Changes in nitric oxide synthesis and epileptic activity in the contralateral hippocampus of rats following intrahippocampal kainate injection

PURPOSE: To investigate the effects of nitric oxide (NO) on seizure activity observed in brain areas that are remote from a primary epileptic focus. METHODS: Following an injection of kainate (concentration 1 mg/ml, volume 1 microl) in the rat hippocampus, we measured NO synthesis in the contralateral hippocampus and epileptic activity by electroencephalogram (EEG). The NO end products, nitrite and nitrate, were measured by in vivo microdialysis combined with an automated NO end-product analyzer and then used as indices of NO synthesis. We also assessed the effect of a specific inhibitor of neuronal NO synthase (NOS) on both the epileptic activity and NO synthesis in the contralateral hippocampus. For this assessment, we administered 7-nitroindazole (7-NI) (50 mg/kg) intraperitoneally 30 min before the kainate injection. RESULTS: Epileptic discharges in the contralateral hippocampus were frequently observed 90 min after unilateral hippocampus kainate injection. The duration of these discharges gradually increased until 240 min after the kainate injection. The NO end-product levels increased immediately after kainate injection and continued to increase gradually throughout the experiments, to a maximum of 213% of the base level. This elevation of NO end products was followed by epileptic discharges. Both the seizure activity and the elevation of contralateral hippocampus NO end-product levels were markedly attenuated in the animals that received 7-NI. CONCLUSIONS: The results suggest that remote seizure activity caused by the transneuronal spread of kainate-induced discharges may be related to NO derived from neuronal NOS.     

Repeated exposure to hyperbaric oxygen sensitizes rats to oxygen-induced seizures

Repeated exposure to increased partial pressure of oxygen (Po₂) is the standard of care for several medical conditions. The side-effects of repeated exposure to hyperbaric oxygen (HBO), however, are not well defined. Previous studies have demonstrated that acute exposure of rats to HBO causes hypothermia that precedes convulsions. In the present studies, rats that were repeatedly exposed to 100% oxygen at 4 atmospheres absolute (ATA) pressure developed convulsions earlier than naive controls. There was also a trend toward less hypothermia in the rats repeatedly exposed to oxygen. The purpose of this study was to test the hypothesis that repeated exposure to HBO increases sensitivity to convulsions induced by HBO and to determine if the time to onset of convulsions is affected by the hypothermia caused by exposure to HBO. Rats were repeatedly exposed to 2 ATA oxygen for a total of 10 days. After 72 h, these rats were challenged by exposure to 100% oxygen at 4 ATA pressure. Rats repeatedly exposed to HBO had convulsions significantly earlier than the naive controls (84±8min compared to 147±11min), and they developed significantly less hypothermia. Control studies suggested that the decrease in the degree of hypothermia was caused by both repeated exposure to oxygen and adaptation to the mild restraint used during oxygen re-exposures. Adaptation to restraint eliminated the hypothermia induced by oxygen but did not change the time to onset of convulsions. Increased sensitivity to convulsions was present after five exposures to 2 ATA oxygen and persisted for 10 days after the last 2 ATA oxygen re-exposure. Kindling is an animal model of epilepsy which is caused by repeated exposure to subthreshold doses of convulsant stimuli. This repeated dosing causes an increase in the sensitivity of the animal to the convulsant stimuli that can persist for weeks. The increased sensitivity of rats to convulsions during subsequent HBO challenge that is induced by repeated exposures to subconvulsant ‘doses’ of HBO (2 ATA) is similar to kindling. At present the mechanism that causes either kindling or the increased sensitivity to seizures induced by HBO are unknown, and therefore may not be the same.     

亚低温对颅脑创伤大鼠脑血流量及组织间液乳酸水平的影响

目的 利用微透析技术观察颅脑创伤大鼠伤后2.5h内邻近受伤区侧脑室透析液中乳酸、脑血流量的变化及亚低温治疗效果。方法 28只Wistar雌性大鼠随机分为正常对照组、常温创伤组和亚低温治疗组。亚低温组动物于创伤后用冰袋进行全身降温,在脑温降至30℃并保持1h后,加热复温至37℃。将透析管插入邻近受伤区的侧脑室区,位于前囱后1.5mm,中线旁2.5mm,深度3.5mm;灌流速度为4μl/min,每30min采1管样本,后两组均于收集第2管样本后制作颅脑创伤动物模型,继续透析至2.5h。应用激光多普勒血流仪监测脑血流量的变化。结果 (1)对照组大鼠透析液中乳酸水平在各时限内差异无显著性意义(P>0.05);常温创伤组于伤后1.5h内乳酸水平明显升高,与对照组比较差异有显著性意义(P<0.01);亚低温组经低温治疗后,乳酸水平降低,与其余两组相比差异有显著性意义(均P<0.01)。(2)常温创伤组大鼠,仅在伤后20min时出现脑血流量减少,与对照组比较差异有显著性意义(P<0.05);而其余各时限与对照组比较差异均无显著性意义(P>0.05)。亚低温组经治疗后2h内脑血流量持续降低,与常温创伤组比较差异有显著性意义(P<0.05)。结论 亚低温治疗可降低损伤区周围脑组织血流量,加速大鼠神经细胞对乳酸的代谢,从而起到神经细胞保护作用。     

The effect of hyperbaric oxygen on regional brain and spinal cord levels of nitric oxide metabolites in rat

Hyperbaric oxygen (HBO(2)) therapy is reported to be beneficial in transient brain ischemia. The present study was conducted to determine the influence of HBO(2) on metabolites of nitric oxide (NO) in brain and spinal cord of rats. Rats were exposed to room air (RA), normobaric air (NBA), normobaric oxygen (NBO(2)), hyperbaric air (HBA) or HBO(2), the last two conditions at 2.5ATA (atmosphere absolute) for 60 min. The results demonstrate that, compared to the NBA control, oxygen alone generally reduced tissue levels of NO(x)(-) (nitrite plus nitrate). On the other hand, 2.5ATA alone tended to have a slight, if any, effect on tissue levels of NO(x)(-). The combination of oxygen and pressure (i.e., HBO(2)) generally led to an increase in tissue levels of NO(x)(-). Based on these findings, it is concluded that HBO(2) appears to markedly increase NO function most notably in the corpus striatum, brainstem, cerebellum and spinal cord.     

一氧化氮及一氧化氮合酶抑制剂在大鼠局灶性脑缺血时作用研究

目的：观察一氧化氮及一氧化氮合酶抑制剂在大鼠局灶性脑缺血时的作用方法;民凝大鼠大脑中动脉制成脑缺血模型,选择脑缺血３０ｍｉｎ,６０ｍｉｎ,１２０ｍｉｎ,１８０ｍｉｎ为研究时点,观察各时点用选择性,非选择性一氧化氮合酶抑制剂亚硝酸盐含量测定,缺血脑组织坏死体积测定及损伤海马ＣＡ１电镜观察。     

蛛网膜下腔出血并迟发性脑血管痉挛大鼠脑能量代谢与脑血流量变化的实验研究

目的探讨实验性蛛网膜下腔出血导致的迟发性脑血管痉挛时脑能量代谢的变化规律，及其与皮质脑血流量（CBF）变化的相关性。方法50只雄性SD大鼠随机分为对照组和蛛网膜下腔出血第1、3、5、7天组．应用非抗凝自体动脉血枕大池两次注血法制备蛛网膜下腔出血大鼠模型；另5只大鼠用于颅内血液大体分布的观察。应用微透析仪监测各组大鼠脑组织间液中葡萄糖（Glu）、乳酸（Lac）及丙酮酸（Pyr）含量，并计算乳酸／丙酮酸比值；激光多普勒血流仪监测各组大鼠皮质脑血流量；于光学显微镜及透射电子显微镜下观察基底动脉血管壁的病理学改变。结果（1）5只用于观察颅内血液大体分布的大鼠中4只蛛网膜下腔出血模型鼠血液主要分布于基底池、脚间池及额叶底面，亚甲蓝与血液混合物主要沉积于颅底．同时在脑室及纵裂池亦可见血液分布；余1只注入人工脑脊液后未发生颅内出血及血液沉积。表明蛛网膜下腔出血动物模型制备成功。（2）与对照组相比，蛛网膜下腔出血组大鼠葡萄糖及丙酮酸含量明显降低（P〈0．01）；乳酸／丙酮酸比值明显升高（P〈0．01），以第5、7天组升高最为明显（P〈0．01）；第5天组乳酸含量亦明显升高（P〈0．01）。（3）激光多普勒监测显示，蛛网膜下腔出血组大鼠脑血流量减少率高于对照组（P〈0．05），其中第5天组减少最为显著。与其他各亚组比较差异有统计学意义（尸〈0．05）；脑血流量变化与乳酸／丙酮酸比值、乳酸水平的改变呈正相关（r=0．721，0．477；均P〈0．01），与葡萄糖、丙酮酸水平的改变呈负相关（r=-0．447，-0．579；均P〈0．01）。结论微透析指标与脑血流量变化具有一致性，可作为蛛网膜下腔出血脑组织间液生化指标的动态监测，用于预测迟发性脑血管痉挛的发生。     

Methylmalonate-induced seizures are attenuated in inducible nitric oxide synthase knockout mice

Methylmalonic acidemias consist of a group of inherited neurometabolic disorders caused by deficiency of methylmalonyl-CoA mutase activity clinically and biochemically characterized by neurological dysfunction, methylmalonic acid (MMA) accumulation, mitochondrial failure and increased reactive species production. Although previous studies have suggested that nitric oxide (NO) plays a role in the neurotoxicity of MMA, the involvement of NO-induced nitrosative damage from inducible nitric oxide synthase (iNOS) in MMA-induced seizures are poorly understood. In the present study, we showed a decrease of time spent convulsing induced by intracerebroventricular administration of MMA (2 μmol/2 μL; i.c.v.) in iNOS knockout (iNOS^−/−) mice when compared with wild-type (iNOS^+/+) littermates. Visual analysis of electroencephalographic recordings (EEG) showed that MMA injection induced the appearance of high-voltage synchronic spike activity in the ipsilateral cortex which spreads to the contralateral cortex while quantitative electroencephalographic analysis showed larger wave amplitude during MMA-induced seizures in wild-type mice when compared with iNOS knockout mice. We also report that administration of MMA increases NOx (NO₂ plus NO₃ content) and 3-nitrotyrosine (3-NT) levels in a greater extend in iNOS^+/+ mice than in iNOS^−/− mice, indicating that NO overproduction and NO-mediated damage to proteins are attenuated in iNOS knockout mice. In addition, the MMA-induced decrease in Na⁺, K⁺-ATPase activity, but not in succinate dehydrogenase (SDH) activity, was less pronounced in iNOS^−/− when compared with iNOS^+/+ mice. These results reinforce the assumption that metabolic collapse contributes for the secondary toxicity elicited by MMA and suggest that oxidative attack by NO derived from iNOS on selected target such as Na⁺, K⁺-ATPase enzyme might represent an important role in this excitotoxicity induced by MMA. Therefore, these results may be of value in understating the pathophysiology of the neurological features observed in patients with methylmalonic acidemia and in the development of new strategies for treatment of these patients.     

一氧化氮在新生鼠缺氧缺血性脑损伤中的变化及高压氧的治疗机制

目的 探讨围产期缺氧缺血引起新生鼠脑损伤后脑组织一氧化氮(NO)含量的变化,以及高压氧(HBO)干预治疗的机制.方法 结扎孕鼠双侧子宫动脉复制围产期缺氧缺血性脑损伤(HIBD)的动物模型,并于生后6h、24h、72h、7d断头取脑,测定NO含量变化及一氧化氮合酶(NOS)的表达,应用TUNEL染色观察细胞凋亡的变化,设立对照组及HBO干预组.结果 HIBD后NO水平有明显上升,NOS表达增强,随缺氧时间的延长而增强,并伴有凋亡细胞显著增加.HBO治疗后可降低脑组织NOS的表达及NO的水平,明显减轻了脑细胞的凋亡.结论 在HIBD时存在NO的过量生成,NO参与HIBD的病理过程,HBO保护损伤脑组织的治疗机制可能与抑制NO生成有关.

Abstract：

Objective To examine the dynamic variation of plasma nitric oxide (NO) level in newborn rats with hypoxic isehemie brain damage (HIBD) and their relation with hyperbaric oxygen (HBO) treatment. Methods To produce HIBD animal model by ligating the uterine arteries of pregnant rats. We detected the plasma NO level and nitric oxide synthetase (NOS) expression by radioimmunoassay and immunohistochemistry before operation and 6h, 24h, 72h,7d after HIBD and treatment with HBO. The pattern of neuronal cell death after HIBD was examined using TUNEL staining. Results Plasma NO level markedly elevated at 24h after HIBD, and NOS expression reached peak at 72h. The two parameters were significantly fell in the same groups treated with HBO. HBO also minimized eminently apoptosis of cerebral cells.Conclusions HIBD may be associated with the abnormal excretion of NO, and the mechanism of HBO treatment of newborn rats with HIBD may be associated with the reduction of plasma NO.     

实验性脑血管痉挛时一氧化氮与超氧化物歧化酶对脑血流的作用研究

目的 探讨一氧化氮（ＮＯ）、超氧化物歧化酶（ＳＯＤ）分别及联合使用对大鼠实验性蛛网膜下腔出血（ＳＡＨ）后脑血管痉挛（ＣＶＳ）时脑血流（ＣＢＦ）的作用。方法 将３０只大鼠随机分成５组（每组６只）。Ａ组：假手术＋盐水,Ｂ组：ＳＡＨ＋盐水;Ｃ组：ＳＡＨ＋ＳＯＤ;Ｄ组：ＳＡＨ＋ＮＯＣ１２;Ｅ组：ＳＡＨ＋ＳＯＤ、ＮＯＣ１２。模拟制成４８ｈ后,通过Ｌａｓｅ－Ｄｏｐｐｌｅｒ血液仪观察各种药物持续静脉注射１ｈ内Ｃ     

Biphasic modulation of GABA release by nitric oxide in the hippocampus of freely moving rats in vivo

The effect of altering hippocampal nitric oxide (NO) levels on basal and N-methyl- -aspartate receptor-evoked release of GABA has been studied in freely moving rats. N-Methyl- -aspartate (NMDA) increased extracellular GABA in a concentration-dependent manner. The nitric oxide synthase inhibitor t-nitro-arginine-methyl ester ( -NAME; 100 μM) increased basal GABA release, and also enhanced release of GABA evoked by NMDA (100 μM) compared with the same concentration of NMDA infused alone. 200 μM -NAME increased basal dialysate GABA, but to a lesser extent than the 100 μM concentration of the drug, and the NMDA-induced release of GABA was decreased. 1.0 MM -NAME significantly decreased basal extracellular GABA, while abolishing the NMDA-evoked release of the amino acid. The actions of -NAME were not mimicked by its much less active isomer -nitro-arginine-methyl ester. The NO donor S-nitroso-N-acetylpenicillamine decreased dialysate GABA at a 500 μM concentration but increased the extracellular level of the transmitter when infused at 1.0 and 2.0 mM concentrations. These data suggest that NO may mediate both excitatory and inhibitory functions in vivo.     

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。     

Increased neuronal nitric oxide synthase (nNOS) activity triggers picrotoxin-induced seizures in rats and evidence for participation of nNOS mechanism in the action of antiepileptic drugs

Increased neuronal nitric oxide synthase (nNOS) activity was observed during the prodromal period of seizures in various rat brain regions following administration of GABA(A) receptor antagonist, picrotoxin (PCT). Pretreatment with the selective nNOS inhibitor 7-nitroindazole (7-NI), dose- and time-dependently delayed the onset of clonus with a corresponding decrease in nNOS activity. The threshold dose of antiepileptic drugs (AEDs; diazepam, phenobarbitone and gabapentin) have potentiated the anticonvulsant action by pretreatment with graded doses of 7-NI. The increase in efficacy of anticonvulsant action correlated with a corresponding decrease of PCT-evoked increase in nNOS activity. The present data support a role for abnormal nNOS activity in mechanisms that trigger seizures and suggest a possible NO-mediated interplay between GABA(A) and glutamate receptors. The results of the present study provide evidence for a trigger role of neuronally produced NO in epileptogenesis induced by PCT and the participation of nNOS inhibitory mechanisms in the action of AEDs.     

Release of arginine, glutamate and glutamine in the hippocampus of freely moving rats: Involvement of nitric oxide

Using in vivo microdialysis, we have monitored the release of three amino acids (arginine, glutamate and glutamine) in the hippocampus of freely moving rats in response to various drugs. In response to N-methyl-d-aspartate (NMDA) infusion, extracellular glutamate was increased, glutamine was decreased and arginine remained unchanged. By contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) elicited an increase in arginine release but had no effect on either glutamate or glutamine. When S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, was infused into the hippocampus, an increase in glutamate, a decrease in glutamine and no change in arginine were recorded. The effect of SNAP on extracellular glutamine levels was reversed by prior infusion of the guanylate cyclase inhibitor oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), however its effect on glutamate release was unchanged. Interestingly, SNAP was found to promote the release of arginine in the presence of ODQ. We also assessed the effect of two nitric oxide synthase inhibitors, N-nitro-l-arginine methylester (l-NAME) and 7-nitroindazole (7-NI), on the release of these amino acids. l-NAME was found to increase arginine and glutamate levels but decrease those of glutamine. In contrast, 7-NI reduced the release of all three amino acids. The results presented here confirm some but not all of the findings previously obtained using in vitro preparations. In addition, they suggest that complex relationships exist between the release of these amino acids, and that endogenous NO plays an important role in regulating their release.     

7-nitroindazole reduces cerebral blood flow following chronic nitric oxide synthase inhibition

Blood flow and glucose utilization were measured in rat brain after chronic L-NAME treatment followed by acute 7-nitroindazole. Following chronic L-NAME, blood flow was not significantly different from control. Treatment with acute 7-nitroindazole reduced blood flow to the same extent in both chronic saline and L-NAME groups. Glucose utilization was unaffected. These results suggest that residual NOS activity in brain is sufficient to provide tonic, NO-dependent cerebrovascular dilator tone.     

迟发性运动障碍患者血浆超氧化物歧化酶和一氧化氮及一氧化氮合酶的变化

目的 探讨血浆超氧化物歧化酶(SOD)、一氧化氮(NO)及NO合酶(NOS)的变化与抗精神病药所敛的迟发性运动障碍(TD)的关系。方法 对42例长期使用抗精神病药治疗伴有TD的男性精神分裂症患者的血浆锰SOD(MnSOD)、铜-锌SOD(CuZnSOD)、NO及NOS的活性进行测定,以59例不伴有TD男性精神分裂症患者和50例健康男性作对照组,使用异常不自主运动量表(AIMS)进行临床评估。结果 TD组MnSOD、CuZnSOD和NO分别高于非TD组(P<0.05)或正常对照组(P<0.05),TD组NOS明显低于正常对照组(P<0.05)、略高于非TD组(p<0.05)。TD组MnSOD浓度越高则TD的症状越严重、NOS则相反(P<0.01),且在TD组MnSOD与NO呈显著的负相关(p<0.05)、在非TD组或正常对照组却呈正相关(前者P<0.01)。分层后发现TD组MnSOD浓度在NO较低时显著高于非TD组(P<0.01),而在NO浓度较高时则略低于非TD组(P>0.05)。结论 抗精神病药所致的TD,可能与患者血浆SOD尤其是MnSOD活性增高以及血浆NO浓度升高密切相关,两者可能来源于不同病理过程,但均提示自由基活动异常。     

局灶性鼠脑缺血时一氧化氮及一氧化氮合酶抑制剂的作用机制

目的研究一氧化氮在鼠脑局灶性脑缺血再灌注损伤中的作用。方法用线栓法建立大鼠大脑中动脉区缺血再灌注模型，分别用选择性和非选择性诱导型一氧化氮合酶抑制剂对鼠脑局灶性缺血再灌注过程中脑组织一氧化氮的变化规律及可能作用进行探讨。结果非选择性一氧化氮合酶抑制剂（Ｌ－ＮＡＭＥ）可加重局灶性脑缺血性损害，而选择性诱导型一氧化氮合酶抑制剂（ａｍｉｎｏｇｕａｎｉｄｉｎｅ，ＡＧ）具有明确的脑保护作用。结论不同类型的一氧化氮合酶所产生的一氧化氮在脑局灶性缺血性损害中具有不同的作用。     

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。     

美解眠诱发癫痫大鼠大脑皮质NO和SOD的变化研究

目的：观察癫痫大鼠脑中的一氧化氮（NO）和超氧化物歧化酶（SOD）的变化,并探讨NO的氧化还原状态对癫痫的作用。方法：通过美解眠诱发致痫的大鼠模型,分别取癫痫发作时及癫痫发作刚停止时的大脑运动区皮质,匀浆后测定NO的含量和SOD活力。结果：癫痫发作组及癫痫发作刚停止组,大脑运动皮质的NO含量均较正常对照组明显升高;癫痫发作组,脑内SOD活力反而下降,癫痫发作刚停止组,脑内SOD活性力明显升高,结论：癫痫发作组和短暂发作刚停止组,SOD活力／NO含量比值具有显著差异,这些结果支持NO的不同氧化还原状态在癫痫发作中起到不同的作用。     

The role of nitric oxide in the regulation of cerebral blood flow

The role of nitric oxide in the cerebral circulation under basal conditions and when exposed to hypoxic, hypercapnic and hypotensive stimuli, was studied in mechanically ventilated rats using a venous outflow technique, by examining the effects of inhibition of nitric oxide synthase with N-nitro-l-arginine methyl ester (l-NAME).l-NAME (10 or 30 mg/kg injected intravenously) raised mean arterial blood pressure by 14% and 24%, and increased cerebrovascular resistance (CVR) by 20% and 24%, respectively. Cerebral blood flow (CBF) was unaltered, as were blood gases and pH. The increases in MABP and CVR were attenuated byl-arginine (300 mg/kg). Following the administration ofl-NAME, the increases in CBF elicited by ventilation with 8% oxygen for 25 s were unaltered, in comparison to control responses.l-NAME attenuated the increases in CBF and reduced the time for recovery to basal flow rates evoked by ventilation with 10% carbon dioxide. These effects were reversed byl-, but not byd-, arginine. Autoregulation by CBF during hypotensive episodes, as measured by comparisons of CVR values, was unaffected byl-NAME. The results suggest that endogenous nitric oxide is involved in the responses of the cerebral vasculature to elevated levels of CO₂ in the arterial blood. Nitric oxide does not appear to play a major role in autoregulation to increases or decreases in MABP, or in hypoxia-evoked vasodilation.