脑桥呼吸调整中枢对黑伯反射和低氧呼吸反应的调控作用

发布时间：2018-04-21 06:30

本文选题：vl-pons + dl-pons　；参考：《山东大学》2008年博士论文

【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%O_2-92%N_2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。
[Abstract]:The main structure of the pons breathing adjustment center consists of two structures of the lateral lateral pontine (dl-pons) and the lateral ventral pontine (vl-pons). The main structure of the.Dl-pons is the KF nucleus and parobulobial nucleus (PB). Vl-pons mainly refers to the two-way fiber connection between.Dl-pons and vl-pons in the A5 region. It is known that the pontine is the location of the respiratory adjustment center, and the respiratory rhythm is initiated. The study is divided into five parts: the first part of the study is divided into five parts: the first part of the study is to observe some neurotransmitter antagonists in dl-pons and vl-pons to the black eye reflex (lung distraction reaction) The second part observed the effects of dl-pons and vl-pons on hypoxic respiration; the third part observed the axon projection of dl-pons and vl-pons neurons to the brainstem respiratory reflex (hypoxia respiratory response and black Bo reflex) related neurons by nerve fiber projection tracing and immunohistochemical technique; fourth partial views were observed. The effect of NMDA receptor on the mouse reflex was observed, and the fifth part observed the regulation of electric stimulation of vl-pons on the black Bo reflex and the role of NMDA receptor in the process.
1, the effects of NMDA, GABA_A and AMPA receptor antagonists on the dl-pons and vl-pons responses to the black reflex.
The experiment was carried out on 35 adult SD rats with an intraperitoneal injection of ethyl carbamate, pancuronium bromide muscle relaxation and artificial ventilation. The electrical stimulation of the phrenic nerve was used as an observation index. Electric stimulation of the vagus nerve was used to simulate the HB reflex. The electrical stimulation parameters were the intensity of 20-80 Mu, the frequency 80 Hz, the width of the wave width of 0.1 ms, and the stimulation time of 60 sec. sustained electric stimulation. The first appearance of the nerve was the intensity dependent apnea, and the phrenic discharge stopped in the expiratory phase (Stage-1). With the continuation of the stimulation, the rhythmic PHRENIC DISCHARGE reappeared and the respiratory frequency was gradually accelerated to the baseline level still lower than the baseline level (Stage-2). The transient respiratory frequency was accelerated and higher than the stimulus after the prickling stop. The pre baseline level, mainly caused by shortening of the expiratory time, is called PR (Stage-3). NMDA receptor blockers D-2- amino -5- phosphonate (D-AP5,10 mM), GABA receptor blocker (bicuculline, BIC, 5) and receptor blockers are microinjected in dl-pons and vl-pons, respectively. -nitroquinoxaline-2,3-dione, 10 mM), the injection dose of 20-50nl. was used to reflect the change of the black Bo reflex after injection. The results were as follows: (1) the microinjection of AP5 in dl-pons could enhance the respiratory inhibition effect of the HB reflex, showing the decrease of respiratory frequency in the first second stage of the HB reflex and the prolonged exhalation time. The amplitude and the shortening of the inspiratory time were all increased; the third stage of the respiration rate was still lower than the baseline, the phenomenon of the jump disappeared, and the increase in the breathing time increased obviously at this stage, and there was a significant difference in the above changes. (2) the microinjection of vl-pons could reduce the respiratory inhibition effect of the black root reflex, which was manifested by the black root reaction. The respiratory frequency decreased in the first second stages, and the expiratory time was reduced. The third stage of the black Bo reflex was higher than the baseline, but the changes were significantly lower than the previous control. (3) the microinjection of BIC in dl-pons could weaken the respiratory inhibition effect of the black root reflex, which was characterized by black uncle. The respiratory frequency decreased in the first stage, the duration of expiration and the shortening of inspiratory time decreased, the duration of expiration and the shortening of inspiratory time decreased in the second stage, and the shortening of expiratory time decreased in the third stage, while the inhalation time was longer than the second stage, but it was still lower than the baseline level. The third stage of respiratory frequency is higher than the baseline level, and there is a reverse jump phenomenon, which weakens but has no significant difference with the previous picture. (4) the microinjection of vl-pons can enhance the respiratory inhibition effect of the black light reflex, which shows the decrease of the respiratory frequency in the first second stage of the black Bo reflex and the prolongation of the expiratory time. There was a significant difference in the shortening of the degree and the inspiratory time, and there was a significant difference in the change of the respiration rate in the third stage of the black light reflex, which was higher than the baseline level, and there was a reverse jump phenomenon. (5) the inhibitory effect of the phrenic discharge in the three stages of the black Bo reflex after the dl-pons microinjection of AMPA receptor blocker CNQX. There should be no significant change. (6) after vl-pons injection of CNQX, the inhibitory effect of phrenic nerve in three stages of black reflex has not changed significantly.
These results suggest that the synaptic transmission mediated by the NMDA receptor and the GABA receptor in dl-pons and vl-pons participates in the regulation of the intensity of the reflexes, and the AMPA receptor does not affect the process.
2, the effects of NMDA, GABA_A and AMPA receptor antagonists on dl-pons and vl-pons on hypoxic respiratory responses.
The experiment was carried out on 35 adult SD rats. Hypoxic ventilation was 8%O_2-92%N_2 mixed gas and hypoxic ventilation time was 30-50 seconds. With phrenic discharge as the observation index, NMDA receptor blocker D-AP5, GABA receptor blocker BIC, AMPA receptor blocker CNQX, were injected into dl-pons and vl-pons, and the hypoxia was observed before and after injection. The changes in phrenic discharge in gas show that the phrenic discharge is increased first, the amplitude of the phrenic nerve increases, the expiratory time and the expiratory time are shortened, which is called acute hypoxia breathing response. The frequency of phrenic discharge can be reduced and lower than the hypoxic stimulation after the recovery of normal ventilation. Baseline level, this phenomenon is called hypoxic respiratory frequency (PHFD). Before microinjection of microinjection as a pre control, (1) there is no significant change in phrenic discharge in acute hypoxia after microinjection of D-AP5 in dl-pons, but the decrease in frequency of phrenic discharge in PHFD phase decreases, that is, the phenomenon of PHFD is weakened. (2) after vl-pons microinjection of D-AP5, There was no significant change in the acute hypoxic reaction and PHFD PHRENIC DISCHARGE. (3) after dl-pons microinjection of BIC, the amplitude of phrenic discharge and the shortening of the expiratory time increased in the acute hypoxia response period, and there was no significant change in the phrenic discharge at PHFD stage. () after the vl-pons microinjection of BIC, the phrenic deity in the acute hypoxia reaction period was not changed. There was no significant change in discharge, but the decrease of respiratory frequency decreased in PHFD phase. (5) after dl-pons microinjection of CNQX, the frequency of phrenic discharge in acute hypoxia reaction was not significantly changed, but the shortening of expiratory time decreased, and the amplitude of shortening of inhalation time increased in the.PHFD PHRENIC DISCHARGE frequency decrease. (6) in vl-pons After microinjection of CNQX, the shortening of the inhalation time in the acute hypoxia response period increased, but the frequency of phrenic discharge and the expiratory time had no significant changes in the amplitude of the decrease in the frequency of phrenic discharge in the.PHFD phase.
These results suggest that both dl-pons NMDA receptor, GABA receptor and AMPA receptor are involved in the regulation of hypoxia respiration. The NMDA receptor mainly affects the decrease of respiratory rate after hypoxia, and the GABA receptor is mainly affected by the acute hypoxia response. The dl-pons AMPA receptor is more complex. It affects both the acute hypoxia response and the respiratory rate after hypoxia. The reduction of.Vl-pons GABA receptor and AMPA receptor is involved in the regulation of hypoxia respiration. The GABA receptor mainly affects the decrease of respiratory rate after hypoxia. The AMPA receptor affects both the acute hypoxia response and the decrease of respiratory frequency after hypoxia, and the NMDA receptor does not participate in the regulation of the reaction.
3, Vl-pons is related to the fiber connections between the peripheral chemoreceptor reflex and the black reflex function related neurons in the medulla oblongata.
The experiment was carried out on 8 adult SD rats. Intraperitoneal injection of phenobarbital (50 mg/kg) anesthesia. Under the aseptic operation, the dl-pons KF nucleus was injected with Biotin dextran with pressure injection or microelectrophoresis. The pressure injection amount was 20-30 NL (10%). The microelectrophoretic injection was used as anode direct current, 5 mu A, 15-30 minutes. The animals survived for 10 days. After 8% hours of hypoxia stimulation or end expiratory positive pressure ventilation (PEEP).2-3 hours, the intraperitoneal injection of ethyl carbamate was administered to animals, left ventricular intubation, fixation, and brain stem section (50 m). Brain slices were cultured with Rabbit anti c-Fos polyclonal antibody (one anti) and biotin's Sheep anti rabbit IgG (two anti), and then ABC-DAB method was used to deal with brain slices. It was found that the fibers from the dl-pons KF nucleus projected into the known medullary respiratory related structures. In addition, single fiber tracing showed that multiple.C-Fos neurons dominated by KF nucleus neurons were mainly distributed in the medial and ventral subnuclei of the nucleus of the nucleus of the solitary tract, the lateral reticular nucleus of the ventrolateral medulla, the nucleus and the surrounding area. Domain. The labelled fiber terminals from dl-pons overlap with the regions of the above c-Fos immunoreactive neurons, suggesting that there may be synaptic connections between the dl-pons projecting fiber and the neurons involved in the peripheral chemoreceptor reflex and the black Bo reflex in the medulla.
4, the characteristics of black reflex in normal wild type and MECP2 knockout mice and the regulation of NMDA receptors on them.
Rett's syndrome (Rett syndrome) is a nervous system disease that seriously affects children's psychomotor development. Children behave normally in 6 to 18 months, followed by rapid degeneration and developmental retardation, and breathing abnormalities such as intermittent breathing. About 26% of patients with Rett syndrome die from respiratory dysfunction. The disease may be related to the mutation of the methyl-CpG binding protein 2 (MECP2) gene. We speculate that the respiratory abnormalities manifested by the sick child may be related to the abnormality of the abnormality of the reflexes. In the fourth part of this study, the control of the common wild type (+ / +) mice and MECP2 knockout mice (+ / -) and the regulation of the reflex by the NMDA receptor The experiment was conducted on 13 anesthetized, muscular and artificially ventilated adult mice. An electrical stimulation of the phrenic nerve discharge was used as an indicator of respiratory observation and the stimulation of the central terminal of the vagus nerve to simulate the HB reflex. The results were as follows: (1) in a + / + mouse, the first occurrence of the continuous electrical stimulation of the vagus nerve was the intensity dependent apnea, and the phrenic discharge stopped in expiratory phase. (Stage-1). With the continuation of the stimulation, the rhythmic PHRENIC DISCHARGE reappears and the respiratory frequency increases gradually (Stage-2). At the end of the 1 minute stimulus, the expiratory time is shorter than the baseline level before the vagal stimulation, and the respiratory frequency is higher than the baseline level (that is, the "delayed frequency increase"). (2) the vagus nerve is stimulated in 8 + / - mice. The results showed that the Stage-1 respiration reaction and the + / + mice showed no distinct difference in.Stage-2 respiration. 4 + / - mice showed Stage-2 "delayed frequency increase", similar to that of + / + mice. However, in the remaining 4 + / - mice, the phenomenon was not observed. (3) in + / + mice and "delayed frequency increase" + / - mice, abdominal cavity The "delayed frequency increase" of the Stage-2 respiratory response after the injection of the NMDA receptor antagonist MK-801 all disappeared. The experiment showed that the Rett syndrome may be associated with the abnormality of the black abbo reflex.
5, electrical stimulation of dl-pons and microinjection of NMDA receptor blocker MK-801 into vl-pons can regulate the reflex of the brain.
This part of the experiment was carried out on 38 adult Wistar rats. The female and male were not confined to the abdominal anesthesia, with three iodide quaternary ammonium phenol muscle relaxant, artificial ventilation, and phrenic discharge as the observation index. Electric stimulation of the vagus nerve stimulates the electrical stimulation parameters of.Vl-pons: 50-80 mu A, 80 Hz, wave width 0.3 ms, stimulation time 20 sec, electric stimulation of vagus nerve. Parameters: 20-40 mu A (1.5 x threshold), 80 Hz, wave width 0.1 msec, stimulation time 60 sec. to electric stimulation of the black beb reflex before vl-pons as the pre control, electrical stimulation of vl-pons in 5 sec to stimulate the vagus nerve to guide the black root reflex, and found that compared with the previous control, the phrenic inhibition effect of the black Bo reflex was enhanced and the respiratory frequency decreased. Prolonged expiratory time, shortened inhalation time, and decreased rebound after stimulation; microinjection.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R332;R748

【共引文献】