内吗啡肽在导水管周围灰质发挥镇痛效应的机制

发布时间：2018-02-01 04:11

本文关键词： 吗啡导水管周围灰质发挥镇痛效应机制　出处：《第四军医大学》2007年博士论文　论文类型：学位论文

【摘要】： 导水管周围灰质(periaqueductal gray,PAG)位于中脑,是哺乳动物脑干内下行痛抑制系统(descending pain inhibitory system,DPIS)内的重要组成。PAG的镇痛作用主要是通过激活其腹外侧区/柱(ventrolateral column of the PAG,vlPAG)内的5-HT能神经元的活性而实现的。vlPAG内的5-HT能神经元兴奋后,可以通过直接或间接的方式抑制脊髓的伤害性感受神经元,从而产生镇痛效应。在PAG内同时存在一个调控5-HT能神经元活性的局部环路。其基本构成如下:PAG内大量存在的γ-氨基丁酸(γ-aminobutyric acid,GABA)能中间神经元可以对5-HT能神经元产生持续性的抑制效应,而阿片类物质通过与表达在GABA能神经元上的μ型阿片受体(MOR)结合,能够抑制GABA能神经元的活性,从而使5-HT能神经元间接激活(脱抑制,disinhibition),最终产生镇痛效应。内吗啡肽(endomorphin,EM)是新近发现的一种内源性阿片肽,也是MOR高选择性的内源性配体。其镇痛效应与吗啡相似,但其副作用却远小于吗啡,在临床镇痛治疗方面有广泛的应用前景。内吗啡肽可以分为内吗啡肽1( endomorphin 1, EM1)和内吗啡肽2(endomorphin 2, EM2)两种亚型。内吗啡肽的胞体在脊髓上位脑区主要分布于下丘脑和孤束核,而纤维则遍布全脑,特别是在PAG的不同亚区内都分布有比较密集的EM能纤维和终末,提示EM可能参与了PAG内功能的调控。但是迄今为止,关于EM参与PAG内镇痛效应机制的研究还未见报道。因此,本论文综合应用当代神经科学研究方法,对以下几个问题进行了探讨: 1. PAG内大量存在的EM能纤维和终末的来源部位在哪里,其来源部位和PAG的不同亚区之间是否存在一定的对应关系? 2. vlPAG是5-HT能DPIS的起源部位,而该部位的EM是否参与了对DPIS局部调控的环路? 3. EM参与DPIS调控的机制是什么? 主要结果: 1.PAG内EM阳性纤维和终末的来源将荧光逆行追踪剂荧光金(Fluoro-Gold,FG)分别电泳入大鼠PAG的不同亚区,通过结合EM1或EM2的免疫荧光组织化学染色技术,我们观察到EM1/FG和EM2/FG双重标记的神经元主要位于下丘脑的不同核团/区域内,孤束核内未见到双标神经元分布。其中,下丘脑结节乳头体区内侧的背内侧核( dorsomedial hypothalamic nucleus , DMH )、DMH及腹内侧核( ventromedial hypothalamic nucleus)之间的中央内侧区(centromedial hypothalamic region,CMH)以及弓状核(arcuate nucleus of the hypothalamus,Arc)内包含了绝大部分双标神经元。它们的分布特点如下: 1)将FG注入vlPAG后,DMH内18.0%的EM1(15.7±6.0)和14.0%的EM2(8.2±2.6)阳性神经元,CMH内15%的EM1(30.7±5.9)和18.6%的EM2(17.2±4.7)阳性神经元以及Arc内10.5%的EM1(6.3±1.9)和12.1%的EM2(5.7±2.0)阳性神经元同时被FG逆行标记; 2)将FG注入PAG外侧区(lateral column of the PAG, lPAG)后,DMH内14.8%的EM1(12.2±4.3)和10.8%的EM2(6.7±2.7)阳性神经元,CMH内11.1%的EM1(23.7±7.9)和10.3%的EM2(9.0±3.2)阳性神经元以及Arc内9.2%的EM(15.2±2.6)和5.6%的EM(22.8±1.2)阳性神经元同时被FG逆行标记; 3)将FG注入PAG背外侧区(dorsolateral column of the PAG, dlPAG)后,DMH内7.0%的EM1(6.2±3.3)和6.2%的EM2(3.8±2.3)阳性神经元,CMH内5.3%的EM(111.6±3.6)和3.4%的EM(23.0±1.6)阳性神经元以及Arc内2.0%的EM(11.2±0.8)和2.4%的EM(21.0±0.7)阳性神经元同时被FG逆行标记; 4)将FG注入PAG背内侧区(dorsomedial column of the PAG,dmPAG)后,DMH内8.5%的EM1(7.8±2.9)和7.0%的EM2(4.6±1.8)阳性神经元,CMH内9.5%的EM(119.2±8.1)和5.7%的EM(24.8±2.4)阳性神经元以及Arc内3.5%的EM1(2.0±1.2)和3.9%的EM(21.8±1.8)阳性神经元同时被FG逆行标记。综合以上结果,可以看出:①下丘脑内EM1/FG双标神经元的数量远多于EM2/FG双标神经元的数量(802 vs. 392);②在下丘脑的不同核团和区域中,CMH内的EM/FG双标神经元数目最多(480 EM1/FG;196 EM2/FG),随后是DMH(237 EM1/FG;131 EM2/FG)及Arc(85 EM1/FG;65 EM2/FG);③将FG注入vlPAG后,在下丘脑内的EM/FG双标神经元数目最多,说明下丘脑内的EM能神经元向vlPAG发出最多的投射,随后是lPAG及dmPAG,最少的是dlPAG。以上结果说明:①PAG的不同亚区内的EM能纤维和终末主要来源于下丘脑而不是孤束核;②下丘脑的不同核团和区域与PAG内的不同亚区之间存在明确的对应关系;③来源于下丘脑的EM能纤维和终末可能参与了PAG的多种功能活动,特别是痛觉的调制。 2.对于EM参与vlPAG内痛觉调控环路的机制问题,我们分别从形态学和行为药理学角度进行了论证。 2.1 EM参与vlPAG内痛觉调控环路的电镜观察通过使用电镜双重标记技术,我们观察了大鼠vlPAG内EM1或EM2阳性的轴突终末与谷氨酸脱羧酶( glutamate decarboxylase,GAD)、MOR以及5-HT阳性的神经元胞体及树突之间的突触联系。GAD是GABA合成中的限速酶,也是GABA能神经元的标志,其分布与GABA相一致,GAD阳性结构即可认为是GABA阳性结构。结果显示: 1) vlPAG内含有大量的EM1和EM2阳性的轴突以及轴突终末以及GAD、MOR以及5-HT阳性的胞体和树突存在; 2) EM1以及EM2阳性的轴突终末能够与MOR、GAD及5-HT阳性的胞体和树突形成突触。其中: EM1及EM2阳性轴突终末与MOR阳性胞体和树突主要形成对称性/抑制性突触(EM1:非对称/对称=37.8/62.2×100%;EM2:非对称/对称=41.4/58.6×100%)。 EM1及EM2阳性轴突终末与GAD阳性胞体和树突也主要形成对称性突触(EM1:非对称/对称=17.5/82.5×100%;EM2:非对称/对称=13.6/86.4×100%)。 EM1及EM2阳性轴突终末与5-HT阳性胞体和树突则主要形成非对称性/兴奋性突触(EM1:非对称/对称=58.3/41.7×100%;EM2:非对称/对称=56.7/43.3×100%)。 3) EM2阳性轴突终末间也可形成以非对称性突触(92%)为主的突触联系。以上结果从电镜水平证实了vlPAG内EM能够对GABA能神经元产生抑制效应,且对5-HT能神经元存在直接兴奋效应,而以上的效应可能是通过MOR介导。 2.2 EM参与vlPAG内痛觉调控环路的共聚焦显微镜观察通过使用谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠,我们进一步观察了vlPAG内EM1及EM2阳性纤维和终末以及表达GFP的GAD阳性胞体与MOR或5-HT阳性胞体之间的共存情况。结果显示: 1) GFP阳性胞体与MOR存在广泛的共存关系,几乎所有的GFP阳性神经元(98%)都表达MOR。而EM1或EM2阳性纤维和终末能够与GFP/MOR双标神经元形成密切接触; 2)部分GFP阳性胞体发出纤维与5-HT能神经元形成密切接触,而EM1或EM2阳性纤维和终末又能够与该GFP阳性胞体发生密切接触。以上结果从形态学光镜水平为vlPAG内EM抑制GABA能神经元(通过MOR介导),从而间接兴奋5-HT能神经元的局部环路的存在提供了形态学依据。 2.3 EM参与vlPAG内痛觉调控环路的行为学观察大鼠vlPAG区埋管制备动物模型,经管给予EM、GABAA受体的激动剂和拮抗剂以及MOR受体的拮抗剂,观察了大鼠给药前后对于热刺激反应潜伏期以及机械刺激反应阈值的变化,探讨EM在vlPAG内作用的机制。结果如下: 1)不同剂量的EM1(4、8、16、32 nmol/0.5μl)以及EM2(2、4、8、16 nmol/0.5μl)可以引发实验动物产生明显的镇痛效应。而该效应可以被MOR拮抗剂完全翻转; 2) GABAA受体的拮抗剂与EM能够产生强力的协同镇痛效应,而GABAA受体的激动剂则可以完全抑制EM的镇痛效应。以上结果从行为学角度说明了EM的作用通过抑制GABA能神经元活性实现,而该效应通过MOR介导。总之,上述形态学和行为学实验说明vlPAG内EM可以产生明确的镇痛效应,而该效应主要是通过抑制表达MOR的GABA能神经元的活性,进而使表达GABAA受体的5-HT能神经元摆脱GABA抑制而间接实现的。
[Abstract]:The periaqueductal gray (periaqueductal gray PAG) is located in the midbrain, mammalian brain stem descending inhibitory system (descending pain inhibitory system, DPIS) is an important component in the analgesic effect of.PAG is mainly through the activation of the ventrolateral column (ventrolateral / column of the PAG, vlPAG) and 5-HT in neuron activity the.VlPAG in 5-HT neurons after excitation, can inhibit the spinal cord through direct or indirect way to nociceptive neurons, resulting in analgesic effect.
A 5-HT can control local circuit neuron activity exist simultaneously in PAG. The basic structure is as follows: PAG in the presence of a large number of gamma amino butyric acid (GABA -aminobutyric acid, GABA) interneurons can 5-HT can inhibit the effect of neuronal persistent, and opioid substances with expression in GABA mu opioid receptor neurons (MOR) combined with GABA, can inhibit the neuron activity, so that the indirect activation of 5-HT neurons (antisuppression, disinhibition), eventually produce analgesic effect.
Endomorphin (endomorphin, EM) is a newly discovered endogenous opioid peptide, is the endogenous ligand for the MOR. The analgesic effect similar to morphine, but its side effect is far lower than that of morphine, and has wide application prospect in clinical analgesic treatment. Endomorphins can be divided into endomorphine 1 (endomorphin 1, EM1) and endomorphin 2 (endomorphin 2, EM2 two) subtypes. The soma of endomorphins in supraspinal brain areas are mainly distributed in the hypothalamus and the nucleus of the solitary tract, and fibers throughout the whole brain, especially in the different sub area of PAG is distributed more intensive the EM fibers and terminals, suggesting that EM may be involved in the regulation of PAG function. But so far, the research on EM in PAG in the mechanism of analgesic effect has not been reported. Therefore, the scientific research method of comprehensive application of the contemporary nerve, the following problems are discussed:
In 1. PAG, where is the source of a large number of EM fibers and terminals, and whether there is a certain correspondence between the sources of the source and the different subregions of the PAG?
2. vlPAG is the origin of 5-HT DPIS, and is the EM involved in the loop of local regulation of DPIS?
3. what is the mechanism that EM participates in DPIS regulation?
Main results:
The source of EM positive fibers and terminals in 1.PAG
The fluorescent retrograde tracer Fluorogold (Fluoro-Gold, FG) in different sub regions respectively into the rat PAG electrophoresis technology, via immunofluorescence histochemistry combined with EM1 or EM2, different nucleus / region we observed EM1/FG and EM2/FG double labeled neurons were mainly located in the hypothalamus, the NTS did not see double labeled neurons distributed. The tuberomammillary hypothalamic dorsomedial nucleus of the medial zone (dorsomedial hypothalamic, nucleus, DMH, DMH) and the ventromedial nucleus (ventromedial hypothalamic nucleus) between the central medial area (centromedial hypothalamic region CMH (arcuate) and nucleus of the in arcuate nucleus hypothalamus, Arc) contains most of the double the distribution characteristics of their neurons:
1) FG after injection of vlPAG, DMH 18% EM1 (15.7 + 6) and 14% EM2 (8.2 + 2.6) CMH positive neurons within 15% EM1 (30.7 + 5.9) and 18.6% EM2 (17.2 + 4.7) positive neurons and Arc within 10.5% EM1 (6.3 + 1.9) and 12.1% the EM2 (5.7 + 2) positive neurons were retrogradely labeled with FG;
2) FG into PAG (lateral column of the lateral region of PAG, lPAG, DMH) in 14.8% EM1 (12.2 + 4.3) and 10.8% EM2 (6.7 + 2.7) CMH positive neurons within 11.1% EM1 (23.7 + 7.9) and 10.3% EM2 (9 + 3.2) positive neurons and Arc in 9.2% EM (15.2 + 2.6) and 5.6% EM (22.8 + 1.2) positive neurons were retrogradely labeled with FG;
3) FG was injected into the dorsal lateral region of PAG (dorsolateral column of the PAG, dlPAG DMH), 7% EM1 (6.2 + 3.3) and 6.2% EM2 (3.8 + 2.3) CMH positive neurons within 5.3% EM (111.6 + 3.6) and 3.4% EM (23 + 1.6) positive neurons and Arc 2% EM (11.2 + 0.8) and 2.4% EM (21 + 0.7) positive neurons were retrogradely labeled with FG;
4) FG was injected into the dorsal medial area (PAG dorsomedial column of the PAG, dmPAG DMH), 8.5% EM1 (7.8 + 2.9) and 7% EM2 (4.6 + 1.8) CMH positive neurons within 9.5% EM (119.2 + 8.1) and 5.7% EM (24.8 + 2.4) positive neurons and Arc 3.5% EM1 (2 + 1.2) and 3.9% EM (21.8 + 1.8) positive neurons were retrogradely labeled with FG.
Based on the above results, we can see that the number of EM1/FG double labeled neurons in the hypothalamus of far more than EM2/FG of double labeled neurons (802 vs. 392); in the hypothalamic nuclei and different regions, the number of EM/FG double labeled neurons in CMH (up to 480 EM1/FG; 196 EM2/FG), followed by DMH (237 EM1/FG; 131 EM2/FG) and Arc (85 EM1/FG; 65 EM2/FG); 3 FG after injection of vlPAG, the number of EM/FG double labeled neurons in the hypothalamus in most EM neurons in the hypothalamus that issued the most projecting to vlPAG, followed by lPAG and dmPAG, the least is dlPAG.
The above results showed that: the PAG of different sub region EM fibers and terminals mainly come from the hypothalamus but not the nucleus of the solitary tract; there is a clear relationship between different sub regions of hypothalamic nuclei and different regions and within PAG; the source in the hypothalamus EM fibers and terminals may participate in the a variety of functional activities of PAG, especially in pain modulation.
2. we demonstrate the mechanism of EM participation in the mechanism of the vlPAG internal pain control loop from the morphological and behavioral pharmacology point of view.
2.1 EM participates in the electron microscope observation of the internal pain control loop of vlPAG
By using electron microscope double labeling technique, we observed the rat vlPAG in EM1 or EM2 positive axon terminals and glutamate decarboxylase (glutamate decarboxylase, GAD), between MOR and 5-HT positive neurons and dendrites of the synaptic connections between.GAD is the rate limiting enzyme in the synthesis of GABA, but also a symbol of GABA neurons and its distribution consistent with GABA, GAD positive structures can be considered GABA positive results show that structure:
1) vlPAG contained a large number of EM1 and EM2 positive axons and end of axons, as well as GAD, MOR and 5-HT positive cells and dendrites.
2) EM1 and EM2 positive axonal terminals are capable of forming synapses with MOR, GAD and 5-HT positive cells and dendrites.
EM1 and EM2 positive axon terminals were mainly formed with symmetric and inhibitory synapses with MOR positive cells and dendrites (EM1: asymmetric / symmetric =37.8/62.2 * 100%; EM2: asymmetric / symmetric =41.4/58.6 * 100%).
EM1, EM2 positive axon terminals and GAD positive cell bodies and dendrites also form symmetrical synapses (EM1: asymmetric / symmetric =17.5/82.5 * 100%; EM2: asymmetric / symmetric =13.6/86.4 * 100%).
EM1 and EM2 positive axon terminals and 5-HT positive cell bodies and dendrites mainly form asymmetric / excitatory synapses (EM1: asymmetric / symmetric =58.3/41.7 * 100%; EM2: asymmetric / symmetric =56.7/43.3 * 100%).
3) the synaptic connections with asymmetric synapses (92%) can also be formed between the EM2 positive axons.
The above results confirm that EM can inhibit GABA neurons and have direct excitation effect on 5-HT neurons in vlPAG, and the above effects may be mediated by MOR.
Confocal microscope observation of 2.2 EM involved in the internal pain control loop of vlPAG
Through the use of glutamic acid decarboxylase 67- green fluorescent protein (GAD67-GFP) gene knock in mice, we further investigate the colocalization between vlPAG EM1 and EM2 positive fibers and terminals and GAD positive cell expression of GFP and MOR or 5-HT positive neurons. The results showed that:
1) there is a wide range of coexistence between GFP positive cell bodies and MOR. Almost all GFP positive neurons (98%) express MOR., while EM1 or EM2 positive fibers and terminals can form close contacts with GFP/MOR double labeled neurons.
2) some GFP positive cells emit close contact with 5-HT neurons, while EM1 or EM2 positive fibers and terminals can also be closely related to the GFP positive cell body.
The above results provide a morphological basis for indirectly stimulating the existence of the local loop of 5-HT neurons from the level of morphological light microscopy, which inhibits GABA neurons in vlPAG (mediated by MOR), and thus indirectly excite the existence of the local loop of EM neurons.
2.3 EM participates in the behavioral observation of the internal pain control loop of vlPAG
The rat vlPAG control area buried animal model, and give EM, GABAA receptor agonists and antagonists of MOR receptor antagonist, rats were observed before and after administration for the thermal stimulus response latency and changes in response to mechanical stimulation threshold, to explore the mechanism of EM in vlPAG. The results are as follows:
1) different doses of EM1 (4,8,16,32 nmol/0.5 and L) and EM2 (2,4,8,16 nmol/0.5 L) can induce obvious analgesic effect in experimental animals, and this effect can be completely reversed by MOR antagonists.
2) the antagonist of GABAA receptor and EM can produce a strong synergistic analgesic effect, and the agonist of GABAA receptor can completely inhibit the analgesic effect of EM.
The above results show that the role of EM is achieved by inhibiting the activity of GABA neurons, which is mediated by MOR.
In conclusion, the above morphological and behavioral experiments show that EM can produce a clear analgesic effect in vlPAG, and this effect is mainly achieved by inhibiting the activity of GABA neurons that express MOR, thereby enabling the expression of GABAA receptor 5-HT neurons to get rid of GABA inhibition.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R338

【参考文献】