下丘脑室旁核对胃食管反流豚鼠咳嗽敏感性的调控机制
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摘要
目的:探究下丘脑室旁核(paraventricular nucleus,PVN)在胃食管反流(gastro-esophageal reflux,GER)豚鼠咳嗽敏感性增高中的作用及其可能的调控途径。方法:利用健康雄性白化Hartley豚鼠制备GER模型,并通过柠檬酸咳嗽激发试验测定咳嗽敏感性;应用免疫荧光组织化学染色方法分别观察PVN内Fos蛋白和P物质(SP)的表达情况,以及Fos/SP双标神经元的分布;利用假狂犬病毒(PRV)示踪剂进行气管壁逆行神经束路追踪,并结合免疫荧光组织化学染色方法观察追踪剂的分布情况。结果:模型组与对照组相比,咳嗽敏感性明显增高。免疫荧光染色结果显示:Fos表达于PVN神经元的细胞核内,SP免疫阳性物质主要表达于PVN神经元的细胞浆内,模型组Fos和SP的表达显著增多(P<0.05),且PVN内部分Fos阳性神经元同时呈SP免疫阳性,这些Fos/SP免疫双标神经元大约占SP单标神经元数量的77.25%。神经束路追踪结果显示:气管壁内注射PRV 48 h后,在延髓迷走复合体(dorsal vagal complex,DVC)内可观察到PRV标记的神经元胞体,72 h后PRV标记的神经元胞体可见于PVN内。结论:PVN在GER豚鼠咳嗽敏感性增高中发挥重要作用,可能是PVN内SP能神经元通过PVN-DVC-气道神经通路调控实现的。
Objective: To investigate the role of the hypothalamic paraventricular nucleus( PVN) in cough hypersensitivity of guinea pigs with gastro-esophageal reflux( GER) and the regulating pathway between airway and PVN. Methods:The experiment was carried out in Hartley guinea pigs with GER and followed by the evaluation of their cough sensitivity using citric acid cough provocation tests. The expressions of Fos,substance P( SP) and Fos / SP double-labeled neurons in PVN of the models were observed by immunofluorescence histochemistry; the pathway between the airway and PVN was studied by retrograde tracing using injecting Pseudorabies virus( PRV) and PRV-labeled neurons were observed under fluorescence microscope. Results: The cough sensitivity was increased in model groups compared with control groups.The immunofluorescence; histochemical staining results show that Fos was observed in the neuronal nucleus in PVN and SP was observed in the neuronal cytoplasm in PVN. The expression of Fos and SP in PVN significantly increased in model groups compared with control groups( P < 0. 05). There were Fos / SP double-labeled neurons in PVN,accounting for77. 25% in SP single labeled neurons. The retrograde tracing results showed that PRV-labeled neurons were observed in dorsal vagal complex( DVC) after 48 hours and in PVN after 72 hours injecting PRV. Conclusion: PVN plays animportant role in cough hypersensitivity of guinea pigs with GER and the SP in PVN can regulate cough sensitivity through PVN-DVC-airway pathway.
Objective: To investigate the role of the hypothalamic paraventricular nucleus( PVN) in cough hypersensitivity of guinea pigs with gastro-esophageal reflux( GER) and the regulating pathway between airway and PVN. Methods:The experiment was carried out in Hartley guinea pigs with GER and followed by the evaluation of their cough sensitivity using citric acid cough provocation tests. The expressions of Fos,substance P( SP) and Fos / SP double-labeled neurons in PVN of the models were observed by immunofluorescence histochemistry; the pathway between the airway and PVN was studied by retrograde tracing using injecting Pseudorabies virus( PRV) and PRV-labeled neurons were observed under fluorescence microscope. Results: The cough sensitivity was increased in model groups compared with control groups.The immunofluorescence; histochemical staining results show that Fos was observed in the neuronal nucleus in PVN and SP was observed in the neuronal cytoplasm in PVN. The expression of Fos and SP in PVN significantly increased in model groups compared with control groups( P < 0. 05). There were Fos / SP double-labeled neurons in PVN,accounting for77. 25% in SP single labeled neurons. The retrograde tracing results showed that PRV-labeled neurons were observed in dorsal vagal complex( DVC) after 48 hours and in PVN after 72 hours injecting PRV. Conclusion: PVN plays animportant role in cough hypersensitivity of guinea pigs with GER and the SP in PVN can regulate cough sensitivity through PVN-DVC-airway pathway.
引文
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[15]刘春丽,姚卫民,陈如冲,等.反复食管内灌注盐酸诱导豚鼠食管支气管反射发生可塑性改变[J].实用医学杂志,2013,10:1569-1572.
[16]Hokfelt T,Kellerth JO,Nilsson G,et al.Substance P:localization in the central nervous system and in some primary sensory neurons[J].Science,1975,190:889-890.
[17]薛德彬,董榕,刘悦.下丘脑室旁核微量注射P物质对豚鼠呼吸的影响[J].东南大学学报(医学版),2004,23:89-92.
[18]肖剑,董榕,刘佳,等.下丘脑室旁核经迷走神经复合体对哮喘大鼠的神经调控研究[J].神经解剖学杂志,2007,23:299-304.
[19]Joad JP,Munch PA,Bric JM,et al.Passive smoke effects on cough and airways in young guinea pigs:role of brainstem substance P[J].Am J Respir Crit Care Med,2004,169:499-504.
[20]董榕,刘悦,薛德彬,等.P物质对哮喘大鼠神经内分泌功能的调节[J].中国药理学通报,2010:26:95-99.
[2]陈如冲,刘春丽,罗炜,等.慢性咳嗽常见病因之间咳嗽敏感性的差异[J].中国呼吸与危重监护杂志,2013,12:384-389.
[3]马千里,龙舟,张巧,等.慢性咳嗽患者咳嗽激发试验的意义[J].中华内科杂志,2011,50:668-671.
[4]叶新民,钟南山,刘春丽,等.副流感病毒感染豚鼠咳嗽反射敏感性变化及其神经源性炎症机制探讨[J].中国病理生理杂志,2011,27:1-8.
[5]Qiu Z,Yu L,Xu S,et al.Cough reflex sensitivity and airway inflammation in patients with chronic cough due to non-acid gastro-oesophageal reflux[J].Respirology,2011,16:645-652.
[6]Otsuka K,Niimi A,Matsumoto H,et al.Plasma substance P levels in patients with persistent cough[J].Respiration,2011,82:431-438.
[7]Chung KF,Pavord ID.Prevalence,pathogenesis,and causes of chronic cough[J].Lancet,2008,371:1364-1374.
[8]Liu C,Chen R,Luo W,et al.Neurogenic airway inflammation induced by repeated intra-esophageal instillation of HCl in guinea pigs[J].Inflammation,2013,36:493-500.
[9]Liu Q,Fujimura M,Tachibana H,et al.Characterization of increased cough sensitivity after antigen challenge in guinea pigs[J].Clin Exp Allergy,2001,31:474-484.
[10]Chung KF,Mc Garvey L,Mazzone SB.Chronic cough as a neuropathic disorder[J].Lancet Respir Med,2013,1:414-422.
[11]Shannon R,Baekey DM,Morris KF,et al.Production of reflex cough by brainstem respiratory networks[J].Pulm Pharmacol Ther,2004,17:369-376.
[12]Mc Govern AE,Davis-Poynter N,Farrell MJ,et al.Transneuronal tracing of airways-related sensory circuitry using herpes simplex virus 1,strain H129[J].Neuroscience,2012,207:148-166.
[13]Jakus J,Poliacek I,Halasova E,et al.Brainstem circuitry of tracheal-bronchial cough:c-fos study in anesthetized cats[J].Respir Physiol Neurobiol,2008,160:289-300.
[14]Chowdreyxa HS,Larsen PJ,Harbuz MS,et al.Endogenous substance P inhibits the expression of corticotropin-releasing hormone during a chronic inflammatory stress[J].Life Sci,1995,57:2021-2029.
[15]刘春丽,姚卫民,陈如冲,等.反复食管内灌注盐酸诱导豚鼠食管支气管反射发生可塑性改变[J].实用医学杂志,2013,10:1569-1572.
[16]Hokfelt T,Kellerth JO,Nilsson G,et al.Substance P:localization in the central nervous system and in some primary sensory neurons[J].Science,1975,190:889-890.
[17]薛德彬,董榕,刘悦.下丘脑室旁核微量注射P物质对豚鼠呼吸的影响[J].东南大学学报(医学版),2004,23:89-92.
[18]肖剑,董榕,刘佳,等.下丘脑室旁核经迷走神经复合体对哮喘大鼠的神经调控研究[J].神经解剖学杂志,2007,23:299-304.
[19]Joad JP,Munch PA,Bric JM,et al.Passive smoke effects on cough and airways in young guinea pigs:role of brainstem substance P[J].Am J Respir Crit Care Med,2004,169:499-504.
[20]董榕,刘悦,薛德彬,等.P物质对哮喘大鼠神经内分泌功能的调节[J].中国药理学通报,2010:26:95-99.