大鼠脑出血周边组织谷氨酸、脑水含量和细胞凋亡的变化及氯美噻唑的影响

目的研究氯美噻唑对脑出血(ICH)大鼠神经功能损伤、脑水肿的影响,氯美噻唑对大鼠脑出血周边组织一谷氨酸(Glu)含量及细胞凋亡的影响。方法 Wistar大鼠112只,随机分为脑出血组、脑出血+氯美噻唑(CMZ)组,两组各分为(出血前,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Glu含量,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。并在脑出血后24h、48h、72h、7d测定大鼠神经功能评分和脑含水量。结果脑出血后24h开始大鼠出现明显的神经功能缺失症状,氯美噻唑治疗后神经功能缺失症状得到明显改善,在1w内的各个时间点两组比较差异有显著性(P0.01)。脑出血大鼠24h即出现脑水肿,脑水肿形成的高峰时间在ICH后48h～7d左右,ICH大鼠在氯美噻唑治疗后脑水肿形成明显减轻,两组间比较差异有显著性(P0.05;P0.01)。大鼠脑出血后4h血肿周边脑区Glu含量开始升高(P0.01),在血肿形成的高峰期12h达峰值。大鼠脑出血周边组织6h出现凋亡细胞,12h上升显著(P0.01),3d凋亡细胞达峰值,7d时仍存在较多凋亡细胞。氯美噻唑干预后Glu含量及凋亡细胞数量与脑出血组对应时间点比较显著下降(P0.01)。结论脑出血大鼠出现明显脑水肿和神经功能损伤,脑出血周边组织Glu含量增高,脑出血周边组织神经细胞存在长时间凋亡,Glu可以促进其凋亡;氯美噻唑干预后大鼠神经功能改善,脑水肿减轻,Glu含量降低,减少大鼠脑出血周边组织神经细胞凋亡。这与氯美噻唑激动γ-氨基丁酸受体、使神经元产生突触前或突触后抑制、降低大脑谷氨酸能活性等作用有关。     

bFGF对大鼠脑出血周边组织NO含量、HSP70表达及细胞凋亡的影响

目的 研究碱性成纤维生长因子(bFGF)对大鼠脑出血周边组织一氧化氮(NO)含量、热休克蛋白70(HSP70)表达及细胞凋亡的影响.方法 Wistar大鼠112只,随机分为脑出血组、脑出血+bFGF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,免疫组化法测定HSP70的表达,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4h开始升高(P＜0.05),24h到7d显著升高(P＜0.01),大约72h左右NO达峰值.大鼠脑出血周边组织HSP70表达4h开始升高(P＜0.01),大约24h左右HSP70表达达峰值.大鼠脑出血周边组织6h出现凋亡细胞,12h上升显著(P＜0.01),72h左右凋亡细胞达峰值,与NO峰值对应,7d时仍存在较多凋亡细胞.bFGF干预后,NO含量、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);HSP70表达与脑出血组对应时间点比较显著升高(P＜0.01).结论 大鼠脑出血周边组织神经细胞存在长时间凋亡,NO可以促进其凋亡,HSP70可以抑制其凋亡,bFGF能使NO含量降低,HSP70表达增高,从而抑制神经细胞凋亡.     

一氧化氮合酶抑制剂对脊髓损伤后运动功能的影响

目的观察诱导型和神经型一氧化氮合酶(iNOS,nNOS)抑制剂对大鼠脊髓损伤(SCI)后运动功能的影响和机理。方法大鼠脊髓压迫伤后分别给予iNOS和nNOS抑制剂—氨基胍(AG)和7-硝基吲唑(7-NI)进行治疗,24h后用分光光度法测定组织中一氧化氮(NO)含量和一氧化氮合酶(NOS)活性,72h后用流式细胞仪检测神经细胞凋亡情况,4周后用电生理和动物行为学等指标评价运动功能的恢复情况。结果AG和7-NI均可以抑制组织中的NO含量,并使NOS活性下降,同时降低神经细胞的凋亡比率,对运动功能的恢复前者优于后者。结论脊髓损伤后应用NOS抑制剂可以使伤后运动功能得到改善,AG的作用似乎更明显,提示iNOS活性变化可能对脊髓损伤的恢复更具决定作用。     

大鼠脑出血周边组织脑水含量、细胞凋亡和Bax表达的变化及粒细胞集落刺激因子的影响

目的研究粒细胞集落刺激因子(G-CSF)对脑出血(ICH)大鼠神经功能损伤、脑水肿的影响,粒细胞集落刺激因子对大鼠脑出血周边组织Bax表达及细胞凋亡的影响。方法 Wistar大鼠112只,随机分为脑出血组、脑出血+G-CSF组,两组各分为(出血前、出血后4 h、6 h、12 h、24 h、72 h、7 d)7个时间点。免疫组化法测定大鼠脑出血周边组织Bax的表达,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。并在脑出血后24 h、48 h、72 h、7 d测定大鼠神经功能评分和脑含水量。结果脑出血后24 h开始大鼠就出现明显的神经功能缺失症状,G-CSF治疗后神经功能缺失症状得到明显改善,在1 w内的各个时间点两组比较差异有显著性(P<0.01)。脑出血大鼠24 h即出现脑水肿,脑水肿形成的高峰时间在ICH后48 h~7 d左右,ICH大鼠在G-CSF治疗后脑水肿形成明显减轻,两组间比较差异有显著性(P<0.05;P<0.01)。大鼠脑出血周边组织Bax表达4 h开始升高(P<0.01),大约24 h左右Bax表达达峰值。大鼠脑出血周边组织6 h出现凋亡细胞,12 h上升显著(P<0.01),3 d达峰值,7 d时仍存在较多凋亡细胞。G-CSF干预后,Bax表达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)。结论脑出血大鼠出现明显脑水肿和神经功能损伤,脑出血周边组织神经细胞存在长时间凋亡。G-CSF可抑制大鼠脑出血后Bax蛋白表达,从而抑制神经细胞凋亡。     

促红细胞生成素对大鼠脑出血周边组织谷氨酸及脑水含量的影响

目的研究促红细胞生成素(Epo)对大鼠脑出血(ICH)周边组织谷氨酸(Glu)、脑水含量及细胞凋亡的影响。方法 Wistar大鼠112只,随机分为脑出血组、脑出血+Epo组,均分为(出血前、出血后4h、6h、12h、24h、72h、7d)7个时间点。测定大鼠脑出血周边组织Glu含量、大鼠脑含水量及脑出血周边组织中神经细胞凋亡情况。结果大鼠脑出血24h后含水量明显增高,48h～7d左右达到高峰,在Epo干预后,脑水肿形成明显减轻(P<0.05;P<0.01)。脑出血周边组织Glu含量在4h开始升高,分别在12h、72h达到高峰。脑出血周边组织6h出现凋亡细胞,12h上升显著(P<0.01),3d达峰值,Epo干预后,G lu含量及凋亡细胞数与脑出血组对应时间点比较显著下降(P<0.01)。结论大鼠脑出血后出现明显脑水肿,脑出血周边组织Glu含量增高,脑出血周边组织神经细胞存在长时间凋亡,Glu可以促进其凋亡;Epo干预后,大鼠脑水肿减轻,Glu含量降低,Epo减少大鼠脑出血周边组织神经细胞凋亡。     

大鼠脑出血周边组织谷氨酸、脑水含量和细胞凋亡的变化及碱性成纤维生长因子的影响

目的研究碱性成纤维生长因子(bFGF)对脑出血(ICH)大鼠神经功能损伤、脑水肿的影响,碱性成纤维生长因子对大鼠脑出血周边组织谷氨酸(Glu)含量及细胞凋亡的影响。方法 Wistar大鼠112只,随机分为脑出血组、脑出血+bFGF组,两组各分为(出血前、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Glu含量,利用原位末端标记法测定出血周边组织神经细胞的凋亡情况。并在脑出血后24h、48h、72h、7d测定大鼠神经功能评分和脑含水量。结果脑出血后24h开始大鼠就出现明显的神经功能缺失症状,bFGF治疗后神经功能缺失症状得到明显改善,在1w内的各个时间点两组比较差异有显著性(P<0.01)。脑出血大鼠24h即出现脑水肿,脑水肿形成的高峰时间在ICH后48h～7d左右,ICH大鼠在bFGF治疗后脑水肿形成明显减轻,两组间比较差异有显著性(P<0.05;P<0.01)。大鼠脑出血后4h血肿周边脑区Glu含量开始升高(P<0.01),在血肿形成的高峰期12h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,7d时仍存在较多凋亡细胞。bFGF干预后Glu含量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)。结论脑出血大鼠出现明显脑水肿和神经功能损伤,脑出血周边组织Glu含量增高,神经细胞存在长时间凋亡,Glu可以促其凋亡;bFGF干预后,大鼠神经功能改善,脑水肿减轻,Glu含量降低,bFGF减少大鼠脑出血周边组织神经细胞凋亡。     

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。     

颅脑损伤后局部组织一氧化氮合酶表达的实验研究

目的研究一氧化氮合酶(nitricoxidesynthase,NOS)基因在实验性大鼠颅脑创伤后局部组织中表达.方法应用免疫组化技术和高清晰度彩色病理图像分析系统,对大鼠脑组织神经细胞中诱导型一氧化氮合酶(iNOS)的表达进行了检测.结果颅脑创伤后大鼠局部及周围神经细胞中有NOS阳性产物表达并具时程特点,伤后2h平均积分光密度(ODI)较0.5h升高不显著(P0.05),伤后6h、12h、24hODI较0.5h升高非常显著(P<0.01).结论在急性脑损伤后的病理过程中,脑组织中诱导型一氧化氮合酶被大量合成,可能是造成机体一氧化氮(Nitricoxide,NO)升高的直接原因.     

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。     

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。     

Endothelial nitric oxide synthetase (eNOS) in astrocytes: another source of nitric oxide in neocortex.

The distribution of the endothelial form of nitric oxide synthetase (eNOS) was examined in the visual cortex of three species of primate and in the rat using immunocytochemistry. Labeled cells were found in both the gray and white matter. These cells were stellate in appearance and labeled cell processes were seen contacting blood vessels or the pia, suggesting that, by morphological criteria, the cells were astrocytes. All eNOS positive cells were double labeled with an antibody against S100beta. Although all cells were double labeled in the white matter, in the gray matter, some S100beta positive cells did not contain detectable levels of eNOS. eNOS positive astrocytic processes appeared to form prominent and distinctive structures next to neurons, especially in cortical layer IIIC. We postulate that these eNOS-positive structures form astrocytic perisynaptic sheaths on neuronal somas in the cortex. If this is true, then nitric oxide can influence neuronal transmission directly at axosomatic synapses in the cortex. In addition, the presence of eNOS in astrocytes and in their processes that contact blood vessels suggests that the link between local cortical activity and changes in cerebral blood flow could be mediated by astrocytic release of nitric oxide.     

A nitric oxide donor (nitroglycerin) triggers genuine migraine attacks

Supersensitivity to induction of headache and arterial dilatation by a donor of nitric oxide (nitroglycerin) has recently been demonstrated in migraine sufferers. The aims of the present study were to examine whether the nitric oxide donor nitroglycerin may induce a typical migraine attack, to exclude placebo-related effects and to describe the relation between middle cerebral artery dilatation and provoked migraine. Nitroglycerin (0.5 μg/kg/min for 20 min) or placebo was infused into 12 migraine patients in a double-blind cross-over trial. Blood velocity in the middle cerebral artery was measured with transcranial Doppler and characteristics of headache and accompanying symptoms were recorded frequently. Headache occurred during the nitroglycerin infusion as previously described but peak headache intensity did first occur 5.5 h after infusion. At this time the induced headaches in 8 of 10 completing patients fulfilled the diagnostic criteria for migraine without aura of the International Headache Society. Furthermore, all patients who normally had unilateral spontaneous migraine attacks also had unilateral headaches after nitroglycerin. Only one subject developed migraine after placebo (p < 0.03). The time pattern of headache and estimated middle cerebral artery dilatation corresponded well. The study therefore demonstrates that activation of the nitric oxide cGMP pathway may cause typical migraine attacks.     

Interaction between prostaglandin E1 and nitric oxide (NO)

A synergistic antiplatelet effect between prostaglandin I2 (PGI2), a cAMP-stimulator, and nitric oxide (NO), a cGMP-stimulator, has been described. Data on a PGE1-NO interaction, however, are lacking so far. We therefore examined the question in healthy volunteers, whether a similar synergism exist between PGE1 and NO on human platelets in vitro. Each of the substances alone caused a dose-dependent inhibition of ADP-induced platelet aggregation, PGE1 (1ng/mlPRP) reduced platelet aggregation by 27.8 +/- 14.6%, NO (0.3%) by 26.7 +/- 25.5%. The combination of these compounds caused an additive effect resulting in a reduction of 40.6 +/- 23.5%. The findings indicate that PGE1 (like PGI2) and NO have a synergistic antiplatelet action. The concomitant treatment with both compounds offers an interesting concept for clinical therapy.     

The role of nitric oxide (NO) in central nervous system (CNS)

Nitric oxide (NO), the smallest known bioactive product of mammalian cells, can be produced by most cell types. In the central nervous system NO acts as intracellular and extracellular messenger of a novel generation. NO plays important physiologic role, mediates learning, memory, regulates food intake, drinking and other. Under conditions of excessive formation, NO is emerging as important mediator of neurotoxicity in a variety disorders of the nervous system.     

A nitric oxide (NO) synthase inhibitor accelerates amygdala kindling.

In response to NMDA receptor activation, hippocampal, striatal and cerebellar neurons synthesize nitric oxide (NO), which in turn elevates cGMP levels via guanylate cyclase. NO is increasingly being considered as a transsynaptic retrograde messenger, involved in neuronal plasticity. The effect of an inhibitor of NO synthase, L-NG-nitroarginine (NOArg), was studied on amygdala kindling and on kindled seizures in rats. NOArg increased kindling rate, particularly in its initial period, but did not modify seizure severity in previously kindled rats, although we have no definitive explanation for this effect. However, an enhanced post-synaptic excitability could be attributed to the blockade of the negative feed-back exerted by NO on the NMDA receptor.     

Inducible nitric oxide synthase and nitric oxide production by oligodendrocytes

It has been previously demonstrated that microglia and astrocytes produce micromolar amounts of nitric oxide in vitro. In this study, we demonstrate that primary rat oligodendrocytes can be stimulated to produce iNOS mRNA as detected by Northern blot and in situ hybridization analysis and a 131-kDa iNOS protein by Western blot analysis; protein was also detected in cells by single- and double-label immunohistochemistry for iNOS and the oligodendrocyte-specific marker CNPase. NO/NOS are produced as a consequence of activation of the gene encoding the inducible nitric oxide synthase as determined by inhibition with actinomycin D and cyclohexamide. The iNOS is functional, leading to calcium/calmodulin-independent NO production in these in vitro cultures. J. Neurosci. Res. 48:372–384, 1997. © 1997 Wiley-Liss, Inc.     

Neuroprotection via inhibition of nitric oxide synthase by bis(7)-tacrine

Here we report that bis(7)-tacrine, a novel acetylcholinesterase inhibitor, exerts neuroprotective effects by inhibition of nitric oxide synthase. In cortical neurons at 12 days in vitro, bis(7)-tacrine concentration-dependently reduced cell death induced by glutamate, beta-amyloid and L-arginine, but not by nitric sodium nitroprusside. N-monomethyl-L-arginine, a nitric oxide synthase inhibitor, also prevented the former three types but not the last type of the cytotoxicity; however, nitric oxide scavengers blocked all of these insults, indicating that nitric oxide mediated these neuronal injuries. Furthermore, with nitric oxide synthase activity assays, it was found that bis(7)-tacrine not only suppressed the activation of nitric oxide synthase caused by glutamate in cortical neurons, but also directly inhibited the activity of nitric oxide synthase in vitro.     

Effects of deltamethrin on nitric oxide synthase and poly(ADP-ribose) polymerase in rat brain

The effects of deltamethrin on the activities of nitric oxide synthase (NOS) and poly(ADP-ribose) polymerase (PARP) and the protein expression of neuronal NOS (nNOS) and PARP in rat brain were investigated in the present study. The activity of NOS was significantly increased in cortex and hippocampus at 5 h after deltamethrin treatment, and maintained at an increased level at 24 h. The activity of PARP was also elevated at the same time points in the same brain regions of treated rats. By immunohistochemical analysis, it was demonstrated that the nNOS-immunoreactive cells were markedly increased at 24 h after treatment in the cortex and hippocampus, whereas few nNOS-immunoreactive cells were observed in the same brain regions of control and treated rats at 5 h after treatment. The immunoreactivity for PARP was also increased in the same brain regions, showing the similar time course of the induction of nNOS by deltamethrin. These results indicate that deltamethrin increases the activities of NOS and PARP and initiates the protein expression of nNOS and PARP, suggesting that NOS and PARP might play important roles in neurotoxicity of deltamethrin.     

The effect of nitric oxide donors on nitric oxide synthase-expressing myenteric neurones in culture.

Previously, we demonstrated that intestinal inflammation leads to a postinflammatory loss of nitric oxide synthase (NOS)-expressing myenteric neurones and motility disturbances. Here, we investigated whether high NO concentrations could be responsible for the decrease in NOS neurones. Myenteric neurone cultures, prepared from guinea-pig small intestine, were incubated with NO donors [sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1)]. After fixation, NOS neurones were identified by NADPH diaphorase staining and neurone-specific enolase (NSE)-positive neuronal content was assessed with an enzyme-linked immunosorbent assay (ELISA)-based method. Twenty-four hours incubation with SIN-1 (10(-3) mol L(-1)) or SNP (10(-4) mol L(-1) or higher) reduced the number of NADPH diaphorase-positive neurones. SNP incubation did not affect the NSE-positive neuronal content. Shorter incubations (SNP: 4 and 12 h) had no significant effect. The SNP-induced reduction was reversed by glutathione (GSH), but not by NO- or O-scavengers, whereas GSH depletion enhanced the decrease. The NO-dependent guanylate cyclase-blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) did not affect the SNP effect. This reduction can be explained by either specific apoptosis of NOS neurones or downregulation of NOS activity. However, TdT-mediated X-dUTP nick end labelling (TUNEL stainings argue in favour of the latter. In conclusion, the NO donor SNP decreases the number of NOS-expressing myenteric neurones time and concentration dependently, without affecting the amount of neuronal material. Glutathione plays an important protective role.     

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。