异丙酚对缺血再灌注损伤大鼠海马氨基酸递质水平变化及神经元凋亡的影响

目的： 观察异丙酚对脑缺血再灌注损伤大鼠海马组织氨基酸递质水平变化及神经元凋亡的影响。方法：雄性Wistar大鼠60只，随机等分为假手术（A）组、缺血再灌注对照（B）组和异丙酚处理（C）组，后者按异丙酚用量又分为50 mg/kg（C₁）、100 mg/kg（C₂）和150 mg/kg（C₃）3个亚组。全脑缺血10 min再灌注60 min时，各组断头处死大鼠6只，检测海马组织中氨基酸水平的变化；于再灌注72 h时，断头处死余下大鼠，用流式细胞仪检测海马组织神经细胞凋亡指数（AI），光镜检测海马CA1区神经元凋亡的密度。结果：与假手术组比较，各组海马组织谷氨酸（Glu）、天冬氨酸（Asp）含量有不同程度的增高，γ-氨基丁酸（GABA）、甘氨酸（Gly）的含量有不同程度的降低(P<0.05或P<0.01)。异丙酚组海马组织Glu、Asp含量低于对照组，GABA、Gly的含量高于对照组（P<0.05或P<0.01）。再灌注72 h时，各组海马组织神经细胞AI有不同程度的增高，海马CA1区神经元凋亡密度明显增加。异丙酚组海马组织神经细胞凋亡数量明显减少。结论：异丙酚对脑缺血再灌注损伤的拮抗作用可能与其抑制兴奋性氨基酸的释放、降低抑制性氨基酸的耗竭、继而减少神经元的凋亡、提高神经元的存活数目有关。     

白松片对应激大鼠模型海马Glu、Asp、GABA含量的影响

目的:探讨中药白松片对应激大鼠海马谷氨酸、天冬氨酸、γ-氨基丁酸含量的影响.方法:将健康成年雄性大鼠48只,随机分为:正常对照、模型对照、氟西汀对照及白松片3个剂量6组,每组8只,通过强迫游泳建立应激大鼠模型.采用预防加治疗给药,高效液相色谱仪-荧光检测法检测大鼠海马Glu、Asp及GABA含量.结果:应激7天后,模型对照组海马Asp、Glu含量(分别为:1.522±0.294、5.123±0.491μmol/g),高于正常对照组,差异有显著性(P均<0.01);白松片等效、3倍、5倍剂量组海马Asp、Glu含量[Asp分别为:(1.268±0.237)μmol/g,(1.253±0.240)μmol/g,(1.168±0.135)μmol/g,Glu分别为(4.051±0.752)μmol/g,(3.565±0.671)μmol/g(3.435±0.443)μmol/g]均低于模型对照组,差异有显著性,(P<0.05、P<0.01).结论:白松片能降低应激大鼠海马Asp、Glu的含量.     

树鼩皮层局部脑缺血时海马细胞微环境及氨基酸含量动态变化的研究

目的：揭示血栓性脑缺血后，海马神经细胞微环境及其细胞外液兴奋性与抑制性氨基酸含量的动态变化，并探讨其相互关系及可能的机制。方法: 建立光化学反应诱导树鼩局部脑缺血模型，进行微灌流并收集海马细胞外液，用高效液相色谱-PITC衍生法和IL-1306型血气分析仪分别测量天冬氨酸（Asp）、谷氨酸（Glu）、甘氨酸（Gly）和γ-氨基丁酸（GABA）含量和微环境相关指标pH值、PCO2、PO2和HCO3－的变化。结果: 局部脑缺血后，海马细胞外液中上述各种氨基酸的含量与对照组比均升高（P<0.01）；pH值、PO2、HCO3－均下降（P<0.01）；PCO2在6 h升高（P<0.05）。结论: 局部脑缺血后海马细胞外液氨基酸平衡紊乱参与了脑损伤的发生，氨基酸的代谢改变与缺血后细胞微环境的变化有关。     

羚羊角与钩藤联合用药抑制热性惊厥大鼠脑损伤作用机制研究

目的:探讨羚羊角与钩藤联合用药对热性惊厥所致发育期大鼠脑损伤的保护作用及其作用机制。方法:采用热水浴建立热性惊厥大鼠模型,实验分为5 组,空白对照组、模型组、羚羊角组、钩藤组、羚羊角与钩藤联合用药组(简称联用组),每组10 只,共50 只。通过HE 染色法观察组织病理情况;ELISA 法检测各组大鼠脑组织TNF-α、IL-1β、Glu 及Asp 含量;免疫组化法检测大鼠海马区TNF-α、IL-1β表达情况。结果:联用组、羚羊角组、钩藤组脑组织Asp、Glu、TNF-α及IL-1β及海马区TNF-α、IL-1β含量显著低于模型组(P<0.01,P<0.05)。联用组脑组织Asp、TNF-α及IL-1β含量较钩藤组显著降低(P<0.05)。联用组海马区TNF-α较羚羊角组、钩藤组显著降低(P<0.05),IL-1β较钩藤组表达显著降低(P<0.05)。结论:羚羊角与钩藤联合用药抑制热性惊厥所致大鼠脑损伤,效果优于羚羊角及钩藤单用,其作用机制可能与其抑制促炎症因子TNF-α、IL-1β及兴奋性氨基酸Asp、Glu 表达有关。     

三种中药复方对慢性束缚应激大鼠皮层和海马BDNF、TrkB的影响

目的： 研究慢性束缚应激时大鼠皮层和海马BDNF、TrkB的变化以及逍遥散、四君子汤、金匮肾气丸3种中药复方对其影响。方法： 用特制束缚架连续束缚7 d与21 d，每天3 h的方法制作大鼠束缚应激模型，用免疫组织化学方法结合图像分析检测大鼠皮层和海马CA1区BDNF、TrkB的变化。结果： 连续束缚7 d、21 d后大鼠大脑额叶皮层与海马CA1区的BDNF均显著低于正常对照组（P<0.05, P<0.01）,尤以21 d模型组明显。连续束缚7 d、21 d后大鼠大脑额叶皮层与海马CA1区的TrkB分别显著高于正常对照组（P<0.05，P<0.01）。3个中药复方均能升高皮层BDNF的积分吸光度和海马中BDNF的阳性细胞数；逍遥散能降低海马和皮层中的TrkB阳性细胞数和海马TrkB的积分吸光度；四君子汤和金匮肾气丸能降低皮层TrkB的积分吸光度；金匮肾气丸能降低皮层TrkB的阳性细胞数；逍遥散升高皮层和海马中BDNF的作用比四君子汤和金匮肾气丸明显。结论： 皮层和海马CA1区BDNF下降参与慢性应激的变化，疏肝、健脾、补肾的中药复方均有一定程度的逆转作用，但以逍遥散的作用较强，优于四君子汤和金匮肾气丸。     

束缚浸水应激对大鼠脑组织中游离氨基酸含量的影响

为探讨脑中神经递质与动物应激反应之间的关系,本研究运用双向上行层析法,测定了大鼠束缚浸水应激5 h后,脑组织中几种游离氨基酸的含量。结果表明:大鼠5 h束缚浸水应激后,脑中天冬氨酸(Asp)、谷氨酸(Glu)、谷氨酰胺(Gln)3种兴奋性氨基酸的含量,较对照组分别增加了69.19%(P<0.05)、77.42%(P<0.01)、136.84%(P<0.01)。但抑制性氨基酸γ-氨基丁酸(GABA)的含量变化不显著(P>0.05)。     

海洛因慢性给药戒断期大鼠海马CA1区神经元电生理研究

目的：研究海洛因戒断期大鼠海马CA1区锥体细胞的生物电活动，以分析海洛因慢性给药戒断期，海马在“毒瘾”产生中的作用和可能机制。方法: 复制海洛因慢性给药戒断大鼠模型，并设生理盐水组和正常对照组，取各组离体海马脑片进行细胞内生物电记录。结果： 戒断期大鼠海马CA1锥体细胞的被动电学性质与各组无显著差异，但锋电位的半幅时程、10%-90%衰减时间显著缩短（P<0.01）、去极化后电位减小(P<0.05)；给予0.4-1.4 nA刺激时,细胞放电频率低于对照组（P<0.05）,1.6-2.0 nA刺激时,放电频率大于对照组(P<0.01)；此外,可见CA1区神经元兴奋性突触后电位（EPSP）有显著增强(P<0.01)。结论： 海洛因慢性给药使大鼠海马CA1区细胞在不改变被动电学特性前提下，对动作电位和EPSP有明显的作用。     

c-Jun氨基末端激酶在慢性低O2高CO2大鼠海马损伤中的作用

目的： 探讨c-Jun氨基末端激酶（JNK）在慢性低O2高CO2大鼠海马损伤中的作用。方法：采用慢性低O2高CO2肺动脉高压大鼠模型。30只SD大鼠随机分为正常对照（NC）组、低O2高CO2 2周（2WH）组和低O2高CO2 4周（4WH）组,每组10只。 Morris水迷宫检测行为学,用半定量逆转录-聚合酶链反应（RT-PCR）动态观察海马p-JNK mRNA的转录情况,免疫组织化学法测定p-JNK在海马CA1/CA3区的变化,TUNEL法检测海马CA1/CA3区神经细胞凋亡。结果：与NC组相比,低O2高CO22WH、4WH组大鼠寻找站台的平均逃避潜伏期延长、游泳总距离增加(P<0.01或P<0.05),且4WH组比2WH组潜伏期延长及游泳总距离增加更加明显(P<0.05);NC组大鼠海马仅见少量JNK mRNA及蛋白表达,低O2高CO2各组较NC组JNK表达明显增加（P<0.01或P<0.05）,且4WH组比2WH组增加更明显(P<0.05)。低O2高CO22WH、4WH组的TUNEL阳性细胞数均明显多于NC组（P<0.01或P<0.05）,且4WH组比2WH组增多明显（P<0.01）。结论：低O2高CO2能导致大鼠学习记忆障碍,可能与激活海马神经细胞内JNK有关。     

急性乙醇中毒大鼠学习记忆行为的改变与脑组织NO和nNOS含量变化

目的：通过观察急性乙醇中毒对大鼠学习记忆的影响并测定脑组织中一氧化氮（NO）与神经型一氧化氮合酶（nNOS）含量的变化，探讨乙醇中毒影响学习记忆的分子机制。方法:成年SD大鼠随机分为2组，模型组腹腔1次性注射乙醇2.5 g/kg［用生理盐水配成含20%乙醇(W/V)溶液］制备急性乙醇中毒大鼠模型；对照组注射等容量的生理盐水。Y-型迷宫检测大鼠学习记忆成绩、硝酸还原酶法检测鼠脑海马CA1、新纹状体中NO的含量、免疫组化方法检测鼠脑海马CA1、纹状体、小脑中nNOS的含量。结果:（1）模型组大鼠达到学会标准所需要的训练次数（34.33±13.04）明显大于对照组（27.50±8.79），P<0.05；（2）海马CA1区NO的含量在模型组为23.09±9.60，明显高于对照组（8.46±5.67）（P<0.01）；新纹状体（尾壳核）NO的含量在模型组（19.46±8.25）也明显高于对照组（8.22±4.46），P<0.01；（3）海马CA1区nNOS阳性神经元的数量在模型组为18.22±7.47，明显高于对照组（10.15±4.24）（P<0.05）；新纹状体（尾壳核）nNOS阳性神经元的数量在模型组（11.38±5.00）也明显高于对照组（6.15±3.69），P<0.05。结论:乙醇的神经毒性作用可能与脑组织中nNOS和 NO信号通路有关。     

甜菜碱对戊四氮致痫大鼠海马GFAP、甘氨酸和甘氨酸受体表达的影响

 目的：研究甜菜碱对癫痫大鼠海马胶质细胞原纤维酸性蛋白（GFAP）、甘氨酸(Gly)及甘氨酸受体(GlyR)表达的影响。方法：将健康雄性Wistar大鼠随机分为：正常对照组（腹腔注射生理盐水,1.0 mL生理盐水灌胃);癫痫组（腹腔注射戊四氮,1.0 mL生理盐水灌胃）;甜菜碱高、中、低浓度组（腹腔注射戊四氮,甜菜碱灌胃）;丙戊酸钠组（腹腔注射戊四氮,丙戊酸钠灌胃）。实验结束后大鼠眼眶取血检测血清中同型半胱氨酸的含量;在低温条件下迅速取脑组织,分析Gly含量的变化,免疫荧光检测GFAP的水平,兔疫荧光和Western bloting检测海马区GlyR表达的变化。结果：各组大鼠大发作潜伏期无显著差异(P＞0.05),但是甜菜碱治疗组较癫痫组的首次大发作持续时间显著缩短(P<0.01)。癫痫组同型半胱氨酸的含量与正常组比较显著降低（P<0.01）,甜菜碱高、低浓度组同型半胱氨酸含量与癫痫组比较明显降低（P<0.05）。癫痫组甘氨酸的含量与正常对照组相比显著下降（P<0.01）。甜菜碱高、中、低浓度组甘氨酸的含量与癫痫组比较显著增高（P<0.01）。免疫荧光检测GFAP结果,癫痫组与正常组比较显著增高（P<0.01）,而甜菜碱高中低浓度与癫痫组相比显著降低（P<0.01）。免疫荧光与Western bloting检测GlyR结果,癫痫组GluR的表达与正常对照组相比显著降低（P<0.01）,甜菜碱高、中、浓度组较癫痫组显著升高（P<0.01）。结论：甜菜碱具有较好的抗癫痫作用。     

大鼠脑缺血再灌注损伤期间脑组织间质各种氨基酸递质水平的变化

目的: 研究清醒大鼠脑缺血再灌注损伤期间海马、纹状体和新皮质细胞外多种氨基酸递质水平的变化。方法: 复制清醒大鼠前脑缺血模型,利用微透析技术,于缺血前、缺血15 min、再灌注15 min、30 min、60 min、120 min及180 min时所收集透析液,测定谷氨酸、天门冬氨酸、γ-氨基丁酸、甘氨酸、牛磺酸、丙氨酸、谷氨酰胺、丝氨酸、苏氨酸等9种氨基酸含量;并计算缺血再灌注期间不同脑区兴奋毒性指数(EI)的变化。结果: 缺血期间细胞外液中不仅兴奋性氨基酸如谷氨酸、天门冬氨酸等以及兴奋性氨基酸受体调质如甘氨酸显著增高,而且抑制性氨基酸如γ-氨基丁酸及抑制性氨基酸受体调质如牛磺酸和丙氨酸亦明显升高;但是反映兴奋性神经递质与抑制性神经递质平衡综合指数的EI值在缺血期间明显升高。结论: 脑缺血期间单纯谷氨酸、天门冬氨酸等升高可能不足以造成缺血性损伤,其它神经递质或其它因素协同兴奋性氨基酸在介导神经细胞内一系列病理生化反应而引起延迟性神经细胞损伤的过程中可能起重要作用。     

Changes of some amino acid concentrations in the medial vestibular nucleus of conscious rats following acute hypotension

Microdialysis and high performance liquid chromatography (HPLC) were used to measure the changes of certain amino acids in the medial vestibular nucleus (MVN) of conscious rats in order to understand whether those amino acids are involved in the regulation of blood pressure. Acute hypotension was induced by infusing sodium nitroprusside (SNP) into the femoral vein. In the control group, glutamate (Glu) release increased, though γ-aminobutyric acid (GABA) and taurine (Tau) release decreased in the MVN following acute hypotension. In the unilateral labyrinthectomy group, the levels of Glu, GABA, and Tau were unchanged in the ipsilateral MVN to the lesion following acute hypotension. Furthermore, in the contralateral MVN to the lesion, Glu release increased, and GABA and Tau release decreased following acute hypotension. These results suggest that SNP-induced acute hypotension can influence the activity of neurons in the MVN through afferent signals from peripheral vestibular receptors, and that certain amino acid transmitters in the MVN are involved in this process.     

An experimental arthritis in rats: dorsal horn aspartate and glutamate increases.

Amino acid release in the dorsal horn of awake rats was examined by microdialysis during the development of arthritis induced by injection of 3% kaolin and 3% carrageenan into the knee joint. The following amino acids were measured by HPLC at baseline and for the first 8 h of arthritis: Asp, Glu, Asn, Gln, Ser, Gly and Tau. An initial increase in all amino acids examined was observed on injection of the knee joint with kaolin and carrageenan. Subsequently, there was a peak increase in Asp (184%), Glu (188%) and Gln (146%) during a prolonged release phase which began at 3.5 h and persisted at least 8 h. While Asn showed no changes from baseline, extracellular fluid concentrations of Ser, Gly and Tau were variable. This data indicates that the induction of arthritis is accompanied by an increased release of excitatory amino acids Asp and Glu which may be important in the generation of acute arthritis.     

GABA,glycine, glutamate,aspartate and taurine in the perihypoglossal nuclei: an immunocytochemical investigation in the cat with particular reference to the issue of amino acid colocalization

Summary The differential distribution of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA) and taurine (Tau) was investigated in the cat's perihypoglossal nuclei. Serial semi-thin (0.5 m) sections through the perihypoglossal nuclei were incubated with antisera raised against the mentioned amino acids with the aim of studying possible co-localization. In each experiment different measures were undertaken in order to screen for possible cross-reactivities, and all sections were processed together with test conjugates in order to ascertain the specificity of the antisera used. A very high proportion of the neurons in the perihypoglossal nuclei (about 90%) shows strong immunostaining for Asp and also displays distinct immunoreactivity for Glu in neighbouring sections. About 25% of the cells in the perihypoglossal nuclei are intensely immunostained for Gly, but very few cells show immunoreactivity for GABA. Only glial cells appear to be immunostained for Tau. Neurons that are Gly(+) also display Glu and Asp immunoreactivities. The neuropil of the perihypoglossal nuclei shows a high density of GABA(+), Gly(+) and Glu(+) puncta mainly representing stained axons and terminals. Fewer Asp(+) puncta and very few Tau(+) nerve terminal-like puncta are seen. Details of the regional distribution of immunopositive neurons and puncta within the perihypoglossal nuclei are described. The findings are discussed with particular reference to the possible role of the mentioned amino acids as transmitter substances in the known synaptic circuitry of the perihypoglossal nuclei.Performed during leave of absence from Laboratory of Neurobiology, Center for Neuro-Behavioural Biology, Mahidol University, Salaya Campus, Bangkok, Thailand     

Glutamine depletion in patients with Crimean-Congo hemorrhagic fever

Crimean-Congo hemorrhagic fever (CCHF) is a viral disease. There is not enough knowledge about plasma amino acid levels in CCHF. Therefore, we investigated plasma amino acid levels in patients with CCHF and the association between the levels of these amino acids and disease severity. The plasma amino acid levels (including glutamate [Glu], aspartate [Asp], glutamine [Gln], asparagine [Asn] and gamma-aminobutyric acid [GABA]) in CCHF patients and controls were measured by using liquid chromatography-mass spectrometry. Plasma levels of Gln were lower while Asp, Glu, and GABA levels were higher in patients. In fatal CCHF patients, we found the plasma level of Asn was increased whereas the plasma level of GABA was decreased. This study is the first in the literature to evaluate the plasma Gln, Glu, Asn, Asp, and GABA levels in CCHF patients. We found that the plasma Gln levels were significantly lower in CCHF patients while Asp, Glu, and GABA levels were elevated. Considering that these amino acids are important for immune cells, the plasma amino acid levels of CCHF patients may contribute to the understanding of the pathophysiology of disease and it can be important for supportive treatment of CCHF.     

Changes in free amino acids in Peripheral Blood (PB) lymphocytes and Polymorphonuclear (PMN) leukocytes after treatment with diazepam

The effect of single and chronic (15 days) i.p. injections (1.0 and 8.0 mg/kg) of diazepam (DZ) on free amino acid profile in peripheral blood (PB) lymphocytes and polymorphonuclear (PMN) leukocytes of male Wister Albino rats were investigated. Depletion of some free amino acids was observed in the lymphocytes (mixed T- and B-lymphocytes) and PMN leukocytes (91–95%) neutrophils especially after chronic DZ-treatment. A dose-dependent depletion in the lymphocyte amino acids, Tau, Gly, Ala, Met and Ile, was found after both acute and chronic DZ-treatment. A similar depletion of Tau, Asp, Glu and Met appeared in the PMN leukocytes after single doses as well as chronic DZ-treatment. These results suggest that administration of 1.0 – 8.0 mg/kg of DZ in single dose or after chronic administration may interfere with the transport of certain important amino acids and/or protein turnover in PB lymphocytes and PMN leukocytes.On the other hand, the basic amino acids Lys, His and Arg were significantly increased in PMN leukocytes after chronic administration of 1.0 mg/kg DZ. It was suggested that the increased levels of the basic amino acids in the neutrophils may interact with the intracellular changes in pH that normally accompany the respiratory burst.     

Pathological relationships between microglial cell activity and tau and amyloid beta protein in patients with Alzheimer's disease

The present study directly examined the axon terminal (synaptosomal) level of amino acid neurotransmitters in rats following chronic constriction injury (CCI) to the sciatic nerve. Dorsal horn, synaptosomal content of aspartate (Asp), glutamate (Glu), glycine and gamma-aminobutyric acid (GABA) was assessed in these rats, and in normal age-matched and younger rats. The synaptosomal content of Asp and Glu in CCI rats was increased by 44-46% compared with control rats (P<0.016). The synaptosomal content of GABA in younger rats was 33% lower than that observed in control rats (P<0.005). Altered axon terminal levels of amino acid neurotransmitters accompany the painful symptoms of neuropathy. The lower axon terminal level of GABA in younger rats may help to explain the age-dependency of pain development in animal models of nerve injury.