丘脑网状核内部亚核团在丙泊酚全麻过程中的作用及机制研究
发布时间：2019-07-08 21:17
【摘要】：研究背景及目的:经美国FDA批准,全身麻醉药丙泊酚于1989年正式上市,是具有里程碑意义的事件。自此丙泊酚凭借其起效快、无蓄积、恢复迅速等优势,在临床上广泛应用。目前世界范围内每年有上千万例外科手术在丙泊酚麻醉下进行。丙泊酚已成为临床应用最为广泛且不可或缺的全身静脉麻醉药物。然而和其他全麻药一样,丙泊酚的麻醉作用机制至今仍然不清楚。揭示全麻机理是神经科学和麻醉学的梦想。2005年《Science》杂志主编Donald Kennedy将全麻机理列为学术界今后100年面临的125个世纪难题之一[1],攻破该堡垒亦将成为神经科学和麻醉学领域里程碑式的飞跃。全麻引起的可逆性意识消失作为大脑的特殊非生理状态,与学习记忆、睡眠觉醒等大脑高级功能息息相关[2]。近年来越来越多的研究结果支持全麻药物的作用机制并非以往认为的——麻醉药物对大脑整体的非特异性广泛抑制,而是由各解剖位置、功能特点不同的神经回路构成的复杂网络协同参与麻醉药物引起的大脑功能可逆性改变[3-4],即麻醉药物通过作用于特定大脑区域,阻断不同脑区的功能联系,打破信息在高级中枢的正、负反馈平衡而发挥麻醉作用。Ying的研究提示丘脑网状核(thalamic reticular nucleus,TRN)在丙泊酚的全麻机理中发挥了重要作用[5]。然而,丙泊酚是否以及如何作用于TRN并产生麻醉作用仍然不清楚。TRN在大脑信息调控中的地位极其特殊。它由GABA能抑制性神经元组成,调控丘脑-皮层环路,是网状上行激动系统的一部分。自2011年起,陆续有文献报道通过光遗传、电生理等技术发现TRN内部存在不同的功能性子网,即TRN各个亚核团的GABA能神经元投射至丘脑及皮层的不同区域并发挥不同的生理作用[6]。由于TRN特殊的解剖位置、生理功能,并且和睡眠、觉醒、意识等大脑功能密切相关,启发并促使我们探索它在麻醉这个无知觉、无意识、类似而有别于睡眠的特殊非生理状态中所起的作用。本课题致力于探索TRN内部亚核团参与丙泊酚发挥麻醉作用的具体分子、细胞、网络及行为机制,为丙泊酚中枢麻醉的网络学说及相关核团定位提供新的依据和方向。研究方法:(1)使用鹅膏蕈氨酸分别对TRN各亚核团进行化学性毁损,检测核团毁损后大鼠对丙泊酚麻醉的反应及敏感性改变。(2)利用即刻表达基因C-Fos和GAD-67免疫荧光共标检测TRN头端(anterior thalamic reticular nucleus,a TRN)和TRN尾端(posterior thalamic reticular nucleus,p TRN)的GABA能神经元在清醒、丙泊酚麻醉、麻醉复苏三个阶段的功能状态。(3)通过分子免疫学方法验证丙泊酚的主要分子靶点GABAa受体β3亚基(GABAa receptor-β3 subunit,GABAa R-β3)在TRN各亚核团中的存在和分布。(4)在大鼠双侧a TRN处注射GABAa受体竞争性拮抗剂荷包牡丹碱(bicuculline,BIC)阻断该受体,观察其行为改变。待大鼠状态平稳后,检测其对丙泊酚麻醉的反应及敏感性改变。(5)制备针对GABAa R-β3的小干扰RNA(small interfering RNA,si RNA)病毒,下调a TRN处GABAa R-β3的表达。免疫印迹法验证病毒干扰效果。待病毒起效后检测大鼠对丙泊酚麻醉的反应及敏感性改变。研究结果:(1)TRN各亚核团被化学性毁损后,仅a TRN毁损组大鼠对丙泊酚的敏感性有明显且持续的提高(1)a TRN毁损组大鼠相较于对照组大鼠在接受丙泊酚静脉泵注后翻正反射消失(lost of righting reflex,LORR)的时间缩短并维持该状态,即a TRN毁损组大鼠对丙泊酚的敏感性有明显且持续的提高;a TRN毁损组大鼠对性质不同的静脉麻醉药物(右美托咪定)的敏感性无明显变化。(2)p TRN毁损组大鼠相较于对照组大鼠接受丙泊酚及右美托咪定静脉泵注后LORR时间均无明显变化。(2)a TRN与p TRN的GABA能神经元在清醒、麻醉和复苏三个阶段呈现相反的功能状态(1)a TRN的GABA能神经元在清醒时活跃,而在丙泊酚麻醉后处于相对抑制状态。(2)p TRN的GABA能神经元在丙泊酚麻醉后被明显激活,而撤药苏醒后则处于相对抑制状态。(3)a TRN和p TRN均表达丙泊酚作用靶点GABAa R-β3且a TRN的表达量较p TRN更为丰富且密集(1)采用免疫印迹法验证抗GABAa R-β3抗体的特异性并使用该抗体检测GABAa R-β3在a TRN、p TRN、丘脑、海马、皮层等脑组织的表达。结果显示a TRN和p TRN区域均存在GABAa R-β3的蛋白表达。(2)免疫荧光检测结果显示,GABAa R-β3在a TRN和p TRN均有一定分布;在相同视野下,GABAa R-β3在a TRN的表达量较p TRN更为丰富且密集。(4)拮抗a TRN的GABAa R及下调a TRN处GABAa R-β3的表达使大鼠对丙泊酚敏感性下降(1)大鼠双侧a TRN处注射GABAa受体拮抗剂BIC阻断该受体,动物即刻行为活跃,活动度明显增加。(2)BIC注射组大鼠相较于对照组大鼠在接受丙泊酚静脉泵注后LORR时间明显延长,即BIC注射组大鼠对丙泊酚敏感性下降。(3)针对GABAa R-β3的si RNA病毒下调a TRN处该蛋白的表达,病毒干扰组大鼠较对照组大鼠在接受丙泊酚静脉泵注后LORR时间明显延长,即病毒干扰组大鼠对丙泊酚敏感性下降。结论:a TRN的神经元被化学性毁损后,动物对丙泊酚的敏感性有显著且持续的提高,而毁损其它部位则无类似变化。这提示a TRN极有可能参与丙泊酚对中枢神经系统发挥麻醉作用的过程。C-Fos和GAD-67免疫荧光共标发现a TRN与p TRN的GABA能神经元在清醒、麻醉、复苏三个阶段显示出相反的功能状态,表明TRN各亚核团在丙泊酚麻醉过程中发生了不同的功能变化。BIC阻断a TRN的GABAa R后动物即刻表现出亢奋状态,即a TRN的GABA能神经元兴奋性升高使动物呈现与麻醉相反的行为学表现。BIC注射组相较于对照组,大鼠对丙泊酚敏感性下降。推测一方面a TRN处的GABAa R可能是丙泊酚的作用靶点,其被拮抗剂阻断后使丙泊酚麻醉效能降低;另一方面a TRN的GABA能神经元兴奋性升高可能对抗丙泊酚的麻醉作用。免疫荧光结果显示GABAa R-β3在a TRN处相较于p TRN有着更为丰富且密集的表达,在形态学角度支持a TRN作为丙泊酚麻醉神经网络相关核团的特殊性。利用si RNA病毒下调GABAa R-β3的表达,病毒干扰组大鼠相较于对照组大鼠对丙泊酚敏感性下降。进一步证明a TRN处的GABAa R-β3对丙泊酚发挥麻醉作用至关重要。综上所述,我们推测丙泊酚极有可能通过与位于a TRN的GABAa R-β3结合后,抑制了该处GABA能神经元(a TRN的GABA能神经元兴奋性下降),从而使其下游、处于p TRN的GABA能神经元去抑制。p TRN的GABA能神经元兴奋性升高,其轴突末梢发放更多的抑制性信号到丘脑和皮层,最终产生麻醉镇静作用(参见假说示意图)。
文内图片：
图片说明：假说示意图
[Abstract]:The background and purpose of the study were that, with the approval of the U.S. FDA, the general anesthetic propofol was officially launched in 1989 and was a landmark event. Since then, the propofol has the advantages of quick action, no accumulation, rapid recovery and the like, and is widely applied in clinic. In that present world, there are ten million surgical procedures each year under the anesthesia of propofol. Propofol has become the most widely and indispensable general intravenous anesthesia drug in clinical application. The anesthesia mechanism of propofol, however, is still not clear to date, as is the case with other panacea. The mechanism of general anesthesia is the dream of neuroscience and anesthesiology. The reversible consciousness caused by general anesthesia disappears as a special non-physiological state of the brain, and is closely related to the high-level functions of the brain such as learning memory and sleep wakefulness[2]. In recent years, more and more research results support the mechanism of the general anesthesia drug, which is not the general non-specific inhibition of the whole brain, but by the various anatomical positions, the complex network of the neural circuits with different functional characteristics can participate in the reversible change of the function of the brain caused by the anesthesia drug (3-4], that is, the narcotic drug acts on a specific brain region, blocks the functional contact of different brain regions, and breaks the positive of the information in the high-level center, And a negative feedback balance is used to play the role of anesthesia. The study of Ying suggested that thalamic reticulate nucleus (TRN) played an important role in the general anesthesia mechanism of propofol[5]. However, whet or not that propofol and how to act on the TRN is still not clear. The status of TRN in the regulation of brain information is extremely special. It is composed of GABA-capable inhibitory neurons, and the control of the thalamus-cortical loop is a part of the mesh up-up system. Since 2011, it has been reported that there are different functional sub-networks in TRN, that is, the GABA-energy neurons of the TRN subnuclei are projected to different regions of the thalamus and the cortex and play different physiological functions[6]. Because of the special anatomy of TRN, the physiological function, and the function of the brain, such as sleep, wakefulness, consciousness, and so on, we have inspired and led us to explore the role of it in the special non-physiological state of anesthesia, unconsciousness, unconsciousness, and the like. The purpose of this study is to explore the specific molecular, cellular, network and behavioral mechanism of the participation of the internal subnuclei of TRN in the anesthesia of propofol, and provide a new basis and direction for the network theory of the central anesthesia of propofol and the location of the related nuclear clusters. Methods: (1) The rats of TRN were chemically damaged by using the goose extract, and the response and the sensitivity of the rats to the anesthesia of propofol were detected. (2) The functional states of the GABA-ergic neurons of the TRN head-end (a TRN) and the tail-end of the TRN were detected by the immediate expression of the C-Fos and the GAD-67 immunofluorescence. (3) The existence and distribution of GABAa receptor-3 subunit (GABAa receptor-Sup3 subunit, GABAa R-Sup3) in the TRN subnuclei were verified by molecular immunology. (4) A competitive antagonist of the GABAa receptor (bicuculline, BIC) was injected at the two-side a TRN of the rat to block the receptor and the behavior of the receptor was observed. After the state of the rat was stable, the response and sensitivity of propofol anesthesia were detected. (5) Preparation of small interfering RNA (si RNA) virus for GABAa R-Sup3 and down-regulation of the expression of GABAa R-Sup3 at a TRN. And the effect of the virus interference is verified by the immunoblotting method. And the response and the sensitivity of the rat to the propofol anesthesia were detected after the effect of the virus. The results of the study: (1) After the chemical damage of the TRN subnuclei, the sensitivity of only a TRN damaged group to the propofol was significantly and continuously increased (1) a (1) a TRN damaged group was reflected and disappeared after the injection of the propofol intravenous pump compared with the control group (lost of right reflex, The time of LORR was shortened and maintained, i.e., the sensitivity of a TRN damaged group to propofol was significantly and continuously increased; a TRN damaged group of rats had no significant change in the sensitivity of the different intravenous anesthesia drugs (dexmedetomidine). (2) There was no significant change in the time of LORR after injection of propofol and dexmedetomidine in the p-TRN damaged group compared with the control group. (2) The GABA-ergic neurons of a TRN and p-TRN exhibited the opposite functional states (1) a TRN in the three stages of conscious, anesthesia and resuscitation, while the GABA-ergic neurons of a TRN were active in the awake period, and were in a state of relative inhibition after the anesthesia of propofol. (2) The GABA-energy neurons of p-TRN were significantly activated after the anesthesia of propofol, and were in a state of relative inhibition after the withdrawal of the drug. (3) The expression of a TRN and p TRN was more abundant and the expression of a TRN was more abundant and the expression of a TRN was more abundant and dense (1) The specificity of the anti-GABAa R-Sup3 antibody was verified by the immunoblotting method and the antibody was used to detect the specificity of the anti-GABAa R-Sup3 antibody and to use the antibody to detect the expression of a TRN, p TRN, thalamus, and hippocampus. The expression of the brain tissue, such as the cortex. The results showed that the expression of GABAa R-Sup3 in a TRN and p TRN regions. (2) The results of immunofluorescence test showed that GABAa R-CD3 had a certain distribution in a TRN and p TRN; in the same visual field, the expression of GABAa R-CD3 in a TRN was more abundant and dense. (4) The expression of GABAa R and the down-regulation a TRN of a TRN inhibited the expression of the GABAa receptor antagonist BIC at the two-side a TRN of the rat on the decrease of the sensitivity of propofol (1). (2) The LRR time of the BBIC injection group was significantly prolonged compared with that of the control group in the control group, that is, the sensitivity of the BBIC injection group to the propofol was decreased. (3) The expression of the protein at a TRN was down-regulated for the si-RNA virus of the GABAa R-Sup3, and the time of the LORR in the control group of the virus-interference group was significantly prolonged after the injection of the propofol intravenous pump, that is, the sensitivity of the group of the virus to the propofol was decreased. Conclusion: After the neurons of a TRN were chemically damaged, the sensitivity of the animals to propofol was significantly and continuously increased, and no similar changes were observed in other parts. This suggests that a TRN is most likely to be involved in the anesthesia of the central nervous system by propofol. C-Fos and GAD-67 immunofluorescence co-standard showed that the GABA-ergic neurons of a TRN and p-TRN showed the opposite functional states in the three stages of conscious, anesthesia and resuscitation, indicating that the TRN subnuclei had different functional changes in the course of propofol anesthesia. After the BIC block a TRN, the animals showed a hyperactive state immediately after the GABAa R of a TRN. In the BIC injection group, the sensitivity of propofol to propofol was decreased compared with the control group. It is suggested that, on the one hand, the GABAa R at a TRN may be the target of the action of propofol, which is blocked by the antagonist, so that the anesthesia efficiency of propofol is reduced; on the other hand, the excitability of the GABA of a TRN can antagonize the anesthesia effect of propofol. The results showed that GABAa R-CD3 had a more abundant and intensive expression at a TRN than p TRN, and it was supported a TRN at a morphological angle as the particularity of the related nuclear group of propofol anesthesia neural network. The expression of GABAa R-Sup3 was down-regulated by the si-RNA virus, and the sensitivity of propofol to propofol was decreased in the virus-disturbed group compared with the control group. It is further proved that the GABAa R-Sup3 at a TRN is essential for the anesthesia of propofol. In conclusion, we have speculated that, after the combination of the GABAa R-Sup3 located in a TRN, it is possible to inhibit the GABA-energy neurons (a TRN in the GABA-energy neurons of a TRN) from decreasing the excitability of the GABA-ergic neurons in the region, so that the GABA-energy neurons in the downstream and in the p-TRN can be inhibited. The GABAergic neurons of p-TRN increase the excitability of the neurons, and the axons of their axons release more inhibitory signals to the thalamus and the cortex, resulting in the anesthesia and sedation (see the hypothesis).
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614.2
本文编号：2511885
文内图片：
图片说明：假说示意图
[Abstract]:The background and purpose of the study were that, with the approval of the U.S. FDA, the general anesthetic propofol was officially launched in 1989 and was a landmark event. Since then, the propofol has the advantages of quick action, no accumulation, rapid recovery and the like, and is widely applied in clinic. In that present world, there are ten million surgical procedures each year under the anesthesia of propofol. Propofol has become the most widely and indispensable general intravenous anesthesia drug in clinical application. The anesthesia mechanism of propofol, however, is still not clear to date, as is the case with other panacea. The mechanism of general anesthesia is the dream of neuroscience and anesthesiology. The reversible consciousness caused by general anesthesia disappears as a special non-physiological state of the brain, and is closely related to the high-level functions of the brain such as learning memory and sleep wakefulness[2]. In recent years, more and more research results support the mechanism of the general anesthesia drug, which is not the general non-specific inhibition of the whole brain, but by the various anatomical positions, the complex network of the neural circuits with different functional characteristics can participate in the reversible change of the function of the brain caused by the anesthesia drug (3-4], that is, the narcotic drug acts on a specific brain region, blocks the functional contact of different brain regions, and breaks the positive of the information in the high-level center, And a negative feedback balance is used to play the role of anesthesia. The study of Ying suggested that thalamic reticulate nucleus (TRN) played an important role in the general anesthesia mechanism of propofol[5]. However, whet or not that propofol and how to act on the TRN is still not clear. The status of TRN in the regulation of brain information is extremely special. It is composed of GABA-capable inhibitory neurons, and the control of the thalamus-cortical loop is a part of the mesh up-up system. Since 2011, it has been reported that there are different functional sub-networks in TRN, that is, the GABA-energy neurons of the TRN subnuclei are projected to different regions of the thalamus and the cortex and play different physiological functions[6]. Because of the special anatomy of TRN, the physiological function, and the function of the brain, such as sleep, wakefulness, consciousness, and so on, we have inspired and led us to explore the role of it in the special non-physiological state of anesthesia, unconsciousness, unconsciousness, and the like. The purpose of this study is to explore the specific molecular, cellular, network and behavioral mechanism of the participation of the internal subnuclei of TRN in the anesthesia of propofol, and provide a new basis and direction for the network theory of the central anesthesia of propofol and the location of the related nuclear clusters. Methods: (1) The rats of TRN were chemically damaged by using the goose extract, and the response and the sensitivity of the rats to the anesthesia of propofol were detected. (2) The functional states of the GABA-ergic neurons of the TRN head-end (a TRN) and the tail-end of the TRN were detected by the immediate expression of the C-Fos and the GAD-67 immunofluorescence. (3) The existence and distribution of GABAa receptor-3 subunit (GABAa receptor-Sup3 subunit, GABAa R-Sup3) in the TRN subnuclei were verified by molecular immunology. (4) A competitive antagonist of the GABAa receptor (bicuculline, BIC) was injected at the two-side a TRN of the rat to block the receptor and the behavior of the receptor was observed. After the state of the rat was stable, the response and sensitivity of propofol anesthesia were detected. (5) Preparation of small interfering RNA (si RNA) virus for GABAa R-Sup3 and down-regulation of the expression of GABAa R-Sup3 at a TRN. And the effect of the virus interference is verified by the immunoblotting method. And the response and the sensitivity of the rat to the propofol anesthesia were detected after the effect of the virus. The results of the study: (1) After the chemical damage of the TRN subnuclei, the sensitivity of only a TRN damaged group to the propofol was significantly and continuously increased (1) a (1) a TRN damaged group was reflected and disappeared after the injection of the propofol intravenous pump compared with the control group (lost of right reflex, The time of LORR was shortened and maintained, i.e., the sensitivity of a TRN damaged group to propofol was significantly and continuously increased; a TRN damaged group of rats had no significant change in the sensitivity of the different intravenous anesthesia drugs (dexmedetomidine). (2) There was no significant change in the time of LORR after injection of propofol and dexmedetomidine in the p-TRN damaged group compared with the control group. (2) The GABA-ergic neurons of a TRN and p-TRN exhibited the opposite functional states (1) a TRN in the three stages of conscious, anesthesia and resuscitation, while the GABA-ergic neurons of a TRN were active in the awake period, and were in a state of relative inhibition after the anesthesia of propofol. (2) The GABA-energy neurons of p-TRN were significantly activated after the anesthesia of propofol, and were in a state of relative inhibition after the withdrawal of the drug. (3) The expression of a TRN and p TRN was more abundant and the expression of a TRN was more abundant and the expression of a TRN was more abundant and dense (1) The specificity of the anti-GABAa R-Sup3 antibody was verified by the immunoblotting method and the antibody was used to detect the specificity of the anti-GABAa R-Sup3 antibody and to use the antibody to detect the expression of a TRN, p TRN, thalamus, and hippocampus. The expression of the brain tissue, such as the cortex. The results showed that the expression of GABAa R-Sup3 in a TRN and p TRN regions. (2) The results of immunofluorescence test showed that GABAa R-CD3 had a certain distribution in a TRN and p TRN; in the same visual field, the expression of GABAa R-CD3 in a TRN was more abundant and dense. (4) The expression of GABAa R and the down-regulation a TRN of a TRN inhibited the expression of the GABAa receptor antagonist BIC at the two-side a TRN of the rat on the decrease of the sensitivity of propofol (1). (2) The LRR time of the BBIC injection group was significantly prolonged compared with that of the control group in the control group, that is, the sensitivity of the BBIC injection group to the propofol was decreased. (3) The expression of the protein at a TRN was down-regulated for the si-RNA virus of the GABAa R-Sup3, and the time of the LORR in the control group of the virus-interference group was significantly prolonged after the injection of the propofol intravenous pump, that is, the sensitivity of the group of the virus to the propofol was decreased. Conclusion: After the neurons of a TRN were chemically damaged, the sensitivity of the animals to propofol was significantly and continuously increased, and no similar changes were observed in other parts. This suggests that a TRN is most likely to be involved in the anesthesia of the central nervous system by propofol. C-Fos and GAD-67 immunofluorescence co-standard showed that the GABA-ergic neurons of a TRN and p-TRN showed the opposite functional states in the three stages of conscious, anesthesia and resuscitation, indicating that the TRN subnuclei had different functional changes in the course of propofol anesthesia. After the BIC block a TRN, the animals showed a hyperactive state immediately after the GABAa R of a TRN. In the BIC injection group, the sensitivity of propofol to propofol was decreased compared with the control group. It is suggested that, on the one hand, the GABAa R at a TRN may be the target of the action of propofol, which is blocked by the antagonist, so that the anesthesia efficiency of propofol is reduced; on the other hand, the excitability of the GABA of a TRN can antagonize the anesthesia effect of propofol. The results showed that GABAa R-CD3 had a more abundant and intensive expression at a TRN than p TRN, and it was supported a TRN at a morphological angle as the particularity of the related nuclear group of propofol anesthesia neural network. The expression of GABAa R-Sup3 was down-regulated by the si-RNA virus, and the sensitivity of propofol to propofol was decreased in the virus-disturbed group compared with the control group. It is further proved that the GABAa R-Sup3 at a TRN is essential for the anesthesia of propofol. In conclusion, we have speculated that, after the combination of the GABAa R-Sup3 located in a TRN, it is possible to inhibit the GABA-energy neurons (a TRN in the GABA-energy neurons of a TRN) from decreasing the excitability of the GABA-ergic neurons in the region, so that the GABA-energy neurons in the downstream and in the p-TRN can be inhibited. The GABAergic neurons of p-TRN increase the excitability of the neurons, and the axons of their axons release more inhibitory signals to the thalamus and the cortex, resulting in the anesthesia and sedation (see the hypothesis).
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614.2
【参考文献】
相关期刊论文 前1条
1 ;GABA_A receptor partially mediated propofol-induced hyperalgesia at superspinal level and analgesia at spinal cord level in rats[J];Acta Pharmacologica Sinica;2004年12期
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