Viable Astrocytes Research Articles

G6PDi-1 is a Potent Inhibitor of G6PDH and of Pentose Phosphate pathway-dependent Metabolic Processes in Cultured Primary Astrocytes

Glucose-6-phosphate dehydrogenase (G6PDH) catalyses the rate limiting first step of the oxidative part of the pentose phosphate pathway (PPP), which has a crucial function in providing NADPH for antioxidative defence and reductive biosyntheses. To explore the potential of the new G6PDH inhibitor G6PDi-1 to affect astrocytic metabolism, we investigated the consequences of an application of G6PDi-1 to cultured primary rat astrocytes. G6PDi-1 efficiently inhibited G6PDH activity in lysates of astrocyte cultures. Half-maximal inhibition was observed for 100 nM G6PDi-1, while presence of almost 10 µM of the frequently used G6PDH inhibitor dehydroepiandrosterone was needed to inhibit G6PDH in cell lysates by 50%. Application of G6PDi-1 in concentrations of up to 100 µM to astrocytes in culture for up to 6 h did not affect cell viability nor cellular glucose consumption, lactate production, basal glutathione (GSH) export or the high basal cellular ratio of GSH to glutathione disulfide (GSSG). In contrast, G6PDi-1 drastically affected astrocytic pathways that depend on the PPP-mediated supply of NADPH, such as the NAD(P)H quinone oxidoreductase (NQO1)-mediated WST1 reduction and the glutathione reductase-mediated regeneration of GSH from GSSG. These metabolic pathways were lowered by G6PDi-1 in a concentration-dependent manner in viable astrocytes with half-maximal effects observed for concentrations between 3 and 6 µM. The data presented demonstrate that G6PDi-1 efficiently inhibits the activity of astrocytic G6PDH and impairs specifically those metabolic processes that depend on the PPP-mediated regeneration of NADPH in cultured astrocytes.

Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes

Brain astrocytes are considered as glycolytic cell type, but these cells also produce ATP via mitochondrial oxidative phosphorylation. Exposure of cultured primary astrocytes in a glucose-free medium to extracellular substrates that are known to be metabolised by mitochondrial pathways, including pyruvate, lactate, beta-hydroxybutyrate, alanine and acetate, revealed that among the substrates investigated extracellular pyruvate was most efficiently consumed by astrocytes. Extracellular pyruvate was consumed by the cells almost proportional to time over hours in a concentration-dependent manner with apparent Michaelis–Menten kinetics [Km = 0.6 ± 0.1 mM, Vmax = 5.1 ± 0.8 nmol/(min × mg protein)]. The astrocytic consumption of pyruvate was strongly impaired in the presence of the monocarboxylate transporter 1 (MCT1) inhibitor AR-C155858 or by application of a 10-times excess of the MCT1 substrates lactate or beta-hydroxybutyrate. Pyruvate consumption by viable astrocytes was inhibited in the presence of UK5099, an inhibitor of the mitochondrial pyruvate carrier, or after application of the respiratory chain inhibitor antimycin A. In contrast, the mitochondrial uncoupler BAM15 strongly accelerated cellular pyruvate consumption. Lactate and alanine accounted after 3 h of incubation with pyruvate for around 60% and 10%, respectively, of the pyruvate consumed by the cells. These results demonstrate that consumption of extracellular pyruvate by astrocytes involves uptake via MCT1 and that the velocity of pyruvate consumption is strongly modified by substances that affect the entry of pyruvate into mitochondria or the activity of mitochondrial respiration.

Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes

BackgroundThe crosstalk and reactivity of the cell type glia, especially microglia and astrocytes, have progressively gathered research attention in understanding proper brain function regulated by the innate immune response. Therefore, methods to isolate highly viable and pure glia for the analysis on a cell-specific level are indispensable. New methodWe modified previously established techniques: Animal numbers were reduced by multiple microglial harvests from the same mixed glial culture, thereby maximizing microglial yields following the principles of the 3Rs (replacement, reduction, and refinement). We optimized Magnetic-activated cell sorting (MACS®) of microglia and astrocytes by applying cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspension. ResultsWe generated highly viable and pure microglia and astrocytes derived from a single mixed culture with a purity of ~99%, as confirmed by FACS analysis. Field emission scanning electron microscopy (FESEM) demonstrated integrity of the MACS-purified glial cells. Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) ELISA confirmed pro- and anti-inflammatory responses to be functional in purified glia, but significantly weakened compared to non-purified cells, further highlighting the importance of cellular crosstalk for proper immune activation. Comparison with existing method(s)Unlike previous studies that either isolated a single type of glia or displayed a substantial proportion of contamination with other cell types, we achieved isolation of both microglia and astrocytes at an increased purity (99–100%). ConclusionsWe have created an optimized protocol for the efficient purification of both primary microglia and astrocytes. Our results clearly demonstrate the importance of purity in glial cell cultivation in order to examine immune responses, which particularly holds true for astrocytes. We propose the novel protocol as a tool to investigate the cell type-specific crosstalk between microglia and astrocytes in the frame of CNS diseases.

The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism.

The reduction of water-soluble tetrazolium salts (WSTs) is frequently used to determine the metabolic integrity and the viability of cultured cells. Recently, we have reported that the electron cycler menadione can efficiently connect intracellular oxidation reactions in cultured astrocytes with the extracellular reduction of WST1 and that this menadione cycling reaction involves an enzyme. The enzymatic reaction involved in the menadione-dependent WST1 reduction was found strongly enriched in the cytosolic fraction of cultured astrocytes and is able to efficiently use both NADH and NADPH as electron donors. In addition, the reaction was highly sensitive towards dicoumarol with Kic values in the low nanomolar range, suggesting that the NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyzes the menadione-dependent WST1 reduction in astrocytes. Also, in intact astrocytes, dicoumarol inhibited the menadione-dependent WST1 reduction in a concentration-dependent manner with half-maximal inhibition observed at around 50 nM. Moreover, the menadione-dependent WST1 reduction by viable astrocytes was strongly affected by the availability of glucose. In the absence of glucose only residual WST1 reduction was observed, while a concentration-dependent increase in WST1 reduction was found during a 30 min incubation with maximal WST1 reduction already determined in the presence of 0.5 mM glucose. Mannose could fully replace glucose as substrate for astrocytic WST1 reduction, while other hexoses, lactate and the mitochondrial substrate β-hydroxybutyrate failed to provide electrons for the cell-dependent WST1 reduction. These results demonstrate that the menadione-mediated WST1 reduction involves cytosolic NQO1 activity and that this process is strongly affected by the availability of glucose as metabolic substrate.

Microglia Sculpt Sex Differences in Social Behavior

Microglia are increasingly recognized as developmental sculptors of neural circuits. In this issue of Neuron, VanRyzin etal. (2019) demonstrate a novel mechanism by which endocannabinoids drive microglia to phagocytose newborn astrocytes in the medial amygdala of male rats, promoting sex differences in social play behavior.

Alginate Hydrogel Modified with a Ligand Interacting with α3β1 Integrin Receptor Promotes the Differentiation of 3D Neural Spheroids toward Oligodendrocytes in Vitro.

In this study, we established a long-term three-dimensional (3D) culture system by using integrin ligand modified alginate hydrogels to encapsulate and differentiate neural progenitor cells (NPCs) toward oligodendrocyte (OL) lineage cells. The porosity of the hydrogel was optimized by varying the alginate concentrations and then characterized by scanning electronic microscopy (SEM). The surface plasmon resonance (SPR) test was used to confirm the ligand-integrin interactions indicating adherence between the NPC surfaces and the hydrogels. Following encapsulation in the hydrogels, both mouse and human NPC sphere cultures could be maintained up to 90 days. Mouse NPC spheres were differentiated into viable neurons, astrocytes and mature OLs by day 60 in all groups whereas human NPC spheres were differentiated into neurons and later into GFAP positive astrocytes and O4 positive pre-OL within 90 days. The species difference in the timeline of OL development between mouse and human was reflected in this system. The ligand LXY30 interacting with the α3β1 integrin receptor was more effective in promoting the differentiation of hNPCs to OL lineage cells compared with the ligand LXW64 interacting with the αvβ3 integrin receptor, hyaluronic acid interacting with CD44 receptor or without any ligand. This study is the first to differentiate O4+ pre-OLs from hNPCs in a LXY30-α3β1 (integrin-ligand) modified alginate 3D hydrogel culture. This 3D platform could serve as a valuable tool in disease modeling, drug discovery, and NPC transplantation.

Abstract 5197: Single-cell analysis of the brain metastatic microenvironment in breast cancer

Abstract Breast cancer brain metastasis affects 10-20% of patients, and there are no effective treatments. Activated infiltrating microglia and astrocytes are a major component of metastatic breast tumors in the brain, but it is not known whether these cells are involved in the promotion or rejection of breast cancer cells. To characterize the brain metastatic microenvironment, we used an FACS-based approach to capture pure and viable microglia and astrocytes from normal and metastatic mouse brains for single-cell mRNA sequencing analysis. Using this approach, we have captured more than 10,000 microglia and astrocytes from normal and metastatic brains. Applying our analysis pipelines to this data revealed distinct pathways and markers associated with microglia and astrocytes in metastatic brains. Future work will aim to identify spatial locations and function of specific microglia and astrocyte subpopulations in the metastatic brain. Citation Format: Katrina Evans, Quy Nguyen, Kerrigan Blake, Devon Lawson. Single-cell analysis of the brain metastatic microenvironment in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5197.

Simulated blast overpressure induces specific astrocyte injury in an ex vivo brain slice model.

Exposure to explosive blasts can produce functional debilitation in the absence of brain pathology detectable at the scale of current diagnostic imaging. Transient (ms) overpressure components of the primary blast wave are considered to be potentially damaging to the brain. Astrocytes participate in neuronal metabolic maintenance, blood–brain barrier, regulation of homeostatic environment, and tissue remodeling. Damage to astrocytes via direct physical forces has the potential to disrupt local and global functioning of neuronal tissue. Using an ex vivo brain slice model, we tested the hypothesis that viable astrocytes within the slice could be injured simply by transit of a single blast wave consisting of overpressure alone. A polymer split Hopkinson pressure bar (PSHPB) system was adapted to impart a single positive pressure transient with a comparable magnitude to those that might be present inside the head. A custom built test chamber housing the brain tissue slice incorporated revised design elements to reduce fluid space and promote transit of a uniform planar waveform. Confocal microscopy, stereology, and morphometry of glial fibrillary acidic protein (GFAP) immunoreactivity revealed that two distinct astrocyte injury profiles were identified across a 4 hr post-test survival interval: (a) presumed conventional astrogliosis characterized by enhanced GFAP immunofluorescence intensity without significant change in tissue area fraction and (b) a process comparable to clasmatodendrosis, an autophagic degradation of distal processes that has not been previously associated with blast induced neurotrauma. Analysis of astrocyte branching revealed early, sustained, and progressive differences distinct from the effects of slice incubation absent overpressure testing. Astrocyte vulnerability to overpressure transients indicates a potential for significant involvement in brain blast pathology and emergent dysfunction. The testing platform can isolate overpressure injury phenomena to provide novel insight on physical and biological mechanisms.

Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes.

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.

Respiration-Deficient Astrocytes Survive As Glycolytic Cells In Vivo.

Neurons and glial cells exchange energy-rich metabolites and it has been suggested, originally based on in vitro data, that astrocytes provide lactate to glutamatergic synapses ("lactate shuttle"). Here, we have studied astrocytes that lack mitochondrial respiration in vitro and in vivo A novel mouse mutant (GLASTCreERT2::Cox10flox/flox) was generated, in which the administration of tamoxifen causes mutant astrocytes to fail in the assembly of mitochondrial cytochrome c oxidase (COX). Focusing on cerebellar Bergmann glia (BG) cells, which exhibit the highest rate of Cre-mediated recombination, we found a normal density of viable astrocytes even 1 year after tamoxifen-induced Cox10 gene targeting. Our data show that BG cells, and presumably all astrocytes, can survive by aerobic glycolysis for an extended period of time in the absence of glial pathology or unspecific signs of neurodegeneration.SIGNIFICANCE STATEMENT When astrocytes are placed into culture, they import glucose and release lactate, an energy-rich metabolite readily metabolized by neurons. This observation led to the "glia-to-neuron lactate shuttle hypothesis," but in vivo evidence for this hypothesis is weak. To study astroglial energy metabolism and the directionality of lactate flux, we generated conditional Cox10 mouse mutants lacking mitochondrial respiration in astrocytes, which forces these cells to survive by aerobic glycolysis. Here, we report that these mice are fully viable in the absence of any signs of glial or neuronal loss, suggesting that astrocytes are naturally glycolytic cells.

Consequences of a Chronic Exposure of Cultured Brain Astrocytes to the Anti-Retroviral Drug Efavirenz and its Primary Metabolite 8-Hydroxy Efavirenz.

Efavirenz is a widely prescribed non-nucleoside reverse transcriptase inhibitor for the treatment of HIV infections. To test for potential long-term consequences of efavirenz on brain cells, cultured primary astrocytes were incubated with this substance or with its primary metabolite 8-hydroxy efavirenz for up to 7days. Both, efavirenz and 8-hydroxy efavirenz caused time- and concentration-dependent cell toxicity and stimulated in subtoxic concentrations the glycolytic flux (glucose consumption and lactate release) in astrocytes. As 8-hydroxy efavirenz was less toxic than efavirenz and stimulated glycolysis in lower concentrations we tested for a potential hydroxylation of efavirenz to 8-hydroxy efavirenz in astrocytes. Analysis of media and cell lysates by HPLC-UV and mass spectrometry revealed that after 3days of incubation viable astrocytes had accumulated about 17 and 7 % of the applied efavirenz and 8-hydroxy efavirenz, respectively. However, in cultures treated with efavirenz neither 8-hydroxy efavirenz nor any other known metabolite of efavirenz was detectable. These data demonstrate that cultured rat astrocytes efficiently accumulate, but not metabolize, efavirenz and 8-hydroxy efavirenz and that the observed chronic stimulation of glycolysis is mediated by both efavirenz and 8-hydroxy efavirenz.

Impact of acquired and innate immunity on spinal cord ischemia and reperfusion injury

The aim of this study was to clarify the impact of acquired and innate immunity on spinal cord ischemia and reperfusion injury using a mouse model of spinal cord ischemia. To define the ischemic duration that caused paraplegia, wild-type and severe combined immunodeficiency (SCID) mice were subjected to cross-clamping of the aorta for 7, 9, 9.5, or 10.5 min with ischemic preconditioning for intestinal protection. In wild-type and SCID mice with paraplegia, histological analyses were performed to investigate viable neurons, inflammatory cells, and reactive astrocytes at 12, 24, 48, and 72 h as well as 7 days after reperfusion. In both wild-type and SCID mice, immediate paraplegia was induced by occlusion for 10.5 min. In both wild-type and SCID mice, no infiltration of T or B lymphocytes was observed at any point after reperfusion, but reactive astrocytes were clearly visible at 7 days after reperfusion, and the number of activated microglia peaked at 12 and 48 h after reperfusion. Although there was no significant difference, wild-type mice had a tendency to have more activated microglia than SCID mice at 12 h after reperfusion, and to have less viable neurons than SCID mice at 12, 24, 48, and 72 h after reperfusion. There was a tendency that the frequency of immediate paraplegia in wild-type mice was more than SCID mice though no statistical difference was observed. Innate immunity, rather than acquired immunity, may be involved in the developing immediate paraplegia in our mouse model.

Copper oxide nanoparticles stimulate glycolytic flux and increase the cellular contents of glutathione and metallothioneins in cultured astrocytes.

Copper oxide nanoparticles (CuO-NPs) are frequently used for industrial or medical applications and are known for their high toxic potential. As little is known so far on the consequences of an exposure of brain cells to such particles, we applied CuO-NPs to cultured primary rat astrocytes and investigated whether such particles affect cell viability and alter their metabolic properties. Astrocytes efficiently accumulated CuO-NPs in a time- and concentration-dependent manner. The cells remained viable during a 24 h incubation with 100 µM copper in the form of CuO-NPs, while higher concentrations of CuO-NPs severely compromised the cell viability. Astrocytes that were exposed for 24 h to 100 µM CuO-NPs showed significantly enhanced extracellular lactate concentrations and increased cellular levels of glutathione and metallothioneins. The CuO-NP-induced increase in lactate release and metallothionein content were prevented by the presence of the membrane-permeable copper chelator tetrathiomolybdate, while this chelator increased already in the absence of CuO-NPs the cellular glutathione content. After removal of the CuO-NPs following a 24 h pre-incubation with 100 µM CuO-NPs, astrocytes maintained during a further 6 h incubation an elevated glycolytic lactate release and exported significantly more glutathione than control cells that had been pre-incubated without CuO-NPs. These data suggest that copper ions which are liberated from internalized CuO-NPs stimulate glycolytic flux as well as the synthesis of glutathione and metallothioneins in cultured viable astrocytes.

Reactive Changes in the Cellular Elements of the Rat Brain in Different Conditions of Circulatory Hypoxia

The aim of the present work was to identify structural, spatial, and quantitative changes in elements of the paranigral nucleus (PNN) of the midbrain and anterior cingulate field (ACF) of the telencephalon in different conditions of circulatory hypoxia. The anterior medial part of the PNN and layers V–VI of the ACF were studied in healthy rats seven days (n = 4) after occlusion of both common carotid arteries and in intact (control 1, n = 4) and sham-operated (control 2, n = 4) rats. Histological sections stained with cresyl violet by the Nissl method and processed for detection of glial fibrillary acidic protein and Iba1 protein were used to identify the ratio of essentially unchanged, hypochromic, pyknomorphic, and ghost neurons, along with the numbers of astrocytes, oligodendrocytes, microgliocytes, and endotheliocytes. The areas of neurons and gliocyte bodies were measured, along with the distances from these cells to capillary walls, and gliocyte:neuron indexes were calculated. The results showed that after exposure to different conditions of circulatory hypoxia, the cellular elements of the brain showed a spectrum of characteristic pathological changes. Neurons showed signs of nuclear pyknosis, lysis, and conversion to “ghost” cells. Cells in the nuclear zone of hypoxia tended to undergo death or pyknosis. Neurons located outside the hypoxic area and exposed only to the humoral actions of the responses of the glutamate-calcium cascade often showed acute swelling. The reactions of microgliocytes, with minor increases in numbers and structural signs of activation, represented early diffuse signs of focal hypoxia in the forebrain. Endotheliocyte proliferation seen at seven days of ischemia was not associated with the chain of cascade reactions, but occurred only in the focus of hypoxia. The concentrations of viable neurons and astrocytes close to blood capillaries and the increase in the number of satellite types of glial cells represent an adaptive mechanism and a constitute a condition for cell survival in the conditions of various types of cerebral ischemia.

Uptake and toxicity of arsenite and arsenate in cultured brain astrocytes

Inorganic arsenicals are environmental toxins that have been connected with neuropathies and impaired cognitive functions. To investigate whether such substances accumulate in brain astrocytes and affect their viability and glutathione metabolism, we have exposed cultured primary astrocytes to arsenite or arsenate. Both arsenicals compromised the cell viability of astrocytes in a time- and concentration-dependent manner. However, the early onset of cell toxicity in arsenite-treated astrocytes revealed the higher toxic potential of arsenite compared with arsenate. The concentrations of arsenite and arsenate that caused within 24h half-maximal release of the cytosolic enzyme lactate dehydrogenase were around 0.3mM and 10mM, respectively. The cellular arsenic contents of astrocytes increased rapidly upon exposure to arsenite or arsenate and reached after 4h of incubation almost constant steady state levels. These levels were about 3-times higher in astrocytes that had been exposed to a given concentration of arsenite compared with the respective arsenate condition. Analysis of the intracellular arsenic species revealed that almost exclusively arsenite was present in viable astrocytes that had been exposed to either arsenate or arsenite. The emerging toxicity of arsenite 4h after exposure was accompanied by a loss in cellular total glutathione and by an increase in the cellular glutathione disulfide content. These data suggest that the high arsenite content of astrocytes that had been exposed to inorganic arsenicals causes an increase in the ratio of glutathione disulfide to glutathione which contributes to the toxic potential of these substances.

8-Hydroxy-efavirenz, the Primary Metabolite of the Antiretroviral Drug Efavirenz, Stimulates the Glycolytic Flux in Cultured Rat Astrocytes

In active antiretroviral therapy antiretroviral drugs are employed for the restoration of a functional immune system in patients suffering from the acquired immunodeficiency syndrome. However, potential adverse effects of such compounds to brain cells are discussed in connection with the development of neurocognitive impairments in patients. To investigate potential effects of antiretroviral drugs on cell viability and the glycolytic flux of brain cells, astrocyte-rich primary cultures were exposed to various antiretroviral compounds, including the non-nucleoside reverse transcriptase inhibitor efavirenz. In a concentration of 10μM, neither efavirenz nor any of the other investigated antiretroviral compounds acutely compromised the cell viability nor altered glucose consumption or lactate production. In contrast, the primary metabolite of efavirenz, 8-hydroxy-efavirenz, stimulated the glycolytic flux in viable astrocytes in a time- and concentration-dependent manner with half-maximal and maximal effects at concentrations of 5 and 10μM, respectively. The stimulation of glycolytic flux by 8-hydroxy-efavirenz was not additive to that obtained for astrocytes that were treated with the respiratory chain inhibitor rotenone and was abolished by removal of extracellular 8-hydroxy-efavirenz. In a concentration of 10μM, 8-hydroxy-efavirenz and efavirenz did not affect mitochondrial respiration, while both compounds lowered in a concentration of 60μM significantly the oxygen consumption by mitochondria that had been isolated form cultured astrocytes, suggesting that the stimulation of glycolytic flux by 8-hydroxy-efavrienz is not caused by direct inhibition of respiration. The observed alteration of astrocytic glucose metabolism by 8-hydroxy-efavirenz could contribute to the adverse neurological side effects reported for patients that are chronically treated with efavirenz-containing medications.

Acute isolation and transcriptome characterization of cortical astrocytes and microglia from young and aged mice

Astrocytes and microglia become reactive in many neurological disorders resulting in phenotypic and functional alterations. Both cell types might also display functional changes during normal aging. To identify gene signatures and changes in basal cellular functions of astrocytes and microglia in relation to aging, we isolated viable astrocytes and microglia from young adult and aged mouse cortices and determined their gene expression profile. Aged astrocytes, compared with young astrocytes, showed an increased inflammatory phenotype and increased ‘zinc ion binding.’ Young astrocytes showed higher expression of genes involved in ‘neuronal differentiation’ and hemoglobin synthesis. Astrocyte expression of genes involved in neuronal signaling remains high throughout age. Aged microglia had higher expression of genes involved in ‘vesicle release,’ ‘zinc ion binding,’ and genes within the tumor necrosis factor-ligand family and young microglia had increased transcript levels of C-C motif chemokines. These data provide a transcriptome database of cell-type enriched genes of astrocytes and microglia from adult mice and give insight into the differential gene signature of astrocytes and microglia in relation to normal aging.

Characterization of Arsenate Uptake by Cultured Primary Rat Astrocytes

Arsenate is known to be accumulated by cultured astrocytes and to stimulate astrocytic glutathione export, but the arsenate uptake into astrocytes has not been characterized so far. To address this topic, we have exposed primary rat astrocyte cultures to arsenate and determined the cellular arsenic content by atomic absorption spectroscopy. Viable astrocytes accumulated arsenate in a time- and concentration-dependent manner. Their cellular arsenic content increased almost proportional with time for up to 60min after application of arsenate. Analysis of the concentration-dependent increase in the specific arsenic content of the cells after 30min of arsenate exposure revealed that cultured astrocytes take up arsenate with saturable kinetics by a transport process that has apparent KM- and Vmax-values of 1.7±0.2mM and 28±4nmol/(mg protein×30min), respectively. Arsenate uptake in viable astrocytes was strongly inhibited by the presence of phosphate or by lowering the incubation temperature to 4°C and was completely abolished in a sodium ion-free medium. These results strongly suggest that the saturable temperature-dependent arsenate uptake into astrocytes is mediated by a sodium-dependent phosphate cotransporter.

Arsenate accumulation and arsenate-induced glutathione export in astrocyte-rich primary cultures

Arsenate is a toxic compound that has been connected with neuropathies and impaired cognitive functions. To test whether arsenate affects the viability and the GSH metabolism of brain astrocytes, we have used primary astrocyte cultures as model system. Incubation of astrocytes for 2h with arsenate in concentrations of up to 10mM caused an almost linear increase in the cellular arsenic content, but did not acutely compromise cell viability. The presence of moderate concentrations of arsenate caused a time- and concentration-dependent loss of GSH from viable astrocytes which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for arsenate in a concentration of about 0.3mM. The arsenate-induced stimulated GSH export from astrocytes was prevented by MK571, an inhibitor of the multidrug resistance protein 1. Exposure of astrocytes to arsenite increased the specific cellular arsenic content and stimulated GSH export to values that were similar to those observed for arsenate-treated cells, while dimethylarsinic acid was less efficiently accumulated by the cells and did not modulate cellular and extracellular GSH levels. The observed strong stimulation of GSH export from astrocytes by arsenate suggests that disturbances of the astrocytic GSH metabolism may contribute to the observed arsenic-induced neurotoxicity.

Fast astrocyte isolation by sedimentation field flow fractionation

Astrocytes play a key role during central nervous system (CNS) repair and glial scar formation. After CNS damage, an extensive deposition of the extracellular matrix produced by the activated astrocytes limits the extension of the lesion but impairs axon outgrowth and functional recovery. Until now, methods to obtain astrocytes need long culture period and laborious cell culture conditions and do not allow the isolation of pure astrocyte preparation. In this study, we used sedimentation field flow fractionation (SdFFF) to rapidly sort well preserved astrocyte population. Four main cell fractions, the total eluted population (TP), and fractions F1, F2, and F3, were isolated by SdFFF from rat newborn cortex. After elution, cells were cultured for one week, and analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (GFAP), O4, β-III tubulin, and CD 68, labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. SdFFF eluted cells were compared with the cells obtained with the classical method. Results showed that SdFFF appeared to be a rapid (one week) and effective method to sort enriched populations of viable and functional astrocytes. In particular, F1 and F3 fractions contained high percentage of GFAP expressing cells (95.6% and 98.0%, respectively). Results also showed that F1 derived cell cultures contained large astrocytes that spread in the culture dish while in fraction F3 derived cell cultures, astrocytes were small, showing a tendency to aggregate and displaying higher migratory capacities than those of fraction F1. Thanks to SdFFF, isolation of almost pure astrocyte populations was rapidly obtained. In addition, the isolation of different astrocyte subpopulations showing different behaviors offers a new perspective to better understand the glial scar formation and remodeling after CNS damage.