【作者】 张灏；

【导师】 石学银；

【作者基本信息】 第二军医大学 ， 麻醉学， 2013， 博士

【摘要】 背景：环磷酸腺苷（cAMP或cyclic AMP，3’-5’-cyclic adenosine monophosphate）作为胞内第二信使，主要负责传递G蛋白偶联受体（GPCR，G-protein coupled receptor）信号。cAMP由腺苷酸环化酶（AC，adenylyl cyclase）分解ATP产生，通过磷酸二酯酶（PDE，phosphodiesterase）降解。在中枢神经系统，cAMP能够通过蛋白激酶A（PKA，protein kinase A）-cAMP反应元件结合蛋白（CREB，cAMP responseelement-binding protein）、环核苷酸控制的离子通道（CGN channels， cyclicnucleotide-gated channels，特别是超极化激活的CGN通道（HCN通道，hyperpolarization-activated CGN channels））和cAMP活化的交换蛋白（Epac，exchangeprotein directly activated by cAMP）等信号通路，参与学习记忆、睡眠和昼夜节律调控等多种功能。静脉全麻药异丙酚和吸入性全麻药异氟烷和七氟烷均能够调节中枢和外周GPCR/G蛋白/cAMP信号通路。本课题拟研究调控脑cAMP信号通路对全麻药导致的遗忘和睡眠损害效应的影响。第一部分cAMP/CREB通路介导异丙酚对空间记忆巩固的损害目的：静脉全麻药异丙酚（propofol）在低于麻醉浓度时，即可损害情景记忆的巩固，导致顺行性遗忘，而对记忆编码和短时记忆无明显影响。情景记忆为人类独有，包含了事件（what）、时间（when）、地点（where）、情绪（emotion）和背景环境（context）等要素。大鼠空间记忆模型（Morris水迷宫，non-cued MWM）能够模拟情景记忆的事件和地点要素，被称为“类情景记忆”（episodic-like memory）。本研究假设一：与临床研究相似，镇静剂量的异丙酚能够选择性损害大鼠空间记忆巩固，而对空间学习和短时记忆无影响。已有研究发现，大鼠空间记忆的巩固依赖于海马cAMP/CREB信使系统。假设二：异丙酚损害空间记忆巩固的同时，抑制了海马cAMP/CREB通路。假设三：选择性磷酸二酯酶-4（PDE-4）抑制剂rolipram能够通过抵消异丙酚对cAMP/CREB通路的抑制，逆转异丙酚的遗忘效应。方法：实验分为四组：C（赋形剂对照组）；P（异丙酚组，25mg/kg，腹腔注射）；R（rolipram组，0.3mg/kg，腹腔注射）；R+P（rolipram联合异丙酚组，异丙酚注射前25分钟注射rolipram）。实验一：为排除异丙酚和rolipram的镇静效应对异丙酚遗忘的影响，分别在异丙酚注射后5、15和25分钟测定了大鼠的镇静/麻醉评分。实验二：为排除运动能力对异丙酚遗忘的影响，分别在异丙酚注射后5、15和25分钟，采用转棒实验测定了大鼠的运动技巧和协调能力。实验三：为排除非空间因素（运动能力、恐惧感、工作记忆等）对大鼠空间记忆和异丙酚遗忘的影响，在异丙酚注射后5分钟和24小时实施显露平台的水迷宫实验（cued MWM）。实验四：空间学习记忆实验（non-cued MWM）和cAMP/CREB通路活化情况测定。在异丙酚注射后5分钟行定位航行训练（隐藏平台实验），测定空间学习和短时空间记忆情况；在异丙酚注射后24小时，行空间探索实验，测定长时间记忆水平，以反映空间记忆巩固情况。在异丙酚注射后5分钟、50分钟和24小时测定海马cAMP/CREB通路活化情况，包括cAMP水平；ERK1/2、AKT、CaMKII和CREB磷酸化活化程度；CaMKIV、BDNF、c-Fos和Arc蛋白表达水平。结果：实验一：与对照组相比，异丙酚25mg/kg注射后，大鼠表现出部分镇静效应。rolipram预注射对异丙酚的镇静效应无影响。实验二：异丙酚和rolipram均不损害大鼠的运动能力。实验三：异丙酚对非空间记忆无影响。异丙酚注射后5分钟和24小时的显露平台实验中，各组大鼠潜伏期无统计学差异。实验四：异丙酚不损害空间学习和短时记忆能力，但损害空间记忆巩固。预先注射rolipram能够对抗异丙酚的遗忘效应。与对照组相比，异丙酚注射后海马cAMP水平降低，CREB和CaMKII磷酸化水平降低，BDNF、Arc蛋白表达下降。单独注射rolipram增加cAMP水平，但对空间记忆和其他信号通路无影响。预先注射rolipram能够对抗异丙酚对cAMP/CREB通路的抑制。结论：异丙酚选择性损害空间记忆巩固，导致顺行性遗忘。cAMP/CREB通路可能介导了异丙酚的遗忘效应。第二部分RGS/Gα_i2/cAMP通路介导吸入性全麻药对睡眠的损害目的：术后睡眠障碍的发生率约为100%，主要表现为清醒次数增加，NREM睡眠时间减少，REM睡眠时间先抑制后反跳性增加，且伴随有严重的昼夜节律紊乱。术后睡眠障碍严重影响手术疗效，增加术后并发症和死亡率。研究发现，全麻药可能是术后睡眠障碍的独立危险因素。不同于异丙酚，吸入性麻醉药麻醉后，小鼠REM睡眠反跳性增加。研究全麻药导致睡眠障碍的相关机制，并探索相关的有效干预措施，可能有助于改善患者术后预后。不同于静脉麻醉药，异氟烷和七氟烷能够作用于Gα_i2/cAMP通路。RGS蛋白（regulators of G protein signaling protein）作为GPCR信号通路的负向调节因子，能够通过加速降解GTP，抑制Gα_i2/cAMP通路。此前研究发现RGS蛋白失敏感的Gα_i2^G184S小鼠对异氟烷更加敏感。异氟烷麻醉后，该基因敲入小鼠翻正反射消失的时间缩短，恢复时间延长。本研究进一步假设一：RGS蛋白对Gα_i2/cAMP通路的抑制有助于维持正常的睡眠结构；假设二：RGS蛋白对Gα_i2/cAMP通路的抑制参与了吸入麻醉导致的睡眠障碍。方法：研究采用了基因敲入（gene knock-in）的Gα_i2^G184S小鼠。该小鼠Gα_i2蛋白第184位甘氨酸残基（G）被突变为丝氨酸残基（S）后，失去与RGS蛋白结合的能力，导致RGS蛋白催化活化型GTP-Gα_i2转变为非活化型GDP-Gα_i2的作用消失，即RGS失敏感的Gα_i2亚单位^G184S等位基因突变(RGS-insensitive^G184S Gα_i2allele mutation)。Gα_i2^G184S基因敲入小鼠的Gα_i2相关信号通路增强，即Gα_i2持续抑制cAMP水平和Gβγ信号通路持续活化。实验一测定了野生型（WT）、杂合子Gα_i2^G184S基因敲入小鼠（+/GS）和纯合子基因敲入小鼠（GS/GS）的24小时基础睡眠结构，以确定RGS/Gα_i2相互作用在维持正常睡眠中的作用。实验二观察给予3小时异氟烷（1.3%）或七氟烷（2.8%）麻醉后18.5小时内，三种基因型小鼠的睡眠结构损害情况，以确定RGS/Gα_i2是否参与了吸入性麻醉药导致的睡眠紊乱。结果：实验一：WT小鼠和+/GS小鼠在24小时觉醒时间、NREM睡眠时间和REM睡眠时间无统计学差异。与WT小鼠相比，纯合子GS/GS小鼠24小时NREM睡眠时间增加了13.95%，而觉醒时间减少了11.85%。WT和GS/GS小鼠的觉醒/睡眠时间比例也不相同。与WT小鼠相比，GS/GS小鼠白昼时NREM睡眠和REM睡眠减少，而夜晚时NREM睡眠和REM睡眠增加。同时，GS/GS小鼠的NREM睡眠和觉醒维持存在障碍，表现为NREM睡眠和觉醒次数增加而平均每次持续时间下降。FFT功率密度谱分析发现，GS/GS小鼠觉醒和NREM睡眠期delta活动（0.5-4Hz）增加，而REM睡眠期theta活动（5-8Hz）减少。以上结果提示RGS/Gα_i2参与了小鼠正常睡眠结构的维持。实验二：与对照组（纯氧组）相比，异氟烷或七氟烷麻醉导致了深度睡眠结构紊乱，主要表现为觉醒时间先增加后减少，NREM睡眠时间先减少后增加和REM睡眠反跳性增加。异氟烷或七氟烷麻醉时，WT小鼠恢复正常睡眠所需时间长于GS/GS小鼠。NREM睡眠期delta活动减少仅发生于WT和+/GS小鼠，而非GS/GS小鼠。以上结果提示RGS/Gα_i2参与了吸入性全麻药导致的睡眠结构损害。结论：RGS/Gα_i2/cAMP通路参与了小鼠正常睡眠的维持，并部分介导了异氟烷和七氟烷吸入导致的睡眠障碍。更多还原

【Abstract】 Background:Cyclic adenosine monophosphate （cAMP, cyclic AMP or3’-5’-cyclic adenosinemonophosphate） is an intracellular second messenger of G-protein coupled receptor（GPCR） signaling. cAMP is synthesized from ATP by adenylyl cyclase （AC） which isactivated by stimulatory G （Gs）-protein-coupled receptors and inhibited by inhibitory G（Gi）-protein-coupled receptors. It’s decomposition into AMP relies on thephosphodiesterase （PDE）. In the central nervous system, cAMP regulates the followingthree main pathways: the protein kinase A （PKA）-CREB （cAMP response element-bindingprotein） pathway, the cyclic nucleotide-gated channels （CNG channels, especially thehyperpolarization-activated CGN channels, HCN channels） pathway and exchangeproteins activated by cAMP （Epac） pathway. GPCR/G protein/cAMP signaling is found toparticipate in the actions of both intravenous anesthetics （for example propofol） andinhaled anesthetics （for example isoflurane and sevoflurane）. We intend to investigate therole of cAMP in general anesthetics-induced amnesia and sleep disorder in the currentstudy.Section1: Rescue of cAMP/CREB signaling reversed spatial memory retentionimpairments induced by subanesthetic dose of propofolObjective：The intravenous anesthetic propofol causes episodic memory impairments independent ofsedation and unconsciousness in human. Episodic memory is the human-unique memory ofautobiographical events （what） which occurs in specified time （when） and place （where）and is mixed with emotion and other contextual knowledge （context）. The non-cued MorrisWater Maze （MWM） is a hippocampus dependent “episodic-like memory” training taskthat gives information on “what-where” elements. We hypothesized that propofol causedepisodic-like spatial memory retention but not acquisition impairments in rats. Theimpaired memory consolidation was accompanied with reduced hippocampalcAMP/CREB signaling. Furthermore, rescuing cAMP/CREB signaling using selectivephosphodiesterase-4inhibitor rolipram could reverse propofol-induced amnesia andcAMP/CREB signaling inhibition. Methods：Male Sprague-Dawley rats were randomized into four groups: vehicle control （C）;propofol （P,25mg/kg, intraperitoneal）; rolipram （R） and rolipram+propofol （R+P,pretreatment of rolipram25minutes before propofol,0.3mg/kg, intraperitoneal）. Sedationand motor coordination were evaluated5,15and25minutes after propofol injection toresist the possibility that inefficient learning and memory after propofol injection duringthe non-Cued MWM was due to sedation and sedation-caused motor incoordination.MWM spatial navigation training （memory acquisition） and probe test （memory retention）were performed5minutes and24hours after propofol injection. Visible cued MWMtraining was simultaneously performed to exclude non-spatial effects. Hippocampal CREBsignaling including cAMP levels; ERK1/2, CaMKII and CREB phosphorylation;CaMKIV, BDNF, c-Fos and Arc protein expression were detected5minutes,50minutesand24hours after propofol administration.Result：Rolipram did not change propofol-induced anesthetic/sedative states. Both propofol androlipram did not impair motor skills evaluated by accelerated rotarod tests. No differencewas found on the latency to the platform during the visible MWM. Propofol impairedspatial memory retention but not acquisition in the non-cued MWM. Rolipram alone didnot change non-cued MWM learning and memory consolidation. Pretreatment withrolipram reversed propofol-induced impairments of spatial memory retention andsuppression on cAMP levels, CaMKII and CREB phosphorylation, BDNF and Arcprotein expression.Conclusion：Propofol caused spatial memory retention impairments but not acquisition inabilitypossibly by inhibiting cAMP/CREB signaling. Section2: RGS/Gα_i2/cAMP signaling modulated volatile anesthesia-induced sleepdisruptionsObjective:Postoperative sleep disorders are commonly seen in almost every patient. The changes insleep architecture and circadian rhythms substantially influence surgery effects, causemore complications and are connected with sudden death. Different from intravenousanesthesia, sleep is disrupted after volatile anesthesia with a rebound in REM sleep. Theunderlying mechanisms remain elusive and a better understanding of the relationshipbetween sleep and anesthesia might be useful to help investigate new strategies to improvesleep and facilitate the recovery of surgical patients. Volatile anesthetics but notintravenous anesthetics act on metabotropic, G-protein coupled receptor （GPCR） signaling.GPCR ligands such as acetylcholine, adenosine, serotonin and histamine; GPCRs; effectors（for example cAMP and Gβγ signaling） and regulators all play a role in the benefcial andadverse effects of volatile anesthetics. Regulators of G protein signaling （RGS） proteinsare negative regulators of GPCR signaling and have been shown to modulate the inductionand emergence from isoflurane anesthesia. Specifically, RGS/Gα_i2signaling was found tomodulate isoflurane-induced loss of consciousness. The current study further tested thehypothesis that RGS/Gα_i2/cAMP signaling modulated normal sleep and disrupted sleepafter isoflurane and sevoflurane anesthesia.Methods:Wild-type （WT）, heterozygous （+/GS） and homozygous （GS/GS） mice with a geneticablation of RGS action at Gα_i2were subjected to electroencephalographic andelectromyographic recordings:1) under ad libitum sleep;2) after3-hour isoflurane （1.3%in pure oxygen） exposure;3) after3-hour sevoflurane （2.8%in pure oxygen） anesthesia.The analogous mutation （Gly184to Ser） prevented the GTPase accelerating protein （GAP）activity of all RGS proteins targeting Gα_i2, thus downstream Gα_i2/cAMP and Gβγ signalingwas enhanced and lasted longer. Sleep architecture （%state; mean episode duration andepisode numbers） and power spectra were collected and analyzed as a function of bothgenotypes and conditions for each time period. Results:The homozygous RGS-insensitive （GS/GS） mice showed less wakefulness time （-11.85%）and more NREM sleep time （+13.95%） at baseline compared to the wild-type mice. Thenumbers of NREM sleep and wakefulness episodes were increased and mean episodedurations of NREM sleep and wakefulness were decreased in the GS/GS mice. The GS/GSmice also showed abnormal circadian distribution of sleep and wakefulness. Increasedwakefulness and NREM sleep delta power and decreased REM sleep theta power wereobserved in the GS/GS mice compared to that in the wild-type and+/GS mice. Isofluraneand sevoflurane caused prolonged disruptions of wakefulness, NREM sleep and REMsleep in the wild-type mice. REM sleep changes persisted for more than18.5hours afteranesthesia in the wild-type mice. NREM sleep delta power was also decreased by volatileanesthesia in the wild-type mice. Complete silencing of RGS/Gα_i2interaction resulted inless changes in sleep architecture and no changes in power spectra after anesthesia.Recovery of sleep/wake cycle was faster in the GS/GS mice than the wild-type mice.Conclusions:RGS/Gα_i2interface regulates sleep amount, sleep consolidation and circadian timing ofsleep. Sleep disruptions after volatile anesthesia partially depend on the inhibition of Gα_i2signaling by RGS proteins.更多还原