Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the `glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.     

Distribution of glutamine-like immunoreactivity in the cerebellum of rat and baboon (Papio anubis) with reference to the issue of metabolic compartmentation

Summary The cellular and subcellular localization of glutamine, a major glutamate precursor, was studied by means of an antiserum raised against glutaraldehydefixed glutamine. Ultrathin sections from the cerebellar cortex of rat and baboon (Papio anubis) were incubated sequentially in the primary antiserum and in a secondary antibody coupled to colloidal gold particles. The labelling intensity was quantified by computer-aided calculation of gold particle densities. High levels of immunoreactivity occurred in glial cells (Bergmann fibres, astrocytes, and oligodendrocytes), intermediate levels in cell bodies and processes of granule cells, and low levels in terminals of presumed GABAergic or glutamatergic fibres (terminals of basket and Golgi cells, and of parallel, mossy, and climbing fibres). The labelling intensity of Purkinje cells showed some variation, but never exceeded that in glial cells. Within the nerve fibre terminals, the glutamine-like immunoreactivity showed some preference for mitochondria, but was otherwise evenly distributed. The predominant glial localization of glutamine was also obvious in light microscopic preparations processed according to the postembedding peroxidase-antiperoxidase procedure. Gold particle densities over different types of profile in glutamine immunolabelled sections were compared with particle densities over the corresponding types of profiles in neighbouring sections labelled with an antiserum to glutaraldehyde-fixed glutamate. The glutamate/glutamine ratio, expressed arbitrarily by the ratio between the respective gold particle densities, varied by a factor of about 6, with the highest ratio in the putative glutamatergic mossy and parallel fibre terminals, and the lowest ratio in glial elements. The remaining tissue components displayed intermediate ratios. The present study provides direct morphological evidence for the existence in the brain of distinct compartments with differing glutamate/glutamine ratios.This paper is dedicated to Professor Fred Walberg on the occasion of his 70th birthdayOn leave of absence from Department of Anatomy, Capital Institute of Medicine, You An Men Street, Beijing, China     

氟尿嘧啶对大鼠小肠的损伤作用研究

目的：探讨氟尿嘧啶对大鼠小肠的损伤作用,方法：通过胃管连续2d给予大鼠尿嘧啶125mk/kg.d,观察给药后第1天－第7天大鼠每日饮食量的变化,给药前和给药后的第8天分别称大鼠体重,给药后第8天测定门静脉血流量及门静脉压,分析小肠形态学变化,观察小肠对色氨酸吸收能力,测定动脉血谷氨酰胺的浓度,结果：给药后第1天－第7天大鼠饮食量均明显低于正常,尤其是第2天－第4天最为严重,第8天大鼠体重下降,小肠结构有明显损伤,门静脉血流量减少,门静脉压下降,小肠对色氨酸的吸收及动脉血谷氨酸胺浓度明显下降,与对照组相比的均有显著差异,结论：氟尿嘧啶可导致大鼠小肠结构和功能的明显障碍。     

短肠综合征的康复治疗

目的总结27例短肠综合征病人康复治疗的经验,介绍短肠康复治疗的具体方法,探讨改善短肠病人营养状况、促进肠功能代偿的措施。方法短肠康复治疗包括营养支持、应用谷氨酰胺和生长激素促进肠粘膜生长、富含膳食纤维的短肠康复饮食、减轻短肠临床症状以及预防和治疗短肠并发症等措施。1997年1月至2000年7月间共27例短肠患者接受了29次康复治疗,患者平均年龄38.5岁±19.3岁。剩余小肠长度范围15～80cm,平均46.8cm±23.4cm,15例有回盲瓣。从肠切除至接受康复治疗的平均时间为86d±105d。结果治疗后病人的营养状况明显改善,肠道吸收功能有所增强。随访超过2年者8例,4例完全脱离肠外营养,随访时间超过1年者13例,有10例完全脱离肠外营养。结论短肠康复治疗能够有效地改善短肠病人的营养状况、并能促进肠功能代偿,治疗效果与残留小肠长度、治疗开始的时间和病人年龄有关,及早进行康复治疗能够促进肠功能代偿,减少病人对肠外营养的依赖。     

谷氨酰胺和5—氨尿嘧啶对荷瘤大鼠肿瘤生长的影响

目的:探讨联合应用谷氨酰胺(Gln)和5-氟尿嘧啶(5-Fu)对肿瘤生长的影响。方法:制备荷Walker-256癌肉瘤大鼠模型,第6天随机分为A、B、C、D四组,分别腹腔注射生理盐水、丙氨酰谷氨酰胺二肽(Ala-Gln)、5-Fu、Ala-Gln和5-Fu。第14天检测瘤重、PCNA指数、凋亡指数、淋巴细胞转化率(LTR)及NK细胞活性(NKCA)。结果:治疗后B组瘤重(13.1±2.5)g与C组(12.0±1.2)g,无明显差异(P>0.05),B组PCNA、凋亡较C组无明显差异(P>0.05),B组LTR、NKCA较C组增强(P<0.05);治疗后D组(10.7±0.8)瘤重较B、C组明显降低(P<0.05),凋亡表达无明显差异(P>0.05),D组LTR较B、C组明显增高(P<0.05),NKCA较C组明显增高(P<0.05)。结论:联合应用Gln和5-Fu治疗肿瘤与单独应用Gln或5-Fu相比能够显著抑制肿瘤生长,且提高化疗后机体的免疫功能。     

Simultaneous action of MK-801 (dizclopine) on dopamine, glutamate, aspartate and GABA release from striatum isolated nerve endings

The simultaneous effect of MK-801 on the baseline- and depolarization (20 microM veratridine or 30 mM high K+)-evoked release of endogenous dopamine, glutamate (Glu), aspartate (Asp), and GABA is investigated in the same preparation of rat striatum isolated nerve endings. MK-801, in the microM range, selectively increases the baseline and high K+ depolarization-evoked release of dopamine, without causing any effect on the baseline or on the high K+-evoked release of Glu, Asp and GABA. In addition to this selective action on dopamine release, MK-801 inhibits the veratridine depolarization-evoked release of all the neurotransmitters tested, including dopamine. In SBFI and fura-2 preloaded striatal synaptosomes, MK-801 inhibits the elevation of internal Na+ (Na(i)) and the elevation of internal Ca2+ (Ca(i)) induced by veratridine depolarization. The elevation of Ca(i) induced by high K+ depolarization is unchanged by MK-801. This study reveals two separate MK-801 actions. (1) The voltage-independent action, which increases dopamine release selectively, and might contribute to the effects of MK-801 on motor coordination. (2) The voltage-dependent action, which inhibits all the veratridine-evoked responses including the evoked release of the excitatory amino acids (which are particularly concentrated in striatum nerve endings), and might contribute to the anticonvulsant and neuroprotective effects of MK-801.     

谷氨酰胺在早产儿肠外营养中的应用研究

目的研究谷氨酰胺(Gln)对早产儿的生长发育、胃肠功能成熟及感染发生率的影响。方法将35例早产儿分为两组,Gln组给予经静脉添加Gln的肠外营养(PN),对照组常规 PN,PN时间均大于2周。监测两组生长发育、喂养耐受情况、胃肠功能及感染发生率。结果 Gln 组生后4周时尿素氮(BUN)水平较对照组高(P=0．044),但仍在正常范围内。平均PN及平均住院时间Gln组均明显短于对照组(P=0．031;P=0．020)。血清胃动素水平Gln组生后2周较生后 3天明显升高(P=0．037);Gln组生后2周较生后3 d胃电节律中节律过快的百分数明显增加 (P=0．017)。Gln组发生感染的次数较对照组明显减少(P=0．001)。结论初步观察提示Gln 有助于早产儿胃肠功能的成熟,减少院内感染的发生。     

谷氨酰胺强化肠内营养对炎症性肠病幼鼠模型结肠黏膜细胞凋亡作用的研究

目的 探讨谷氨酰胺强化肠内营养对炎症性肠病（IBD）幼鼠模型肠黏膜细胞凋亡的调控及促进黏膜愈合的作用。方法 将80只4~5周龄Sprague-Dawley雄性大鼠随机分为空白对照组、IBD模型组、短肽组和短肽+谷氨酰胺（Gln）组,每组20只。采用一次性结肠灌注三硝基苯磺酸建立IBD模型,造模3 d后,短肽组给予短肽制剂（100 mL/kg）,短肽+Gln组给予短肽制剂（100 mL/kg）+Gln（0.5 g/kg）,干预1周。实验结束后观察幼鼠的一般情况,并留取肠黏膜,苏木素-伊红（HE）染色观察肠黏膜组织病理情况;RTPCR法检测肠黏膜凋亡调控基因（bax、bcl-2）及凋亡信号转导因子（Caspase-3、Caspase-9）的表达;Westernblot法检测结肠黏膜IGF-1表达水平。结果 IBD模型组一般情况均较其他组差,短肽+Gln组一般情况优于IBD模型组和短肽组。模型组bax mRNA表达水平高于空白对照组、短肽组和短肽+Gln组（P < 0.05）;bcl-2、Caspase-3、Caspase-9 mRNA水平在各组比较差异均无统计学意义（P > 0.05）。短肽组IGF-1水平明显高于短肽+Gln组、空白对照组及IBD模型组（P < 0.05）。结论 Gln强化肠内营养能有效改善IBD模型幼鼠的一般营养状况,但在抑制结肠黏膜细胞凋亡及刺激结肠黏膜IGF-1合成方面并未优于专一肠内营养。     

Hepatocellular adenoma: Classification,variants and clinical relevance

Hepatocellular adenomas are benign tumors with two major complications, bleeding and malignant transformation. The overall narrative of hepatocellular adenoma has evolved over time. Solitary or multiple hepatocellular developing in the normal liver of women of child bearing age exposed to oral contraceptives still represents the most frequent clinical context, however, new associations are being recognized. Hepatocellular adenoma is discovered on a background of liver diseases such as non-alcoholic steatohepatitis, vascular diseases, and alcoholic cirrhosis. Hepatocellular adenoma is also reported in men, young or older adults, and even in infants. On the morpho-molecular side, the great leap forward was the discovery that hepatocellular adenoma was not a single entity and that at least 3 different subtypes exist, with specific underlying gene mutations. These mutations affect the HNF1A gene, several genes leading to JAK/STAT3 pathway activation and the CTNNB1 gene. All of them are associated with more or less specific histopathological characteristics and can be recognized using immunohistochemistry either with specific antibodies or with surrogate markers. Liver pathologists and radiologists are the key actors in the identification of the different subtypes of hepatocellular adenoma by the recognition of their specific morphological features. The major impact of the classification of hepatocellular adenoma is to identify subjects who are at higher risk of malignant transformation. With the development of new molecular technologies, there is hope for a better understanding of the natural history of the different subtypes, and, particularly for their mechanisms of malignant transformation.     

Transcranial direct current stimulation of prefrontal cortex: An auditory event-related potential and proton magnetic resonance spectroscopy study

Transcranial direct current stimulation (tDCS) is a non-invasive intervention altering neural plasticity by modulating neuronal excitability of pre- and postsynaptic neuron populations, which has been shown to improve depression symptoms and cognition. We investigated the effects of a single session of 20 min of 2 mA left-prefrontal anodal versus sham stimulation on auditory event-related potentials (ERPs) in 11 male and 5 female healthy subjects (mean age of 28.6 [SD 6.2] years) by employing a randomized single-blind crossover design. Stimulation effects on cortical glutamate (Glu) and glutamine (Glx) levels were subsequently measured in 12 of the 16 healthy subjects in a 3 T proton magnetic resonance spectroscopy scan. tDCS was associated with a significant increase of N1 amplitudes while smaller P3b amplitudes correlated with higher cortical Glu and Glx levels in the stimulated brain area when performing an auditory go/no-go discrimination task. tDCS did not change mismatch negativity, nor task performance or cortical Glu/Glx levels which, together with N1 amplitudes, depended on stimulation order (“sham” versus “active”). Increased N1 amplitudes are consistent with higher levels of cortical excitability following prefrontal anodal tDCS. The failure to replicate Glu/Glx changes with tDCS may have been masked by between-session carry-over effects while ceiling effects may have masked tDCS effects on task performance.