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Izhodišča: Zastrupitev z ogljikovim monoksidom (CO) močno okrne funkcijo astrocitov in nevronov. Pozne nevropsihološke posledice zastrupitve lahko preprečimo z zdravljenjem s hiperbaričnim kisikom (HBO). V raziskavi smo preučevali učinek CO in HBO na zgodnje procese celične smrti v nevronski in mešani kulturi ter ugotavljali, ali se raven glutationa v astrocitih po izpostavljenosti CO in HBO spremeni in ali bi lahko le-ta bil možna nova tarča za zdravljenje zastrupitve s CO. Metode: Primarne astrocitne, nevronske in mešane celične kulture možganske skorje podgane smo izpostavili 3.000 ppm CO v zraku, nato pa jih v obdobju 24-urne normoksije v različnih časovnih presledkih za 1 uro izpostavili 100-odstotnemu kisiku pri tlaku 3 barov. V celicah mešane in nevronske kulture smo merili aktivnost laktat dehidrogenaze (LDH) in kaspaz 3/7, v astrocitih pa raven glutationa. Rezultati: CO je povzročil zvišanje aktivnosti LDH in kaspaz 3/7 v nevronskih kulturah, v mešanih pa le zvišanje aktivnosti kaspaz 3/7. Po izpostavitvi CO in HBO se je zvišala aktivnost LDH v nevronskih kulturah in znižala aktivnost kaspaz 3/7 v mešanih kulturah. CO je v astrocitih povzročil znižanje celokupnega glutationa (GSHt), zvišanje glutation disulfida (GSSG) in znižanje GSH/GSSG, po izpostavitvi CO in HBO pa se je zvišala GSHt, znižala GSSG in zvišala GSH/GSSG. Zaključek: Razlike v citotoksičnem delovanju CO in zaščitni vlogi HBO v nevronski, mešani in astrocitni kulturi kažejo, da so nevroni, ki rastejo brez astrocitov, v primerjavi z mešano kulturo dovzetnejši za škodljive učinke CO ter nakazujejo, da astrociti ob oksidativnem stresu poskušajo ščititi nevrone, ki so pri sintezi glutationa odvisni od njih.

Background Astrocytes maintain central nerve system homeostasis and are relatively resistant to cell death. Dysfunction of cell death mechanisms may underlie glioblastoma genesis and resistance to cancer therapy; therefore more detailed understanding of astrocytic death modalities is needed in order to design effective therapy. The purpose of this study was to determine the effect of VAS2870, a pan-NADPH oxidase inhibitor, on staurosporine-induced cell death in astrocytes. Materials and methods Cultured rat astrocytes were treated with staurosporine as activator of cell death. Cell viability, production of reactive oxygen species (ROS), and mitochondrial potential were examined using flow cytometric analysis, while chemiluminescence analysis was performed to assess caspase 3/7 activity and cellular ATP. Results We show here for the first time, that VAS2870 is able to prevent staurosporine-induced cell death. Staurosporine exerts its toxic effect through increased generation of ROS, while VAS2870 reduces the level of ROS. Further, VAS2870 partially restores mitochondrial inner membrane potential and level of ATP in staurosporine treated cells. Conclusions Staurosporine induces cell death in cultured rat astrocytes through oxidative stress. Generation of ROS, mitochondrial membrane potential and energy level are sensitive to VAS2870, which suggests NADPH oxidases as an important effector of cell death. Consequently, NADPH oxidases activation pathway could be an important target to modulate astrocytic death.

Astrocytes support the brain through numerous functional interactions in health and disease. The recent advances in our knowledge of astrocyte involvement in various neurological disorders raised up several questions about their role and functioning in the central nervous system. From the evidence discussed in this review, we show that histamine importantly influences the main astrocytic activities such as ion homeostasis, energy metabolism, neurotransmitter clearance, neurotrophic activity and immune response. These processes are mediated through at least three histamine receptor subtypes, H1, H2 and H3, expressed on the astrocyte surface. Thus, we recognize histamine as an important player in the modulation of astrocytic functions that deserves further considerations in exploring involvement of astrocytes in neurological disorders.

In astrocytes, carbon monoxide (CO) poisoning causes oxidative stress and mitochondrial dysfunction accompanied by caspase and calpain activation. Impairment in astrocyte function can be time-dependently reduced by hyperbaric (3bar) oxygen (HBO). Due to the central role of astrocytes in maintaining neuronal function by offering neurotrophic support we investigated the hypothesis that HBO therapy may exert beneficial effect on acute CO poisoning-induced impairment in intrinsic neurotrophic activity. Exposure to 3000ppm CO in air followed by 24-72h of normoxia caused a progressive decline of gene expression, synthesis and secretion of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) to different extent. 1h treatment with 100% oxygen disclosed a pressure- and time-dependent efficacy in preserving astrocytic neurotrophic support. The beneficial effect was most evident when the astrocytes were exposed to HBO 1-5h after exposure to CO. The results further support an active role of hyperbaric, not normobaric, oxygenation in reducing dysfunction of astrocytes after acute CO poisoning. By preserving endogenous neurotrophic activity HBO therapy might promote neuronal protection and thus prevent the occurrence of late neuropsychological sequelae.

Background Metrifonate is an inhibitor of acetylcholinesterase (AChE). Several studies confirmed its positive effects on cognitive impairment in Alzheimer's disease but it was due to adverse events withdrawn from clinical trials. Based on the importance of astrocytes in physiological and pathological brain activities we investigated the impact of metrifonate and, for comparison, acetylcholine on intrinsic neurotrophic activity in these cells. Methods Metabolic activity, intracellular adenosine 5′-triphosphate (ATP) levels and lactate dehydrogenase (LDH) release was measured to examine the impact of metrifonate on viability and integrity of cultured rat cortical astrocytes. The influence of metrifonate, acetylcholine and selective cholinergic ligands on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) synthesis and secretion was determined by specific two-site enzyme immunoassays. Results Exposure of cultured astrocytes to metrifonate displayed no toxic effects on cell viability. Metrifonate and acetylcholine potently and transiently elevated NGF and BDNF, but not NT-3, protein levels and secretion with different intensity and time frame of their maximal response. Stimulatory effect on NGF was mimicked by selective nicotinic receptor agonist nicotine and completely blocked by nicotinic antagonist mecamylamine. The impact on BDNF synthesis was mimicked by muscarinic receptor agonist pilocarpine and abolished by selective muscarinic antagonist scopolamine. Conclusions Metrifonate up-regulates astrocytic NGF and BDNF synthesis in the same manner as acetylcholine, their effect depends on different cholinergic pathways. These results suggest a trophic role of metrifonate, based on a well-known neurotrophic activity of NGF and BDNF in vivo.

Interactions involved in the regulation of nerve growth factor (NGF) release by inflammatory cytokines: interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) were examined in rat neonatal cortical astrocytes in primary culture. Abstract Exposure of cultured astrocytes to IL-1β, IL-6, and TGF-β1 resulted in the stimulation of NGF secretion. Treatment of cells for 24 h with IL-1β (10 U/ml), IL-6 (5 ng/ml) and TGF-β1 (5 ng/ml) caused 3-, 1.8-, and 2.8-fold increase in NGF secretion as compared to the control cells. In contrast, TNF-α (30 ng/ml) by itself had no stimulatory action on NGF release whereas co-stimulation of astrocytes with IL-1β and TNF-α showed a synergistic interaction. Co-treatment of astrocytes with IL-1β and TGF-β1 increased NGF secretion in an additive way, whereas simultaneous application of IL-1β and IL-6 resulted in the inhibitory effect on NGF secretion. Our results suggest that interactions between cytokines used cause the stimulation of NGF secretion through different regulatory mechanisms.

Microcystins (MCs) comprise a group of cyanobacterial toxins with hepatotoxic, nephrotoxic and, possibly, neurotoxic activity in mammals. In order to understand the development of their neurotoxicity we investigated the toxic effects of MC variants, MC-LR, MC-LW and MC-LF, in astrocytes that play a central role in maintaining brain homeostasis. 24h exposure of cultured rat cortical astrocytes to MCs revealed dose-dependent toxicity of MC-LF and MC-LW, but not of MC-LR, observed by significant reduction in cell number, declined viability monitored by MTT test and an increased percentage of apoptotic cells, confirmed by Annexin-V labelling. The cultured astrocytes expressed organic anion-transporting polypeptides (Oatp) Oatp1a4, Oatp1c1 and Oatp1a5, but not Oatp1b2. Intracellular localisation of MC-LF and MC-LW, proven by anti-Adda primary antibody, demonstrated transport of tested MCs into cultured astrocytes. Acute MC-LW and MC-LF intoxication induced cytoskeletal disruption as seen by the degradation of glial fibrillary acid protein (GFAP), actin and the tubulin network. In this in vitro study, MC-LF and MC-LW, but not MC-LR, are shown to cause the dysfunction of astrocytic homeostatic capabilities, already at low concentrations, suggesting that astrocyte atrophy, with loss of function, could be expected in the brain response to the toxic insult.

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.

Astrocytes as an active part of the tripartite synapse can respond to the synaptically released neurotransmitters. Because brain-derived neurotrophic factor (BDNF) is produced by astrocytes, in addition to neurons, we focused our present study on the regulatory effects of monoamines noradrenaline (NA), serotonin (5-HT), and dopamine (DA) on the synthesis of BDNF protein in rat neonatal astrocytes from specific brain regions (cortex, cerebellum). All tested neurotransmitters are able to potently and transiently increase BDNF cellular contents; their maximal effects are dose and time dependent and differ between the two brain regions. In cultured cortical astrocytes, NA (1 microM; 6 h) elevates BDNF levels by a 4-fold, 5-HT (1 microM; 4 h) by a 2.3-fold, and DA (150 microM; 4 h) by a 2.2-fold. In cerebellar astrocytes, NA (1 microM; 4 h) increases BDNF content by a 4.7-fold, 5-HT (1 microM; 4 h) by a 1.7-fold, and DA (150 microM; 4 h) by a 1.4-fold. The initial increase in the BDNF levels return to basal levels when incubation with monoamines is extended beyond 12 h (for 5-HT) or 24 h (for NA and DA). Our results confirm the involvement of monoaminergic systems in the regulation of BDNF production in astrocytes and suggest the existence of a positive reciprocal interaction between monoaminergic neuronal activity and astrocytic neurotrophic support in neuron-astrocyte crosstalk, which has a dynamic role in mediating neuronal plasticity and trophic functions in the brain.

Carbon monoxide (CO) poisoning causes neuronal and glial apoptosis that can result in delayed neurological symptoms. The damage of brain cells can be prevented by oxygen therapy. Based on the central role of astrocytes in maintaining neuronal function and viability we investigated the toxic effects of 3000ppm CO in air followed by 24h of normoxia and evaluated the possible protective influence of 100% normobaric oxygen or 100% oxygen at a pressure of 3bar (hyperbaric) against CO poisoning in these cells. CO/normoxia caused a progressive decline of viability, increase in reactive oxygen species and decline of mitochondrial membrane potential and intracellular ATP levels in cultured rat astrocytes. Increased caspase-9, caspase-8 and calpain activity converged in activation of caspase-3/7. 1h treatment with oxygen disclosed pressure- and time-dependent efficacy in restoring astrocytic mitochondrial function and the prevention of apoptosis. The protective effect was most evident when the astrocytes were exposed to hyperbaric oxygen, but not normobaric oxygen, 1-5h after exposure to CO.

Recently we reported that cultured rat cortical astrocytes express histamine H 3 receptor that is functionally coupled to G i/o proteins and participates to the stimulatory effect of histamine. Due to the lack of data on the distribution of histamine H 3 receptors on glial cells we further investigated their presence in cultured astrocytes from different brain regions. Real-time PCR was performed to examine the expression of native histamine H 3 receptor in cultured rat astrocytes from cortex, cerebellum, hippocampus and striatum. Double-antigen immunofluorescence staining and [ 3 H]N-α-methylhistamine ([ 3 H]NαMH) binding studies were utilized to specifically identify and characterize receptor binding sites in astrocytes. Histamine H 3 receptor mRNA was detected in rat astrocytes from all the regions under investigation with the highest levels in striatal astrocytes followed by hippocampal astrocytes and approximately equal levels in cerebellar and cortical astrocytes. Double-antigen immunofluorescence confirmed the presence of histamine H 3 receptors on the membrane of all examined astroglial populations. [ 3 H]NαMH bound with high affinity and specificity to an apparently single class of saturable sites on cortical astrocytic membranes ( K D =4.55±0.46 nM; B max =5.63±0.21 fmol/mg protein) and competition assays with selective agonists and antagonists were consistent with labeling of histamine H 3 receptor (range of p K i values 7.50–8.87). Our study confirmed the ability of cultured astrocytes from different rat brain regions to express histamine H 3 receptors. The observed diverse distribution of the receptors within various astrocytic populations possibly mirrors their heterogeneity in the brain and indicates their active involvement in histamine-mediated effects.

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1 beta (IL-1 beta). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1 alpha and IL-1 beta) and the antagonist (IL-1ra)-members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [(125)I] IL-1 beta to cultured astrocytes was saturable and of high affinity. Mean values for the K(D) and B(max) were calculated to be 60.7+/-7.4 pM and 2.5+/-0.1 fmol mg(-1) protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1 alpha (K(i) of 341.1 pM) and IL-1 beta (K(i) 59.9 pM), as well as the antagonist IL-1ra (K(i) 257.6 pM), displaced specific [(125)I] IL-1 beta binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [(125)I] IL-1 beta binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1 alpha and IL-1 beta revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1 beta (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1 alpha by itself had no stimulatory action on NGF release. The simultaneous application of IL-1 alpha and IL-1 beta elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1 beta. We concluded that IL-1 beta-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1 alpha and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.

Neurotrophin-3 (NT-3) is produced by astrocytes, in addition to neurons, and monoamine neurotransmitters play a role in controlling NT-3 synthesis. The impact of histamine (HA) on the regulation of NT-3 synthesis in cultured astrocytes has not been studied. We evaluated the involvement of histamine receptors and intracellular mechanisms in the regulation of NT-3 production by HA. Real-time PCR was performed to examine the expression of all known histamine receptor subtypes in cultured rat cortical astrocytes. Pharmacological tools, selective for the H1, H2 and H3 receptors and intracellular systems, were utilized to confirm functional properties of HA receptors in histaminergic up-regulation of astrocytic NT-3 synthesis. HA potently and transiently elevated NT-3 expression and protein levels by more than twofold. In addition to H1 and H2 receptors, cultured astrocytes also express H3 receptors, which activate Gi/o proteins to inhibit adenylyl cyclase and modulate MAP kinase activity. Histaminergic stimulation was partly inhibited by selective H1, H2, and H3 antagonists whereas selective H1, H2, and H3 agonists or mediators of the intracellular histaminergic pathways increased NT-3 levels. Inhibitors of PKA, PKC, and CaMK II significantly reduced the HA-induced increase in NT-3 cellular levels whereas the MAP kinase cascade inhibitor completely blocked the stimulatory action of HA and all selective agonists. In conclusion, the synthesis of astrocytic NT-3 stimulated by HA is a receptor-mediated process, which is fine-tuned via subtle modulation of parallel histaminergic H1, H2, and H3 pathways that converge at the level of MAP kinase activity. This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.

The cytokine interleukin-1 (IL-1) is one of the key mediators in the body’s response to microbial invasion, inflammation, immunological reactions and tissue injury (1) and also acts as a growth factor for a variety of cells (2). In the brain, two major sources of IL-1 are neurons and glial cells (3). Administration of IL-1 produces a variety of brain-mediated effects which are presumed to be induced by binding of IL-1 to specific sites with high affinity for type I receptor binding. This binding takes place on the surface of neuronal and non-neuronal (glial and endothelial) cells in the CNS (4), while IL-1 signal transduction occurs through different second messenger pathways that further regulate transcription factors (5,6).