兴奋毒性在肌萎缩侧索硬化的发病机制中具有重要作用

目的:分析比较肌萎缩侧索硬化病人和神经系统正常的受试者脑脊液中的谷氨酸水平,以明确由谷氨酸介导的“兴奋毒性”是否在ALS的发病机制中具有作用。方法:肌萎缩侧索硬化病人15例,神经系统正常的外科手术腰麻病人20例,采集脑脊液后用氨基酸自动分析仪进行检测。结果:肌萎缩侧索硬化病人脑脊液中谷氨酸水平与对照比较增高有极显著差异(两组分别为48.81±31.67μmol/L和15.85±6.70μmol/L)(P(0.01)。结论:由谷氨酸介导的“兴奋毒性”在肌萎缩侧索硬化的发病过程具有重要作用。     

肌萎缩侧索硬化脑脊液中兴奋性氨基酸的测定

运动神经元病是选择性侵犯上和 (或 )下运动神经元的神经系统变性疾病 ,肌萎缩侧索硬化 (ＡＬＳ)是其最常见的一种类型 ,近年来的研究表明 ,ＡＬＳ脑脊液中谷氨酸 (ＧＬＵ)含量明显增高 ,同时存在转运异常 ,为了进一步证实这一点 ,我们对ＡＬＳ病人进行脑脊液氨基酸的测定。资料和方法 :ＡＬＳ组 14例 ,男性 10例 ,女性 4例 ,平均年龄 4 5岁 ,对照组 6名 ,男性 4名 ,女性 2名 ,平均年龄 4 1岁 ,均为神经系统查体正常健康者。ＡＬＳ组和对照组腰穿后获得的脑脊液立即储存在 - 80℃冰箱中。标本的细胞总数均小于 5 ,糖、氯化物、蛋白均正常。…     

兴奋性氨基酸毒性与肌萎缩侧索硬化症的研究进展

兴奋性氨基酸毒性是肌萎缩侧索硬化症的主要发病机制之一,主要涉及以下几个环节:突触间隙兴奋性氨基酸水平的升高,谷氨酸转运蛋白异常导致的谷氨酸摄取减少,谷氨酸受体缺陷及钙离子结合蛋白的异常导致的运动神经元细胞内钙离子稳态失衡。对于各环节在发病机制中的具体作用仍存在争论。     

谷氨酸的兴奋性神经毒性作用与中枢神经系统损伤

谷氨酸(Glu)具有兴奋性神经毒性,其毒性作用与促进Na~+、Cl~-、水内流、Ca~(++)超载、自由基及一氧化氮的介导有关,参与多种急慢性脑损伤的发生。本文重点综述了Glu的神经毒性作用和机制及其在各种急慢性中枢神经系统损伤中的可能作用。     

运动神经元病的谷氨酸中毒学说

肌萎缩侧索硬化 (AmyotrophicLateralSclerosis,ALS)是选择性侵犯上下运动神经元的神经系统变性疾病 ,病因不明 ,目前尚无有效治疗。主要的发病机制假说之一为谷氨酸中毒学说。目前已经证实肌萎缩侧索硬化存在谷氨酸代谢、摄取异常 ,认为其选择性运动神经元损伤可能是通过Ca2 + -AMPA/Kanait受体介导的兴奋性中毒发生的 ,此外运动神经元保护机制异常可能也发挥了一定作用     

肌萎缩侧索硬化患者血浆和脑脊液谷氨酸改变及利鲁唑对血浆谷氨酸的影响

目的 :了解肌萎缩侧索硬化 (ALS)患者血浆和脑脊液谷氨酸浓度改变 ,以及药物干预对谷氨酸水平的影响。方法 :应用HPLC方法对ALS患者进行血浆和脑脊液谷氨酸测定。结果 :①患者血浆中存在兴奋性谷氨酸显著增高 ,谷氨酸浓度与性别、年龄、病程均无关。②力鲁唑 (力如太 )治疗 1个月后 ,血浆谷氨酸浓度下降 ,而环磷酰胺治疗变化不明显。③脑脊液中谷氨酸浓度未见明显变化。结论 :ALS患者存在谷氨酸代谢异常 ,但谷氨酸可能不是ALS致病的惟一途径。     

海马神经元兴奋性毒性模型的建立及其意义

目的探讨不同浓度谷氨酸对海马神经元的损伤作用,流式细胞仪在建立理想的兴奋性毒性模型中的应用。方法在体外原代培养10d的海马神经元中分别加入不同浓度的谷氨酸(100、200、400、600、800μmol/L),24h后相差显微镜下观察细胞形态变化,用MTT法检测存活率和流式细胞仪检测凋亡率以评定谷氨酸对海马神经元的损伤程度。结果不同浓度的谷氨酸组与对照组的细胞存活率差异有显著性(P<0.01),并呈浓度依赖性,随着谷氨酸浓度的升高,神经元的存活率降低;谷氨酸终浓度(100、200、400μmol/L)组的细胞凋亡率与对照组比较差异有显著性(P<0.01);600和800μmol/L谷氨酸组的细胞凋亡率与对照组比较差异无显著性(P>0.05),但细胞凋亡率随着谷氨酸浓度的增加而降低。结论过高浓度的谷氨酸导致细胞急性坏死而非迟发性凋亡,运用流式细胞仪检测体外培养海马神经元凋亡率是一种特异性高的检测方法,值得推广。     

Riluzole治疗肌萎缩侧索硬化

Riluzole是谷氨酸受体拮抗剂。现介绍Riluzole治疗ALS的作用机制和临床疗效。     

粒细胞集落刺激因子治疗肌萎缩侧索硬化：增加细胞外谷氨酸摄取和抗自由基的作用

兴奋性氨基酸毒性和自由基氧化损伤在肌萎缩侧索硬化的发生发展中起到重要的作用。一种新的神经保护类药物粒细胞集落刺激因子（Granulocyte colony stimulating factor,G-CSF）被认为具有抗兴奋性氨基酸毒性、抗氧化应激、抗凋亡等作用。既往研究发现G-CSF可以对暴露于高浓度谷氨酸盐的神经细胞起到保护作用,并且拮抗脑缺血再灌注后的氧化应激反应。由此,在慢性谷氨酸中毒诱导的运动神经元损伤的体外模型中,我们（1）使用生物化学方法测定G-CSF对体外模型细胞外谷氨酸浓度的影响;（2）测定G-CSF对运动神经元胞内超氧化物歧化酶、谷胱甘肽过氧化物酶活性以及组织内丙二醛、一氧化氮含量的影响;（3）免疫组织化学染色方法测定G-CSF对体外模型运动神经元存活的影响。结果显示G-CSF有显著的抗自由基和抗兴奋性毒性作用并明显减少脊髓前角运动神经元的凋亡和缺失,可以为肌萎缩侧索硬化提供新的治疗方向。     

肌萎缩侧索硬化症患者血浆和脑脊液谷氨酸含量变化的研究

目的 了解肌萎缩侧索硬化症(ALS)患者血浆、脑脊液谷氨酸浓度的改变,为治疗提供依据。方法应用毛细管电泳-激光诱导荧光检测器,测定ALS患者和对照组的血浆及脑脊液谷氨酸含量,比较两者的不同,并对不同病情ALS患者的谷氨酸浓度进行比较。结果 ALS患者血浆、脑脊液谷氨酸的浓度均较对照组明显升高(P<0.05)。不同病情患者间的谷氨酸浓度也有区别,谷氨酸水平升高程度与病情轻重相关。结论 ALS患者血浆和脑脊液的谷氨酸水平较正常高,脑脊液谷氨酸浓度比血浆浓度敏感,临床病情越重,谷氨酸浓度越高。     

The ratio of N-acetyl aspartate to glutamate correlates with disease duration of amyotrophic lateral sclerosis

Glutamate (Glu)-induced excitotoxicity has been implicated in the neuronal loss of amyotrophic lateral sclerosis. To test the hypothesis that Glu in the primary motor cortex contributes to disease severity and/or duration, the Glu level was investigated using MR spectroscopy. Seventeen patients with amyotrophic lateral sclerosis were diagnosed according to the El Escorial criteria for suspected, possible, probable or definite amyotrophic lateral sclerosis, and enrolled in this cross-sectional study. We measured metabolite concentrations, including N-acetyl aspartate (NAA), creatine, choline, inositol, Glu and glutamine, and performed partial correlation between each metabolite concentration or NAA/Glu ratio and disease severity or duration using age as a covariate. Considering our hypothesis that Glu is associated with neuronal cell death in amyotrophic lateral sclerosis, we investigated the ratio of NAA to Glu, and found a significant correlation between NAA/Glu and disease duration (r = −0.574, p = 0.02). The “suspected” amyotrophic lateral sclerosis patients showed the same tendency as possible, probable and definite amyotrophic lateral sclerosis patients in regard to correlation of NAA/Glu ratio with disease duration. The other metabolites showed no significant correlation. Our findings suggested that glutamatergic neurons are less vulnerable compared to other neurons and this may be because inhibitory receptors are mainly located presynaptically, which supports the notion of Glu-induced excitotoxicity.     

Riluzole increases high-affinity glutamate uptake in rat spinal cord synaptosomes

The purpose of this study was to examine the effect of the anti-convulsant agent, riluzole, on high-affinity glutamate uptake as measured in rat spinal cord synaptosomes. The rate of glutamate uptake was significantly increased in the presence of 0.1 microM and 1.0 microM riluzole, but not at the higher concentrations examined. Kinetics analysis demonstrated that riluzole (0.1 microM) decreased the apparent K(m) by 21% and increased the V(max) by 31%. Glutamate uptake also was significantly increased in spinal cord synaptosomes obtained from rats treated with 8 mg/kg (i.p.) of riluzole and sacrificed 4 h later. The increase in glutamate uptake in vitro was not affected by pretreatment either with H7, an inhibitor of PKA and PKC, or with the PKC activating phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Previous studies have shown that some of the actions of riluzole are mediated by G proteins sensitive to pertussis toxin. Surprisingly, treatment of synaptosomes with pertussis toxin alone increased the rate of glutamate uptake, while having no effect on uptake in the presence of riluzole. However, pretreatment with cholera toxin was found to completely block the effects of riluzole on glutamate uptake. These results reveal an additional mechanism by which riluzole can affect glutamatergic neurotransmission, and provides further support that riluzole may prove beneficial in the treatment of traumatic central nervous system injuries involving the excitotoxic actions of glutamate.     

Polymorphisms in the glutamate transporter gene EAAT2 in European ALS patients

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder characterised by degeneration of upper and lower motor neurons. Whilst the primary pathogenic trigger is unknown in most cases, evidence is mounting to implicate a role for glutamate-mediated neurotoxicity in the disorder. Recent studies have shown reduced levels of the mainly astroglial glutamate transporter EAAT2 in ALS motor cortex and spinal cord and multiple abnormal EAAT2 mRNA species in ALS brain tissue. One cause of the low EAAT2 levels may be that point mutations in the EAAT2 gene, EAAT2, result in an abnormal unstable protein. To test this hypothesis we analysed EAAT2 in 128 sporadic and 23 familial European ALS cases. No variants within the coding sequence of EAAT2 to affect the protein sequence nor in the consensus splice sites of the flanking intronic sequences were found in any cases, similar to findings in other reports. Frequent polymorphisms within the flanking intronic sequences of both exons 2 and 4 were seen but at similar frequencies in controls. Mechanisms other than mutations within the coding region of EAAT2 must therefore be responsible for the low levels of EAAT2 seen in most cases of ALS. Received: 26 February 1999 Received in revised form: 2 June 1999 Accepted: 2 July 1999     

Cerebral neurons of transgenic ALS mice are vulnerable to glutamate release stimulation but not to increased extracellular glutamate due to transport blockade

Mechanisms of motor neuron loss in amyotrophic lateral sclerosis (ALS) are unknown, but it has been postulated that excitotoxicity due to excessive glutamatergic neurotransmission by decreased efficiency of glutamate transport may be involved in both familial (FALS) and sporadic ALS. Using microdialysis in vivo, we tested the effects of the glutamate transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC) and of 4-aminopyridine (4-AP), which stimulates glutamate release from nerve endings, in the hippocampus and motor cortex of wild type (WT) and transgenic SOD1/G93A mice, an established model of FALS. Perfusion of 4-AP induced convulsions, expression of the inducible stress-marker heat-shock protein 70 (HSP70) and hippocampal neuronal loss. These effects were similar in both WT and G93A mice, and, in both groups, they were prevented by the previous systemic administration of the NMDA receptor antagonist MK-801. In contrast, perfusion of PDC resulted in a large and long-lasting (2 h) increase of extracellular glutamate, but no convulsions, neuronal damage or HSP70 expression were observed in either the WT or the G93A mice. Our results demonstrate that SOD1 G93A mutation does not enhance the vulnerability to endogenous glutamate-mediated excitotoxicity in brain, neither by blocking glutamate transport nor by stimulating its release. Therefore, these data do not support the possibility that glutamate transport deficiency may be an important factor of brain neuronal degeneration in familial ALS.     

Tumour associated epilepsy and glutamate excitotoxicity in patients with gliomas

Tumour associated epilepsy (TAE) is common, debilitating and often not successfully controlled by surgical resection of the tumour and administration of multiple anti-epileptic drugs. It represents a cause of significant lost quality of life in an incurable disease and is therefore an important subject for ongoing research. The pathogenesis of TAE is likely to be multifactorial and involve, on the microscopic level, the interaction of genetic factors, changes in the peritumoural microenvironment, alterations in synaptic neurotransmitter release and re-uptake, and the excitotoxic effects of glutamate. On a macroscopic level, the occurrence of TAE is likely to be influenced by tumour size, location and interaction with environmental factors. The optimal treatment of TAE requires a multi-disciplinary approach with input from neurosurgeons, neurologists, radiologists, pathologists and basic scientists. This article reviews the current literature regarding the incidence, treatment, and aetiology of TAE.     

G-CSF对肌萎缩侧索硬化细胞外谷氨酸摄取和抗自由基作用的影响

目的研究在肌萎缩侧索硬化的体外模型中,粒细胞集落刺激因子(G-CSF)是否可以通过促进细胞外谷氨酸摄取和抗自由基作用发挥对运动神经元的保护作用。方法取8日龄SD乳鼠断头处死,取脊髓切片后放入培养液中构建脊髓器官模型。将构建的脊髓器官模型随机分为对照组、肌萎缩侧索硬化症(ALS)模型组和G-CSF组。对照组不做任何处理,仍常规培养;ALS模型组按照Matyja等人的方法加入THA和PDC构建ALS模型;G-CSF组在ALS模型组处理的基础上再加入重组人粒细胞集落刺激因子。在培养的1w、2w、3w、4w时对各组培养液中的Glu进行测定,并在第4w测定培养液中超氧化物歧化酶(SOD)、丙二醛(MDA)含量,免疫组化染色计数各组前角存活的运动神经元。结果对照组培养液中Glu浓度较低,并且随时间变化(1w、2w、3w、4w)不大,基本维持在恒定水平;ALS模型组和G-CSF组培养液中Glu浓度随时间延长逐渐增加,2w后G-CSF组Glu浓度显著低于ALS组(P0.05)。第4周时ALS模型组脊髓前角中SOD活性显著低于对照组(P0.05),G-CSF组SOD活性高于ALS模型组(P0.05),但仍低于对照组(P0.05)。结论 G-CSF有显著的抗自由基和抗兴奋性毒性作用并明显减少脊髓前角运动神经元的凋亡和缺失,可能为肌萎缩侧索硬化提供新的治疗方向。     

GLT-1 glutamate transporter levels are unchanged in mice expressing G93A human mutant SOD1.

A decrease in expression of the glutamate transporter GLT-1 is thought to be responsible for the increase in extracellular glutamate observed in patients with amyotrophic lateral sclerosis (ALS) and in a transgenic mouse model of ALS. We examined protein levels of the glutamate transporters GLT-1, GLAST and EAAC1 in the G93A (SOD1) transgenic mouse model of ALS. GLT-1 was detected in two bands (72 and 150 kD). Semi-quantitative analysis of Western blots showed that GLT-1 levels in sensorimotor cortex, brain stem, and cervical and lumbar spinal cord of G93A mice did not differ significantly from controls, either at end stage or at 60- or 90-days old. Nevertheless, other differences were found in GLT-1 at end stage. The percentage of total GLT-1 in the 150 kD band increased significantly (p<0.05) in the spinal cord and was elevated in the brain stem and cortex. Furthermore, brain stem and spinal cord GLT-1 from G93A mice showed retarded mobility on gels compared to controls (M(r) approximately equal to 77.3+/-2.3 and 164.3+/-3.1 vs. 72.2+/-2.4 and 153.6+/-4.7, respectively). GLAST and EAAC1 were unchanged in both amount and mobility. These results show that a loss of GLT-1 protein is not necessary for ALS-like neurodegeneration in G93A mice. However, the changes in GLT-1 mobility and distribution indicate that GLT-1 is altered in mice with the SOD1 mutation.