Cultured Brain Astrocytes Research Articles

Vitamin D3 Inhibits Phagocytic Activity of Rat Brain Astrocytes in Primary Culture

Vitamin D3 Inhibits Phagocytic Activity of Rat Brain Astrocytes in Primary Culture

ВЛИЯНИЕ ОКИСЛИТЕЛЬНОГО СТРЕССА НА ПРОЦЕСС ФАГОЦИТОЗA АПОПТОТИЧЕСКИХ СУБСТРАТОВ АСТРОЦИТАМИ МОЗГА КРЫС

Brain astrocytes are commonly believed to be relatively tolerant to oxidative stress. In this work, we studied the effect of oxidative stress on the ability of rat brain astrocytes in primary culture to ingest and phagocytose apoptotic substrates. Using fluorescent microscopy, we demonstrated that the ingestion of rat retinal photoreceptor outer segments (POS), used here as a phagocytic substrate, dramatically decreased in astrocytes preincubated with various hydroperoxides. The ability of primary astrocytes to ingest POS also depended on the substrate oxidative status. The ingestion of “oxidized” POS preparations preincubated with hydrogen peroxide was sharply reduced compared to “non-oxidized” POS. We also showed that natural and synthetic antioxidants ( α -tocopherol, Trolox) efficiently protected the phagocytic function of astrocytes from oxidative damage. These data suggest that the phagocytic function of rat brain primary astrocytes is extremely vulnerable to oxidative stress, while oxidative stress tolerance characterizes not all functions of these glial 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.

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.

Brain astrocyte‐derived EETs and 20‐EHTE elicit opposing actions on calcium movement and KCa channel current activities in astrocytes

Cultured brain astrocytes incubated with arachidonic acid (AA) produce four CYP epoxygenase metabolites epoxyeicosatrienoic acids (EETs) 5,6‐, 8,9‐, 1,12‐and 14,15‐EET. The formation and action of 20‐hydroxyeicosatrienoic acid (20‐HETE) in brain astrocytes is not known. We investigated the capacity of brain astrocytes to express CYP‐4A‐ù‐hydroxylase protein and to convert AA to 20‐HETE. We also investigated the effects of 20‐HETE on membrane potential, KCa single‐channel activity, and on intracellular Ca2+ ([Ca2+]i) levels in cultured astrocytes. We found that astrocytes express CYP‐4A‐ù‐hydroxylase at the transcript and protein levels and produce 20‐HETE when incubated with AA. Treatment of astrocytes with 20‐HETE (100 nM) inhibited the openings of KCa single‐channel currents in cell‐attached patches and caused astrocyte membrane potential depolarization, whereas it had no effect on [Ca2+]i levels in astrocytes loaded with the Ca2+ probe Fura‐2AM. In contrast, treatment of cultured astrocytes with one of the EETs, 11.12‐EET increased [Ca2+]i level and activated astrocyte KCa channel currents. These findings indicate that the EETs and 20‐HETE produced and released by astrocytes could have opposing physiological actions at astrocyte end feet, which might be crucial for the maintenance of constant hyperemic cerebral blood flow during increased neuronal activation.

Abstract WP433: Haemostatic and Neuroprotective Effects of Xe-loaded Echogenic Liposomes in a Subarachnoid Hemorrhagic Stroke Model

Background: We have developed Xe-containing echogenic liposomes (Xe-ELIP). Using this delivery agent, we have demonstrated neuroprotective effects in an ischemic stroke model. Given the common mediators of cellular injury in ischemic and early-stage hemorrhagic stroke, the goal of this study was to evaluate the neuroprotective effects of Xe-ELIP on early brain injury after subarachnoid hemorrhage (SAH). Methods: Xe-ELIP was prepared by a pressurization-freeze method. The neuroprotective effects of Xe-ELIP on cultured brain astrocytes and microglia were evaluated by a lactate dehydrogenase (LDH) assay. For in vivo evaluation, rats (n=35) were randomly divided into sham, SAH, SAH with Xe-ELIP, ELIP treatment, and Xe saturated saline treatment groups. SAH was induced by an endovascular puncture method. Treatments were administrated intravenously in combination with ultrasound application over the common carotid artery to trigger Xe release from circulating Xe-ELIP at 30 minutes after SAH. Mortality assessments, neurological evaluations, and behavioral tests were conducted for three days following surgery. Brain tissue was harvested for SAH grading, H&E staining, TUNEL staining, and brain water content measurement on the third day after surgery. Results: Treatment with Xe-ELIP protected brain astroacytes and microglia against H 2 O 2 (10 mM) induced cytotoxicity. SAH grading revealed effective reduction of bleeding in association with decreased mortality. Moreover, Xe-ELIP significantly reduced apoptotic neuronal death and improved both general neurological and motor function. Conclusions: Xe-ELIP alleviates early SAH brain injury by inhibiting neuronal death and bleeding. This novel delivery approach, using Xe-ELIP with ultrasound-controlled cerebral drug release, provides a relatively noninvasive strategy for therapeutic gas delivery in SAH.

The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood–brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 ± 2 to 107 ± 6 nm and the zeta-potential of the particles from −22 ± 5 to −9 ± 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

BackgroundMatrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Moreover, bradykinin (BK) induces the expression of several inflammatory proteins in brain astrocytes. Recent studies have suggested that increased oxidative stress is implicated in the brain inflammation and injury. However, whether BK induced MMP-9 expression mediated through oxidative stress remains virtually unknown. Herein we investigated the role of redox signals in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).ResultsIn the study, we first demonstrated that reactive oxygen species (ROS) plays a crucial role in BK-induced MMP-9 expression in cultured brain astrocytes (in vitro) and animal brain tissue (in vivo) models. Next, BK-induced MMP-9 expression is mediated through a Ca2+-mediated PKC-α linking to p47phox/NADPH oxidase 2 (Nox2)/ROS signaling pathway. Nox2-dependent ROS generation led to activation and up-regulation of the downstream transcriptional factor AP-1 (i.e. c-Fos and c-Jun), which bound to MMP-9 promoter region, and thereby turned on transcription of MMP-9 gene. Functionally, BK-induced MMP-9 expression enhanced astrocytic migration.ConclusionsThese results demonstrated that in RBA-1 cells, activation of AP-1 (c-Fos/c-Jun) by the PKC-α-mediated Nox2/ROS signals is essential for up-regulation of MMP-9 and cell migration enhanced by BK.

Ferritin up-regulation and transient ROS production in cultured brain astrocytes after loading with iron oxide nanoparticles

To investigate the cellular consequences of a prolonged cellular presence of large amounts of iron oxide nanoparticles (IONPs) as well as the fate of such particles in brain cells, cultured primary astrocytes were loaded for 4h with dimercaptosuccinate-coated IONPs. Subsequently, the IONP-treated cells were incubated for up to 7days in IONP-free medium and the cell viability, metabolic parameters and iron metabolism of the cells were investigated. Despite an up to 100-fold elevated specific cellular iron content, IONP-loaded cells remained viable throughout the 7day main incubation and did not show any substantial alteration in glucose and glutathione metabolism. During the incubation, the high cellular iron content of IONP-loaded astrocytes remained almost constant. Electron microscopy revealed that after 7days of incubation most of the cellular iron was still present in IONP-filled vesicles. However, the transient appearance of reactive oxygen species (ROS) as well as a strong increase in cellular levels of the iron storage protein ferritin suggest that at least some low-molecular-weight iron was liberated from the accumulated IONPs. These results demonstrate that even the prolonged presence of large amounts of accumulated IONPs does not harm astrocytes and that these cells store IONP-derived iron in ferritin.

Survivin prevents apoptosis by binding to caspase-3 in astrocytes infected with the BeAn strain of Theiler’s murine encephalomyelitis virus

This paper reports the upregulation of the gene coding for the apoptosis regulator family member "surviving" in SJL/J mouse brain astrocyte cultures infected with the BeAn strain of TMEV. cRNA from mock- and TMEV-infected SJL/J astrocytes was hybridised to an Affymetrix whole murine genome DNA microarray. Analysis revealed the upregulation of two sequences coding for the survivin protein in infected cells; this was confirmed by RT-PCR and qPCR. Western blotting showed an increase in the synthesis of survivin and caspase-3 after infection. Unexpectedly, no enzymatic activity was detected in BeAn-infected cell lysates in caspase-3-specific colorimetric assays. Cross-linking experiments showed survivin and caspase-3 to exist as a complex containing one molecule of caspase-3 (17 kDa) and one of either 16 kDa or 14 kDa survivin. The neutralization of caspase-3 by survivin-containing lysates was demonstrated using recombinant caspase-3. Brains from TMEV-infected mice, but not from naïve mice, contained survivin mRNA during the acute phase of encephalitis. The present results suggest that astrocytes infected by the BeAn strain do not undergo apoptosis due to the production of survivin.

Valproic Acid Enhances Glucose Transport in the Cultured Brain Astrocytes of Glucose Transporter 1 Heterozygous Mice

Glucose transporter 1 facilitates glucose transport across the blood-brain barrier. By increasing histone acetylation at the SLC2A1 promotor, valproic acid could increase SLC2A1 gene expression. This study was designed to evaluate the effects of valproic acid on glucose transport in astrocyte cultures derived from SLC2A1 heterozygous mice. Primary astrocyte cultures were prepared from the cerebral cortex of 1-day-old neonatal mice. Cultured astrocytes were incubated with valproic acid (0.05, 0.5, and 5 mM) for 48 hours. On day 3, the glucose uptake capacity of the astrocytes was measured by using (14)C-2-Deoxy-d-glucose under zero-trans conditions. The heterozygous astrocyte glucose uptake treated with valproic acid (0.05 and 0.5 mM) for 48 hours was significantly increased compared with the untreated control heterozygous astrocytes. Our findings demonstrate that valproic acid increased glucose transport capacity in SLC2A1 heterozygous cerebral astrocytes.

The antiretroviral protease inhibitors indinavir and nelfinavir stimulate Mrp1‐mediated GSH export from cultured brain astrocytes

Combinations of antiretroviral drugs are successfully used for the treatment of acquired immune deficiency syndrome and reduce the incidence of severe human immunodeficiency virus (HIV)-associated dementia. To test whether such drugs affect the GSH metabolism of brain cells, we have exposed astrocyte-rich primary cultures to various antiretroviral compounds. Treatment of the cultures with the protease inhibitors indinavir or nelfinavir in low micromolar concentrations resulted in a time- and concentration-dependent depletion of cellular GSH from viable cells which was accompanied by a matching increase in the extracellular GSH content. In contrast, the reverse transcriptase inhibitors zidovudine, lamivudine, efavirenz or nevirapine did not alter cellular or extracellular GSH levels. Removal of indinavir from the medium by washing the cells terminated the stimulated GSH export immediately, while the nelfinavir-induced accelerated GSH export was maintained even after removal of nelfinavir. The stimulation of the GSH export from viable astrocytes by indinavir or nelfinavir was completely prevented by the application of MK571, an inhibitor of the multidrug resistance protein 1. These data demonstrate that indinavir and nelfinavir stimulate multidrug resistance protein 1-mediated GSH export from viable astrocytes and suggest that treatment of patients with such inhibitors may affect the GSH homeostasis in brain.

Magnetic field‐induced acceleration of the accumulation of magnetic iron oxide nanoparticles by cultured brain astrocytes

Magnetic iron oxide nanoparticles (Fe-NPs) are considered for various biomedical and neurobiological applications that involve the presence of external magnetic fields. However, little is known on the effects of a magnetic field on the uptake of such particles by brain cells. Cultured brain astrocytes accumulated dimercaptosuccinate-coated Fe-NP in a time-, temperature-, and concentration-dependent manner. This accumulation was strongly enhanced by the presence of the magnetic field generated by a permanent neodymium iron boron magnet that had been positioned below the cells. The magnetic field-induced acceleration of the accumulation of Fe-NP increased almost proportional to the strength of the magnetic field applied, increasing the cellular-specific iron content from an initial 10 nmol/mg protein within 4 h of incubation at 37°C to up to 12,000 nmol/mg protein. However, presence of a magnetic field also increased the amounts of iron that attached to the cells during incubation with Fe-NP at 4°C. These results suggest that the presence of an external magnetic field promotes in cultured astrocytes both the binding of Fe-NP to the cell membrane and the internalization of Fe-NP.

Accumulation of silver nanoparticles by cultured primary brain astrocytes

Silver nanoparticles (AgNP) are components of various food industry products and arefrequently used for medical equipment and materials. Although such particles enter thevertebrate brain, little is known on their biocompatibility for brain cells. To study theconsequences of an AgNP exposure of brain cells we have treated astrocyte-richprimary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubationof cultured astrocytes with micromolar concentrations of AgNP for up to 24 hresulted in a time- and concentration-dependent accumulation of silver, but didnot compromise the cell viability nor lower the cellular glutathione content. Incontrast, the incubation of astrocytes for 4 h with identical amounts of silver asAgNO3 already severely compromised the cell viability and completely deprived the cells ofglutathione. The accumulation of AgNP by astrocytes was proportional to theconcentration of AgNP applied and significantly lowered by about 30% in thepresence of the endocytosis inhibitors chloroquine or amiloride. Incubation at4 °C reducedthe accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed toAgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulatePVP-coated AgNP in a temperature-dependent process that most likely involvesendocytotic pathways.

Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brainastrocytes

Magnetic iron oxide nanoparticles (Fe-NP) are currently considered for variousdiagnostic and therapeutic applications in the brain. However, little is known onthe accumulation and biocompatibility of such particles in brain cells. We havesynthesized and characterized dimercaptosuccinic acid (DMSA) coated Fe-NPand have investigated their uptake by cultured brain astrocytes. DMSA-coatedFe-NP that were dispersed in physiological medium had an average hydrodynamicdiameter of about 60 nm. Incubation of cultured astrocytes with these Fe-NPcaused a time- and concentration-dependent accumulation of cellular iron, but didnot lead within 6 h to any cell toxicity. After 4 h of incubation with 100–4000 µM iron supplied as Fe-NP, the cellular iron content reached levels between 200 and 2000 nmol mg − 1 protein. The cellular iron content after exposure of astrocytes to Fe-NP at4 °C was drastically lowered compared to cells that had been incubated at37 °C. Electronmicroscopy revealed the presence of Fe-NP-containing vesicles in cells that were incubated with Fe-NPat 37 °C, but not in cells exposed to the nanoparticles at4 °C. These data demonstrate that cultured astrocytes efficiently take up DMSA-coated Fe-NPin a process that appears to be saturable and strongly depends on the incubationtemperature.

Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88‐dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways

There is increasing evidence that astrocytes play important roles in immune regulation in the brain. Astrocytes express toll-like receptors (TLR) and build up responses to innate immune triggers by releasing proinflammatory molecules. We investigate signaling pathways and released molecules after astrocyte TLR4 activation. Purified rodent brain astrocyte cultures were treated with the TLR4 activator bacterial lipopolysaccharide (LPS). Tools used to interfere with this system include small interference RNA, inhibitory drugs, and MyD88 or Stat1 deficient mice. LPS induced early activation of the transcription factor NFκB, through the MyD88 adaptor, and expression of TNF-α, VCAM-1, IL-15, and IL-27. LPS also induced delayed Jak1/Stat1 activation, which was MyD88-independent but was not mediated by IFN-β. Jak1/Stat1 activation induced the expression of negative cytokine regulator SOCS-1 and CXCL10 chemokine (IP-10). Mitogen-activated protein kinases (MAPK) were also involved in TLR4 signaling in a MyD88-independent fashion. p38 exerted a strong influence on LPS-induced gene expression by regulating the phosphorylation of Stat1 and the transcriptional activity of NFκB, while JNK regulated the Jak1/Stat1 pathway, and ERK1/2 controlled the expression of Egr-1 and influenced MyD88-dependent MMP-9 expression. Interplay between these signals was evidenced by the increased induction of MMP-9 in Stat1-deficient cells challenged with LPS, suggesting that Stat1 negatively regulates the expression of MMP-9 induced by LPS. Therefore, astrocytes are responsive to TLR4 activation by inducing a complex set of cell-dependent molecular reactions mediated by NFκB, MAPK and Jak1/Stat1 signaling pathways. Here we identified cross-talking signals generating a proinflammatory environment that will modulate the response of surrounding cells.

Advanced Biomaterials Accumulation of Citrate‐Coated Magnetic Iron Oxide Nanoparticles by Cultured Brain Astrocytes

AbstractMagnetic iron oxide nanoparticles (Fe‐NP) are considered for various applications in the brain. However, little is known so far on the uptake and the metabolism of such nanoparticles in brain cells. Since astrocytes are strategically localized between capillaries and neurons, astrocytes are of particular interest concerning uptake and fate of nanoparticles in the brain. Using astrocyte‐rich primary cultures as model system we have investigated the accumulation of citrate‐coated Fe‐NP by astrocytes. Viable cultured astrocytes accumulate iron from citrate‐coated Fe‐NP in a time‐, concentration‐, and temperature‐dependent manner. The cellular iron content determined after 4 h of incubation increases proportional to the concentration of Fe‐NP, if the particles were applied in concentrations of up to 1000 × 10−6 M of total iron. The iron accumulation from 500 or 1000 × 10−6 M iron as Fe‐NP is significantly slowed by lowering the incubation temperature from 37 to 4 °C. Transmission electron microscopy of the cells revealed that most of the cellular Fe‐NP are present in intracellular vesicles. These data demonstrate that astrocytes accumulate efficiently citrate‐coated Fe‐NP, most likely by an endocytotic pathway.

Interferon-α/β Genes Are Up-Regulated in Murine Brain Astrocytes After Infection With Theiler’s Murine Encephalomyelitis Virus

This article reports the production of interferon alpha/beta (IFN-alpha/beta) by SJL/J mouse brain astrocyte cultures infected with Theiler's murine encephalomyelitis virus (TMEV). cRNA from mock- and TMEV-infected SJL/J astrocytes was hybridized to the Affymetrix whole murine genome DNA microarray. Analysis revealed the up-regulation of 3 sequences coding for the IFN-alpha/beta domain. Increased expression of mRNA coding for IFN-alpha was shown by conventional RT-PCR and quantitative real-time RT-PCR. According to ELISA, the concentration of IFN-alpha in the supernatants of infected astrocyte cultures varied with the multiplicity of infection and post-infection time. The IFN-alpha/beta secreted was biologically active, as shown by a virus-based IFN bioassay involving Cocal virus and TMEV infection. The contribution to total interferon activity was 29% +/- 3.0% for IFN-alpha and 52% +/- 3.6% for IFN-beta. IFN-alpha/beta was induced by whole TMEV virions; induction was not achieved with either purified isolated virion capsid proteins or UV-inactivated virus. Further, induction was inhibited by specific anti-TMEV antibodies. The receptor for IFN-alpha/beta, which is absent in uninfected astrocytes, was up-regulated after infection, as suggested by DNA hybridization analysis. The brains of infected mice contained IFN-alpha/beta mRNA during the acute encephalitis phase, peaking at day 5 post-infection. Our findings could have significance for human diseases such as viral encephalitis and multiple sclerosis.

Accumulation of Iron Oxide Nanoparticles by Cultured Brain Astrocytes

Magnetic iron oxide nanoparticles are considered for various diagnostic and therapeutic applications in brain including their use as contrast agent for magnetic resonance imaging or as tool for magnetic drug delivery. However, little is known so far on the consequences of a treatment of brain cells with such nanoparticles. In order to study the biocompatibility of iron oxide nanoparticles and the accumulation of iron from such particles in brain cells, we have used astrocyte-rich primary cultures as model system. Iron oxide nanoparticles were chemically synthesized with a yield of about 80% regarding the iron content. Transmission electron microscopy revealed that the synthesized iron oxide nanoparticles had a diameter of about 10 nm. Exposure of astrocyte cultures to 100 microM iron that were supplied as citrate-coated iron oxide nanoparticles did not cause any acute loss in cell viability but resulted in an almost linear increase in the cellular iron content that reached after 6 h of incubation at 37 degrees C a total cellular iron content of about 300 nmol/mg protein. The rate of iron accumulation from iron oxide nanoparticles was significantly higher than that from the low molecular weight iron complex ferric ammonium citrate (FAC). Lowering the incubation temperature from 37 degrees C to 4 degrees C reduced the iron accumulation rate from iron oxide nanoparticles or FAC by about 60%. In addition, presence of ferric or ferrous iron chelators did not affect the cellular iron accumulation from iron oxide nanoparticles. These data suggest that cultured astrocytes are able to accumulate intact iron oxide nanoparticles.

Mitochondrial glutathione uptake: characterization in isolated brain mitochondria and astrocytes in culture

Glutathione in the mitochondria is an important determinant of cellular responses to oxidative stress. Mitochondrial glutathione is maintained by uptake from the cytosol, a process that has been little studied in brain cells. In the present study, measurements using isolated rat brain mitochondria showed a rapid uptake of [3H]-glutathione that was strongly influenced by the mitochondrial glutathione content. [3H]-glutathione incorporated into the mitochondria was not rapidly released. Uptake was inhibited by substrates and inhibitors for several known mitochondrial anion transporters. Citrate, isocitrate and benzene-1,2,3-tricarboxylate were particularly effective inhibitors, suggesting a possible role for a tricarboxylate carrier in the glutathione transport. The properties of uptake differed greatly from those reported previously for mitochondria from kidney and liver. In astrocytes in primary culture, diethylmaleate or hydrogen peroxide treatment resulted in depletion of cytosolic and mitochondrial glutathione. The pattern of restoration of glutathione content in the presence of glutathione precursors following treatment with diethylmaleate was consistent with uptake into mitochondria being controlled primarily by the glutathione gradient between the cytosol and mitochondria. However, following hydrogen peroxide treatment, recovery of glutathione in the mitochondria initially preceded comparable proportional restoration in the cytosol, suggesting the possibility of additional controls on glutathione uptake in some conditions.