Effects of Lamotrigine on brain nitrite and cGMP levels during focal cerebral ischemia in rats

Glutamate receptor antagonists are protective in animal models of focal cerebral ischemia. Lamotrigine (3,5-diamino-6-[2,3-dichlorophenyl]-1,2,4-triazine) is an anticonvulsant drug that blocks voltage-gated sodium channels and inhibits the ischemia-induced release of glutamate. Experiments in primary neuronal cultures implicate nitric oxide (NO) as a mediator of glutamatergic neurotoxicity acting via N-Methyl- d -Aspartate (NMDA) receptors. The effect of glutamate release inhibitor, Lamotrigine upon NO and cGMP production has been examined in focal cerebral ischemia in rats. Focal cerebral ischemia was produced by the permanent occlusion of right middle cerebral artery (MCA) in urethane anesthetized rats. A number of indicators of brain NO production (nitrite, cGMP) were determined in ipsilateral and contralateral cerebral cortex and cerebellum after 0, 10, 60 min of focal cerebral ischemia. The same parameters were measured in rats treated with Lamotrigine (20 mg/kg, i.p.) 30 min before or just after the occlusion of the right MCA.     

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。     

The effects of NMDA receptor antagonist MK-801 on lipid peroxidation during focal cerebral ischemia in rats

The effect of MK-801 on ischemic neuronal damage was studied in a rat model of permanent focal cerebral ischemia in terms of ipsilateral and contralateral cortical and cerebellar tissue lipid peroxides. Forty-five male Swiss Albino rats were assigned into one of four groups: sham operated as controls, subjected to right middle cerebral artery occlusion (MCAO) or injection of MK-801 (0.5 mg/kg i.p.) either 30 min before or just after right MCAO. Changes in lipid peroxides were expressed as nmol malondialdehyde (MDA) and conjugated diene (CD)/mg protein. MDA values after 60 min of ischemia relative to contralateral cortex and CD levels in 0, 10 and 60 min after ischemia were found to be higher in ipsilateral cortex than those in contralateral cortex. On the other hand, contralateral cerebellar MDA levels in 0 and 60 min of ischemia and CD levels in 0, 10 and 60 min after ischemia were found to be higher than those in ipsilateral cerebellum. Pharmacological inhibition of glutamate receptor by MK-801 before or just after permanent MCAO decreased significantly the MDA and CD levels in both cortex and cerebellum. Although no significant differences found in MDA values between rats pre- and posttreated with MK-801, CD levels in posttreated group seemed significantly lower than those in pretreated group. On the whole, these results suggest that MDA and CD represent early biochemical marker of lipid peroxidation in ischemic tissues, reflecting the radical-mediated tissue damage.     

脑缺血对局部超氧化物岐化酶、丙二醛水平的影响

建立大鼠右大脑中动脉栓塞（ＲｉｇｈｔＭｉｄｄｌｅＣｅｒｅｂｒａｌＡｒｔｅｒｙＯｃｌｕｓｉｏｎＲ－ＭＣＡＯ）模型,检测局部缺血后不同时间左右脑组织超氧化物岐化酶（ＳｕｐｅｒｏｘｉｄｅＤｉｓｍｕｔａｓｅＳＯＤ）、丙二醛（ＭＤＡ）的含量,发现Ｒ－ＭＣＡＯ１０分钟始,缺血脑组织ＳＯＤ即降低,至３０分钟后恢复,而ＭＤＡ则在Ｒ－ＭＣＡＯ６０分钟后持续升高。说明缺血后ＳＯＤ活力下降、致ＭＤＡ蓄积,是造成脑组织损伤的机制之一。     

Modulation by nitric oxide of cerebral neutrophil accumulation after transient focal ischemia in rats.

A beneficial role of nitric oxide (NO) after cerebral ischemia has been previously attributed to its vascular effects. Recent data indicate a regulatory role for NO in initial leukocyte-endothelial interactions in the cerebral microcirculation under basal and ischemic conditions. In this study, the authors tested the hypothesis that endogenous NO production during and/or after transient focal cerebral ischemia can also be neuroprotective by limiting the process of neutrophil infiltration and its deleterious consequences. Male Sprague-Dawley rats were subjected to 2 hours occlusion of the left middle cerebral artery and the left common carotid artery. The effect of NG-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg, intraperitoneally), an NO synthase inhibitor, was examined at 48 hours after ischemia on both infarct size and myeloperoxidase activity, an index of neutrophil infiltration. L-NAME given 5 minutes after the onset of ischemia increased the cortical infarct volume by 34% and increased cortical myeloperoxidase activity by 60%, whereas administration of L-NAME at 1, 7, and 22 hours of reperfusion had no effect. Such exacerbations of infarction and myeloperoxidase activity produced when L-NAME was given 5 minutes after the onset of ischemia were not observed in rats rendered neutropenic by vinblastine. These results suggest that after transient focal ischemia, early NO production exerts a neuroprotective effect by modulating neutrophil infiltration.     

Early reperfusion improves the recovery of contralateral electrophysiological diaschisis following focal cerebral ischemia in rats

Abstract

Objectives: We evaluated electrophysiological benefits of reperfusion following ischemic stroke.

Methods: Rats received either transient proximal occlusion of the right middle cerebral artery for 30 (Group I, n=8) or 90 minutes (Group II, n=8) or permanent thermocoagulation of the distal right middle cerebral artery (Group III, n=6). Neurobehavioral outcome and somatosensory evoked potentials (SSEPs) were examined before and at 7 days after the onset of brain ischemia. Brain infarction was assessed after the rats were euthanized.

Results: Before ischemia, stable SSEPs were consistently recorded. At 7 days post-insult, Group III (permanent occlusion) had the greatest reduction in the SSEPs recorded ipsilaterally and contralaterally. Groups I and II (transient ischemic groups) also had depressant SSEPs recorded from the ipsilateral ischemic and the contralateral intact brain (electrophysiological diaschisis). However, prolonged ischemia resulted in greater brain infarction and increased neurological deficits in addition to greater reductions in the ipsilateral and the contralateral SSEPs.

Conclusion: Early reperfusion facilitates the electrophysiological recovery in both ipsilateral lesional and the contralateral intact brain, which may be closely relevant to post-injury brain rewiring. We also demonstrated that contralateral electrophysiological diaschisis could be greatly reversed by early reperfusion and is independent of the lesion size of striatum.     

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。     

大鼠急性脑缺血后Hephaestin的表达变化

目的 研究大鼠急性局灶性脑缺血后皮质和纹状体Hephaestin表达的变化。方法 线栓法制备大鼠急性大脑中动脉阻塞(MCAO)再灌注模型,在再灌注后的不同时间点应用免疫组化、图像分析以及SDS-PAGE Werstern blot方法检测缺血侧皮质和纹状体的表达变化。结果 MCAO再灌注后大鼠出现大脑中动脉梗死的神经系统损害体征,TTC染色有白色梗死区。Hephaestin在正常大鼠的皮质、纹状体有表达,在急性脑缺血再灌注后12h缺血侧皮质、纹状体表达明显增加并持续到再灌注后48h,在24h时到达高峰(P<0.01),至1周时表达较正常明显减少(P<0.01)。结论 大鼠急性脑缺血再灌注后Hephaestin的表达出现明显的变化,在急性脑缺血的病理生理变化中可能起着重要的作用。     

Decreased neuronal nitric oxide synthase expression and cell migration in the peri-infarction after focal cerebral ischemia in rats

Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in the normal developing brain, but the role of nNOS in neurogenesis of the adult ischemic brain remains unclear. The aim of this study was to investigate the temporal and spatial relationship between cell migration from the ependymal/subventricular zone (SVZ) to peri-infarction and nNOS expression in the rat. Ependymal/subventricular zone cells were prelabeled with fluorescence dye DiI. Focal cerebral ischemia was induced by occlusion of the left middle cerebral artery. At 1, 3, 7, 14 and 21 days after ischemia, the rats were killed in order to determine the number of migrating cells, the colocalization of DiI and nNOS as well as nNOS quantity in specific regions. Compared to non-ischemic control and 1 day post-ischemia, the number of DiI-labeled cells in the selected regions increased at 3 days and peaked 14 days following ischemia. During 3–7 days post-ischemia, none of the migrating cells expressed nNOS and decreased nNOS expression was observed in the regions where migrating cells passed through. These results suggest the possible association between ependymal/SVZ cell migration and decreased nNOS expression within the areas including the migrating routes towards the peri-infarction.     

脑缺血再灌流大鼠海马中一氧化氮与自由基的含量变化

目的　探讨脑缺血再灌流损伤中一氧化氮与自由基所起的作用及二者间的相互关系。方法　通过暂时夹闭大鼠两侧颈总动脉 ,造成大鼠脑缺血再灌流模型。取各组大鼠海马组织 ,测定其一氧化氮和丙二醛的含量 ,观察二者在脑缺血再灌流不同时间的动态变化。结果　脑缺血再灌流组大鼠与假手术组相比 :大鼠海马组织中一氧化氮含量明显升高 (P <0 0 1) ,其中在灌流 6h时的一氧化氮含量最高 ;大鼠海马组织中丙二醛含量也显著升高 (P <0 0 1)。结论　在急性脑缺血再灌流损伤过程中 ,一氧化氮既具有保护作用又具有细胞毒性作用。它主要通过介导自由基的大量产生 ,发挥其细胞毒性作用     

脑红蛋白在脑梗死大鼠表达及丁苯酞干预效应

目的探讨脑红蛋白在脑梗死大鼠中的表达和丁苯酞干预在氧化应激损伤中作用。方法将90只雄性SD大鼠随机分为假手术组、模型组、治疗组。每组再分为3个亚组:1 d组、3 d组和7 d组,每亚组10只。模型组和治疗组采用线栓法制作大鼠大脑中动脉闭塞模型(MCAO),假手术组只分离不结扎。治疗组在动物苏醒后以丁苯酞植物油灌胃,假手术组和模型组同法给予等量植物油灌胃。每只大鼠在术后3h和处死前行神经功能评分,应用RT-PCR和免疫组化检测脑组织脑红蛋白(NGB)表达,化学比色法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果 (1)神经功能评分,模型组和治疗组术后3 h评分差异无统计学意义(P>0.05),各时间点处死前评分差异均有统计学意义(P<0.05)。(2)NGB mRNA和免疫组化表达随时间延长逐渐降低,各时间点模型组较假手术组、治疗组较模型组高,差异均有统计学意义(P<0.05)。(3)SOD活性和MDA含量随时间延长逐渐减少。SOD活性假手术组较治疗组、治疗组较模型组高,差异均有统计学意义(P<0.05);MDA含量假手术组较治疗组、治疗组较模型组低,差异均有统计学意义(P<0.05)。结论丁苯酞促进脑缺血大鼠脑红蛋白表达,减少氧化应激损伤。     

缺血性脑梗死大鼠大脑皮质与海马Fas介导磷酸酯酶的表达

目的研究Fas介导磷酸酯酶(FAP-1)在缺血性脑梗死大鼠脑组织中的表达。方法应用免疫组化方法观察大鼠局灶性脑缺血后脑组织中FAP-1蛋白免疫反应阳性细胞数量及分布。结果脑梗死大鼠大脑皮质和海马FAP-1阳性细胞数分别是10.76±1.23、9.24±0.87,极显著高于假手术组(4.12±0.36、3.83±0.43)(均P<0.001),且阳性细胞以脑皮质锥体细胞树突、轴突较明显,分布于患侧大脑中动脉缺血区域。结论缺血性脑梗死大鼠脑组织的FAP-1表达明显增高,其表达增高可以提示脑组织有缺血性损伤。     

bFGF对大鼠局灶性缺血再灌注脑组织的保护作用

目的探讨bFGF对大鼠脑缺血再灌注损伤后神经细胞凋亡和脑组织中SOD、MDA含量变化的影响。方法应用线栓法制作大鼠局灶性脑缺血再灌注模型,大脑中动脉阻塞1h再灌注损伤24h,检测假手术组、缺血再灌注组和bFGF组的凋亡细胞数和脑组织中SOD、MDA的含量。结果假手术组偶见凋亡细胞,缺血再灌注组和bFGF组凋亡细胞数分别是26.35±5.67和18.65±5.91,与缺血再灌注组相比,bFGF组缺血区皮质凋亡神经元明显减少(P<0.05)。SOD,MDA测定结果显示三组间比较,具有显著性差异(P<0.01和P<0.05)。结论抗氧化作用可能是bFGF减少大鼠脑缺血再灌注损伤后细胞凋亡的分子机制之一。     

Chronic administration of rotenone increases levels of nitric oxide and lipid peroxidation products in rat brain

The complex I inhibitor rotenone is a neurotoxin that has been proposed to induce Parkinson-like degeneration. As the mechanisms of rotenone toxicity are not fully understood, the present study addresses the question of whether rotenone induces NO production and lipid peroxidation-like products, that is, thiobarbituric acid reactive substances (TBARS). Rotenone at a dose of 1.5 mg kg(-1) i.p. was administered to rats daily for 10, 20, 30, and 60 days, and NO and TBARS were measured in the frontal cortex and in the striatum. On the 1st and 10th day, there were no increases in NO and TBARS levels, after 20 days, the NO and TBARS levels were increased in the striatum. After 30 and 60 days, NO and TBARS levels were increased in striatum and frontal cortex. Behaviorally, on days 30 and 60, the rats exhibited akinesia and rigidity in the catalepsy test. These results show that chronic administration of rotenone over a long period is capable of increasing NO and TBARS in the cortex and striatum and mimics Parkinson's disease (PD)-like behavioral symptoms that are akinesia and rigidity in rats.     

The role of nitric oxide in the development of cortical spreading depression-induced tolerance to transient focal cerebral ischemia in rats

Cortical spreading depression (CSD) has been documented to confer ischemic tolerance on brain. Although nitric oxide (NO) is a crucial mediator in preconditioning under certain circumstances, the role of NO in CSD-induced neuroprotection is unclear. We examined the effect of L-NAME, an inhibitor of NO synthase, on CSD-induced tolerance against transient focal cerebral ischemia. A solution of 0.5 M KCl was applied for 2 h on the right hemisphere to induce CSD. Animals received either vehicle or L-NAME (4 mg/kg, iv) 30 min before CSD. Temporary occlusion (120 min) of the right middle cerebral artery was induced 4 days after preconditioning and the infarct volume was measured. Additionally, ERK 1/2 activation and cyclooxygenase-2 (COX-2) expression in the cerebral cortex were examined by Western blotting analysis immediately after cessation of CSD, or at 1, 2, 4, 8, and 24 h after CSD. CSD reduced infarct volume from 275 +/- 15 mm3 (mean +/- SEM) in the non-CSD group to 155 +/- 14 mm3 in the CSD group (P < 0.05). L-NAME abolished this protection (281 +/- 14 mm3; P < 0.05 vs. CSD group). Elevated ERK activation and COX-2 expression were observed immediately after or 8 h after preconditioning, respectively. Those responses are significantly augmented by L-NAME (3-fold for ERK and 4-fold for COX-2). These results suggest a crucial role of NO in the establishment of preconditioning with CSD.     

Time dependent changes of striatal interneurons after focal cerebral ischemia in rats

Summary. The cellular damage over time and the alterations of neuronal subtypes was characterized in the striatum after 90-min middle cerebral artery occlusion and reperfusion in rats. We investigated the immunohistochemical alterations of choline acetyltransferase (ChAT)-positive (cholinergic-positive), γ-aminobutyric acid (GABA)ergic parvalbumin (PV)-positive, GABAergic nNOS (neuronal nitric oxide synthase)-positive interneurons, neuronal nuclei (NeuN)-positive spiny projection neurons, glial fibrillary acidic protein (GFAP)-positive strocytes and microglial response factor-1 (MRF-1)-positive microglia in the striatum after focal cerebral ischemia in rats. In the present study, transient focal cerebral ischemia in rats caused severe damage against interneurons as well as spiny projection neurons in the striatum. In contrast, a significant increase in the number of GFAP-immunopositive astrocytes was observed in the ipsilateral striatum 15 days after focal cerebral ischemia. Furthermore, a significant increase of MRF-1 immunoreactivity was observed in microglia of the ipsilateral striatum 7 days and 15 days after focal cerebral ischemia. Among three types of cholinergic interneurons, GABAergic PV-positive interneurons and GABAergic nNOS-positive interneurons, the severe damage of cholinergic and GABAergic PV-positive interneurons was more pronounced than that of GABAergic nNOS-positive interneurons after transient focal cerebral ischemia in rats. Furthermore, the present results suggest that GABAergic nNOS-positive interneurons in the striatum after focal cerebral ischemia undergo cellular death in a delayed manner. Correspondence: Tsutomu Araki, Department of Neurobiology and Therapeutics, Graduate School and Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan     

Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model★

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry. RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P< 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P<0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.