【作者】 赵东芹；

【导师】 艾洪滨；

【作者基本信息】 山东师范大学 ， 动物学， 2011， 博士

【摘要】 束缚-浸水应激(restraint water-immersion stress,RWIS)是一种强度较大的复合刺激,能在数小时内产生迷走神经介导的胃运动机能亢进、胃酸分泌增多、急性胃粘膜损伤等现象,该应激模型被广泛用来研究应激性胃溃疡的发生机制及其临床治疗药物的筛选。Fos蛋白是即刻早期基因(IEG)c-fos的表达产物,神经元有Fos表达,表明神经元处于活动状态。我们课题组前期的研究发现束缚-浸水应激不同时间段(30、60、120 min),迷走复合体(DVC)、疑核(NA)以及下丘脑前部的视上核(SON)、室旁核(PVN)、视交叉上核(SCh)等均有不同程度的Fos表达,说明上述核团参与束缚-浸水应激致胃粘膜损伤的中枢调控。束缚-浸水应激诱导的胃损伤效应能被预先切断膈肌下迷走神经或给予阿托品所抑制,而预先切除垂体、肾上腺或给予肾上腺素能α-受体阻断剂酚苄明对束缚-浸水应激引起的胃运动机能亢进、胃酸分泌增多没有影响。这说明胃机能的神经调控主要是副交感控制,而且这种副交感信息主要来自迷走神经背核(DMV),部分来自疑核。迷走神经背核、疑核过度活动可能是导致大鼠胃粘膜损伤的初级中枢机制之一,但电刺激、化学刺激迷走神经背核、疑核均引起胃运动抑制。是不是束缚-浸水应激过程中,高位中枢——下丘脑前部的活动解除了延髓内脏中枢对胃的抑制作用,从而导致胃运动亢进、胃酸分泌增多的呢?如果是,那么:1.束缚-浸水应激过程中,延髓迷走复合体、疑核等核团(称为延髓内脏中枢)内被激活的神经元是什么性质的神经元?2.视上核、室旁核是下丘脑前部最显著的2个核团,主要由大细胞精氨酸加压素能神经元和催产素能神经元组成,参与多种应激反应。那么,在束缚-浸水应激过程中,下丘脑前部视上核、室旁核中被激活的神经元是不是催产素能神经元和精氨酸加压素能神经元?3.如果下丘脑前部室旁核和视上核催产素能神经元和精氨酸加压素神经元的轴突末梢除了分布到垂体后叶之外,还下行到延髓内脏中枢,与这里的神经元形成突触联系,以神经递质的形式调控这些神经元的活动。那么束缚-浸水应激后迷走复合体、疑核的神经元胞体膜或树突膜上的精氨酸加压素受体、催产素受体(胞体膜上分布有精氨酸加压素受体、催产素受体的神经元我们称为精氨酸加压素敏感、催产素敏感神经元)会发生什么变化?4.神经系统是生命活动中起整合和调节作用的信息系统,神经元是神经系统的结构和功能单位,具有接受、整合和传递信息的功能。突触是神经元之间信息交流最关键的连接部位,在反射活动中各种神经冲动都要通过突触进行传导,可以说突触是整个神经系统传递的最小功能单位,突触的数量及结构的完整性对维持脑功能的正常发挥起重要作用。突触膨体素(SYN)是一种广泛分布于突触前囊泡膜上的钙结合酸性糖蛋白,参与突触囊泡的导入、转运和神经递质的释放、突触囊泡再循环、突触发生和稳定等生理过程,其含量与突触密度呈正相关。突触蛋白I主要分布在神经末梢的小型突触前囊泡外侧,磷酸化后可作用于囊泡释放池释放囊泡、囊泡与突触前膜的融合以及囊泡的再循环等几个环节,在神经递质释放及突触可塑性等生理活动过程中具有重要作用。因此,突触膨体素和突触蛋白I可作为突触前终末的特异性标记物,用来检测突触的密度和分布,从而反映突触可塑性的变化情况。那么,在束缚-浸水应激过程中,下丘脑前部、延髓内脏中枢部位的突触膨体素和突触蛋白I的表达会发生什么样的变化?为解决上述问题,设计实验如下:(1)根据束缚-浸水时间将雄性Wistar大鼠随机分为2组,分别是对照组(unstressed group)和应激1h组。以Fos作为神经元活动的标志,采用Fos与儿茶酚胺能神经元(CA)标志酶酪氨酸羟化酶(TH)、胆碱能神经元(ACh)标志酶胆碱乙酰转移酶(ChAT)以及催产素(OT)、精氨酸加压素(AVP)等神经递质和催产素受体(OTR)、精氨酸加压素Ⅰ型b亚型受体(V1bR)免疫组织化学双标技术,研究大鼠下丘脑前部、延髓内脏中枢等核团内相应神经元的活动情况,探讨这些核团中哪类神经元参与应激状态的胃机能调控,哪类神经元作用大一些,为进一步阐明束缚-浸水应激致胃机能紊乱的中枢调控机制提供实验依据和基础资料。(2)根据束缚-浸水时间将雄性Wistar大鼠随机分为4组,分别是对照组(unstressed group)和应激1h、2h、4h组。采用免疫组织化学技术及Western-blotting技术观察束缚-浸水应激不同时间段下丘脑前部-延髓内脏中枢主要核团中突触膨体素和突触蛋白I的变化,旨在探讨束缚-浸水应激过程中上述部位是否发生突触可塑性的改变,如有,哪些核团发生改变,从而为研究束缚-浸水应激致胃粘膜损伤后突触的重塑打下基础。本文研究发现:1.迷走神经背核、疑核中胆碱能神经元过度活动是束缚-浸水应激致胃粘膜损伤中枢机制之一乙酰胆碱转移酶免疫反应阳性(ChAT-IR)神经元分布于DVC吻-尾全段。RWIS 1h组DMV和NA中单位面积内的ChAT-IR神经元较对照组显著减少;Fos和乙酰胆碱转移酶双标阳性(Fos+ChAT-IR)神经元在DMV吻段、中段、尾段和NA中分别是对照组的3.60、16.00、4.00和3.29倍,占ChAT-IR神经元的比例均显著高于对照组。而在NTS中ChAT-IR神经元、Fos+ChAT-IR神经元在RWIS 1h组和对照组间几无变化,Fos+ChAT-IR神经元占ChAT-IR神经元的比值组间差异不显著。这说明DMV和NA中ACh能神经元的过度活动,尤其是DMV中段ACh能神经元的过度活动是RWIS致胃粘膜损伤的主要机制之一,NTS中的胆碱能神经元不参与RWIS致胃粘膜损伤的中枢调控过程。2.延髓内脏中枢主要核团中的儿茶酚胺能神经元参与束缚-浸水应激致胃粘膜损伤的调控,而下丘脑前部核团中儿茶酚胺能神经元很少甚或没有,因此不是下丘脑前部参与束缚-浸水应激反应的主要神经元,但核团内神经元的活动可能受延髓儿茶酚胺能神经元的反馈调节Fos和酪氨酸羟化酶双标阳性(Fos+TH-IR)神经元主要分布于NTS(A2/C2细胞群)和VLM(A1/C1细胞群),AP中也有较多分布,DMV中只有中段和尾段有少量酪氨酸羟化酶免疫反应阳性(TH-IR)神经元,NA中只有TH阳性终末,未见TH-IR神经元胞体存在。RWIS 1h组Fos+TH-IR神经元占TH-IR神经元的比例在DMV、NTS、AP和VLM分别为38.0%、34.4%、18.6%和45.7%,分别是对照组(分别为14.3%、9.7%、4.5%、18.9%)的2.7倍、3.6倍、4.1倍和2.4倍,组间差异均极显著(P<0.01),说明延髓内脏中枢上述核团中的CA能神经元参与了RWIS致胃机能紊乱的中枢调控。PVN中只有少量TH-IR神经元胞体分布于,SON中没有TH阳性胞体,但两核团中都有大量TH阳性终末,提示在RWIS过程中PNV、SON中的CA能神经元并不起主要作用或不起作用,但核团中神经元的活动可能受延髓CA能神经元的反馈调控。3.下丘脑前部主要核团中的催产素能、精氨酸加压素能神经元参与束缚-浸水应激反应,且精氨酸加压素能神经元活动强于催产素能神经元催产素免疫阳性(OT-IR)神经元主要分布于PVN内侧大细胞部及SON背侧;精氨酸加压素免疫阳性(AVP-IR)神经元主要分布于PVN大细胞部和SON的腹侧。RWIS 1h组,PVN中有约34%的OT-IR神经元、40%的AVP-IR神经元表达Fos;SON中有约28%的OT-IR神经元、53%的AVP-IR神经元表达Fos。束缚-浸水应激激活了PVN、SON中的OT和AVP能神经元,且AVP能神经元活动强于OT能神经元。4.延髓内脏中枢多数活动神经元的胞体膜上分布有催产素受体、精氨酸加压素V1b受体,提示催产素、精氨酸加压素在束缚-浸水过程中通过OT受体、V1b受体调节延髓内脏中枢神经元活动催产素受体免疫阳性(OTR-IR)神经元、V1b受体免疫反应阳性(V1bR-IR)神经元都几近均匀地分布于DVC吻-尾全段。DMV中RWIS 1h组超过10%的OTR-IR和V1bR-IR神经元表达Fos,而对照组只约3%;NTS中RWIS 1h组约10%的OTR-IR神经元和8%V1bR-IR神经元为双标神经元,而对照组则分别为5%和4%,这些RWIS 1h组和对照组的显著差异说明DMV、NTS中的神经元的活动受OT、AVP的调控。RWIS 1h组Fos与OTR双标免疫阳性(Fos+OTR-IR)、Fos与V1bR双标免疫阳性(Fos+V1bR-IR)神经元神占Fos-IR神经元的比值,在DMV中分别约58%和72%,在NTS中约45%和52%,说明OT、AVP对这些核团中神经元活动的调控主要是通过OT受体和V1b受体,尤其是V1b受体。5.短时间的束缚-浸水应激改变了突触膨体素和突触蛋白I在下丘脑前部和延髓内脏中枢中的含量及其分布在RWIS 0h到4h过程中,下丘脑前部中突触膨体素表达在不同应激时间段差异不显著;但突触蛋白I,尤其是突触蛋白Ib的含量发生了显著性变化,这说明短时间的RWIS应激引起下丘脑前部突触可塑性的改变。在RWIS 0h到4h过程中,延髓中的突触膨体素表达量呈现一个先增加后减少的趋势,突触蛋白I含量也发生了显著性变化,说明延髓内脏中枢在RWIS致胃粘膜损伤中枢调控过程中也发生了突触可塑性的变化。综上所述,束缚-浸水应激诱导DMV、NA中胆碱能神经元Fos显著表达,说明DMV、NA中胆碱能神经元的过度活动是RWIS诱导胃粘膜损伤的中枢机制之一。DVC、VLM中的CA能神经元可能投射到下丘脑前部,激活下丘脑前部的OT、AVP能神经元,尤其是AVP能神经元,OT、AVP能神经元继而投射到DMV、NTS,通过与OT受体、V1b受体结合激活这些核团中的OT、AVP敏感神经元,进而参与束缚-浸水应激导致胃粘膜损伤的调控。短时间的束缚-浸水应激不影响突触膨体素在下丘脑前部的含量和分布,但显著影响突触膨体素在延髓内脏中枢中含量及其分布;短时间的束缚-浸水应激影响突触蛋白I在下丘脑前部和延髓内脏中枢中含量及其分布,说明RWIS过程中下丘脑前部、延髓内脏中枢发生了突出可塑性的改变。更多还原

【Abstract】 Restraint water-immersion stress (RWIS), considered to be a mixture of physical and psychological stressors, induces vagally-mediated gastric hypercontractility, hypersecretion of gastric acid and acute gastric erosions within a few hours. It is widely used to study the pathogenesis of stress-induced gastric lesions and to filtrate the medicine that can cure this disease. Using Fos as a marker of the activation of neurons, we found that after different durations of RWIS, the most intense Fos expression was observed in specific brain areas, such as the medullary visceral zone [dorsal motor nucleus of the vagus (DMV), nucleus of solitary tract (NTS), area postrema (AP) and nucleus ambiguous (NA)] and the anterior hypothalamus [paraventricular nucleus (PVN) and supraoptic nucleus (SON)]. These results indicate that the neuronal hyperactivity of the DMV, NTS, AP, NA, PVN and SON may play an important role in the gastric erosions induced by RWIS. Pretreatment with bilateral subdiaphragmatic vagotomy or atropine inhibited gastric hypercontractility, hypersecretion of gastric acid and prominently alleviated gastric erosions under RWIS, while pretreatment with hypophysectomy, adrenalectomy or phenoxybenzamine failed to affect gastric hypercontractility, hypersecretion of gastric acid and gastric erosions under RWIS, which innervates the stomach, and the abnormalities of gastric functions induced by RWIS are not due to the hyperactivity of the hypothalamo-pituitary-adrenal (HPA) axis, but due to the hyperactivity of vagal parasympathetic efferents, which largely originating in the DMV and partly in the NA. It seems that the hyperactivity of the DMV and NA leads to gastric lesions,but, electrical and chemical stimulations of the DMV and NA inhibited gastric motility. So, whether the hyperactivity of the higher centre, mainly the PVN and SON, relieves the inhibition of gastric motility mediated by the primary nerve centre? If so:1. Would the brainstem circuits regulating gastric function change during RWIS? What are the phenotype natures of activated neurons during RWIS?2. The PVN and SON are the main nuclei of the anterior hypothalamus, which might be stimulus-dependent. Vasopressin (AVP) and oxytocin (OT) are two structurally related nonapeptides synthesized mainly in the magnocellular neurons of the PVN and SON, and may act as neurotransmitters and/or neuromodulators which are considered to be related to gastric functions and the regulation of stress response. Whether the activated neurons in the PVN and SON of rats induced by RWIS were vasopressinergic and oxytocinergic neurons, and whether the activated neurons would display certain typical topographic features?3. Supposed that the activated neurons in the anterior hypothalamus not only projected to the candal part of pituitary but also to the medulla oblongata gastric nerve centre, and then may act as neurotransmitters to innervate the activity of the primary nerve centres of regulating gastric functions. If so, whether the phenotypic natures of activated neurons in the medullary visceral zone were AVP sensitive or OT sensitive neurons, where AVP or OT receptors located?4. Synapses are connections between neurons; biological signals are transferred from the presynaptic membrane by the synaptic transmission to the postsynaptic membrane. Synaptophysin (SYN or p38) and SynapsinI are the specific component of presynaptic membrane and the small synaptic vesicles (SSVs). The proteins were clearly found in vesicles in all efferent terminals, in both medial and lateral efferent systems, where they are believed to be involved in synaptic vesicle formation and exocytosis, the exocytosis of stored neurotransmitter. So, SYN and SynapsinI are considered to be the sensitive neuritic markers for synaptic degeneration and regenerative response.Thus, in the present study:(1) Male Wistar rats were randomly divided into 2 groups designated according to their duration of RWIS, respectively 0h and 1h. The rats stressed for 0h were considered as a control group. Additionally, to evaluate the role of catecholaminergic neurons, cholinergic neuron, oxytocinergic neurons ( or OT sensitive neurons) and vasopressinergic (or AVP sensitive neurons) in the anterior hypothalamus and the medulla oblongata gastric nerve centre during RWIS, the phenotype nature of activated neurons was determined by a double immunohistochemical method for collocations of Fos with one of Tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), Vasopressin (AVP), oxytocin (OT) and oxytocin receptor (OTR), V1-type AVP receptors (V1bR). (2) Male Wistar rats were randomly divided into 4 groups designated according to their duration of RWIS, respectively 0h, 1h, 2h and 4h. The rats stressed for 0h were considered as a control group. Additionally, in order to detect whether synaptic plasticity changed, immunohistochemistry staining technique and Western blotting technique were used to determine the expression and amounts of SYN and SynapsinI in the anterior hypothalamus and the medulla oblongata gastric nerve centre during RWIS.The present data demonstrate that:1. The hyperactivity of cholinergic neurons in the DMV and NA but the NTS might be one of the primary central mechanisms of the gastric dysfunctions induced by the RWIS. ChAT-IR neurons were observed evidently from the rostral to the caudal portions of the DVC either in the RWIS rats or the unstressed rats. In the DMV and NA, RWIS for 1h induced a robust decrease in ChAT-IR neurons. In the RWIS 1h rats, the number of Fos+ChAT-IR neurons was evidently increased compared to the unstressed rats, 3.60-fold in the rostral DMV, 16.00-fold in the intermedial DMV, 4.00-fold in the caudal DMV. Meanwhile, the percentage of Fos+ChAT-IR neurons in ChAT-IR neurons was 3% and 21% in the unstressed and RWIS 1h rats, respectively (P<0.01). In the NA, the number of Fos+ChAT-IR neurons was evidently increased 3.29-fold compared to the unstressed rats, and the percentage of Fos+ChAT-IR neurons in ChAT-IR neurons was 36% (i.e. 4.78-fold VS the unsressed rats) (P<0.01). But, in the NTS, the number of ChAT-IR neurons and Fos+ChAT-IR neurons was hardly increased compared to the unstressed rats, and the percentage of Fos+ChAT-IR neurons in ChAT-IR neurons between the RWIS 1h and unstress rats (P>0.05). These data strongly suggest that ChAT-neurons in the DMV and NA, but not the NTS, are related to stress-responsive signal transduction.2. Catecholaminergic neurons in the medulla oblongata gastric nerve centre may be related to the stress-responsive signal transduction, while those in the anterior hypothalamus were not but activated by Catecholaminergic neurons.In the brainstem, the marked activation of catecholaminergic neurons induced by RWIS encompasses mainly the NTS (A2/C2), VLM (A1/C1) cell groups and AP along with a few stained cells were found in the intermediate and the rostral DMV, while no TH-IR neurons were observed, although the CA-nerve ending were relatively abundant in the NA. In RWIS rats and unstressed rats, the percentage of Fos+TH-IR neurons in TH-IR neurons was respectively 38.0% and 14.3% in the DMV (i.e. 2.7-fold), 34.4% and 9.7% in the NTS (i.e. 3.6-fold), 18.6% and 4.5% in the AP (i.e. 4.1-fold), 45.7% and 18.9% in the VLM (i.e. 2.4-fold) (all the P<0.01). These data strongly suggest that CA-neurons in the medullary visceral zone are related to stress-responsive signal transduction.In the anterior hypothalamus, TH-labeled immunoreactive perikarya represented the fairly lowest population of cells in the PVN, and in the SON the presence of TH-immunoreactive perikarya was completely absent either in the control rats or the rats stimulated with RWIS, which indicate that the CA-neurons in the PVN and SON may be not related to the stress-responsive signal transduction, but the neurons may be activated by CA-neurons.3. The hypothalamic oxytocinergic and vasopressinergic neurons were involved in the regulation of a variety of central neural functions, but not the main type neurons activated by RWIS.OT-IR neurons were mostly located in the magnocellular portion of the PVN (including PaMM and PaLM), while mainly in dorsal part of the SON; AVP-IR neurons mainly located in the magnocellular portion of the medial part of the PVN (PaMM), while mainly in dorsal part of the SON. RWIS for 1h results in the activations of a large population of OT- and AVP- containing neurons, 31% and 40% in the PVN, 28% and 53% in the SON, respectively. These data indicate that the hypothalamic oxytocinergic and vasopressinergic neurons may be involved in the regulation of a variety of central neural functions, and more vasopressinergic neurons than the oxytocinergic ons were activated during RWIS.4. The phenotypic natures of activated neurons in the medullary visceral zone were AVP sensitive or OT sensitive neurons, where OTR or V1bR located in the medulla oblongata gastric nerve centre, which indicate that OT and AVP might mediate the activity of these neurons by OTR and V1bR.OTR-IR and V1bR-IR neurons were observed evidently from the rostral to the caudal portions of the DVC either in the RWIS rats or the unstressed rats. In the present study, more than 10% of OTR-IR and V1bR-IR neurons in the DMV were activated in the RWIS rats while less than 3% in the nonstressed rats. In the NTS, the percentages of Fos-IR neurons in the OTR-IR and V1bR-IR neurons were 10% and 8%, while 5% and 4% in the nonstressed rats, respectively. The significant difference of the ratio between the RWIS and nonstressed groups indicates that the OT-sensitive and AVP-sensitive neurons may be involved in the modulation of the gastric dysfunction during the RWIS. In addition, in RWIS 1h rats, the percentage of Fos+OTR-IR and Fos+V1bR-IR neurons in Fos-IR nuclei was more than 58% and 72% in the DMV, 35% and 52% in the NTS, which indicated that OT and AVP mediate the activity of these neurons mainly by OTR and V1bR.5. Amounts and distributions of SYN did not significantly differ in the anterior hypothalamus but the medulla oblongata with different durations of RWIS; and amountsand distribution of SynapsinI in the anterior hypothalamus and the medulla oblongata gastric nerve centre were changedDuring RWIS 0h to 4h, the SYN and SynapsinI immunoreactivity and amounts were examined using immunohistochemistry and Wstern blotting. Amounts and distributions of SYN did not significantly differ in the anterior hypothalamus but the medulla oblongata with different durations of RWIS (P<0.05). Amountsand distribution of SynapsinI in the anterior hypothalamus and the medulla oblongata gastric nerve centre were changed (P<0.05). These data indicate that RWIS effect the distribution and contents of SYN and SynapsinI in the medulla oblongata and/or the anterior hypothalamus. Since changes of SYN and SynapsinI correlated with brain dysfunction, the synaptic plasticity was changed in the anterior hypothalamus and the medulla oblongata gastric nerve centre in rats exposed to RWIS.In conclusion, RWIS for 1h results in the activations of a large population of ChAT-IR neurons in the DMV and NA, which indicates that the hyperactivity of cholinergic neurons in the DMV and NA leads to gastric lesions. OT- and AVP-containing neurons in the PVN and SON, which may be activated by Catecholaminergic neurons, would project to the NTS or DMV, mediated by OTR and V1bR, and then the DMV in turn provide the preganglionic efferent fibers to regulate gastric information. RWIS effects the distribution and contents of SynapsinI but SYN in the anterior hypothalamus, while in the medulla oblongata gastric nerve centre, the distribution and contents of SYN and SynapsinI changed during the RWIS. These data indicate that the synaptic plasticities were changed in the anterior hypothalamus and the medulla oblongata gastric nerve centre in rats exposed to RWIS.更多还原