硫化氢复合浅低温可选择性激活突触内NMDARs及其下游PI3K/Akt信号通路

目的观察硫化氢（H₂S）复合浅低温对大鼠全脑缺血-再灌注后海马N-甲基-D-天冬氨酸受体（NMDARs）的亚单位NR2A、NR2B、磷酸化蛋白激酶B（p-Akt）及磷酸化糖原合成酶激酶3β（p-GSK 3β）的影响,旨在探讨其发挥脑复苏作用的潜在机制。方法雄性SD大鼠100只,随机均分为五组:假手术组（Ⅰ组）、模型组（Ⅱ组）、浅低温组（Ⅲ组）、硫氢化钠（NaHS）组（Ⅳ组）、浅低温+NaHS组（Ⅴ组）。采用Pulsinelli-Brierley四血管阻塞法建立大鼠全脑缺血再灌注损伤模型,缺血15 min再灌注即刻Ⅳ组和Ⅴ组腹腔注射14μmol/kg NaHS,Ⅲ组和Ⅴ组行体表降温至肛温32～33℃。6 h后断头取海马,分别采用分光光度计法测H₂S的含量,western blot法测NR2A、NR2B、p-Akt及p-GSK 3β的表达,每组分别取4只于再灌注24 h取脑行Tunel染色观察CA1区锥体细胞凋亡情况。结果与Ⅰ组相比,Ⅱ、Ⅲ、Ⅳ、Ⅴ组海马组织H₂S含量均升高（P<0.05）;与Ⅱ组相比,Ⅳ和Ⅴ组H₂S含量显著升高（P<0.05）;与Ⅰ组相比,Ⅱ、Ⅲ、Ⅳ、Ⅴ组NR2A、NR2B的表达、凋亡指数（AI）均增高（P<0.05）,且Ⅱ和Ⅲ组NR2A/NR2B<1,Ⅰ、Ⅳ和Ⅴ组NR2A/NR2B>1;与Ⅰ组相比,Ⅱ、Ⅲ、Ⅳ、Ⅴ组海马p-Akt及p-GSK 3β的表达均上调（P<0.05）;与Ⅱ组相比,Ⅲ、Ⅳ、Ⅴ组海马p-Akt及p-GSK 3β的表达均上调,AI降低（P<0.05）;与Ⅲ和Ⅳ组相比,Ⅴ组海马p-GSK 3β的表达上调（P<0.05）。与Ⅱ组相比,Ⅲ、Ⅳ、Ⅴ组CA1区锥体细胞凋亡程度均明显减轻,尤以Ⅴ组效果最明显。结论 H₂S复合浅低温可能通过选择性作用于突触内的NMDARs,进而激活其下游促存活磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt）信号通路,抑制锥体细胞凋亡,从而发挥脑复苏的作用。     

N-甲基-D-天冬氨酸受体和突触后致密物质95在血管性痴呆大鼠中作用机制的研究

目的观察血管性痴呆(VaD)大鼠N-甲基-D-天冬氨酸-2B亚基受体(NR2B)和突触后致密物质95(PSD-95)在VaD发生、发展中的作用。方法采用永久性结扎双侧颈总动脉方法将96只Wistar大鼠随机分为假手术组(32只)、VaD模型组(模型组,32只)和美金刚治疗组(治疗组,32只)。用免疫组织化学法检测术后4、8、12、16周大鼠海马NR2B和PSD-95的表达,并同时采用Morris水迷宫测试大鼠的学习记忆水平。结果随着缺血时间的延长,与假手术组比较,模型组大鼠术后4、8、12、16周学习记忆能力下降,差异显著(P<0.01);术后4周时NR2B和PSD-95的表达明显高于假手术组(P<0.01),此后逐渐减少,显著低于假手术组(P<0.01),术后16周时表达最少。治疗组大鼠术后4周时学习记忆水平及NR2B、PSD-95的表达与假手术组比较无显著差异,术后8、12、16周时,上述指标较模型组显著好转但仍差于假手术组(P<0.05,P<0.01)。结论大鼠海马NR2B和PSD-95作为复合体参与VaD的形成和发展,适量的美金刚通过调节NR2B的表达进而改善VaD大鼠的认知功能。     

α-突触核蛋白促进原代神经元及转基因小鼠神经细胞表面NMDA受体的内在化

目的在原代培养神经元及转基因动物水平上,研究α-突触核蛋白（α-synuclein,α-Syn）对神经细胞表面N-甲基-D-天门冬氨酸（N-methyl-D-aspartate,NMDA）受体的影响及其机制。方法免疫荧光标记法和Western blotting测定NMDA受体（NMDA receptor,NMDAR）和Rab5B质量分数。Rab5B反义寡核苷酸抑制Rab5B基因表达。以原代培养神经细胞及转基因小鼠为模型,观察α-Syn蛋白增多对细胞膜NMDAR质量分数的影响以及Rab5B的作用。结果在细胞及动物水平,α-Syn明显上调Rab5B表达,促进NMDAR的内在化。而抑制Rab5B表达可消除α-Syn致NMDAR的内在化。结论α-Syn通过上调Rab5B的表达促进NMDAR的内在化。     

姜黄素对神经病理性痛大鼠脊髓背角及背根神经节神经元大麻素受体1和NR2B表达的影响

目的 探讨姜黄素对神经病理性痛大鼠脊髓背角及背根神经节(DRG)神经元大麻素受体1 (CBR1)及含NR2B亚基N-甲基-D-天冬氨酸(NMDA)受体表达的影响.方法 雄性SD大鼠72只,体重200～230 g,采用随机数字表法,将其随机分为4组(n=18):假手术组(S组)、慢性压迫性损伤组(CCI组)、姜黄素组(Cur组)和溶媒对照组(SC组).S组仅分离、暴露坐骨神经,其余3组采用慢性压迫性损伤法制备神经病理性痛模型.Cur组术后腹腔注射姜黄素100 mg·kg-1·d-1,连续14 d,SC组给予等容量二甲基亚砜.分别于术前2d、术后1、3、7、10、14 d时测定机械缩足反应阈值(MWT)和热缩足潜伏期(TWL).分别于术后3、7、14 d时处死6只大鼠,取脊髓背角和DRG,采用免疫组化法检测神经元CBR1和NR2B的表达.结果 与S组比较,其他3组术后MWT降低,TWL缩短,脊髓背角和DRG神经元CBR1和NR2B表达上调(P＜0.05);与CCI组比较,Cur组术后MWT升高,TWL延长,脊髓背角和DRG神经元CBR1表达上调,NR2B表达下调(P＜0.05),SC组上述指标差异无统计学意义(P＞0.05).结论 姜黄素可减轻大鼠神经病理性痛,其机制与脊髓背角及DRG神经元CBR1表达上调、NR2B表达下调有关.     

The effects of d-cycloserine, a partial agonist at the glycine binding site, on spatial learning and working memory in scopolamine-treated rats

Summary The present study investigated the effect of d-cycloserine, a partial agonist at the glycine binding site on NMDA receptor complex, on the performance of scopolamine-treated adult rats in a water maze task assessing spatial learning and in a delayed non-matching to position task assessing working memory in a spatial context. In the spatial learning task, scopolamine (0.4 mg/kg, i.p.) impaired acquisition (increased escape latency and distance) and increased swimming speed of rats. D-cycloserine (1.0 mg/kg, i.p.) reversed the deficits in acquisition performance but not the increases in behavioral activity. In the working memory task, scopolamine (0.2 mg/kg, i.p.) produced deficits on nonmnemonic rather than on mnemonic performance factors; scopolamine delay-independently decreased the percent correct responses and reduced behavioral activity of rats. D-cycloserine (1.0, 3.0 and 10 mg/kg, i.p.) did not reverse these performance deficits. When administered alone, the moderate to higher doses of d-cycloserine had no effects on working memory but the lower dose produced slight deficits in mnemonic performance factors; the 1.0 mg/kg dose delay-dependently decreased the percent correct responses without affecting behavioral activity of rats. In the water maze task, d-cycloserine had no effects on acquisition performance or behavioral activity of rats. These results suggest that acute, systemic administration of d-cycloserine does not improve spatial learning or working memory. However, at appropriate doses this agent may be efficacious in disease states of central cholinergic hypofunction since 1.0 mg/kg d-cycloserine was able to reverse the scopolamine-induced deficits in acquisition.     

The cytochromes P-450 are not involved in the modulation of the N-methyl-D-aspartate response by sigma ligands in the rat CA3 dorsal hippocampus

Recent in vitro radioligand binding studies have shown that several cytochrome P-450 inhibitors can displace [³H] sigma ligands, suggesting that these ligands might bind to the cytochrome P-450 superfamily of enzymes. Using an in vivo electrophysiological model of extracellular recordings performed in the CA₃ region of the rat dorsal hippocampus, we have previously shown that intravenous administration of low doses of several sigma ligands, such as 1, 3-di(2-tolyl) guanidine (DTG), JO-1784, and (+)pentazocine potentiate the neuronal response induced by microiontophoretic applications of N-methyl-D-aspartate (NMDA) without affecting those induced by quisqualate and kainate, suggesting that they act as sigma agonists. Conversely, the sigma ligands haloperidol, (+)3-PPP, and BMY-14802, which have no effect by themselves on the NMDA response, prevent and suppress the potentiating effect of sigma agonists on the NMDA response, suggesting that they act as sigma antagonists. The present studies were undertaken to determine if cytochromes P-450 could be involved in the modulation of the NMDA response by sigma ligands. For this purpose, two cytochrome P-450 inhibitors, proadifen (SKF-525A) and piperonyl butoxide (PB), have been tested in our model. Unlike sigma agonists, at low doses, neither SKF-525A nor PB affected the NMDA response of CA₃ dorsal hippocampus pyramidal neurons. Unlike sigma antagonists, neither of these drugs reversed or prevented the DTG-induced potentiation of the NMDA response. In addition, following high doses of SKF-525A or PB, sufficient to induce a complete inactivation of cytochromes P-450, DTG still potentiated the NMDA response. The present data suggest that cytochrome P-450 inhibitors do not modulate the NMDA response like sigma agonists or antagonists do in this brain region. Furthermore, they rule out the involvement of cytochromes P-450 in the modulation of the NMDA response by sigma ligands. © 1993 Wiley-Liss, Inc.     

Competitive and noncompetitive N-methyl-D-aspartate antagonists protect dopaminergic and serotonergic neurotoxicity produced by methamphetamine in various brain regions

Summary Protective effects of NMDA antagonists on dopaminergic and serotonergic neurotoxicity produced by methamphetamine (MA) were examined. Four injections of MA (7.5 mg/kg, s.c., at 2 h intervals) caused significant decrements (40–60% of control values) in levels of dopamine (DA) and its metabolites in the rat striatum and levels of serotonin (5-HT) and its metabolite in the medial prefrontal cortex, nucleus accumbens, striatum, anterior hypothalamus, amygdala and hippocampus. These decreases in DA, 5-HT and their metabolites were prevented by pretreatment with MK-801, a noncompetitive N-methyl-D-aspartate (NMDA) antagonist, or D-CPP-ene (SDZ EAA 494), a competitive NMDA antagonist. The results suggest that NMDA receptors play a role for MA-induced serotonergic damage in various brain regions as well as dopaminergic damage in the striatum.     

N-甲基-D-门冬氨酸受体重组肽抗血清对谷氨酸兴奋毒性导致神经元坏死和凋亡的保护作用

目的 观察N-甲基-D-门冬氯酸受体(NMDAR，NR)主亚基(NR1)M3M4环重组肽自身抗血清对不同浓度谷氨酸诱导的神经元死亡的保护作用。方法 用NR1 M3M4环重组肽免疫Balh／C(H-2^d)小鼠制备抗血清，用锥虫蓝染色法和原位末端标记(TUNEL)法观察抗血清对原代培养海马神经元兴奋毒性坏死和凋亡的保护效应。结果 在高浓度谷氨酸(500μmoL／L)条件下，阳性抗血清可以使神经元坏死减少16％；在低浓度谷氨酸(50μmol／L)作用下，阳性抗血清保护可使神经元凋亡率减少13％。结论 NMDAR重组肽自身免疫抗血清具有抗兴奋毒性作用，这一结论为免疫防治兴奋毒性脑损伤策略的建立提供了坚实的体外基础。     

P77PMC和Wistar大鼠发育中不同脑区NR1 mRNA表达的差异

目的：比较发育中遗传性癫痫易感大鼠P77PMC和癫痫不易感大鼠Wistar不同脑区NMDA受体（N－methyl－D－aspartatereceptor，NR）一型亚基（NR1）基因表达特点，探讨其与惊厥易感性的关系。方法：Northern印迹杂交方法。结果：两系大鼠发育中，大脑皮层、海马、下丘3个脑区NR1mRNA表达趋势一致，并有共同规律，即出生后表达量逐渐增加，在发育的某一阶段到高峰，成年下降并稳定于某一水平，在整个发育过程中，P77PMC大鼠NR1mRNA的表达量高于Wistar大鼠，并在某一阶段显著增高。结论：提示发育中P77PMC大鼠脑内NR1亚基基因表达增高可能是造成P77PMC大鼠“低惊厥阈值”及对听源性惊厥易感的原因之一。     

Activation of Class II or III Metabotropic Glutamate Receptors Protects Cultured Cortical Neurons Against Excitotoxic Degeneration

Trans-1-aminocyclopentane-1,3-dicarboxylic acid, a mixed agonist of all metabotropic glutamate receptor (mGluR) subtypes, is known to produce either neurotoxic or neuroprotective effects. We have therefore hypothesized that individual mGluR subtypes differentially affect neurodegenerative processes. Selective agonists of subtypes which belong to mGluR class II or III, such as (2_s, 1′_R,2′_R,3′_R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV) (specific for subtypes mGluR2 or 3) or L-2-amino-4-phosphonobutanoate and L-serine-O-phosphate (specific for subtypes mGluR4, 6 or 7), were highly potent and efficacious in protecting cultured cortical neurons against toxicity induced by either a transient exposure to N-methyl-D-aspartate (NMDA) or a prolonged exposure to kainate. In contrast, agonists that preferentially activate class I mGluR subtypes (mGluR1 or 5), such as quisqualate or trans-azetidine-2,3-dicarboxylic acid, were inactive. DCG-IV was still neuroprotective when applied to cultures after the toxic pulse with NMDA. This delayed rescue effect was associated with a reduction in the release of endogenous glutamate, a process that contributes to the maturation of neuronal damage. We conclude that agonists of class II or III mGluRs are of potential interest in the experimental therapy of acute or chronic neurodegenerative disorders.