Effect of peptide Semax and its C-terminal fragment PGP on Vegfa gene expression during incomplete global cerebral ischemia in rats

Vascular endothelial growth factor (VEGF-A) is hypoxia-inducible signal glycoprotein. VEGF-A induces vascular endothelial cell proliferation, which leads to the reconstitution of the vascular network in brain regions damaged by ischemia. However, this protein is also involved in the processes of inflammation and edema in early stages of ischemia. The synthetic peptide Semax has neuroprotective and anti-inflammatory properties and is actively used in the treatment of cerebral ischemia. We have previously shown that Semax reduces vascular injury and activates the mRNA synthesis of neurotrophins and their receptors during global cerebral ischemia in rats. In this work, we studied the effect of Semax and its C-terminal Pro-Gly-Pro tripeptide on Vegfa mRNA expression in different regions of the rat brain after 0.5, 1, 2, 4, 8, 12 and 24 h, which is the irreversible occlusion of the common carotid arteries. It was shown that ischemia increases the levels of Vegfa mRNA in rat brains (4 h after occlusion in cerebellum, cerebral cortex, and hippocampus; 8 h after occlusion in the cortex and hippocampus; and 24 h after occlusion in the cortex). Treatment with Semax reduces the levels of Vegfa mRNA in the frontal cortex (4, 8 and 12 h after occlusion) and the hippocampus (2 and 4 h after occlusion). The effect of PGP on the Vegfa gene expression was almost negligible. It was shown that Semax prevents the activating effect of hypoxia on the expression of the Vegfa gene at early stages of global cerebral ischemia. In turn, an increase in the level of Vegfa mRNA in the hippocampus 24 h after occlusion and Semax administration apparently reflects the neuroprotective properties of the drug.     

Enhanced expressions of arachidonic acid-sensitive tandem-pore domain potassium channels in rat experimental acute cerebral ischemia

To further explore the pathophysiological significance of arachidonic acid-sensitive potassium channels, RT-PCR and Western blot analysis were used to investigate the expression changes of TREK channels in cortex and hippocampus in rat experimental acute cerebral ischemia in this study. Results showed that TREK-1 and TRAAK mRNA in cortex, TREK-1 and TREK-2 mRNA in hippocampus showed significant increases 2 h after middle cerebral artery occlusion (MCAO). While the mRNA expression levels of the all three channel subtypes increased significantly 24 h after MCAO in cortex and hippocampus. At the same time, the protein expressions of all the three channel proteins showed significant increase 24 h after MCAO in cortex and hippocampus, but only TREK-1 showed increased expression 2 h after MCAO in cortex and hippocampus. Immunohistochemical experiments verified that all the three channel proteins had higher expression levels in cortical and hippocampal neurons 24 h after MCAO. These results suggested a strong correlation between TREK channels and acute cerebral ischemia. TREK channels might provide a neuroprotective mechanism in the pathological process.     

Localization of aldolase C mRNA in brain cells

The expression of aldolase C and aldolase A mRNA was assessed by Northern blot hybridization using RNAs purified from cultured rat and mouse brain neurons and astroglial cells. Neurons were found to contain about 4-fold more aldolase C mRNA and about twice as much aldolase A mRNA than astroglia. Analysis of the cellular localization of aldolase C mRNA by in situ hybridization to brain slices showed a predominantly neuronal labeling with an irregular distribution. A strong signal was observed in Purkinje cell somata and a weaker signal in subpopulations of neurons in cerebral cortex, striatum, hippocampus, hypothalamic nuclei and primary olfactory cortex.     

Cold shock response in mammalian cells

Compared to bacteria and plants, the cold shock response has attracted little attention in mammals except in some areas such as adaptive thermogenesis, cold tolerance, storage of cells and organs, and recently, treatment of brain damage and protein production. At the cellular level, some responses of mammalian cells are similar to microorganisms; cold stress changes the lipid composition of cellular membranes, and suppresses the rate of protein synthesis and cell proliferation. Although previous studies have mostly dealt with temperatures below 20 degrees C, mild hypothermia (32 degrees C) can change the cell's response to subsequent stresses as exemplified by APG-1, a member of the HSP110 family. Furthermore, 32 degrees C induces expression of CIRP (cold-inducible RNA-binding protein), the first cold shock protein identified in mammalian cells, without recovery at 37 degrees C. Remniscent of HSP, CIRP is also expressed at 37 degrees C and developmentary regulated, possibly working as an RNA chaperone. Mammalian cells are metabolically active at 32 degrees C, and cells may survive and respond to stresses with different strategies from those at 37 degrees C. Cellular and molecular biology of mammalian cells at 32 degrees C is a new area expected to have considerable implications for medical sciences and possibly biotechnology.     

Ischemia induces metallothionein III expression in neurons of rat brain

Metallothionein III (MT-III) is a brain-specific member of the metallothionein family and binds zinc in vivo. In order to confirm the precise localization of MT-III in normal rat brain and the change of MT-III expression after transient whole brain ischemia, we raised a high affinity phagemid-antibody specific for rat MT-III. Immunohistochemical analysis revealed that MT-III in normal brain is localized abundantly in neuronal cell bodies in CA1-3 regions of hippocampus, dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. This expression pattern of MT-III was similar to that of MT-III mRNA observed by in situ hybridization studies. ELISA and Northern blot analysis revealed that MT-III protein as well as mRNA levels were up-regulated in cerebrum soon after ischemic stress. Immunohistochemical analysis also demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain. These results suggest that MT-III plays an important role in protecting neurons from ischemic insult by reducing neurotoxic zinc levels and inhibits uncontrolled growth of neurites after ischemia.     

bFGF对脑缺血再灌注大鼠海马及顶叶皮质神经元的保护作用

目的本实验应用大脑中动脉栓塞（MCAO）模型,观察bFGF对脑缺血再灌注损伤后海马及顶叶皮质中Wnt通路抑制因子Dickkopf-1（DKK-1）和Wnt通路中pCatenin的表达作用的影响,以探讨Wnt通路对缺血性脑损伤的作用机制,为临床治疗缺血性脑血管病提供实验依据。方法应用线栓法制作大鼠局灶性脑缺血再灌注模型,大脑中动脉阻塞1h再灌注损伤24h,采用免疫组织化学SABC法及RT-PCR法检测海马及顶叶皮质CA1区神经元β-Catenin和DKK-1mRNA的表达。结果正常sham组,大鼠海马组织DKK-1 mRNA表达较少,β-Catenin阳性产物在细胞质内有所表达;I／R组,DKK-1 mRNA表达明显增多,β-Catenin在胞质内表达明显减少;bFGF组,大鼠海马组织DKK-1 mRNA表达较I／R组明显减少,而海马细胞质内β-Catenin表达较I／R组明显增加。结论bFGF抑制缺血神经元凋亡,参与DKK-1 mRNA和β-Catenin的调节,对缺血神经元有保护作用。     

Hepcidin is involved in iron regulation in the ischemic brain

Oxidative stress plays an important role in neuronal injuries caused by cerebral ischemia. It is well established that free iron increases significantly during ischemia and is responsible for oxidative damage in the brain. However, the mechanism of this ischemia-induced increase in iron is not completely understood. In this report, the middle cerebral artery occlusion (MCAO) rat model was performed and the mechanism of iron accumulation in cerebral ischemia-reperfusion was studied. The expression of L-ferritin was significantly increased in the cerebral cortex, hippocampus, and striatum on the ischemic side, whereas H-ferritin was reduced in the striatum and increased in the cerebral cortex and hippocampus. The expression level of the iron-export protein ferroportin1 (FPN1) significantly decreased, while the expression of transferrin receptor 1 (TfR1) was increased. In order to elucidate the mechanisms of FPN1 regulation, we studied the expression of the key regulator of FPN1, hepcidin. We observed that the hepcidin level was significantly elevated in the ischemic side of the brain. Knockdown hepcidin repressed the increasing of L-ferritin and decreasing of FPN1 invoked by ischemia-reperfusion. The results indicate that hepcidin is an important contributor to iron overload in cerebral ischemia. Furthermore, our results demonstrated that the levels of hypoxia-inducible factor-1α (HIF-1α) were significantly higher in the cerebral cortex, hippocampus and striatum on the ischemic side; therefore, the HIF-1α-mediated TfR1 expression may be another contributor to the iron overload in the ischemia-reperfusion brain.     

暂时性脑缺血诱导c-fos原癌基因和鸟氨酸脱羧酶基因表达

应用大鼠椎动脉与颈内动脉结扎造成暂时性脑缺血再灌注以及RNA点杂交方法观察c-fos基因与鸟氨酸脱羧酶(ODC)基因表达的动力学过程。结果表明:大鼠大脑皮质c-fos基因在再灌后0.5至3小时表达,ODC基因在6小时至14小时表达;海马的c-fos基因则自36至72小时呈现高水平表达,ODC基因表达与之相对同步或稍延后。但是暂时性脑缺血却不能诱导c-Myc基因表达。上述结果提示c-fos及ODC可能在缺血型脑损伤后具有特殊作用。文中对缺蛋再灌引起cfos-及ODC基因表达的机制进行了分析与讨论,并提出“神经元应激状态”这一概念以描述神经元对伤害性刺激的反应历程。     

暂时性脑缺血诱导c-fos原癌基因和鸟氨酸脱羧酶基因表达

应用大鼠椎动脉与颈内动脉结扎造成暂时性脑缺血再灌注以及RNA点杂交方法观察c-fos基因与鸟氨酸脱羧酶(ODC)基因表达的动力学过程。结果表明:大鼠大脑皮质c-fos基因在再灌后0.5至3小时表达,ODC基因在6小时至14小时表达;海马的c-fos基因则自36至72小时呈现高水平表达,ODC基因表达与之相对同步或稍延后。但是暂时性脑缺血却不能诱导c-Myc基因表达。上述结果提示c-fos及ODC可能在缺血型脑损伤后具有特殊作用。文中对缺蛋再灌引起cfos-及ODC基因表达的机制进行了分析与讨论,并提出“神经元应激状态”这一概念以描述神经元对伤害性刺激的反应历程。     

Neuroprotective Potential of Fasudil Mesylate in Brain Ischemia-Reperfusion Injury of Rats

We previously reported that inhibition of Rho-kinase (ROCK) by hydroxyl fasudil improves cognitive deficit and neuronal damage in rats with chronic cerebral ischemia (Huang et al., Cell Mol Neurobiol 28:757–768, 2008). In this study, fasudil mesylate (FM) was investigated for its neuroprotective potential in rats with ischemia following middle cerebral artery occlusion (MCAO) and reperfusion. The effect of fasudil mesylate was also studied in rat brain cortical and hippocampal slices treated with oxygen-glucose deprivation (OGD) injury. Gross anatomy showed that cerebral infarct size, measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in the FM-treated than in the non-FM-treated ischemic rats. In the brain regions vulnerable to ischemia of ischemic rats, fasudil mesylate was also found to significantly restore the enzyme protein expression level of endothelial nitric oxide synthase (eNOS), which was decreased in ischemia. However, it remarkably reduced the protein synthesis of inducible nitric oxide synthase (iNOS) that was induced by ischemia and reperfusion. In rat brain slices treated with OGD injury, fasudil mesylate increased the neuronal cell viability by 40% for cortex and by 61% for hippocampus, respectively. Finally, in the presence of OGD and fasudil mesylate, superoxide dismutase (SOD) activity was increased by 50% for cortex and by 58% for hippocampus, compared to OGD only group. In conclusion, our in vivo study showed that fasudil mesylate not only decreased neurological deficit but also reduced cerebral infarct size, possibly and at least partially by augmenting eNOS protein expression and inhibiting iNOS protein expression after ischemia-reperfusion. Xian-Ju Huang contributed equally to this article.     

Down-regulation of cold-inducible RNA-binding protein does not improve hypothermic growth of Chinese hamster ovary cells producing erythropoietin

Discovery of the cold-inducible RNA-binding protein (CIRP) in mouse fibroblasts suggests that growth suppression at hypothermic conditions is due to an active response by the cell rather than due to passive thermal effects. To determine the effect of down-regulated CIRP expression on cell growth and erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells at low culture temperature, stable CHO cell clones with reduced CIRP expression level were established by transfecting (rCHO) cells with the CIRP siRNA vector with a target sequence of TCGTCCTTCCATGGCTGTA. For comparison of the degree of specific growth rate (micro) reduction at low culture temperature, three CIRP-reduced clones with different mu and three control clones transfected with null vector were cultivated at two different temperatures, 32 degrees C and 37 degrees C. Unlike mouse fibroblasts, alleviation of hypothermic growth arrest of rCHO cells by CIRP down-regulation was insignificant, as shown by statistical analysis using the t-test (P<0.18, n=3). The ratios of mu at 32 degrees C to micro at 37 degrees C of CIRP-reduced clones and control clones were 0.29+/-0.03 and 0.25+/-0.03 on an average, respectively. Furthermore, it was also found that overexpression of CIRP did not inhibit rCHO cell growth significantly at 37 degrees C. Taken together, the data obtained show that down-regulation of only CIRP in rCHO cells, unlike mouse fibroblasts, is not sufficient to recover growth arrest at low-temperature culture (32 degrees C).     

Inductin of neuron-derived orphan receptor-1 in the dentate gyrus of the hippocampal formation following transient global ischemia in the rat

Neuron-derived orphan receptor (NOR-1) is a member of the thyroid/steroid receptor superfamily that was originally identified in forebrain neuronal cells undergoing apoptosis. In addition to apoptotic stimuli, activation of several signal transduction pathways including direct neuronal depolarization regulates the expression of NOR-1. In this study we tested whether the expression of NOR-1 is changed following transient ischemic injury in the adult rat brain. NOR-1 mRNA increased rapidly in the dentate gyrus of the hippocampal formation and piriform cortex 3 h after transient global ischemia and returned to basal level at 6 h. On the other hand, oxygen-glucose deprivation of cultured cerebral cortical neurons did not alter the expression of NOR-1. These results suggest that expression of NOR-1 is differentially regulated in different brain regions in response to globally applied brain ischemia, but that hypoxia is not sufficient to induce its expression.     

Cold-inducible RNA-binding protein mediates neuroinflammation in cerebral ischemia

Background

Neuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA-binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke.

Methods

Stroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-α (TNF-α) levels were assessed by RT-PCR and Western blot analysis.

Results

CIRP is elevated along with an upregulation of TNF-α expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-α, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-α levels. Addition of recombinant murine (rm) CIRP directly induces TNF-α release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-κB signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP.

Conclusion

Extracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia.

General significance

Development of an anti-CIRP therapy may benefit patients with brain ischemia.     

Effect of long-term cold adaptation on the mRNA expression of serotonin 5-HT1A and 5-HT2A receptors

The mRNA expression of serotonin receptors 5-HT1A and 5-HT2A was investigated by the quantitative method RT-PCR in rats adapted to cold (5 weeks at +4 -(+6) degrees C) and in control (5 weeks at +20-22 degrees C). Four brain regions were examined: frontal cortex, hypothalamus, hippocampus, and midbrain. The influence of cold adaptation on the mRNA expression of 5-HT15 receptor was not found to be absent. The mRNA expression of 5-HT2A receptor changed under long-term cold exposure. These changes in different brain regions were various: in hypothalamus, there was an increase of the 5-HT2A receptor mRNA expression; in the cortex, a decrease; in the hippocampus and midbrain, significant changes of the mRNA expression were absent. The findings appear bo te adaptive and, according to their localization in the central nervous system, regulatory. They also suggest involvement of brain serotoninergic system in mechanism of adaptive reorganization of temperature regulation.     

Cloning and characterization of amphibian cold inducible RNA-binding protein

Gene expression of cold inducible RNA-binding protein (CIRP) was examined in the frog. In Xenopus laevis, expression of CIRP (XCIRP) was observed in both brain and liver at 24 degrees C. Circadian expression of XCIRP was observed in brain. Expression of XCIRP in brain was induced by cold treatment and gradually decreased to the control level at 24 degrees C, but no significant changes were observed in liver. Employing the sequence of murine CIRP, bullfrog (Rana catesbeiana) CIRP gene was cloned. The bullfrog CIRP gene, designated BFCIRP, was 706 bp in length and encoded a putative protein of 164 amino acid residues. The deduced protein contained one consensus sequence of RNA-binding domain (CS-RBD) and a glycine rich domain (GRD). The amino acid sequence of BFCIRP was 78.4% identical to XCIRP. Expression of BFCIRP in brain was stronger in winter than that in summer. These findings suggest that BFCIRP expression in brain may link to hibernation.     

The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain

The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.