摘要
目的：呼吸运动是一种受呼吸中枢调节的节律性的活动。中枢化学感受器(central chemoreceptors)感受细胞外pH和CO2的浓度变化,将化学信号转化电信号,传导给呼吸中枢的运动神经元,完成对呼吸运动的调节。中枢化学感受器主要存在于延髓腹外侧区(ventrolateral medulla, VLM),近几年下丘脑外侧区(lateral hypothalamus, LH)的Orexin神经元在中枢化学感受中的作用也逐渐引起大家的关注。目前,中枢化学感受器感受细胞外H+浓度变化的离子通道机制还不明确,也一直是呼吸生理学研究的重点。1997年,Waldmann,R.克隆了第一个酸敏感离子通道(acid-sensing ion channels, ASICs),这是一类H+门控的阳离子通道。研究发现,ASICs参与机体许多生理和病理生理过程,如：学习记忆,痛觉、视觉、听觉、味觉的感受,脑缺血损伤等,但是ASICs在呼吸的中枢调节中的作用却知之甚少。本课题假设：1ASICs在VLM和LH都有表达并参与对呼吸的调节；2.LH的Orexin神经元通过其胞膜上的ASICs通道感受细胞外pH值的变化,并调节呼吸运动。中枢的化学感受调节是快速调节维持机体酸碱平衡、内环境稳定的关键环节。中枢性呼吸衰竭、中枢性呼吸节律紊乱、先天性中枢性肺换气不足综合征等呼吸中枢调节紊乱的疾病对机体造成的危害是致命的,在
一些肺和呼吸道病变所引发的疾病如慢性阻塞性肺疾病、阻塞性睡眠呼吸暂停低通气综合征等疾病的病理过程中也伴有呼吸中枢调节功能的异常。因此,探讨呼吸中枢调控机制不仅是呼吸生理学的理论突破,对呼吸系统疾病的病理过程研究和临床预防与治疗也具有重要的意义。
方法：1.实验用新生(1～5d)和成年(6～8w)SD大鼠,雌雄不计,取延髓部位,用免疫组织化学ABC方法、免疫荧光双标、confoca1、westernblot和图像分析技术,观察和比较ASIC1和ASIC2a亚单位在大鼠延髓和下丘脑的表达,Orexin A神经元在下丘脑的表达,Orexin A神经纤维在延髓的投射,Orexinl型受体(orexin type1receptor, OX1R)在延髓的表达以及ASIC1,ASIC2a亚单位与神经纤维丝-H的共表达、ASIC1与ASIC2a亚单位的共表达、ASIC1与Orexin的共表达。2.成年(6～8w)SD大鼠腹腔麻醉后,应用电生理实验,采用脑立体定位和中枢核团微量注射技术,首先观察在延髓腹外侧区(VLM)和下丘脑外侧区(LH)微量注射不同pH值的人工脑脊液对膈神经放电活动、血压和心率的影响。然后在LH微量注射ASICs的非特异性阻断剂Amiloride或ASICla选择性阻断剂PcTX1后,观察酸化LH对呼吸和心血管系统的调节作用。观察在侧脑室微量注射Orexin A对膈神经放电活动、血压和心率的影响。3.成年(6～8w)SD大鼠腹腔麻醉后,应用电生理实验,采用脑立体定位和中枢核团微量注射技术,在LH注射酸化人工脑脊液激活ASICs,同时在延髓的孤束核微量注射SB408124阻断OX1R后,观察膈神经放电活动、血压、心率的变化。4.采用脑立体定位和中枢核团微量注射技术,成年(6-8w)SD大鼠的LH区微量注射Orexin-SAP特异性损毁该核团的Orexin能神经元,观察大鼠摄食和体重的变化；应用电生理实验观察核团损毁后,动物的基础呼吸频率、血压和心率的变化,以及酸化刺激LH区对心血管呼吸系统的调节。
结果
1. ASIC1和ASIC2a在成年和新生SD大鼠延髓的表达：(1)免疫组化的结果显示：ASIC1和ASIC2a免疫反应阳性细胞在成年鼠的延髓有广泛分布：ASIC1和ASIC2a免疫反应阳性细胞在第四脑室旁的中间核(intercalated nuclus of medulla, In)以及延髓背侧的楔束副核(external cuneate nuclus, ECu)都有散在分布,在延髓的头端腹外侧区则有着广泛分布。ASIC1和ASIC2a免疫反应阳性细胞在新生鼠延髓的表达略有不同：ASIC1免疫反应阳性细胞主要分布在VLM区,在延髓背侧的ECu也有散在表达；ASIC2a免疫反应阳性细胞也主要分布在VLM区,但是在延髓背侧未观察到ASIC2a免疫反应阳性细胞。在VLM区,成年鼠组的ASIC2a免疫反应的相对光密度(relative optical density, ROD)和阳性细胞数值均低于新生鼠组的(p<0.001,n=6)；成年鼠组的ASIC1免疫反应的ROD值高于新生鼠组(p<0.001,n=6)而细胞计数统计结果显示成年鼠组的ASIC1免疫反应阳性细胞数少于新生鼠组(p<0.001,n=6)。(2)应用Western blot的方法：在成年鼠和新生鼠延髓都检测到ASIC1和ASIC2a蛋白的表达,ASIC1和ASIC2a在成年鼠组延髓的蛋白含量均低于新生鼠组(p<0.01,p<0.001,n=6)。(3)应用免疫荧光双染和免疫荧光共聚焦实验方法,在VLM区观察到了ASIC1和ASIC2a与神经纤维丝-H的共表达,以及ASIC1和ASIC2a的共表达。
2. ASIC1和ASIC2a在成年SD大鼠下丘脑的表达：免疫组织化学实验结果显示：ASIC1和ASIC2a两个亚单位在成年SD大鼠的下丘脑广泛表达：ASIC1和ASIC2a免疫反应阳性细胞在下丘脑背侧区(dorsal hypothalamus area, DA)和下丘脑外侧区(LH)都有分布,但是两者的分布存在差异性。细胞计数结果显示：LH区的ASIC1免疫反应阳性细胞数多于DA区(p<0.001,n=7),ASIC2a免疫反应阳性细胞数在DA区多于在LH的分布(p<0.001,n=7)；在DA区ASIC1免疫反应阳性细胞数少于ASIC2a (p<0.01,n=7),LH区ASIC1免疫反应阳性细胞数多于ASIC2a(p<0.001,n=7)。对ASIC1和ASIC2a免疫反应阳性细胞的相对光密度(ROD)值的分析未发现两者在DA区和LH区存在明显差异。
3.在VLM区和LH区微量注射酸化人工脑脊液对呼吸及心血管系统的调节作用：(1)在SD大鼠的单侧VLM区注射不同酸化程度的人工脑脊液(0.1μ1),pH值分别为pH7.4、6.5、6.0、5.5、5.0、4.5,与pH7.4组相比,观察到pH6.5和pH6.0的人工脑脊液可以增加膈神经放电强度(p<0.05,n=12),对呼吸频率、血压、心率均没有明显的影响。(2)在SD大鼠的单侧LH区注射不同酸化程度的人工脑脊液(0.1μ1),pH值分别为pH7.4、6.5、5.5、4.5,与pH7.4组相比,观察到pH6.5的人工脑脊液可以明显增大膈神经放电强度(p<0.001,n=6),对平均动脉压、心率和呼吸频率没有明显影响。(3)在LH区微量注射ASICs非特异性阻断剂Amiloride (10mM,0.1μl)或ASIC1a特异性阻断剂PcTX1(10nM,0.1μl)之后立刻注射pH6.5人工脑脊液(0.1μ1),明显阻断了pH6.5人工脑脊液的增大膈神经的效应(p<0.001,n=6),而单独注射Amiloride或PcTX1对呼吸没有明显抑制作用。各个用药组对呼吸频率、血压、心率没有明显的影响。
4. Orexin A对呼吸和心血管系统的调节作用：(1)免疫组化结果显示：Orexin A免疫反应阳性纤维存在于延髓的孤束核(nucleus tractus solitary, NTS),并且在NTS也观察到OX1R免疫反应阳性神经元的存在。(2)单侧侧脑室微量注射不同浓度的Orexin A(10、100、10001μg/ml,5μl),剂量依赖性地增强膈神经放电强度、升高动脉血压、加快心率,但对呼吸频率没有影响。三个浓度组的膈神经放电强度分别与生理盐水对照组相比都有统计意义(p<0.05,p<0.01,p<0.001,n=6)。100、1000μg/ml两个浓度组的平均动脉压与生理盐水对照组相比有统计意义(p<0.05,p<0.01,n=6)。100、1000μg/ml浓度组的心率与生理盐水对照组相比有统计意义(p<0.01,p<0.01,n=6)。
5. ASIC1与Orexin A在LH区的共表达,以及它们在呼吸调节中的相互作用：(1)应用免疫组化实验在LH区观察到Orexin A免疫反应阳性细胞。应用免疫荧光双染和免疫荧光共聚焦实验方法,在LH区观察到ASIC1亚单位与Orexin A的共表达。(2)在单侧LH区注射pH6.5人工脑脊液(0.1μl),同时在同侧NTS注射SB408124(0.1μl)与注射pH7.4的人工脑脊液(0.1μl)相比,明显阻断了pH6.5人工脑脊液的增加呼吸强度的效应(p<0.01,n=6),而单侧LH区注射pH7.4的人工脑脊液,同时同侧NTS注射SB408124对呼吸强度没有影响。(3)双侧LH区注射Orexin-SAP(0.43mg/ml,400nl/侧)两周后,观察到损毁组大鼠的体重明显下降,应用尼氏染色和免疫组化方法,观察到两周后LH区的Orexin A神经元数量明显减少,仅有少量残存的Orexin A神经元。应用电生理的实验技术,观察到损毁组大鼠与Blank-SAP对照组相比平均动脉压下降,心率减慢(p<0.01,n=6),呼吸频率没有变化。在损毁组大鼠的单侧LH区注射pH6.5人工脑脊液,对膈神经放电强度不再有兴奋作用。
结论：
1.AISC1和ASIC2a亚单位在SD大鼠的延髓呼吸相关核团VLM区和下丘脑的LH区都有表达。随着发育成熟,ASIC1和ASIC2a亚单位蛋白表达量在成年鼠延髓的比在新生鼠减少,虽然ASIC1细胞数量减少,但是ASIC1免疫反应阳性细胞的相对光密度值(ROD)增加,提示成年鼠延髓单个细胞上的ASIC1表达增加。ASIC2a的ROD和细胞数量在成年鼠VLM区都比在新生鼠减少。ASICs亚单位表达量的变化可能与不同发育阶段中枢对化学刺激的敏感性不同有关。ASIC1和ASIC2a在LH区都有表达,但是以ASIC1亚单位为主。提示下丘脑的LH区可能是新发现的中枢化学敏感区。实验结果为探讨ASICs在呼吸中枢调节中的作用提供了形态学基础。
2.在延髓VLM区和下丘脑LH区适度的酸化刺激都可以增加呼吸强度。观察到酸化LH区引起的呼吸兴奋效应更明显,其有效刺激的酸化程度为pH6.5,与ASIC la的pHo.5相符。ASICs的非特异性阻断剂Amiloride和ASIC1a的特异性阻断剂PcTX1几乎都可以完全阻断酸化LH区引起的兴奋呼吸的效应。在生理pH值范围内,单独阻断LH区的ASICs通道对呼吸并没有抑制作用。提示ASICs不仅在LH区存在,并且感受局部的化学变化,然后调节呼吸活动,在此调节过程中ASIC1a亚单位起主要作用。
3. ASIC1在下丘脑LH区的Orexin能神经元上表达。LH区的Orexin(?)神经元损毁后,酸化刺激LH区不再有兴奋呼吸的作用。提示LH区的Orexin(?)神经元可以感受局部的化学变化,可能是一类新发现的中枢化学感受器。而ASICs在下丘脑对呼吸的调节作用正是通过Orexin(?)神经元实现的。侧脑室微量注射外源性的OrexinA可以增加呼吸强度,Orexin能神经纤维和OX1R在延髓NTS有表达。提示Orexin有可能通过向NTS的投射发挥对呼吸的调节作用。在NTS阻断OX1R可以抑制酸化LH区增加呼吸强度的效应,而单独阻断NTS的OX1R对呼吸活动没有影响。说明酸化刺激LH区,同时阻断NTS的OX1R所产生的效应并非两种相反作用的相互抵消,进一步说明酸化LH区是通过兴奋Orexin神经元,释放Orexin与延髓NTS的OX1R结合,发挥兴奋呼吸的效应。
Purpose Respiratory movementknown as a kind of hythmic aetivity can be regulated by ptratory canter. The central respiratory chemoreceptors (CRCs) sense the changes of extrocellular pH and the concentration of CO2, and transduct the chemical signal the electrical signal to excite the motoneuron of respiratory center. The CRCs is mainly distributed in ventrolateral medulla (VLM). Recenty, the Orexinergic neurons in the lateral hypothalamus (LH) have been noticed for its acid-sensing properties. However, the ionic mechanism of acid sensing of CRCs is unclear.1997, one of the acid sensing ion channels (ASICs) subunits which is belong to a family of proton-gating cation channel was been cloned firstly. Researches show that ASICs are involved in several physiological and pathophysiological functions such as learning and memory, nociception, sour taste, hearing and visual perception, acidosis-mediated neuronal injury. However, the function of ASICs on central regulation of respiration has not been addressed. We hypothses:1. ASICs expressed in VLM and LH, and taken part in the central regulation of breathing.2. The central mechanism of acid-sengsing ion channnels (ASICs) on respiratory regulation is that ASICs channels on the membrane of Orexin containing neurons percepted the decreace of excellular pH to excite the neurons and then respiroration. There are many fatal diseases such as central respiratory failture, central respiratory rhythm disturbance, congenital central hypoventialation syndrome (CCHS), which are due to dysfunction of respiratory center. Additionally, many pulmonary and airway diseases such as chronic obstructive pulmonary disease (COPD), obstructive sleep apnea syndromes (OSAS) also complicated with the abnormality of respiratory center. So this work is not only meanful in physiology but also in clinical medicine. Maybe ASICs is a pharmacological target for disease of respiratory system.
Methods1.Experiments were performed on neonatal (1～5d) and adult (6～8w) SD rats. Expression and distribution of ASIC1-like-immunoreactivity (ASIC1-like-ir) and ASIC2a-like-immunoreactivity (ASIC2a-like-ir) in the medulla and hypothalamus, Orexin A-like-immunoreactivity (OXA-like-ir) in hypothalamu and medulla, Orexin1receptor-like-immunoreactivity (OX1R-like-ir) and the co-location of ASIC1/AISC2a with Neurofiber-H, ASIC1with ASIC2a, ASIC1with Orexin A in the medulla or hypothalamus were observed and compared by using immunohistochemistry ABC method, double immunofluorescence technique, confocal, westernblot and software of ImageMeasure.2. Adult (6-8w) SD rats were anesthetized intraperitoneally. The rats were placed on a stereotaxic frame in a prostrate position and the injection site on the brain surface was exposed. Finally, the phrenic nerve discharge (PND), respiratory rate (RR), artery blood pressure (BP) and heart rate (HR) was recorded pre-and post-aCSF with different pH microinjected into ventrolateral medulla (VLM) or lateral hypothalamus (LH) respectively. The effective dose was routinely given together with one of the test agents to the LH, which included a nonselective ASICs inhibitor, amiloride or a selective ASIC1a inhibitor, tarantula venom PcTX1.The co-microinjection was performed as follow: the inhibitors were administrated first and then the effective pH, and there is no time interval between them. The effect of post-Orexin administration into lateral ventricle (LV) was also observed.3. Adult (6-8w) SD rats were anesthetized intraperitoneally, the phrenic nerve discharge (PND), respiratory rate (RR), artery blood pressure (BP) and heart rate (HR) was recorded pre-and post-the simultaneous microinjection of the aCSF with effective pH into LH and SB408124into the nucleus tractus solitarius (NTS).4. The Orexin containing neurons of LH were lesioned by microinjection of Orexin-SAP. Two weeks after the treatment, weight of the dhult was measured and the respiratory rate (RR), artery blood pressure (BP) and heart rate (HR) was recorded. The effect of microinjection of the aCSF with effective pH on PND was also observed.
Results
1. The expression of ASIC1and ASIC2a positive neurons in medulla of adult and neonatal SD rats:(1) The ASIC1and ASIC2a positive neurons mainly expressed in VLM of medulla and slightly in the dorsal area of medulla (intercalated nuclus of medulla, In; external cuneate nuclus, ECu) in the adult. The location of ASIC1positive neurons in the neonatal was similar with that in the adult. But the ASIC2a positive neurons merely expressed in VLM of medulla in the neonatal. Relative optical density (ROD) and cell count of ASIC1and ASIC2a positive neurons in VLM of adult and neonatal rats was measured. ROD of ASIC1positive neurons in VLM of adult rats was higher than that of the neonatal rats. But the ROD of ASIC2a in adult rats was less than in neonatal rats (p<0.001, n=6). Cell count of ASIC1and ASIC2a positive neurons in VLM of adult rats was less than in neonatal rats (p<.001, n=6).(2) Western blot analysis of ASIC1and ASIC2a expression in the medulla of adult and neonatal SD rats showed that ASIC1/ASIC2a immunoreactivity was present in medulla. The relative levels of ASIC1and ASIC2a protein expression were less in the adult rats. Data were normalized by the mean value for control (p<0.01, p<0.001, n=6).(3) ASIC1and ASI2a summits were co-espression with Neurofilament-H in VLM of adult SD rats. ASIC1and ASI2a were also co-located with each other.
2. The expression of ASIC1and ASIC2a positive neurons in hypothalamus of adult SD rats:Both of ASIC1-like-ir neurons and ASIC2a-like-ir neurons ditributed in dorsal hypoth area (DA) and in the LH. But the expression of them was different. The cell number of ASICl-like-ir neurons was larger in LH than in DA (p<0.001, n=6). Contrarily, ASIC2a-like-ir neurons were mainly in DA (p<0.001, n=6). Additionally, there were more ASIC1-like-ir neurons than ASIC2a-like-ir neurons in LH, and more ASIC2a-like-ir neurons in DA (p<0.01, p<0.001, n=6).
3. Unilateral microinjection of acidosis aCSF in VLM and LH enlarged PND and ASICs antagonist inhibited the accretion effect:(1) The chemical stimulation of VLM using aCSF with different pH (pH=7.4,7.0,6.5,6.0,5.5,5.04.5,0.1μl) showed that pH6.5and pH6.0resulted in a greater increase in iPND than pH7.4(p<0.05, n=12).(2)The chemical stimulation of LH using aCSF with different pH (pH=7.4,6.5,5.5,4.5,0.1μl) showed that pH6.5resulted in a greater increase in iPND than pH7.4(p<0.001, n=6).(3) Co-microinjection into LH of the nonselective ASICs inhibitor, amiloride (10mM,0.1μl) or selective ASIC1a inhibitor, PcTX-1(10nM,0.1μl) with pH6.5almost totally blocked the iPND enhancement promoted by pH6.5(both p<.001, n=6). And singly administration of the two inhibitors did not result in alterations in PND. All the gradient of pH and agents did not elicit changes in RR, MAP and HR.
4. The effect of Orexin A on the regulation of cardiorespiratory system: Expression of Orexin A-like-ir fibers and OX1R-like-ir neurons were found in the nucleus tractus solitarius (NTS). Unilateral microinjection of Orexin A at different concentrations (10,100,1000μg/ml,5μl) into LV resulted in augmentation of PND, BP and HR. The effects were dose-dependent. The acceleration of PND at all concentrations was significant (p<0.05, p<0.01, p<0.001vs N.S., n=6). The increase of RR at the three concentrations was not significant. The acceleration of HR was notable at100,1000μg/ml concentrations (p<0.01, p<0.01vs N.S., n=6). The increase of MAP was significant at100,1000μg/ml concentrations (p<0.05, p<0.01vs N.S., n=6).
5. Co-expression of ASIC1and Orexin A in LH, and the synergistic effect of them in regulation of respiration:(1) Orexin A-like-ir neurons were distributed in LH and co-located with ASIC1.(2) Simultaneous microinjection of SB408124in NTS and aCSF with pH6.5in LH had no effect on PND. Simultaneous microinjection of SB408124in NTS and aCSF with pH7.4in LH didn't decrease amplitude of PND.(3) There was significant loss of Nissl bodies and Orexin neurons in the LH of Orexin-SAP-treated rats relative to the blank-SAP-treated rats. Few residual Orexin neurons remain in Orexin-SAP-treated rats, two weeks after treatment. There was decrease in MAP and HR in LH lesion group (both p<0.01v.s. blank-SAP treated group, p<0.01, p<0.05v.s. control, respectively), but no change in RR. And microinjection of aCSF with pH6.5in LH no longer increased in LH lesioned rats.
Conclusion
1. ASIC1and ASIC2a subunits are expressed in the VLM and LH. There is a decrease of ASICs peptides in medulla as mature. The ROD and cell count of ASIC2a-like-ir both reduced in VLM of adult SD rats compared to the neonatal rats. Interestingly, there is a contradictory finding in ASIC1:cell count downgrade, but rod upgrade in adult VLM. Perhaps, the difference expression of ASICs means the distinct sensitivity of central nerve system to chemical stimulation. In LH, ASIC1and ASIC2a are found, especially ASIC1. The result of immunohistochemistry laid the groundwork for the research on the role of ASICs on the central regulation of respiration.
2. Acidosis in VLM and LH excited breathing. The distinguished effect is elicited by stimulating LH with artificial cerebrospinal fluid with pH6.5. The effective dose is in accordance with the pHo.sof ASIC1a. And the stimulating effect of pH6.5on respiration was almost completely blocked by ASICs inhibitors (nonselective inhibitor, Amiloride or ASIC1a selective inhibitor, PcTX1), in LH. The result show ASICs take part in central regulation of respiration, and ASIC1a is highlighted.
3. ASIC1co-located with orexin in LH, and lesion of Orexin neurons distressed the excitation of respiration provoked by ASICs, additionally, microinjection of Orexin A into LV increased the amplitude of respiration, which show that ASICs contribute to the excitation of respiration via neurotransmitters (Orexin A). Orexin-like-ir fiber and OXiR-like-ir neurons are located in NTS, so we blocked OX1R of NTS, and observed that the effect of acidosis in LH is inhibited. But blocked OX1R of NTS alone had no effect on respiration. This further proofed that ASICs lying in LH via orexin through medulla pathway to regulate respiration.
引文
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