P2X3受体在感觉神经节的表达

用免疫组织化学与原位杂交研究P2X3受体在背根神经节,三叉神经节和结状神经节的分布。结果显示：1、原位杂交;在三种感觉神经节中,95%左右的神经节细胞为P2X3mRNA阳性,中、小型神经节细胞的杂交信号一般要比大型的神经节细胞强一些,2、免疫组织化学;免疫组织化学结果与原位杂交结果基本一致。此外,在各神经节内,均显示出许多P2X3免疫阳性神经纤维,在足掌表皮也显示许多P2X3免疫反应阳性纤维,结果提示：P2X3不仅参与机体的痛觉的形成,还可能参与其它感觉,如本体感觉等的形成。     

大鼠初级感觉神经元P2X3受体的表达及其与SP的关系

目的研究在大鼠初级感觉神经元细胞上P2X3受体的表达情况及其与P物质的关系。方法取SD大鼠背根神经节(DRG)和三叉神经节(TG)固定后切片;用抗P2X3受体抗体和抗SP抗体进行免疫组织化学反应,并通过两种不同的显色方法同时进行P2X3受体和SP的双标。结果P2X3免疫反应阳性细胞主要集中在小细胞和中等细胞(其中在TG,P2X3-ir阳性神经元约占整个细胞的24.8%;在DRG约31.7%的神经元是P2X3-ir阳性),并且在DRG和TG细胞上均存在有P2X3受体和SP共存(TG上的双标细胞占P2X3-ir阳性细胞总数的36.26%,DRG上占46.81%)。结论由于ATP门控阳离子通道受体P2X3本身就与伤害性感受的初级传入有关,而它与SP的共存可提示当组织中的ATP释放时可以通过P2X3受体作用于含SP的伤害性感觉神经末梢上,促使SP释放引起痛觉过敏。     

化学损毁交感神经后大鼠心内神经节生长抑素的变化

应用免疫组织化学和原位杂交方法研究了大鼠心内神经节细胞中SS的分布及其mRNA表达。结果发现,大鼠心内神经节中有SS－IR阳性神经纤维和细胞,心内神经部分细胞浆中有SSmRNA表达,表明大鼠心内神经节细胞有SS合成和贮存。用小剂量6－OH－DA选择性损毁心内交感神经纤维后,心内神经节中SS－IR阳性神经纤维和细胞的积分光密度均有不同程度增强,反映了心内交感神经和付交感神经的相互抑制作用。     

心内神经元血管加压素的基因表达-原位杂交与免疫组织化学结合法研究

取中华蟾蜍心房后壁腔静脉窦部位组织作冰冻切片（30μm）,以原位杂交和免疫组织化学双标法进行心内神经节细胞的血管加压素mRNA(VPmRNA)及血管加压素免疫反应（VP-IR）染色观察,结果在心内神经节发现了3种标记细胞;（1）VP-IR阳性神经元;（2）VPmRNA阳性神经元;（2）VPmRNA和VP-IR双阳性神经元,VPmRNA和VP-IR双标记神经元证明部分心内神经节的细胞元内既有血管加压素（VP）的基因表达,又储存VP,从而说明VP系心内神经节的一种内源性神经递质,为心内神经节以VP为递质直接参与血液循环的调节提供了直接的分子生物学和化学神经解剖学证据。     

大鼠坐骨神经结扎后疼痛受体P2X3在背根神经节内的表达变化

目的:研究坐骨神经结扎损伤后疼痛受体P2X3在相应背根神经节(dorsal root ganglia,DRG)内的表达变化情况。方法:选取健康成年SD大鼠35只,建立右侧坐骨神经结扎损伤模型,采用免疫组织化学和图像分析技术检测相应L4-6DRG内P2X3的表达情况。结果:正常大鼠L4-6DRG内有大量P2X3免疫阳性神经元,坐骨神经结扎后3d P2X3表达即下调,3,7,14,21和28d其表达呈进行性下降趋势,各时间点与正常和假手术对照比较差异均有统计学意义(P＜0．05)。结论:坐骨神经结扎后P2X3在L4-6DRG内表达明显下调,提示其可能在神经源性疼痛中发挥一定的作用。     

CCR7和B7-2在抗原负载树突状细胞诱导特异性CTL抗肿瘤效应中的表达和意义

目的:研究CCR7(趋化因子受体7)和B7-2(白细胞分化抗原86)与抗原负载树突状细胞(dentritic cell,DC)诱导特异性CTL(细胞毒性T淋巴细胞)抗肿瘤效应的关系.方法:分离和培养DC,制备B16黑色素瘤细胞抗原,进行共培养,即为抗原负载的DC,建立B16黑色素瘤小鼠模型,于肿瘤周围皮下注射抗原负载的DC.应用原位杂交和免疫组织化学方法检测CCR7和B7-2的表达情况.结果:原位杂交和免疫组织化学染色显示,CCR7和B7-2阳性细胞主要分布于肿瘤周围组织,随着注射抗原负载DC时间的进展,CCR7和B7-2呈强阳性表达.结论:CCR7和B7-2的表达与抗原负载树突状细胞诱导特异性CTL抗肿瘤效应有关.     

大鼠面部慢性炎症痛导致三叉神经节表达P2X3神经元表型的转变

目的研究大鼠面部慢性炎症痛与三叉神经节内表达P2X3受体亚型神经元细胞大小和表型变化之间的关系。方法参照Neumann(1996)报道的研究方法,采用大鼠面部皮下注射松节油建立慢性炎症痛模型,用热测痛的方法测定面部皮肤的痛阈值,每天一次,连续测15d。用免疫组织化学技术观察大鼠面部慢性炎症后第5d三叉神经节内感觉神经元P2X3受体的表达。采用体视学的方法测量表达P2X3神经节细胞大小及表型的变化。结果炎症侧大鼠面部痛阈值与对照组相比明显降低,在第5d达到最低值,以后逐渐恢复,第13d开始痛阈恢复正常水平。炎症侧三叉神经节内表达P2X3神经元的平均细胞表面积(721±12μm2)与对照组(616±8μm2)相比明显增大(P<0·01)。进一步观察发现表达P2X3小细胞群(<950μm2)的表面积由炎症前的537±13μm2增加到炎症后的582±15μm2(P<0·05)而且小细胞占总细胞的数量比例由炎症前的42·2±3·2%增加到炎症后的51·8±3·5%(P<0·05);而表达P2X3受体的大细胞(>950μm2)的数量比例由炎症前的6·5±1·9%增加到炎症后的12·8±2·2%(P<0·05)。结论面部慢性炎症痛时,其三叉神经节内表达P2X3受体神经元的表型可发生改变,这可能与面部痛觉过敏和触诱发痛的形成有密切关系。     

慢性炎症痛引起DRG感觉神经元P2X3受体表达的改变

目的探讨脊髓背根神经节（dorsal root ganglia,DRG）P2X3受体参与大鼠足底慢性炎症痛相关的热痛觉过敏机制。方法 1）用行为学的研究方法,以大鼠右侧后脚掌注射松节油加石蜡（各占50％）0．1ml建立后脚掌慢性痛模型,用热测痛的方法测量后脚掌皮下注射松节油后的痛阈,每天1次,连续测15d。2）用免疫组织化学技术观察大鼠后脚掌慢性炎症后第2天和第7天,炎症侧脊髓背根神经节（L4—6）神经元中P2X,受体阳性细胞类型的分布变化;以及正常脊髓背根神经节（L4—6）神经元中P2X,受体阳性细胞类型的分布作为对照。结果1）炎症后大鼠后脚掌侧痛阈出现降低,在第2天痛阈达到最低,后逐渐恢复,14d后恢复正常痛阈值。2）正常大鼠P2X,主要表达于DRG的中小神经元上,炎症后DRG（L4—6）,中小型P2X,受体阳性细胞数比对照组明显增加。细胞平均面积增大。结论后脚掌慢性炎症痛可以引起大鼠对伤害性热刺激的痛觉过敏,并导致脊髓背根神经节（L4—6）神经元qbP2X3受体阳性细胞数目增加,表明P2X3在DRG的中小神经元的改变可能对松节油引起脚掌炎症痛时热痛觉过敏的形成与维持起重要作用。     

IAPP与SS在大鼠和家兔背根神经节细胞内分布与共存的免疫组织化学研究

应用免疫组织化学ABC法,观察到在大鼠和家兔背根神经节内存在胰岛淀粉样多肽免疫阳性反应细胞.用相邻镜像邻片免疫双标记技术,证实胰岛淀粉样多肽存在于背根神经节内那些生长抑素免疫反应阳性的细胞中,说明胰岛淀粉样多肽在大鼠和家兔背根神经节内与生长抑素共存.实验结果为胰岛淀粉样多肽的胰腺外研究提供了新的资料,为进一步研究背根神经节内生物活性物质间的关系及生理作用提供了形态学证据.     

P物质受体在大鼠纹状体边缘区内的表达

我们以前的工作观察到纹状体边缘区内有密集的P物质纤维及终末分布,本用原位杂交和免疫组织化学方法研究了大鼠纹状体边缘区内P物质受体（SPR）的表达及分布,原位杂交结果发现P物质mRNA阳性杂交信号在纹状体内的分布不均匀,尾壳核内只有少量中等大小的阳性胞体,苍白球内只有少量较大的阳性胸体,而在尾壳核和苍白球之间的边缘区部位则可见许多中等大小的梭形阳性神经元胞体,并呈现密集的带关分布。免疫组织化学结果观察到P物质阳性神经元胞体在纹状体内的分布与原位杂交结果一致。推测大鼠纹体边缘区内可以合成P物质受体,具有接受和整合P物质神经递质的功能,推测边缘区内SPR神经元可能对SP递质的接受、调节有重要作用。     

Localisation of P2Y<Subscript>1</Subscript> and P2Y<Subscript>4</Subscript> receptors in dorsal root,nodose and trigeminal ganglia of the rat

The presence and distribution of P2Y (nucleotide) receptor subtypes in rat sensory neurons has been investigated. RT-PCR showed that P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptor mRNA is expressed in sensory ganglia [dorsal root ganglion (DRG), nodose ganglion (NG) and trigeminal ganglion (TG)]. The regional and cellular distribution of P2Y₁ and P2Y₄ receptor proteins in these ganglia was investigated using immunohistochemistry. P2Y₁ polyclonal antibodies stained over 80% of the sensory neurons, particularly the small-diameter (neurofilament-negative) neurons. The P2Y₄ receptor antibody stained more medium- and large- (neurofilament-positive) diameter neurons than small-diameter neurons. P2Y₁ and P2Y₄ receptor immunoreactivity (P2Y₁-IR and P2Y₄-IR) was often coexpressed with P2X₃ receptor immunoreactivity (P2X₃-IR) in subpopulations of neurons. Double immunohistochemistry showed that 73–84% of P2X₃ receptor-positive neurons also stained for the P2Y₁ receptor in DRG, TG and NG while only 25–35% also stained for the P2Y₄ receptor. Subpopulations of P2Y₁-IR neurons were coexpressed with NF200, CGRP and IB₄; most P2Y₄-IR neurons were coexpressed with NF200, while only a few neurons were coexpressed with CGRP (10–20%) or with IB₄ (1–2%). The results suggest that P2Y as well as P2X receptor subtypes contribute to purinergic signalling in sensory ganglia.     

The involvement of P2X3 receptors of rat sympathetic ganglia in cardiac nociceptive transmission

In this work we have examined the effects of P2X3 receptor antagonist A-317491 on P2X3 expression in superior cervical ganglion (SCG) from naive and myocardial ischemic rats to observe the effect of P2X3 receptors in cardiac nociceptive transmission. A-317491 improved nociceptive behavior. In the ganglia neurons of rats at 14 days after myocardial ischemic injury, the staining of P2X3 receptor in myocardial ischemic groups appeared to be more intense than those of naive rats detected by immunohistochemistry. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 immunoreactivity was lower than that in myocardial ischemic rats. The signals of P2X3 and its protein and mRNA in myocardial ischemic groups were higher than those in control group measured by western blotting and in situ hybridization. After myocardial ischemic rats treated with A-317491, the intensity of the P2X3 and its mRNA was lower than that in myocardial ischemic rats. These results suggest the involvement of P2X3 receptors in cardiac nociceptive transmission and A-317491 may inhibit the transmission mediated by P2X3 receptors in rat SCG after myocardial ischemia.     

Comparative Analysis of P2X1, P2X2, P2X3, and P2X4 Receptor Subunits in Rat Nodose Ganglion Neurons

Nodose ganglion (NG) neurons are visceral primary sensory neurons. The transmission and regulation of visceral sensation is mediated mainly by the P2X purinoceptor (P2X receptor). Although the characteristics of different P2X receptor subunits in the NG have been studied previously, comprehensive analyses have not been performed. In this study, we used immunohistochemistry, immunocytochemistry, and whole cell patch clamp techniques to compare the expression and function of P2X1, P2X2, P2X3, and P2X4 receptor subunits in adult rat NG neurons. Polyclonal antibodies against the four P2X subunits labeled different subpopulations of NG neurons. P2X1 and P2X3 were expressed mainly in small-to-medium sized NG neurons, whereas P2X2 and P2X4 were located mostly in medium- and larger-sized NG neurons. Over 36% of NG neurons were P2X3 positive, which was higher than the other three P2X subunits. In addition, different types of currents were recorded from neurons expressing different P2X subunits. The fast type of ATP current was recorded from neurons containing P2X1–4 subunits, the intermediate type of current was recorded from neurons containing the P2X1, P2X3, and P2X4 subunits, the slow type was recorded from neurons expressing P2X1–3, and/or P2X4 subunits, whereas the very slow type was recorded from neurons containing the P2X2 and P2X3 subunits. These comparative results provide an anatomical verification of the different subunits in NG neurons, and offer direct support for the idea that various functional NG populations have distinct responses to ATP, which might be in part due to the different expression profiles of diverse P2X subunits.     

大鼠脊神经节内交感神经支配的荧光组织化学与HRP示踪观察

本研究应用乙醛酸诱发儿茶酚胺（ＣＡ）荧光技术观察大鼠肾上腺素（ＮＡ）能神经在脊神经节内的分布;并应用ＨＲＰ顺、逆行追踪技术对脊神经节内ＮＡ能神经纤维的起源及其与脊神经节神经元的关系进行了探讨。荧光组织化学观察发现、有些神经节神经元胞体周围分布有带膨体的ＮＡ能神经末梢;有的紧密围绕脊神经节细胞——卫星细胞复合体。颈上交感神经节内注射霍乱毒素Ｂ亚单位结合ＨＲＰ（ＣＢ┐ＨＲＰ）,在同侧Ｃ３～６节段脊神经节内可见标记的点状纤维末梢紧邻于节细胞旁。Ｔ１１～Ｌ２节段脊神经节内注射ＨＲＰ后,在同侧椎旁交感链（Ｔ９～Ｌ１）内可见标记的交感节后神经元胞体。上述实验结果表明,交感节后神经元发出节后纤维可直接到达脊神经节内,与节细胞发生接触。本研究提示、交感神经在脊神经节水平可能参与躯体初级传入信息的调制     

P2X2 receptors differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and esophagus

The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neuron's embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to alpha,beta-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to alpha,beta-methylene ATP (30 microM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of alpha,beta-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X2/3 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate.     

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.     

Differential Trk expression in explant and dissociated trigeminal ganglion cell cultures

During embryonic development, expression of neurotrophin receptor tyrosine kinases (Trks) by sensory ganglia is continuously and dynamically regulated. Neurotrophin signaling promotes selective survival and axonal differentiation of sensory neurons. In embryonic day (E) 15 rat trigeminal ganglion (TG), NGF receptor TrkA is expressed by small diameter neurons, NT-3 receptor TrkC and BDNF receptor TrkB are expressed by large diameter neurons. Organotypic explant and dissociated cell cultures of the TG (and dorsal root ganglia) are commonly used to assay neurotrophin effects on developing sensory neurons. In this study, we compared Trk expression in E15 rat TG explant and dissociated cell cultures with or without neurotrophin treatment. Only a subset of TG cells express each of the three Trk receptors in wholemount explant cultures as in vivo conditions. In contrast, all TG neurons co-express all three Trk receptors upon dissociation, regardless of neurotrophin treatment. Neurons cultured in low concentrations of one neurotrophin first, and switched to higher concentrations of another after 1 day, survive and display morphological characteristics of neurons cultured in a mixture of both neurotrophins for 3 days. Our results indicate that wholemount explant cultures of sensory ganglia represent in vivo conditions in terms of Trk expression patterns; whereas dissociation dramatically alters Trk expression by primary sensory neurons.     

Pituitary adenylate cyclase activating polypeptide is expressed in autonomic neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide, which is present in neuronal elements of several peripheral organs, and thus a putative neurotransmitter/modulator. In the present study, the expression of PACAP in two parasympathetic ganglia (otic, sphenopalatine) and one mixed parasympathetic/sensory ganglion (jugular-nodose) in rat was characterized by use of in situ hybridization and immunocytochemistry and compared to that of VIP and calcitonin gene-related peptide (CGRP). PACAP and VIP were expressed in virtually all nerve cell bodies in the otic and sphenopalatine ganglia; PACAP and VIP were also expressed in subpopulations of nerve cell bodies in the jugular-nodose ganglion. CGRP was expressed in numerous nerve cell bodies in the jugular-nodose ganglion and in a few, scattered, nerve cell bodies in the sphenopalatine ganglion. In the otic and sphenopalatine ganglia, PACAP- and VIP-like immunoreactivities were frequently co-localized; in the jugular-nodose ganglion, PACAP-like immunoreactivity was frequently co-localized with CGRP-like immunoreactivity in presumably sensory neurons and to a lesser extent with VIP in parasympathetic neurons. Thus, PACAP is synthesized and stored in autonomic parasympathetic neurons as well as in vagal sensory neurons, which provides an anatomical basis for the diverse effects of PACAP previously described.