白藜芦醇后处理对大鼠脑缺血再灌注损伤海马CA1区Bax、Bcl-2的影响

摘要 目的：探讨白藜芦醇后处理对大鼠脑缺血再灌注损伤Bax、Bcl-2表达的影响。方法：清洁级雄性SD大鼠60只随机分为假手术组（n=12）、I/R组（n=12）、白藜芦醇组（n=36）,白藜芦醇组按不同剂量分为低剂量、中剂量、高剂量组（10 mg/kg、20 mg/kg、40 mg/kg）,每组12只。假手术组：仅暴露大鼠颈外动脉,不做缺血处理;I/R组：采用改良线栓法制备大鼠大脑中动脉缺血再灌注损伤模型（缺血2 h,再灌注24 h）;白藜芦醇组：造模方法同I/R组,在大鼠缺血2h后,将不同剂量白藜芦醇腹腔注射入大鼠体内,比较各组SD大鼠神经功能缺损评分、采用Western blotting法、免疫组化法对大鼠脑组织缺血侧海马CA1区Bax和Bcl-2表达进行比较。结果：白藜芦醇低、中、高剂量组神经功能缺损评分均低于I/R组,随着白藜芦醇剂量的增加,神经功能缺损评分逐渐降低,其中白藜芦醇高剂量组神经功能缺损评分降低最为明显;白藜芦醇组与I/R组相比,不同剂量白藜芦醇组Bax表达逐渐减少,而Bcl-2表达明显增加,其中以白藜芦醇高剂量组改变最为明显。结论：高剂量白藜芦醇可以降低大鼠神经功能缺损评分,减轻脑缺血再灌注损伤,对大鼠脑缺血再灌注损伤具有保护作用,其机制与Bax、Bcl-2的表达有关。     

Persistent Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia

Abstract: The influence of brain ischemia on the subcellular distribution and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca²⁺/calmodulin-dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.     

Damage to Neurons and Oligodendrocytes in the Hippocampal CA1 Sector after Transient Focal Ischemia in Rats

Focal brain lesions such as transient focal cerebral ischemia can lead to neuronal damage in remote areas, including the ipsilateral substantia nigra and hippocampus, as well as in the ischemic core. In this study, we investigated acute changes in the ipsilateral hippocampus from 1 up to 7 days after 90 min of transient focal cerebral ischemia in rats, using anti-NeuN (neuronal nuclei), anti-Cu/Zn-superoxide dismutase (Cu/Zn-SOD), anti-Mn-SOD, anti-neuronal nitric oxide synthase (nNOS), anti-inducible NOS (iNOS), anti-glial fibrillary acidic protein (GFAP), anti-ionized calcium-binding adaptor molecule 1(Iba 1) and anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) antibodies. In our western blot and histochemical analyses, present results show that transient focal cerebral ischemia in rats can cause a severe and acute damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector. The present findings also demonstrate that the expression of iNOS produced by Iba 1-immunopositive microglia precedes the damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector after transient focal cerebral ischemia. In contrast, our results suggest that increased reactive oxygen species (ROS) production during reperfusion cannot lead to damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector after transient focal cerebral ischemia, because of an insufficient expression of Cu/Zn-SOD and Mn-SOD. Our double-labeled immunohistochemical study demonstrates that the overexpression of iNOS produced by Iba 1-immunopositive microglia may play a pivotal role in the damage of neurons and oligodendrocytes in the ipsilateral hippocampal CA1 sector at an acute stage after transient focal cerebral ischemia.     

NMDA and Non-NMDA Receptor Gene Expression Following Global Brain Ischemia in Rats: Effect of NMDA and Non-NMDA Receptor Antagonists

Abstract: Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous sludy, we have shown that expression of GIuR2, the kainate/a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca' permeability, is preferentially reduced in CA1 at a time point proceeding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), protects CAI neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor anltagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDARI. In ischemic rats treated with saline, GIuR2 and (31uR3 mRNAs were markedly reduced in CAI but were unchanged in CA3 or dentate gyrus. GluRl and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modificatian of GIuR2 expression changes     

凋亡相关基因bcl-2和Bax在大鼠应激性溃疡中的表达及作用

目的探讨凋亡相关基因bcl-2和Bax在应激性溃疡中表达的变化及中药组方应激宁对其变化的影响。方法Wistar大鼠水浸-束缚应激(WRS)4h,用免疫组织化学方法检测胃黏膜组织bcl-2和Bax蛋白的表达。结果bcl-2和Bax在正常大鼠胃黏膜组织中有散在的表达;应激宁组bcl-2阳性细胞数目较应激组增多,两者具有明显的差异(P<0.05);应激组Bax阳性细胞数目较应激宁组增多,两者也具有明显的差异(P<0.05);在应激组和应激宁组中,Bax阳性细胞数目均比bcl-2增加(P<0.05)。结论WRS后胃黏膜组织bcl-2和Bax的表达差异很可能参与了胃黏膜损伤和修复过程,应激宁对胃黏膜的损伤具有一定的保护作用。     

Effects of Apigenin on the Expression of LOX-1, Bcl-2, and Bax in Hyperlipidemia Rats

We aimed to investigate the impact of apigenin on LOX-1, Bcl-2, and Bax expression in hyperlipidemia rats and explore the possible molecular pathological mechanism of apigenin in improving hyperlipidemia and preventing atherosclerosis. In hyperlipidemia models, the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c) and the LOX-1 protein expression were apparently increased (P<0.01), while the high-density lipoprotein cholesterol (HDL-c) levels and the ratio of Bcl-2/Bax were reduced significantly (P<0.01) in comparison with the standard control group. After the treatment of apigenin, the levels of TC, TG, LDL-c, and the LOX-1 protein expression were noticeably decreased (P<0.01), while the levels of HDL-c and the Bcl-2/Bax ratio were increased (P<0.01). The intima was thickened and had protrusions in the hyperlipidemia model group compared to the normal control group. In comparison with the atherosclerosis model group, the degree of aortic lesions in the low-dose, middle-dose, high-dose groups was alleviated. Apigenin can reduce the level of blood lipid, improve hyperlipidemia, and prevent atherosclerosis in hyperlipidemia rats. The molecular mechanism may be related to inhibiting LOX-1 gene expression and increasing the Bcl-2/Bax ratio.     

Expression of Bcl-2, Bax and Caspase-3 in Nerve Tissues of Rats Chronically Exposed to 2,5-hexanedione

Occupational exposure and experimental intoxication with n-hexane or its metabolite 2,5-hexanedione (HD) produce a central-peripheral neuropathy. However, the mechanism remains unknown. We hypothesized that HD affected the expression of Bcl-2, Bax and Caspase-3 in the central nervous system (CNS) and the peripheral nervous system (PNS). Male adult Wistar rats were administered by intraperitoneal injection at a dosage of 200 or 400 mg/kg HD, five days per week for 8 weeks. Samples of the cerebral cortex, cerebellum, spinal cord and sciatic nerves were collected and examined for Bcl-2, Bax and Caspase-3 expression using Western blotting. Subchronic exposure to HD resulted in significantly increased expression of both anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax and Caspase-3 in cerebral cortex and cerebellum, which exhibited a dose-dependent pattern. Though little change was detected in spinal cord, our results showed that the expression of Bcl-2, Bax and Caspase-3 was markedly enhanced in the sciatic nerves. These findings suggested that the changes of apoptosis-related protein level in rat nerve tissues were associated with the intoxication of HD, which might be involved in early molecular regulatory mechanism of apoptosis in the HD-induced neuropathy.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型,研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化.基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征——DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14、0.49和1.43,与妊娠早期相比,妊娠中、晚期胎盘基因组DNA断裂值有显著性增加.TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层.免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax∶Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势.实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax∶Bcl-2的比例相关.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型。研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化,基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征-DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14,0.49和1.43,与妊娠早期相比,妊娠中,晚期胎盘基因组DNA断裂值有显著性增加,TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层,免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax:Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势,实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax:Bcl-2的比例相关。     

Ultrastructural Morphology of Neuronal Death Following Reversible Focal Ischemia in the Rat

Electron microscopy and terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labelling (TUNEL) were used to illustrate the different stages and subcellular alterations of cell degeneration that occur in the striatum of the rat after transient focal ischemia. Degenerating neurons exhibited different morphological types: apoptosis Type 1 (aggregation of dense masses of chromatin beneath the 'intact' nuclear membrane) and Type 2 (high cytoplasmic vacuolization), and necrosis. These profiles were localized in different part of the striatum. Type 1 was found in the head of the caudate putamen, Type 2 in the middle part of the striatum and necrosis in the striatal core. These ultrastructural results demonstrated that apoptosis occurs in neurons following focal ischemia in the striatal penumbra. In contrast, necrosis can be observed in the ischemic core, the region maximally affected by the ischemia. Finally, the presence of astrocytes throughout both the penumbra and ischemic core displaying numerous cytoplasmic vacuoles suggested an activation of glial cells.     

丰富环境对脑缺血再灌注小鼠神经细胞凋亡和XIAP基因表达的影响

该研究主要探讨丰富环境干预对脑缺血再灌注小鼠神经细胞凋亡的影响。实验采用健康雄性ICR 小鼠,随机分为丰富环境缺血组(IE)、标准环境缺血组(IS),同时分别设丰富环境假手术组(SE)、标准环境假手术组(SS)。通过双侧颈动脉重复结扎建立小鼠全脑缺血再灌注模型,分别在缺血后在丰富环境和标准环境饲养3 d或7 d 后,进行开场行为和水迷宫空间记忆行为检测;同时进行神经细胞损伤的组织学观测,并采用琼脂糖凝胶电泳技术分析DNA 片段化,用RT-PCR 检测X 染色体连锁的凋亡抑制蛋白（X-linked inhibitor of apoptosis protein,XIAP） mRNA的表达。结果表明,丰富环境干预能有效改善脑缺血导致的自发活动、探究行为减少和空间学习记忆能力减退,并对正常小鼠也有促进作用。缺血再灌注4 d的海马神经细胞损伤较严重,神经元密度显著减少,脑组织DNA 片段化明显增强,丰富环境作用使神经细胞损伤和DNA 片段化程度均有所减轻。同时丰富环境作用可抑制反复脑缺血再灌注导致的XIAP mRNA 表达下调。可见,丰富环境干预可改善脑缺血小鼠的自发活动、探究行为和空间学习记忆能力,该作用可能与其抑制神经细胞XIAP基因表达下调、减弱脑缺血再灌注诱导的神经细胞凋亡有关。     

Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ-Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat

We evaluated whether regional differences in the magnitude of glutamate, gamma-aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia-induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10- and 20-min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathological neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathological outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10-min insult but severely damaged after 20 min of ischemia. Ischemia-induced increase in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different. Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10- and 20-min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20-min insult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Transient Cerebral Ischemia on the Expression of DNA Methyltransferase 1 in the Gerbil Hippocampal CA1 Region

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferases (Dnmt). Increasing evidences suggest that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. In the present study, we investigated the changes in DNA methyltransferases 1 (Dnmt1) immunoreactivity and its protein levels in the gerbil hippocampal CA1 region after 5 min of transient global cerebral ischemia. CA1 pyramidal neurons were well stained with NeuN (a neuron-specific soluble nuclear antigen) antibody in the sham-group, Four days after ischemia–reperfusion (I–R), NeuN-positive (⁺) cells were significantly decreased in the stratum pyramidale (SP) of the CA1 region, and many Fluro-Jade B (a marker for neuronal degeneration)⁺ cells were observed in the SP. Dnmt1 immunoreactivity was well detected in all the layers of the sham-group. Dnmt1 immunoreactivity was hardly detected only in the stratum pyramidale of the CA1 region from 4 days post-ischemia; however, at these times, Dnmt1 immunoreactivity was newly expressed in GABAergic interneurons or astrocytes in the ischemic CA1 region. In addition, the level of Dnmt1 was lowest at 4 days post-ischemia. In brief, both the Dnmt1 immunoreactivity and protein levels were distinctively decreased in the ischemic CA1 region 4 days after transient cerebral ischemia. These results indicate that the decrease of Dnmt1 expression at 4 days post-ischemia may be related to ischemia-induced delayed neuronal death.     

Transient Elevation of Interstitial N-Acetylaspartate in Reversible Global Brain Ischemia

Abstract: We evaluated the changes of interstitial N -acetylaspartate (NAA) concentration ([NAA]_e) in rat striatum by microdialysis following transient global ischemia and depolarization. The dialysate NAA concentration ([NAA]_d) values were corrected for the in vivo recovery to obtain [NAA]_e, by the use of [³H]mannitol in the perfusion fluid. During global ischemia the relative loss (RL) of [³H]mannitol decreased to 40% of preischemic values, reflecting the decrease in extracellular volume fraction. During reperfusion RL of [³H]mannitol quickly normalized. The [NAA]_d doubled during transient ischemia, which, after correction for in vivo recovery, corresponds to a fivefold increase in [NAA]_e ( p < 0.05). Reperfusion induced a >10-fold increase of [NAA]_e ( p < 0.01) with subsequent normalization after 45 min. KCI at 100 m M caused a reversible 50% reduction in RL of [³H]mannitol and a three times increase in [NAA]_e ( p < 0.05) but no further increase when normal perfusate was reintroduced. The mechanisms of NAA release from neurons are unknown but may involve the activation of unknown channels/carriers—possibly in relation to a volume regulatory response. The present study shows that the distribution of NAA in brain is dynamically regulated in acute ischemia and suggests that changes of NAA levels could be caused by other means than neuronal loss.     

Expression of the Baculovirus p35 Gene Inhibits Mammalian Neural Cell Death

Expression of the apoptosis suppressor genep35, derived from the baculovirus Autographa californica nuclear polyhedrosis virus, markedly inhibited the cell death of stably transfected mammalian neural cells whether the cell death was induced by glucose withdrawal, calcium ionophore, or serum withdrawal. The p35 protein, which is required to block virus-induced apoptosis of cultured insect cells, is only the second gene product shown to block mammalian neural cell death, with Bcl-2 being the first. Because there is no apparent homology between p35 and Bcl-2, the existence of a cellular death program that may be modulated at multiple points is suggested/Furthermore, these findings demonstrate that the putative cellular death program is conserved across species and cell types.     

Stimulation of Inositol Phospholipid Hydrolysis by Excitatory Amino Acids Is Enhanced in Brain Slices from Vulnerable Regions after Transient Global Ischemia

Stimulation of inositol phospholipid hydrolysis by transmitter receptor agonists was measured in slices from hippocampus, cerebral cortex, and corpus striatum at various intervals after transient global ischemia in rats. Ischemia was induced through the four-vessel occlusion model. Stimulation of [3H]inositol monophosphate formation by excitatory amino acids was greatly enhanced in hippocampal slices prepared from ischemic rats at 24 h or 7 days after reperfusion. This potentiation was more evident using ibotenic acid and was also observed in cerebral cortex, but not in corpus striatum. This regional profile correlated with the pattern of ischemia-induced neuronal damage observed under our experimental conditions. The enhanced responsiveness to excitatory amino acids was always accompanied by an increase in both basal and norepinephrine-stimulated [3H]inositol monophosphate formation. In contrast, stimulation of [3H]inositol monophosphate formation by carbamylcholine was not modified in hippocampal or cortical slices from ischemic animals.     

大鼠全脑缺血后海马CA1区锥体神经元L型钙通道电流的改变(英)

已有研究表明在脑缺血期间及再灌流后早期,海马CA1锥体神经元细胞内钙浓度明显升高,这一钙超载被认为是缺血性脑损伤的重要机制之一.电压依赖性钙通道是介导正常CA1神经元钙内流的主要途径.实验观察了脑缺血再灌流后早期海马CA1锥体神经元电压依赖性L型钙通道的变化.以改良的四血管闭塞法制作大鼠15 min前脑缺血模型,在急性分离的海马CA1神经元上,采用膜片钳细胞贴附式记录L型电压依赖性钙通道电流.脑缺血后CA1神经元L型钙通道的总体平均电流明显增大,这是由于通道的开放概率增加所致.进一步分析单通道动力学显示,脑缺血后通道的开放时间变长,通道的开放频率增大.研究结果提示L型钙通道功能活动增强可能参与了缺血后海马CA1锥体神经元的细胞内钙浓度升高.