【作者】 钟明奎；

【导师】 朱国庆；

【作者基本信息】 南京医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 背景心交感传入反射（cardiac sympathetic afferent reflex,CSAR）是一种交感兴奋性反射,心脏表面应用缓激肽、辣椒素或过氧化氢可刺激神经末梢,兴奋通过心交感传入神经到达中枢,经中枢整合后反射性地引起交感传出活动增强和动脉血压升高。心肌缺血时产生的化学物质也可以引起CSAR。慢性心力衰竭（chronic heart failure,CHF）是严重危害人类健康的常见病。交感神经系统活动过度增强是CHF的重要特征之一,是加速病情恶化并使存活期缩短的重要因素。CHF时CSAR的病理性增强是导致交感活动亢进的重要原因之一,在CHF发病机制中起了极其重要的作用。室旁核（paraventricular nucleus,PVN）是心血管活动整合的重要中枢结构之一,我们以前研究表明PVN内血管紧张素Ⅱ（AngⅡ）和AT₁受体调节正常大鼠和犬的CSAR,并与CHF状态下过度增强的CSAR有关。最近我们发现PVN内NAD（P）H氧化酶来源的活性氧,尤其是超氧阴离子和过氧化氢介导了AngⅡ对CSAR的调节作用。虽然PVN的信号分子在CSAR调节中起极其重要的作用,但至今对CSAR的中枢传导通路仍不够了解。进一步探讨PVN在CSAR中枢通路中的作用及其调控机制不仅有重要理论意义,而且可为寻找新的防治CHF药物提供线索和奠定理论基础。目的1研究PVN是否为CSAR反射弧中枢通路的重要结构之一2探讨PVN中γ-氨基丁酸（GABA）对CSAR的调节作用及其受体机制方法实验在去压力感受器神经支配和双侧迷走神经切断的麻醉SD大鼠上进行,采用立体定位仪进行大鼠双侧PVN定位、核团损毁和微量注射,利用Powerlab生物信号采集系统,在体记录肾交感神经放电活动（RSNA）、平均动脉压（MAP）和心率（HR）,以心室表面应用缓激肽（BK）和辣椒素引起RSNA的变化值作为评价CSAR的指标。1可逆性抑制或兴奋PVN神经元对CSAR、RSNA和MAP的影响大鼠随机分为3组,每组7只,分别在双侧PVN内注射生理盐水、8.5 nmol利多卡因和1 nmol L-谷氨酸。观察注射前、注射后5 min和30 min的CSAR、RSNA和MAP的变化。2电损毁PVN对CSAR、RSNA和MAP的影响大鼠随机分为PVN损毁组和假损毁组,每组6只,分别观察损毁前和损毁后10 min的CSAR、RSNA和MAP的变化。3选择性损毁PVN神经元胞体对CSAR、RSNA和MAP的影响大鼠随机分为2组,每组6只,分别在双侧PVN内注射生理盐水、2 nmol海人酸,分别观察注射前和注射后90 min的CSAR、RSNA和MAP的变化。另外还观察PVN注射海人酸1周后对CSAR的影响（n=3）。为排除药物扩散至PVN邻近部位产生效应的可能性,另取3只大鼠,观察下丘脑前区微量注射2 nmol海人酸对CSAR的影响。4 GABA_A受体激动剂和拮抗剂对CSAR、RSNA和MAP的影响大鼠随机分为6组,每组6只,分别在双侧PVN内注射生理盐水、3种剂量的GABA_A受体激动剂isoguvacine（0.1 nmol,1 nmol或10nmol）、GABA_A受体拮抗剂gabazine（0.1 nmol）和isoguvacine（10nmol）+gabazine（0.1 nmol）（gabazine预处理10 min后注射isoguvacine）,观察其对CSAR、RSNA和MAP的影响。PVN微量注射药物10 min后测定CSAR。5 GABA_B受体激动剂和拮抗剂对CSAR、RSNA和MAP的影响大鼠随机分为6组,每组6只,分别在双侧PVN内注射生理盐水、3种剂量的GABA_B受体激动剂baclofen（0.01 nmol,0.1 nmol或1nmol）、GABA_A受体拮抗剂CGP-35348（10 nmol）和baclofen（1 nmol）+CGP-35348（10 nmol）（CGP-35348预处理10 min后注射baclofen）,观察其对CSAR、RSNA和MAP的影响。PVN微量注射药物10min后测定CSAR。6 PVN内源性GABA对CSAR、RSNA和MAP的影响大鼠随机分为5组,每组6只,分别在双侧PVN内注射生理盐水、GABA转氨酶抑制剂vigabatrin（10 nmol）、vigabatrin（10 nmol）+gabazine（0.1 nmol）、vigabatrin（10 nmol）+CGP-35348（10 nmol）、和vigabatrin（10 nmol）+gabazine（0.1 nmol）+CGP-35348（10 nmol）,vigabatrin注射30 min后再应用gabazine和/或CGP-35348,观察其对CSAR、RSNA和MAP的影响。PVN微量注射vigabatrin或生理盐水70 min后测定CSAR。结果1双侧PVN内微量注射利多卡因抑制PVN神经元活动对基础RSNA和MAP无显著影响,但可消除CSAR;而应用L-谷氨酸兴奋PVN神经元则增强CSAR、增加基础RSNA和MAP。2双侧电损毁PVN不可逆地消除CSAR,而对基础RSNA和MAP无显著影响。3双侧PVN注射海人酸选择性损毁神经元胞体,引起基础RSNA增加和MAP升高,持续约60 min,继后保持在正常水平,90 min时测定CSAR发现海人酸化学损毁PVN后消除CSAR。下丘脑前部注射海人酸则对CSAR无显著影响。4双侧PVN内微量注射GABA_A受体激动剂isoguvacine引起剂量依赖性的基础RSNA减少、MAP降低和CSAR减弱,其效应可被GABA_A受体拮抗剂gabazine阻断。Gabazine增加基础RSNA和MAP,但对CSAR无显著影响。5双侧PVN内微量注射GABA_B受体激动剂baclofen剂量依赖性减弱RSNA、降低MAP,并抑制CSAR,大剂量baclofen可完全阻断CSAR。GABA_B受体拮抗剂CGP-35348则产生相反的效应,并可阻断baclofen的作用。静脉注射相同剂量的baclofen对RSNA、MAP和CSAR无显著影响。6双侧PVN内微量注射特异性的GABA转氨酶抑制剂vigabatrin,显著降低RSNA和MAP,消除CSAR。应用GABA_A受体拮抗剂gabazine或GABA_B受体拮抗剂CGP-35348预处理,均可部分抑制vigabatrin的作用,而联合应用gabazine和CGP-35348则可完全阻断vigabatrin的作用。结论1应用利多卡因抑制PVN神经元活动可消除CSAR,而应用L-谷氨酸兴奋PVN神经元则增强CSAR。非选择性电损毁PVN或选择性化学损毁PVN神经元胞体均可完全阻断CSAR。表明PVN是CSAR反射弧重要整合中枢之一。2激活PVN中GABA_A受体使CSAR减弱,而激活GABA_B受体则完全消除CSAR。阻断GABA_A受体对CSAR无显著影响,但阻断GABA_B受体增强CSAR。PVN中内源性GABA水平升高可抑制CSAR,其效应是通过GABA_A和GABA_B受体共同介导的。表明PVN中GABA是调控CASR的重要神经递质或调质,其抑制CSAR作用是由GABA_A和GABA_B受体受体共同介导的。更多还原

【Abstract】 BackgroundThe cardiac sympathetic afferent reflex（CSAR）is a sympatho-excitatory reflex.The sympathetic afferent endings innervating the heart are stimulated by epicardial application of bradykinin（BK）, capsaicin,or hydrogen peroxide,the afferent signals from cardiac receptors arrive the central,or then reflexly increases sympathetic tone and arterial pressure.During myocardial ischemia and infarction,CSAR is induced.Chronic heart failure（CHF）is a common disease threatening human’s health with its high mortality.It is well accepted that CHF is characterized by enhanced sympathetic tone which plays a critical role in the pathogenesis of CHF.The degree of sympathoexcitation is positively correlated with mortality in the CHF state.The CSAR is enhanced in CHF and partially contributes to the over-excitation of sympathetic nervous system.The paraventricular nucleus（PVN）is an important integrative region in the control of sympathetic outflow and arterial pressure.Our previous studies have shown that angiotensinⅡ（AngⅡ）and AT₁ receptors in the PVN modulate the CSAR in normal rats and dogs and partially contribute to the enhanced CSAR in CHF.Recently,we have found that NAD（P）H oxidase-derived reactive oxygen species especially superoxide anions and hydrogen peroxide in the PVN contribute to the effect of AngⅡin the PVN on the CSAR in rats.These results suggest the importance of PVN in the central control of the CSAR.However the central pathway of the CSAR is not clear.The present study was designed to more conclusively demonstrate that the PVN is an important component of the central neurocircuitry of the CSAR and further elucidate the central mechanism responsible for modulation of the CSAR.It will lay a foundation in finding new drugs for management of CHF.Objective1.The effects of inhibiting and exciting the PVN,or bilateral electrolytic and chemical lesion of the PVN on the CSAR were respectively determined.2.The roles of GABA_A and GABA_B receptors as well as GABA in the PVN in the control of the CSAR and sympathetic outflow were investigated.MethodsIn anesthetized rats with sinoaortic denervation and cervical vagotomy,renal sympathetic nerve activity（RSNA）,mean arterial pressure（MAP）and heart rate（HR）were recorded in vivo on a PowerLab data acquisition system.The CSAR was evaluated by the RSNA response to epicardial application of bradykinin（BK）or capsaicin.The coordinates for PVN were determined in a stereotaxic instrument according to the Paxinos and Watson rat atlas.All the drugs are bilaterally microinjected into PVN.1.The effects of lidocaine and glutamate,which respectively inhibit and activate the neurons in the PVN on the CSAR,were investigated. Lidocaine（8.5 nmol）,L-glutamate（1 nmol）and saline were respectively microinjected into the bilateral PVN in three groups of rats（n=7 for each group）.The CSAR was determined before microinjection,5 and 30 minutes after microinjection.2.Electrolytic lesion and sham lesion of the PVN were carried out in two groups of rats（n=6 for each group）.The CSAR was respectively determined before lesion and 10 minutes after the lesion.3.Kainic acid（KA）was used to creat a chemical lesion to destroy neuronal perikarya in the PVN but spare axons of passage and terminals in the vicinity of the injection site.The effects of microinjection of KA（2 nmol）and saline into the PVN on the CSAR were determined in two groups of rats（n=6 for each group）.The CSAR was evaluated before microinjection and 90 minutes after microinjection.To exclude the possibility that the effect of KA on the CSAR was caused by diffusion to other brain area,the effect of microinjection of same dose of KA into the anterior hypothalamic area,which is adjacent to the PVN,on the CSAR was also determined（n=3）.Furthermore,the long-term effect of KA on the CSAR was observed at the eighth day after microinjection（n=3）.4.The rats were randomly divided into six groups（n=6 for each group）, which were subjected to the bilateral PVN microinjection of saline,three doses of GABA_A receptor agonist isoguvacine（0.1,1 or 10 nmol）, GABA_A receptor antagonist gabazine（0.1 nmol）,or gabazine plus the high dose of isoguvacine（pretreatment with gabazine 10 min before isoguvacine）.The CSAR was evaluated 10 minutes after the microinjection.5.The rats were randomly divided into six groups（n=6 for each group）, which were subjected to the bilateral PVN microinjection of saline,three doses of GABA_B receptor agonist baclofen（0.01,0.1 or 1 nmol）,GABA_B receptor antagonist CGP-35348（10 nmol）,or CGP-35348 plus the high dose of baclofen（pretreatment with CGP-35348 10 min before baclofen）. The CSAR was evaluated 10 minutes after the microinjection.6.The rats were randomly divided into five groups（n=6 for each group）, which were subjected to the bilateral PVN microinjection of saline, GABA-transminase inhibitor vigabatrin（10 nmol）,vigabatrin plus gabazine,vigabatrin plus CGP-35348,and vigabatrin plus combined gabazine and CGP-35348.The gabazine and/or CGP-35348 were microinjected into the PVN 30 minutes after microinjection of vigabatrin. The CSAR was evaluated 70 minutes after microinjection of saline or vigabatrin. Result1.The effects of inhibiting and exciting the neurous in PVN on the CSAR and sympathetic outflow.Microinjection of lidocaine into the PVN abolished the CSAR without significant effects on the baseline RSNA and MAP,while glutamate which excites the neurons in the PVN enhanced the CSAR and increased the baseline RSNA and MAP.2.The effects of electrolytic lesion of the PVN on the CSAR and sympathetic outflow.Electrolytic lesions of the PVN irreversibly abolished the CSAR without significant effects on the baseline RSNA and MAP.3.The effects of selective lesion of the neuronal perikarya in PVN on the CSAR and sympathetic outflow.Selective lesion of the neuronal perikarya in the PVN with kainic acid induced rapid and great increases in both RSNA and MAP,which lasted about 60 minutes and then remained at the baseline levels.At the 90th minute after kainic acid,epicardial application of bradykinin or capsaicin failed to induce the CSAR.4.The effects of GABA_A receptor agonist and antagonist in the PVN on the CSAR and sympathetic outflow.The PVN microinjection of isoguvacine,a GABA_A receptor agonist, induced dose-dependently decreases in RSNA and MAP,and inhibited the CSAR.The high dose of isoguvacine caused great decreases in the baseline RSNA and MAP,but only showed a weakly inhibitory effect on the CSAR.These effects of isoguvacine were completely abolished by gabazine,a GABA_A receptor antagonist.However,gabazine alone did not cause any significant effect on the CSAR,but increased the baseline RSNA and MAP.5.The effects of GABA_B receptor agonist and antagonist in the PVN on the CSAR and sympathetic outflow.The PVN microinjection of baclofen,a GABA_B receptor agonist, dose-dependently caused decreases in RSNA and HR,and inhibited the CSAR evoked by either capsaicin or BK.The high dose of baclofen caused great decreases in the baseline RSNA and MAP,and almost completely abolished the CSAR.These effects of baclofen were completely antagonized by CGP-35348,a GABA_B receptor antagonist. Moreover,CGP-35348 alone enhanced the CSAR,but only slightly increased the baseline RSNA and MAP.Intravenous administration of same dose of baclofen had no significant effect on the CSAR,baseline RSNA and MAP.6.The effects of GABA-transaminase inhibitor in the PVN on the CSAR and sympathetic outflow.The PVN microinjection of vigabatrin,a selective GABA-transaminase inhibitor which increases endogenous GABA level, caused great decreases in baseline RSNA,MAP and HR,and almost completely abolished the CSAR.These effects of vigabatrin were partially inhibited by either GABA_A receptor antagonist gabazine or GABA_B receptor antagonist CGP-35348,and were completely blocked by gabazine plus CGP-35348.Conclusions1.PVN microinjection of lidocaine to inhibit the neural activity of the PVN abolishes the CSAR,but excitatory amino acid L-glutamate enhances the CSAR.Either nonselective electrolytic lesion of the PVN or selective lesion of the neuronal perikarya in the PVN with KA abolishes the CSAR.The results indicate that the PVN is a pivotal component of the central neurocircuitry of the CSAR.2.Activating the GABA_A receptors in the PVN attenuates the CSAR, while activating the GABA_B receptors abolishes the CSAR.The blockade of the GABA_A receptors in the PVN has no significant effect on the CSAR,while blockade of the GABA_B receptors enhances the CSAR.The increased endogenous GABA level in the PVN caused by the GABA-transaminase inhibitor inhibits the CSAR,which is mediated by both the GABA_A and GABA_B receptors.These results indicate that the GABA in the PVN plays an important role in inhibiting the CSAR,which is involved in both the GABA_A and GABA_B receptors in the PVN.更多还原