Biomedical subjects

Bozena Gabryel

Publications and source records attributed to Bozena Gabryel.

13 recordsLinked to original sources

PubMed2006

Calcineurin and Erk1/2-signaling pathways are involved in the antiapoptotic effect of cyclosporin A on astrocytes exposed to simulated ischemia in vitro.

The present study focused on mechanisms involved in the anti-apoptotic effect of cyclosporin A (CsA) towards ischemic injured astrocytes in vitro [under combined oxygen glucose deprivation (OGD)]. We investigated whether this action might be mediated through activation of extracellular signal regulated kinases 1 and 2 (Erk1/2) or attenuation of calcineurin (CaN) by immunosuppressant in ischemic astrocytes. Additionally, the influence of CsA on phosphorylation of Akt kinase was determined. After 21 days of in vitro culture, astrocytes were subjected to OGD (for 8 h) and CsA (0.25-10 microM); 0.25 microM CsA distinctly stimulated the Erk1/2 pathway in astrocytes exposed to OGD. This protective effect of CsA was strongly associated with CaN inhibition, increased expression of anti-apoptotic factors such as Bcl-X(L) and NF-kappaB, as well as suppression of caspase-3 activity. Maximum p-Akt kinase expression was observed following treatment with 1 microM CsA. Finally, we also demonstrated that the beneficial effect of CsA at a concentration of 10 microM is related mainly to strong CaN inhibition. The results obtained suggest that, depending on the concentration used, CsA might act as a protective agent towards ischemia-injured astroglial cells through alternative intracellular pathways associated with increased p-Erk1/2 and p-Akt expression or CaN inactivation.

Androstadienes↗

PubMed2006

Inhibition of arachidonic acid release by cytosolic phospholipase A2 is involved in the antiapoptotic effect of FK506 and cyclosporin a on astrocytes exposed to simulated ischemia in vitro.

In the present study, we investigated whether the protective effect of FK506 and cyclosporin A (CsA) against in vitro ischemic injury of astrocytes might be mediated through attenuation of cytosolic isoform of phospholipase A(2) (cPLA(2)) expression and activity as well as inhibition of arachidonic acid (AA) release. On the 21st day in vitro, cultures of rat astrocytes were subjected to ischemia-simulating conditions (combined oxygen glucose deprivation) for 8 h and exposed to FK506 (10 - 1,000 nM) and CsA (0.25 - 10 microM). Obtained data suggest the cross-talk between the action of 0.25 - 10 microM CsA as well as 1 microM FK506 on calcineurin (CaN) and cPLA(2) in anti-apoptotic signal transduction pathways. Moreover, we have shown that immunosuppressants at these concentrations protected glial cells against ischemia-induced apoptosis through the increase of cell viability, mitochondrial function restoration, and attenuation of oxidative stress. Finally, in our study, low concentrations of FK506 (10 and 100 nM) exerted limited effects on the assessed parameters. Our findings document a key role either for CaN or cPLA(2) expression attenuation and AA release inhibition in the antiapoptotic effect of FK506 and CsA in ischemic astrocytes.

Animals↗

PubMed2005

Chlorpromazine and loxapine reduce interleukin-1beta and interleukin-2 release by rat mixed glial and microglial cell cultures.

The cytokines IL-1beta and IL-2 are released from activated glial cells in the central nervous system and they are able to enhance catecholaminergic neurotransmission. There is no data concerning influence of antipsychotics on glial cell activity. Antipsychotics reaching the brain act not only on neurons but probably also on glial cells. The aim of this study was to evaluate the effect of chlorpromazine and loxapine on release of IL-1beta and IL-2 by mixed glial and microglial cell cultures. Chlorpromazine in concentrations 2 and 20 muM, and loxapine 0.2, 2 and 20 microM reduced IL-1beta secretion by LPS-activated mixed glia cultures after 1 and 3 days of exposure. Chlorpromazine in concentrations of 0.2, 2 and 20 microM reduced the IL-2 secretion in mixed glial cultures after 3 days of exposure. Loxapine in concentrations of 0.2, 2 and 20 microM reduced IL-2 secretion in mixed glia cultures after 1 and 3 days of exposure, and additionally loxapine decreased IL-1beta and IL-2 secretion in LPS-induced microglia cultures in concentrations of 2, 10 and 20 muM. Quinpirole-a D2 dopaminergic agonist increased LPS-induced IL-1beta and IL-2 secretion in mixed glia cultures only in the highest dose of 20 microM. These findings suggest the absence of functional dopamine receptors on cortical microglial cells. Mixed glia cultures deprived of microglia (by shaking and incubating with L-leucine methyl ester) did not release IL-1beta and IL-2. This observation suggests that microglia can be a source of assessed cytokines. Results of the present study support the view that antipsychotics act not only on neurons but also on glial cells. However, the clinical significance of these observations still remains unclear.

Analysis of Variance↗

PubMed2005

Immunosuppressive immunophilin ligands attenuate damage in cultured rat astrocytes depleted of glutathione and exposed to simulated ischemia in vitro. Comparison with N-acetylcysteine.

The aim of the present study was to test the hypothesis that exposure of astrocytes depleted of glutathione (GSH) to simulated ischemia conditions in vitro and treated with immunosuppressant immunophilin ligands (cyclosporin A (CsA) and FK506) can increase intracellular GSH levels and that such mechanism may be responsible, at least in part, for their protective effects. In addition, we also compared the antioxidant properties of these immunosuppressants with N-acetylcysteine (NAC), a precursor of GSH synthesis. GSH depletion was induced by 24 h pretreatment with L-buthionine sulfoximine (BSO). Cultures of rat astrocytes were exposed to CsA (1-50 microM) and FK506 (1-1000 nM) and NAC (100 or 200 microM). We examined the effects of these compounds on apoptosis, cell viability, reactive oxygen species production and GSH content. Our study demonstrated that toxicity of simulated ischemia conditions were enhanced when intracellular GSH was depleted, and immunosuppressants (especially 100 nM FK506 and 10 microM CsA) effectively prevented ischemia toxicity in GSH depleted astrocytes. In addition, we have shown that interfering with the generation of GSH and attenuation, the rise of oxidative stress level by NAC may be a powerful tool for prevention of ischemia-induced glial cell damage.

Acetylcysteine↗

PubMed2004

Erk1/2 and Akt kinases are involved in the protective effect of aniracetam in astrocytes subjected to simulated ischemia in vitro.

The present study focused on the mechanism of cytoprotective effect of aniracetam on the primary rat astrocyte cultures exposed to simulated ischemia conditions in vitro. To study these mechanisms, the aniracetam-mediated modulation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/Akt kinase pathways was determined. Simulated in vitro ischemia caused death of approximately 35% of astrocytes via apoptosis and decreased cell viability about 50% at 8 h. Exposure to aniracetam at concentrations of 0.1-10 microM in these conditions significantly decreased the number of apoptotic cells. Moreover, the intensification of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion and the decrease of lactate dehydrogenase (LDH) release after 1 and 10 microM aniracetam treatment were observed indicating a significant increase in cell viability. When cultured astrocytes were incubated during 8 h simulated ischemia with [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] (U0126), an extracellular regulated kinase 1 and 2 (Erk1/2) inhibitor or wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase)/Akt inhibitor, the cell apoptosis was accelerated. These effects of used kinase inhibitors (both U0126 and wortmannin) were antagonized by adding 1 and 10 microM aniracetam to the culture medium. In addition, aniracetam significantly stimulated of phospho-Erk1/2 kinase and phospho-Akt expression. Maximum levels of Erk1/2 and Akt activation were observed as a result of treatment with 10 microM aniracetam. U0126 and wortmannin markedly attenuated the effects of aniracetam on expression of activated kinases. Results of the present study indicate that both Erk1/2 and PI 3-K/Akt kinase pathways are vital for cytoprotective effect of aniracetam.

Animals↗

PubMed2004

[Culture of the corneal epithelium--comparison of the mitotic potential of limbal cells from living and cadaveric donors].

PURPOSE: We analyse effectiveness of corneal limbal cells' culture prepared from heart-beating organ donors, that include living donors and from cadaver donors buttons following 3 days storage in 4 degrees C in Likorol. MATERIAL AND METHODS: For experiment 12 adults (living and heart-beating organ donors) aged 28-63 (mean 46.3) and 12 corneal buttons of cadaver (aged 18-51, mean 34.1) were qualified. Tissue samples (1 mm2) were taken from superior corneal limbus. Sample from living donor obtained during routine operation was sent immediately to laboratory, as well as from heart-beating organ donor. The limbal biopsy of preserved cornea was taken after 3 days storage in 4 degrees C (Likorol). Samples were treated with trypsin/EDTA solution before culture. Collected cells in similar density in 1 ml of medium laid on dishes inserts, covered with fibrin (Tissucol) and cultured in presence of feeder layer of fibroblasts (L929 line). Epithelial cells were cultured for 14 days at 37 degrees C in humidified 5% CO2 atmosphere in supplemented 2:1 mixture of DMEM and Ham's F12. On the 14th day cells were collected. Number of cells per 1 ml of medium was counted by cytometer. Immunostaining for epithelium type (Keratin 3) was performed. RESULTS: The number of cells obtained from cadaver donors reached 184.2+/-14.9% whereas from living donors revealed 1013.1+/-104.2%, increase in relation to number of delivered cells. We observed only 0.83+/-0.3 colonies per microscopic area in cultures from preserved tissue versus 6.67+/-0.6 colonies in cultures from living donor. CONCLUSIONS: The preservation in 4 degrees C in Likorol significantly decreases proliferative potential of the corneal limbus.

Adult↗

PubMed2002

Methionine-enkephalin and leucine-enkephalin increase interleukin-1 beta release in mixed glia cultures.

Interleukin-1 beta (IL-1 beta) is synthesized in the brain in response to LPS. Excessive IL-1 beta expression is observed in neurodegenerative diseases. The aim of this study was to evaluate the effects of methionine-enkephalin (ME) and leucine-enkephalin (LE) on the baseline and LPS-activated release of IL-1 beta in rat mixed glia cultures. ME and LE increased LPS-induced IL-1 beta release, which was not blocked by naloxone. Both ME and LE increased the baseline release of IL-1 beta, which was completely blocked by naloxone pretreatment. Mixed glia cultures deprived of microglia (by shaking and incubating with L-leucine methyl ester) did not release IL-1 beta, which indicates microglia as a source of the changes in IL-1 beta release. The results of the study suggest that neurons may regulate glial activity through releasing enkephalins.

Animals↗

PubMed2002

Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation.

The aim of the present study was to establish whether piracetam (2-pyrrolidon-N-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP) are capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we observed the cells treated with PIR and VINP during and after in vitro simulated hypoxia. Cell viability was determined by Live/Dead Viability/Cytotoxicity Assay Kit, LDH release assay and MTT conversion test. Apoptotic cell death was distinguished by a method of Hoechst 33342 staining underfluorescence microscope and caspase-3 colorimetric assay. In addition the intracellular levels of ATP and phosphocreatine (PCr) were evaluated by bioluminescence method. Moreover, the effect of the drugs on the DNA synthesis was evaluated by measuring the incorporation of [3H]thymidine into DNA of astrocytes. PIR (0.01 and 1 mM) and VINP (0.1 and 10 microM) were added to the medium both during 24 h normoxia, 24 h hypoxia or 24 h reoxygenation. Administration of 1 mM PIR or 0.1 microM VINP to the cultures during hypoxia significantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also confirmed by their stimulation of mitochondrial function, the increase of intracellular ATP, and the inhibition of the caspase-3 activity. The prevention of apoptosis was accompanied by the increase in ATP and PCr levels and increase in the proliferation of astrocytes exposed to reoxygenation. The higher concentration of VINP (10 microM) was detrimental in hypoxic conditions. Our experiment proved the significant cytoprotective effect of 1 mM PIR and 0.1 microM VINP on astrocytes in vitro.

Animals↗

PubMed2002

Aniracetam attenuates apoptosis of astrocytes subjected to simulated ischemia in vitro.

The aim of the present study was to establish whether aniracetam is capable of protecting cultured rat astrocytes against ischemic injury. Treatment of the cultures with aniracetam (1, 10 and 100 mM) during 24 h ischemia simulated in vitro significantly decreased the number of apoptotic cells. The antiapoptotic effects of the drug were confirmed by the increase of intracellular ATP and phosphocreatine (PCr) levels and the inhibition of the caspase-3 activity. Aniracetam also attenuated cellular oxidative stress by decreased production of reactive oxygen species (ROS). These effects were associated with the decrease in levels of c-fos and c-jun mRNA in primary astrocyte cultures exposed to 24 h ischemia. When cultured astrocytes were incubated during 24 h simulated ischemia with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor or PD98059, a mitogen-activated protein (MAP)/extracellular signal regulated kinase (ERK) (MEK) inhibitor the cell apoptosis was accelerated. This effect was antagonized by adding 100 mM aniracetam to the culture medium. These findings suggest that the protective effect of aniracetam is mediated by PI 3-kinase and MEK pathways in the downstream mechanisms.

Adenosine Triphosphate↗

PubMedDate not supplied

Effect of antidepressants on the phospholipase A2 activity in plasma membranes of the rat brain cortex.

The aim of the present study was to establish whether antidepressants (ADs) of potentially different chemical structure and mechanisms of action affected the phospholipase A2 (PLA2) activity in plasma membranes of the rat brain cortex. It was decided to evaluate the influence of imipramine (IMI), amitriptyline (AMI), fluvoxamine (FLU), mianserin (MIA) and tianeptine (TIA) on PLA2 activity after an acute and long-term (4 weeks) drug administration. To study the time-related effects of FLU on PLA2 activity, animals were treated for 1, 7, 14 and 28 days. The experiments were performed on male Wistar rats. The PLA2 activity was determined by the method of Strosznajder and Strosznajder as well as Jelsema with slight modifications. It was shown that ADs significantly changed the PLA2 activity in plasma membranes of the rat brain cortex and the effects depended on the dose, time of administration and the structure of the drug. Tricyclic ADs, both classic (IMI and AMI) as well as atypical (e.g. TIA) inhibited PLA2 activity. It seems that FLU was the only antidepressant, which induced either inhibition or activation of PLA2 depending on time of administration. It may be suggested that PLA2 appears to be a common target for drugs showing quite different mechanisms of action.

Animals↗

PubMedDate not supplied

Immunophilin ligands decrease release of pro-inflammatory cytokines (IL-1beta, TNF-alpha and IL-2 in rat astrocyte cultures exposed to simulated ischemia in vitro.

The aim of present study was to evaluate the effects of immunophilin ligands (cyclosporin A, FK506 and rapamycin) on the simulated ischemia-induced release of pro-inflammatory cytokines (IL-1beta, TNF-alpha and IL-2) in rat primary astrocyte cell cultures. Astrocytes were exposed to cyclosporin A (CsA) (0.25, 0.5, 1, 10, 20 and 50 microM), FK506 (1, 10, 100, 1000 nM) and rapamycin (10, 100, 500 and 1000 nM). In vitro simulated ischemia significantly increased secretion of IL-1beta, TNF-alpha and IL-2 by astrocyte cultures deprived of microglia (by shaking and incubating with L-leucine methyl ester). CsA (at concentrations of 10-50 microM), FK506 (at all used concentrations) and rapamycin (in dose-dependent manner) significantly attenuated IL-1beta release after 24 h exposure to ischemic conditions. Immunophilin ligands at all used concentrations significantly decreased TNF-alpha levels in culture media after 24 h exposure to ischemia. Moreover, significant decrease in IL-2 secretion at 0.25, 0.5, 1 and 50 microM CsA and FK506 at concentrations of 100 and 1000 nM were observed. The results suggest that immunophilin ligands may regulate glial activity during ischemia by affecting the release of pro-inflammatory cytokines.

Animals↗

PubMedDate not supplied

Effect of aniracetam on phosphatidylinositol transfer protein alpha in cytosolic and plasma membrane fractions of astrocytes subjected to simulated ischemia in vitro.

Brain ischemia affects phosphoinositide metabolism and the level of lipid-derived second messengers. Phosphatidylinositol transfer proteins (PI-PTs) are responsible for the transport of phosphatidylinositol (PI) and other phospholipids through membranes. Isoform of PI-TPs (PI-TPalpha) is an essential component in ensuring substrate supply for phospholipase C (PLC). The current study was conducted to examine potential effect of aniracetam on PI-TPalpha expression and to characterize the PI-TPalpha isoform distribution between membrane and cytosol fractions of astrocytes exposed to simulated ischemia in vitro. After 8 h period of ischemia, the level of PI-TPalpha was significantly higher in cytosol (by about 28%) as well as in membrane fraction (by about 80%) in comparison with control. We have found that aniracetam treatment of astrocytes in normoxia significantly increased the level of PI-TPalpha in membrane fraction with a maximal effect at 0.1 microM concentration of aniracetam (by about 195% of control). In membrane fractions of ischemic cells, aniracetam increased PI-TPalpha expression in a concentration-dependent manner. In ischemic cells, aniracetam (10 microM) has elevated PI-TPalpha expression up to 155% and 428% in cytosolic and membrane fractions in comparison with ischemic untreated cells, respectively. The study has shown that aniracetam significantly activates PI-TPalpha in cell membrane fraction and this effect might be connected with previously described activation of MAP kinase cascade.

Animals↗

PubMedDate not supplied

Ebselen attenuates oxidative stress in ischemic astrocytes depleted of glutathione. Comparison with glutathione precursors.

In this study, we investigated the protective effect of ebselen, a seleno-organic compound with antioxidant activity, towards astrocyte degeneration caused by exposure to simulated in vitro ischemic conditions and simultaneous depletion of glutathione (GSH). Depletion of GSH was induced by 24 h pretreatment with L-buthionine-(S,R)-sulfoximine (BSO). In this experimental paradigm, we examined the effects of ebselen (1-40 microM) on apoptosis, mitochondrial function, reactive oxygen species (ROS) production, intracellular GSH level and mitochondrial transmembrane potential (MTP). In addition, we also compared the antioxidant potential of ebselen with cystine and methionine as precursors of GSH synthesis as well as with GSH ethyl ester. Our study demonstrated that toxicity of simulated ischemia conditions was enhanced when intracellular GSH was depleted. Treatment with ebselen, especially at concentrations of 20 and 40 microM prevented ischemia-induced cytotoxicity. Our study has shown that antiapoptotic effect of ebselen is associated with its strong antioxidant properties, preservation of MTP and possibly conservation of mitochondrial GSH during cytoplasmatic GSH depletion caused by oxidative damage. Also, promoting GSH synthesis by the delivery of its substrates, like cystine or inhibition of the efflux by methionine may be a powerful strategy to minimize cell damage in the nervous tissue after ischemia.

Animals↗