重症肌无力患者IgG对大鼠脑内NOS不同时间表达的影响

目的观察重症肌无力(myasthenia gravis,MG)患者IgG(AchRab)对大鼠脑内一氧化氮合酶(NOS)表达的影响,探讨NOS在MG中造成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,1次/d,连续4次.免疫组化方法观察不同时间点大鼠脑皮质、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)的表达变化.结果侧脑室注射后1周实验组大鼠皮质、海马神经元nNOS表达量明显减少,后2周实验组皮质、海马神经元nNOS表达下降更为明显,同时杏仁核神经元nNOS表达量也减少;实验组及对照组脑内细胞均未见iNOS表达.结论AchRab侧脑室内注射可引起大鼠皮质、海马及杏仁核神经元nNOS表达量减少,且2周内这种减少效应随时间延长而增强,但未能诱导脑内细胞iNOS表达,提示AchRab尚可通过抑制大鼠中枢神经系统nNOS表达,降低脑内正常的一氧化氮浓度,减弱一氧化氮对脑组织的保护作用,增加神经元的易损性.     

Aβ_(1-40)海马注射对大鼠脑内一氧化氮合酶表达的影响

目的　探讨一氧化氮合酶 (NOS)在 β淀粉样蛋白 (Aβ)神经毒性及阿尔茨海默病 (AD)病理机制中的作用。方法　应用免疫组化方法 ,观察大鼠海马齿状回Aβ1 4 0 注射后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　正常大鼠海马齿状回区含nNOS神经元计数为 8 96± 0 35个 /视野 ;生理盐水注射后局部含nNOS神经元无明显变化 (8 97± 0 2 9个 /视野 ) ;Aβ1 4 0 注射后 ,注射区周围含nNOS神经元数目显著减少 (2 98± 0 2 4个 /视野 )。正常及生理盐水注射组脑内未见iNOS表达 ;Aβ1 4 0 注射后 2d、10d和 30d ,注射区持续出现大量含iNOS的胶质细胞 (主要为星形胶质细胞 ) ,反应面积分别为 0 90 5± 0 0 82、0 96 2± 0 16 1、0 935± 0 12 5mm2 。结论　Aβ1 4 0 海马注射可损伤局部含nNOS神经元及诱导胶质细胞iNOS表达 ,NOS在Aβ神经毒性和AD发病中有重要作用。     

低剂量伽玛刀照射对致(癎)大鼠皮质及海马神经元c-fos和nNOS表达的影响

目的 通过观察低剂量伽玛刀照射对致(癎)大鼠大脑皮质及海马神经元c-fos和脑型一氧化氮合酶(nNOS)表达的影响,探讨伽玛刀治疗癫(癎)的作用机制.方法 将44只青霉素致(癎)大鼠模型等分为实验组和实验对照组大鼠各22只,另取4只正常大鼠作为正常对照组.实验组行伽玛刀照射(周边剂量12 Gy)后,应用免疫组化方法,观察大脑皮质及海马神经元c-fos和nNOS表达的变化.结果 无论是皮质还是海马,c-fos和nNOS在实验组与实验对照组动物之间,表达均有明显的差别,实验组表达明显少于实验对照组,而后者呈现双高峰现象.结论 c-fos和nNOS在伽玛刀治疗癫(癎)的机制中发挥了重要作用.     

Aβ1—40海马注射对大鼠脑内一氧化氮合酶表达的影响

目的探讨一氧化氮合酶(NOS)在β淀粉样蛋白(Aβ)神经毒性及阿尔茨海默病(AD)病理机制中的作用.方法应用免疫组化方法,观察大鼠海马齿状回Aβ1-40注射后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.结果正常大鼠海马齿状回区含nNOS神经元计数为8.96±0.35个/视野;生理盐水注射后局部含nNOS神经元无明显变化(8.97±0.29个/视野);Aβ1-40注射后,注射区周围含nNOS神经元数目显著减少(2.98±0.24个/视野).正常及生理盐水注射组脑内未见iNOS表达;Aβ1-40注射后2d、10d和30d,注射区持续出现大量含iNOS的胶质细胞(主要为星形胶质细胞),反应面积分别为0.905±0.082、0.962±0.161、0.935±0.125mm2.结论Aβ1-40海马注射可损伤局部含nNOS神经元及诱导胶质细胞iNOS表达,NOS在Aβ神经毒性和AD发病中有重要作用.     

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。     

低剂量伽玛刀照射对致大鼠皮质及海马神经元c-fos和nNOS表达的影响

目的 通过观察低剂量伽玛刀照射对致痫大鼠大脑皮质及海马神经元c—fos和脑型一氧化氮合酶（nNOS）表达的影响，探讨伽玛刀治疗癫痫的作用机制。方法将44只青霉素致痫大鼠模型等分为实验组和实验对照组大鼠各22只，另取4只正常大鼠作为正常对照组。实验组行伽玛刀照射（周边剂量12Gy）后，应用免疫组化方法，观察大脑皮质及海马神经元c-fos和nNOS表达的变化。结果无论是皮质还是海马，c—fos和nNOS在实验组与实验对照组动物之间，表达均有明显的差别，实验组表达明显少于实验对照组，而后者呈现双高峰现象。结论c—fos和nNOS在伽玛刀治疗癫痫的机制中发挥了重要作用。     

重症肌无力中枢神经系统损害与Fas关系

目的 探讨细胞凋亡与重症肌无力（MG）中枢神经系统（CNS）损害之间关系。方法 将MG患者血中提取的IgG注入大鼠脑室系统，建立大鼠MGCNS损害模型。用免疫细胞化学方法观察Fas在脑组织中的表达。结果 在MG组脑内Fas抗原表达于大脑皮质区和海马的神经元胞膜和树突上，而正常对照组脑内无阳性Fas抗原表达。结论 Fas-FasL凋亡过程在MG CNS损害中可能起一定作用。     

慢性间断性缺氧诱导一氧化氮合酶表达的研究

目的:建立大鼠缺氧模型,检测神经元型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(iNOS)的表达情况。方法:1.建立缺氧模型:将SD大鼠置于常压低氧舱中,充入氮气调节氧浓度至所需氧浓度。2.动物分组:(1)急性缺氧组:在低氧舱中缺氧1.5小时。(2)慢性间断性缺氧组:每日在低氧舱中6小时。每周缺氧6天,共缺氧28天。3.采用免疫组化法检测nNOS和iNOS的表达。4.统计学分析检验。结果:急性缺氧后,iNOS、nNOS阳性神经元增加;慢性缺氧后,iNOS、nNOS阳性神经元仍持续增多,慢性缺氧时增加iNOS-IR细胞远远多于nNOS-IR细胞。结论:我们的研究表明缺氧可引起iNOS、nNOS阳性神经元增加,NOS亚型表达时间的不同说明其脑损伤具有阶段性。     

丁苯酞对慢性脑缺血大鼠海马iNOS及MG表达的影响

目的观察丁苯酞对慢性脑缺血大鼠海马区诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、小胶质细胞(microglia,MG)表达的影响,探讨丁苯酞脑保护作用机制。方法 48只雄性SD大鼠随机分为4组:正常大鼠组为A组,假模型组为B组,模型组为C组,丁苯酞干预组为D组。行双侧大鼠颈总动脉永久结扎的方法制作大鼠慢性脑缺血模型。丁苯酞干预组在慢性脑缺血模型成功后给予丁苯酞软胶囊0.2g·kg~(-1)治疗12周,采用Y迷宫测定各组大鼠学习记忆能力,免疫组化染色测定iNOS及OX42(小胶质细胞的标志)表达,特异性尼氏染色方法测定海马CA1区神经元。结果 A、B组大鼠学习记忆能力正常,海马区iNOS、OX42少量表达,神经元数目正常。C、D组大鼠学习记忆能力下降,海马区iNOS、OX42表达增多,神经元数目减少。与C组相比,D组大鼠学习记忆能力明显增加(P0.01),海马区iNOS、OX42表达减少(P0.01),神经元数目增加(P0.01)。结论丁苯酞可减少慢性脑缺血大鼠海马区iNOS、OX42表达,减少MG的过度激活,减少海马区神经元坏死,提高大鼠的学习记忆能力,具有脑保护作用。     

NOS反义RNA重组腺相关病毒载体体内提高脑细胞耐缺血能力的实验研究

目的 探讨二种分别携带神经元型一氧化氮合酶(nNOS)、诱导型一氧化氮合酶(iNOS)反义RNA重组腺相关病毒载体(rAAV-AsnNOS和rAAV-AsiNOS)提高脑细胞耐受缺血能力的作用机制.方法 应用立体定位技术将预处理好的病毒载体转染至将要梗死侧的基底节区,转染病毒滴度为2×109mL-1,并将SD大鼠分为4组即rAAV-AsnNOS组,rAAV-AsiNOS组,rAAV-LacZ组和对照组;处理后运用MACO建立缺血模型,每组分为缺血早期和缺血晚期,流式细胞术(FCM)检测NT阳性细胞百分比和细胞凋亡率,逆转录反应系统(RT-PCR)分析nNOS、iNOS,p38MAPK,Caspase-3 mRNA的表达.结果 一定剂量的重组病毒载体转染到大鼠海马区域,无神经损伤症状;转染rAAV-AsnNOS病毒载体的脑神经细胞在缺血早期(缺血1～6 h),NT阳性细胞百分比、细胞凋亡率以及nNOS、p38MAPK和Caspase-3 mRNA表达量均较对照组、rAAV-LacZ组和rAAV-AsiNOS组降低;转染rAAV-AsiNOS病毒载体的脑神经细胞在缺血晚期(缺血24～72 h),NT阳性细胞百分比、细胞凋亡率以及nNOS、p38MAPK和caspase-3 mRNA表达量均较对照组、rAAV-LacZ组和rAAV-AsnNOS组降低,差异有统计学意义.结论 转染重组病毒载体后动物模型脑神经细胞能够耐受缺血损伤,转染rAAV-AsnNOS病毒载体的脑神经细胞能够在缺血早期抑制nNOS、p38MAPK和Caspase-3的表达,转染rAAV-AsiNOS病毒载体的脑神经细胞能够在缺血晚期抑制iNOS、p38MAPK和Caspase-3的表达,从而在缺血后抑制神经细胞凋亡的发生.     

去松果体对大鼠学习能力及大脑皮质NOS表达的影响

目的探讨去松果体后其功能减退致褪黑素（ＭＴ）分泌减少对大鼠学习能力及大脑皮质一氧化氮合酶（ＮＯＳ）表达的影响。方法将大鼠进行Ｙ型迷宫测试,淘汰学习障碍的大鼠,将学习正常的大鼠随机分二组,实验组手术摘除松果体,对照组给予假手术,饲养４０天后再行Ｙ型迷宫测试,ＳＡＢＣ法检测神经元型一氧化氮合酶（ｎＮＯＳ）表达。结果实验组大鼠学习能力明显低于对照组大鼠（Ｐ〈０．０１）,而大脑皮质、内侧隔核－斜角带核（Ｓ     

Expression of nitric oxide system in clinically evaluated cases of Alzheimer's disease

The expression of neuronal nitric oxide (nNOS) and inducible nitric oxide (iNOS) as isoforms of the nitric oxide synthase (NOS) as well as nitrotyrosine as an end product of protein nitration was analyzed in sections of temporal cortex taken from postmortem brains of patients with Alzheimer's disease (AD). The patients were evaluated by the Clinical Dementia Rating scale (CDR0-CDR3) and studied in the Memory and Aging Project (MAP) of the Washington University Alzheimer Disease Research Center (ADCR). With the use of immunocytochemical procedures, neurons immunoreactive to nNOS were found to show large and small multipolar and pyramidal morphologies over the entire chronic AD evolution. The iNOS and nitrotyrosine immunoreactivities were also found in pyramidal-like cortical neurons and glial cells. Here, we speculate on the interaction among all specific neurodegenerative changes in AD and nitric oxide as an additional contribution to neuronal death in AD.     

Neuronal and inducible nitric oxide synthase immunoreactivity following serotonin depletion

Serotonin (5HT) modulates the development and plasticity of its innervation areas in the central nervous system (CNS). Astrocytic 5HT(1A) receptors are involved in the plastic phenomena by releasing the astroglial-derived neurotrophic factor S-100beta. Several facts have demonstrated that nitric oxide (NO) and the nitric oxide synthase enzyme (NOS) may also be involved in this neuroglial interaction: (i) NO, S-100beta and 5HT are involved in CNS plasticity; (ii) micromolar S-100beta concentration stimulates inducible-NOS (iNOS) expression; (iii) neuronal NOS (nNOS) immunoreactive neurons are functionally and morphologically related to the serotoninergic neurons; (iv) monoamines level, including 5HT, can be modulated by NO release. We have already shown that 5HT depletion increases astroglial S-100beta immunoreactivity, induces neuronal cytoskeletal alterations and produces an astroglial reaction, while once 5HT level is recovered, a sprouting phenomenon occurs [Brain Res. 883 (2000) 1-14]. To further characterize the relationship among nNOS, iNOS and 5HT we have analyzed nNOS and iNOS expression in the CNS after 5HT depletion induced by parachlorophenylalanine (PCPA) treatment. Studies were performed immediately after ending the PCPA treatment and during a recovery period of 35 days. Areas densely innervated by 5HT fibers were studied by means of nNOS and iNOS immunoreactivity as well as NADPH diaphorase (NADPHd) staining. All parameters were quantified by computer-assisted image analysis. Increased nNOS immunoreactivity in striatum and hippocampus as well as increased NADPHd reactivity in the striatum, hippocampus and parietal cortex were found after PCPA treatment. The iNOS immunoreactivity in the corpus callosum increased 14 and 35 days after the end of PCPA treatment. These findings showed that nNOS immunoreactivity and NADPHd activity increased immediately after 5HT depletion evidencing a close functional interaction between nitrergic and serotoninergic systems. However, iNOS immunoreactivity increased when 5HT levels were normalized, which could indicate one of the biological responses to S-100beta release.     

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.     

Importance of endogenous nitric oxide synthase in the rat hypothalamus and amygdala in mediating the response to capsaicin

Although capsaicin has been shown to activate certain neuronal groups in the hypothalamus and amygdala, the neurotransmitters involved and the exact mechanism of action are not clearly understood at present. The aim of this study was to examine the hypothesis that the effect of capsaicin in the rat hypothalamus and amygdala primarily involves direct activation of the endogenous nitric oxide synthase (NOS) neurons responsible for the synthesis of nitric oxide (NO). Subcutaneous capsaicin injection in male rats, compared with vehicle, caused a significant increase in Fos expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON), and medial and cortical amygdala. The expression of nicotinamide adenine dinucleotide phosphate diaphorase, a histochemical marker for NOS, was also increased in these brain areas in addition to the periventricular and lateral hypothalamic area and central amygdaloid nucleus. Also, capsaicin significantly increased the expression of neuronal NOS messenger RNA and protein in the PVN, SON, and medial amygdala as demonstrated by in situ hybridization and immunohistochemistry, respectively. A higher proportion of the NOS neurons in the PVN, periventricular region, SON and amygdala showed Fos expression in response to capsaicin than vehicle injection. There was little, if any, Fos activation in the NOS-positive neurons in the lateral hypothalamic area. The capsaicin-induced activation of the hypothalamic PVN and SON neurons and the medial amygdaloid nucleus was attenuated in the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) -pretreated animals in comparison with the inactive enantiomer D-NAME. These observations indicate that activation of the endogenous NOS system and production of NO constitute a major pathway through which capsaicin exerts its effect within the hypothalamus and amygdala.     

重症肌无力CNS损害和神经细胞关系

目的　探讨神经细胞凋亡与重症肌无力 ( MG)中枢神经系统 ( CNS)损害之间的关系。方法　MG患者血中提取纯化的 Ig G注入 SD大鼠脑室系统 ,然后用透射电镜观察大鼠中枢神经细胞形态学变化。结果　侧脑室注射 MG患者 Ig G后 ,在大鼠大脑皮层、海马神经细胞中观察到神经细胞凋亡现象。结论　 MG中枢损害过程中 ,神经细胞凋亡可能起重要的作用。 MG患者乙酰胆碱受体抗体 ( ACh RAb)与 CNS神经 -ACh R结合 ,诱发神经细胞凋亡 ,从而可能致 MG CNS功能障碍。     

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.     

Immunohistochemical study of the expression of cytokines and nitric oxide synthases in brains of patients with dementia with Lewy bodies

Regional expression of cytokines (IL‐1α, TNF‐α), inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) was immunohistochemically investigated in the brains of patients with dementia with Lewy bodies (DLB), compared with those of patients with Alzheimer's disease (AD) and non‐demented elderly persons. It has been reported that inflammatory responses by cytokines and oxygen free radicals such as nitric oxide (NO) are associated with damaged neurons, degenerative neurites or amyloid deposits in AD brains. In the present study, overexpression of IL‐1α, TNF‐α and iNOS was demonstrated in the amygdala, hippocampus, entorhinal and insular cortices of DLB brains, which are pathologically the most vulnerable regions in DLB brains as well as AD brains. In addition, some Lewy body (LB)‐bearing neurons were involved by the processes of IL‐1α‐ and TNF‐α‐positive microglia, and most extracellular LB were associated with the processes of TNF‐α‐ and iNOS‐positive astroglia. Glial involvement was also found around neuritic plaques and extracellular neurofibrillary tangles. In contrast, the expression of nNOS was reduced in the amygdala of DLB brains showing severe Lewy pathology. These findings suggest that cytokines and NO are significantly implicated in neuronal damage and death including LB formation in DLB brains.     

Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurons in vitro

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non-nNOS neurons. Hence, NO or the NO-cGMP cascade appears to have a neuroprotective action in trophic factor-deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l-arginine completely abolished this effect in almost all nNOS neurons and 8-bromo-cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase-9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO-cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase-3 and -9 activation.