支配肺的心内神经节一氧化氮能阳性神经元的研究-神经追踪与组织化学结合法

目的 :探索大鼠支配肺的心内神经节细胞是否含有递质一氧化氮。方法 :以荧光追踪剂 /一氧化氮合酶组织化学结合技术将快兰 (fastblue ,FB)注入大鼠肺内 ,在心内神经节区切片进行一氧化氮合酶组织化学染色反应。结果 :发现部分FB标记的心内神经节细胞含递质一氧化氮 (nitrogenmonoxide,NO)。 6只动物发现FB/NO双阳性神经细胞 1 2 8个 ,占NO阳性神经细胞的 3 5± 1 8% ,散在分布于心内神经节。细胞多为卵圆及梭形。以中、小型为主。讨论 :本实验证实支配肺的部分心内神经节细胞以NO为递质 ,实现对肺的调节。     

局部皮质内注射海人酸对皮质和海马一氧化氮合酶阳性神经元的影响

本实验用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶组织化学方法结合ＧＦＡＰ免疫组化方法，对微量海人酸皮质内注射后的一氧化氮合酶阳性神经元和胶质细胞等的变化进行了研究．结果发现：注射区内的一氧化氮合欧阳性神经元很快消失；注射侧皮质和海马的旧阳性神经元和神经末梢溃变；至注射海人酸后８ｈ，这些变化波及到对侧皮质和海马．随着皮质内一氧化氮合酶阳性神经元的溃变，在注射侧皮质和海马内的一些神经元、星形胶质细胞出现新的一氧化氮合酶活性，这些新的酶活性可能是诱导型的一氧化氮合酶．这些结果表明：表达一氧化氮合酶的皮质和海马神经元对海人酸的毒性很敏感，神经细胞和非神经细胞中出现的诱导型的一氧化氮合酶可能是海人酸引起脑损伤时机体的适应性反应．     

大鼠三叉神经半月神经节内一氧化氮合酶阳性神经元的分布,投射及其与...

用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶组织化学方法观察了大鼠三叉神经半月神经节内一氧化氮合酶阳性神经元的分布。结果发现的５￣８％的节细胞呈一氧化氮合酶阳性，几乎均是中、小细胞，弥散地分布在神经节各处。结合荧光金逆行追踪技术观察，发现荧光金注入三叉神经脊束核尾侧亚核浅层后，约９０％的一氧化氮合酶阳性节细胞被逆行标记，提示半月神经节内的一氧化氮合酶阳性神经元主要投射于三叉神经脊束核尾侧亚核浅层。用一氧化     

大鼠三叉神经半月神经节内一氧化氮合酶阳性神经元的分布、投射及其与CGRP、SP的关系

用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶组织化学方法观察了大鼠三叉神经半月神经节内一氧化氮合酶阳性神经元的分布．结果发现约５～８％的节细胞呈一氧化氮合酶阳性，几乎均是中、小细胞，弥散地分布在神经节各处．结合荧光金逆行追踪技术观察，发现荧光金注入三叉神经脊束核尾侧亚核浅层后，约９０％的一氧化氮合酶阳性节细胞被逆行标记，提示半月神经节内的一氧化氮合酶阳性神经元主要投射于三叉神经脊束核尾侧亚核浅层．用一氧化氮合酶组织化学反应结合荧光金逆行追踪技术及ＣＧＲＰ、ＳＰ免疫荧光技术进行重合处理的结果显示，在向三叉神经脊束核尾侧亚核投射的一氧化氮合酶阳性半月节神经元中，约６０％呈ＣＧＲＰ样免疫阳性，约６５％呈ＳＰ样免疫阳性．结合文献及本文结果，可以认为半月神经节向三叉神经脊束核尾侧亚核浅层投射的一氧化氮合酶与ＣＧＲＰ或ＳＰ共存的神经元可能在面口部伤害性信息的传递和调控中起重要作用．     

豚鼠窦房结受一氧化氮能神经支配的形态学观察

目的：观察豚鼠窦房结受一氧化氮能神经元及神经纤维支配的形态分布方式。方法：利用硫辛酰胺脱氢酶(NADPH diaphorase，NADPH-d)还原氮蓝四唑(nitro blue tetrazolium，NBT)，生存蓝色沉淀物显示一氧化氮合酶神经。结果：(1)窦房结周围含一氧化氮能神经节主要有三处：①右心房后壁处的窦房结心外膜下；②腔耳角处心外膜下；③腔静脉窦壁处窦房结心外膜下。结内未见NADPH—d阳性神经节或神经元；(2)窦房结内见有丰富的NADPH-d阳性神经纤维，呈细束状或嘭体串珠状，由外膜编织成网状到达内膜，部分直接起源于周围NADPH—d阳性神经元。结论：豚鼠窦房结一氧化氮能神经分布丰富，作为一种特殊的递质，NO可能直接或间接的调节着窦房结冲动的产生、释放和传递。     

单眼缝合,双眼缝合对猫视觉中枢中一气体氮合酶阳怀神经元数？…

用ＮＡＤＰＨ－ｄ组织化学方法观察了在生后一周内即施行单眼缝合和双眼缝合成活至１年的猫视觉中枢（上丘表层，外膝体和视皮层１７区）中的一氧化氮合酶阳性神经元数量及形态。结果显示：（１）单眼缝合或双眼缝合并不改变上丘表层中一氧化氮合酶阳性细胞的分布模式，也不影响该神经元的数量，但单眼缝合使对侧上丘表层一氧化氮合酶阳性神经元的树突野最大半径减少，树突总长度减少；而双眼缝合可使双侧上丘中一氧化氮合酶阳性细胞     

LPS性肺损伤大鼠一氧化氮合酶表达和肺细胞凋亡变化

目的: 观察内毒素性肺损伤过程中肺细胞凋亡和一氧化氮合酶（NOS）mRNA表达的时程性变化及关系,探讨LPS性肺损伤（ALI）的发病机制。方法: 健康雄性SD大鼠48只,随机分成2组:①对照组：静注等量生理盐水；②模型组(LPS组)：静注LPS复制ALI模型,分别于给药1、3、6、9及12h后采集样品；逆转录聚合酶链反应（RT-PCR）法测定肺组织中NOSmRNA表达变化；电镜、流式细胞术检测肺细胞凋亡率；免疫组化法测定Bcl-2和Bax；光镜、电镜观察肺组织病理变化。结果: 与对照组比较，LPS组iNOSmRNA表达随时间延长而增强，给予LPS 3 h后有显著差异(P<0.05)，eNOSmRNA随时间延长而降低，给LPS 3 h时有显著差异(P<0.05)，nNOSmRNA在观察时间内没有变化；光镜和电镜下可见在观察时间内1 h起肺损伤随时间延长加重；流式细胞术显示LPS组凋亡细胞随时间延长增多，9 h达高峰，12 h有所降低；免疫组化结果显示，LPS组随时间延长Bcl-2减少，主要表现在肺上皮细胞，Bax增多；对照组无明显变化。结论: 不同一氧化氮合酶在ALI中表达强度不同；抗凋亡蛋白Bcl-2和促凋亡蛋白Bax是ALI时调节细胞凋亡的途径之一；一氧化氮合酶可能通过调节Bcl-2和Bax平衡而影响凋亡。     

大鼠腰神经根受压后背根神经节及脊髓后角神经元型一氧化氮合酶阳性神经元

目的：探讨大鼠腰神经根受压后后背根神经节和脊髓后角神经元型一氧化氮合酶阳性神经元的变化规律。方法：选用１２只Ｗｉｓｔａｒ大鼠,随机分为实验组和正常组,采用免疫组织化学ＡＢＣ法结合图像分析系统进行研究。结果：大鼠腰神经根受压后４周,实验组背根神经节中神经元型一氧化氮合酶阳性神经元细胞数、平均面积明显增多,脊髓后角神经元型一氧化氮合酶阳性神经末梢也明显增多,与正常组相比均有显著性差异。神经元形态以中、小型细胞为主。结论：大鼠腰神经根受压后感觉神经元神经元型一氧化氮合酶表达上调,提示神经元型一氧化氮合酶可能与根性腰腿痛的发生有关。     

豚鼠耳蜗缺血再灌注损伤后iNOS活性与细胞凋亡关系

目的：探讨椎基底动脉缺血再灌注耳蜗组织损伤方式及可能的损伤机制。方法：健康豚鼠72只，随机分成正常组、假手术组、模型组，每组各8只。用组织化学方法观察缺血再灌注不同时间耳蜗各部位的组织改变；用免疫组织化学法（ABC）检测各组动物耳蜗中诱导型一氧化氮合酶的表达和变化；用原位凋亡法（TUNEL法）观察耳蜗的细胞凋亡情况。结果：缺血再灌注的内外毛细胞变形缺损、血管纹变薄等；诱导型一氧化氮合酶在缺血及再灌注的表达增强；正常组及假手术组没有或仅有个别部位出现细胞凋亡，缺血及再灌注各组内外毛细胞及螺旋神经节部位凋亡细胞明显增多。结论：再灌注期间细胞凋亡数较缺血期间明显增多，其原因为包括一氧化氮在内的多种氧自由基表达增高所致。     

NG-硝基-L-精氨酸对大鼠内毒素性肺线粒体损伤的影响

目的： 观察非选择性一氧化氮合酶(NOS)抑制剂NG-硝基-L-精氨酸(L-NA)对急性肺损伤大鼠肺线粒体功能的影响，并探讨其改善急性肺损伤的作用机制。方法: 将SD大鼠随机分为空白对照组、急性肺损伤组、L-NA治疗组，采用舌静脉注射脂多糖(LPS)复制大鼠急性肺损伤模型，于大鼠急性肺损伤3h后给L-NA治疗3h，断头放血处死大鼠，迅速取出肺脏，匀浆器混匀后，低温差速离心法提取肺线粒体，测定线粒体总ATP酶、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、总一氧化氮合酶（T-NOS）、诱生型一氧化氮合酶（iNOS）、结构型一氧化氮合酶（cNOS）的活性，以及线粒体肿胀度、膜流动性和线粒体一氧化氮（NO）、丙二醛（MDA）含量；电镜观察大鼠肺线粒体超微结构的改变及治疗药对此改变的影响。结果: 在大鼠内毒素性急性肺损伤后，肺脏组织中线粒体表现为肿胀、膜流动性降低，线粒体中的T-NOS和iNOS活性显著升高，线粒体NO生成明显增加，而cNOS活性无明显变化；线粒体总ATP酶、SOD、GSH-Px活性均明显下降，线粒体MDA含量明显升高。急性肺损伤3h给予L-NA治疗3h,与急性肺损伤组相比，一氧化氮合酶活性有所改变，NO生成显著下降，总ATP酶、SOD、GSH-Px活性均显著升高，MDA含量下降。电镜结果显示内毒素性急性肺损伤后肺脏组织细胞水肿，线粒体肿胀、嵴断裂、溶解、消失；L-NA能改善内毒素性急性肺损伤引起的细胞水肿、线粒体肿胀和空泡化。结论: L-NA能明显抑制急性肺损伤后线粒体一氧化氮合酶活性，减少NO生成，改善线粒体能量供应，增加线粒体抗氧化作用，从而减轻急性肺损伤。     

Nitric oxidergic neurons of the respiratory organs

Using histochemical NADPH-diaphorase reaction NO-ergic neurons distribution in human tracheobronchial tree was studied in man and mammals. Psedounipolar sensory neurons forming microganglia mark in adventitia of the trachea posterior surface from superior border up to trachea bifurcation. It was suggested that protoneurons are the constant structural formations of respirator organs. Their capacity to exert effector influence on the tissue through axon-reflex mechanism, apart from their sensory function is under discussion.     

NO-ergic rat brain commissural neurons in the norm and during opiate administration

Using histochemical reaction demonstrating NADPH-diaphorase (NADPH-d), the dynamics of NO synthesis was studied in the rat brain raphe nuclei following intravenous injection of morphine hydrochloride. In normal conditions NADPH-d activity was demonstrated in neurons of all raphe nuclei. Acute and chronic administration of morphine in different doses (0.5 mg/kg and 5 mg/kg) was found to inhibit NO-ergic activity of the major part of raphe nuclei neurons. The depression of NADPH-d activity was unequal in different nuclei. The NO-ergic changes are caused by an activation of opiate receptors, as they depend on morphine dose, while the application of opiate antagonist naloxone restores the NO-ergic function of raphe neurons. Formation of tolerance to opiate analgetic effect is accompanied by a significant, though short-lasting increase of NO synthesis activity. It is suggested that the changes in NO-ergic function of raphe neurons may influence brain serotonin balance after opiate administration.     

Structure and neurochemistry of the raphe nuclei in teleosts

NO is an important modulator of the brain serotoninergic function as it is co-localized with 5-hydroxytryptamine in the neurons of raphe nuclei in the brain of all the higher vertebrates. In the brain of fishes this property of NO is poorly studied. The purpose of this work was to study the neuromorphological peculiarities and distribution of NO-ergic neurons in raphe nuclei of Teleost species--carps and perch-like fishes. Using standard neuromorphological methods and the histochemical reaction demonstrating NADPH-diaphorase activity, the comparative analysis of NO-ergic neurons was performed and their localization was studied in the raphe complex of five teleost species. Within the raphe complex in fishes two main nuclei--nucleus raphe superior and nucleus raphe inferior were demonstrated. NADPH-diaphorase activity was observed in practically all the neurons of raphe niclei in species examined. Some intergroup morpho-adaptational neurochemical differences were found in the of the ventro-medial reticular formation adjacent to the raphe complex.     

No-Ergic Neurons of the Cervical Nucleus of the Rat Brain in Normal Conditions and After Administration of Opiates

Histochemical reactions for NADPH-diaphorase (NADPH-d) were used to study the dynamics of NO synthesis activity in the cervical nuclei of the rat brain after i.v. administration of morphine hydrochloride. In normal conditions, NADPH-d activity was found in neurons in all the cervical nuclei. Acute and chronic administration of morphine at different doses (0.5 and 5 mg/kg) suppressed the NO-ergic activity of most cervical neurons. Decreases in the level of NADPH-d activity were different in different nuclei. NO-ergic changes were due to activation of opiate receptors, as they were dependent on the dose of morphine used, and treatment with the opiate antagonist naloxone restored the NO-ergic function of cervical neurons. Formation of tolerance to the analgesic effect of opiates was accompanied by significant but short-lived increases in NO synthesis activity. It is suggested that changes in the NO-ergic functions of cervical neurons may affect the balance of serotonin in the brain during opiate treatment.     

Incipient cauda equina syndrome as a model of somatovisceral pain in dogs: spinal cord structures involved as revealed by the expression of c-fos and NADPH diaphorase activity

Segmental and laminar distribution of Fos-like immunoreactive, reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting and double-labeled (Fos-like immunoreactive and NADPHd-exhibiting) neurons was examined in lower lumbar and sacral segments of the dog spinal cord using the model of multiple cauda equina constrictions. NADPHd histochemistry was used as marker of nitric oxide synthase-containing neurons. The appearance and the time-course of Fos-like immunoreactive, NADPHd and double-labeled neurons was studied at 2 h and 8 h postconstriction characterized as the incipient phase of cauda equina syndrome. The occurrence of Fos-like immunoreactive and NADPHd-exhibiting neurons in fully developed cauda equina syndrome was studied at five days postconstriction. An increase in Fos-like immunoreactivity in superficial laminae (I–II) and an enhanced NADPHd staining of lamina VIII neurons were found. A statistically significant increase in Fos-like immunoreactive neurons was found in laminae I–II and VIII–X 8 h postconstriction, and in contrast, a prominent decrease in Fos-like immunoreactive neurons was found in laminae I–II, accompanied by a statistically significant increase in Fos-like immunoreactive neurons in more ventrally located laminae VII–X at five days postconstriction. Quantitative analysis of laminar distribution of constriction-induced NADPHd-exhibiting neurons revealed a considerable increase in these neurons in laminae VIII–IX 8 h postconstriction and a statistically highly significant increase in NADPHd-exhibiting neurons in laminae VII–X five days postconstriction. Concurrently, the number of NADPHd-exhibiting neurons in laminae I–II was greatly reduced. While a low number of double-labeled neurons was found throughout the gray matter of lower lumbar and sacral segments at 2 h postconstriction, a statistically significant number of double-labeled neurons was found in lamina X 8 h and in laminae VII–X five days postconstriction. The course and distribution of anterograde degeneration resulting five days after multiple cauda equina constrictions are compared with segmental and laminar distribution of Fos-like immunoreactive and NADPHd-exhibiting neurons.Prominent involvement of the spinal cord neurons appearing in the lumbosacral segments at the early beginning and in fully developed cauda equina syndrome results in a Fos-like immunoreactivity and strongly enhanced NADPHd staining of some neuronal pools. Under such circumstances, an early cauda equina decompression surgery is advisable aimed at decreasing or preventing the derangement of the neural circuits in the lumbosacral segments.     

Incipient cauda equina syndrome as a model of somatovisceral pain in dogs: spinal cord structures involved as revealed by the expression of c-fos and NADPH diaphorase activity

Segmental and laminar distribution of Fos-like immunoreactive, reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting and double-labeled (Fos-like immunoreactive and NADPHd-exhibiting) neurons was examined in lower lumbar and sacral segments of the dog spinal cord using the model of multiple cauda equina constrictions. NADPHd histochemistry was used as marker of nitric oxide synthase-containing neurons. The appearance and the time-course of Fos-like immunoreactive, NADPHd and double-labeled neurons was studied at 2 h and 8 h postconstriction characterized as the incipient phase of cauda equina syndrome. The occurrence of Fos-like immunoreactive and NADPHd-exhibiting neurons in fully developed cauda equina syndrome was studied at five days postconstriction. An increase in Fos-like immunoreactivity in superficial laminae (I-II) and an enhanced NADPHd staining of lamina VIII neurons were found. A statistically significant increase in Fos-like immunoreactive neurons was found in laminae I-II and VIII-X 8 h postconstriction, and in contrast, a prominent decrease in Fos-like immunoreactive neurons was found in laminae I-II, accompanied by a statistically significant increase in Fos-like immunoreactive neurons in more ventrally located laminae VII-X at five days postconstriction. Quantitative analysis of laminar distribution of constriction-induced NADPHd-exhibiting neurons revealed a considerable increase in these neurons in laminae VIII-IX 8 h postconstriction and a statistically highly significant increase in NADPHd-exhibiting neurons in laminae VII-X five days postconstriction. Concurrently, the number of NADPHd-exhibiting neurons in laminae I-II was greatly reduced. While a low number of double-labeled neurons was found throughout the gray matter of lower lumbar and sacral segments at 2 h postconstriction, a statistically significant number of double-labeled neurons was found in lamina X 8 h and in laminae VII-X five days postconstriction. The course and distribution of anterograde degeneration resulting five days after multiple cauda equina constrictions are compared with segmental and laminar distribution of Fos-like immunoreactive and NADPHd-exhibiting neurons. Prominent involvement of the spinal cord neurons appearing in the lumbosacral segments at the early beginning and in fully developed cauda equina syndrome results in a Fos-like immunoreactivity and strongly enhanced NADPHd staining of some neuronal pools. Under such circumstances, an early cauda equina decompression surgery is advisable aimed at decreasing or preventing the derangement of the neural circuits in the lumbosacral segments.     

Nitric oxide synthase distribution in the cat superior colliculus and co-localization with choline acetyltransferase

Nitric oxide and acetylcholine are important neuromodulators implicated in brain plasticity and disease. We have examined the cellular and fiber localization of nitric oxide in the cat superior colliculus (SC) and its degree of co-localization with ACh using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and an antibody to neuronal nitric oxide synthase. ACh was localized using an antibody against choline acetyltransferase. We also made injections of biocytin into the region of the parabrachial brainstem to confirm that this region is a source of nitric oxide containing fibers in SC. NADPHd labeled neurons within the superficial layers of the superior colliculus included pyriform, vertical fusiform, and horizontal morphologies. Labeled neurons in the intermediate gray layer were small to medium in size, and mostly of stellate morphology. Neurons in the deepest layers had mostly vertical or stellate morphologies. NADPHd labeled fibers formed dense patches of terminal boutons within the intermediate gray layer and streams of fibers within the deepest layers of SC. Choline acetyltransferase antibody labeling in adjacent sections indicated that many fibers must contain both labels. Over 94% of neurons in the pedunculopontine tegmental and lateral dorsal tegmental nuclei were also labeled by both NADPHd and choline acetyltransferase. In addition, biocytin labeled fibers from this region were localized in the NADPHd labeled patches. We conclude that nitric oxide is contained in a variety of cell types in SC and that both nitric oxide and ACh likely serve as co-modulators in this midbrain structure.     

Reduced number of intrinsic pulmonary nitrergic neurons in Fawn-Hooded rats as compared to control rat strains

The Fawn-Hooded rat (FHR) strain reveals a congenital predisposition to primary (idiopathic) pulmonary hypertension (PPH), and can therefore be regarded as an animal model in which to study possible mechanisms underlying an inherited susceptibility to pulmonary hypertension. Pulmonary hypertension can be induced in FHRs after a short exposure to mild hypoxia, presumably because of an altered peripheral oxygen sensitivity. Given the presence of pulmonary nitrergic neurons in rat lungs, the observed link between airway hypoxia and the expression of pulmonary neuronal nitric oxide synthase (nNOS), and the fact that nNOS appears to be involved in peripheral chemoreceptor sensitivity, we examined the intrinsic pulmonary nitrergic innervation in the FHR. In the present study the number of intrapulmonary nitrergic nerve cell bodies, detected by NADPH diaphorase (NADPHd) histochemistry, was quantified in the FHR and three control rat strains. Compared to the control rat strains, the FHR lungs revealed a highly significantly lower number of intrinsic nitrergic neurons, while no apparent differences were found in the number of enteric nitrergic neurons in the esophagus. In conclusion, the possible links between neuronal NO, hypersensitivity to airway hypoxia, and the development of PPH clearly deserve further investigation.     

Dynamics of NADPH Diaphorase Activity in Raphe Neurons during Chronic Treatment with Opiates

Systemic administration of diacetylmorphine considerably reduced the number of NADPH diaphorase-positive (NO-synthesizing) neurons in rat brain raphe nuclei. This effect was blocked by naloxone. In animals with the withdrawal syndrome NO-ergic activity in raphe neurons increased and surpassed the normal.