Susana Tchernin Wofchuk

Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology.

Primary astrocyte cultures prepared from neonatal hippocampus, cerebral cortex and cerebellum were morphologically distinct. Cells from hippocampus and cortex were almost entirely protoplasmic, whereas cerebellar astrocytes had many processes; in the absence of serum these differences were accentuated. We compared the immunocontent and secretion of the mitogenic protein S100B in these cultures. Immunocontent was 2.5 times higher in cerebellar astrocytes than in hippocampal or cortical astrocytes. Cells from all three regions secreted S100B under basal conditions, but the secretion rate was higher in cerebellar astrocytes. Secretion depended on protein synthesis and was increased by incubation with forskolin or lysophosphatidic acid in mechanisms which were additive. The stellate morphology induced by forskolin was reversed by lysophosphatidic acid in hippocampal but not in cerebellar cultures, suggesting that S100B secretion was not associated with a process‐bearing phenotype of astrocytes.

In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model

BackgroundATP is an important signalling molecule in the peripheral and central nervous system. Both glioma growth and tumor resection induces cell death, thus liberating nucleotides to the extracellular medium. Nucleotides are hydrolyzed very slowly by gliomas when compared with astrocytes and induce neuronal cell death and glioma proliferation. The objective of the present study was to test the involvement of extracellular ATP in glioblastoma growth in a rat glioma model.MethodsTo deplete the extracellular ATP, the enzyme apyrase was tested on the treatment of gliomas implanted in the rats CNS. One million glioma C6 cells in 3 microliters of DMEM/FCS were injected in the right striata of male Wistar rats, 250–270 g. After 20 days, the rats were decapitated and the brain sectioning and stained with hematoxylin and eosine. We performed immunohistochemical experiments with Ki67, CD31 and VEGF. Total RNA was isolated from cultured glioma C6 cells and the cDNA was analyzed by Real Time-PCR with primers for the NTPDase family.ResultsC6 glioma cells effectively have a low expression of all NTPDases investigated, in comparison with normal astrocytes. The implanted glioma co-injected with apyrase had a significant reduction in the tumor size (p < 0.05) when compared with the rats injected only with gliomas or with gliomas plus inactivated apyrase. According to the pathological analysis, the malignant gliomas induced by C6 injection and co-injected with apyrase presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. Reduction of proliferation induced by apyrase co-injection was confirmed by counting the percentage of Ki67 positive glioma cell nuclei. According to counts with CD31, vessel density and neoformation was higher in the C6 group 20 days after implantation. Confirming this observation, rats treated with apyrase presented less VEGF staining in comparison to the control group.ConclusionThese results indicate that the participation of extracellular ATP and the ecto-nucleotidases may be associated with the development of this type of brain tumor in an in vivo glioma model.

Ontogenetic profile of glutamate uptake in brain structures slices from rats: sensitivity to guanosine

The excitotoxicity of the neurotransmitter glutamate has been shown to be connected with many acute and chronic diseases of the CNS. High affinity sodium-dependent glutamate transporters play a key role in maintaining adequate levels of extracellular glutamate. In the present study, we used slices of striatum, hippocampus and cortex from rat brain to describe the in vitro profile of glutamate uptake during development and ageing, and its sensitivity to guanosine. In all structures, glutamate uptake was higher in immature animals. There was a maximum decrease in glutamate uptake in striatum and hippocampus in 15-month-old rats, which later increased, while in cortex there was a significant decrease in rats aged 60 days old. The effect of guanosine seems to be age and structure dependent since the increase in basal glutamate uptake was only seen in slices of cortex from 10-day-old animals.

Quinolinic acid promotes seizures and decreases glutamate uptake in young rats: reversal by orally administered guanosine

Quinolinic acid (QA) has been used as a model for experimental overstimulation of the glutamatergic system. Glutamate uptake is the main mechanism involved in the maintenance of extracellular glutamate below toxic levels. Guanosine systemically administered prevents quinolinic acid-induced seizures in adult mice and increases basal glutamate uptake by cortical astrocyte culture and slices from young rats. The immature brain differs from the adult brain in its susceptibility to seizures, seizure characteristics, and responses to antiepileptic drugs (AED). Here we investigated the effect of guanosine p.o. on QA-induced seizures in young rats (P12-14) and upon ex vivo glutamate uptake by cortical slices from these animals. I.c.v. infusion of 250 nmol QA induced seizures in all animals and decreased glutamate uptake. I.p. injection of MK-801 and phenobarbital 30 min before QA administration prevented seizures in all animals. Guanosine (7.5 mg/kg) 75 min before QA prevented seizures in 50% of animals as well as prevented the decrease of glutamate uptake in the protected animals. To investigate if the anticonvulsive effect of guanosine was specific for QA-induced seizures, the picrotoxin-induced seizures model was also performed. Pretreatment with phenobarbital i.p. (60 mg/kg-30 min) prevented picrotoxin-induced seizures in all animals, whereas guanosine p.o. (7.5 mg/kg-75 min) and MK-801 i.p. (0.5 mg/kg-30 min) had no effect. Thus, guanosine protection on the QA-induced seizures in young rats and on the decrease of glutamate uptake showed some specificity degree towards the QA-induced toxicity. This points that guanosine could be considered for treatments of epilepsy, and possibly other neurological disorders in children.

Glutamate uptake in cultured astrocytes depends on age: a study about the effect of guanosine and the sensitivity to oxidative stress induced by H2O2

Relatively few studies have been conducted to investigate the relationship between glutamate and development and/or aging. Rat cortical astrocyte cultures were used as a model to investigate glutamate uptake during development. The immunocontent of the markers glial fibrillary acidic protein (GFAP) and S100B increased, while basal secretion of S100B decreased, in astrocytes from 10 to 40 days in vitro (DIV). Basal glutamate uptake increased with age. Exposure to hydrogen peroxide decreased glutamate uptake more potently at 40 than 10 DIV. Moreover, 40 DIV astrocytes showed earlier loss of integrity (at 6 h) than 10 DIV astrocytes (at 24 h) after H(2)O(2) exposure. Addition of guanosine stimulated glutamate uptake only in 10 DIV astrocytes. The present work shows that mature astrocytes in culture present some neurochemical alterations also observed in astrocytes of aged animals. These results can contribute to the understanding of some consequences of the excitotoxicity and oxidative stress during brain aging.

Glutamate uptake is stimulated by extracellular S100B in hippocampal astrocytes

Summary1.S100B is a calcium-binding protein expressed and secreted by astrocytes, which has been implicated in glial-neuronal communication. Extracellular S100B appears to protect hippocampal neurons against toxic concentrations of glutamate. Here we investigated a possible autocrine role of S100B in glutamate uptake activity.2.Astrocyte cultures were prepared of hippocampi from neonate Wistar rats. [3H] Glutamate uptake was measured after addition of S100B protein, antibody anti-S100B or TRTK-12, a peptide that blocks S100B activity mediated by the C-terminal region.3.Antibody anti-S100B addition decreased glutamate uptake measured 30 min after medium replacement, without affecting cell integrity or viability. Moreover, low levels of S100B (less than 0.1 ng/mL) stimulated glutamate uptake measured immediately after medium replacement.4.This finding reinforces the importance of astrocytes in the glutamatergic transmission, particularly the role of S100B neuroprotection against excitotoxic damage.

Resveratrol protects against oxidative injury induced by H2O2 in acute hippocampal slice preparations from Wistar rats

There is a current interest in dietary compounds (such as trans-resveratrol) that can inhibit or reverse oxidative stress, the common pathway for a variety of brain disorders, including Alzheimers disease and stroke. The objective of the present study was to investigate the effects of resveratrol, under conditions of oxidative stress induced by H(2)O(2), on acute hippocampal slices from Wistar rats. Here, we evaluated cell viability, extracellular lactate, glutathione content, ERK(MAPK) activity, glutamate uptake and S100B secretion. Resveratrol did not change the decrease in lactate levels and in cell viability (by MTT assay) induced by 1mM H(2)O(2), but prevented the increase in cell permeability to Trypan blue induced by H(2)O(2). Moreover, resveratrol per se increased total glutathione levels and prevented the decrease in glutathione induced by 1mM H(2)O(2). The reduction of S100B secretion induced by H(2)O(2) was not changed by resveratrol. Glutamate uptake was decreased in the presence of 1mM H(2)O(2) and this effect was not prevented by resveratrol. There was also a significant activation of ERK1/2 by 1mM H(2)O(2) and resveratrol was able to completely prevent this activation, leading to activity values lower than control levels. The impairments in astrocyte activities, induced by H(2)O(2), confirmed the importance of these cells as targets for therapeutic strategy in brain disorders involving oxidative stress. This study reinforces the protective role of resveratrol and indicates some possible molecular sites of activity of this compound on glial cells, in the acute damage of brain tissue during oxidative stress.

Hypoxic-ischemic insult decreases glutamate uptake by hippocampal slices from neonatal rats: Prevention by guanosine

Brain injury secondary to hypoxic-ischemic disease is the predominant form of damage encountered in the perinatal period. The impact of neonatal hypoxia-ischemia (HI) in 7-day-old pups on the high-affinity [3H] glutamate uptake into hippocampal slices at different times after insult was examined. Immediately following, and 1 day after the insult there was no effect. But at 3 to 5 days after the HI insult, glutamate uptake into the hippocampus was markedly reduced; however, after 30 or 60 days the glutamate uptake into hippocampal slices returned to control levels. Also, this study demonstrated the effect of the nucleoside guanosine (Guo) on the [3H] glutamate uptake in neonatal HI injury, maintaining the [3H] glutamate uptake at control levels when injected before and after insult HI. We conclude that neonatal HI influences glutamate uptake a few days following insult, and that guanosine prevents this action.

Guanine nucleotides inhibit the stimulation of GFAP phosphorylation by glutamate.

Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by guanine nucleotides. Recent work has shown that guanine nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that guanine nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.

Regulation of protein phosphorylation in astrocyte cultures by external calcium ions: specific effects on the phosphorylation of glial fibrillary acidic protein (GFAP), vimentin and heat shock protein 27 (HSP27)

The effect of external Ca2+ ([Ca2+]e) on the incorporation of [32P] into total protein, cytoskeletal proteins and the heat shock protein HSP27, was studied in primary cultures of astrocytes from the rat hippocampus. Zero [Ca2+]e increased total 32P-incorporation into astrocyte protein and when this was normalized to 100%, incorporation was significantly increased into glial fibrillary acidic protein (GFAP), vimentin (VIM) and HSP27. The difference in total 32P-incorporation between zero [Ca2+]e and 1 mM [Ca2+]e was reversed by incubation of the cells with the protein phosphatase inhibitor okadaic acid in the range 1-10 nM; higher concentrations of okadaic acid (50-100 nM) further increased total 32P-incorporation. In zero [Ca2+]e the non-specific channel blocker Co2+ (1 mM) decreased total 32P-incorporation by approximately 30%. The results were compared with a previous study [S.T. Wofchuk, R. Rodnight, Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res. 85 (1995) 181-186] in which it was shown that in immature hippocampal slices zero [Ca2+]e compared with 1 mM [Ca2+]e increased 32P-incorporation into GFAP without changing total incorporation. The difference between the results for total 32P-incorporation obtained in cultured astrocytes and immature brain tissue was found to be related to the concentration of [Ca2+]e in the medium since in slices concentrations of [Ca2+]e higher than 1 mM progressively decreased total incorporation. The difference may reflect a higher Ca2+-permeability of the plasma membrane in cultured astrocytes and/or to the complex structure of the slice tissue. In two-dimensional electrophoresis HSP27, in contrast to GFAP and VIM, was separated into 3 immunodetectable isoforms only two of which were normally phosphorylated. After labelling in the presence of okadaic acid both immunodetectable and phosphorylated HSP27 focussed as a single polypeptide. Phorbol dibutyrate (1 microM) and zero [Ca2+]e stimulated the phosphorylation of both isoforms, but in the case of zero [Ca2+]e the effect on the more acidic isoform was proportionally greater.