急性缺氧小鼠海马CAl区NOS活力和nNOS蛋白表达的时程变化

目的:观察急性缺氧小鼠海马CAl区一氧化氮合酶(NOS)和神经元型一氧化氮合酶(nNOS) 阳性神经元的时程变化,探讨NO在脑缺氧中的作用并为抗脑缺氧提供依据。方法:复制小鼠急性缺氧模型,采用NADPH-d组织化学和nNOS免疫组织化学方法,研究急性缺氧后不同时程点小鼠海马CAl区NADPH-d 和nNOS阳性神经元数量的变化。结果:与正常对照组相比较,急性缺氧后0.5h组小鼠海马CAl区NADPH-d 和nNOS阳性神经元的数量无明显变化,差异无显著性(P>0.05),3h、6h和12h组逐渐增多并于12h升高达到最高峰,差异有显著性(P<0.05),而于24h后开始降低,48h恢复正常。结论:急性缺氧后早期海马CAl区NOS和nNOS水平明显增多,NO在缺氧所致早期脑损伤中起重要作用。     

急性缺氧小鼠海马CA1区NOS活力和nNOS蛋白表达的时程变化

目的观察急性缺氧小鼠海马CA1区一氧化氮合酶(NOS)和神经元型一氧化氮合酶(nNOS)阳性神经元的时程变化,探讨NO在脑缺氧中的作用并为抗脑缺氧提供依据.方法复制小鼠急性缺氧模型,采用NADPH-d组织化学和nNOS免疫组织化学方法,研究急性缺氧后不同时程点小鼠海马CA1区NADPH-d和nNOS阳性神经元数量的变化.结果与正常对照组相比较,急性缺氧后0.5h组小鼠海马CA1区NADPH-d和nNOS阳性神经元的数量无明显变化,差异无显著性(P＞0.05),3h、6h和12h组逐渐增多并于12h升高达到最高峰,差异有显著性(P＜0.05),而于24h后开始降低,48h恢复正常.结论急性缺氧后早期海马CA1区NOS和nNOS水平明显增多,NO在缺氧所致早期脑损伤中起重要作用.     

低水平铅暴露大鼠齿状回nNOS阳性神经元的时程变化

目的:了解齿状回nNOS阳性神经元在铅影响学习记忆神经毒机理中的作用。方法:采用免疫组织化学ABC法,观察了大鼠腹腔注射130mg/kg醋酸铅溶液染毒3h,6h,12h和24h后齿状回nNOS阳性神经元数目的变化。结果:与对照组相比,染铅12h时,齿状回nNOS阳性神经元数目均明显减少(P<0.01),染铅24h时,齿状回uNOS阳性神经元数目仍处于较低水平(P<0.05)。结论:本研究提示齿状回nNOS阳性神经元的时程变化可能为揭示铅对学习记忆影响的分子机制提供了一定的实验依据。     

大鼠局灶性脑缺血—再灌注后尾壳核神经型一氧化氮合酶表达的变化

目的：探讨神经型一氧化氮合酶（Neuronal nitric oxide synthase,nNOS)在大鼠脑缺血后不同时程尾壳核内的变化。方法：应用大鼠大脑中动脉阻塞模型（MCAO），结合免疫细胞化学ABC法和图像定量分析技术进行研究。结果：缺血－再灌注早期（0h,1h,6h)尾壳核内nNOS免疫反应阳性神经元数量减少，细胞形态变圆，树突缩短，形态学参数开始下降，缺血－再灌注中期（24h,48h,72h)nNOS阳性神经元数量和形态学参数较前有一过性升高，但随后仍持续降低，缺血－再灌注后期（1W）nNOS阳性神经元数量继续下降，胞体浓缩，树突消失，细胞崩解，形态学参数降到最低，结论：脑缺血－再灌注后尾壳核内nNOS免疫反应阳性神经元损伤较重，持续时间长，后期更为严重，这可能会影响尾壳核功能的恢复。     

反复发热惊厥大鼠脑内NOS/NO体系的变化

目的研究一氧化氮合酶(NOS),一氧化氮(NO)体系与反复发热惊厥(febrile seizures,FS)的关系。方法采用热水浴诱导大鼠FS,隔日1次,每次大鼠进行热水浴的时间不超过5min,共10次。大鼠随机分为2组：正常对照组和发热组,后者又根据惊厥与否进一步分为发热对照组和反复FS组。用原位杂交法观察大脑皮层神经元型NOS(nNOS)mRNA的变化,用分光光度计检测大鼠脑组织及血浆中NO含量,用放射免疫法检测大鼠脑组织cGMP含量。结果在大脑皮层深层,FS组nNOS表达阳性的神经元明显增高,而发热对照组仅出现少量nNOS阳性神经元,正常对照组偶见nNOS阳性神经元;脑组织及血浆中NO含量各组间无统计学意义;FS组脑组织cGMP含量吸显高于正常对照组及高热对照组。结论大鼠反复FS后24h脑内nNOS mRNA表达增高,但此时NO不见增多,脑组织cGMP水平增高,可能由于其他途径调节所致。     

心肌缺血大鼠心内神经节神经生长因子表达的变化

目的观察神经生长因子在急性心肌缺血大鼠心内神经节表达及其变化,探讨神经生长因子与心肌缺血的关系。方法采用免疫组织化学方法动态观察了正常大鼠以及大鼠冠状动脉结扎后1、6、12、24h心内神经节神经生长因子的表达及变化。结果各组大鼠的心内神经节均存在神经生长因子阳性神经元,并且神经生长因子阳性神经元的数目在冠状动脉结扎后1、6、12、24h后增多,表达也增强。结论心肌缺血大鼠心内神经节神经生长因子表达持续显著升高,提示神经生长因子可能参与心肌缺血的病变过程。     

秋水仙碱对基底前脑巢蛋白阳性和阴性胆碱能神经元的影响

目的:通过研究侧脑室注射秋水仙碱前后基底前脑巢蛋白阳性和阴性胆碱能神经元变化,探讨巢蛋白表达对基底前脑神经元机能的影响及其可能机制。方法:成年健康雌性SD大鼠随机分为正常对照组和侧脑室注射秋水仙碱组,术后分别于24 h、48 h、3 d、7 d、14 d和28 d取脑行冷冻切片与免疫组织化学显色,比较秋水仙碱注射后不同时间点基底前脑巢蛋白~+和巢蛋白~-胆碱能神经元的数目变化。结果:大鼠侧脑室秋水仙碱注射后24 h,基底前脑的内侧隔核(MS)、斜角带核垂直支(vDB)和水平支(hDB)的巢蛋白~+和巢蛋白的胆碱能神经元数目都急骤下降。随着时间的推移巢蛋白~+神经元数目逐渐恢复,术后14 d,巢蛋白~+神经元数目基本恢复至正常水平,但巢蛋白~-胆碱能神经元数目一直维持较低水平。结论:侧脑室注射秋水仙碱后,基底前脑巢蛋白~+和巢蛋白~-的胆碱能神经元都急骤减少,但巢蛋白~+神经元在减少后可逐渐恢复,而巢蛋白~-神经元则不能恢复。     

老年性痴呆模型大鼠海马结构nNOS神经元的变化

目的　研究老年性痴呆模型大鼠海马结构nNOS神经元的变化情况。方法　用 15月龄老年Wistar大鼠以D 半乳糖腹腔注射 4周加上鹅膏蕈氨酸 (ibotenicacid ,IBO)脑内Meynert核注射造模 ,然后运用免疫组织化学方法检测大鼠海马CA1、CA3和齿状回nNOS神经元数目。结果　老年性痴呆模型大鼠海马CA1、CA3和齿状回nNOS阳性神经元数目及其积分光密度较正常老年组和正常青年组有明显减少的趋势 ,组间比较有差异显著性。结论　大鼠海马结构nNOS神经元的减少是老年性痴呆病的主要病理变化之一     

糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元的变化

目的：观察糖尿病大鼠下丘脑视上核神经元型一氧化氮合酶(nNOS)免疫阳性神经元数量的变化。方法：用链脲佐菌素诱导建立糖尿病大鼠模型；免疫细胞化学染色显示nNOS免疫阳性神经元，并进行定量分析。结果：糖尿病视上核nNOS免疫阳性神经元着色深浅不一，着色较深的阳性神经元散在分布，神经元的形态多样，突起较少。对照组大鼠视上核nNOS免疫阳性神经元较稀疏，各时期无明显改变。糖尿病2w，nNOS免疫阳性神经元数量与对照组无显著差异；7w，nNOS阳性神经元较密集，明显多于对照组；12w，nNOS免疫阳性神经元数量略低于7w，但仍多于对照组。结论：糖尿病大鼠下丘脑视上核nNOS免疫阳性神经元数量明显增多。     

癫痫大鼠海马结构谷氨酸和nNOS神经元的动态变化

的：探讨谷氨酸(Glu)和一氧化氮(NO)二者在癫痫模型中的作用及其相关性。方法：戊四唑化学点燃癫痫大鼠，分为Ⅰ、Ⅲ、Ⅴ级组和Ⅴ级后24h组。采用免疫组织化学方法和图像分析技术。结果：(1)Ⅲ、Ⅴ级组Glu免疫反应阳性神经元数目和平均光密度值升高；但Ⅴ级比Ⅲ级组有所下降；Ⅴ级后24h组恢复到对照组水平。(2)Ⅴ级组和Ⅴ级后24h组nNOS免疫反应阳性神经元数目和平均光密度值升高。结论：戊四唑点燃癫痫模型中，随着点燃级别的进展，Glu的含量是呈先增加后减少的趋势，而NO的含量是逐渐增加的，提示二者在癫痫发作中既有相关性又有独立性。     

The Expression of Neuronal Nitric Oxide Synthase in the Brain of the Mouse During Embryogenesis

The distribution of neuronal nitric oxide synthase (nNOS) in the process of normal mouse brain growth from embryonic (E) Day 11 to postnatal (P) Day 1 was investigated by means of immunohistochemical and immunofluorescent methods. Our results demonstrated that nNOS positive neurons appeared early in superficial cortex at E11. At E13, nNOS positive neurons were located in lateral hypothalamus and amygdala, and temporarily in medullar and ventral hypothalamic neuroepithelia. From E15 to P0, nNOS positive neurons were distributed in superior and inferior colliculi, positive staining could also be seen in superior and inferior tectal neuroepithelium at E15. From E17 to birth, the medial geniculate nucleus had a high density of nNOS labeling. The distribution of nNOS gradually increased and extended laterally in embryo brain, which in turn implies that NO might be involved in the development of mouse brain. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

局灶性脑缺血大鼠皮质和尾壳核神经干细胞增殖分化与nNOS的表达

本研究旨在探讨局灶性脑缺血再灌注大鼠皮质和尾壳核神经干细胞的增殖分化与nNOS表达的关系。大脑中动脉线栓法制作大鼠局灶性脑缺血再灌注模型,5溴脱氧尿核苷(BrdU)标记分裂增殖细胞,免疫组化单标和双标记技术检测各组大鼠缺血侧皮质和尾壳核BrdU阳性细胞和nNOS的表达。模型组大鼠皮质和尾壳核BrdU阳性细胞再灌注后3d开始增多,14d达高峰,nNOS阳性细胞再灌注后7d表达增强,28d达高峰,BrdU/nNOS双标细胞在14d达高峰,占皮质BrdU阳性细胞数的42.95%,尾壳核内占42.56%。新生细胞分化组BrdU阳性细胞和BrdU/nNOS双标细胞显著多于模型组(P<0.05),皮质双标细胞占BrdU阳性细胞数的54.08%,尾壳核内占47.84%。提示局灶性脑缺血可增强大鼠皮质和尾壳核的增殖能力,部分增殖细胞分化为nNOS阳性神经元,参与神经网络的重建。     

Further characterization of sleep-active neuronal nitric oxide synthase neurons in the mouse brain

We recently demonstrated that Fos is induced in a subpopulation of cortical neuronal nitric oxide synthase (nNOS)-immunoreactive neurons in three rodent species both during spontaneous sleep (SS) and recovery sleep (RS) after a period of sleep deprivation (SD); the proportion of cortical Fos⁺/nNOS neurons was significantly correlated with non-REM (NREM) sleep delta energy. The present study was undertaken to evaluate the specificity of this state-dependent activation of cortical nNOS cells. The percentage of nNOS neurons that expressed Fos during SD and RS was determined in nine subcortical brain regions and the cortex of the mouse brain; a significantly greater proportion of Fos⁺/nNOS neurons was observed during RS only in the cortex and in none of the nine subcortical regions. The proportion of calretinin-, calbindin- and parvalbumin-immunoreactive cortical interneurons that expressed Fos during SD and RS was also determined. In contrast to cortical nNOS neurons, a higher percentage of Fos⁺/calbindin neurons was found during SD than RS; there were no differences in the proportions of Fos-expressing parvalbumin or calretinin neurons between these conditions. Since the nNOS and calretinin cortical interneuron populations overlap extensively in the mouse brain, triple-labeling with these two phenotypic markers and Fos was undertaken in mice from the RS group to determine which combination of markers could best identify the rare “sleep-active” cortical interneuron population. The proportions of both Fos⁺/nNOS neurons and Fos⁺/nNOS/calretinin neurons far exceeded the proportion of Fos⁺/calretinin neurons during RS, but the proportions of these two cell types were not significantly different during RS. Thus, functional activation of nNOS neurons during sleep appears to be restricted to the cerebral cortex and cortical nNOS cells and nNOS/calretinin cells collectively define a cortical interneuron population that is activated during sleep.     

Exposure to aluminium changes the NADPH-diaphorase/NPY pattern in the rat cerebral cortex

Aluminium (Al) impairs the glutamate-nitric oxide-cGMP pathway and reduces the number of nitroxidergic neurons in the rat somatosensory cortex. To understand better the effect of the time of exposure, we monitored the effect of aluminium administration on nitroxidergic neurons, identified by NADPH-diaphorase (NADPH-d) or by nitric oxide synthase (NOS) staining, after 0.5, 1, 2, 3, 6 and 12 months of aluminium administration. Since neuropeptide Y (NPY) is known to be colocalised with nitric oxide synthase in cortical neurons, the aim of this work was to study the effects of Al administration on the cortical expression of NADPH-d, nNOS, and NPY. NADPH-d or NOS positive neurons were found scattered in the cortex where they constituted about 1% of all neurons. Double staining using NADPH-d and NPY showed that almost all nitroxidergic neurons were co-localised with NPY neurons (NADPH-d/NPY double stained neurons) whereas some neurons were stained only with NPY (NPY single stained neurons) ; these were more numerous than NADPH-d/NPY double stained neurons. Al significantly reduced NADPH-d and nNOS positive neurons in the cerebral cortex time dependently, with the greatest effect appearing after 3 months. Also measured was the integrated optical density (IOD) of nNOS positive neurons showing a significant decrease of NOS immunostaining even in the remaining NOS positive neurons. The double staining experiment exhibited a decrease in NADPH-d/NPY double stained neurons with an apparent increase in NPY single stained neurons; these then decreased after 6-12 months. On the whole, the results confirm that Al impairs nitroxidergic pathways time dependently; moreover, the transient increase in NPY single stained neurons from 1 to 3 months suggests that there is an intraneuronal down-regulation of NOS, without affecting neuronal viability. In addition, the decrease in the NPY system found at 6 and 12 months may indicate that Al affected nitroxidergic and NPY systems at different times.     

Inhibition of neuronal nitric oxide synthase-mediated activation of poly(ADP-ribose) polymerase in traumatic brain injury: neuroprotection by 3-aminobenzamide

Focal traumatic injury to the cerebral cortex is associated with early activation of the neuronal isoform of nitric oxide synthase (nNOS), where high concentrations of nitric oxide-derived free radicals elicit extensive DNA damage. Subsequent activation of the nuclear repair enzyme poly(ADP-ribose) polymerase (PARP) causes a severe energy deficit leading to the ultimate demise of affected neurons. Little is known about the temporal relationship of nNOS and PARP activation and the neuroprotective efficacy of their selective blockade in traumatic brain injury. To determine the relationship of nNOS and PARP activation, brain injury was induced by cryogenic lesion to the somatosensory cortex applying a pre-cooled cylinder after trephination for 6 s to the intact dura mater. Pre-treatment with 3-bromo-7-nitroindazole (BrNI; 25 mg/kg, i.p.), and pre- or combined pre- and post-treatment with 3-aminobenzamide (AB; 10 mg/kg (i.c.v.) or 10 mg/kg/h (i.p.)) were used to inhibit nNOS and PARP, respectively. Cold lesion-induced changes in the somatosensory cortex and neuroprotection by BrNI and AB were determined using immunocytochemistry and immunodot-blot for detection of poly(ADP-ribose; PAR), the end-product of PARP activation, and the triphenyltetrazolium-chloride assay to assess lesion volume. PAR immunoreactivity reached its peak 30 min post-lesion and was followed by gradual reduction of PAR immunolabeling. BrNI pre-treatment significantly decreased the lesion-induced PAR concentration in damaged cerebral cortex. Pre-treatment by i.c.v. infusion of AB markedly diminished cortical PAR immunoreactivity and significantly reduced the lesion volume 24 h post-injury. In contrast, i.p. AB treatment remained largely ineffective.In conclusion, our data indicate early activation of PARP after cold lesion that is, at least in part, related to nNOS induction and supports the relevance of nNOS and/or PARP inhibition to therapeutic approaches of traumatic brain injury.     

宫内缺氧对新生鼠顶叶皮质神经元内nNOS表达及对成年小鼠学习记忆能力的影响

目的探讨孕鼠宫内缺氧对生后小鼠发育时期顶叶皮质神经元内神经性一氧化氮合酶(nNOS)表达及对成年小鼠学习记忆能力的影响。方法用低张性缺氧模型致胎龄13d、15d、17d小鼠宫内缺氧,将新生后P1、P7、P14、P28、P90鼠脑组织作Nissl染色及nNOS免疫组织化学反应,观察顶叶皮质神经元数量、形态及nNOS神经元表达,P90小鼠行Morris水迷宫实验。结果与正常组比较,宫内缺氧组小鼠顶叶皮质神经元数量明显减少(P<0.05),顶叶皮质神经元内nNOS表达明显减弱(P1,P<0.01;P7,P<0.05;P14,P<0.05;P28,P<0.01;P90,P<0.01)。宫内缺氧组小鼠逃避潜伏期延长(1d、2d,P<0.05;3d、4d,P<0.01)及穿环次数明显减少(P<0.05)。结论宫内缺氧导致生后小鼠发育时期顶叶皮质神经元数量明显减少,顶叶皮质神经元内nNOS表达明显减弱;宫内缺氧引起成年小鼠学习记忆能力降低。     

一氧化氮合酶阳性神经元在小鼠脑内的分布

目的 :研究一氧化氮合酶 (NOS)在小鼠脑内的分布。方法 :用NADPH 黄递酶组织化学技术 ,观察了NOS阳性神经元在小鼠脑内的分布和形态。结果 :在大脑皮质、纹状体、基底前脑、杏仁核、下丘脑和脑干等处有较多一氧化氮合酶阳性神经元的分布。结论 :表明NO与中枢神经系统的诸多功能有关     

Focal,but not global,cerebral ischaemia causes loss of myenteric neurons and upregulation of vasoactive intestinal peptide in mouse ileum

Reduced blood flow to the brain induces cerebral ischaemia, potentially causing central injury and peripheral complications including gastrointestinal (GI) dysfunction. The pathophysiology behind GI symptoms is suspected to be neuropathy in the enteric nervous system (ENS), which is essential in regulating GI function. This study investigates if enteric neuropathy occurs after cerebral ischaemia, by analysing neuronal survival and relative numbers of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) expressing neurons in mouse ileum after three types of cerebral ischaemia. Focal cerebral ischaemia, modelled by permanent middle cerebral artery occlusion (pMCAO) and global cerebral ischaemia, modelled with either transient occlusion of both common carotid arteries followed by reperfusion (GCIR) or chronic cerebral hypoperfusion (CCH) was performed on C56BL/6 mice. Sham‐operated mice for each ischaemia model served as control. Ileum was collected after 1–17 weeks, depending on model, and analysed using morphometry and immunocytochemistry. For each group, intestinal mucosa and muscle layer thicknesses, neuronal numbers and relative proportions of neurons immunoreactive (IR) for nNOS or VIP were estimated. No alterations in mucosa or muscle layer thicknesses were noted in any of the groups. Loss of myenteric neurons and an increased number of VIP‐IR submucous neurons were found in mouse ileum 7 days after pMCAO. None of the global ischaemia models showed any alterations in neuronal survival or relative numbers of VIP‐ and nNOS‐IR neurons. We conclude that focal cerebral ischaemia and global cerebral ischaemia influence enteric neuronal survival differently. This is suggested to reflect differences in peripheral neuro‐immune responses.