氯美噻唑对大鼠脑出血周边组织一氧化氮含量、一氧化氮合酶活性及细胞凋亡的干预作用

目的 研究氯美噻唑对大鼠脑出血周边组织一氧化氮(NO)含量、一氧化氮合酶(NOS)活性及细胞凋亡的影响.方法 Wistar大鼠112只,随机分为脑出血组和脑出血+氯美噻唑(CMZ)组,两组各分为(出血前和出血后4h、6h、12h、24h、72h、7d)7个时间点.利用化学方法测定大鼠脑出血周边组织NO含量、NOS活性;利用原位末端标记法测定出血周边组织中神经细胞的凋亡情况.结果 大鼠脑出血周边组织NO含量、诱导型一氧化氮合酶(iNOS)、一氧化氮合酶(NOS)4h开始升高(P<0.05),24h到7d显著升高(P<0.01),大约72h左右NO、iNOS、NOS达峰值.大鼠脑出血周边组织6h出现凋亡细胞,12h上升显著(P<0.01),3d凋亡细胞达峰值,与NO、iNOS、NOS峰值对应,7d时仍存在较多凋亡细胞.氯美噻唑干预后,NO含量、iNOS和NOS活性及凋亡细胞数量与脑出血组对应时间点比较显著下降(P<0.01).结论 大鼠脑出血周边组织NO含量增高,iNOS、NOS活性增强,脑出血周边组织神经细胞存在长时间凋亡,NO、iNOS可以促进其凋亡;氯美噻唑干预后NO含量降低,iNOS、NOS活性下降,减少大鼠脑出血周边组织神经细胞凋亡.     

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。     

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。     

一氧化氮合酶对脑损伤后神经细胞凋亡的影响

目的探讨脑损伤后神经型一氧化氮合酶（nNOS）和诱生型一氧化氮合酶（iNOS）对神经细胞凋亡的影响及其相关机制。方法320只SD大鼠随机分成以下4组：假手术组、脑创伤组、7-硝基吲唑组（nNOS抑制剂）和氨基胍组（iNOS抑制剂），此4组分别划分为伤后3h、6h、12h、24h、2d、3d、7d、14d8个时相组，每个时相组均为10只大鼠，采用Marmarou法制造大鼠重型弥漫性颅脑创伤模型，运用末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口标记法（TUNEL）和免疫组化法，观察4组不同时相点海马CA1区的神经细胞凋亡情况和Bcl-2、Fas的表达情况。结果①假手术组偶见TUNEL阳性凋亡细胞及Bcl-2、Fas阳性细胞；②脑创伤组，伤后各时相点均出现TUNEL阳性凋亡细胞及Bcl-2阳性细胞，先上升后下降；③7-硝基吲唑（7-NI）组，伤后6h、12h、24h凋亡细胞明显下降而Bcl-2阳性细胞却明显上升（同脑创伤组比较P〈0．05）；④氨基胍（AG）组，伤后2～7d，凋亡细胞及Fas阳性细胞明显下降（同脑创伤组比较P〈0．01）。结论在脑损伤的早期，nNOS能够通过抑制Bcl-2的表达来促进神经细胞的凋亡；在脑损伤的晚期，iNOS能够通过诱导Fas的表达来促进神经细胞的凋亡。     

急性脊髓损伤后诱生型一氧化氮合酶免疫组化分析

目的探讨大鼠急性脊髓损伤后诱生型一氧化氮合酶(iNOS)活性表达变化的规律及其意义. 方法按改良的Gruner法将大鼠作成T9脊髓损伤模型,分别于伤后4 h、1、3、7、14 d取出损伤段脊髓,采用免疫组化法和免疫印迹分析检测iNOS活性的变化. 结果免疫组化显示:正常脊髓组织内iNOS的表达为(4.34±1.15)%,脊髓损伤后24 h表达上升为(9.03±3.73)%(P<0.05),伤后7 d表达最强为(47.57±8.62)%(P<0.01).免疫印迹结果显示正常脊髓组织扫描条带微弱,伤后24 h逐渐增强,伤后7 d扫描最强.结论 iNOS不参与正常脊髓组织的生理调节,而与脊髓损伤后的继发性炎症反应相关.     

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。     

神经节苷脂GM1对大鼠脑液压伤后行为和记忆的影响

目的:探讨大鼠脑液压伤后GM1与学习记忆、脑内一氧化氮、突触素和细胞凋亡的关系.方法:液压损伤法建立大鼠脑损伤模型,随机分为治疗组、损伤组和对照组.观察伤后学习记忆改变,检测一氧化氮合酶(NOS)、一氧化氮(NO)、突触素和海马、皮质及基底节区细胞凋亡指数.结果:治疗组学习记忆成绩高于损伤组,NOS、NO明显降低,治疗组海马CA1区突触素显著增多,皮质、海马和基底节的凋亡细胞数明显减少.结论:GM1能减少海马和皮质细胞凋亡,可能有利于促进脑损伤后神经行为和记忆的恢复.     

L-NIL在大鼠创伤性脑损伤中的应用研究

目的探讨选择性诱导型一氧化氮合酶(iNOS)抑制剂L-N6-亚氨乙基-赖氨酸(L-NIL)在大鼠创伤性脑损伤(TBI)模型中对大鼠伤后学习记忆功能、海马神经元的影响.方法 24只SD大鼠随机分为假创伤性脑损伤组、创伤性脑损伤组、非选择性一氧化氮合酶(NOS)抑制剂N-硝基-L-精氨酸(L-NNA)组、选择性诱导型一氧化氮合酶抑制剂L-NIL组.其中L-NNA组、L-NIL组分别于伤后即刻、24 h、48 h经腹腔注射L-NNA或L-NIL.进行避暗回避试验以评价伤后各组大鼠的学习记忆功能恢复情况.伤后第7 d处死大鼠取脑,采用Nissl染色及神经元抗核抗体(NeuN)免疫组化染色观察测量伤侧海马CA2区锥体细胞层面积.结果①L-NIL组避暗回避试验结果显著好于创伤性脑损伤组(P<0.05).②L-NIL组海马CA2区锥体细胞层面积与创伤性脑损伤组比较显著增加(P<0.05).③L-NIL组避暗回避试验结果及海马CA2区锥体细胞层面积值均优于L-NNA组.结论适当地应用选择性iNOS抑制剂L-NIL可促进大鼠脑损伤后学习记忆功能的恢复,保护海马神经元,其效果优于非选择性NOS抑制剂.     

神经节苷脂GMl对大鼠脑液压伤后行为和记忆的影响

目的探讨大鼠脑液压伤后GM1与学习记忆、脑内一氧化氮、突触素和细胞凋亡的关系.方法液压损伤法建立大鼠脑损伤模型,随机分为治疗组、损伤组和对照组.观察伤后学习记忆改变,检测一氧化氮合酶(NOS)、一氧化氮(NO)、突触素和海马、皮质及基底节区细胞凋亡指数.结果治疗组学习记忆成绩高于损伤组,NOS、NO明显降低,治疗组海马CA1区突触素显著增多,皮质、海马和基底节的凋亡细胞数明显减少.结论GM1能减少海马和皮质细胞凋亡,可能有利于促进脑损伤后神经行为和记忆的恢复.     

神经元型一氧化氮合酶在学习记忆过程中的变化和作用

目的　探讨神经元型一氧化氮合酶 (neuronalnitricoxidesynthase,nNOS)及一氧化氮 (nitricoxide ,NO)在学习记忆机制中的相关作用。方法　采用免疫组化方法观察Y迷宫空间辨别学习训练后大鼠不同脑区nNOS表达变化 ,并探讨特异性nNOS抑制剂 7 nitroindozal(7 NI)、钙拮抗剂尼莫通 (nimotop)腹腔注射对大鼠学习获得和记忆再现能力的影响。结果　学习训练后海马各亚区nNOS样神经元数量及染色强度明显增加 ,而皮层和纹状体区则无显著变化 ;7 NI以剂量依赖方式损伤大鼠的学习获得能力 ,但不影响记忆再现 ,尼莫通则对这两种能力均有破坏。结论　提示学习记忆过程可能伴有nNOS合成及活性增加 ,nNOS/NO在学习获得阶段具有重要作用。     

Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis.

Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25-1 mM) provoked long-lasting reactive morphological changes characterized by process-bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase-3 and nitrotyrosine. Motor neuron death was largely prevented by NOS inhibitors and peroxynitrite scavengers but not by trophic factors that otherwise will support motor neuron survival in the absence of astrocytes. The bacterial lipopolysaccharide, a well-known inflammatory stimulus that induces iNOS expression in astrocytes, provoked the same effects on astrocytes as peroxynitrite. Thus, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitrite-dependent mechanisms.     

Nicotine attenuates iNOS expression and contributes to neuroprotection in a compressive model of spinal cord injury

Primary impact to the spinal cord results in stimulation of secondary processes that potentiate the initial trauma. In the present study, we hypothesized that the altered expression of nitric oxide synthase (NOS) may contribute to these effects. Recent evidence indicates that nicotine can exert potent antioxidant and neuroprotective effects in spinal cord injury (SCI). Therefore, the aim of the present study was to evaluate whether the administration of nicotine can influence expression of inducible NOS (iNOS) and/or neuronal NOS (nNOS) in injured spinal cords. Adult male Long-Evans rats were subjected to a moderate contusion model of SCI and received a single intraperitoneal injection of either saline or nicotine (0.35, 3.5, or 7 mg/kg) 2 hr after trauma. SCI dramatically increased iNOS (but not nNOS) mRNA and protein levels in microglial cells in the thoracic and lumbar regions of spinal cords. iNOS overexpression resulted in increased nitrotyrosine formation, decreased number of NeuN (neuronal nuclei)-immunoreactive cells, and up-regulation of inflammatory genes. Most importantly, these effects were markedly attenuated by nicotine acting via a receptor-mediated mechanism. These data may have significant therapeutic implications for the targeting of nicotine receptors in the treatment of compressive spinal cord trauma.     

Nitric oxide in the injured spinal cord: synthases cross-talk, oxidative stress and inflammation

Nitric oxide (NO) is a unique informational molecule involved in a variety of physiological processes in the central nervous system (SNS). It has been demonstrated that it can exert both protective and detrimental effects in several diseases states of the CNS, including spinal cord injury (SCI). The effects of NO on the spinal cord depend on several factors such as: concentration of produced NO, activity of different synthase isoforms, cellular source of production and time of release. Basically, it has been shown that low NO concentrations may play a role in physiologic processes, whereas large amounts of NO may be detrimental by increasing oxidative stress. However, this does not explain all the discrepancies evidenced studying the effects of NO in SCI models. The analysis of the different synthase isoforms, of their temporal profile of activation and cellular source has shed light on this topic. Two post-injury time intervals can be defined with reference to the NO production: immediately after injury and several hours-to-days later. The initial immediate peak of NO production after injury is due to the up-regulation of the neuronal NO synthase (nNOS) in resident spinal cord cells. The late peak is due primarily to the activity of inducible NOS (nNOS) produced by inflammatory infiltrating cells. High NO levels produced by up-regulated nNOS and iNOS are neurotoxic; the down-regulation of nNOS corresponds temporally to the expression of iNOS. On the bases of the evidence, therapeutic approaches should be aimed: (1) to reduce the NO-elicited damage by inhibition of specific synthases according to the temporal profile of activation; (2) by maintaining physiologic amount of NO to keep the induction of iNOS.     

The differential role of NOS inhibitors on stress-induced anxiety and neuroendocrine alterations in the rat

The inhibitors of nitric oxide synthase (NOS) have been shown to possess antidepressant- and anxiolytic-properties in animal model. In order to examine the involvement of nitric oxide (NO) on stress-induced neurobehavioral changes and the concomitant alterations of neuroendocrinological factors, we studied the effects of the nonselective NOS inhibitor, N(ω)-Nitro l-arginine methyl ester hydrochloride (l-NAME) and the specific neuronal NOS inhibitor, 7-nitroindazole (7-NI) on restraint stress-induced anxiety in the elevated plus maze (EPM) test and biochemical analysis. Restraint stress significantly reduced the latency time in open arm and the percentage of open arm entries of the plus maze. Pretreatment with l-NAME (10mg/kg) or 7-NI (10mg/kg) significantly attenuated stress-induced anxiety response. In addition, administration of l-NAME (10mg/kg) reversed stress-induced increase in corticosterone and NO metabolites (NO(x)) in plasma. The administration of 7-NI, but notl-NAME, reversed stress-induced NO(x) in paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus (LC), accompanying with decrease of NADPH-d reactivity in the PVN and lateral dorsal tegmental nucleus (LTDg). These results showed that l-NAME influences HPA axis activity such as corticosterone levels and NO(x) in plasma, whereas 7-NI produced anxiolytic-like effects through the direct reduction in NO(x) in the brain. The results of this study demonstrated that NOS inhibitors have differential effect on stress responses and inhibition of NO could be responsible for the beneficial effect on regulation of stress.     

Effects of MPSS and a potent iNOS inhibitor on traumatic spinal cord injury

ONO-1714, a selective inhibitor of inducible nitric oxide synthetase (iNOS) attenuated the increase of apoptosis and improved the functional outcome of urinary bladder after traumatic spinal cord injury. These findings suggest that iNOS plays a role in the process of SCI. Early treatment with 30 mg/kg methylprednisolone sodium succinate (MPSS) could also inhibit the expression of iNOS gene, apoptosis and the loss of urinary bladder function. We confirmed that early MPSS treatment may prevent injury associated with apoptosis and urinary bladder disability by reducing iNOS mRNA. However, delayed single MPSS treatment 8 h after spinal cord injury was not effective. Early repeated MPSS treatment might allow greater recovery from acute spinal cord injury.     

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.     

一氧化氮合酶活性与抑郁症的相关性

目的通过对抑郁症患者一氧化氮合酶（NOS）活性进行检测，从而研究和探讨一氧化氮合酶、一氧化氮（NO）与抑郁症之间的关系。方法采用分光光度法检测抑郁症患者治疗前后的一氧化氮合酶NOS及其亚型（结构型cNOS、诱导型iNOS）的活性，并与正常对照组比较。结果抑郁症组的NOS、cNOS活性显著低于正常对照组；治疗组的NOS、cNOS活性高于抑郁症（无显著性），但治疗后缓解组的NOS、cNOS活性均显著高于治疗前。各组iNOS的活性无显著差异。结论抑郁症病人的NOS活性下降，而且主要是结构型cNOS活性下降，经治疗缓解后有所提高。因此，NOS和NO很有可能在抑郁症的发病过程中起着重要作用。     

Prolonged suppression of brain nitric oxide synthase activity by 7-nitroindazole protects against cerebral hypoxic-ischemic injury in neonatal rat

Nitric oxide mediates glutamate-induced excitotoxicity associated with cerebral hypoxia-ischemia through production in the brain by several isoforms of nitric oxide synthase (NOS). We examined the influence of the selective neuronal NOS inhibitor, 7-nitroindazole (7-NI), on brain NOS activity and its neuroprotective effects against cerebral hypoxic-ischemic injury in the postnatal day (PND) 7 rat. In the first set of experiments, 7-NI (50 mg/kg) administered intraperitoneally (i.p.) transiently inhibited NOS activity to 40% below the vehicle control level at 1 h after injection (P<0.001, analysis of variance (ANOVA)). In contrast, 7-NI (100 mg/kg, i.p.) inhibited NOS activity to 56% below the control level at 1 h with prolonged suppression of NOS activity at 3, 6, 9 and 12 h after injection. Two-factor ANOVA revealed an overall effect on NOS activity of 7-NI treatment (P<0.001) and time after injection (P<0.001). In the second set of experiments, 7-NI (50, 100 mg/kg) or an equal volume of vehicle was administered after unilateral carotid artery ligation, but 30 min before hypoxia in PND 7 rats. 7-NI (100 mg/kg) significantly protected against cerebral hypoxic-ischemic injury (100 mg/kg of 7-NI, 1.7+/-1.0% damage; control, 8.7+/-1.6%,P<0.05). 7-NI administered 15 min after cerebral hypoxia-ischemia was not neuroprotective. The data suggest that the protective effect of 7-NI is dose dependent, and is related to the duration of suppressed NOS activity.     

The role of nitric oxide in the pathophysiology of thromboembolic stroke in the rat

Although nitric oxide (NO) has been shown to play an important role in the pathophysiology of cerebral ischemia, its contribution to the pathogenesis of experimentally induced thromboembolic stroke is unknown. In this study, we pharmacologically manipulated NO levels in the acute post-thrombotic stage and determined the effects on behavior and histopathology. The following drugs were used: nitro-l-arginine-methyl ester (l-NAME), a non-specific endothelial and neuronal nitric oxide synthase (eNOS and nNOS) inhibitor, 3-bromo-7-nitroindazole (7-NI), a specific inhibitor for nNOS, the NO precursor, exogenous l-arginine and the NO-donor, 3-morpholino-sydnonimine (SIN-1). Male Wistar rats (n=76) were randomly assigned to receive vehicle or drug immediately after common carotid artery thrombosis (CCAT). Regional measurements of cortical NOS activity using the [³H]l-arginine to [³H]l-citrulline conversion assay were decreased 1 h after treatment with l-NAME and 7-NI by 50 and 65%, respectively; hippocampal NOS activity was reduced with l-NAME by 35% and with 7-NI by 65%. l-NAME significantly worsened forelimb placing as compared to other groups. 7-NI accelerated sensorimotor recovery. Water maze retention deficits were noted 48 h after CCAT and these were exacerbated by l-NAME treatment. Histopathological protection was conferred in the hippocampus by 7-NI and SIN-1; conversely, l-NAME increased neuronal injury in the contralateral cortex. l-arginine had no effect on these outcomes. In conclusion, both structural and functional consequences of CCAT can be aggravated by limiting endothelial NO production in the acutely post-thrombotic brain. In contrast, inhibition of nNOS and infusion of an NO donor has a beneficial effect on pathology.     

Inhibition of neuronal nitric oxide synthase increases adrenalectomy-induced granule cell death in the rat dentate gyrus

Recent studies have shown that the expression of neuronal nitric oxide synthase (NOS) mRNA is increased after adrenalectomy (ADX). However, the role of increased NO production after ADX in the dentate gyrus is unknown. In this study, the relationship between NO inhibition and apoptosis in the dentate gyrus after ADX was examined. 7-Nitroindazole (7-NI; 30 mg/kg, i.p.), a selective inhibitor of neuronal NOS, was injected 1 day before ADX and subsequently once every 24 h. Then 4 days after ADX, dentate granule cell death was evaluated using silver impregnation and Nissl staining methods. Inhibition of neuronal NOS by 7-NI increased the number of dying granule cells approximately 4-fold in the dentate gyrus of the ADX rats, compared to vehicle-injected ADX controls. These results suggest that increased NO production after ADX may play an endogenous neuroprotective role in the dentate gyrus.