降(P0.05)；在FST中,和对照组子鼠相比,氯胺酮组子鼠不动时间显著延长(P0.05)；在OFT中,两组子鼠运动总距离和平均运动速度的差异均无统计学意义(P0.05),但是氯胺酮组子鼠穿越中心区的次数和逗留于中心区的时间显著减少(P0.05)；在WMT中,氯胺酮组子鼠找到平台的潜伏期显著延长(P0.01)。这些行为学结果表明,孕中期母体氯胺酮麻醉可致子鼠抑郁和焦虑样表现及学习记忆力损害。 3.在P0和P30时,和对照组子鼠相比,氯胺酮组子鼠海马CA3区细胞密度显著下降,P0时下降了23%,P30时下降了21%(P0,P0.01；P30,P0.05),两组子鼠CA1区椎体细胞密度差异均无统计学意义(P0.05)；P30时,氯胺酮组子鼠海马CA3区椎体细胞树突总长度显著短于对照组子鼠(P0.01),锥体细胞树突分支数目显著减少(P0.01)及突触棘密度显著下降(P0.05),Sholl分析示氯胺酮组子鼠CA3椎体细胞树突与各半径的同心圆交点数目显著减少(P0.01)。这些数据表明,CA3区对氯胺酮的损害更加敏感,孕中期母体氯胺酮麻醉致海马发育异常,海马CA3区(而不是CA1区)椎体细胞缺失及锥体细胞发育成熟障碍,突触形成障碍。 4.PO和P30时,和对照组子鼠相比,氯胺酮组子鼠DG和SVZ区BrdU阳性细胞数目显著低于对照组子鼠(PO-DG, P0.05, PO-SVZ, P0.01; P30-DG,P0.05,P30-SVZ,P0.05)。在P30时,氯胺酮组子鼠DG和SVZ区BrdU阳性细胞数目显著低于对照组,且BrdU/DCX双标阳性细胞数目和BrdU阳性细胞数目比值显著低于对照组子鼠,但是只有DG区的差异有统计学意义(P0.01)。孕中期母体氯胺酮麻醉可致子鼠细胞增殖及神经元增殖下降。 5.P30时,和对照组子鼠相比,氯胺酮组子鼠海马NMDA受体亚基NR2A表达显著升高(P0.05),NR2B表达显著下降,少于对照组的1/4(P0.01),NRl差异无统计学意义(P0.05),而且氯胺酮组子鼠海马BDNF、PSD-95的蛋白表达显著下降,其中PSD-95的蛋白表达少于对照组的三分之-(BDNF,P0.05; PSD-95, P0.01)。说明孕中期母体氯胺酮麻醉导致NMDA受体亚基表达紊乱,从而影响其功能,并导致其下游效应因子BDNF、PSD-95表达下调,抑制mTOR信号转道通路。实验结论：在孕中期,母体氯胺酮麻醉可导致胎儿大脑损伤,子鼠出生后直至成年齿状回及室管膜下区细胞增殖减少,海马CA3区细胞缺失,且成年后CA3区锥体细胞发育障碍；成年后的神经行为学异常,呈抑郁和焦虑样表现及记忆力损害。其机制可能与海马NMDA亚基受体紊乱致NMDA受体功能低下,致其下游效应因子BDNF、PDS-95表达下降,下调nTOR信号转道通路,导致海马神经元发育异常有关。
[Abstract]:Research background:
In recent years, a large number of experimental studies have confirmed that common anesthetics such as ketamine, sevoflurane and isoflurane have neurotoxicity to the brain of rodents and primates, which can lead to a series of changes in pathomorphology and neurophysiology: (1) the homeostasis of intracellular calcium homeostasis, mitochondrial dysfunction, and multi brain region deity can be caused. Apoptosis; (2) inhibiting the proliferation of nerve cells; (3) damage to the skeleton structure of neurons, hindering the formation of the skeleton structure, affecting the development of synapses, causing synapse formation obstacles; (4) hindering the growth of glial cells and affecting its maturation; (5) affecting the long term enhancement of the hippocampus (LTP), and in accordance with the morphological and neurophysiological damage. The result is that total anesthetics can lead to long-term cognitive impairment. Clinical retrospective studies also show that children who have been anesthetized and operated before four years of age have increased risk of long-term reading and writing and calculation disorders, and people are increasingly concerned about the safety of the common anesthetics for the development of the brain. The study of long-term neurobehavioral abnormalities caused by toxic damage mainly focuses on cognitive impairment, such as memory, reading and writing and the impairment of computational ability, while neglecting another important aspect of behavioral disorders, namely, emotional disorders. The research on the relationship between emotion and cognition has aroused great concern in recent years. The relationship between emotional disorders and cognitive impairment is very complex. The two can exist and exist alone, and they interact and interact with each other. The pathogenesis of the causes of emotional disorders and cognitive impairment and the order of the development of the two are not clear, and cognitive impairment (such as memory impairment) may be a symptom of emotional disorder. Or it is the result of a certain degree of development. In addition, the study of the neurotoxicity of total anesthetics on the developmental stage of the brain is mostly concentrated in the postnatal period (similar to the human neonatal period, childhood) of the animal brain, while the maternal anaesthesia has fewer studies on the effects of the maternal anaesthesia on the intrauterine fetal neurodevelopment. The effects of the teratogenicity of total anesthetic in the early pregnancy and the maternal anesthetic medication at the stage of childbirth on the state of the organ function of the newborns, and few studies on the influence of the mid trimester total anesthetics on the fetal nerve development. It is a period of relative safety. In fact, the proliferation and migration of human neurons begin to accelerate in the middle of the pregnancy and reach the peak after the mid pregnancy. The synaptic formation period, that is, the "vulnerable period" begins in the middle of the pregnancy to a few years after birth. Most of the clinical total anesthetics are fat soluble and easy to enter the fetal blood circulation through the placenta. The blood circulation system acts on the fetal brain and may have a variety of adverse effects on the growth and development of the fetus. At present, about 0.75%-2% of pregnant women need surgical treatment (such as acute appendix and gallbladder inflammation, ovarian tumors, trauma, etc.) during pregnancy. These hands are mostly unavoidable operations. The exhibition, laparoscopic surgery and fetal surgery is increasing gradually to carry out surgical indications during pregnancy is increasing, the number will be more and more. So the second trimester maternal anesthesia on fetal brain development caused by the harm of particular concern.
Ketamine is a commonly used general anesthesia drug, a classic NMDA receptor antagonist. Although it is no longer used in developed countries, it still plays a significant role in clinical anesthesia and analgesia in developing countries. In recent years, the abuse of ketamine in entertainment places in the Asia Pacific region, especially in China and India, has increased dramatically. In addition, its complex pharmacological effects and side effects, such as schizophrenia, hallucination and cognitive impairment, have attracted much attention.
This study simulated the non obstetric anesthesia in the midtrimester of pregnant women, given ketamine anesthesia in the mid-term rats, given consideration to many factors such as maternal body, placenta, pregnancy cycle, and surgical stimulation, and observed the effects of ketamine anesthesia on the cognitive function and emotion of the adult rats in the middle of pregnancy and the effect on the development of the hippocampus after birth, and studied the possible toxic molecules. The mechanism is of great significance to the selection of anesthetic drugs for general safety during the second trimester.
Objective:
Through a series of behavioural studies, such as sugar water preference test (SPT), open field experiment (OFT) test, water maze test (WMT) and forced swimming test (FST), the effects of ketamine intravenous anesthesia on the cognitive function and emotion of the rats in the mid-term pregnancy were observed. The Western Blot was used to detect the NMDA receptor subunit NRl, NR2A, NR2B and the Western Blot, and The downstream effect factor brain derived nutrition factor (BDNF) and the expression of postsynaptic density protein -95 (PSD-95): using bromodeoxypyrimidine (BrdU) labeling technique to mark the hippocampal dentate gyrus (DG) and subventricular subregion (SVZ) neurons of adult offspring to observe the proliferation of neurons, and observe Zi Shuhai with Golgi leaching and Nissl staining. The morphological changes of neurons in the hippocampus were studied to explore the mechanism of the neurotoxicity of maternal ketamine anesthesia on offspring rats.
12 adult female SD rats, weighing about 180-220g, were bred for one week after adaptation, at 7:00 p. m. and sexually experienced male SD rats. The next morning, a vaginal picture was performed (the sperm was found to be 0 days of pregnancy, that is, GO). The pregnant rats were fed to G14 randomly and divided into 2 groups: the control group (C, n=6) and the ketamine group (K, n=6).C group did not do it. In any treatment, the pregnant rats in group K were injected with ketamine 40mg/kg from the posterior muscle group of the right thigh, then the 24G cannula was inserted from the caudal vein to the microinjection pump, and the intravenous infusion of ketamine (diluted to 10mg/ml) 2h. at a rate of 40 to 60mg/kg.h (10mg/ml) 2h. at any time to adjust the rate of infusion at any time so that pregnant rats were in deep sedation to mild surgical anesthesia. The state of the pregnant rats was unautonomously movement, muscle relaxation, reaction to pain stimulation, and eyelid reflex disappearing. During the operation, electric heating plate was used to keep the body temperature of the mother rats at 36.5-37.5 C, while observing the amplitude, frequency and color of the lip mucous membrane. 3 pregnant rats were intraperitoneally injected with bromodeoxypyrimidine (50mg/kg). After 2h, the number of BrdU positive cells in DG and SVZ regions was detected by immunohistochemistry. The remaining pregnant rats were waiting for natural childbirth (P0). After birth, the brains of 2 rats in each mouse were stained with Nissl, and the remaining rats were fed to 21 days of weaning and continued to be kept to P25 days to P25, The rats were randomly divided into two groups (n=11/10,3-4 / female rats) for 3 consecutive days of behavioral test, a batch of water maze (WMT) test and sugar water preference test (SPT), a batch of open field experiments (OFT) and forced swimming (FST). After the end of the behavior, 6 rats (2 / mother rats) were randomly selected to take the brain to prepare the paraffin section for Nissl The density of neurons in the hippocampal CA1 and CA3 region was detected by staining, and 5 rats (1-2 rats) were randomly selected to be intraperitoneally injected with bromodeoxypyrimidine (50mg/kg) for 3 days. After the last injection of 12h, the frozen section was prepared by the end of 12h, and the number of BrdU positive cells in DG and SVZ region was detected by immunohistochemistry. After the death of 1 mice (1 / mother mice), the number of positive cells was taken quickly. The brain was stained with Golgi silver staining to observe the number of dendritic branches of the hippocampal CA1, CA3 pyramidal cells, the length of the total dendrites, the spatial distribution of dendrites, and the density of synaptic spines. After the 3 rats (1 rats) were randomly selected, the NMDA receptor subunit NR1, NR2A, NR2B, and the downstream effect of NMDA receptor subunit NR1, NR2A, NR2B, and the downstream effect were detected by Western Blot on the ice surface. In addition, in another group, 3 female G14 rats and 3 female rats of the same age were treated with the same ketamine intravenous anesthesia in another group of experiments. The blood gas analysis of the left ventricular arterial blood was done immediately after the anesthesia. This group of mice did not participate in the follow-up experiment. The results of the experiment were: 3
1. of the female rats with ketamine intravenous anesthesia, the anaesthesia and revival process were smooth and no respiratory inhibition appeared. The blood gas analysis showed that all the physiological indexes were in the normal range, and there was no significant difference between the two groups of rats (P0.05).P28, the weight of the ketamine group was significantly lower than that of the control group (P0.01). Two low weight (36g, 44g) mice were no longer involved in subsequent experiments.
At 2.P25-P30, the behavioral results showed that the rats in the ketamine group showed depression and anxiety like emotional disorders and the impairment of learning and memory. The specific manifestation was that there was no significant difference in total liquid drinking in the two groups of rats in SPT (P0.05), but compared with the control group, the drinking rate of sugar water in the ketamine group decreased significantly (P0. 05); in FST, the time of inactivity of rats in the ketamine group was significantly longer than that of the control group (P0.05); in OFT, there was no significant difference in the total distance between the two groups of rats and the average speed of movement (P0.05), but the times of passing through the central area in the ketamine group and the time in the central area decreased significantly (P0.05); in WMT, Ketamine group were found the latency was significantly prolonged (P0.01). These behavioral results indicate that ketamine anesthesia can cause fetal second trimester maternal depression and anxiety related behavior and learning memory impairment.
3. at P0 and P30, compared with the control group, the cell density in the hippocampal CA3 area of the rats in the ketamine group decreased significantly, the P0 decreased by 23%, and the P30 decreased by 21% (P0, P0.01; P30, P0.05), and there was no significant difference in the density of the vertebral cells in the CA1 area of the two groups (P0.05), and the total length of the dendritic cells in the hippocampal vertebral body of the ketamine group was significant. Shorter than the control group (P0.01), the number of dendritic branches in pyramidal cells decreased significantly (P0.01) and the density of synaptic spines decreased significantly (P0.05). Sholl analysis showed that the number of concentric circles of the CA3 vertebral cell dendrites in the CA3 vertebral body of the group of ketamine group decreased significantly (P0.01). These data showed that the CA3 region was more sensitive to the damage of ketamine and the mother body in the middle pregnancy. Ketamine anesthesia induced hippocampal dysplasia, and CA3 cells in the hippocampus (not CA1) were deficient in vertebral cells and dysplasia in pyramidal cells.
4.PO and P30, compared with the control group, the number of BrdU positive cells in the DG and SVZ regions of the ketamine group was significantly lower than the control group (PO-DG, P0.05, PO-SVZ, P0.01; P30-DG, P0.05, P30-SVZ, and P30-SVZ). The number of U positive cells was significantly lower than the control group rats, but the difference was statistically significant only DG zone (P0.01). The second trimester maternal ketamine anesthesia can cause proliferation and neuronal cell proliferation were decreased.
5.P30, compared with the control group, the expression of NMDA receptor subunit NR2A in hippocampus of ketamine rats increased significantly (P0.05), NR2B expression decreased significantly, less than 1/4 (P0.01) in control group, NRl difference was not statistically significant (P0.05), and the expression of PSD-95 protein in hippocampal BDNF and PSD-95 was significantly decreased in ketamine group, and the expression of PSD-95 protein was less than that of control. A group of three cent - (BDNF, P0.05; PSD-95, P0.01). The second trimester maternal ketamine anesthesia resulted in NMDA receptor subunit expression disorder, which affects its function, and lead to the downstream effectors of BDNF, down-regulation of PSD-95 expression, inhibition of mTOR signal transduction pathway.
Experimental conclusions:
In the middle of the pregnancy, the maternal fetal brain damage can be caused by the maternal ketamine anesthesia. The proliferation of the cells in the dentate gyrus and the subependyma region of the adult rats is reduced, the cells in the hippocampal CA3 region are missing, and the pyramidal cell development disorder in the adult CA3 region; the adult neurobehavioral abnormalities are depressed and anxiety like and memory impairment. The mechanism can be found. With the hippocampal NMDA subunit receptor disorder induced by NMDA receptor dysfunction, caused by the downstream effectors of BDNF, decrease the expression of PDS-95 and downregulation of nTOR signal transduction pathway, resulting in abnormal development of hippocampal neurons
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

【共引文献】