P物质参与小鼠中枢内源性下行抑制系统镇痛效应的形态学研究

发布时间：2018-03-09 07:36

本文选题：P物质　切入点：神经激肽-1受体　出处：《第四军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 延髓吻段腹内侧部(RVM)是中枢内源性下行痛抑制系统的一个重要组成部分,RVM内的神经元不但接受多个脑区的上位调节,还向脊髓背角浅层发出下行投射,参与外周伤害性信息向中枢传递的调控。动物行为学实验研究表明,电刺激中缝大核(RMg)可以观察到动物对热刺激和机械性刺激的反应降低,反应阈值增大,对伤害性信息的传递有抑制作用。破坏RMg对伤害性信息传递的抑制作用降低或消失。RVM内神经元的活动受到多种机制的调节,其中最经典的例子就是RVM内局部给予吗啡后观察到RVM内神经元的活动水平升高,并产生镇痛效应。已经证明,多种神经活性物质和受体均可以通过调节RVM内向脊髓背角投射的神经元参与中枢内源性镇痛,而对这些物质及受体的研究和探讨,无疑对进一步揭示中枢内源性下行抑制系统发挥镇痛效应的机制具有重要的意义。 P物质(SP)是一种重要的,与伤害性信息调节相关的神经激肽,主要通过神经激肽-1受体(NK-1R)在中枢发挥调节作用。尽管SP在外周对伤害性信息向中枢传递的过程中起易化作用,但在中枢神经系统内却主要发挥镇痛作用。免疫组织化学研究表明有大量的SP免疫阳性纤维和终末分布于RVM内,与该处的神经元形成复杂的突触联系;局部给予SP可以使RVM内的神经元发放动作电位的频率升高,这些研究结果提示SP可能参与了RVM内神经元活动的调节。但是,RVM内SP能纤维和终末的来源、SP能纤维和终末与NK-1R阳性神经元的联系及RVM内可能受到SP调节的NK-1R阳性神经元向脊髓背角的下行投射状况等问题均尚未阐明。本研究采用免疫荧光组织化学染色、荧光束路追踪以及免疫电镜技术,观察了小鼠RVM内:①SP能纤维和终末的来源,②SP能终末与NK-1R阳性神经元之间的突触联系,③NK-1R阳性神经元的下行投射状况。本研究的结果将为SP参与中枢内源性下行镇痛系统的机能活动提供形态学证据。第一部分延髓吻段腹内侧部内P物质阳性纤维和终末的来源本研究采用顺行、逆行束路追踪与免疫荧光组织化学染色相结合的方法,观察了分布于RVM内的SP能纤维和终末的来源部位。将逆行性束路追踪剂荧光金(FG)注射到中缝大核(RMg)后,结合SP的免疫荧光组织化学染色,我们观察到FG逆行标记的SP阳性神经元主要分布于楔形核(CnF),中缝背核(DR)与中脑导水管周围灰质外侧部(lPAG)。将顺行示踪剂生物素化葡聚糖胺(BDA)注射到相应的CnF、DR和lPAG后,SP、NK-1R和BDA的免疫荧光组织化学三重染色结果显示,在RMg和旁巨细胞核(PGi)内,BDA标记的SP阳性终末与表达NK-1R的神经元形成密切接触。结果提示:RVM内的SP能纤维和终末主要来源于CnF、DR和lPAG,SP能终末能够与相应的表达NK-1R的神经元细胞发生密切接触,鉴于RVM内神经元是内源性下行抑制系统的主要组成,该结果从形态学上提示了SP参与中枢内源性下行痛抑制系统的可能性。第二部分中脑导水管周围灰质下行投射的SP能终末与中缝大核内神经激肽-1受体阳性神经元的突触联系本研究采用顺行追踪与免疫组化双重染色相结合的三标方法,观察了起源于PAG的SP能阳性终末与RMg内NK-1R阳性神经元之间的突触联系。结果显示:将BDA注入小鼠PAG的外侧区后,BDA顺行标记终末可见于脑干许多区域,主要位于RMg内,其中的部分BDA顺行标记终末呈SP阳性。RMg内可见到散在分布的NK-1R阳性神经元。在电镜下可见来自PAG的SP/BDA双标终末与RMg内的NK-1R阳性神经元的胞体和树突形成以非对称性为主的突触联系。本研究的结果提示,起源于PAG的下行投射终末所释放的SP可能参与中枢内源性镇痛系统,通过激活RMg内表达NK-1R的下行投射神经元发挥镇痛作用,在电镜水平进一步证实了第一部分的实验结果。第三部分延髓吻段腹内侧部内神经激肽-1受体阳性神经元向脊髓背角的投射本研究采用FG逆行束路追踪和NK-1R免疫荧光组织化学相结合的方法,观察了RVM内NK-1R阳性神经元向脊髓背角的下行投射状况。将FG注射入脊髓背角后,FG逆行标记的神经元主要分布于RVM内。其中,部分神经元同时呈NK-1R免疫阳性。以上结果提示:在RVM内,部分表达NK-1R的神经元向脊髓背角发出下下行投射,参与脊髓背角浅层神经元活动的调节。该下行投射可能与伤害性信息的传入调控有关。
[Abstract]:The rostral ventromedial part of the medulla oblongata (RVM) is an important part of the central endogenous descending pain suppression system. The neurons in RVM not only receive superordinate regulation in multiple brain regions, but also send down projections to the superficial part of the spinal dorsal horn, and participate in the regulation and control of peripheral nociceptive information to the central nervous system.
Experimental results show that the animal behavior, electrical stimulation of nucleus raphe magnus (RMg) can be observed in animal to reduce the heat and mechanical stimulus response, response threshold increases, has inhibitory effect on the transmission of nociceptive information. RMg damage is regulated by multiple mechanisms of inhibition of nociceptive transmission decreased or disappeared in.RVM the activity of neurons, one of the most classic example is the RVM in the local administration of morphine was observed after RVM in neuronal activity levels, and produce analgesic effect. It has been proven, neuroactive substances and receptors are mediated by the RVM spinal projecting neurons in the central endogenous pain, and study of the materials and their receptors the discussion is important to further reveal the central endogenous descending inhibitory system play analgesic effect mechanism.
Substance P (SP) is a kind of important, and nociceptive information related to modulation of neurokinin, mainly through neurokinin -1 receptor (NK-1R) play a regulatory role in the CNS. Although SP in the peripheral of the afferent nociceptive information in facilitation, but in the central nervous system is mainly play analgesic. Immunohistochemical studies show that a large number of SP immunoreactive fibers and terminals are distributed in the RVM, make complicate synaptic connections with the neurons; local administration of SP can make RVM neurons within the spike frequency increases, these results suggest that SP may be involved in the modulation of neurons in the RVM but. RVM, SP fibers and terminals, SP NK-1R may be regulated by SP positive neurons to the spinal dorsal horn of the descending projection of contact and ask RVM fibers and terminals and NK-1R positive neurons The questions have not been clarified.
This study used immunofluorescence staining, fluorescence tracing and immunoelectron microscopy, observed in RVM mice: SP fibers and terminals of the source, the SP synaptic connections between terminals and NK-1R positive neurons and NK-1R positive neurons in the descending projection condition. The results of this study will provide morphological evidence for that SP participates in the central endogenous descending pain control system.
The source of substance P positive fibers and terminals in the ventromedial ventromedial part of the medulla oblongata
In this study, anterograde retrograde tracer tracing and immunofluorescence histochemical staining were used to observe the location of SP energy fibers and terminals in RVM.
The retrograde tract tracing of fluoro gold (FG) injected into the nucleus raphe magnus (RMg), combined with immunohistochemical SP staining, we observed that FG labeled SP positive neurons were mainly distributed in the cuneate nucleus (CnF), dorsal raphe nucleus (DR) and lateral (periaqueductal gray lPAG). The anterograde tracer biotinylated dextran amine (BDA) was injected into the corresponding CnF, DR and lPAG, SP, NK-1R and BDA immunofluorescence histochemical staining showed that in three, RMg and paragigantocellular nucleus (PGi), BDA labeled SP positive terminals and NK-1R expressing neurons the formation of close contact.
The results suggest that RVM in SP fibers and terminals mainly derived from CnF, DR and lPAG, SP neurons and the corresponding expression of NK-1R has close contact to the end, in view of the neurons in RVM is a major component of endogenous descending inhibitory system, the results suggest the possibility that SP participates in the central endogenous descending inhibitory system from the morphology.
The second part of the periaqueductal gray of the descending projection terminals and SP synapses in the nucleus raphe magnus neurokinin -1 receptor positive neurons
Three standard methods used in this study anterograde tracing and immunohistochemical double staining combined with observation, originated from the PAG SP to synaptic connections between NK-1R positive terminals and RMg positive neurons. The results showed that BDA was injected into the lateral region of mouse PAG, BDA anterogradely labeled terminals can be found in many regions of the brain stem. Mainly located in the RMg, BDA anterogradely labeled terminal which were positive for SP.RMg can be seen scattered in the distribution of NK-1R positive neurons in the electron microscope. From PAG SP/BDA double labeled terminals and RMg in NK-1R positive cell bodies and dendrites of neurons form synapses with non symmetry based.
The results of this study suggest that the SP released from the descending projection terminals originating from PAG may participate in the central endogenous analgesia system. It plays an analgesic role by activating the down projection neurons expressing NK-1R in RMg, and further confirms the experimental results in the first part at the electron microscope level.
The third part of neurokinin -1 receptor positive neurons in the medulla oblongata kiss section within the Department of the ventral part of the medial projection to the dorsal horn of spinal cord
In this study, FG retrograde tracing and NK-1R immunofluorescence histochemistry were used to observe the descending projection of NK-1R positive neurons in RVM to the dorsal horn of spinal cord.
After injecting FG into the dorsal horn of the spinal cord, the FG retrograde labeled neurons were mainly distributed in RVM, and some of the neurons were NK-1R immunoreactive at the same time.
The above results suggest that in RVM, some neurons expressing NK-1R are descending projections to the dorsal horn of the spinal cord, which are involved in the regulation of neurons in the superficial dorsal horn of spinal cord. This downward projection may be related to the afferent regulation of noxious information.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R322

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