【作者】 李慧；

【导师】 艾洪滨；

【作者基本信息】 山东师范大学 ， 动物学， 2010， 硕士

【摘要】 束缚-浸水应激(Restraint Water-Immersion Stress, RWIS;水温21±1℃)是一种心理和躯体的复合应激模型,刺激强度较大。该模型可在短时间内导致大鼠情绪改变、体温降低和胃肠机能紊乱,包括胃运动机能亢进、胃酸分泌增多、急性胃粘膜损伤等。传统观点认为应激(stress)主要启动“下丘脑-腺垂体-肾上腺皮质”轴,通过引发一系列生物学反应进而改变器官系统的功能状态,从而使机体内环境保持稳态以应对应激性刺激。而众多研究表明RWIS主要诱导副交感神经系统的活动加强。延髓是胃机能初级调控中枢,其内的迷走神经背核和疑核发出迷走神经中的副交感纤维到胃,支配胃的活动。延髓的孤束核是内脏感觉信息的接受核团,来自胃肠的感觉信息经孤束核中继向更高级中枢传导。由此可见,延髓是直接对胃机能状态进行调控的神经中枢。下丘脑是调节内脏活动的较高级中枢。形态学研究发现,RWIS在导致大鼠产生应激性胃粘膜损伤的同时,还引起大鼠延髓迷走神经背核、孤束核、最后区及下丘脑室旁核、视上核内Fos表达加强。可见延髓、下丘脑胃机能调控中枢内神经元活动加强与应激性胃粘膜损伤的产生具有密切关联。众多研究表明,室旁核与迷走神经背核、孤束核间均存在双向的纤维联系。室旁核和视上核都是位于下丘脑视前区的两个非常重要的神经核团,其大细胞性分泌神经元分泌的抗利尿激素和催产素分别经室旁垂体束和视上垂体束运输至神经垂体。研究表明室旁核和视上核是下丘脑在RWIS状态下参与胃机能调控的两个神经核团。既然室旁核与延髓部位纤维联系密切,那么视上核也应该与延髓部位的核团之间存在纤维联系,有研究已经证明它和孤束核之间是有直接纤维联系,那么还有一个问题需要解决:视上核与迷走神经背核之间是否存在纤维联系?越来越多的研究表明应激能改变轴浆运输和突触的机能状态,在一定情况下甚至还能使“沉默突触”激活。那么RWIS这种强大的复合型应激是否也会对神经通路产生类似的影响,从而促使应激性胃粘膜损伤的产生?另一个问题是,如果膈肌下切断迷走神经,消除来自外周(胃肠)的应激反馈信息后,RWIS状态下大鼠延髓和下丘脑对胃肠的调控会发生怎样的变化?针对以上提出的问题,本文分两部分。第一部分应用辣根过氧化物酶逆行追踪方法探讨两个问题:(1)视上核与迷走神经背核间是否存在纤维联系。(2)RWIS是否对延髓、下丘脑两个胃机能调控中枢间的神经通路产生影响。将雄性Wistar大鼠随机分为2组:束缚-浸水应激组,对照组。应激组动物,向迷走神经背核内微量注射HRP,动物清醒后给予RWIS 3h,注射HRP 48~60h后,过量戊巴比妥钠腹腔注射麻醉动物,快速心脏灌流固定脑组织、脱水处理,然后进行冰冻切片。漂浮切片进行HRP组化显色。对照组动物除不进行RWIS外,其余操作均同应激组。结果显示,无论是对照组还是应激组,在下丘脑室旁核、视上核均发现HRP标记的神经元胞体;应激组室旁核内HRP标记的神经元胞体数目较对照组显著增多(P<0.01),而视上核内无显著性变化;同侧室旁核HRP标记的神经元胞体数较对侧室旁核多(P<0.01)。这些结果提示:视上核有向迷走神经背核投射的神经元,说明视上核和室旁核在胃机能调控中都起重要作用;RWIS使室旁核到迷走神经背核的神经纤维末梢(指“沉默末梢”被激活成“活性末梢”的数目)增多了,说明室旁核与迷走神经背核之间神经通路在RWIS过程中对胃肠调控起重要的作用。第二部分应用免疫组织化学方法探讨切断膈肌下迷走神经阻断来自胃肠的应激反馈信息和传出指令后,在束缚-浸水应激状态下,大鼠延髓、下丘脑神经元Fos的表达情况,从另一方面研究应激反馈信息对中枢调控的影响。本研究将雄性Wistar大鼠随机分2组:手术组、对照组。手术组切断双侧膈肌下迷走神经,对照组同样暴露神经但不切断。术后6 d进行实验,将大鼠束缚-浸水应激3 h后处死,测腹腔温度;取胃,观察胃粘膜损伤程度;取脑,应用免疫组化染色方法观察两组动物延髓、下丘脑神经元的Fos表达。结果发现,手术组动物延髓迷走神经背核、孤束核、下丘脑室旁核Fos表达较对照组减弱(P<0.05),延髓最后区减弱最为显著(P<0.01);腹腔温度低于对照组(P<0.05);胃粘膜损伤程度较对照组减轻(P<0.05)。这些结果提示切断膈肌下迷走神经后进行束缚-浸水应激,延髓迷走神经背核、孤束核、最后区、下丘脑室旁核神经元Fos表达均减弱,结合第一部分结果,说明了切断膈肌下迷走神经消除应激反馈信息后,延髓与下丘脑之间跨突触信息传递呈减弱趋势。更多还原

【Abstract】 Restraint water-immersion stres(sRWIS; 21±1℃), considered to be a mixture of physical and psychological stressors, has very strong effect on body. It can induce lower body temperature, emotion change and gastric disfunction, which include vagally-mediated gastric hypercontractility, hypersecretion of gastric acid and acute gastric erosions.It is well known that stress can activate hypothalamus-pituitary-adrenocortical(HPA) axis,which change the functional state of organ system through a series of biological reactions,and keep steady-state. However, according to the former research, RWIS mainly induced the hyperactivity of the parasympathetic nervous system.Medulla is the primary centre of gastric function. The vagal parasympathetic neurons innervating the stomach are largely located in the dorsal motor nucleus of the vagus(DMV) and partly in the nucleus ambiguus(NA). The nucleus of solitary tract receive the sensory information from the stomach. Therefore, medulla is a nerve center which dominate gastrointestinal function directly. Hypothalamus is a higher nerve centre of gastric function. Research on morphology shows that Fos expression in medullary DMV, NTS and hypothalamic SON, PVN were enhanced markedly when stress ulcer were arisesed by RWIS. From above we see that enhanced activity of nerve centre of gastric function are closely associated with appearance of stress ulcer.Many studies show that there is a two-way connection of nerve fiber between PVN and DMV, so as PVN and NTS is similar. PVN and NTS are very important nuclei in preoptic region of hypothalamus.Magnocellular neurosecretory neurons distributed both in PVN and SON, and this kind of cell can secret vasopressin and oxytocin. The hormones released by PVN and SON were transported to neurohypophysis through paraventriculohypophyseal tract and supraopticohypophyseal tract respectively, and both of the two nuclei took part in the regulation of gastric function. Since PVN and medullary have close link, SON may have close fiber link with medullary. It has already been proved that there is nerve fiber contact between SON and NTS, but the situation is unhnow between SON and DMV.More and more studies show that stress can change the state of axonal transport and synapse, and in some cases, silent synapse can even be activated. While RWIS, as a powerful compound stress, whether has function to change neural pathway and leads to stress ulcer eventually?In the context of having solved the above problems and learned the effect of stress on neural pathway , we cut off the vagus nerve to block the feedback information of stress and dictate from nerve centre,then the approach to influence neural pathway of stress is decrease. under the state of RWIS,the Fos expression in medulla and hypothalamus is unknow.Aimed at the above questions, this research were divided into two parts。Part1:We applied retrograde tracing method of horse radish(HRP) to discuss two qustions. (1) If there is direct nerve fiber connection between SON and DMV? (2)If RWIS influence neural pathway in nerve centre of gastric function?Male Wistar rats were randomly divided into 2 groups designated according to suffering RWIS or not. In stress group, we inject HRP into DMV. After operation, rats were replaced in their home cages for 60 min before restraint water-immersion stress(RWIS)to recover consciousnes. Rats were then restrained and immersed in cold water(21±1℃) for 3 h. 48-60h after injection, the rat was anesthetized with pentobarbital sodium,and then was perfused quickly with buffer. Afterward, the brain was removed immediately and cryoprotected overnight in 20% sucrose in 0.1 mol/l PB at 4℃until sectioning. Histochemical detection of HRP were applied in sections. In control group, rats were operate in the same way as the above besides not suffering RWIS. We found: Cells labelled by HRP were found in SON; The number of cells labelled by HRP in stress group was more than that of control group(P <0.01);The amount of labelled cells in the PVN of the same side as jinjection site is larger than the opposite(P <0.01).These show that there is direct nerve fiber connection between SON and DMV; RWIS can change neural pathway between PVN and DMV; PVN have more nerve fiber project to the same side DMV.Part 2: In this part we applied immuno-histochemical methods to observe the Fos expression in medulla and hypothalamus under RWIS after bilateral subdiaphragamatic vagotomy. Since the operation can block the feedback information of stress, from Fos expression we can investigate the influence of this information on Information transfer across synapses. Male Wistar rats weighting 180-220 g were randomly divided into two groups: Operation group, Control group. Bilateral subdiaphragamatic vagotomy was performed in rats of operation group, the vagus were uncovered but were not cut off in control group. Six days after operation, rats were put into death after restraint water-immersion stress(3 h). The abdominal cavity temperature was measured, the gastric mucosal ulcer index was determined according to Guth criteria, and the immuno-histochemical methods were used to detect the Fos expression in medulla and hypothalamus. We found: Expression of Fos was decreased significantly in DMV, NTS and PVN after bilateral subdiaphragamatic vagotomy(P<0.05), and the decrease in AP was the most prominent (P<0.01); the abdominal cavity temperature was lower than that of the control group(P<0.05); the gastric mucosal ulcer index was reduced significantly(P<0.05). The results suggest that the feedback regulation information from the gastrointestinal tract, rather than lower body temperature and psychological stressor, play a dominant role in inducing the Fos expression in DMV, NTS, AP and PVN. Since Fos expression is a mark of neuronal excitability, combining the results of part 1,the results show that the transfer of information among medulla and hypothalamus tend to be weaker after the feedback information of stress were bated.更多还原