急性脑缺血后NO与ET代谢的相互关系

在兔大脑中动脉阻断(MCAo)型局灶脑缺血前后分别应用外源性-氧化氮合成酶(NOS)抑制剂L-NNA和一氧化氮(NO)合成底物L-Arginine,观察缺血4h后脑组织内皮素(ET)含量的变化。结果发现L-NNA组较缺血对照组脑组织ET含量明显增加(1262.9±387.6 vs789.3±188.4pg/mg·pro,P<0.01),脑水肿显著;而L-Arginine用药组脑组织ET含量较缺血对照组明显减少(P<0.05),且脑水肿减轻。本研究提示促进NO代谢能抑制缺血脑组织ET的产生。NO影响早期脑缺血可能与此途径有关。     

NOS活性在局灶脑缺血后的时相变化

一氧化氮(NO)在脑缺血中起重要作用,一氧化氮合成酶(NOS)作为NO合成的关键酶,其活性变化直接调节NO的生成量及生物学效应,本文在建立兔MCAO局灶脑缺血模型基础上,测定缺血不同时间缺血区和正常脑组织的NOS活性。结果显示脑缺血早期(1小时内)NOS活性突然升高,随之下降;脑缺血后24小时NOS活性又回升,48小时、96小时明显升高。     

局灶脑缺血后早,晚期一氧化氮合成酶的活性变化

一氧化氮(NO)在脑缺血中起重要作用,一氧化氮合成酶(NOS)作为NO合成的关键酶,其活性变化直接调节NO的生成量及生物学效应。本文在建立兔MCAO局灶脑缺血模型基础上,测定缺血后不同时间缺血区和正常脑组织的NOS活性。结果显示,脑缺血早期(1h内)NOS活性突然升高,随之下降;脑缺血后24h NOS活性又升高,至48h、96h明显升高。     

NO对兔局灶脑缺血脑水肿和脑梗塞灶的影响

本文在兔MCAO局灶脑缺血模型基础上,利用外源性一氧化氮(NO)前体物质L-精氨酸和NOS抑制剂L-NNA研究NO对脑缺血后脑水肿和脑梗塞的影响。NZW兔42只随机分为7组,每组6只,计对照组、假手术组、MCAO组、MCAO+NS组、MCAO+L-Arg组、MCAO+D-Arg组及MCAO+L-Arg+L-NNA组。结果提示:与单纯MCAO组比较,L-Arg组的脑组织H_2O、Na~+、Ca~(2+)含量明显下降(P<0.01),脑梗塞灶亦明显缩小(P<0.01)。从而显示L-Arg系通过产生NO促使血管舒张而减轻脑水肿和缩小梗塞灶,为临床改进脑缺血的治疗提供依据。     

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。     

NO增加兔局灶脑缺血后缺血脑组织VEGF表达

目的一氧化氮(NO)和血管内皮生长因子(VEGF)参与血管再生并且可能在脑缺血相互影响,本实验探讨NO对局灶脑缺血后VEGF表达的影响。方法兔局灶脑缺血后应用NO合成底物:左旋-精氨酸(L-Arg),荧光RT-PCR分析缺血脑组织VEGF mRNA表达,ELISA分析VEGF蛋白,脑组织含水率评价脑水肿。结果L-Arg明显增加缺血区VEGF蛋白(1.180±0.433ng/ml vs0.649±0.274ng/ml,P<0.05)和mRNA表达(0.3402±8.876×10-3vs0.2025±0.0413,P<0.05),同时减轻脑水肿(P<0.01)。结论外源性NO增加缺血区VEGF表达,减轻脑水肿,提示联合应用NO合成底物和VEGF对脑缺血后神经保护可能起到更好的协同作用。     

脑缺血后脑组织NO含量和NOS活性变化及三七总皂甙的影响

为了探讨一氧化氮 (NO)在缺血神经元坏死中的作用机制及三七总皂甙 (PNS)是否通过影响NO的变化而起脑保护作用。　　方法　用栓线法建立大鼠局灶性脑缺血模型 ,测定脑缺血后不同时间脑组织NO含量和一氧化氮合成酶 (NOS)活性变化及PNS对其的影响。　　结果　缺血后 3 0分钟脑组织NO含量和NOS活性均显著升高 (P <0 0 1 ) ,而PNS能防止脑缺血后两者的升高。NO含量与NOS活性呈显著正相关。　　结论　NO在缺血神经元损伤中起重要作用 ,PNS是通过降低NO的含量而起保护脑组织作用     

脑缺血后脑组织NOS基因表达的变化与巴曲酶的影响

近年来发现脑缺血后脑组织一氧化氮(NO)含量增高,NO能显著影响缺血后脑水肿及脑梗塞灶的大小,但一氧化碳合成酶(NOS)拮抗剂对脑缺血的影响尚有争论。巴曲酶(batroxobin)为蛇毒酶制剂,我们用巴曲酶治疗急性缺血性脑血管病取得了较好疗效,动物实验也表明巴曲酶可减轻沙土鼠脑缺血后脑水肿程度和卒中症候,但巴曲酶治疗脑缺血是否能直接或间接影响缺血脑组织的NOS基因表达尚未见报道。     

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。     

大鼠脑缺血再灌流后海马区一氧化氮合酶活性的变化

目的 :观察鼠全脑缺血再灌流后海马区NOS活性的变化。方法 :采用大鼠 4血管关闭方法制作全脑缺血再灌流模型。实验动物分为假手术组、缺血 10min组、再灌注 1、2、3d组 ,测定脑缺血再灌流后海马区NOS活性的变化。结果 :全脑缺血再灌注后海马组织NOS活性被激活上调。结论 :NO可能参与了海马CA1区迟发性神经元死亡 (DND)的发生。     

局灶性脑缺血和再灌注期一氧化氮合成酶的活性和局部脑血流量的动态变化

研究局灶性脑缺血和再灌注期一氧化氮合酶的活性和局部脑血流量的动态变化.用改良Koizumi’s大鼠局灶性脑缺血和再灌注模型及改良Bredt和Snyder’s法测定了缺血和再灌注期缺血侧脑组织一氧化氮合成酶(NOS)总活性,并同步以激光多普勒血流仪(LDF)对缺血周边区局部脑血流量(ICBF)进行了测定.结果:缺血早期,缺血侧半球脑组织NOS活性急剧升高至缺血前2～3倍(P<0.01),缺血后期NOS活性降至缺血前水平(P>0.05);缺血10～20 min缺血周边区ICBF回升至缺血前的30%左右(P<0.05);再灌注10min出现高灌,30min后持续下降.提示:缺血期因为钙内流,使NOS活性剧增,有利于维持缺血区和周边区的脑血流量,但过量产生的NO可造成细胞损伤.再灌注期由于表达增加,加之钙超载使NOS活性再度升高,NO可与再灌注期大量产生的超氧阴离子反应生成毒性自由基而损伤细胞.     

NO代谢变化对缺血性脑组织内皮素产生的影响

在兔大脑中动脉阻断（ＭＣＡｏ）型局灶脑缺血前后分别应用外源性一氧化氮合成酶（ＮＯＳ）抑制剂Ｌ－ＮＮＡ和ＮＯ合成底物Ｌ－ａｒｇｉｎｉｎｅ，观察缺血４小时后脑组织内皮素（ＥＴ）含量的变化。结果发现Ｌ－ＮＮＡ组较缺血对照组脑组织ＥＴ含量明显增多（１２６２．９±３８７．６ｖｓ７８９．３±１８８．４ｐｇ／ｍｇ·ｐｒｏ，Ｐ值＜０．０１），脑水肿显著；而Ｌ－ａｒｇｉｎｉｎｅ用药组脑组织ＥＴ含量较缺血对照组明显减少（Ｐ值＜０．０５），且脑水肿减轻。本研究提示ＮＯ能抑制缺血脑组织ＥＴ的产生。ＮＯ对脑缺血影响可能与此途径有关。     

Nitric oxide synthase in cerebral ischemia

The results of our continuing studies on the role of nitric oxide (NO) in cellular mechanisms of ischemic brain damage as well as related reports from other laboratories are summarized in this paper. Repetitive ip administration ofN ^G-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor, protected against neuronal necrosis in the gerbil hippocampal CA1 field after transient forebrain ischemia with a bell-shaped response curve, the optimal dose being 3 mg/kg. Repeated ip administration of L-NNA also mitigated rat brain edema or infarction following permanent and transient middle cerebral artery (MCA) occlusion with a U-shaped response. The significantly ameliorative dose-range and optimal dose were 0.01–1 mg/kg and 0.03 mg/kg, respectively. Studies using a NO-sensitive microelectrode revealed that NO concentration in the affected hemisphere was remarkably increased by 15–45 min and subsequently by 1.5–4 h after MCA occlusion. Restoration of blood flow after 2 h-MCA occlusion resulted in enhanced NO production by 1–2 h after reperfusion. Administration of L-NNA (1 mg/kg, ip) diminished the increments in NO production during ischemia and reperfusion, leading to a remarkable reduction in infarct volume. In brain microvessels obtained from the affected hemisphere, Ca²⁺-dependent constitutive NOS (cNOS) was activated significantly at 15 min, and Ca²⁺-independent inducible NOS (iNOS) was activated invariably at 4 h and 24 h after MCA occlusion. Two hour reperfusion following 2 h-MCA occlusion caused more than fivefold increases in cNOS activity with no apparent alterations in iNOS activity. Thus, we report here based on available evidence that there is good reason to think that NOS activation in brain microvessels may play a role in the cellular mechanisms underlying ischemic brain injury.     

NOS抑制剂对缺血再灌注脑组织rCBF及白细胞浸润的影响研究

目的应用非选择性NOS抑制剂L－NAME和选择性iNOS抑制剂AG治疗鼠大脑中动脉缺血再灌注损伤。通过对脑梗死体积、rCBF和白细胞浸润程度的观察，研究探讨不同类型NOS抑制剂治疗脑梗死的机制。方法采用线检法制作鼠大脑中动脉缺血再灌注模型，不同缺血及再灌注时间测定脑梗死体积、rCBF、缺血脑组织MPO酶活性。结果应用L－NAME（15mg／kg，ip）不但阻碍再灌注后rCBF的恢复，也增加缺血脑组织MPO酶活性（中性白细胞浸润增加），致脑梗死体积增加，脑损害加重；而AG（150mg／kg，ip）可有效降低脑梗死体积，且不影响rCBF的恢复和中性白细胞浸润。结论早期过度抑制神经元和内皮细胞NO对血流量的调节和抗白细胞粘附浸润作用可能是L-NAME加重缺血再灌注脑损害的重要原因之一，而选择性iNOS抑制剂有确切的脑保护作用。     

Nitric oxide synthase expression and enzymatic activity in human brain tumors

Nitric oxide (NO) is synthesized by NO synthases (NOS), existing in 3 isoforms. NO influences a great variety of vital functions including vascular tone and neurotransmission. Under conditions of excessive formation, NO emerges as an important mediator of neurotoxicity in a variety of disorders of the central nervous system (CNS). Inhibitors of NOS are available that may modify the activity of all isoforms, which may be of clinical relevance. The expression of the 3 NOS isoforms nNOS, iNOS and eNOS and NOS enzymatic activity was examined in 40 patients with primary CNS tumors (gliomas WHO grades I - IV and meningeomas WHO grades I - III) and in 13 patients with metastases from adenocarcinomas or malignant melanomas. A polyclonal antibody directed against nNOS and monoclonal antibodies directed against iNOS and eNOS were used for immunohistochemical staining. NOS enzymatic activity, measured by labeled arginine to citrulline conversion, was assessed in tissue specimens obtained from the same tumors. NOS data were compared with clinical variables and the degree of edema as judged from MR scanning. nNOS expression was increased in tumor cells of glial neoplasms and most pronounced in high-grade tumors, WHO grades III and IV, and in the carcinoma and melanoma metastases. Low-grade gliomas, WHO grades I and II and meningeomas expressed no or only little nNOS. iNOS was only expressed in a few tumors. eNOS was expressed sporadically in the tumor cells while the expression was increased in vascular endothelial cells in both the tumor itself and the peritumoral area of glial neoplasms, and in metastases. eNOS expression was sporadic in endothelial cells of meningeomas. NOS enzymatic activities were heterogeneous among tumor types (0 - 13.8 pmol/min/mg of protein) without correlation to the NOS expression found by immunohistochemical techniques. Likewise, NOS activity and expression was not correlated to the clinical scores or brain edema. In conclusion, nNOS expression may be a putative useful indicator of brain tumor differentiation and malignancy. The enhanced expression of eNOS in vascular endothelial cells of glial neoplasms and metastases raises the possibility that NO production in tumor endothelial cells may contribute to tumor blood flow regulation and possibly brain edema.     

一氧化氮供体及前体对大鼠脑缺血再灌注损伤保护机制的探讨

目的 观察介入给药一氧化氮（NO）供体硝酸甘油（Nitroglycerine，NG）及前体L-精氨酸（L-Arginine，ARG）对大鼠脑缺血再灌注后海马区星形胶质细胞表达的胶质纤维酸性蛋白（GFAP）的影响，探讨NG及ARG的脑保护机制。方法 采用大鼠大脑中动脉阻塞（MCAO）法建立局灶性脑缺血模型。将大鼠随机分为假手术组、MCAO组、NG组和ARG组。MCAO组、NG组和ARG组于缺血2 h再灌注同时分别局部介入给予生理盐水、NG和ARG，于再灌注3 h或24 h时，荧光法检测血清NO含量。并在3 h或24 h时处死大鼠，病理分析脑梗死体积以及免疫组织化学法检测海马区GFAP表达情况。结果 缺血再灌注后3 h血清NO升高（P <0.01），治疗组较MCAO组明显（P <0.01），GFAP表达阳性细胞数增加，但治疗组较MCAO组减少（P <0.01），各组大鼠脑组织未出现肉眼可见梗死灶；缺血再灌注后24 h，血清NO治疗组较3 h降低，而MCAO组较3 h升高（P <0.05），GFAP表达阳性细胞数较3 h增加（P <0.01），治疗组较MCAO组减少（P <0.01），TTC染色显示脑梗死体积治疗组较MCAO组减小（P <0.05）。结论 脑缺血再灌注后海马区脑组织GFAP表达增强，通过局部介入给予NG、ARG增加NO合成，抑制GFAP高表达，减小脑梗死体积。提示NG、ARG抗脑缺血性损伤的保护机制可能与抑制星形胶质细胞过度表达有关。     

Nitric oxide synthase inhibition prevents neuronal death in the developing visual cortex

During postnatal development of the visual cortex of golden hamster, there is a transient increase in both the expression and the activity of nitric oxide synthase (NOS), which coincides temporally with the formation of ipsilateral retino-collicular and retino-geniculate projections and the functional differentiation of primary visual cortex, suggesting the involvement of NO in the maturation of the visual cortex. In the present study, an inhibitor of NOS, N-nitro-L-arginine (L-NNA) was used to block the NOS activity of newborn golden hamster, and effects on development were examined. L-NNA treatment caused an increase in mortality, and suppression of both body weight gain and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in the early phase of treatment (before postnatal day 14, PD14). The growth of NADPH-d-positive neurons in the visual cortex was also suppressed by the treatment. In control animals, significant numbers of apoptotic neurons were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay on PD14, and this apoptosis mainly affected cells in cortical layers II and III. NOS inhibition largely rescued neurons from undergoing apoptosis, indicating that NO may serve as a signal triggering apoptosis and play a role in the maturation of the visual cortex.