一氧化氮合酶在并发癫(癎)的脑膜瘤中的表达及其生物学意义

目的探讨一氧化氮在脑膜瘤并发癫癇的发病机制中的作用。方法sABC法研究一氧化氮合酶的3种亚型在并发术前癫癇的脑膜瘤患者的肿瘤切除标本中的表达。结果脑膜瘤的组织切片中。nNOS呈散在分布,eNOS沿肿瘤内的血管壁分布,iNOS则呈点状或片状分布;按WHO的脑膜瘤恶性程度分类标准,nNOS和iNOS的表达有显著性差异;按WH0的脑膜瘤组织病理分类标准,eNOS在血管瘤型脑膜瘤中的表达与其在其他组织病理类型的脑膜瘤的表达之闻有显著性差异;按脑膜瘤的不同原发部位划分。nNOS的表达在额叶、顸叶和颞叶有显著性差异,iNOS的表达在各部位均有显著性差异。结论NO参与脑膜瘤术前并发癫癇的发作病理过程。iNOS是诱发脑膜瘤术前并发癫癇的内源性NO的主要合酶,nNOS增强iNOS的诱导作用。     

一氧化氮合酶在继发癫痫的脑膜瘤中表达及其生物学意义

目的探讨一氧化氮在脑膜瘤继发癫痫的发病机制中的作用。方法运用SABC免疫组化方法研究了一氧化氮合酶三种亚型在继发术前癫痫的脑膜瘤标本中的表达。结果在实验组与对照组脑膜瘤的组织切片中,诱生型一氧化氮合酶(iNOS)表达有非常显著性差异(P<0.01),神经元型一氧化氮合酶(nNOS)的表达有显著性差异(P<0.05),内皮组织型一氧化氮合酶(eNOS)的表达没有统计学意义(P>0.05)。结论一氧化氮参与了脑膜瘤继发的术前癫痫发作病理过程,iNOS是诱发脑膜瘤继发术前癫痫的内源性一氧化氮的主要合酶,nNOS可以增强iNOS的诱导作用。     

一氧化氮和一氧化氮合酶在大鼠局灶性脑缺血中的表达特点

目的建立脑缺血SD大鼠模型,探讨一氧化氮(NO)和一氧化氮合酶(NOS)在脑缺血模型中的表达特点。方法应用线栓法制作大脑中动脉阻塞(MCAO)局灶性脑缺血模型,根据缺血不同时间分为8组,设立假手术组和正常对照组,每组各有6只大鼠。应用硝酸还原酶法测定脑组织NO的含量,流式细胞术(FCM)定量检测硝基酪氨酸(NT)表达,化学比色法测定脑组织NOS的活性,免疫组织化学方法定位检测eNOS、nNOS和iNOS表达位置,逆转录反应系统(RT-PCR)半定量分析eNOS、nNOS和iNOS的 mRNA在脑缺血区域的表达。结果神经功能缺失评分发现缺血时间越长,神经功能缺失越明显;脑组织中NO含量与缺血时间正相关;缺血1h后NT阳性细胞百分比开始明显升高(9.50%);缺血0.5h时NOS的活性开始升高,缺血3d达到高峰[0.94nmol/(g.min)];免疫组织化学提示eNOS在神经细胞和血管内皮细胞胞浆均有表达,nNOS和iNOS抗体主要在神经细胞胞浆中表达;RT-PCR半定量分析在缺血早期(0.5h~6h),随着缺血时间延长,eNOS和nNOS表达增加,iNOS未见表达或低表达;缺血中晚期(6h~5d),iNOS高表达,并与缺血时间呈正相关,eNOS和nNOS表达明显减少。结论脑缺血时间延长,NOS的活性升高,NO在体内特异性代谢产物NT量增加,神经功能缺失越明显;NOS在缺血早期以eNOS和nNOS为主,在缺血晚期以iNOS为主。     

热射病大鼠中eNOS和iNOS基因的表达

目的研究热射病大鼠中内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)基因的表达。方法构建经典型和劳力型热射病大鼠模型,利用实时荧光定量PCR方法检测不同热射病大鼠模型的脑、肝、肾和心脏组织中eNOS和iNOS基因的表达情况。结果 2种热射病大鼠中,eNOS和iNOS mRNA在脑、肝、肾和心脏组织中的表达量均高于对照组;eNOS mRNA在劳力型热射病大鼠各器官中的表达量高于经典型热射病大鼠(P0.05);iNOS mRNA在经典型热射病大鼠的脑、肝和肾组织中的表达量低于劳力型热射病大鼠(P0.05),在心脏组织中,二者无显著性差异(P0.05)。结论eNOS和iNOS mRNA的上调表达在热射病发病机制中发挥重要作用。     

一氧化氮合酶及其抑制剂与脑缺血

一氧化氮合酶(NOS)在脑缺血中具有双重作用,nNOS介导缺血早期神经元损伤,iNOS介导缺血晚期神经元损伤,eNOS则介导神经保护作用。对NOS抑制剂,尤其是选择性nNOS和iNOS抑制剂的研究,无疑将为缺血性脑损伤的治疗提供新途径。     

一氧化氮合酶在脑缺血再灌注中的双重作用

目的 探讨短暂脑缺血再灌注后大鼠脑内3型一氧化氮合酶(nitric oxide synthase，NOS)的表达及作用，为脑缺血治疗提供理论依据。方法 采用免疫组织化学方法，用3型NOS的多克隆抗体检测大鼠局灶性脑缺血2h再灌注15min及22h NOS在脑内的表达情况。结果 大鼠脑缺血2h再灌注15min，在脑缺血边缘区的血管壁及神经细胞出现内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)上调表达；脑缺血2h再灌注22h，在脑梗死区内表达神经元型一氧化氮合酶(neuronal mitric oxide synthase，nNOS)的神经细胞减少，并出现表达诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)的胶质细胞，同时梗死边缘区血管及神经细胞出现eNOS及iNOS的上调表达。结论 在短暂脑缺血再灌注早期，缺血区周围可能有eNOS相关的保护机制；亚急性期eNOS及iNOS的保护及损伤机制并存；因此，在短暂脑缺血早期恢复灌注后予选择性iNOS抑制剂及促进eNOS活性有可能减少迟发性神经损伤。     

一氧化氮与癫癎的研究进展

癫癎是严重危害人类健康的常见病、多发病,引起癫(?)的病理机制复杂。目前一氧化氮(NO)参与癫(?)的发作已得到公认,但具体机制不明,现就NO与癫(?)的最新研究进展综述如下。1 NO的生物学特性 NO是L精氨酸(L-Arg)在一氧化氮合酶(NOS)作用下生成的,NOS主要有三种类型:Ⅰ型神经元型NOS(nNOS);Ⅱ型诱导型NOS(iNOS);Ⅲ型内皮型NOS(eNOS),nNOS和eNOS属于结构型NOS(cNOS),依赖ca2 或钙调蛋白,快速短暂生成NO。研究     

一氧化氮与Alzheimer病(综述)

目的 概述了在Alzheimer病（AD）脑内一氧化氮（NO）可能介导胶质细胞激活后对神经元的损伤，参与炎性变化的毒性机制。诱导型一氧化氮合酶（iNOS）在AD脑的胶质细胞和微血管内高表达，同时又在神经元内出现。而反应性胶质细胞内既有iNOS的激活又有神经元型NOS（nNOS）激活，共同产生过量的、具有神经毒性的NO。     

大剂量伽玛刀照射后脑组织一氧化氮合酶亚型表达改变及其与急性脑水肿的关系

目的探讨大剂量伽玛刀(γ刀)照射后脑组织一氧化氮合酶(NOS)亚型表达改变及其与急性脑水肿的关系。方法200Gy量γ刀照射正常大鼠脑,采用光镜、电镜、免疫组化及原位杂交技术研究照射后急性脑水肿的发生发展及脑组织NOS亚型表达变化。结果①照射后30min出现急性脑水肿病理改变,照射后2h出现明显血管源性脑水肿,照射后6h出现明显细胞性脑水肿,照射后3d急性脑水肿达高峰。②脑组织NOS亚型表达在照射后30min开始增高,内皮型一氧化氮合酶(eNOS)及诱导型一氧化氮合酶(iNOS)表达分别于照射后2h及6h显著增高,神经元型一氧化氮合酶(nNOS)表达亦增高,但nNOS阳性表达细胞数量较少。3种NOS亚型表达增高持续时间长,伴行于脑水肿的急性发展阶段。结论大剂量γ刀照射后脑组织NOS亚型表达增高与急性脑水肿的发生发展有关。     

脑室内注射乙酰胆碱受体抗体IgG对大鼠神经元凋亡及一氧化氮合酶表达的影响

目的研究乙酰胆碱受体抗体(AchRab)对大鼠脑内神经元的损害及一氧化氮合酶(NOS)在损害中所起的作用，探讨重症肌无力(MG)中枢神经系统损害的机制。方法将AchRab IgG或健康人的IgG注入大鼠侧脑室。HE染色、TUNEL法检测细胞凋亡；免疫组化方法观察大鼠皮质、海马及杏仁核神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)表达变化。结果2周后实验组皮质、海马及杏仁核凋亡细胞明显增多，对照组仅见少量凋亡。实验组皮质、海马及杏仁核nNOS神经元数目明显减少。实验组及对照组脑内细胞均来见iNOS表达。结论AchRab脑内注射可诱导神经元凋亡；损伤皮质。海马及杏仁核nNOS神经元；但未能诱导脑内细胞iNOS表达。神经元凋亡损害参与了AchRab对中枢神经损害的机制；nNOS神经元的减少，可能与MG认知功能障碍有密切关系；而神经元的损伤可能与NO的毒性作用无关。     

Nitric oxide synthase and arginase expression in the vestibular nucleus and hippocampus following unilateral vestibular deafferentation in the rat

The aim of this study was to investigate the possible relationship between changes in neuronal and endothelial nitric oxide synthase (nNOS and eNOS) and arginase expression in the vestibular nucleus complex and the hippocampus (CA1, CA2/3 and the dentate gyrus (DG) at 10 h or 2 weeks following a unilateral vestibular deafferentation (UVD) in rats. There were no significant differences in nNOS or arginase II expression in the ipsilateral or contralateral VNC at either 10 h or 2 weeks post-UVD. For eNOS, there was only a significant decrease in expression in the ipsilateral VNC at 2 weeks post-UVD (P<0.01). In the hippocampus, the only significant difference in nNOS expression was a decrease in the ipsilateral DG at 2 weeks post-UVD (P<0.05). There was a significant decrease in eNOS expression in the contralateral CA2/3 region at 10 h post-UVD (P<0.01). The only other significant change in eNOS was an increase in the contralateral DG at 10 h post-UVD (P<0.01). Although arginase II was expressed in all regions of the hippocampus, there were no significant differences in arginase II expression at any time point following UVD. These results suggest that the changes in NOS expression that occur in the VNC and hippocampus following UVD are not correlated with one another or with changes in arginase II.     

Time-related changes in constitutive and inducible nitric oxide synthases in the rat striatum in a model of Huntington's disease

Excitotoxicity and oxidative stress are mechanisms involved in the neuronal cell death induced by the intrastriatal injection of quinolinic acid (QUIN) as a model of Huntington's disease. Production of nitric oxide by nitric oxide synthase (NOS) has been proposed to participate in QUIN-induced neurotoxicity; however, the precise role of NOS in QUIN-induced toxicity still remains controversial. In order to provide further information on the role of NOS isoforms in QUIN toxicity, we performed real time RT-PCR and immunohistochemistry of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) and determined Ca(2+)-dependent and Ca(2+)-independent NOS activity in a temporal course (3-48h), after an intrastriatal injection of QUIN to rats. NOS isoforms exhibited a transitory expression of mRNA and protein after QUIN infusion: eNOS increased between 3 and 24h, iNOS between 12 and 24h, while nNOS at 35 and 48h. Ca(2+)-independent activity (iNOS) did not show any change, while Ca(2+)-dependent activity (constitutive NOS: eNOS/nNOS) exhibited increased levels at 3h. Our results support the participation of Ca(2+)-dependent NOS isoforms during the toxic events produced at early times after QUIN injection.     

Role of Nitric Oxide in the Epileptogenesis of EL Mice

Summary: Purpose : To understand the role of nitric oxide (NO) in the regulation of seizures, we measured the extracellular levels of the NO metabolites nitrite and nitrate as indices of NO generation in the parietal cortex, hippocampus, and temporal cortex of EL mice. Furthermore, alterations of neuronal, endothelial, and inducible nitric oxide synthetase (nNOS, eNOS, and iNOS, respectively) were observed to correlate them with epileptogenesis.
Methods : EL mice of 20 weeks and 30 weeks of age (before and after the establishment of epileptogenesis, respectively) were used. Nitrite was quantified using the specific absorbancy of diazo dye. NOS isoenzymes (nNOS, iNOS, and eNOS) were also investigated in the hippocampus during development until mice were 30 weeks old. Samples (total protein, 8·33 to 8·43 μg) were separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and identified by immunoblotting.
Results : EL mice that experienced repetitive seizures showed a remarkable increase in nitrite in the hippocampus at 30 weeks of age compared with EL mice that had no experience of seizures. nNOS and iNOS were major and minor components, respectively, and both increased in parallel with the development of epileptogenesis. eNOS was not detectable.
Conclusions : Excess iNOS (and subsequent increase in harmful NO) and deficient eNOS (and subsequent decrease in NO identified as an endothelium-derived relaxing factor) may work together to form a focus complex.     

Methamphetamine administration causes overexpression of nNOS in the mouse striatum

The accumulated evidence suggests that the overproduction of nitric oxide (NO) is involved in methamphetamine (METH)-induced neurotoxicity. Using NADPH-diaphorase histochemistry, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) antibody immunohistochemistry, the possible overexpression of nNOS and iNOS was investigated in the brains of mice treated with METH. The number of positive cells or the density of positive fibers was assessed at 1 h, 24 h and 1 week after METH injections. There were no clear positive iNOS cells and fibers demonstrated in the brains of mice after METH treatment. In contrast, METH caused marked increases in nNOS in the striatum and hippocampus at 1 and 24 h post-treatment. The nNOS expression normalized by 1 week. There were no statistical changes in nNOS expression in the frontal cortex, the cerebellar cortex, nor in the substantia nigra. These results provide further support for the idea that NO is involved in the neurotoxic effects of METH.     

Expression of endothelial and inducible NOS-isoforms is increased in Alzheimer’s disease, in APP23 transgenic mice and after experimental brain lesion in rat: evidence for an induction by amyloid pathology

The nitric oxide-synthesizing enzyme nitric oxide synthase (NOS) is present in the mammalian brain in three different isoforms, two constitutive enzymes (i.e., neuronal, nNOS, and endothelial eNOS) and one inducible enzyme (iNOS). All three isoforms are aberrantly expressed in Alzheimer’s disease giving rise to elevated levels of nitric oxide apparently involved in the pathogenesis of this disease by various different mechanisms including oxidative stress and activation of intracellular signalling mechanisms. It still is a matter of debate, however, whether the abnormal expression of NOS isoforms has some primary importance in the pathogenetic chain and might thus be a potential therapeutic target or only reflects a secondary effect that occurs at more advanced stages of the disease process. To tackle this question, we analysed the expression of both eNOS and iNOS in patients with sporadic AD, in transgenic mice expressing human amyloid precursor protein (APP) with the Swedish double mutation under control of the Thy1 promotor (APP23 mice), and after electrolytic cortical lesion in rat, an experimental paradigm associated with elevated expression of APP. In all three conditions, an astrocytosis was induced accompanied by a strong increase of both iNOS and eNOS. Both NOS isoforms were frequently though not always colocalized. Thus, based on the expression pattern of NOS isoforms three types of astrocytes, expressing only one of the two isoforms or both together could be distinguished. In both AD and transgenic mice eNOS-expressing astrocytes exceeded iNOS-expressing astrocytes in number. Astrocytes with elevated levels of iNOS or eNOS were constantly seen in direct association with Aβ-deposits in AD and transgenic mice and were found in the vicinity of the lesion site in the rat cortex. The results of the present study show that expression of both iNOS and eNOS is increased in activated astrocytes under experimental conditions associated with elevated expression of APP (electrolytic brain lesion) or Aβ-deposition (APP23 transgenic mice). Therefore, it is suggested that altered expression of these NOS isoforms being part of AD pathology is secondary to the amyloid pathology and might not be primarily involved in the pathogenetic chain though it might contribute to the maintenance, self-perpetuation and progression of the neurodegenerative process.     

神经源型一氧化氮合酶C276T基因多态性与抑郁症相关分析

目的测定抑郁症患者抗抑郁剂治疗前后血浆一氧化氮（NO）水平变化,旨在探讨神经源型一氧化氮合酶（nNOS）基因C276T多态性与血浆NO浓度及抑郁症发病相关性。方法采用硝酸盐还原酶法测定正常对照组及抑郁症患者治疗前后血浆NO水平;全部受试者取全血标本提取基因组DNA,并采用PCR-RFLP方法对nNOS基因C276T多态性进行基因分型。结果患者组疗前血浆NO水平为（76.8±31.6）μmol/L显著高于疗后[（66.9±25.7）μmol/L,P=0.044]及正常对照组[（64.2±33.3）μmol/L,P=0.02],两组疗后血浆NO水平相比差异无显著性（P=0.588）;根据PCR-RFLP结果,nNOS基因可见两种等位基因条带C、T,组成三种基因型CC、CT、TT,两组等位基因及基因型分布频率差异无显著性（均P〉0.05）,且携带不同基因型者之间血浆NO水平差异亦无显著性（均P〉0.05）。结论血浆NO浓度增高可能是抑郁症发病的影响因素;nNOS基因C276T多态性可能不直接影响血浆NO浓度,也不是抑郁症发病的主要基因因素。     

In situ localization of nitric oxide synthase and direct evidence of NO production in rat retinal ganglion cells

The expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) during synaptogenesis for various phases of the pre- and postnatal developmental periods were investigated. The retinas from prenatal, lactating, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pups were immunostained for constitutive isoforms of NOS (n and eNOS) and observed with a confocal laser scanning microscope. Synthesis of NO in the RGCs was achieved by in vitro stimulation with glutamate. The intracellular NO levels were measured in real time using diaminofluorescein-2 diacetate, a fluorescence indicator of NO. Immunohistochemical analysis revealed nNOS and eNOS expressed in retinal ganglion cells during the first 2 postnatal weeks. Cultured RGCs also expressed nNOS and eNOS in vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, L-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited (P<0.001) the increase of intracellular NO 6 and 8 min after the introduction of L-arginine and glutamate to the medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glutamate in cultured RGCs.     

Expression and distribution of the nitric oxide synthases in idiopathic inflammatory myopathies

Different immune effector mechanisms have been characterised in the idiopathic inflammatory myopathies (IIM): in polymyositis (PM) and sporadic inclusion body myositis (sIBM), T-cell-mediated cytotoxicity targets nonnecrotic muscle fibres, whereas in dermatomyositis (DM) the complement-mediated immune response is directed against the microvasculature. As nitric oxide (NO) has an important function in cell signalling and in the cytotoxicity displayed by activated macrophages, it is potentially involved in the immunopathogenesis of IIM. Using immunohistochemical, in situ hybridisation and Western blotting techniques, we visualised the three isoforms of NO synthase (NOS) in muscle tissues from normal controls and from patients diagnosed with IIM. The levels of both constitutive isoforms of NOS (endothelial, i.e., eNOS, and neuronal, i.e., nNOS) were unchanged in IIM as compared with normal muscle. Both protein and mRNA of the inducible form (iNOS) were detected in half of the control biopsies. Constant and increased iNOS protein expression was found in endomysial infiltrates of PM and sIBM, whereas perimysial inflammatory cells in DM were largely negative. We developed a quantitative Western blotting protocol which confirmed the constitutive nature of nNOS and eNOS and the significant induction of iNOS in PM. Our results appoint iNOS with a dual function: a limited and transient role in normal muscle physiology and an active cytotoxic role in PM and sIBM.