Hypovolemia upregulates the expression of neuronal nitric oxide synthase gene in the paraventricular and supraoptic nuclei of rats

We have examined the effects of isotonic hypovolemia on the expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) of the rat, using in situ hybridization histochemistry with a ³⁵S-labelled oligodeoxynucleotide probe complementary to nNOS mRNA. Intraperitoneal (i.p.) administration of polyethylene glycol (PEG) (MW 4000, 20 ml/kg body weight) dissolved in 0.9% saline (20% w/v) induced isotonic hypovolemia. The expression of the nNOS gene in the PVN and SON 6 h after i.p. administration of PEG was increased significantly in comparison with controls. The dual staining for NADPH diaphorase activity and Fos-like immunoreactivity (Fos-LI) showed that at 3 and 6 h after i.p. administration of PEG, a subpopulation of NADPH diaphorase-positive cells in the PVN and SON exhibited nuclear Fos-LI. These results suggest that NO in the PVN and SON may be involved in the neuroendocrine and autonomic responses to non-osmotic hypovolemia.     

Region-specific alterations of neuronal nitric oxide synthase (nNOS) expression in the amygdala of the aged rats

Although many amygdalar functions are altered by aging, little is known about their mechanisms. As these functions are related with nitric oxide (NO), we examined neuronal nitric oxide synthase (nNOS) expression in the amygdala of the aged rats via immunohistochemical technique. We found that nNOS immunoreactive neurons are decreased in almost all amygdalar areas of the aged rats, while nNOS immunoreactivity of the neuropil is significantly increased in the amygdalar nuclei related with main and accessory olfactory system. These suggest altered levels of NO might provide region-specific mechanisms of many physiological and behavioral deficits of the amygdala developed by aging. However, exact effects of these changes require further elucidation.     

Localization of nitric oxide synthase in the brain of the frog,Xenopus laevis

Nitric oxide synthase was localized in the brain of the South african clawed frog by NADPH diaphorase histochemistry and immunohistochemistry. All structures stained by the antiserum also displayed NADPH diaphorase activity. The fiber bundles of the terminal nerve, however, were positive for NADPH diaphorase but were not immunoreactive. In the forebrain, neurons expressing nitric oxide synthase were concentrated to the pallium, striatum, nucleus accumbens and anterior entopeduncular nucleus. Strongly stained neurons in the diencephalon were detected in the lateral thalamus, the tuberculum posterior and in the ventral hypothalamus. In the mesencephalon, the tectum and the magnocellular nucleus of the torus semicircularis contained many positive cells. Farther caudally, intensely stained neurons were abundant in an area corresponding to the anuran locus coeruleus, in the descending nucleus of the trigeminus and the inferior reticular nucleus. In the cerebellum, Purkinje cells were weakly stained. In summary, the expression pattern of nitric oxide synthase in the anuran brain reveals similarities to that of other vertebrates. The strongly positive cell group in the locus coeruleus may correspond to cholinergic cell groups in the mesopontine area in mammals.     

海人酸致痫动物模型脑内一氧化氮,一氧化氮合酶的变化

目的探讨一氧化氮（ＮＯ）、一氧化氮合酶（ＮＯＳ）在癫痫发生中的作用及ＮＯＳ抑制剂的作用。方法采用海人酸致痫大鼠模型并应用ＮＯＳ抑制剂Ｌ－硝基精氨酸甲酯（Ｌ－ＮＡＭＥ）,分别在致痫后３０分钟、６０分钟取海马组织,匀浆后测定ＮＯ及ＮＯＳ水平。结果致痫３０分钟后海马ＮＯ含量显著升高,至６０分钟恢复正常;ＮＯＳ活性水平增高＞５０％;Ｌ－ＮＡＭＥ明显抑制大鼠的痫性发作,应用ＮＯＳ抑制剂组大鼠海马ＮＯ、ＮＯＳ含量明显下降。结论癫痫发作后脑内ＮＯ、ＮＯＳ活性增强,ＮＯＳ抑制剂通过抑制酶活性使ＮＯ生成降低,并完全抑制痫性发作。ＮＯＳ活性受抑制＞４８％即可产生明显效果。提示ＮＯ可能有内源性致痫作用。     

Changes in the distribution of nitric oxide synthase immunoreactive nerve fibers in the chronically hypoxic rat carotid body

The distribution of nitric oxide synthase (NOS) immunoreactive nerve fibers in the carotid body was compared between normoxic and chronically hypoxic rats (10% O₂ and 3.0–4.0% CO₂ for 3 months). NOS immunoreactive fibers appeared as thin processes with many varicosities. They were distributed predominantly around small arteries and arterioles, and around clusters of glomus cells. When expressed by the density of varicosities per unit area in the parenchyma, the density of NOS fibers associated with the vasculature and with the glomus cells in the chronically hypoxic carotid bodies was significantly decreased. Because nitric oxide (NO) is an inhibitory neuronal messenger in the normoxic carotid body, the present findings suggest that the sensory mechanisms in the hypoxic carotid body may be involved in `disinhibition' resulting from reduced NO synthesis.     

Immunohistochemical study on the distribution of nitrotyrosine and neuronal nitric oxide synthase in aged rat cerebellum

In the present study, we examined age-related changes in 3-nitrotyrosine (NT) and neuronal nitric oxide synthase (nNOS) in rat cerebellum using immunohistochemistry. No immunoreactivity for NT was found in any layers of adult cerebellar cortex. In aged cerebellar cortex, the most prominent labeling of NT was found in the Purkinje cell layers and molecular layers. In aged cerebellar nuclei, NT immunoreactivity was observed in the surrounding neuropil. In aged rat cerebellum, nNOS immunoreactivity was significantly decreased in the molecular layer, while it was slightly increased in the granular layer. Image analysis showed no significant age-related changes in nNOS immunoreactivity in the cerebellar nuclei. In summary, this report has demonstrated that NT increases with age in the cerebellum, and suggests that NO production by the neuronal form of NOS may not be the rate limiting step in NT formation in the aged brain. Further work is needed to examine the mechanisms underlying the increased immunoreactivity for NT, and the functional implications of this increase.     

一氧化氮和一氧化氮合酶在鼠脑内形成吗啡依赖的作用研究

目的：研究一氧化氮（NO）和一氧化氮合酶（NOS）在吗啡依赖形成中的作用。方法;对吗啡依赖和戒断大鼠脑内NO含量和NOS活力进行测定。结果：未发现吗啡依赖和戒断大鼠脑内NO含量和NOS活力有改变。结论：对NO／NOS与吗啡依赖的关系还有待进一步研究。     

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.     

NADPH diaphorase activity and nitric oxide synthase immunoreactivity in lordosis-relevant neurons of the ventromedial hypothalamus

The distribution of the enzymes NADPH diaphorase and nitric oxide synthase in the ventromedial nucleus of the hypothalamus of cycling and ovariectomized/estrogen-treated and control female rats was demonstrated using histochemical and immunocytochemical methods. Serial section analysis of vibratome sections through the entire ventromedial nucleus showed that NADPH diaphorase cellular staining was localized primarily in the ventrolateral subdivision. NADPH diaphorase staining was visible in both neuronal perikarya and processes. Light microscopic immunocytochemistry using affinity-purified polyclonal antibodies to brain nitric oxide synthase revealed a similar pattern of labelling within the ventromedial nucleus and within neurons of the ventrolateral subdivision of the ventromedial nucleus. Control experiments involved omitting the primary antibodies; no labelling was visible under these conditions. Some, but not all, neurons in the ventrolateral subdivision of the ventromedial nucleus contained both NADPH diaphorase and brain nitric oxide synthase as demonstrated by co-localization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain nitric oxide synthase were found in estrogen-binding cells was shown by co-localization of NADPH diaphorase and estrogen receptor and brain nitric oxide synthase and estrogen receptor at the light and ultrastructural levels, respectively. Our studies suggest that brain nitric oxide synthase is present and may be subject to estrogenic influences in lordosis-relevant neurons in the ventrolateral subdivision of the ventromedial nucleus. The hypothalamus is a primary subcortical regulatory center controlling sympathetic function. Therefore, not only is nitric oxide likely to be important for reproductive behavior, but also for the regulation of responses to emotional stress and other autonomic functions.     

Excitability of spinal cord and gracile nucleus neurons in rats with chronically injured sciatic nerve examined by c-fos expression

Low-threshold sensory pathways have been suggested to have an important role in the formation and maintenance of sensory abnormalities which are observed after peripheral nerve injury. Fos-like immunoreactive (Fos-LI) neurons are expressed in spinal cord laminae III-IV and the gracile nucleus by electrically stimulating the injured nerves at Abeta strength after sciatic nerve transection in rats. This suggests that the excitability of these neurons is increased by nerve injury. In this study, we investigated which receptors are involved in the regulation of the increased excitability in spinal and gracile nucleus neurons. The sciatic nerve of Sprague-Dawley rats (150 g) was transected 7 days before the experiment day. The rats were administered morphine, muscimol, baclofen, MK-801, CNQX, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or clonidine i.p., and then electrically stimulated at 0.1 mA to the proximal region to the nerve injury site under urethane anesthesia. Two hours after the stimulation, Fos-LI expression was increased in the spinal cord dorsal horn and the gracile nucleus in control rats. Baclofen inhibited the Fos-LI expression both in the spinal cord and the gracile nucleus. Morphine inhibited only the Fos-LI expression in the posterior cutaneous (PC) nerve territory of laminae I-II, but not in the sciatic nerve (SC) territory, laminae III-IV nor the gracile nucleus. MK-801 had an inhibitory but complicated effect in laminae I-II and the gracile nucleus. The other drugs were not effective on Fos-LI expression. It is suggested that the GABA(B) receptor has a pivotal role in the regulation of Fos-LI expression after electrical stimulation to the injured low-threshold sensory fibers, and other receptors have little effect on the Fos-LI expression.     

癫癎患者血清一氧化氮和一氧化氮合酶活性水平的研究

目的　探讨一氧化氮 (NK)和一氧化氮合酶 (NOS)在癫患者中血清活性水平及意义。方法　采用化学比色法对 10 0例癫患者血清中NO和NOS活性水平进行检测。结果　癫患者间歇期血清中NO和NOS活性水平显著高于对照组 (P <0 0 1)。结论　NO和NOS在癫病理过程中起重要作用。     

Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. N^GMMA and N^G-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.     

一氧化氮／一氧化氮合酶与神经创伤

一氧化氮是一种简单的气体分子，可在哺乳类神经细胞内经一氧化氮合酶作用产生。ＮＯ在神经创伤修复中的多重作用近年来已受到越来越多的重视。本文对ＮＯ／ＮＯＳ与神经创伤和再生之间的关系作一综述。     

Distinct distribution and time-course changes in neuronal nitric oxide synthase and inducible NOS in the paraventricular nucleus following lipopolysaccharide injection

Nitric oxide (NO) is known to be involved in the modulation of neuroendocrine function. To clarify the role of different isoforms of NO synthase (NOS) in the neuroendocrine response to immune challenge, the expressions of neuronal NOS (nNOS) and inducible NOS (iNOS) genes in the hypothalamus following lipopolysaccharide (LPS) injection were examined using in situ hybridization. NOS activity was also determined by NADPH-diaphorase (NADPH-d) histochemistry. LPS (25 mg/kg) or sterile saline was injected intraperitoneally to male Wistar rats and the rats sacrificed 30 min, or 1, 2, 3, 5, 12 or 24 h after injection. nNOS mRNA expression in the paraventricular nucleus (PVN) was significantly increased 2 h after LPS injection. iNOS mRNA, which was not detected until 2 h after LPS injection, was significantly increased in the PVN 3 h after LPS injection. Both RNA expressions had returned to basal levels by 12 h after LPS injection. The number of NADPH-d positive cells was significantly increased 5 h after LPS injection. iNOS expression was more robust in parvocellular PVN, while nNOS was distributed mainly in the magnocellular PVN. Double in situ hybridization histochemistry revealed that some of the iNOS- (48.4%) or nNOS-positive cells (34. 3%) in the parvocellular PVN expressed CRF mRNA. The results demonstrate that LPS-induced sepsis causes significant increases in nNOS and iNOS gene expression with different time-courses and distributions, and that iNOS mRNA was more frequently co-localized with CRF-producing parvocellular neurons in the PVN. Thus, NO produced by iNOS and nNOS may play an important role in the neuroendocrine response to an immune challenge. Distinct differences in the distribution and time-course changes of iNOS and nNOS suggest different roles for the hypothalamic-pituitary-adrenal axis and/or neurohypophyseal system.     

Induction of microglial reaction and expression of nitric oxide synthase I in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection

In the present study, immunohistochemical stainings for OX-6, OX-42, nitric oxide synthase I and II as well as nitrotyrosine were used to investigate possible correlation among microglial reactivity, nitric oxide synthase upregulation, peroxynitrite involvement and neuronal death in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection. Significant neuronal loss was found in the ipsilateral nucleus dorsalis and contralateral red nucleus after cord hemisection. A distinctive microglial reaction for OX-42 could be observed from one to four weeks post axotomy in the ipsilateral nucleus dorsalis; by contrast, it was observed on both sides of the red nucleus from one to three weeks following cord hemisection. The activated microglial cells showed some degree of hypertrophy. From the microglial immunoreactivity as well as their appearance, it was speculated that microglial activation might be beneficial or protective to the axotomized neurons. In normal and sham-operated rats, neurons of the nucleus dorsalis were not nitric oxide synthase I reactive. Three weeks after cord hemisection, neurons in the ipsilateral nucleus dorsalis below the lesion showed strong immunoreactivity. Neurons in the red nucleus that normally displayed weak nitric oxide synthase I immunoreactivity showed an increase on both sides of the nucleus. These results suggested that nitric oxide synthase I expression in the nucleus dorsalis following axotomy was synthesized de novo and might act as a neurotoxic agent. However, the bilateral increase in expression of nitric oxide synthase I in the red nucleus after lower thoracic cord hemisection was due to up-regulation of the constitutive enzyme and might have some neuroprotective function. Our results also suggested that peroxynitrite played no or little role in the neurodegeneration in the nucleus dorsalis and red nucleus following axotomy.     

儿童癫癎患者血清一氧化氮及一氧化氮合酶活性水平的研究

目的　探讨一氧化氮 (NO)及一氧化氮合酶 (NOS)在儿童癫患者中血清活性水平及意义。方法　采用化学比色法对12 4例儿童癫患者血清中NO及NOS活性水平进行检测。结果　儿童癫患者发作期及间歇期血清中NO及NOS活性水平均显著高于对照组 (P <0 0 1) ,发作期血清中NO及NOS活性水平均高于间歇期 (P <0 0 5 )。结论　NO及NOS在儿童癫病理过程中起重要作用     

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。     

Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase

This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS). which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some infiltrating inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases.     

Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.     

一氧化氮合酶、谷氨酸在局灶脑缺血中的变化

目的　观察一氧化氮合酶 (NOS)和谷氨酸 (Glu)在脑缺血时的改变。方法　应用大鼠大脑中动脉闭塞局灶脑缺血模型 ,观察脑缺血 1h后NOS和Glu含量的变化。结果　缺血 1h后NOS活性显著升高(P <0 .0 5 )、Glu含量亦显著升高 (P <0 .0 1) ;用L NMMA处理后 ,NOS活性显著降低 (P <0 .0 1) ,Glu含量亦降低 (P <0 .0 5 )。结论　Glu生成过多可激活NOS ;抑制NOS活性可减少Glu的生成。