【作者】 汪慧；

【导师】 宋刚； 张衡； Chi-Sang Poon；

【作者基本信息】 山东大学 ， 生理学， 2008， 博士

【摘要】 脑桥呼吸调整中枢由脑桥背外侧（dl-pons）和脑桥腹外侧（vl-pons）两个结构组成。Dl-pons的主要结构即KF核及臂旁核（PB）,vl-pons则主要是指A5区。Dl-pons和vl-pons之间存在双向纤维联系。众所周知,脑桥是呼吸调整中枢所在地,在呼吸节律的发起、呼吸时相转换以及呼吸运动协调中发挥重要作用。本课题试图通过研究脑桥呼吸调整中枢对呼吸反射的调节作用探讨脑桥呼吸运动调控机制。研究分为五部分:第一部分观察了某些神经递质拮抗剂在dl-pons及vl-pons对黑伯反射（肺牵张反射）的影响;第二部分观察了它们在dl-pons及vl-pons对低氧呼吸反应的影响;第三部分应用神经纤维投射追踪和免疫组织化学技术观察了dl-pons和vl-pons神经元向脑干呼吸反射（低氧呼吸反应和黑伯反射）相关神经元的轴突投射;第四部分观察了小鼠的黑伯反射,并观察了NMDA受体对小鼠黑伯反射的影响;第五部分观察了电刺激vl-pons对黑伯反射的调控及NMDA受体在该过程中的作用。1、NMDA、GABA_A及AMPA受体拮抗剂在dl-pons及vl-pons对黑伯反射的影响实验在35只成年SD大鼠上进行,氨基甲酸乙酯腹腔注射麻醉,巴夫龙（pancuronium bromide）肌松,人工通气。以膈神经放电为观察指标。电刺激迷走神经模拟黑伯反射。电刺激参数为:强度20-80μA,频率80 Hz,波宽0.1 ms,刺激时间60 sec。持续电刺激迷走神经首先出现的是强度依赖性的呼吸暂停、膈神经放电停止于呼气相（Stage-1）。随着刺激的延续,节律性膈神经放电重新出现,呼吸频率逐渐加快向基线水平恢复仍然低于基线水平（Stage-2）。刺激停止后出现短暂的呼吸频率加快并高于刺激前基线水平,该现象主要是由呼气时间的缩短引起的,称为刺激后反跳效应（PR,Stage-3）。分别在dl-pons及vl-pons微量注射NMDA受体阻断剂D-2-氨基-5-膦酸基戊酸（D-AP5,10 mM）、GABA受体阻断剂荷包牡丹碱（bicuculline,BIC,5 mM）及AMPA受体阻断剂CNQX（6-cyano-7-nitroquinoxaline-2,3-dione,10 mM）,注射剂量20-50nl。以注射前黑伯反射为前对照,观察注射后黑伯反射的改变。结果发现:（1）在dl-pons微量注射AP5可以增强黑伯反射的呼吸抑制效应,表现为黑伯反射第一第二阶段呼吸频率下降幅度、呼气时间延长幅度及吸气时间缩短幅度均增大;黑伯反射第三阶段呼吸频率仍然低于基线水平,反跳现象消失,该阶段吸气时间增加的幅度明显增大,上述变化均有显著性差异。（2）在vl-pons微量注射AP5可以削弱黑伯反射的呼吸抑制效应,表现为黑伯反射第一第二阶段呼吸频率下降幅度、呼气时间延长幅度均减小,黑伯反射第三阶段呼吸频率高于基线水平出现反跳现象,但比前对照削弱,上述变化均有显著性差异。（3）在dl-pons微量注射BIC可以削弱黑伯反射的呼吸抑制效应,表现为黑伯反射第一阶段呼吸频率下降幅度、呼气时间延长幅度及吸气时间缩短幅度均减小,第二阶段呼气时间延长幅度及吸气时间缩短幅度也减小,第三阶段呼气时间缩短的幅度减小,吸气时间虽比第二阶段有所延长但仍低于基线水平,上述变化均有显著性差异。第三阶段呼吸频率高于基线水平,出现反跳现象,与前对照相比有所削弱但无显著性差异。（4）在vl-pons微量注射BIC可以增强黑伯反射的呼吸抑制效应,表现为黑伯反射第一第二阶段呼吸频率下降幅度、呼气时间延长幅度及吸气时间缩短幅度均增大,变化均有显著性差异;黑伯反射第三阶段呼吸频率高于基线水平,出现反跳现象,与前对照相比有所削弱但无显著性差异。（5）在dl-pons微量注射AMPA受体阻断剂CNQX后,黑伯反射三个阶段的膈神经放电抑制效应无明显变化;（6）在vl-pons微量注射CNQX后,黑伯反射三个阶段的膈神经放电抑制效应也没有显著改变。这些结果表明存在于dl-pons及vl-pons的由NMDA受体及GABA受体介导的突触传递参与了对黑伯反射强度的调节,AMPA受体不影响该过程。2、NMDA、GABA_A及AMPA受体拮抗剂在dl-pons及vl-pons对低氧呼吸反应的影响实验在35只成年SD大鼠上进行。动物制备同前。低氧通气为8%O₂-92%N₂混合气体,低氧通气时间30-50秒。以膈神经放电为观察指标,分别向dl-pons及vl-pons内微量注射NMDA受体阻断剂D-AP5、GABA受体阻断剂BIC、AMPA受体阻断剂CNQX,观察注射前后低氧通气时膈神经放电的变化。结果显示给予大鼠低氧刺激后,膈神经放电首先出现频率升高、吸气相幅度增大、呼气时间及吸气时间均缩短,此谓急性低氧呼吸反应。终止低氧恢复正常通气后即可出现膈神经放电频率降低并低于低氧刺激前基线水平,此现象称为低氧后呼吸频率下降（PHFD）。以微量注射前低氧反应为前对照,（1）在dl-pons微量注射D-AP5后,急性低氧反应膈神经放电无显著性改变,但PHFD期膈神经放电频率下降幅度减小,即PHFD现象被削弱。（2）在vl-pons微量注射D-AP5后,急性低氧反应期及PHFD期膈神经放电均无显著性改变。（3）在dl-pons微量注射BIC后,急性低氧反应期膈神经放电频率升高的幅度及呼气时间缩短的幅度均增大,PHFD期膈神经放电无显著性变化。（4）在vl-pons微量注射BIC后,急性低氧反应期膈神经放电无显著性变化,但PHFD期呼吸频率下降幅度减小。（5）在dl-pons微量注射CNQX后,急性低氧反应期膈神经放电频率无明显变化,但呼气时间缩短的幅度减小,吸气时间缩短的幅度增大。PHFD期膈神经放电频率降低的幅度减小。（6）在vl-pons内微量注射CNQX后,急性低氧反应期吸气时间缩短的幅度增大,但膈神经放电频率及呼气时间均无显著性变化。PHFD期膈神经放电频率降低的幅度减小。这些结果表明dl-pons NMDA受体、GABA受体及AMPA受体均参与了对低氧呼吸反应的调节,NMDA受体主要影响低氧后呼吸频率下降,GABA受体主要影响急性低氧反应,dl-pons AMPA受体作用则表较复杂,它既影响急性低氧反应又影响低氧后呼吸频率下降。vl-pons GABA受体及AMPA受体参与了低氧呼吸反应的调节,GABA受体主要影响低氧后呼吸频率下降,AMPA受体既影响急性低氧反应又影响低氧后呼吸频率下降,NMDA受体没有参与该反应的调节。3、Vl-pons与延髓外周化学感受性反射及黑伯反射功能相关神经元之间的纤维联系实验在8只成年SD大鼠上进行。腹腔注射苯巴比妥（50 mg/kg）麻醉。在无菌手术条件下应用压力注射或微电泳方法向dl-pons KF核内微量注射Biotin dextran,压力注射量为20-30 nl（10%）。微电泳注射采用阳极直流电,5μA,15-30分钟。动物存活10天后给予动物8%的低氧刺激或呼气末正压通气刺激（PEEP）。2-3小时后,腹腔注射氨基甲酸乙酯将动物麻醉,左心室插管灌流、固定,取脑干切片（50μm）,用兔抗c-Fos多克隆抗体（一抗）及结合生物素的羊抗兔IgG（二抗）培育脑片,再用ABC-DAB方法处理脑片,用甲酚紫染色方法染色。结果发现发自dl-pons KF核的纤维投射到已知的延髓呼吸相关结构。另外单纤维追踪显示KF核神经元支配多个结构。c-Fos免疫阳性神经元主要分布在孤束核内侧及腹外侧亚核、延髓腹外侧的外侧网状核、疑核及其周围区域。发自dl-pons的标记纤维末梢与上述c-Fos免疫阳性神经元集中存在的区域重叠,提示dl-pons投射纤维与延髓中参与外周化学感受器反射及黑伯反射的神经元可能存在突触联系。4、普通野生型及MECP2基因敲除小鼠黑伯反射特点及NMDA受体对其调节作用雷特氏综合症（Rett综合症）是一种严重影响儿童精神运动发育的神经系统疾病,儿童在6到18个月表现正常,随后病童会有快速退化及发育迟缓的现象,并出现阵发喘气等呼吸异常现象,大约26%的Rett综合症患者死于呼吸功能紊乱。研究发现该病可能与methyl-CpG结合蛋白2（MECP2）基因的突变有关。我们推测病童所表现出来的呼吸异常症状可能与黑伯反射功能异常有关。本课题第四部分研究了普通野生型（+/+）小鼠及MECP2基因敲除小鼠（+/-）黑伯反射及NMDA受体对该反射的调控作用。实验在13只麻醉、肌松、人工通气的成年小鼠上进行。以膈神经放电为呼吸观察指标,电刺激迷走神经中枢端模拟黑伯反射。结果发现:（1）在+/+小鼠,持续电刺激迷走神经首先出现的是强度依赖性的呼吸暂停、膈神经放电停止于呼气相（Stage-1）。随着刺激的延续,节律性膈神经放电重新出现,呼吸频率逐渐加快（Stage-2）。在1分钟刺激将要结束时,呼气时间短于迷走刺激前的基线水平,呼吸频率亦高于基线水平（即“延迟频率增加”现象）。（2）在8只+/-小鼠上刺激迷走神经,结果显示Stage-1呼吸反应与+/+小鼠无明显区别。Stage-2呼吸反应表现出个体差异。有4只+/-小鼠表现出Stage-2“延迟频率增加”现象,与+/+小鼠类似。然而在剩余4只+/-小鼠未观察到该现象。（3）在+/+小鼠和表现出“延迟频率增加”的+/-小鼠,腹腔注射NMDA受体拮抗剂MK-801后Stage-2呼吸反应的“延迟频率增加”现象均消失。该组实验显示Rett综合症可能伴随黑伯反射异常。5、电刺激dl-pons及向vl-pons内微量注射NMDA受体阻断剂MK-801对黑伯反射的调控作用该部分实验在38只成年Wistar大鼠上进行,雌雄不拘。氨基甲酸乙酯腹腔麻醉,用三碘季铵酚肌松,人工通气,以膈神经放电为观察指标。电刺激迷走神经模拟黑伯反射。Vl-pons电刺激参数:50-80μA,80 Hz,波宽0.3 ms,刺激时间20 sec,迷走神经电刺激参数:20-40μA（1.5×阈值）,80 Hz,波宽0.1 msec,刺激时间60 sec。以电刺激vl-pons之前的黑伯反射为前对照,电刺激vl-pons后5 sec内再次电刺激迷走神经引导出黑伯反射,发现与前对照相比,黑伯反射的膈神经抑制效应增强,表现为呼吸频率下降现象、呼气时间延长现象、吸气时间缩短现象更明显,刺激后反跳现象减弱;以微量注射前黑伯反射为前对照,向vl-pons内微量注射MK-801内再次电刺激迷走神经引导出黑伯反射,发现与前对照相比,黑伯反射的抑制效应减弱,表现为呼吸频率下降现象、呼气延长现象、吸气相缩短现象减弱。结果表明vl-pons可以调制黑伯反射的呼吸抑制作用,并可以调节黑伯反射的刺激后反跳现象,并且vl-pons对黑伯反射的调节作用是由NMDA受体介导的神经递质传递介导的。本部分结果发表于Adv Exp Med Biol,vol.605,2008。更多还原

【Abstract】 The rostrolateral pontine pneumotaxic center comprised of a dorsal（dl-）and a ventral（vl-）subdivision.The anatomical substrates are KF and parabrachial nuclei for the dl-pons,and A5 for the vl-pons.These two pneumotaxic subdivisions are reciprocally interconnected both functionally and anatomically.The pontine pneumotaxic center participated in respiratory rhythm genesis,respiratory phase-switching,and the coordination of respiratory movements with other somatic activities.The aim of the present study is to decipher the cellular mechanism of pneumotaxic function by analyzing the influences of dl-and vl-pons on two fundamental respiratory reflexes:the pulmonary stretch reflex（also called Hering-Breuer reflex,HBR）as simulated by electrical vagal stimulation,and the peripheral chemoreflex as evoked by a brief hypoxic ventilation.This systemic work comprised of 5 parts.Part-1 and Part-2:influences of certain neurotransmitter antagonists in the dl-and vl-pons on Hering-Breuer reflex（part-1）and on the peripheral chemoreflex or hypoxic respiratory response（part-2）.Part-3:Descending axonal projections from dl-pons to brainstem respiratory-related structures as marked by hypoxia-evoked and HBR-evoked c-Fos immunohistology.Part-4:The influences of NMDA receptor antagonist MK-801 on the Hering-Breuer reflex in wild-type or MECP2 knock-out mice.Part-5:Effects of pre-stimulation at vl-pons and MK-801 on the Hering-Breuer reflex.1.Influences of NMDA,GABA and AMPA receptor antagonists in the dl-and vl-pons on Hering-Breuer reflexExperiments were performed on 35 adult SD rats that were anesthetized with urethane,paralyzed with pancuronium,and ventilated artificially.Phrenic nerve discharge was recorded as central respiratory output.Hering-Breuer reflex was simulated with electrical vagal stimulation at the central end with low-intensity （20-80μA）,high frequency（80 Hz）and short-pulse duration（0.1 ms）pulses.One episode of stimulation lasted 60 sec.Typically,such electrical vagal stimulation produced immediate phrenic inhibition and the respiration ceased at expiratory phase（Te）.This is called HBR stage-1 Te prolongation.The duration of this vagal evoked expiration（stage-1）was stimulation intensity-dependent.However with the continuation of vagal stimulation,rhythmic phrenic discharge reappeared and gradually adapted toward pre-stimulation baseline level.This gradual adaptation of phrenic discharge to vagal stimulation is called the HBR stage-2.At the off-set of the vagal stimulation,the respiratory frequency showed a temporary rebound-like increase,which is called HBR stage-3.After a control HBR was recorded, microinjections of neurotransmitter antagonists were made and another HBR was recorded.The following antagonists were used:NMDA receptor antagonist D-AP5 （10 mM）;GABA_A receptor antagonist bicuculline（BIC,5 mM）and AMPA receptor antagonist CNQX（10 mM）.The volume of injection was 20-50 nl.Results are as follows:1,the HBR was enhanced following 1)microinjection of D-AP5 into dl-pons,and 2)microinjection of BIC into vl-pons.In these cases the stage-1 Te was significantly longer than control HBR and the adaptation of stage-2 was significantly slower and incomplete.In addition,the shortening of Ti was stronger. The stage-3 rebound was blocked after D-AP5 into dl-pons,but remained unchanged after BIC into vl-pons.2,the HBR was weakened following 1) microinjection of D-AP5 into vl-pons,and 2)microinjection of BIC into dl-pons.In these cases the stage-1 Te was significantly shorter than control HBR and the adaptation of stage-2 was more complete.The shortening of Ti was weaker.The stage-3 rebound was weaker after BIC into dl-pons,but remained unchanged after D-AP5 into vl-pons.3,microinjection of CNQX had no significant influence on HBR.This part of study showed that the pontine NMDA receptor and GABA_A receptor mediated neurotransmissions are involved in the modulation of Hering-Breuer reflex. 2.Influences of NMDA,GABA and AMPA receptor antagonists in the dl-and vl-pons on peripheral chemoreflexThe animal preparations were the same as in part-1.Peripheral chemoreflex was evoked by ventilating the animal with nitrogen-balanced 8%O₂ for 30-50 sec. Under control condition,such hypoxic ventilation caused rapid increase in respiratory frequency and inspiratory amplitude,and shortening of inspiration time. This abrupt increase in respiratory frequency is largely due to the parallel shortening of Te.At the resumption of normal ventilation,the respiratory frequency showed abrupt decline that was mainly due to the parallel prolongation of Te.This abrupt dropping in respiratory frequency is called PHFD（post-hypoxic frequency decline）.The peripheral chemoreflex was tested again following microinjections （see part-1 for details concerning the microinjection）.Results are as follows:1,the rapid increase in respiratory frequency and the parallel shortening of Te was enhanced after microinjection of BIC in dl-pons.2,the rapid shortening of Te was weakened after microinjection of CNQX into dl-pons,however,the parallel weakening of rapid respiratory frequency increase was not statistically significant, which was due to the enhanced hypoxic shortening of Ti.3,the hypoxic shortening of Ti was enhanced after microinjection of CNQX into vl-pons.4,the PHFD became smaller after microinjection D-AP5 or CNQX into dl-pons,and BIC or CNQX into vl-pons,respectively.5,hypoxic changes in respiratory parameters after microinjections other than those described above were not statistically significant.6, the hypoxic increase in inspiratory amplitude was not affected in any case.This part of study showed that the pontine neurotransmissions mediated by NMDA receptor,GABA_A receptor,and AMPA receptor participated in the modulation of peripheral chemoreflex.In the dl-pons,the NMDA receptor is mainly involved in the PHFD,while the GABA_A receptor mainly in acute hypoxic responses.In the vl-pons,the GABA_A receptor is mainly involved in PHFD and the AMPA receptor is involved in both acute and PHFD.NMDA receptor did not play significant role at vl-pons in modulating peripheral chemoreflex.3.Pontine projections to brainstem structures that are involved in HBR and peripheral chemoreflexExperiments were done on 8 adult SD rats that were anesthetized with pentobarbital.The anterograde tracer biotin dextran（BDA）was injected into dl-pons with pressure（volume 20-30 nl,10%）or electroiontophoresis（positive current,5μA,15-30 min.）under sterile condition.After a post-surgery survival period of 10 days,the animals were either challenged with hypoxia or PEEP （positive end-expiratory pressure）for 2-3 hours.The animals were anesthetized, perfused,post-fixed and the brains were cut into serial sections at thickness of 50μm.Sections were incubated in rabbit anti-cFos primary antibody and biotinated goat anti-rabbit IgG.The c-Fos and BDA labeling were visualized using avidin-biotin-HRP histochemistry with DAB as chromogen.It was found that c-Fos immunopositive neurons mainly localized in NTS（medial,commissural, ventrolateral）and ventrolateral medulla following either hypoxia or PEEP. Anterogradely labeled axonal terminals from dl-pons were found to reach well-established brainstem respiratory related structures and overlap with c-Fos immunopositive neurons in NTS and ventrolateral medulla in and around the ambiguus nucleus,where the ventral group of respiratory neurons were recorded.In addition,single axon from KF nucleus sent out branches to innervate multiple brainstem structures.This study revealed anatomical connections between dl-pons and HBR or peripheral chemoreflex related neurons,underscoring our findings in part-1 and part-2 of the present research.4.Hering-Breuer reflex in wild-type and MECP2 knock-out miceRett syndrome is a severe neurodegenerative disease that afflicts tens of thousand children.One of the most prominent symptoms of Rett syndrome is the apneusis or inspiratory withholding.About 26%Rett syndrome patients died of respiratory failure.It was revealed that Rett syndrome is caused by mutation of MECP2 gene that encodes methyl-CpG combining protein.We hypothesize that the respiratory abnormality is caused by the malfunction of Hering-Breuer reflex.This part of research was performed on wild type（+/+）and heterozygous MECP2 knock-out（+/-）mice.Mice were anesthetized with urethane,paralyzed,and artificially ventilated.Phrenic nerve discharge was recorded as central respiratory output.Hering-Breuer reflex was simulated with sustained electrical stimulation at the central cut-end of cervical vagus nerve.Stimulation parameters were the same as in part-1.The results are as follows:1,in +/+ mice,sustained vagal stimulation produced similar stage-1 HBR as in rats as described in part-1.However,the stage-2 adaptation led to net increase in respiratory frequency higher than pre-stimulation value.We call this net increase in respiratory frequency in stage-2 "delayed frequency increase",which is largely due to the progressive shortening of Te in stage-2.Stage-3 rebound was much weaker than in rats thus not analyzed in this part of study.2,in 8 +/-mice,the stage-1 HBR was similar to that of +/+ mice. However,4 of them failed to exhibit stage-2 "delayed frequency increase".The other 4 exhibited "delayed frequency increase" as in +/+ mice.3,Following systemic application of the NMDA receptor antagonist MK-801,the stage-2 "delayed frequency increase" was not observed in either +/+ or +/-mice.This part of study suggested that the HBR was defective in at least part of MECP2 knock-out mice,which might contribute to the respiratory failure observed in patients suffering from Rett syndrome.5.Pre-electrical stimulation and microinjection of MK-801 at vl-pons modulated the Hering-Breuer reflexExperiments were performed on 38 adult Wistar rats under urethane anesthetization,gallamine triethiodide paralysis and artificial ventilation.Phrenic nerve discharge was recorded as central respiratory output.HBR was simulated with electrical vagal stimulation at the following parameters:intensity 20-40μA （1.5 threshold）,frequency 80 Hz,pulse duration 0.1 ms,stimulation time 60 sec. Parameters for vl-pons stimulation:intensity 50-80μA,frequency 80 Hz,pulse duration 0.3 ms,stimulation time 20 sec.Immediately following vl-pons stimulation, another vagal stimulation caused stronger HBR than control,with stage-1 Te increased,adaptation weakened,and stage-3 rebound weakened too.Following microinjection of MK-801 at the vl-pons,another vagal stimulation caused weaker HBR than control,with stage-1 Te shortened,adaptation more complete,and stage-3 rebound weakened.This part of study supplied another proof that the vl-pons modulates the Hering-Breuer reflex.This part of research has been published in Adv Exp Med Biol,vol.605,2008).更多还原