Glutamine Synthetase Levels Research Articles

Pathological Changes of Astrocytes under Seizure

Multiple lines of studies support the view that defective functions of astrocytes contribute to neuronal hyper-excitability in the epileptic brain. Autopsy and surgical resection specimens find that post-traumatic seizures and chronic temporal lobe epilepsy may originate from glial scars. Astrogliosis, a component of glial scar, which involves structural and metabolic changes in astrocytes, is often a prominent feature of temporal epilepsy and most animal models of recurrent seizures. Although glial scar formation has been recognized for over 120 years, fundamental aspects of the cellular mechanisms are poorly understood. We here provide an overview of the experimental findings about astrogliosis in seizures, which involves the changes in astrocytic structure and loss of domain. These structure changes are paralleled by functional changes, including expression levels of glutamate transporter and glutamine synthetase. Reactive astrocytes have also been shown to down- regulate the activities of K + channels, leading to impairment of clearance of K+. The functional remodeling may contribute to

Enhanced Ca2 +-dependent glutamate release from astrocytes of the BACHD Huntington's disease mouse model

Huntington's disease (HD) causes preferential loss of a subset of neurons in the brain although the huntingtin protein is expressed broadly in various neural cell types, including astrocytes. Glutamate-mediated excitotoxicity is thought to cause selective neuronal injury, and brain astrocytes have a central role in regulating extracellular glutamate. To determine whether full-length mutant huntingtin expression causes a cell-autonomous phenotype and perturbs astrocyte gliotransmitter release, we studied cultured cortical astrocytes from BACHD mice. Here, we report augmented glutamate release through Ca2+-dependent exocytosis from BACHD astrocytes. Although such release is usually dependent on cytosolic Ca2+ levels, surprisingly, we found that BACHD astrocytes displayed Ca2+ dynamics comparable to those in wild type astrocytes. These results point to a possible involvement of other factors in regulating Ca2+-dependent/vesicular release of glutamate from astrocytes. We found a biochemical footprint that would lead to increased availability of cytosolic glutamate in BACHD astrocytes: i) augmented de novo glutamate synthesis due to an increase in the level of the astrocyte specific mitochondrial enzyme pyruvate carboxylase; and ii) unaltered conversion of glutamate to glutamine, as there were no changes in the expression level of the astrocyte specific enzyme glutamine synthetase. This work identifies a new mechanism in astrocytes that could lead to increased levels of extracellular glutamate in HD and thus may contribute to excitotoxicity in this devastating disease.

Temporospatial expression and cellular localization of glutamine synthetase following traumatic spinal cord injury in adult rats

Glutamine synthetase (GS) is an enzyme involved in an endogenous mechanism of protection against glutamate neurotoxicity and is important in the regulation of astrocyte migration. To date, limited information is available concerning the expression of GS in normal spinal cords and following injury. In the present study, GS expression was identified in astrocytes, oligodendrocytes and microglia in normal rat spinal cords. Following traumatic spinal cord injury (SCI), the glutamate concentration increased rapidly at 1h and returned to baseline rapidly. However, the GS activity and protein levels were found to decrease at 4h and then increase gradually from day3 following SCI. The quantification of astrocytes, oligodendrocytes and activated microglia/macrophages, as well as immunohistochemistry staining of day7 post‑injured spinal cords, indicated that the astrocytes and microglia/macrophages contributed to the increase in GS. Collectively, the results provided evidence for the temporospatial expression and location of GS following SCI and suggested that the changes in GS levels may contribute to glutamate neurotoxicity and glial cell response following SCI.

Changes in glutamate homeostasis cause retinal degeneration in Royal College of Surgeons rats

The aim of the present study was to investigate glutamate homeostasis in retinal degeneration-induced changes and the potential mechanisms of glutamate-mediated excitotoxicity in a rat model. The expression of vesicular glutamate transporter-1 (VGLUT-1) and protein kinase Cα (PKCα) in wild-type and Royal College of Surgeons (RCS) rat retinas, at postnatal Day 15 (P15), P30, P60 and P90, were detected using quantitative real-time polymerase chain reaction and immunohistochemistry. The levels of glutamine synthetase (GS) and L-glutamate/L-aspartate transporter (GLAST) were evaluated by western blotting. Compared with wild-type rats, outer nuclear layer thickness was significantly thinner and VGLUT-1 expression was upregulated in a time-dependent pattern in RCS rats. The ratio of VGLUT-1 to PKCα in RCS rats peaked at P60 (p<0.01) and subsequently decreased by P90 (p<0.01), while it remained constant in wild-type rats. The expression of GS increased gradually from P30 to P90 in RCS rats (p<0.01), while it remained constant in wild-type rats at various time-points. No significant difference in GLAST expression was found between RCS and wild-type rats at all stages of retinal degeneration. Our results confirm the occurrence of glutamate-mediated excitotoxicity to RCS rat retinas and provide an experimental foundation for safeguarding the remnant visual function in retinal degenerative disorders.

Green Tea Is Neuroprotective in Diabetic Retinopathy

Green tea (GT), widely studied for its beneficial properties in protecting against brain ischemia, is a rich source of polyphenols, particularly (-)-epigallocatechin gallate (EGCG). The results presented here demonstrate the beneficial effects of GT in diabetic retinas and in retinal cells under diabetic conditions. Diabetes was induced in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats. Treatment animals received GT orally for 12 weeks. A vehicle was administered orally to the control animals. The protective effects of GT were also evaluated in Müller and in ARPE-19 cells. In diabetic rats, there was an increase in the expression of glial fibrillary acidic protein (GFAP), oxidative retinal markers, and glutamine synthetase levels. In addition, there was a decrease in occludin and glutamate transporter and receptor. Diabetic SHR also demonstrated blood-retinal barrier breakdown and impaired electroretinography results. Müller cells exposed to high-glucose medium produced higher levels of reactive oxygen species (ROS) and glutamine synthetase but reduced levels of glutathione, glutamate transporter, and glutamate receptor. Similarly, ARPE-19 cells exhibited increased ROS production accompanied by decreased expression of claudin-1 and glutamate transporter. Treatment with GT fully restored all the above-mentioned alterations in diabetic animals as well as in retinal cells. GT protected the retina against glutamate toxicity via an antioxidant mechanism. These findings reveal a novel mechanism by which GT protects the retina against neurodegeneration in disorders such as diabetic retinopathy.

Hyposmolality differentially and spatiotemporally modulates levels of glutamine synthetase and serine racemase in rat supraoptic nucleus

Prolonged hyposmotic challenge (HOC) has a dual effect on vasopressin (VP) secretion [Yagil and Sladek (1990) Am J Physiol 258(2 Pt 2):R492-R500]. We describe an electrophysiological correlate of this phenomenon, whereby in vitro HOC transiently reduced the firing activity of VP neurons within the supraoptic nucleus of brain slices, which was followed by a rebound increase of their activity; this was paralleled by changes in the level of proteins relevant to astroglia-neuronal interactions. Hence, in vitro HOC transiently (at 5 min) increased the level of astrocyte-specific glial fibrillary acidic protein (GFAP), which then declined to control or base level (at 20 min); this was blocked by the gliotoxin L-aminoadipic acid, but not by tetanus toxin, which was used to inhibit neurotransmission. Similarly, in vivo HOC led to changes in GFAP level, which after an early increase (10 min) returned to normal (30 min). Immunoassays revealed that neuronal, but not astrocytic, expression of serine racemase (SR) was increased at the late stage of HOC in vivo, whereas at an early stage there was a transient increase in level of the astrocyte-specific glutamine synthetase (GS). Furthermore, there was an increased molecular association between GFAP and GS at 10 min, whereas SR increased its association with the neuronal nuclear antigen NeuN at 30 min. These results suggest that the dual effect of HOC on VP neuronal secretion/activity could be related to metabolic/signaling changes in astrocytes (glutamate-glutamine conversion) and neurons (D-serine synthesis/ammonia production), which may account for the rebound in VP neuronal activity, presumably by promoting the activation of neuronal glutamate receptors.

Genetic studies on the virus-like regions in the genome of hyperthermophilic archaeon, Thermococcus kodakarensis

Four virus-like integrated elements (TKV1, TKV2, TKV3, and TKV4) have been found in the genome of hyperthermophilic archaeon, Thermococcus kodakarensis, but virus particle formation has not been observed in the culture of T. kodakarensis. As the result of growth property analyses, mutants lacking each of the four virus-like regions exhibited decrease in the cell concentration and/or less growth rates compared to growth of parental strain (KU216), when the T. kodakarensis strains were grown at 85°C in nutrient-rich medium. These results indicated that the genes in virus-like regions stimulated the cell growth under the observed growth condition. As the result of transcriptome analyses, genes involved in amino acid, energy or nucleotide metabolisms, and transport systems were up- or down-regulated in the cells of mutant strains. Interestingly, a decrease in transcriptional levels of glutamine synthetase (TK1796) gene (Tk-glnA) was observed in the cells of four mutant strains. Growths of TKV1 disrupted strain and TKV4 disrupted strain have shown no difference compared with that of KU216 by the addition of glutamate or glutamine, and the result suggested that TKV1 and TKV4 contributed to supply of amino acids to the cell.

Does MPTP intoxication in mice induce metabolite changes in the nucleus accumbens? A 1H nuclear MRS study

Using in vivo ¹H NMR spectroscopy in a mouse model of Parkinson's disease, we previously showed that glutamate concentrations in the dorsal striatum were highest after dopamine denervation associated with an increase in gamma-aminobutyric acid (GABA) and (Gln) glutamine levels. The aim of this study was to determine whether the changes previously observed in the motor part of the striatum were reproduced in a ventral part of the striatum, the nucleus accumbens (NAc). This study was carried out on controls and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In vivo spectra were acquired for a voxel (8 μL) in the dorsal striatum, and in the NAc (1.56 μL). NMR acquisitions were first performed 10 days after the last MPTP injection in a basal condition [after saline intraperitoneal (i.p.) injection] and then in the same animal the week after basal NMR acquisitions, after acute levodopa administration (200 mg kg⁻¹, i.p.). Immunohistochemistry was used to determine the levels of (Glu) glutamate, glutamine synthetase (GS) and glutamic acid decarboxylase (GAD) isoform 67 in these two structures. The Glu, Gln and GABA concentrations obtained in the basal state were higher in the NAc of MPTP-intoxicated mice which have the higher dopamine denervation in the ventral tegmental area (VTA) and in the dorsal striatum. Levodopa decreased the levels of these metabolites in MPTP-intoxicated mice to levels similar to those in controls. In parallel, immunohistochemical staining showed that glutamate, GS and GAD67 immunoreactivity increased in the dorsal striatum of MPTP-intoxicated mice and in the NAc for animals with a severe dopamine denervation in VTA. These findings strongly supported a hyperactivity of the glutamatergic cortico-striatal pathway and changes in glial activity when the dopaminergic denervation in the VTA and substantia nigra pars compacta (SNc) was severe.

Investigation of proteomic responses of Streptomyces lydicus to pitching ratios for improving streptolydigin production

Streptomyces lydicus has been reported to produce antibiotic streptolydigin. Pitching ratios play crucial roles in primary and secondary metabolism of Streptomyces bacteria. The higher pitching ratio (30%, v/v) significantly enhanced the levels of streptolydigin products in S. lydicus. Proteome analysis revealed that betaglucosidase and UTP-glucose-1-phosphate uridylyltransferase were up-regulated to accelerate the starch hydrolyzation at the high pitching ratios. Enhancement in the levels of UDPN-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase and glycine cleavage system aminomethyltransferase were involved in the conversion of amino acids into secondary metabolites. Additionally, the expression levels of PfkA2, PfkA3, Zwf2, SucD, GalE1, GatB, TktA1 and ThcA, associated with glycolysis, pentose phosphate pathway, TCA cycle and amino acid metabolism, were dramatically elevated at high pitching ratios, which play important roles in the enhanced streptolydigin production in S. lydicus E9. Interestingly, the levels of proteins (glutamine synthetase I, glutamate synthase subunit beta and glutamine synthetase) were down-regulated with the increases of pitching ratios and fermentation progress, revealing that pitching ratio altered the glutamine synthetase levels and consequently regulated the streptolydigin production of S. lydicus E9. The up-regulation of proteins (eg, aldehyde dehydrogenase and alkyl hydroperoxide reductase) was involved in the redox-based regulation network triggered by an imbalance of the intracellular cell redox homeostasis and by crosstalk with secondary metabolism at the higher pitching ratio. These results settle new insights into physiological facts of S. lydicus E9 in responses to pitching ratios and will eventually improve the antibiotic production schemes in industry.

APOE genotype affects the pre‐synaptic compartment of glutamatergic nerve terminals

Apolipoprotein E (APOE) genotype affects outcomes of Alzheimer's disease and other conditions of brain damage. Using APOE knock-in mice, we have previously shown that APOE-ε4 Targeted Replacement (TR) mice have fewer dendritic spines and reduced branching in cortical neurons. As dendritic spines are post-synaptic sites of excitatory neurotransmission, we used APOE TR mice to examine whether APOE genotype affected the various elements of the glutamate-glutamine cycle. We found that levels of glutamine synthetase and glutamate uptake transporters were unchanged among the APOE genotypes. However, compared with APOE-ε3 TR mice, APOE-ε4 TR mice had decreased glutaminase levels (18%, p < 0.05), suggesting decreased conversion of glutamine to glutamate. APOE-ε4 TR mice also had increased levels of the vesicular glutamate transporter 1 (20%, p < 0.05), suggesting that APOE genotype affects pre-synaptic terminal composition. To address whether these changes affected normal neurotransmission, we examined the production and metabolism of glutamate and glutamine at 4-5 months and 1 year. Using high-frequency (13)C/(1)H nuclear magnetic resonance spectroscopy, we found that APOE-ε4 TR mice have decreased production of glutamate and increased levels of glutamine. These factors may contribute to the increased risk of neurodegeneration associated with APOE-ε4, and also act as surrogate markers for Alzheimer's disease risk.

Effects of short-hairpin RNA-inhibited β-catenin expression on the growth of human multiple myeloma cells in vitro and in vivo

Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of β-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between β-catenin expression and MM remains unclear. In the present study, RNA interference was used to inhibit β-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that β-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of β-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and β-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that β-catenin could be a new therapeutic agent that targets the biology of MM cells.

Dietary protein source significantly alters growth performance, plasma variables and hepatic gene expression in rainbow trout (Oncorhynchus mykiss) fed amino acid balanced diets

The objective of the study was to evaluate the effect of dietary protein source on fish growth, nutrient utilization, plasma variables and hepatic gene expression in juvenile rainbow trout (Oncorhynchus mykiss). Fishmeal (FM) and soy protein isolate (SPI) were used as the main sources of protein in six isonitrogenous, isolipidic and isocaloric diets. The amino acid profiles of the diets were completely balanced to minimize differences between experimental treatments and formulated to contain increasing levels of branched-chain amino acids (BCAA) based upon dietary requirements for trout (NRC, 1993). Dietary protein source more consistently changed the measured variables while BCAA supplementation had an unexpected effect over whole body lipid content. Growth performance and protein retention efficiency were significantly reduced in fish fed SPI diets independently of BCAA supplementation. Total concentration of amino acids as well as circulating indispensable amino acids (IAA) were significantly elevated in the plasma of fish receiving SPI diets compared to fish fed FM diets. The change in IAA was large enough to increase (p<0.05) plasma IAA/DAA (DAA: dispensable amino acids) proportion even when the diets were formulated to have a ratio close to 1. Levels of circulating BCAA and alanine were also elevated in the fish fed SPI diets, possibly indicating a change in protein turnover. The use of SPI caused a reduction (p<0.05) in the hepatic expression levels of alanine amino transferase (alt1) and glutamine synthetase 2 (gls02), while an increase was observed for aspartate aminotransferase (got2), and asparagine synthetase (asns) compared with FM diets. Expression of the gene tor (target of rapamycin) declined over time for all treatments, while expression of a gene known to repress tor function, redd-1, was consistently higher in the liver of fish fed SPI diets. Glucose 6 phosphate dehydrogenase (g6pd) also showed a significantly higher expression in the liver of fish fed SPI diets but only at higher levels of BCAA supplementation.In summary dietary protein source has a significant effect over growth performance, body composition and hepatic gene expression in rainbow trout. We also identified for the first time in fish changes in the expression of redd-1, which may represent another regulatory point in the TOR cascade.

The expression of Dishevelled-3 and glutamine metabolism in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive tumour.1 Biomarker information for MPM is lacking and could prove useful for understanding MPM metastasis and proliferation, and for providing potential targets...

Glutamine Acts as a Neuroprotectant against DNA Damage, Beta-Amyloid and H2O2-Induced Stress

Glutamine is the most abundant free amino acid in the human blood stream and is ‘conditionally essential’ to cells. Its intracellular levels are regulated both by the uptake of extracellular glutamine via specific transport systems and by its intracellular synthesis by glutamine synthetase (GS). Adding to the regulatory complexity, when extracellular glutamine is reduced GS protein levels rise. Unfortunately, this excess GS can be maladaptive. GS overexpression is neurotoxic especially if the cells are in a low-glutamine medium. Similarly, in low glutamine, the levels of multiple stress response proteins are reduced rendering cells hypersensitive to H2O2, zinc salts and DNA damage. These altered responses may have particular relevance to neurodegenerative diseases of aging. GS activity and glutamine levels are lower in the Alzheimer's disease (AD) brain, and a fraction of AD hippocampal neurons have dramatically increased GS levels compared with control subjects. We validated the importance of these observations by showing that raising glutamine levels in the medium protects cultured neuronal cells against the amyloid peptide, Aβ. Further, a 10-day course of dietary glutamine supplementation reduced inflammation-induced neuronal cell cycle activation, tau phosphorylation and ATM-activation in two different mouse models of familial AD while raising the levels of two synaptic proteins, VAMP2 and synaptophysin. Together, our observations suggest that healthy neuronal cells require both intracellular and extracellular glutamine, and that the neuroprotective effects of glutamine supplementation may prove beneficial in the treatment of AD.

The Metabolic Profile of Tumors Depends on Both the Responsible Genetic Lesion and Tissue Type

The altered metabolism of tumors has been considered a target for anticancer therapy. However, the relationship between distinct tumor-initiating lesions and anomalies of tumor metabolism invivo has notbeen addressed. We report that MYC-induced mouse liver tumors significantly increase both glucose and glutamine catabolism, whereas MET-induced liver tumors use glucose to produce glutamine. Increased glutamine catabolism in MYC-induced liver tumors is associated with decreased levels of glutamine synthetase (Glul) and the switch from Gls2 to Gls1 glutaminase. In contrast to liver tumors, MYC-induced lung tumors display increased expression of both Glul and Gls1 and accumulate glutamine. We also show that inhibition of Gls1 kills cells that overexpress MYC and catabolize glutamine. Our results suggest that the metabolic profiles of tumors are likely to depend on both the genotype and tissue of origin and have implications regarding the design of therapies targeting tumor metabolism.

In vitro effect of adenosine A2A receptor antagonist SCH 442416 on the expression of glutamine synthetase and glutamate aspartate transporter in rat retinal M�ller cells at elevated hydrostatic pressure

The aim of this study was to investigate the effect of an adenosine A2A receptor antagonist on the expression of glutamine synthetase (GS) and glutamate aspartate transporter (GLAST) in rat retinal Müller cells at elevated hydrostatic pressure in vitro. Immunofluorescence staining of GS and GFAP was used for the identification of Müller cells. The expression of GS and GLAST in different hydrostatic pressure (0, 20, 40, 60, 80mmHg/24h) was examined by real-time PCR and Western blotting to identify the most suitable pressure. Müller cells treated with 0.1, 1, 10 µM SCH 442416 (A2A receptor antagonist) in the most suitable pressure, and the levels of GS and GLAST were examined by real-time PCR and Western blotting. Significantly increased expression of GS and GLAST at 40 mmHg pressure was observed in Müller cells and treatment with 10µM SCH 442416 in 40 mmHg pressure further promoted the expression of GS and GLAST. A2A receptor antagonist increased the expression of GLAST and GS of Müller cells and accelerated the clearance of extracellular glutamate.

Neuroprotective effect of transcorneal electrical stimulation on ischemic damage in the rat retina

Some previous studies have showed that transcorneal electrical stimulation (TES) could protect retinal neurons in certain rodent models. However, it is not yet clear whether TES could also definitely protect retinal neurons against ischemic insults. In the present study, we hypothesized that TES had such a neuroprotective effect and further investigated its underlying mechanism. Adult female Sprague–Dawley (SD) rats received TES treatment every other day after ocular ischemia was induced by elevating the intraocular pressure to 120mmHg for 60min. Retinal ganglion cells (RGCs) were labeled retrogradely 7 days before ischemia and were counted 7 and 14 days later. At the same time points, retinal function was assessed by scotopic electroretinography (ERG), combined with retinal histological analysis. The glutamine synthetase (GS) immunoreactivity was compared between ischemic retinas with TES and those with sham stimulation under identical confocal laser microscope conditions. The immunohistochemical indications were confirmed by Western blot analysis. Higher mean density of RGCs was quantified in TES treated retinas compared to retinas with sham stimulation on days 7 and 14 after ischemia. Similarly, histological analysis showed that TES better preserved the mean thickness of separate retinal layers. ERG studies indicated that by undergoing TES treatment, the b-wave amplitude was also significantly preserved on day 7 after ischemia and recovered robustly on day 14. Immunohistochemical and Western blot analysis both revealed that GS levels remarkably increased after TES and lasted for at least 7 days. Our results indicate that TES can protect retinal neurons against ischemic insults, probably related to increasing levels of GS localized in Müller cells. These findings suggest a new approach for potential clinical application to ocular ischemic diseases.

Architecture of the hypothalamo-posthypophyseal complex is controlled by monoamines

The hypothalamo-neurohypophyseal system displays significant plasticity when subjected to physiological stimuli, such as dehydration, parturition, or lactation. This plasticity arises at the neurochemical and electrophysiological levels but also at a structural level. Several studies have demonstrated the role of monoaminergic afferents in controlling neurochemical and electrophysiological plasticity of the supraoptic nucleus (SON) and of the neurohypophysis (NH), but little is known about how the changes in structural plasticity are triggered. We used Tg8 mice, disrupted for the monoamine oxidase A gene, to study monamine involvement in the architecture of the SON and of the NH. SON astrocytes in Tg8 mice displayed an active status, characterized by an increase in S100β expression and a significant decrease in vimentin expression, with no modification in glial fibrillary acidic protein (GFAP) levels. Astrocytes showed a decrease in glutamate dehydrogenase (GDH) levels, whereas glutamine synthetase (GS) levels remained constant, suggesting a reduction in astrocyte glutamate catabolism. Tenascin C and polysialic acid-neural cell adhesion molecule (PSA-NCAM) expressions were also elevated in the SON of Tg8 mice, suggesting an increased capacity for structural remodelling in the SON. In the NH, similar date were obtained with a stability in GFAP expression and an increase in PSA-NCAM immunostaining. These results establish monoamine (serotonin and noradrenaline) involvement in SON and NH structural arrangement. Monoamines therefore appear to be crucial for the coordination of the neurochemical and structural aspects of neuroendocrine plasticity, allowing the hypothalamo-neurohypopyseal system to respond appropriately when stimulated.

Effect of Pigment Epithelium Derived Factor on the Expression of Glutamine Synthetase in Early Phase of Experimental Diabetic Retinopathy

Purpose: A predominant function of Müller cells is to regulate glutamate levels, but in diabetic retinopathy (DR) the function is compromised. The present study was performed to investigate the role of pigment epithelial-derived factor (PEDF) on the expression of glutamine synthetase (GS) in retina of diabetic rats.Methods: The levels of interleukin-1ß (IL-1ß), PEDF, and GS in the retina of diabetic rats were analyzed by Western blotting and real-time-RT-PCR, and glutamate concentrations in retina or vitreous body were determined by high-pressure liquid chromatography. After being treated with PEDF in diabetic rats, these proteins (IL-1ß and GS) and their mRNAs, as well as glutamate concentrations, were detected. To confirm the effect of PEDF on GS against the role of IL-1ß, treatments with IL-1ß companied with or without PEDF were performed in the eyes of normal rats.Results: Diabetes increased IL-1ß levels and decreased PEDF and GS levels in retina. Simultaneously, diabetes induced elevated glutamate concentrations in retina and vitreous body. Administration of PEDF ameliorated the characteristic changes in diabetic retinopathy. Moreover, the effect of IL-1ß on the expression of GS was inhibited by PEDF in retina of normal rats.Conclusions: PEDF increases expression of GS against the effect of IL-1ß in early diabetic retinopathy. These findings suggest that PEDF may act as an anti-inflammatory factor against decrease of GS expression in retinal Müller cells in diabetic retinopathy.

Pre-Conditioning Induces the Precocious Differentiation of Neonatal Astrocytes to Enhance Their Neuroprotective Properties

Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia–ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O2). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)-positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.