新生BALB/c小鼠大脑皮质神经元细胞培养方法的建立

目的:经改良和优化,建立高纯度BALB/c小鼠大脑皮质神经元培养的方法.方法:采用L-多聚赖氨酸包被细胞培养板,取新生BALB/c小鼠(出生24 h内)大脑皮质组织,经0.25%胰酶消化后吹打成单个细胞,按1×106/孔接种于35 mm的六孔板中,用神经元细胞培养种植液培养6 h后换神经元细胞培养饲养液,培养40 h时...     

Microarray Analysis of Gene Expression in Rat Hippocampus After Chronic Ethanol Treatment

It is thought that changes in gene expression in the brain mediate chronic ethanol-induced complex behaviors such as tolerance, dependence, and sensitization, and also relate to ethanol-induced brain toxicity. Using high-density filter-based cDNA microarrays (GeneFilters), we analyzed the expression of over 5000 genes in the dorsal hippocampus of rats treated with 12% ethanol or tap water for 15 months. Ethanol-induced changes in gene expression were particularly prominent in two groups of genes. One group consisted of oxidoreductases, including ceruloplasmin, uricase, branched-chain alpha-keto acid dehydrogenase, NADH ubiquinone oxidoreductase, P450, NAD⁺-isocitrate dehydrogenase, and cytochrome c oxidase, which may be related to ethanol-induced oxidative stress. The other group of genes included ADP-ribosylation factor, RAS related protein rab10, phosphatidylinositol 4-kinase, dynein-associated polypeptides, and dynamin-1, which seem to be involved in membrane trafficking. The results may reveal some of the pathways involved in ethanol-induced pathophysiological changes.     

Gene Expression Profiling of Rat Cerebral Cortex Development Using cDNA Microarrays

A large amount of genetic information is devoted to brain development. In this study, the cortical development in rats at eight developmental time points (four embryonic [E15, E16, E18, E20] and four postnatal [P0, P7, P14, P21]) was studied using a rat brain 10K cDNA microarray. Significant differential expression was observed in 467 of the 9,805 genes represented on the microarray. Two major Gene Ontology classes—cell differentiation and cell–cell signaling—were found to be important for cortical development. Genes for ribosomal proteins, heterogeneous nuclear ribonucleoproteins, and tubulin proteins were up-regulated in the embryonic stage, coincidently with extensive proliferation of neural precursor cells as the major component of the cerebral cortex. Genes related to neurogenesis, including neurite regeneration, neuron development, and synaptic transmission, were more active in adulthood, when the cerebral cortex reached maturity. The many developmentally modulated genes identified by this approach will facilitate further studies of cortical functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Loss of Neurons in the Hippocampus and Cerebral Cortex of AMSH-Deficient Mice

AMSH, a molecule that associates with STAM1, is involved in the in vitro cell growth signaling mediated by interleukin 2 and granulocyte-macrophage colony-stimulating factor. To investigate the in vivo functional role of AMSH, we have generated AMSH-deficient mice by gene targeting. The AMSH-deficient mice were morphologically indistinguishable from their littermates at birth, and histopathological examinations revealed normal morphogenesis in all tissues tested. However, all the AMSH-deficient mice exhibited postnatal growth retardation and died between postnatal day 19 (P19) and P23. Examination of brain sections at P6 demonstrated significant loss of neurons and apoptotic cells in the CA1 subfield of the hippocampus. Brain atrophy developed by P16 and was accompanied by complete loss of the CA1 neurons in the hippocampus and marked atrophy of the cerebral cortex. Furthermore, AMSH-deficient hippocampal neuronal cells were unable to survive in vitro, even in the presence of several stimulatory cytokines, while AMSH-deficient cerebellar neurons, thymocytes, and embryonic fibroblasts survived normally. Taken together, these observations indicate that AMSH is an essential molecule for the survival of neuronal cells in early postnatal mice.     

Differential Regulation of GABAA Receptor Gene Expression by Ethanol in the Rat Hippocampus Versus Cerebral Cortex

Abstract: Previous research has shown that chronic ethanol consumption dramatically alters GABA_A receptor α₁ and α₄ subunit gene expression in the cerebral cortex and GABA_A receptor α₁ and α₆ subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABA_A receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABA_A receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABA_A receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABA_A receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABA_A receptor α₄ subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABA_A receptor α₁, α₂, α₃, α_2/3, or γ₂ was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABA_A receptor α₄ subunits and significantly decreased the relative expression of cerebral cortical GABA_A receptor α₁ subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABA_A receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.     

用cDNA表达阵列分析遗传性癫痫易感大鼠海马和大脑皮质的基因表达

采用Atlas^TM Rat cDNA Expression Array建立遗传性癫痫易感性P77PMC大鼠和正常对照Wistar大鼠的海马与大脑皮质基因表达谱,用Eagle EyeⅡStill Video System(Stratagene)图象分析仪分析两者基因表达谱差异.结果发现海马和大脑皮质中各有15个差异表达基因.海马组织中,12个基因在P77PMC大鼠中高表达而在正常对照Wistar大鼠中低表达,3个基因在正常对照Wistar大鼠中高表达,而在P77PMC大鼠中低表达;大脑皮质中,13个基因在P77PMC大鼠中高表达,而在正常对照Wistar大鼠中低表达,2个基因在正常对照Wistar大鼠中高表达,而在P77PMC大鼠中低表达. 结果说明,P77PMC大鼠与正常对照Wistar大鼠海马和大脑皮质存在多个差异表达基因,这些差异表达基因可能在癫痫的发生中扮演了重要角色.     

Gene Expression Profile in the Liver of BALB/c Mice Infected with Fasciola hepatica

Background

Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology.

Methodology

A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21).

Results

We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death.

Conclusion

The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis.     

Loureirin C and Xanthoceraside Attenuate Depression-Like Behaviors and Expression of Interleukin-17 in the Prefrontal Cortex Induced by Chronic Unpredictable Mild Stress in Mice

Neurochemical Research - Major depressive disorder (MDD) is the most prevalent and serious psychiatric disease involving inflammation. Loureirin C and Xanthoceraside are extracts of dragon’s...     

Microarray Analysis of Perinatal-Estrogen-Induced Changes in Gene Expression Related to Brain Sexual Differentiation in Mice

Sexual dimorphism of the behaviors or physiological functions in mammals is mainly due to the sex difference of the brain. A number of studies have suggested that the brain is masculinized or defeminized by estradiol converted from testicular androgens in perinatal period in rodents. However, the mechanisms of estrogen action resulting in masculinization/defeminization of the brain have not been clarified yet. The large-scale analysis with microarray in the present study is an attempt to obtain the candidate gene(s) mediating the perinatal estrogen effect causing the brain sexual differentiation. Female mice were injected with estradiol benzoate (EB) or vehicle on the day of birth, and the hypothalamus was collected at either 1, 3, 6, 12, or 24 h after the EB injection. More than one hundred genes down-regulated by the EB treatment in a biphasic manner peaked at 3 h and 12-24 h after the EB treatment, while forty to seventy genes were constantly up-regulated after it. Twelve genes, including Ptgds, Hcrt, Tmed2, Klc1, and Nedd4, whose mRNA expressions were down-regulated by the neonatal EB treatment, were chosen for further examination by semiquantitative RT-PCR in the hypothalamus of perinatal intact male and female mice. We selected the genes based on the known profiles of their potential roles in brain development. mRNA expression levels of Ptgds, Hcrt, Tmed2, and Nedd4 were significantly lower in male mice than females at the day of birth, suggesting that the genes are down-regulated by estrogen converted from testicular androgen in perinatal male mice. Some genes, such as Ptgds encoding prostaglandin D2 production enzyme and Hcrt encording orexin, have been reported to have a role in neuroprotection. Thus, Ptgds and Hcrt could be possible candidate genes, which may mediate the effect of perinatal estrogen responsible for brain sexual differentiation.     

Using the One-Lung Method to Link p38 to Pro-Inflammatory Gene Expression during Overventilation in C57BL/6 and BALB/c Mice

Introduction

The mechanisms of ventilator-induced lung injury (VILI), including the role of MAP kinases, are frequently studied in different mouse strains. A useful model for such studies is the isolated perfused mouse lung. As a further development we present the one-lung method that permits to continue perfusion and ventilation of the right lung after removal of the left lung. This method was used to compare the effect of high pressure ventilation (HPV) on pro-inflammatory signaling events in two widely used mouse strains (C57BL/6, BALB/c) and to further define the role of p38 in VILI.

Methods

Lungs were perfused and ventilated for 30 min under control conditions before they were randomized to low (8 cm H₂O) or high (25 cm H₂O) pressure ventilation (HPV) for 210 min, with the left lung being removed after 180 min. In the left lung we measured the phosphorylation of p38, JNK, ERK and Akt kinase, and in the right lung gene expression and protein concentrations of Il1b, Il6, Tnf, Cxcl1, Cxcl2, and Areg.

Results

Lung mechanics and kinase activation were similar in both mouse strains. HPV increased all genes (except Tnf in BALB/c) and all mediators in both strains. The gene expression of mRNA for Il1b, Il6, Cxcl1 and Cxcl2 was higher in BALB/c mice. Backward regression of the kinase data at t = 180 min with the gene and protein expression data at t = 240 min suggested that p38 controls HPV-induced gene expression, but not protein production. This hypothesis was confirmed in experiments with the p38-kinase inhibitor SB203580.

Conclusions

The one-lung method is useful for mechanistic studies in the lungs. While C57BL/6 show diminished pro-inflammatory responses during HPV, lung mechanics and mechanotransduction processes appear to be similar in both mouse strains. Finally, the one-lung method allowed us to link p38 to gene expression during VILI.     

Murine Mammary Tumor Virus Expression During Mammary Tumorigenesis in BALB/c Mice

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.     

Young Plasma Induces Antidepressant-Like Effects in Aged Rats Subjected to Chronic Mild Stress by Suppressing Indoleamine 2,3-Dioxygenase Enzyme and Kynurenine Pathway in the Prefrontal Cortex

Pathophysiology of depression in elderlies is linked to aging-associated increase in indoleamine 2,3-dioxygenase (IDO) levels and activity and kynurenine (Kyn) metabolites. Moreover, these aging-induced changes may alter the brain’s responses to stress. Growing evidence suggested that young plasma can positively affect brain dysfunctions in old age. The present study aimed to investigate whether the antidepressant effects of young plasma administration in aged rats subjected to chronic unpredictable mild stress (CUMS) and underlying mechanisms, focusing on the prefrontal cortex (PFC). Young (3 months old) and aged (22 months old) male rats were divided into five groups; young control, aged control, aged rats subjected to CUMS (A?+?CUMS), aged rats subjected to CUMS and treated with young plasma (A?+?CUMS?+?YP), and aged rats subjected to CUMS and treated with old plasma (A?+?CUMS?+?OP). Plasma was injected (1 ml, intravenously) three times per week for four weeks. Young plasma significantly improved CUMS-induced depressive-like behaviors, evidenced by the increased sucrose consumption ratio in the sucrose preference test and the reduced immobility time in the forced swimming test. Furthermore, young plasma markedly reduced the levels of interferon-gamma (IFN-γ), IDO, Kyn, and Kyn to tryptophan (Kyn/Trp) ratio in PFC tissue. Expression levels of the serotonin transporter and growth-associated protein (GAP)-43 were also significantly increased after chronic administration of young plasma. These findings provide evidence for the antidepressant effect of young plasma in old age; however, whether it improves depressive behaviors or faster recovery from stress-induced deficits is required to be elucidated.

     

Intracerebroventricular Administration of Ouabain to Rats Changes the Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus

Ouabain exerts neurotoxic action and activates the population of NMDA receptors. Herein the effect of ouabain on the expression of NMDA subunits was evaluated. Adult Wistar rats were administered intracerebroventricularly with 0.1, 10 and 100 nmol ouabain or saline solution (control). Two days later, membranes of cerebral cortex and hippocampus were isolated. Western blots with antibodies for the NMDA receptor subunits: NR1; NR2A; NR2B; NR2C and NR2D were carried out. In cerebral cortex, NR2D subunit increased 30% with 10 nmol ouabain dose. With 100 nmol ouabain, NR1 and NR2D subunits enhanced 40 and 20%, respectively. In hippocampus, with the dose of 0.1 nmol ouabain, NR1 subunit enhanced roughly 50% whereas NR2B subunit decreased 30%. After administration of 10 nmol ouabain dose, NR2A, NR2B and NR2C subunits decreased 40, 50 and 30%, respectively. With the dose of 100 nmol of ouabain, NR1, NR2A and NR2B subunits diminished 10–20%. It is concluded that ouabain administration led to a differential regulation in the expression of NMDA subunits. These results may be correlated with the modulatory action of ouabain on NMDA receptor.     

Effects of Chronic Introduction of Antidepressants on NMDA-Induced Damage to Neurons of the Rat Hippocampus and Cerebral Cortex

In in vitro studies on superfused slices obtained from the rat hippocampus and cortex, we found that 50 μM N-methyl-D-aspartate (NMDA) applied to the slices in the presence of 10 μM glycine for 15 min exerts a significant damaging action to neurons of these structures. One hour after termination of the action of NMDA, this was manifested in more than a twofold decrease in the synaptic reactivity of pyramidal neurons of the hippocampal СА1 area and layers II/III of the cerebral cortex. The excitotoxic effect of NMDA was prevented by application of competitive (D-2-amino-5-phosphonovaleric acid, 50 μM) and noncompetitive (ketamine, 100 μM) blockers of NMDA receptors. A blocker of glycine-binding sites of NMDA receptors (compound ТСВ 24.15, 10 μM) weakened NMDA-induced damage to the neurons. A competitive blocker of glutamate АМРА receptors, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μM), and a local anesthetic, lidocaine hydrochloride (50 μM), did not modify the excitotoxic effect of NMDA. A blocker of voltagedependent L-type calcium channels, verapamil (20 μM), demonstrated some trend to intensification of NMDA excitotoxic action. An inhibitor of tyrosine-protein phosphatases, sodium vanadate, when i.p. injected into rats in a dose of 15 mg/kg 6 h prior to the electrophysiological experiment, decreased the damaging action of NMDA. Two-hour-long treatment of cerebral slices with 1 μM genistein, an inhibitor of tyrosine kinases, weakened the neuroprotective effect of sodium vanadate. Chronic injections (14 days in daily doses of 20 mg/kg) of antidepressants belonging to different functional classes (imipramine, fluoxetine, and pyrazidol) into rats decreased (similarly to blockers of NMDA receptors) the excitotoxic action of NMDA receptors. Neuroprotective effects of antidepressants were weakened upon the action of genistein. We conclude that the neuroprotective activity of antidepressants under conditions of excitotoxic action of NMDA is mainly determined by an increase in the activity of tyrosine kinases in the cytoplasm and/or neuronal nucleus.     

Experimental Evidence that Phenylalanine Provokes Oxidative Stress in Hippocampus and Cerebral Cortex of Developing Rats

High levels of phenylalanine (Phe) are the biochemical hallmark of phenylketonuria (PKU), a neurometabolic disorder clinically characterized by severe mental retardation and other brain abnormalities, including cortical atrophy and microcephaly. Considering that the pathomechanisms leading to brain damage and particularly the marked cognitive impairment in this disease are poorly understood, in the present study we investigated the in vitro effect of Phe, at similar concentrations as to those found in brain of PKU patients, on important parameters of oxidative stress in the hippocampus and cerebral cortex of developing rats. We found that Phe induced in vitro lipid peroxidation (increase of TBA-RS values) and protein oxidative damage (sulfhydryl oxidation) in both cerebral structures. Furthermore, these effects were probably mediated by reactive oxygen species, since the lipid oxidative damage was totally prevented by the free radical scavengers α-tocopherol and melatonin, but not by L-NAME, a potent inhibitor of nitric oxide synthase. Accordingly, Phe did not induce nitric oxide synthesis, but significantly decreased the levels of reduced glutathione (GSH), the major brain antioxidant defense, in hippocampus and cerebral cortex supernatants. Phe also reduced the thiol groups of a commercial GSH solution in a cell-free medium. We also found that the major metabolites of Phe catabolism, phenylpyruvate, phenyllactate and phenylacetate also increased TBA-RS levels in cerebral cortex, but to a lesser degree. The data indicate that Phe elicits oxidative stress in the hippocampus, a structure mainly involved with learning/memory, and also in the cerebral cortex, which is severely damaged in PKU patients. It is therefore presumed that this pathomechanism may be involved at least in part in the severe cognitive deficit and in the characteristic cortical atrophy associated with dysmyelination and leukodystrophy observed in this disorder.     

iTRAQ-Based Quantitative Proteomics Suggests Synaptic Mitochondrial Dysfunction in the Hippocampus of Rats Susceptible to Chronic Mild Stress

Mounting studies show that hippocampal synaptic transmission and plasticity are abnormal in depression. It has been suggested that impairment of synaptic mitochondrial functions potentially occurs in the hippocampus. Thus, the synaptic mitochondria may be a crucial therapeutic target in the course of depression. Here, we investigated the potential dysregulation of synaptic mitochondrial proteins in the hippocampus of a chronic mild stress (CMS) rat model. Proteomic changes of hippocampal synaptosomes containing synaptic mitochondria were quantitatively examined using the isobaric tag for relative and absolute quantitation labeling combined with tandem mass spectrometry. 45 Proteins were identified to be differentially expressed, of which 21 were found to be putative synaptic mitochondrial proteins based on gene ontology component and SynaptomeDB analyses. Detailed investigations of protein functions and disease relevance support the importance of hippocampal synaptic mitochondria as a key substrate contributing to impairment in synaptic plasticity of stress-related disorders. Interestingly, eight synaptic mitochondrial proteins were specifically associated to the susceptible group, and might represent part of molecular basis of depression. Further analysis indicated that the synaptic mitochondrial oxidative phosphorylation (OXPHOS) system was heavily affected by CMS in the susceptible rats. The present results provide novel insights into the disease mechanism underlying the abnormal OXPHOS that is responsible for energy-demanding synaptic plasticity, and thereby increase our understanding of the role of hippocampal synaptic mitochondrial dysfunction in depression.