苯乙酸对大鼠原代皮质神经元的毒性作用

探讨苯乙酸(phenylacetic acid,PAA)对大鼠原代培养的皮质神经元的毒性作用及其作用机制.实验结果显示,PAA加入无血清Neurobasal培养基可剂量依赖地损伤原代培养的皮质神经元,且干预培养的时间越长,神经元存活率越低.而同浓度(1.1×10-3mol/L)的PAA对皮质神经元的损伤明显高于海马.原代培养的皮质神经元补充一氧化氮合成酶抑制剂L--NAME、NMDA受体拮抗剂MR-801及I-型钙通道阻滞剂尼莫地平可显著地抵抗PAA的毒性作用.以上实验结果提示,PAA可选择性地损伤皮质神经元,其毒性作用可能与NO过量产生、兴奋性氨基酸的堆积及钙超载有关.     

皮质酮对体外培养的大鼠海马神经元膜电位的影响

为探讨应激激素－皮质酮以违法乱纪 马神经细胞膜电位的影响,采用膜片钳全细胞记录的方法,测量了原代培养的大鼠马神经细胞膜的静脉电位。结果发现,在皮质酮的作用下,海马神经静息电位幅值明显降低,细胞膜呈去极化。     

皮质酮对体外培养的大鼠海马神经元膜电位的影响

为探讨应激激素--皮质酮对海马神经细胞膜电位的影响,采用膜片钳全细胞记录的方法,测量了原代培养的大鼠海马神经细胞膜的静息电位.结果发现,在皮质酮的作用下,海马神经细胞膜静息电位幅值明显降低,细胞膜呈去极化.推测,皮质酮可能通过抑制海马细胞的能量代谢,使细胞能量匮乏,导致膜去极化.     

帕罗西汀对皮质酮抑制成年大鼠海马细胞增殖的调制作用

目的慢性糖皮质激素治疗叮能导致认知和情感变化,这或许是由于糖皮质激素对海马神经发牛肢细胞增殖的抑制作用造成。帕罗西汀是一种选择性血清素重摄取抑制剂,临床常用作减轻抑郁症状,近几年来发现它能促进海马神经发生。本研究探讨帕罗西汀与慢性糖皮质激素的相耳作用。方法成年大鼠被分成四绀,分别给予芝麻油、皮质酮、帕罗西汀或皮质酮和帕罗西汀十四天。溴脱氧尿嘧啶核苷（5-bromo-2-deoxyuridine,BrdU）免疫组化法被用于定量齿状回的细胞增殖。结果皮质酮抑制了海马的细胞增殖,帕罗西汀增加了海马的细胞增殖。同时给药组还显示帕罗西汀能逆转皮质酮的抑制作用。结论本研究结果对防止海码在类同醇治疗以后的损害或许有临床意义。     

慢性应激对大鼠海马CA3区锥体细胞顶树突及血清皮质酮浓度的影响

目的探讨慢性应激对大鼠海马CA3区锥体细胞结构和血清皮质酮浓度的影响。方法将20只雄性Sprague-Dawley大鼠按体质量随机分为应激组和对照组,每组10只。采用高尔基染色法及酶联免疫分析方法,观察慢性强迫游泳应激对大鼠海马CA3区锥体细胞顶树突和血清皮质酮浓度的影响。结果应激组大鼠海马CA3区锥体细胞顶树突的总长度[(112±10)μm]短于对照组[(168±34)μm],差异有统计学意义(P<0.01);一级树突直径[(9.0±1.1)μm]大于对照组[(5.7±0.9)μm],差异有统计学意义(P<0.01);血清皮质酮浓度[(13±14)μg/L]低于对照组[(30±16)μg/L],差异有统计学意义(P<0.05)。结论慢性强迫游泳可引起大鼠海马CA3区锥体细胞顶树突及血清皮质酮浓度的改变。     

帕罗西汀对皮质酮抑制成年大鼠海马细胞增殖的调制作用(英文)

目的慢性糖皮质激素治疗可能导致认知和情感变化,这或许是由于糖皮质激素对海马神经发生及细胞增殖的抑制作用造成。帕罗西汀是一种选择性血清素重摄取抑制剂,临床常用作减轻抑郁症状,近几年来发现它能促进海马神经发生。本研究探讨帕罗西汀与慢性糖皮质激素的相互作用。方法成年大鼠被分成四组,分别给予芝麻油、皮质酮、帕罗西汀或皮质酮和帕罗西汀十四天。溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,BrdU)免疫组化法被用于定量齿状回的细胞增殖。结果皮质酮抑制了海马的细胞增殖,帕罗西汀增加了海马的细胞增殖。同时给药组还显示帕罗西汀能逆转皮质酮的抑制作用。结论本研究结果对防止海马在类固醇治疗以后的损害或许有临床意义。     

皮质酮对TNF-α致海马神经元细胞内钙变化的作用

应用荧光影像系统检测了原代培养海马神经元内钙离子的变化情况,并分析了皮质酮、肿瘤坏死因子(TNF-α)对谷氨酸引起的海马神经元内钙升高的调节作用及其皮质酮对TNF-α作用的调节.结果显示:(1)使用不同浓度的皮质酮处理海马神经元48 h后,观察到10-6 mol/L和10-7 mol/L的皮质酮可诱导海马神经元静息钙浓度升高,但10-8 mol/L和10-9 mol/L的皮质酮对海马神经元内钙无影响.(2)10-6 mol/L皮质酮处理海马神经元48 h后,对谷氨酸诱导的海马神经元内钙升高无调节作用.(3)使用100 ngTNF-α处理海马神经元48 h后,既可诱导海马神经元静息钙升高,也可抑制谷氨酸引起的海马神经元内钙升高.(4)皮质酮可逆转TNF-α对海马神经元静息钙浓度的调节作用,但对TNF-α抑制谷氨酸升钙效应无影响.     

实验性癫痫时大鼠海马内兴奋性氨基酸受体对阿片肽含量的影响

应用免疫组织化学方法观察了青霉素致痫及海马内微量注射ＮＭＤＡ受体拮抗剂ＭＫ８０１（５－ｍｅｔｈｙ１－１０，１１－ｄｉｈｙｄｒｏ－５Ｈ－ｄｉｂｅｎｚｏ（ａ，ｄ）ｃｙｃｌｏｈｅｐｔａｎ－５，１０－ｉｍｉｎｅｍａｌｅａｔｅ）和非ＮＭＤＡ受体拮抗剂ＤＮＱＸ＊（６，７－ｄｉｎｉｔｒｏｑｕｉｎｏｘａｌｉｎｅ－２，３－ｄｉｏｎｅ）后，大鼠海马内吗啡肽Ａ１－１７和亮啡肽的含量变化，结果发现，在青霉素致痫４ｈ后，海     

皮质酮对TNF—α致海马神经元细胞内钙变化的作用

应用荧光影像系统检测了原代培养海马神经元内钙离子的变化情况，并分析了皮质酮、肿瘤坏死因子（TNF－α）对谷氨酸引起的海马神经元内钙升高的调节作用及其皮质酮对TNF－α作用的调节。结果显示：（1）使用不同浓度的皮质酮处理海马神经元48h后，观察到10^-6mol/L和10^-7mol／L的皮质酮可诱导海马神经元静息钙浓度升高，但10^-8mol/L和10^-9mol/L的皮质酮对海马神经元内钙无影响。（2）10^-6mol/L皮质酮处理海马神经元48h后，对谷氨酸诱导的海马神经元内钙升高无调节作用。（3）使用100ngTNF－α处理海马神经元48h后，既可诱导海马神经元静息钙升高，也可抑制谷氨酸引起的海马神经元内钙升高。（4）皮质酮可逆转TNF－α对海马神经元静息钙浓度的调节作用，但对TNF－α抑制谷氨酸升钙效应无影响。     

Clenbuterol对海人藻酸所致大鼠海马组织培养损伤的保护作用研究

目的　探讨 β-肾上腺素能受体协同剂 Clenbuterol是否可保护海马神经组织对抗兴奋性氨基酸—海人藻酸 (KA)所致的神经损伤。方法　应用培养的海马组织切片模型 ,将 14d的海马脑片在高钙离子浓度的培养液中加用 5 0 μm ol/ L 的 KA3h,在应用 KA前 2 h、应用的同时及之后的 18h内 ,加用 10 0 μm ol/ L 的 Clenbuterol。KA所致的神经损伤由 Propidium Iodide(PI)的吸收量、乳酸脱氢酶 (L DH)的释放量来评价。结果　 Clenbuterol可以保护海马神经组织由 KA所致的损伤 ,表现在 Clenbuterol和 KA同时用药组比 KA单独用药组 L DH释放量及 PI吸收量均降低 2 5 %～ 30 %左右。并证明 Clenbuterol的神经保护作用 ,是通过 β2 -肾上腺素能受体的激活来完成。结论　 Clenbuterol可保护海马神经组织对抗 KA所致的兴奋性损伤 ,这种神经保护作用可以使它成为一种治疗缺血性卒中很有潜力的药物。     

NGF对体外培养海马神经元氧应激状态的调控作用及机制研究

目的:探讨神经生长因子NGF(ncrve growth factor)对海马神经元氧应激状态的影响及可能机制。方法:利用H2O2诱导建立了海马神经细胞氧应激的实验模型,H2O2选取5个剂量(分别为0.1mM,0.25mM。0.5mM,0.75mM,1.0mM);NGF取3个剂量(分别为1ng/ml,10ng/ml,100ng/ml),通过形态学观察、图像分析细胞光密度MOD(matrix optical density)检测添加NGF对海马神经元氧应激的影响,利用Fluo-3/AM负载培养海马神经元胞内游离钙测定的手段,通过激光扫描共聚焦显微镜观察添加NGF后海马神经元胞内钙的变化。结果:NGF组的海马神经元光密度除一组(H2O2-0.1mM+NGF-10ng/ml)外,均与单纯H2O2组有显著性差异。加入NGF可以延长海马神经细胞胞内游离钙浓度水平正常的时程,降低H2O2引起的神经元胞内游离钙浓度的升高。结论:NGF可以部分逆转H2O2诱导的海马神经元氧应激状态,且可能与可以逆转H2O2引起的胞内游离钙浓度增加有关。先后添加NGF,实验结果未见差异。在有效剂量范围,不同剂量NGF实验结果也未见差异。     

Inhibitory and excitatory amino acids in the cerebrospinal fluid of children with two types of cerebral palsy*

BACKGROUND： Under normal conditions, excitatory amino acids are dynamically balanced with inhibitory amino acids. Excitatory amino acids have been implicated in perinatal brain injury. OBJECTIVE： To investigate differences in the levels of the excitatory amino acids glutamic acid and aspartic acid, and the inhibitory amino acid gamma-aminobutyric acid （GABA） in the cerebrospinal fluid （CSF） of children with spastic cerebral palsy or athetotic cerebral palsy. DESIGN, TIME AND SETTING： Case-control exploratory observation of neurotransmitter in patients. The experiment was performed in the Pediatrics Department of the Second Affiliated Hospital of Changsha Medical College, the Cerebral Palsy Center of Xiangtan Affiliated Hospital of South China University and the Pediatrics Department of Xiangya Hospital, between February 2006 and May 2007. PARTICIPANTS： We selected 27 children with cerebral palsy, including 13 with spastic cerebral palsy and 14 with athetotic cerebral palsy. We selected 10 patients who were not affected by any neurological disease as controls. METHODS： Two mL blood-free CSF was harvested between the third and fourth lumbar vertebrae of each patient after anesthesia, and stored at -70℃. One mL CSF was mixed with 10 mg sulfosalicylic acid and placed in ice-bath for 10 minutes, then centrifuged 2 000 g for 10 minutes. The supernatant was collected for amino acid quantitation. MAIN OUTCOME MEASURES： The concentrations of glutamic acid, aspartic acid and GABA in the CSF were determined by high-performance liquid chromatography and fluorometric method. The correlation of glutamic acid, aspartic acid and GABA levels with muscular tension in children with cerebral palsy was analyzed using linear dependence. RESULTS： The concentration of GABA was significantly lower in both spastic cerebral palsy and athetotic cerebral palsy patients than in the control group （P 〈 0.01）. Glutamic acid and aspartic acid were significantly higher in both cerebral palsy groups than in the control     

Mg~(2 )对小鼠学习记忆的影响及其与海马CA_3区和大脑皮层突触界面结构的相关性

采用行为测试和亚微结构定量分析相结合的方法，研究了Ｍｇ２＋对小鼠分辨学习和记忆巩固的影响，同时测定了海马ＣＡ３区及大脑皮层突触界面结构参数的变化。实验结果表明：慢性给予Ｍｇ２＋后，血浆及海马内Ｍｇ２＋含量有一定程度的升高，小鼠分辨学习能力显著下降，记忆巩固受到破坏，并且海马ＣＡ３区及大脑皮层的突触后致密物质（ｐｏｓｔ—ｓｙｎａｐｔｉｃｄｅｎｓｉｔｙ，ＰＳＤ）显著变薄，同时，皮层突触活性区长度亦显著缩短。以上结果提示，脑内Ｍｇ２＋水平过高造成的学习记忆过程损伤可能与其改变ＰＳＤ厚度和突触活性区长度密切相关。     

Effects of oxysophoridine on amino acids after cerebral ischemic injury in mice

Background:

Our previous studies demonstrated that oxysophoridine (OSR) had neuroprotective effects on mice through antioxidant and anti-apoptotic mechanisms. In this study, we investigated whether OSR could influence the release of amino acids in ischemic mice brains.

Materials and Methods:

Male ICR mice were scheduled to undergo 2 h middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Before MCAO, mice in corresponding groups were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days. After reperfusion, neurological scores were estimated, infarct volume and the brain water content were assessed. The levels of glutamate (Glu), aspartate (Asp), γ-aminobutyric acid (GABA) and Glycine (Gly) were measured by amino acid analyzer.

Results:

OSR significantly decreased neurological scores, reduced infarct volume and the brain water content. After treatment with OSR of 250 mg/kg, the contents of Glu, Asp, GABA and Gly in mice brains could maintain at a normal level compared with MCAO group mice. The Glu/GABA ratio was significantly decreased in OSR group mice.

Conclusion:

These findings indicate that OSR has a protective effect on cerebral ischemic injury and helps to maintain the amino acids homeostasis after reperfusion for a long time.     

Protection Against N-methyl-D-aspartate Receptor-Mediated Neuronal Degeneration In Rat Brain by 7-chlorokynurenate and 3-amino-1-hydroxypyrrolid-2-one,Antagonists at The Allosteric Site for Glycine

7-Chlorokynurenate (7-Cl KYNA) and 3-amino-1-hydroxypyrrolid-2-one (HA-966), two selective antagonists of the glycine site on the N-methyl-D-aspartate (NMDA) receptor, have been used to assess the involvement of this site in the neurodegeneration resulting from injection of excitotoxins in the rat brain. In the rat striatum, reductions in the enzymes choline acetyltransferase (CAT) and glutamate decarboxylase (GAD), occurring 7 days after a unilateral, intrastriatal injection of quinolinate (200 nmol), were prevented in a dose-dependent manner by intrastriatal administration of 7-Cl KYNA (10 - 50 nmol) and HA-966 (200 - 500 nmol) 1 h after the excitotoxin. In the rat hippocampus, degeneration of pyramidal and granule neurons caused by direct injection of quinolinate (60 nmol) was completely prevented by 7-Cl KYNA (50 nmol) and partially by HA-966 (500 nmol) injected intrahippocampally 1 h after the excitotoxin. In the rat striatum, 7-Cl KYNA (50 nmol) and HA-966 (500 nmol) also reduced neurotoxicity caused by intrastriatal injection of NMDA (200 nmol), but not that caused by the 'non-NMDA' receptor agonists DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. The time course of protective effects of 7-Cl KYNA and HA-966 in the striatum was similar to that previously observed with the uncompetitive NMDA receptor antagonist MK-801, indicating that activation of the glycine site contributes to the delayed degeneration of neurons which occurs over the first 5 h following quinolinate injection. The neuroprotective effects of both 7-Cl KYNA and HA-966 in the rat striatum appear to be mediated via the glycine site on the NMDA receptor as they were completely reversed by D-serine, but not L-serine. These results indicate that activation of the glycine site is essential for the expression of the delayed degeneration of neurons resulting from intracerebral injection of an NMDA receptor agonist, a process which bears similarities to the delayed neurodegeneration which results from a period of cerebral ischaemia.     

急性脑梗死患者脑脊液兴奋性氨基酸水平的变化及其临床意义

目的 研究急性脑梗死患者脑脊液（CSF）兴奋性氨基酸含量变化及其临床意义。方法 采用高效液相层析方法测定30例急性脑梗死患者及20例偏头痛患者（对照组）CSF谷氨酸（Glu）、天门冬氨酸（Asp）的含量。结果 脑梗死组患者CSF中Glu、Asp含量显著高于对照组（均P〈0．001）。脑梗死组患者CSF中Glu和Asp含量与梗死体积呈显著正相关（r=0．75，P〈0．01；r=0.437，P〈0．05），CSF中Clu含星与神经功能缺损程度亦呈正相关（r=0．55，P〈0．01）。结论 脑梗死发生后CSF中Glu、Asp含量明显升高提示其参与了脑梗死的病理生理过程CSP中Glu、Asp含量可反映脑梗死患者的病情及梗死灶的大小。     

Evidence for Excitatory Amino Acid Neurotransmitters in Forward and Feedback Corticocortical Pathways within Rat Visual Cortex

It is a commonly accepted notion that cells which make projections between the multiple cortical areas found in the mammalian visual system are excitatory, but there is little direct evidence that this is the case. Here we demonstrate using retrograde tracing with D-[³H]aspartate that connections in the rat which project from lower to higher visual areas (i.e. forward) and those which project from higher to lower areas (i.e. feedback) may use excitatory amino acid neurotransmitters. Following injection into the primary visual cortex, clusters of retrogradely labelled cells were found in several extrastriate areas within the cytoarchitectonic subdivisions 18a (‘areas’ LM, AL, PX, FLX, RL, AX) and 18b (‘area’ MX), and in the retrosplenial cortex. In all of these areas D-[³H]aspartate-labelled cells were surrounded by diffuse label which may represent anterograde labelling of axon terminals. This suggests that both legs of reciprocal intracortical circuits have similar chemospecificity. To directly demonstrate excitatory amino acid localization in forward projections, D-[³H]aspartate was injected into extrastriate area LM. As expected, the results revealed retrogradely labelled neurons within area 17. Outside area 17, LM injections labelled neurons in AL, PX, FLX, RL, AX and MX. Taken in the context of the hierarchy of areas in rat cerebral cortex (Coogan and Burkhalter, J. Neurosci., 13, 3749–3772, 1993), these results show that D-[³H]aspartate labels: (1) forward connections from area 17 to LM, AL, PX, RL, AX and MX, (2) feedback connections from LM, AL, FLX, PX, RL, AX and MX to area 17, (3) feedback connections from AL, PX, RL, AX and MX to LM, and (4) lateral connections between FLX and LM. These findings strongly indicate that both forward and feedback connections as well as lateral connections at several different levels of the cortical hierarchy use excitatory amino acid neurotransmitters.     

Simultaneous blockade of non-NMDA ionotropic receptors and NMDA receptor-associated ionophore partially protects hippocampal slices from protein synthesis impairment due to simulated ischemia

A large body of evidence exists to demonstrate that excitatory amino acids (EAA) and their receptors are involved in the pathophysiological mechanisms linking several acute brain insults, such as cerebral ischemia, to neuronal degeneration and death. Accordingly, the use of EAA receptor antagonists can be beneficial in attenuating or preventing the neuronal irreversible damage subsequent to various neuropathological syndromes. We have investigated the effect of 15 min of simulated ischemic conditions, i. e., oxygen/glucose deprivation, on hippocampal slices preparation measuring, as neurotoxicity indexes, both the amino acids efflux in the incubation medium, detected by HPLC, and the inhibition of protein synthesis, evaluated as ³H-Leucine incorporation into proteins. Accumulation of neurotransmitter amino acids was measured in the medium during the “ischemic” period. Glutamate increased 30-fold over the basal level while aspartate was sevenfold and GABA 12-fold higher than in normal conditions. After a reoxygenation period of 30 min, the rate of protein synthesis of hippocampal slices subjected to “ischemia” was reduced to 35–50% of controls. The non-competitive NMDA antagonist MK-801 (100 μM) and the competitive NMDA antagonist CGP 39551 (100–250 μM) as well as the non-NMDA receptor antagonist NBQX (100 μM) and AP3 (300 μM) were unable to counteract the metabolic impairment when they were present alone in the incubation fluid during simulated “ischemia.” An incomplete, but highly significant (p < 0.001), protection from protein synthesis impairment was achieved in the presence of an equimolar concentration (100 μM) of MK-801 and NBQX. A similar protective effect could be reproduced using 100 μM NBQX in concomitance with a high Mg⁺⁺ (20 μM) voltage-dependent block of the NMDA receptor-associated channel but not exposing the slices to a NBQX (100 μM) and CGP 39551 (100–250 μM) mixture. The recovery of protein synthesis in the presence of the MK-801/NBQX effective combination was not paralleled by a detectable decrease in the amount of amino acids released in the incubation medium during the “ischemic” period. Taken together, the present data allow new insights into neurotoxicity-mediating mechanisms, suggesting that multiple additive processes are involved and that antagonists acting at different sites on excitatory amino acid receptor subtype can show different neuroprotective potency. © 1995 Wiley-Liss, Inc.     

Blockade of NMDA receptors increases cell death and birth in the developing rat dentate gyrus

Excitatory input regulates cell birth and survival in many systems. The granule cell population of the rat dentate gyrus is formed primarily during the postnatal period. Excitatory afferents enter the dentate gyrus and begin to form synapses with granule cells during the first postnatal week, the time of maximal cell birth and death. In order to determine whether excitatory input plays a role in the regulation of cell birth and survival in the developing granule cell layers and their germinal regions, the subependymal layer and hilus, we treated rat pups with the N-methyl D-aspartate (NMDA) receptor antagonists MK-801, CGP 37849, or CGP 43487 during the first postnatal week and examined the numbers of ³H-thymidine-labeled cells, pyknotic cells, and healthy cells in these regions. In order to determine the cell type that was affected, sections from brains of MK-801-treated rats were processed for ³H-thymidine autoradiography combined with immunohistochemistry for the marker of radial glia, vimentin, and the marker of mature astrocytes, glial fibrillary acidic protein (GFAP). Within the dentate gyrus, NMDA receptor blockade resulted in the following changes: (1) the density of ³H-thymidine-labeled cells was increased, (2) the density of pyknotic cells was increased, (3) the density of ³H-thymidine-labeled pyknotic cells was increased, and (4) the density of healthy cells was decreased. The infrapyramidal blade/hilus showed changes throughout its extent, whereas the suprapyramidal blade showed changes only at the rostral level. No change in the numbers of ³H-thymidine-labeled vimentin-immunoreactive or GFAP-immunoreactive cells was observed in the dentate gyrus with MK-801 treatment, indicating that glia are not primarily affected by NMDA receptor blockade. Blockade of NMDA receptors resulted in gross morphologic changes in the dentate gyrus; in most cases, the infrapyramidal blade was indistinguishable from the hilus. Moreover, in several brains of animals treated with CGP 37849 or CGP 43487 on postnatal day (P)5, an abnormal aggregation of cells was observed ventral to the normal location of the infrapyramidal blade. This cellular cluster contained many pyknotic and ³H-thymidine-labeled cells and may represent cells that normally comprise the infrapyramidal blade. Dramatic changes to the subependymal layer were also seen following NMDA receptor blockade. The cross-sectional area of this region was significantly increased with MK-801, CGP 37849, or CGP 43487 treatment and contained a high density of ³H-thymidine-labeled cells and ³H-thymidine-labeled pyknotic cells. These results indicate that NMDA receptor activation is critical for the normal development of the rat dentate gyrus. The finding that blockade of NMDA receptors resulted in increased levels of cell death and birth supports the hypothesis that NMDA receptor activation is a natural signal for the inhibition of these processes in the developing dentate gyrus. © Wiley-Liss, Inc.     

兴奋性氨基酸受体的分子生物学

近年来分子生物学技术的应用使兴奋性氨基酸受体的研究取得重大突破，分属两个受体超家族的二十余种亚基已被克隆。兴奋性氨基酸的复杂作用源于受体的多型性，受体的多型性则来自不同亚基组合方式的变化。对各种亚基氨基酸序列－功能关系和基因表达调控的研究将进一步揭示兴奋性氨基酸受体亚型多样性的生物学意义