【作者】 孙洪兆；

【导师】 艾洪滨；

【作者基本信息】 山东师范大学 ， 动物学， 2010， 博士

【摘要】 大量的形态学和生理学研究证明,调控胃的副交感神经节前纤维主要来自迷走神经背核(DMV),部分来自疑核(NA),这说明NA对胃也具有调控作用。但是NA对胃运动是兴奋作用还是抑制作用,文献报道颇不一致。这提示我们非常有必要就NA对胃运动的调控做进一步研究。本实验室前面的工作用电刺激的方法观察了NA对胃运动的调控作用,结果发现电刺激大鼠NA通过迷走神经对胃运动产生显著的抑制作用,这一结果是由于兴奋了疑核内的神经元胞体所致还是兴奋了过路纤维所致?这是电刺激的方法所不能解决的问题。因此,本实验用神经元胞体兴奋剂L-谷氨酸钠(L-Glu)刺激大鼠的NA观察其对胃运动的影响及其作用机制。初步的研究结果发现,NA内注射L-Glu对胃运动也产生抑制作用。电刺激NA引起的胃运动反应是迷走神经介导的,那么NA内注射L-Glu对胃运动产生的抑制作用是否也是通过迷走神经途径呢?如果预先使胃的收缩活动加强,再在NA内注射神经元胞体兴奋剂L-Glu对胃运动是否仍然产生抑制作用呢?乙酰胆碱(ACh)是调控胃壁平滑肌收缩的一种兴奋性神经递质,因此,我们首先在大鼠腹腔注射ACh使胃的收缩活动加强,再观察NA内注射L-Glu对胃运动的调控作用及其传出神经通路。L-Glu是中枢神经系统中一种重要的兴奋性神经递质,它通过作用于配体门控离子通道型受体(离子型受体)和G蛋白偶联受体(代谢型受体)发挥作用;离子型谷氨酸受体是由4～5个亚基组成的多聚体,根据其药理特性和结构的不同又分成3种类型:NMDA、AMPA和KA受体。NA中注射L-Glu对胃运动产生抑制作用,但是,L-Glu是通过何种受体调控胃运动的未见文献报道。中枢神经元之间由L-Glu介导的兴奋性传递主要是通过NMDA受体和AMPA受体,而且L-Glu在NA中对呼吸、心率、食管等的调控也是通过NMDA受体和AMPA受体。因此,本文探讨了NA中L-Glu对胃运动的调控作用是否也是通过NMDA受体和AMPA受体。DMV中注射L-Glu调控胃收缩和胃舒张的副交感节前神经元均是胆碱能神经元。既然DMV和NA均为调控胃的副交感节前中枢,两个核团有某些相似性。因此,我们设想NA中注射L-Glu调控胃舒张的节前神经元也是胆碱能神经元。本研究也就此问题进行了探讨。NO是胃肠道非胆碱能非肾上腺素能神经所释放的主要抑制性递质,能够介导胃肠平滑肌舒张。有研究表明,NA中含有一氧化氮合酶能神经元,并且NA内内源性的NO能加强延髓呼吸神经网的输出活动,通过迷走神经控制心率,调节胸腺内T细胞的分化和成熟,调控食道和咽部活动。但尚未见关于NA中NO调控胃机能的文献报道。这提示我们NO是否也参与NA对胃机能的调控呢?如果NO参与NA对胃机能的调控,那么其调控途径是否也是通过迷走神经呢?我们初步的研究显示,大鼠NA内注射NO供体SNP和NO合成原料L-精氨酸均通过迷走途径显著地抑制了胃平滑肌的收缩。我们前面的研究也证实,大鼠NA注射L-Glu是通过兴奋了其节前胆碱能神经元对胃运动产生抑制作用,那么NA内外源性NO是否也是通过NA中的节前胆碱能神经元对胃运动产生抑制呢?本研究也对该问题进行了探讨。本研究前期的工作发现,NA中外源性L-Glu和NO均能抑制胃平滑肌收缩,那么这两种递质对胃运动的调控是否存在着某种关联呢?越来越多的研究表明,NMDA受体的激活和NO的合成存在着耦联。有研究报道,向兔DMV注射L-Glu增强了胆囊的运动是通过NMDA receptor-NO-cGMP途径。既然不管是在饮食后还是在饥饿状态下,人类胆囊的运动跟胃肠的运动都非常相似,这就提示我们,NA中注射L-Glu抑制胃平滑肌收缩可能也是通过NMDA receptor-NO途径。因此,本研究也就此问题进行了探讨。用胃腔内放置水囊法和二导生理记录仪全程记录注射药物前、后胃的收缩曲线。分别统计大鼠在注药前、后各5min内胃收缩波的幅度、时程、频率以及胃运动指数。在预先腹腔注射ACh组,根据预实验我们分别统计大鼠在注射前后各3 min内胃运动频率、幅度以及“胃的平均静息压”。结果发现,L-Glu(5,10 and 20 nmol)被注射进右侧NA后,在注射后的5 min内胃运动均受到极显著性的抑制,而且这种抑制作用具有剂量依赖性;但生理盐水注射到右侧NA前后,胃运动没有明显的改变;预先切断双侧膈下迷走神经完全消除了L-Glu被注射到右侧NA后对胃运动产生的抑制作用。预先腹腔注射ACh增强胃平滑肌的收缩活动后,再在右侧NA注射L-Glu仍然通过迷走神经显著地抑制了胃运动。NA内注射L-Glu对胃运动产生的抑制作用可被预先注射选择性的NMDA受体拮抗剂D-AP5所阻断,而不能被预先注射non-NMDA受体竞争性拮抗剂CNQX所阻断。预先股静脉注射六烃季铵完全消除了右侧NA注射L-Glu对胃运动产生的抑制作用。右侧NA注射NO供体硝普钠(SNP)、NO合成前体原料L-精氨酸(L-Arg)均显著性地抑制了胃平滑肌的收缩,而注射一氧化氮合成酶抑制剂L-NAME却极显著地增强了胃平滑肌的收缩。本研究还发现,SNP和L-Arg对胃运动的抑制作用可被切断双侧膈下迷走神经所消除。右侧NA注射SNP对胃运动的抑制作用也被预先股静脉注射六烃季铵所阻断。右侧NA预先注射nNOS抑制剂L-NAME并没有完全消除右侧NA注射L-Glu对胃运动产生的抑制作用,但是与NA内仅注射L-Glu组相比对胃运动的抑制程度有所下降。本研究还发现,NMDA受体拮抗剂D-AP5完全消除了右侧NA注射SNP对胃运动产生的抑制作用。总之,本研究得出结论,右侧NA注射L-Glu剂量依赖性地抑制胃平滑肌的收缩,是通过作用于NMDA受体兴奋了NA中的节前胆碱能神经元再通过迷走神经途径实现的。右侧NA注射SNP和L-Arg引起NO的释放和生成,NO产生后引起具有NMDA受体的节前胆碱能神经元的兴奋,再通过迷走神经途径抑制了胃平滑肌的收缩。NA中注射L-Glu抑制胃运动部分通过NMDA receptor-NO途径,但该途径不是主要途径,大多数L-Glu直接作用于具有NMDA受体的节前胆碱能神经元引起胃运动的抑制。更多还原

【Abstract】 Numerous morphologic and physiological studies indicate that the vagal parasympathetic preganglionic neurons innervating the stomach are largely located in dorsal motor nucleus of the vagus (DMV) and partly in the nucleus ambiguus (NA). This suggests the NA is involved in the modulation of gastric functions. However, there are no unanimous standpoints about the effects of NA on the gastric motility up to now. To clarify the effects of excitation of NA on gastric motility, our research group electrically stimulated the NA in the anterior work and found that electrical stimulation of NA inhibited gastric motility significantly and the vagotomy beneath the diaphragm abolished the inhibitory response in rats. Was the result of electrical stimulation due to exciting neuron bodies or the passed fibres? To illuminate this question, we microinjected of L-Glutamate (L-Glu), a neuron body incitant, into NA to further investigate the effect of exciting NA on gastric motility in this paper. Our primary results showed that L-Glu microinjected into the right NA significantly inhibited gastric motility. Since the electrical stimulation of NA inhibited the gastric motility by the vagus nerves, is the vagal pathway involved in this response in rats either? Did microinjection of L-Glu into NA still inhibit gastric motility significantly in the state of stomach contracting strongly. Acetylcholine (ACh) is a neurotransmitter of modulating contraction of gastric smooth muscles. So we further explored the effect and the pathway of microinjection of L-Glu into NA on gastric motility in the state of stomach contracting strongly with intraperitoneal injection ACh.L-Glu is a major neurotransmitter of the mammalian central nervous system. It activates two families of receptors: the metabotropic and ionotropic receptors. The ionotropic glutamate receptors are nonselective cation permeable receptor channels that are classified in two subtypes according to their most selective agonist: N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainite (non-NMDA). The Glu-mediated excitability transfer among the central neurons is via the NMDA and AMPA receptors. L-Glu in the NA also regulates respiration, heart rate, oesophagus through NMDA and non-NMDA glutamate receptors. These reports clue to us whether the effect of L-Glu microinjected into NA on gastric motility was also through NMDA and AMPA glutamate receptors in rats. L-Glu caused gastric contraction when it was injected into the rostral part of the DMV and relaxation when it was injected into the caudal part of the DMV by activating cholinergic preganglionic neurons in the DMV. The result suggested whether the effect of L-Glu microinjected into NA on gastric motility was also via activating NA cholinergic preganglionic neurons.Nitric oxide (NO) is a nonadrenergic-noncholinergic (NANC) neurotransmitter in the vagus nerve mediating relaxation of the gastrointestinal tract. NO is present in and released from NANC nerves and mediates gastrointestinal relaxation following vagal stimulation. NOS positive neurones and processes were seen in the NA. Endogenous NO may reinforce the output activity of the medullary respiratory network, modulate heart rate by vagus nerves, modulate activity of oesophagus and pharynx. According to these reports we assume whether NO within NA modulates gastrointestinal motility. Is the vagal pathway also involved in the modulation in rats? Our primary results showed that microinjection of sodium nitroprusside (SNP) and L-arginine (L-Arg) into the right NA respectively significantly inhibited gastric motility via vagally mediated pathways. In the previous study we found that microinjection of L-Glu into the right NA significantly inhibited gastric motility by activating the cholinergic preganglionic neurons in the NA. Whether was the effect of NO in the NA on gastric motility also via activating cholinergic preganglionic neurons within NA?We have reported that both L-Glu and NO participated in the regulation of gastric relaxation in the right NA. Whether is there some correlation between the L-Glu and NO? More and more evidences indicate the coupling of NMDA receptor activation with the synthesis of NO. Microinjection of L-Glu into dorsal motor nucleus of the vagus excites gallbladder motility through NMDA receptor-nitric oxide-cGMP pathway. There is a close relationship in humans between gallbladder motility and gastrointestinal motility during the fasting state, as well as in the postprandial period. These reports illume us whether microinjection of L-Glu into NA inhibits gastric motility through NMDA receptor-nitric oxide pathway. Therefore, we explored all the questions in this paper.A latex balloon connected with a pressure transducer was inserted into the pylorus through the forestomach for continuous recording of the gastric motility. The amplitude, duration, frequency, and motility index of gastric contraction waves within 5 minutes before microinjection and after microinjection were measured. In the intraperitoneal injection ACh group, frequency, amplitude, and average resting intragastric pressure of gastric contraction waves within 3 minutes before microinjection and after microinjection were measured.The results showed that L-Glu (5 nmol, 10 nmol and 20 nmol) microinjected into the right NA significantly inhibited gastric motility in a dose-dependent manner, however, microinjection of physiological saline (PS) at the same position and the same volume did not initiate any change of the gastric motility. Bilateral subdiaphragmatic vagotomy abolished the inhibitory effect of microinjection of L-Glu into NA on gastric motility. Microinjection of L-Glu into NA still significantly inhibited gastric motility by vagus nerves with intraperitoneal injection ACh in advance. The pretreatment of D-AP5, the specific NMDA receptor antagonist completely abolished the inhibitory effect of L-Glu on gastric motility. But the pretreatment of CNQX, the non-NMDA receptor antagonist, did not change the inhibitory effect of L-Glu on gastric motility. The pretreatment of intravenous injection hexamethonium bromide abolished the inhibitory effect of L-Glu on gastric motility too.Microinjection of SNP and L-Arg into the right NA significantly inhibited gastric motility by vagus nerves, respectively. However, microinjection of L-NAME, the inhibitor of nitric oxide synthase, into the right NA significantly enhanced gastric motility. The pretreatment of intravenous injection hexamethonium bromide abolished the inhibitory effect of SNP on gastric motility too.The pretreatment of L-NAME did not abolish the inhibitory effect of microinjection of L-Glu into the right NA on gastric motility completely, but the inhibitory effect was lower than that in the Glu group. We also found that the pretreatment of D-AP5 completely abolished the inhibitory effect of SNP on gastric motility.In conclusion, the data of these experiments suggested that microinjection of L-Glu into the right NA inhibited gastric motility through specific NMDA receptor activating the cholinergic preganglionic neurons in the NA and the efferent pathway was the vagal nerves. NO inhibited gastric motility by activating the cholinergic preganglionic neurons including NMDA receptor in the NA and the inhibitory effect was mediated by vagus nerves. Microinjection of L-Glu into NA inhibited gastric motility through NMDA receptor-nitric oxide pathway minorly.更多还原