谷氨酸损伤体外培养大鼠螺旋神经元中凋亡诱导因子的表达与分布

目的：观察不同浓度谷氨酸（Glu）损伤螺旋神经元中凋亡诱导因子（apoptosis inducing factor,AIF）的表达与分布变化,探讨利用谷氨酸损伤体外培养螺旋神经元是否与 AIF 凋亡途径相关。方法取40只出生后0～3天 SD 仔鼠螺旋神经元行体外培养,所得培养细胞平均分为正常组和10 mM、20 mM、40 mM Glu 组,正常组仅给予正常 SGNs 培养液培养,其余三组分别加入相应浓度谷氨酸和 SGNs 培养液,培养48 h 后,应用光学显微镜、免疫荧光染色及实时荧光定量 PCR 方法观察四组中 SGN 细胞形态、SGN 中 AIF 的分布及 AIF、钙蛋白酶（cal-pain）、半胱氨酸天冬氨酸3（caspase 3）的表达变化;用 TUNEL 法观察细胞凋亡。结果20 mM 和40 mM Glu 组SGNs 细胞形态改变和细胞凋亡明显,并伴有 AIF 核转位;不同浓度 Glu 组 AIF 和 calpain 基因表达明显高于正常组（P＜0．05）,但 caspase 3表达无明显升高（P＞0．05）。结论谷氨酸损伤体外培养的螺旋神经元过程中,AIF 凋亡途径参与了细胞凋亡,calpain 的高表达也促进了兴奋性神经递质对 SGNs 的损伤,但 caspase3途径无明显作用。     

A radioautographic study of [3H]L-glutamate and [3H]L-glutamine uptake in the guinea-pig cochlea

Since glutamate has been recently proposed as a possible transmitter of the sensory hair cells in the cochlea, a radioautographic study was performed to look for the in vitro uptake of [³H] l-glutamate and [³H]l-glutamine. Several experimental conditions were applied. The control experimental procedures consisted in an incubation with one of the labelled tracers (10 min), followed by a post-incubation (3 × 10 min) without tracer. In these experiments, either with [³H]l-glutamate or [³H]l-glutamine, the following structures were labelled: inner hair cells, glial cells of the osseous spiral lamina and areas of the inner spiral and tunnel spiral bundles. When these experiments were carried out in absence of Na⁺, these labellings were strongly decreased. When the incubation was not followed by a post-incubation, the results differed depending on the tracer: with [³H]l-glutamate, the glial cells and the areas of inner spiral and tunnel spiral bundles were labelled, whereas with [³H]l-glutamine, mainly the inner hair cells were labelled. An addition of l-methionine-dl-sulfoximine, a glutamine synthetase inhibitor, into the incubation and post-incubation media, produced a decrease of the labelling of the inner hair cells and of the glial cells. An addition of unlabelled glutamine to the post-incubation media decreased the inner hair cell labelling, while a similar addition of unlabelled glutamate did not. In either case, neither the outer hair cells, the second type of sensory cells, nor the spiral ganglion neurons were labelled.These results suggest that in the cochlea, glutamate and glutamine have their metabolisms linked together, as in some parts of the central nervous system. Correlated to biochemical and electro physiological data these results support the hypothesis that glutamate could be the neurotransmitter of the inner hair cells.     

雏菊叶龙胆酮对缺血性脑损伤保护作用的实验研究

[目的]研究雏菊叶龙胆酮（Bellidifolin）对小鼠永久性完全性全脑缺血性脑损伤和大鼠弥散性不完全性全脑缺血性脑损伤的保护作用,并探讨其作用机制。[方法]采用两种方法制备两种动物全脑缺血模型,观察不同剂量Bellidifolin对小鼠断头后张口喘息时间;对大鼠脑缺血后的脑指数,脑SOD（超氧化物歧化酶）活力,脑MDA（丙二醛）含量及脑兴奋性氨基酸-Glu（谷氨酸）和抑制性氨基酸-GABA（γ-氨基丁酸）含量,脑细胞病理组织学变化的影响。[结果]（1）与模型组相比：Bellidifolin 50 mg.kg^-1、75 mg.kg^-1和100 mg.kg^-1组分别延长小鼠断头后张口喘息时间7.78%（P〉0.05）,13.34%（P〈0.01）和10.73%（P〈0.05）。（2）与假手术组相比,大鼠模型组脑指数升高14.0%（P〈0.01）,MDA含量升高80.54%（P〈0.01）,SOD活力下降25.37%（P〈0.01）,Glu含量升高15.91%（P〈0.01）,GABA含量升高26.83%（P〈0.01）。（3）与大鼠模型组相比,Bellidifolin 25 mg.kg-1、50mg.kg-1、75 mg.kg-1组分别降低脑指数1.75%（P〉0.05）、8.77%（P〈0.01）、10.53%（P〈0.01）,降低MDA含量5.31%（P〉0.05）、33.46%（P〈0.01）、10.93%（P〉0.05）,增加SOD活力1.92%（P〉0.05）、15.41%（P〈0.05）、19.46%（P〈0.01）,降低Glu含量3.92%（P〉0.05）、9.80%（P〈0.01）、11.76%（P〈0.01）,降低GABA含量3.85%（P〉0.05）、11.54%（P〈0.01）、13.46%（P〈0.01）。[结论]Bellidifolin具有脑保护作用,其脑保护作用可能与其抗氧化作用和抗兴奋毒性损伤作用有关。     

The unsolved puzzle of neuropathogenesis in glutaric aciduria type I

Glutaric aciduria type I (GA-I) is a cerebral organic aciduria caused by deficiency of glutaryl-Co-A dehydrogenase (GCDH). GCDH deficiency leads to accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA), two metabolites that are believed to be neurotoxic, in brain and body fluids. The disorder usually becomes clinically manifest during a catabolic state (e.g. intercurrent illness) with an acute encephalopathic crisis that results in striatal necrosis and in a permanent dystonic-dyskinetic movement disorder. The results of numerous in vitro and in vivo studies have pointed to three main mechanisms involved in the metabolite-mediated neuronal damage: excitotoxicity, impairment of energy metabolism and oxidative stress. There is evidence that during a metabolic crisis GA and its metabolites are produced endogenously in the CNS and accumulate because of limiting transport mechanisms across the blood-brain barrier. Despite extensive experimental work, the relative contribution of the proposed pathogenic mechanisms remains unclear and specific therapeutic approaches have yet to be developed. Here, we review the experimental evidence and try to delineate possible pathogenetic models and approaches for future studies.     

Anterior cingulate proton spectroscopy glutamate levels differ as a function of smoking cessation outcome

Background

Cigarette smoking is the leading preventable cause of death. Unfortunately, the majority of smokers who attempt to quit smoking relapse within weeks. Abnormal dorsal anterior cingulate cortex (dACC) function may contribute to tobacco smoking relapse vulnerability. Growing evidence suggests that glutamate neurotransmission is involved in mediating nicotine dependence. We hypothesized that prior to a cessation attempt, dACC glutamate levels would be lower in relapse vulnerable smokers.

Methods

Proton magnetic resonance spectra (MRS) were obtained from dACC and a control region, the parieto-occipital cortex (POC), using two-dimensional J-resolved MRS at 4 T and analyzed using LCModel. Nine nicotine-dependent women were scanned prior to making a quit attempt. Subjects then were divided into two groups; those able to maintain subsequent abstinence aided by nicotine replacement therapy (NRT) and those who slipped while on NRT (smoked any part of a cigarette after attaining at least 24 h of abstinence).

Results

Slip subjects exhibited significantly reduced dACC MRS glutamate (Glu/Cr) levels (p < 0.03) compared to abstinent subjects. This effect was not observed in the POC control region.

Conclusions

Our preliminary findings suggest that dACC Glu levels as measured with MRS may help identify and/or be a biomarker for relapse vulnerable smokers. Future research following up on these findings may help clarify the role of dACC Glu in smoking dependence that may lead to new treatment strategies.     

Potential involvement of calcium and nitric oxide in protective effects of puerarin on oxygen-glucose deprivation in cultured hippocampal neurons

The aim of this study was to clarify the mechanisms underlying neuroprotection of puerarin (Pur) against cerebral hypoxia-ischemia. Primary hippocampal cultures were prepared from 2-day-old Sprague-Dawley rats. After 8 days in vitro, the cultures subjected to 3h oxygen/glucose deprivation (OGD). Flow cytometric analysis of annexin-V and propidium iodide (PI) labeling cells found that apoptosis and necrosis were significantly reduced in the cultured hippocampal neurons by addition of Pur during 3h OGD and for the following 24h. Pur (40 and 100 microM) also attenuated glutamate (Glu) induced neuronal damage, suppressing apoptosis and necrosis induced by Glu of 0.5mM. Furthermore, the changes in intracellular Ca(2+) and generation of nitric oxide (NO) were measured by confocal laser scanning microscopy with Fluo-3, a Ca(2+) probe, and diaminofluorescein diacetate (DAF DA), a NO probe, respectively. In agreement with the results from flow cytometric analysis, Pur (40 and 100 microM) markedly slowed down OGD-induced Ca(2+) influx and lowered the intracellular Ca(2+) peak. Meanwhile, NO synthesis induced by OGD was significantly inhibited by Pur. Our findings suggest that Pur can ameliorate hippocampal neuronal death induced by OGD in vitro. The protective effects of Pur are associated with inhibiting the action of glutaminergic transmitter, intracellular Ca(2+) elevation and neuronal NO synthesis.     

High field (1)H MRS of the hippocampus after donepezil treatment in Alzheimer disease

The purpose of this study was to measure metabolite level changes in patients with newly diagnosed Alzheimer Disease (AD) following four months of donepezil treatment. A small number of cognitively normal elderly subjects were also scanned longitudinally (twice within one year) to assess the reproducibility. Short echo-time (1)H magnetic resonance spectra were acquired at 4.0 T in the right hippocampus. Subjects were scanned at the time of first diagnosis (prior to receiving donepezil) and then following four months of donepezil treatment (5 mg/day for the first month, 10 mg/day thereafter). Changes in absolute metabolite levels and metabolite ratios were quantified and compared. There was no change in measured cognitive function following four months of donepezil treatment in the AD patients. Decreased levels of N-acetylaspartate, choline, N-acetylaspartate/creatine, choline/creatine, and myo-inositol/creatine were observed in AD patients after four months of treatment. Cognitively normal elderly subjects showed an increase in myo-inositol/choline ratio following one year. The reduced levels of N-acetylaspartate in AD patients indicates continued decline in neuronal function and/or integrity. However decreased levels of choline and myo-inositol/creatine ratio may indicate a positive treatment effect.     

不同的频率调谐与递质特异性神经元在鼠耳蝠听觉脑干的空间分布

分别用频率为２０,６０kHz,强度为５９－６３或７１－７３dB SPL(Sound pressure level)的纯音（时程４ms,升降时间各0.5ms,重复速率２pulses/s)对9只鼠耳蝠双耳或单耳进行暴露。采用Ｆos免疫组化重标记技术,考察了Ｆos、Fos／谷氨酸（Ｇlu)和Ｆos/γ－氮基丁酸（ＧＡＢＡ）阳性神经元的空间分布。结果表明,在其脑干听核团中,耳蜗核,斜方体核,上橄榄核,外侧丘系核以及下丘分别有不同数量的Ｆos、Fos/Glu或Fos/GABA阳性神经元分布。两和睦 怕频率暴露条件下Ｆos、Fos/Glu和Fos/GABA都具有特定的空间分布模式,从一个侧面证实脑干频率－幅度轴的存在,并提示Ｇlu能和ＧＡＢＡ能神经元在脑干不同层次对听觉信息加工,特别是对频率编码的作用既表现在参与的神经细胞数量上,也表现在参与神经元的空间分布模式上。     

An aggressive form of familial amyloid polyneuropathy caused by a Glu54Gly mutation in the transthyretin gene

We report a patient with familial amyloid polyneuropathy. Gene analysis revealed a heterozygous Glu54Gly substitution (A-to-G change) in the transthyretin gene. This is the first case of a Glu54Gly substitution that was devoid of a Gly6Ser substitution. Compared with the previously reported case with compound heterozygotes of Glu54Gly and Gly6Ser, the age of onset in our case is much younger and another characteristic findings were the amyloid vasculopathy and the multiple organ involvement. A Glu54Gly mutation is amyloidogenic by itself and a Gly6Ser mutation may offer some protection from the Glu54Gly mutant.