电刺激猫上矢状窦区硬脑膜对三叉神经二级神经元兴奋性的影响

目的通过电刺激清醒状态下猫上矢状窦区硬脑膜，制备反应可靠的偏头痛反复发作动物模型，探索偏头痛等血管源性头痛的发病机制。方法体重为2-4kg的短毛猫12只随机分为反复刺激组（n=4）、急性刺激组（n=4）、假手术对照组（n=4）三个组。反复刺激组每天刺激2h，共刺激8d；急性刺激组刺激1次，2h；假手术对照组不予刺激。之后应用免疫组化技术，行延髓和上颈髓冰冻切片c-fos染色。结果c-fos免疫反应阳性神经元主要位于延髓三叉神经脊束核尾侧部浅层和C1后角的Ⅰ、Ⅱ层。假手术对照组动物免疫反应阳性神经元较少，急性刺激组和反复刺激组免疫反应阳性神经元较假手术对照组显著增多（P〈0．05）．急性刺激组和反复刺激组免疫反应阳性神经元数无显著差异（P〉0．05）。结论刺激上矢状窦区硬脑膜可激活在偏头痛痛觉中枢传递中起重要作用的三叉神经二级神经元。     

中脑导水管周围灰质在大鼠血管源性头痛模型中的作用

目的研究中脑导水管周围灰质(PAG)在血管源性头痛如偏头痛涉及的伤害觉信息的传递中的作用。方法以雄性SD大鼠(体重为220~250g)为实验对象,在手术暴露其上矢状窦(SSS)后电刺激SSS区硬脑膜制作血管性头痛的动物模型;应用免疫组织化学染色技术,观察中脑PAG原癌基因蛋白质c-fos(Fos)和一氧化氮合酶(NOS)表达的变化。结果Fos免疫反应阳性神经元和NOS免疫反应阳性神经元主要位于中脑PAG的腹外侧区,阳性细胞数头侧至尾侧逐渐增多。空白对照组、假手术对照组、刺激组每张切片的Fos阳性神经元数分别为7.2±4.2、13.6±4.3、76.0±12.3;NOS阳性神经元数分别为35.0±3.5、42.3±4.2、162.0±11.6。结论刺激大鼠SSS区硬脑膜可激活PAG,提示PAG不但参与对于伤害性感觉信息传入后的下行调节,还通过上行投射纤维与疼痛中枢丘脑发生联系。PAG可能参与血管源性头痛如偏头痛的痛觉中枢调控。     

颈交感神经对中脑导水管周围灰质信号表达的影响

目的：通过电刺激大鼠上矢状窦区硬脑膜，观察颈上交感神经节（SCG）摘除术前、后中脑导水管周围灰质（PAG）一氧化氮合酶（NOS）阳性神经元数日的变化，以探讨交感神经系统在血管源性头痛如偏头痛伤害性信息传递巾的作用。方法：以雄性SD大鼠行颈上交感神经节摘除术后再手术暴露其上矢状窦（SSS），电刺激SSS区硬脑膜，应用免疫组织化学染色技术，观察PAG区域NOS表达的变化。结果：NOS免疫反应阳性神经元主要分布在PAG的腹外侧区，双侧对称。SCG摘除组NOS阳性神经元数目较假手术组明显增加。结论：颈交感神经系统通过PAG对痛觉的中枢调整参与了血管源性头痛如偏头痛中伤害性感觉信息的产生、传导及调节过程。     

颈交感神经对血管源性头痛痛觉传导的影响

目的采用电刺激大鼠上矢状窦(SSS)区硬脑膜,观察颈上交感神经节(SCG)摘除术前后在延髓和上颈段三叉神经脊束核一氧化氮合酶(NOS)阳性神经元数目的变化,以探讨交感神经系统在血管源性头痛(如偏头痛)涉及的伤害觉信息传递中的作用。方法以雄性SD大鼠(体重为220～250g)为实验对象,行颈上交感神经节摘除术后再手术暴露其SSS,然后电刺激SSS区硬脑膜,应用免疫组织化学染色技术,观察延髓和上颈段三叉神经脊束核NOS表达的变化。结果NOS免疫反应阳性神经元主要分布在三叉神经脊束核和上颈段脊髓的第Ⅰ、Ⅱ板层,双侧对称。假手术对照组、SCG摘除组每张切片的NOS阳性神经元数分别为150.2±10.3、223.0±11.6,SCG摘除组NOS阳性神经元数目较假手术对照组明显增加(P<0.05)。结论颈交感神经系统参与了头部血管源性疼痛(如偏头痛)中伤害性感觉信息导致的疼痛的产生、传导及调节过程。     

5-HT1D受体调节大鼠偏头痛模型三叉神经二级神经元兴奋性

目的探索5-HT1D受体在偏头痛发病中的中枢角色.方法应用大鼠电刺激偏头痛模型,观察给予佐米曲普坦和BRL15572后三叉神经脊束核尾段和上颈髓背角的c-fos表达变化.结果佐米曲普坦明显抑制电刺激诱发的三叉神经脊束核尾段和上颈髓背角浅层神经元c-fos表达,BRL15572可拮抗佐米曲普坦的大部分抑制效应,单独BRL15572对c-fos表达无影响.结论5-HT1D受体在偏头痛模型中是调节三叉神经二级神经元兴奋的主要受体,曲坦类药物通过5-HT1D受体抑制该神经元伤害性兴奋.     

舒马曲坦对偏头痛模型大鼠中脑导水管周围灰质区核转录因子-κB表达的影响

目的 探讨舒马曲坦治疗偏头痛的作用机制.方法 应用电刺激大鼠上矢状窦区硬脑膜制作偏头痛动物模型,观察给予舒马曲坦后中脑导水管周围灰质核转录因子-κB(NF-κB)蛋白表达的变化.结果 空白组、假手术组、电刺激组、舒马曲坦组、生理盐水对照组每张切片的NF-κB阳性神经元计数分别为111.7±15.7、112.9±10.7、508.7±30.8、179.5±14.9、497.8±20.6.刺激组与空白组、假手术组及舒马曲坦组比较,差异有统计学意义(F=944.78,P<0.05).结论 电刺激大鼠上矢状窦区硬脑膜后,在中脑导水管周围灰质区出现了NF-κB细胞的激活,舒马曲坦可使其表达降低.     

电刺激三叉神经节对蝶腭神经节内一氧化碳合酶和血管活性肠肽阳性神经元的影响

目的：观察实验性偏头痛大鼠副交感神经蝶腭神经节内一氧化氮合酶（NOS）和血管活性肠肽（VIP）阳性神经元的变化。方法：12只雄性SD大鼠随机分为实验组和对照组（均n=6）。实验组为电刺激三叉神经节建立的偏头痛大鼠模型。对照组仅作手术而不刺激三叉神经节。用组织化学的方法观察蝶腭神经节内NOS阳性神经元的变化,用免疫荧光法观察蝶腭神经节内V1P阳性神经元的变化。结果：实验组和对照组大鼠的蝶腭神经节内均有NOS和VIP阳性神经元,但实验组NOS和VIP阳性神经元均较对照组显著增加（P〈0．01）。结论：电刺激三叉神经节可以通过三叉-副交感神经反射系统,显著升高蝶腭神经节中NOS和VIP神经元的数目。偏头痛发病过程中脑膜及颅内大血管的剧烈扩张很可能与蝶腭神经节中NOS和VIP神经元增加有关。     

刺激大鼠上矢状窦区硬脑膜对中脑导水管周围灰质核因子-κB蛋白表达的影响

目的 探讨核因子(NF)-κB蛋白在偏头痛痛觉信息的传递中的作用.方法 雄性SD大鼠,手术暴露其上矢状窦区(superior sagittal sinus,SSS)后电刺激该区硬脑膜,应用免疫组织化学染色技术观察中脑导水管(PAG)周围灰质NF-κB蛋白表达的变化.结果 空白组、假手术组、刺激组每张切片PAG区的NF-κB蛋白阳性神经元数分别为111.7±15.7、112.9±10.7、508.7±30.8,刺激组较其他组差异有统计学意义(t=-41.52,t=-36.21,均P＜0.05).结论 在PAG区出现NF-κB细胞的激活,表明NF-κB蛋白在偏头痛的发生及发展过程中起到了一定作用.     

天舒胶囊对偏头痛动物模型血浆一氧化氮、一氧化氮合酶、降钙素基因相关肽含量及血流动力学的影响

目的 研究天舒胶囊对偏头痛动物模型血浆及脑组织血管活性物质及血流动力学的影响.方法 皮下注射硝酸甘油分别制作大鼠和兔偏头痛模型.给予天舒胶囊后用放免法和分光光度法测大鼠血浆一氧化氮(NO)和一氧化氮合酶(NOS)、降钙素基因相关肽(CGRP)含量;通过免疫组织化学染色观察三叉神经脊束核神经元型NOS(NOS1)和CGRP表达;用经颅多普勒检测兔颈内动脉血流速度改变.结果 模型组大鼠血浆NO、NOS和CGRP较对照组明显升高;经不同剂量天舒胶囊灌胃后大鼠血浆NO、NOS和CGRP的增加受到抑制,尤以中、高剂量组明显(P<0.05～0.01).模型组兔颈内动脉收缩期峰值流速明显下降,经中剂量天舒胶囊干预后流速下降也受到抑制 (P<0.05).免疫组织化学染色发现灌胃天舒胶囊后,偏头痛大鼠三叉神经脊束核NOS1和CGRP表达增加的程度减小(均P<0.05).结论 天舒胶囊可改善偏头痛发作时血管活性物质和神经递质水平失常,从而缓解偏头痛症状.     

依达拉奉对慢性脑缺血大鼠一氧化氮及一氧化氮合酶阳性神经元数量的影响

目的观察依达拉奉对脑缺血大鼠脑组织NO含量及NOS阳性神经元数量的影响,为临床上防治慢性脑缺血引发的疾病提供指导。方法 54只Wistar大鼠,分为NO含量组和NOS阳性神经元组,每组再分为模型组、治疗组、假手术组,利用结扎大鼠两侧颈总动脉制作大鼠慢性脑缺血模型,硝酸还原酶法测NO含量,NADPH-d组织化学方法染色NOS阳性神经元,光镜下观察。结果治疗组各时间点NO含量和NOS阳性神经元数量较模型组减少。结论依达拉奉对慢性脑缺血大鼠脑组织具有保护作用。     

Stress- and lipopolysaccharide-induced c-fos expression and nNOS in hypothalamic neurons projecting to medullary raphe in rats: a triple immunofluorescent labeling study

Neurons in the rostral raphe pallidus (rRP) have been proposed to mediate experimental stress-induced tachycardia and fever in rats, and projections from the dorsomedial hypothalamus (DMH) may signal their activation in these settings. Thus, we examined c-fos expression evoked by air jet/restraint stress and restraint stress or by systemic administration of lipopolysaccharide (10 microg/kg and 100 microg/kg) as well as the distribution of the neuronal nitric oxide synthase (nNOS) in neurons retrogradely labeled from the raphe with cholera toxin B in key hypothalamic regions. Many neurons in the medial preoptic area and the dorsal area of the DMH were retrogradely labeled, and approximately half of those in the medial preoptic area and moderate numbers in the dorsal DMH were also positive for nNOS. Either stress paradigm or dose of lipopolysaccharide increased the number of c-fos-positive neurons and nNOS/c-fos double-labeled neurons in all regions examined. However, retrogradely labeled neurons positive for c-fos were increased only in the dorsal DMH and adjoining region in both stressed and lipopolysaccharide-treated groups, and triple-labeled neurons were found only in this area in rats subjected to either stress paradigm. Thus, hypothalamic neurons that project to the rRP and express c-fos in response to either experimental stress or systemic inflammation are found only in the dorsal DMH, and many of those activated by stress contain nNOS, suggesting that nitric oxide may play a role in signaling in this pathway.     

Nitric oxide synthase inhibition lowers activity of neurons with meningeal input in the rat spinal trigeminal nucleus

Nitric oxide is thought to control transmitter release and neuronal activity in the spinal dorsal horn and the spinal trigeminal nucleus, where nociceptive information from extra- and intracranial tissues is processed. Extracellular impulse activity was recorded from neurons in the rat spinal trigeminal nucleus with afferent input from the cranial dura mater. In contrast to the inactive isomer D-NAME, infusion of the nitric oxide synthase inhibitor L-NAME (20 mg/kg) significantly reduced neuronal activity and increased systemic blood pressure. It is concluded that nitric oxide production contributes to the ongoing activity of sensitized neurons in the spinal trigeminal nucleus. The results suggest that nitric oxide may be involved in the generation and maintenance of primary headaches such as migraine.     

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat spinal cord

Nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) compose a cyclic pathway to form nitric oxide (NO). These enzymes, however, are localized differentially in most regions of the brain. To find out whether NOS, ASS, and ASL are colocalized in neurons of the spinal cord, we examined the distribution of these enzymes by using a double-labeling procedure combining fluorescent immunohistochemistry with an assay for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). Results indicate that neurons in the dorsal horn, the intermediolateral nucleus, and the central canal region were NADPH-d active (+) and NOS-, ASS-, and ASL-like immunoreactive (-LI). In laminae II and III of the dorsal horn, some NADPH-d (+) neurons were ASL-LI (8–30%) but only a few were ASS-LI (0.5–7%). In the nucleus intermediolateralis, a large portion of NADPH-d (+) neurons were ASL-LI (30–60%), whereas only a small portion of NADPH-d (+) neurons were ASS-LI (10–20%). In the central canal region, some NADPH-d (+) neurons were ASL-LI (15–40%), and a few NADPH-d (+) neurons were ASS-LI (3–16%). Thus, the results suggest that, in the nucleus intermediolateralis and the central canal region, NOS, ASS, and ASL are colocalized and form a cyclic pathway to produce NO, whereas, in the dorsal horn, these enzymes are more characteristically localized in different neurons, which may transport the substrates intercellularly. J. Comp. Neurol. 385:616–626, 1997. © 1997 Wiley-Liss, Inc.     

Coexistence of NADPH diaphorase with GABA,glycine, and acetylcholine in rat spinal cord

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred and twenty-four NADPH diaphorase-positive neurons in midlumbar spinal cord from four rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motorneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies. These results suggest that NADPH diaphorase is largely restricted to GABAergic neurons in the lumbar spinal cord, and that it is mainly present in those neurons in which GABA coexists with glycine or acetylcholine. Since nitric oxide has been implicated in pain processing and hyperalgesia, while GABA, glycine, and acetylcholine are thought to be involved in analgesia and prevention of hyperalgesia, it is likely that nitric oxide synthase-containing GABAergic neurons in dorsal horn have dual actions in transmission of nociceptive information. © 1993 Wiley-Liss, Inc.     

Capsaicin augments synaptic transmission in the rat medial preoptic nucleus

Functional imaging studies and clinical evidence suggest that structures in the brainstem contribute to migraine pathophysiology with a strong association between the brainstem areas, such as periaqueductal gray (PAG), and the headache phase of migraine. Stimulation of the superior sagittal sinus (SSS) in humans evokes head pain. Second-order neurons in the trigeminal nucleus that are activated by SSS stimulation can be inhibited by PAG stimulation. The present study was undertaken to identify pontine and medullary structures that respond to noxious stimulation of the superior sagittal sinus or to ventrolateral PAG stimulation. The distribution of neurons expressing the protein product (fos) of the c-fos immediate early gene were examined in the rostral medulla and caudal pons of the cat after (i) sham, (ii) stimulation of the superior sagittal sinus, (iii) stimulation of the superior sagittal sinus with PAG stimulation, or (iv) stimulation of the PAG alone. The structures examined for fos were the trigeminal nucleus, infratrigeminal nucleus, reticular nuclei, nucleus raphe magnus, pontine blink premotor area, and superior salivatory nucleus. Compared with all other interventions, fos expression was significantly greater in the trigeminal nucleus and superior salivatory nucleus after SSS stimulation. After PAG with SSS stimulation, on the side ipsilateral to the site of PAG stimulation, fos was significantly greater in the nucleus raphe magnus. These structures are likely to be involved in the neurobiology of migraine.     

Systemic nitroglycerin increases nNOS levels in rat trigeminal nucleus caudalis

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraineurs a delayed attack of unknown mechanisms. Subcutaneous nitroglycerin (10 mg/kg) produced a significant increase of nitric oxide synthase (NOS)- and c-fos-immunoreactive neurons in the cervical part of trigeminal nucleus caudalis in rats after 4 h. This effect was not observed in the thoracic dorsal horn. Similar increase of NOS and c-fos was obtained in the brain stem after a somatic nociceptive stimulus, i.e. on the side of the formalin injection in the lip. Nitric oxide is thus able to increase NOS availability in second order nociceptive trigeminal neurons, which may be relevant for central sensitization and the understanding of its effect in migraine.     

Blockade of Nav1.8 Currents in Nociceptive Trigeminal Neurons Contributes to Anti-trigeminovascular Nociceptive Effect of Amitriptyline

Amitriptyline (AMI), a tricyclic antidepressant, has been widely used to prevent migraine attacks and alleviate other various chronic pain, but the underlying mechanism remains unclear. Accumulated evidence suggests that the efficacy of AMI is related to the blockade of voltage-gated sodium channels. The aim of the present study was to investigate the effect of AMI on Na_v1.8 currents in nociceptive trigeminal neurons and trigeminovascular nociception induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus (SSS) in rats, as in the animal model of vascular headaches such as migraines. Using a whole-cell voltage recording technique, we showed that Na_v1.8 currents were blocked by AMI in a concentration-dependent manner, with an IC₅₀ value of 6.82 μM in acute isolated trigeminal ganglion neurons of the rats. AMI caused a hyperpolarizing shift in the voltage-dependent activation and steady-state inactivation and significantly blocked in a use-dependent manner and slowed the recovery from the inactivation of Na_v1.8 currents. In addition, the systemic administration of AMI and A-803467 (a selective Na_v1.8 channel blocker) potently alleviated the nociceptive behaviors (head flicks and grooming) induced by the electrical stimulation of the dura mater surrounding the SSS. Taken together, our data suggest that Na_v1.8 currents in nociceptive trigeminal neurons are blocked by AMI through modulating the activation and inactivation kinetics, which may contribute to anti-nociceptive effect of AMI in animal models of migraines.