面部炎症痛诱发大鼠三叉神经节神经元中辣椒素受体表达的改变(英文)

目的探讨辣椒素受体(transient receptor potential vanilloid receptor 1,TRPV1)参与和面部炎症痛相关的热痛觉过敏与冷痛觉感受的可能机制。方法于大鼠面部皮下注射松节油造成面部炎症痛模型,分别应用热测痛和冷测痛装置测量热缩头潜伏期(head withdrawal thermal latency, HWTL)和冷缩头潜伏期(head withdrawal coldlentency, HWCL)的变化,每天测量一次,连续21天。应用免疫组织化学染色,细胞大小频率分析和平均光密度值分析来研究面部炎症痛后第3、5、7、14、21天支配大鼠面部表皮区三叉神经节(trigeminal ganglion,TG)初级感觉神经元、触须部皮肤末梢神经纤维和投射至三叉神经感觉尾侧亚核(trigeminal sensory nuclei caudalis,Vc)中枢突TRPV1表达的改变。结果注射松节油后第1至14天,热退缩反应潜伏期与冷退缩反应潜伏期均明显下降,分别于注射后第5 天和第3 天达到最低,第21 天恢复到正常水平;注射松节油后第1至14天 ,TRPV1表达的细胞数量增加,并于第7天达到最大,第21 天恢复到正常水平。正常大鼠TRPV1主要表达于TG的中小神经元,触须部皮肤以及三叉神经尾侧亚核含丰富的TRPV1阳性末梢;面部炎症痛后2周内,TG的中小神经元,触须部皮肤末梢以及Vc的Ⅰ和Ⅱ外层均可见明显的TRPV1表达增加。结论面部炎症痛可以引起大鼠对伤害性热刺激和冷刺激的痛觉过敏,并导致三叉神经节中TRPV1阳性神经元和外周与中枢阳性神经纤维末梢数目增加,表明TRPV1在三叉神经节的中小神经元和末梢轴突表型的改变可能对松节油引起面部炎症痛时热痛觉过敏和冷痛觉感受的形成与维持起重要作用。     

WIN55,212-2对大鼠三叉神经节神经元烟碱激活电流抑制作用的研究

目的：应用全细胞膜片钳技术,在培养的大鼠三叉神经节（trigeminal ganglion, TG）神经元上观察大麻素WIN55,212-2对烟碱激活电流（Inic）的调制作用及其机制。方法：培养大鼠三叉神经节神经元,利用全细胞膜片钳技术记录烟碱激活电流。结果：(1)实验中大部分受检细胞对外加烟碱敏感,产生一浓度依赖性内向电流。(2) 与烟碱单独作用相比,同时给予WIN55,212-2（3 µM）和烟碱能明显抑制Inic峰值;此快速抑制作用呈可逆性、浓度依赖性和非电压依赖性;WIN55,212-2使烟碱激活电流量效曲线明显下移,但EC50值无明显改变;WIN55,212-2不改变烟碱激活电流的失敏时间常数。(3) WIN55,212-2对烟碱激活电流的抑制作用不被CB1受拮抗剂AM81、CB2受体拮抗剂AM630和VR1受体拮抗剂capsazepine阻断;不受cAMP类似物8-Br-cAMP(1mM)影响;胞内应用非特异性的G蛋白抑制剂GDP-β-S(1mM) 也不影响WIN55,212-2对烟碱激活电流的抑制作用。结论：WIN55,212-2可能直接作用于nACh受体,通过非竞争性方式抑制烟碱激活电流发挥外周镇痛作用。     

痛刺激后大鼠三叉神经尾侧亚核星形细胞和神经元相互关系的电镜观察

目的：观察大鼠上唇皮下注射福尔马林后，三叉神经尾侧亚核（Sp5C)内反应性星形细胞和神经元之间相互关系的超微结构。方法：用DAB染色的抗胶质原纤维酸性蛋白（GFAP）、抗connexin43(Cx43）和金颗粒标记抗Cx32双标记免疫电镜方法。结果：电镜下观察到Sp5C内反应性星形细胞和神经元之间存在4种联系结构：第一种是突触样结构；第二种是三种成分的突触复合体，第三种是缝隙连接；第四种是由Cx32和Cx43构成的异源性缝隙连接（HGJ）。HGJ表现为两侧膜增厚，Cx43阳性物质和Cx32阳性金颗粒分别位于星形细胞和神经元一侧，痛刺激后HGJ数明明显增加。结论：神经元和星形细胞之间有多种信息通道，HGJ可能是一种快速，适应性信息通道。Sp5C星形细胞可能通过HGJ调节神经元的活动，共同参与中枢神经系统对刺激反应的调节。     

辣椒素对大鼠骶髓后连合核神经元AMPA受体所介导电流的抑制作用

目的研究辣椒素（CAP）在急性分离的大鼠骶髓后连合核（SDCN）神经元的药理学特性。方法采用制霉菌素穿孔膜片钳全细胞记录方法。结果当钳制电压为-40mV时，在所有被检测的SDCN神经元上给予CAP不引起任何电流；CAP（10～3000μmol／L）可以呈浓度依赖方式可逆性的抑制海人藻酸（KA）所激活的AMPA受体介导的电流，IC50为（60．7±19．2）μmol/L；该抑制作用呈电压非依赖性。结论CAP在大鼠SDCN神经元可对AMPA受体激活的电流产生抑制作用，此作用可能和内脏痛的调节有关。     

痛刺激后大鼠三叉神经尾侧亚核星形细胞和神经元相互关系的电镜观察

目的:观察大鼠上唇皮下注射福尔马林后,三叉神经尾侧亚核(Sp5C)内反应性星形细胞和神经元之间相互关系的超微结构.方法:用DAB染色的抗胶质原纤维酸性蛋白(GFAP)、抗connexin43(Cx43)和金颗粒标记抗Cx32双标记免疫电镜方法.结果:电镜下观察到Sp5C内反应性星形细胞和神经元之间存在4种联系结构:第一种是突触样结构;第二种是三成分的突触复合体;第三种是缝隙连接;第四种是由Cx32和Cx43构成的异源性缝隙连接(HGJ).HGJ表现为两侧膜增厚,Cx43阳性物质和Cx32阳性金颗粒分别位于星形细胞和神经元一侧,痛刺激后HGJ数量明显增加.结论:神经元和星形细胞之间有多种信息通道,HGJ可能是一种快速、适应性信息通道.Sp5C星形细胞可能通过HGJ调节神经元的活动,共同参与中枢神经系统对刺激反应的调节.     

刺激大鼠大脑脚对福尔马林诱发的脊髓背角神经元持续性痛反应的影响

在大鼠用玻璃微电极细胞外记录单个脊髓背角神经元的放电,观察了刺激大脑脚(CP)对外周感受野内皮下注射福尔马林(5%,50 μl)诱发的背角广动力范围型(WDR)神经元的晚时相反应的影响,结果表明刺激CP对福尔马林诱发的晚时相反应具有抑制作用,并有明显的强度-效应关系,提示大脑皮质感觉运动区对持续性痛具有下行调控作用.     

刺激大鼠大脑脚对福尔马林诱发的脊髓背角神经元持续性痛反应的影响

在大鼠用玻璃微电极细胞外记录单个脊髓背角神经元的放电 ,观察了刺激大脑脚 ( CP)对外周感受野内皮下注射福尔马林 ( 5%,50 μl)诱发的背角广动力范围型 ( WDR)神经元的晚时相反应的影响 ,结果表明刺激 CP对福尔马林诱发的晚时相反应具有抑制作用 ,并有明显的强度 -效应关系 ,提示大脑皮质感觉运动区对持续性痛具有下行调控作用。     

GABA对大鼠伏隔核痛反应神经元电活动的影响

目的在50只成年Wister大鼠上，观察了侧脑室(icv)注射GABA(γ-氨基丁酸)后，伏隔核(NAc)痛反应神经元放电的变化和荷包牡丹碱(Bic)对GABA作用的阻断效应，从而进一步研究GABA与NAc在痛觉调制中的作用。方法采用icy注射，电脉冲强直刺激坐骨神经作为伤害性痛刺激，玻璃微电极细胞外记录痛反应神经元放电的变化。结果(1)icv注入GABA能够使正常大鼠NAc中痛兴奋神经元(PEN)痛诱发放电频率减少、潜伏期延长，而使痛抑制神经元(PIN)痛诱发放电频率增加、诱发放电完全抑制时程缩短；(2)icv注入GABAA受体拮抗剂Bic能够阻断GABA的上述效应。结论(1)外源性GABA可使正常大鼠NAc中痛反应神经元对伤害性刺激的反应减弱，表现为镇痛效应；(2)GABA的这种镇痛作用主要是通过GABAA受体介导的。该结果揭示，GABA和NAc在痛觉调制中具有非常重要的作用。     

日本猕猴背根节表达阿片μ受体细胞的定量分析

使用免疫组织化学和组织化学结合标记技术,对阿片μ受体样免疫反应阳性物质（ＭＯＲ－Ｌｉ）和ＢＳＩ－Ｂ４植物凝集素组织化学结合标记物在日本猕猴背根节（ＤＲＧ）细胞内的定位形式和特点进行了比较研究。用Ｌｅ－ｉｃａ Ｑ５００ＭＣ图像分析系统,对２６７９个ＭＯＲ－Ｌｉ细胞进行计数和测量表明,９８．７７％（２６４６／２６７９）阳性细胞为直径小于５０μｍ的小细胞亚群;只有１．２３％（３３／２６７９）阳性细胞的直     

大鼠咬肌神经切断后三叉神经运动神经元高亲和性神经营养因子受体表达的变化

目的应用双重标记技术观察咬肌神经切断对支配咬肌的三叉神经运动神经元所含高亲和性神经营养物质受体———Trk受体,即TrkA、TrkB和TrkC表达的影响。方法切断大鼠咬肌神经7和14 d后,对脑切片进行免疫组织化学染色并观察荧光金(FG)标记的三叉神经运动核(Mo5)神经元表达的三种Trk受体。结果(1)荧光金标记神经元中TrkA免疫反应阳性神经元比例无显著性变化(P>0.05);(2)神经切断后7和14 d均观察到TrkB表达上调(P<0.05);(3)神经切断后14 d观察到TrkC表达下调(P<0.05)。结论咬肌神经切断后,三叉神经运动核神经元所含不同Trk受体的表达存在差异性调节。     

Increased gene and protein expressions of the transient receptor potential vanilloid receptor 4 following sustained pure mechanical pressure on rat dorsal root ganglion neurons

Dorsal root ganglion(DRG) neurons from newborn Wistar rats cultured in vitro were pressurized with 20,40,80 or 120 mm Hg compressive loadings(1 mm Hg = 0.133 kPa) for 12,24,48 or 72 hours,respectively.The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide test showed that pressures less than 80 mm Hg had no obvious impact on the activity of DRG neurons.The protein expression levels of transient receptor potential vanilloid receptor 4(TRPV4),transient receptor potential vanilloid receptor 1,transie...     

Orofacial inflammatory pain affects the expression of MT1 and NADPH-d in rat caudal spinal trigeminal nucleus and trigeminal ganglion

Very little is known about the role of melatonin in the trigeminal system, including the function of melatonin receptor 1. In the present study, adult rats were injected with formaldehyde into the right vibrissae pad to establish a model of orofacial inflammatory pain. The distribution of melatonin re- ceptor 1 and nicotinamide adenine dinucleotide phosphate diaphorase in the caudal spinal trigeminal nucleus and trigeminal ganglion was determined with immunohistochemistry and histo- chemistry. The results show that there are significant differences in melatonin receptor 1 expression and nicotinamide adenine dinucleotide phosphate diaphorase expression in the trigeminal ganglia and caudal spinal nucleus during the early stage of orofacial inflammatory pain. Our findings sug- gest that when melatonin receptor 1 expression in the caudal spinal nucleus is significantly reduced, melatonin＇s regulatory effect on pain is attenuated.     

The putative role of vanilloid receptor-like protein-1 in mediating high threshold noxious heat-sensitivity in rat cultured primary sensory neurons

High threshold noxious heat-activated currents and vanilloid receptor-like protein-1 expression were studied in rat cultured primary sensory neurons to find out the molecule(s) responsible for high threshold noxious heat-sensitivity. The average temperature threshold and amplitude of high threshold noxious heat-activated currents were 51.6 +/- 0.13 degrees C and -2.0 +/- 0.1nA (at a holding potential of -60 mV), respectively. The current-voltage relationship of high threshold noxious heat-activated currents was linear at positive membrane potentials, while it showed a weak inward rectification at negative membrane potentials. The average reversal potential measured in control intracellular and extracellular solutions was 4.5 +/- 0.9 mV (n = 6). Ionic substitutions revealed that the high threshold noxious heat-activated current is a nonselective cationic current with calculated ionic permeabilities of Cs+ : Na+ : Ca2+ (1 : 1.3 : 4.5). Consecutive stimuli reduced the heat threshold from 52.2 +/- 1 to 48.4 +/- 1.4 degrees C and then to 44 +/- 0.7 degrees C (n = 3). High threshold noxious heat-activated currents could dose-dependently and reversibly be reduced by ruthenium red (100 nm-10 micro m) but not by capsazepine (10 micro m). The average longest diameter of high threshold noxious heat-sensitive neurons was 31.48 +/- 0.5 micro m (A = approximately 778 micro m2; n = 77). Twenty-three percent of the total neuronal population expressed vanilloid receptor-like protein-1. The average area of the vanilloid receptor-like protein-1-immunopositive cells was 1,696 +/- 65.3 micro m2 (d = approximately 46 micro m). Vanilloid receptor-like protein-1-expressing neurons did not express the vanilloid receptor 1. Comparison of our data with results obtained in vanilloid receptor-like protein-1-expressing non-neuronal cells and previous immunohistochemical findings suggests that high threshold noxious heat-activated currents are produced by vanilloid receptor-like protein-1 and that high threshold heat-sensitive dorsal root ganglion neurons are the perikarya of type I noxious heat-sensitive fibers.     

Neurochemical characterization of insulin receptor-expressing primary sensory neurons in wild-type and vanilloid type 1 transient receptor potential receptor knockout mice

The insulin receptor (IR) is expressed by a subpopulation of primary sensory neurons (PSN), including a proportion of cells expressing the nociceptive transducer vanilloid type 1 transient receptor potential receptor (TRPV1). Recent data suggest functional links between the IR and other receptors, including TRPV1, which could be involved in the development of PSN malfunctions in pathological insulin secretion. Here we used combined immunohistochemical labelling on sections from L4-5 dorsal root ganglia of wild-type (WT) and TRPV1 knockout (KO) mice to examine the neurochemical properties of IR-expressing PSN and the possible effect of deletion of TRPV1 on those characteristics. We found that antibodies raised against the high-molecular-weight neurofilament (NF-200) and the neurofilament protein peripherin distinguished between small and large neurons. We also found that the IR was expressed predominantly by the small peripherin-immunopositive cells both in the WT and in the KO animals. IR expression, however, did not show any preference between the major subpopulations of the small cells, the calcitonin gene-related peptide (CGRP)-expressing and Bandeiraea simplicifolia isolectin B4 (IB4)-binding neurons, either in the WT or in the KO mice. Nevertheless, a significant proportion of the IR-expressing cells also expressed TRPV1. Comparison of the staining pattern of these markers showed no difference between WT and KO animals. These findings indicate that the majority of the IR-expressing PSN are small neurons, which are considered as nociceptive cells. Furthermore, these data show that deletion of the TRPV1 gene does not induce any additional changes in neurochemical phenotype of nociceptive PSN.     

RNA interference-mediated knock-down of transient receptor potential vanilloid 1 prevents forepaw inflammatory hyperalgesia in rat

Transient receptor potential vanilloid (TRPV)1 is a ligand-gated cation channel expressed by primary sensory neurons, including those in the dorsal root ganglia (DRG). TRPV1 plays an essential role in development of inflammatory thermal hyperalgesia after tissue injury and its expression in rat lumbar DRG is increased after hindpaw inflammation. However, the identity of factors mediating forepaw inflammatory hyperalgesia has remained elusive. Here, we examined behavioral responses to noxious thermal stimuli after forepaw inflammation in rats and found that inflammation induced by intraplantar injection of complete Freund's adjuvant significantly reduced hot-plate latency (HPL) at 50 degrees C. TRPV1 expression levels in the ipsilateral cervical DRG were also elevated after forepaw inflammation. By contrast, HPL at 56 degrees C was not shortened after forepaw inflammation and expression of TRPV2, a TRPV1 homolog, in the DRG was not increased. Paratracheal injection of short interfering RNA targeting TRPV1 blocked TRPV1 up-regulation in cervical DRG and abolished inflammation-mediated HPL reductions seen at 50 degrees C. However, thermal hyperalgesia previously established by inflammation was not reversed by short interfering RNA injection. These results indicate that: (i) enhanced TRPV1 expression in cervical DRG is closely associated with development of inflammatory thermal hyperalgesia in the forepaw after tissue injury and (ii) RNA interference targeting TRPV1 prevents inflammatory thermal hyperalgesia after forepaw injuries but does not ameliorate it when already established in a rat model of nociceptive pain representing upper limb injury in humans.     

c-Jun expression after axotomy of corneal trigeminal ganglion neurons is dependent on the site of injury

The proto-oncogene c-Jun has been implicated in the control of neuronal responses to injury and in axonal growth during regenerative processes. We have investigated the expression of c-Jun during normal terminal remodelling in trigeminal ganglion neurons innervating the cornea and after acute injury of epithelial nerve terminals or parent axons. Remodelling and rearrangement, or damage limited to corneal epithelium endings, was not a trigger for activation of c-Jun expression. However, injury of parent axons in the stroma or in the orbital ciliary nerves induced c-Jun expression in 50% of the population of corneal neurons, which included all of the large myelinated and 20% of the small neuropeptide-containing corneal neurons. This suggests that c-Jun expression in trigeminal ganglion neurons is not associated with normal remodelling or regeneration of peripheral nerve terminals, and that it takes place only when parent axons are injured. A substantial number of damaged neurons do not express c-Jun, indicating that in primary sensory neurons, injury and regeneration may not always be coupled to the expression of this proto-oncogene.     

Expression of transient receptor potential ankyrin 1 (TRPA1) in the rat trigeminal sensory afferents and spinal dorsal horn

Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold and pungent compounds, is implicated in the mediation of nociception, but little is known about the processing of the TRPA1‐mediated nociceptive information within the trigeminal sensory nuclei (TSN) and the spinal dorsal horn (DH). To address this issue, we characterized the TRPA1‐positive (+) neurons in the trigeminal ganglion (TG) and investigated the distribution of TRPA1⁺ afferent fibers and their synaptic connectivity within the rat TSN and DH by using light and electron microscopic immunohistochemistry. In the TG, TRPA1 was expressed in unmyelinated and small myelinated axons and also occasionally in large myelinated axons. Many TRPA1⁺ neurons costained for the marker for peptidergic neurons substance P (26.8%) or the marker for nonpeptidergic neurons IB4 (44.5%). In the CNS, small numbers of axons and terminals were immunopositive for TRPA1 throughout the rostral TSN, in contrast to the dense network of positive fibers and terminals in the superficial laminae of the trigeminal caudal nucleus (Vc) and DH. The TRPA1⁺ terminals contained clear round vesicles, were presynaptic to one or two dendrites, and rarely participated in axoaxonic contacts, suggesting involvement in relatively simple synaptic circuitry with a small degree of synaptic divergence and little presynaptic modulation. Immunoreactivity for TRPA1 was also occasionally observed in postsynaptic dendrites. These results suggest that TRPA1‐dependent orofacial and spinal nociceptive input is processed mainly in the superficial laminae of the Vc and DH in a specific manner and may be processed differently between the rostral TSN and Vc. J. Comp. Neurol. 518:687–698, 2010. © 2009 Wiley‐Liss, Inc.     

Research progress in transient receptor potential vanilloid 1 of sensory nervous system

The transient receptor potential vanilloid subfamily member 1 (TRPV1) is a protein mainly expressed in sensory neurons and fibers, such as in trigeminal ganglion and dorsal root ganglion, and has been indicated to be involved in several physiological and pathological processes. Studies on thermal activation have revealed that phosphorylation is involved in TRPV1 activation and 2 putative phosphorylation sites, Ser residues 502 (Ser-502) and Ser residues 800 (Ser-800), have been recently confirmed to possess the capability of resensitizing TRPV1. In addition to acidification, alkalization has also been proved to be a highly effective stimulator for TRPV1. TRPV1 could be regulated by various physical and chemical modulators, as well as the chronic pain. TRPV1 plays a crucial role in the transmission of pain signals, especially under inflammation and the neoplasm conditions, and it can also modulate nociceptive afferents by reinforcing morphine tolerance. The present review mainly focused on the structural and functional complexities of TRPV1, together with its activation and modulation by a wide variety of physical and chemical stimuli. Its pharmacological manipulation (sensitization/desensitization) and therapeutical targets were also discussed.     

Developmental shift of vanilloid receptor 1 (VR1) terminals into deeper regions of the superficial dorsal horn: correlation with a shift from TrkA to Ret expression by dorsal root ganglion neurons

The cloned vanilloid receptor VR1 can be activated by capsaicin and by thermal stimuli. The pattern of nerve terminals that contain VR1 in adult rat spinal cord does not correspond to axons that arise from a single subset of dorsal root ganglion neurons. Thus, we postulated that the basis underlying this complexity might be better understood from a developmental perspective. First, using capsaicin-induced hyperalgesia as a measure of VR1 function, we found that vanilloid receptors were functional as early as postnatal day 10 (P10), although hyperalgesia was of longer duration in adult. Interestingly, the appearance of VR1 protein in terminals of dorsal root ganglion neurons shifts over this postnatal period. From embryonic day 16 to P20, the majority of VR1 protein in the spinal cord was observed in lamina I. As animals matured, VR1 protein became more abundant in lamina II, particularly in the inner portion. Consistent with these observations, the number of dorsal root ganglion neurons coexpressing VR1 and isolectin B4 binding sites doubled while the number of neurons that had both VR1 and substance P remained relatively constant from P2 to P10. In peripheral processes, the number of VR1-positive nerve fibres and terminals in cutaneous structures in postnatal day 10 was half of that in adults. We also show that the association of VR1 with Ret is the reciprocal of the association of VR1 with Trk A. These results suggest that neurotrophins may regulate the extent to which populations of dorsal root ganglion neurons express VR1.