蓝斑去甲肾上腺素递质参与丙泊酚致意识消失及苏醒调控的机制研究

发布时间：2018-04-21 23:02

本文选题：丙泊酚 + 药物遗传学　；参考：《遵义医学院》2017年硕士论文

【摘要】：研究背景与目的:全身麻醉应用于临床已经百余年历史,但全身麻醉药如何致意识消失及苏醒的机制仍不清楚。近年来研究发现,蓝斑(locus coeruleus,LC)核团内去甲肾上腺素能神经元具有调控睡眠和觉醒的重要作用,其递质去甲肾上腺素(Noradrenaline,NE)是中枢觉醒环路信息传递的重要媒介。此外,通过在体、离体电生理等技术发现蓝斑参与了全身麻醉致意识消失或苏醒过程。但目前尚未阐明全身麻醉药如何影响蓝斑内肾上腺素递质变化以及这种变化与麻醉效应的相关性。本实验采用大鼠微透析以及药物遗传学的方法,研究静脉全麻药丙泊酚麻醉实施过程中,LC-NE递质的释放对大鼠意识消失和苏醒调控的作用与机制。方法:(一)随机选取健康成年雄性SD大鼠12只,建立LC微透析模型,分别测定对照组和丙泊酚组LC区域NE含量,每组6只。大鼠神经递质收集检测实验均在建好模型24h后进行。丙泊酚组先收集2h清醒活动大鼠NE作为基础水平,尾静脉给予丙泊酚(11 mg/kg),待大鼠大鼠翻正反射消失(loss of righting reflex,LORR)后,丙泊酚(40 mg/kg/h)持续泵注1h,待翻正反射恢复(resumption of righting reflex,RORR)后持续收集1h,每30min一个样本;对照组给予等量生理盐水持续泵注,利用微透析-高效液相电化学检测技术测定两组NE含量变化;(二)运用DREADDs技术,通过携带特异性激活序列的腺相关病毒(AAV)转染NE神经元,腹腔给予CNO激活神经元后观察LC-NE释放量的改变以及对丙泊酚麻醉下大鼠行为学的影响。构建特异性激活(h M3Dq)及抑制(h M4Di)LC-NE能神经元活性AAV,实验分组,h M3Dq组(n=6)、h M4Di(n=6)、对照组(n=6)。实验组在LC区域微注射AAV 500n L,对照组在LC区域微注射人工脑脊液(ACSF)500n L,2-3周后,待病毒特异性转染NE能神经元,建立LC微透析模型,给予腹腔注射CNO(3mg/kg)激活病毒后,分别测定三组NE的含量变化;(三)行为学观察。实验分两组,h M3Dq组(n=9)、h M4Di(n=8),实验组在LC区域微注射AAV 500n L,对照组在LC区域微注射人工脑脊液(ACSF)500n L,3周后,待病毒成功转染神经元。大鼠尾静脉置管,丙泊酚(800μg/kg/h)持续泵注,待LORR时记录时间为LORR时间,并以同样剂量维持30min,停止丙泊酚泵注,记录翻正反射恢复时间;7d后,大鼠尾静脉置管1h后,腹腔注射CNO(3mg/kg),30min后,再次记录LORR、RORR时间。结果:1.采用微透析技术,对比发现,丙泊酚降低LC细胞外液NE水平(P0.05);丙泊酚降低LC细胞外液DA代谢产物HVA、DOPAC水平,同时降低5-HT水平(P0.05),但对其代谢产物5-HIAA无影响(P0.05);2.DREADDs法特异性激活h M3Dq大鼠,LC细胞外液NE水平升高(P0.05),特异性激活h M4Di大鼠,LC细胞外液NE水平降低(P0.05);3.在丙泊酚麻醉下,LC细胞外液NE水平升高,伴随RORR时间缩短(P0.05),对LORR时间无影响(P0.05);4.在丙泊酚麻醉下,LC细胞外液NE水平降低,伴随LORR时间缩短(P0.05),对RORR时间无影响(P0.05)。结论:1.静脉给予丙泊酚可以降低脑内LC区域NE递质水平;LC NE水平改变,影响丙泊酚麻醉的诱导及苏醒时间;2.LC去甲肾上腺素能神经元参与全身麻醉致意识消失及恢复过程。
[Abstract]:Background and objective: general anesthesia has been used in clinical history for more than a hundred years, but the mechanism of consciousness disappearance and revival of general anesthetics is still unclear. In recent years, it has been found that noradrenergic neurons in locus coeruleus (LC) nuclei have an important role in regulating sleep and arousal, and their neurotransmitter norepinephrine (norepinephrine) Noradrenaline, NE) is an important medium for the transmission of information in the central awakening loop. In addition, in vivo and in vitro electrophysiology, it is found that the locus coeruleus participates in the disappearance or revival of consciousness induced by general anesthesia. However, it has not yet been clarified how the general anesthetics affect the changes in epinephrine delivery and the relationship between the changes and the anesthetic effect. In this experiment, microdialysis and pharmacogenetic methods were used to study the effect and mechanism of the release of LC-NE transmitter on the consciousness disappearance and revival of rats during the process of propofol anesthesia. Methods: (1) 12 healthy adult male SD rats were randomly selected and the LC microdialysis model was established, and the control group and the third group were determined respectively. The content of NE in the LC area of poolol group was 6. The rat neurotransmitter collection test was performed after the model 24h was built. The propofol group first collected NE as the base level of 2H conscious rats and propofol (11 mg/kg) to the tail vein (loss of righting reflex, LORR), and propofol (40 mg/kg/h) continuous pump 1H, after resumption of righting reflex, RORR), 1H was collected continuously, one sample per 30min; the control group was given constant pumping of normal saline, and the microdialysis high performance liquid phase electrochemical detection technique was used to determine the change of NE content in two groups; (two) DREADDs technology was used to carry the adeno-related disease with specific activation sequence. Toxic (AAV) transfection of NE neurons, CNO activation neurons in the abdominal cavity to observe the changes in the release of LC-NE and the effect on the behavior of rats under propofol anesthesia. Construction of specific activation (H M3Dq) and inhibition (H M4Di) LC-NE neuron activity AAV, experimental group, H M3Dq group, control group. V 500N L, the control group microinjected the artificial cerebrospinal fluid (ACSF) 500N L in the LC region. After 2-3 weeks, the virus specific transfection of NE neurons, the LC microdialysis model was established. After the intraperitoneal injection of CNO (3mg/kg) activated virus, the changes in the content of the NE were measured in the three groups. (three) the experimental group was divided into two groups. The region was microinjected with AAV 500N L, and the control group microinjected the artificial cerebrospinal fluid (ACSF) 500N L in the LC region. After 3 weeks, the virus was successfully transfected to the neuron. The rat tail vein was inserted into the tube, the propofol (800 mu g/kg/h) was pumped continuously, and the recording time was LORR time to LORR, and the same dose was maintained, and the propofol pump was stopped, and the recovery time of the reflex reflex was recorded; 7 After D, 1H was injected into the caudal vein of rats and CNO (3mg/kg) was injected intraperitoneally. After 30min, LORR and RORR time were recorded again. Results: 1. using microdialysis technique, propofol decreased the NE level of LC extracellular fluid (P0.05), propofol decreased the level of DA metabolites of LC extracellular fluid and reduced the level of LC. No effect (P0.05); 2.DREADDs specific activation of H M3Dq rats, LC cell liquid NE level increased (P0.05), specifically activated h M4Di rats, LC extracellular liquid NE level (P0.05); under propofol anesthesia, the level of extracellular fluid increased, accompanied by time contraction, no effect on the time; 4. under propofol anesthesia, fine The NE level of extracellular fluid decreased and the time of LORR shortened (P0.05), and there was no effect on the time of RORR (P0.05). Conclusion: 1. intravenous propofol can reduce the level of NE transmitters in the LC region of the brain, the change of LC NE level, the induction of propofol anesthesia and the awakening time; 2.LC normethyrofenenal neurons are involved in the consciousness disappearance and recovery of general anesthesia. Cheng.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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