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Piracetam and Vinpocetine Exert Cytoprotective Activity and Prevent Apoptosis of Astrocytes In Vitro in Hypoxia and Reoxygenation
NeuroToxicology 23 (2002) 19±31
Piracetam and Vinpocetine Exert Cytoprotective Activity and Prevent Apoptosis of Astrocytes In Vitro in Hypoxia and Reoxygenation BozÇena Gabryel1,*, Mariusz Adamek1,2, Anna Pudeøko1, Andrzej Maøecki1, Henryk I. Trzeciak1 1
Department of Pharmacology, Silesian Medical University, 18 MedykoÂw St., PL 40752 Katowice, Poland Department and Clinic of Internal Diseases and Physical Medicine, Center for Laser Diagnostics and Therapy, Silesian Medical University, 15 Batory St., PL 41902 Bytom, Poland 2
Received 23 July 2001; accepted 7 November 2001
Abstract 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 under ¯uorescence 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 mM) 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 mM VINP to the cultures during hypoxia signi®cantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also con®rmed 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 mM) was detrimental in hypoxic conditions. Our experiment proved the signi®cant cytoprotective effect of 1 mM PIR and 0.1 mM VINP on astrocytes in vitro. # 2002 Published by Elsevier Science Inc.
Keywords: Piracetam; Vinpocetine; Astrocytes; Hypoxia; Reoxygenation; Apoptosis
INTRODUCTION Nootropic drugs, including piracetam (2-pyrrolidonN-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP), are used in the treatment of brain injures, cognitive dysfunction caused by hypoxia, hypogycaemia and heavy metal intoxication as well as in senile dementia of the Alzheimer's type (Nicholson, 1990; Gabryel and Trzeciak, 1994). * Corresponding author. Tel.: 48-3225-23835; fax: 48-3225-23835. E-mail address: [email protected] (B. Gabryel).
Results of studies using various models in vivo and in vitro of experimental brain ischemia as well as of clinical trials indicate neuroprotective properties of PIR and VINP (Krieglstein and Rischke, 1991; Erdo et al., 1990; Sauer et al., 1988). Yet, mechanisms through which the drugs exert the protective effects are different and not fully known. The ef®cacy of PIR in ischemia prevention is connected mainly with the drug positive in¯uence on neuronal cell membrane ¯uidity. It has been shown that PIR acts on the cell membrane, to which it binds, being inserted at the level of the polar heads of phospholipids (Muller et al., 1994, 1997; Peuvot et al., 1995). Moreover, PIR exerts
0161-813X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 0 4 - 9
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antithrombotic as well as haemorrheological effects (Moriau et al., 1993; Stockmans et al., 1998). VINP, on the other hand, mainly inhibits intraneuronal adenosine re-uptake and also has vasodilatory effect (Krieglstein and Rischke, 1991; Gabryel and Trzeciak, 1994). Despite a number of experimental data concerning the protective effects of PIR (Poeck, 1998; Coq and Xerri, 2000; Rauca et al., 2000a,b) and VINP (Kiss et al., 1991; Bereczki and Fekete, 1999) on damaged neurons in vivo and in vitro it remains unknown whether they also have the glioprotective activity. It seems particularly important in the context of coexistence of necrotic and apoptotic death of both neurons and astrocytes as a result of ischemic brain damage. Studies on the pathomechanism of brain ischemia indicate that a decrease in neuronal survival during and after ischemia is also associated with astrocytic dysfunction (Aschner, 1998; Gabryel and Trzeciak, 2001). Therefore, using the cell culture model, we decided to investigate if and in what way drugs of potentially different neuroprotective mechanisms protect astrocytes from rat cerebral cortex in primary cell culture during in vitro simulated hypoxia and reoxygenation. For this purpose we decided to examine the effect of PIR and VINP on astrocyte apoptosis by determining the amount of apoptotic nuclei, the percentage of lactate dehydrogenase (LDH) released into the culture medium, caspase-3 activity, mitochondrial function, and by determining intracellular high-energy phosphates concentration (ATP and PCr). Additionally, we evaluated the proliferation of astrocytes treated in vitro with selected drugs in normoxia, hypoxia and reoxygenation. MATERIALS AND METHODS Materials Chemicals and materials were obtained from the following sources: PIR (Polfa, Poland), VINP (Cavinton1, Gedeon Richter, Hungary), Dulbecco's modi®ed Eagle's medium (DMEM), foetal bovine serum (FBS), phosphate buffered saline (PBS), antibioticantimycotic mixture (all from GIBCO Laboratories, USA), DAKO PAP kit system glial ®brillary acidic protein (GFAP) test (DAKO Co., Denmark), Live/Dead Kit (Molecular Probes Inc., USA), Caspase-3 Colorimetric Assay (R&D System, UK), ATP monitoring kit (Labsystems, Finland), creatine kinase (Boehringen Mannheim, Germany), adenosine 50 -pentaphosphate, ADP, poly-D-lysine, 3-(4,5-dimethylthazol-2-yl)-2,5-
diphenyltetrazolinum bromide (MTT), Hoechst 33342, etylenediaminetetraacetic acid disodium salt (EDTA 2 Na), trichloracetic acid (TCA), trypan blue solution (0.4%), NADH, sodium pyruvate and other chemicals all from Sigma (USA), [methyl-3 H] thymidine (2.00 Ci/ mM) (Dupont, NEN, USA), plastic tissue culture Petri dishes (Becton Dickinson, USA). Primary Culture of Rat Astrocytes Astrocytes were isolated and cultured essentially according to the method of Hertz et al. (1985). Brie¯y, hemispheres from one-day old Wistar rat pups were removed aseptically from skulls, freed of the meninges, minced and mechanically disrupted by vortexing in DMEM containing penicillin (100 U/ml) and streptomycin (100 mg/ml). This suspension was ®ltered through sterile nylon screening cloth with pore sizes 70 mm (®rst sieving) and 10 mm (second sieving). Subsequently, cultures were incubated at 37 8C in 95% air and 5% CO2 with relative humidity 95% (CO2-Incubator, Kebo±Assab, Sweden). Before the material was plated onto the plastic dishes the cells were counted in a Coulter Z1 counter (Coulter Counter, UK). Cells destined for bioluminescent and isotopic studies were plated at a density of 3 105 cells per dish (35 mm in diameter). For MTT conversion measurements cells were seeded at 1 104 per well on 96-well plates. Astrocytes for ¯uorescent studies (Live/Dead Kit and Hoechst 33342 staining) were cultured on coverslips covered with poly-D-lysine (100 mg/ml). The cells destined for determination of caspase-3 activity with the colorimetric assay were seeded onto plastic dishes of 100 mm in diameter at the density of 1 106 cells per dish. The culture medium initially contained 20% of FBS and after 4 days was replaced with medium containing 10% FBS. The whole volume of culture medium was changed twice a week. In order to remove contaminating non-astroglial cells, con¯uent cultures were shaken overnight on a rotary platform at 200 rpm (37 8C) (McCarthy and De Vellis', 1980). Cell culture medium was replaced after overnight shaking. To identify astrocytes, cultures (21 days old) were stained immunocytochemically for GFAP. Analysis of the cultures has shown that 90±95% of cells were GFAP-positive. All experiments were performed on 21-day cultures. Experimental Design Control astrocytes (Normoxia) were cultured in DMEM medium with 5.5 mM glucose and 10% FBS
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in 95% air/5% CO2. In order to mimic the in vivo hypoxia, cultures were placed for 24 h in 37 8C in 3% O2/5% CO2/92% N2 (CO2 incubator, Heraeus, Germany) in DMEM without glucose and serum. Osmolarity of the medium was measured and adjusted to 319 mOsm with mannitol. Some astrocyte cultures were exposed for 24 h to hypoxia (Hypoxia) or 24 h hypoxia followed by 24 h reoxygenation (Reoxygenation). Cells were treated with PIR (0.01 and 1 mM) and VINP (0.1 and 10 mM) according to three distinct protocols: (i) for 24 h in normoxia; (ii) for 24 h of hypoxia; (iii) for 24 h of reoxygenation. Treatment of astrocytes with PIR and VINP in normoxia and reoxygenation was conducted in a medium containing glucose and serum at 37 8C in 95% air/5% CO2. Cell Death Cell death was determined by a ¯uorescent method with the use of a Live/Dead Kit (Molecular Probes Inc., USA) containing calcein/AM and ethidium homodimer-1 (EthD-1). Calcein/AM is absorbed by living cells and subsequently converted by cytosolic esterases into a green ¯uorescent product (ex/em 495 nm/ 530 nm), whereas EthD-1 is known to enter only the cell with compromised cell membrane permeability and after being attached to nucleic acids yields red ¯uorescence (ex/em 495/635 nm). After twice washing with PBS both reagents diluted in PBS were applied to the cell cultures immediately after exposure to hypoxia. Astrocytes were incubated with 2 mM of calcein/AM and 4 mM of EthD-1 for 30 min at 37 8C. The number of dead and live cells was determined on at least six randomly selected areas from three coverslips of every experimental group, each containing approximately 200 cells. Cells were examined by ¯uorescence imaging MiraCal Pro III workstation (Life Science Resources Ltd., UK) comprising of inverted microscope Eclipse TE200 (Nikon, Japan) and high-resolution cooled CCD camera (Photonic Science Ltd., UK). The results were expressed as a percentage of dead cells relative to the total number of cells. Assessment of Apoptosis Cell nuclei staining by Hoechst 33342 (Molecular Probes Inc., USA) enables visualization of fragmented and condensed DNA, a characteristic feature of apoptosis. After washing with PBS astrocytes cultured on coverslips were ®xed for 10 min with a 4% paraformaldehyde
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at room temperature. Subsequently, after being washed twice with PBS the samples were dehydrated ®rst in 70% ethanol and then in absolute ethanol. The samples were kept in 20 8C until they were stained with Hoechst 33342 (5 mg/ml in PBS) for 5 min at room temperature. Then the cells were washed again with PBS. Cell nuclei analysis was conducted with the above-described imaging system (ex/em 340/510 nm). A 20 objective was used. The number of apoptotic nuclei was determined on at least six randomly selected areas from three coverslips of every experimental group, each containing approximately 200 cells. The results were expressed as a percentage of apoptotic cells relative to the total number of cells. Measurement of Caspase-3 Activity Enzymatic activity of caspase-3 was determined by Caspase-3 Colorimetric Assay (R&D System, UK), according to the manufacturer's instructions. This assay is based on the release of the ¯uorochrome p-nitroaniline (p-NA) combined with a caspase-3 speci®c peptide substrate (DEVD±p-NA). Peptide cleavage through active caspase-3 releases chromophor p-NA, which can be quanti®ed using colorimetric plate reader (405 nm). Brie¯y, cultured astocytes were lysed and cell extracts were centrifuged to eliminate cellular debris. Aliquots (20 ml of the cell extract) were incubated for 2 h at 37 8C in the presence of the substrate. The level of caspase-3 activity, proportional to the color reaction intensity was expressed as a percentage of control. Mitochondrial Function Mitochondrial function of astrocytes treated with PIR and VINP was evaluated with 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion method (Mosman, 1983). The cells ability to convert MTT indicates mitochondrial integrity and activity, which might in turn indicate cell viability. The cleavage of tetrazoline ring in MTT takes place mainly with the participation of the mitochondrial succinate dehydrogenase and depends on the activity of the respiratory chain and the redox state of the mitochondria (Mosman, 1983; Shearman et al., 1995). MTT (®nal concentrationÐ0.25 mg/ml) was added to the medium three hours before the scheduled end of the experiment and then the cultures were incubated at 37 8C in proper conditions. At the end of the experiment, after being washed twice with PBS, cells were lysed in 100 ml dimethyl sulphoxide which
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enabled the release of the blue reaction productÐ formasan. Absorbance at the wavelength of 570 nm was read on a microplate reader and results were expressed as a percentage of absorbance measured in control cells. Measurement of ATP and PCr Concentrations The ATP and PCr concentrations were determined by high-speci®c ®re¯y luciferin±luciferase bioluminescence assay system using a Fluoroscan Ascent plate reader (Labsystems, Finland). The extraction of ATP and PCr was performed according to procedure described by Fitzpatrick et al. (1988). The conversion of PCr into ATP catalyzed by creatine kinase was performed according to the method of Lowry and Passonneau (1972). ATP concentrations were measured by bioluminescence quanti®cation according to the method of Lust et al. (1981). Brie¯y, 100 ml samples of disrupted cells were added to equal volumes of ice-cold 5% TCA solution with 2 mM EDTA, vortexed rapidly for 5 s and incubated for 30 min at room temperature. Then the samples were 20-fold diluted with Tris±acetate buffer pH 7.75 and 10 ml solution of known ATP concentration was added to each well as internal standard and the total amount of ATP was determined. The results were calculated as nM of ATP or PCr per mg of protein (nM/mg). Lactate Dehydrogenase (LDH) Activity LDH activity was estimated by measuring the decrease in absorbance at 340 nm due to the conversion of enzyme co-factor NADH to NAD (one enzyme activity unit 0:001 DA/min). Brie¯y, 100 ml aliquots of culture media were collected and dissolved in 0.1 M pyruvate buffer (0.25 mg sodium pyruvate in PBS). NADH solution (0.25 mg in PBS) was then added to the samples. Cells were lysed by repeated freezing and thawing and total released LDH was measured. Absorbance was measured immediately after the addition of NADH and 60 s after the initial reading. The data are presented as the percentage of the total releasable LDH. [3H]Thymidine Incorporation The amount of incorporated [3 H]thymidine was measured according to the method of Neary et al. (1994) with slight modi®cations. In brief, astrocytes treated with PIR and VINP in the above-mentioned
experimental conditions were incubated with [3 H]thymidine (20 Ci/mM) 0.5 mCi/ dish (2 ml) for 18 h in DMEM containing 0.5% FBS. At the end of incubation, cells were washed twice with 1 ml of ice-cold phosphate-buffered saline (PBS). Then cells were scraped with a plastic policeman (Costar Co, USA) and centrifuged for 10 min (2000 rpm/min). A total of 1 ml 10% TCA was added to the sediment and samples were incubated at 4 8C for 30 min. The samples were centrifuged again for 10 min (2000 rpm/min). A total of 1 ml of 0.1 N NaOH was then added to each tube and samples were lysed overnight at 4 8C. Next day the samples were neutralized by 10% formic acid and transferred to scintillation vials. After addition of 8 ml of Bray scintillating solution, radioactivity was determined in a Beckman LS 6000 IC counter (Beckman Instruments Inc., USA). The results were calculated in dpm per mg of protein and expressed as percentage of control. Protein Determination Protein content in astrocytes was measured according to the method of Lowry et al. (1951). Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) followed by the Newman±Keuls test. A P-value of 0.05 was considered as statistically signi®cant. All data were expressed as mean S:D. RESULTS Effect of PIR and VINP on Cell Death The number of dead astrocytes in cell cultures treated with PIR and VINP in hypoxia was determined by ¯uorescent staining with the use of the Live/Dead Kit. Nuclei of cells undergoing necrosis stain red whereas calcein identi®es viable cells. In the astrocyte cultures exposed to normoxia or reoxygenation few dead cells were observed, therefore, the Live/Dead kit was only used for determining the increase in necrosis in cultures exposed to hypoxia. The percentage of dead cells stained with EthD-1 in comparison with the cells stained with calcein in particular groups is presented in Fig. 1. Hypoxia signi®cantly increased the number of dead astrocytes in the cultures as compared to the normoxia. Interestingly, adding 1 mM PIR into the culture medium in
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pictures were analyzed in order to determine the percentage of apoptotic cells in particular groups. The results are shown in Fig. 3A. After the 24 h reoxygenation period, we observed a similar amount of apoptotic nuclei as in normoxic conditions (Fig. 3B). The presence of 0.01 mM PIR or 10 mM VINP in the culture medium in these conditions signi®cantly increased the amount of apoptotic nuclei. Exposing astrocytes to 0.1 mM VINP during 24 h reoxygenation period turned out to be the most effective method in the prevention of morphological changes of nuclei induced by hypoxia. Influence of PIR and VINP on Caspase-3 Activity Fig. 1. Effect of piracetam or vinpocetine on astrocyte death induced by hypoxia. The results are shown as a percentage relation of dead cells to the total amount of cells in the field and are a mean S:D. of six randomly selected areas from three coverslips. At least 1000 cells were counted in each experiment. Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. The symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia.
hypoxia signi®cantly reduces the number of dead cells. 0.01 mM PIR or 0.1 mM VINP did not cause statistically signi®cant changes in the amount of dead astrocytes in hypoxic conditions. On the other hand, 10 mM VINP signi®cantly increased the number of dead cells which might indicate the drug toxic activity at this concentration. Influence of PIR and VINP on Apoptosis (Hoechst 33342 Staining) In order to determine the in¯uence of PIR and VINP on the process of apoptosis the astrocyte cell nuclei were visualized by DNA-speci®c ¯uorescent dye Hoechst 33342. Fig. 2 shows microphotographs representative for experimental groups. Most cells from the control group (Normoxia) had big, regular nuclei and few had apoptotic nuclei with condensated chromatin (Fig. 2A). In the cultures of astrocytes exposed to hypoxia many more picnotic nuclei occured (Fig. 2B). Treatment of astrocytes with 0.01, 1 mM PIR or 0.1 mM VINP during hypoxia prevented the process of apoptosis (Fig. 2C, D, E). However, exposure of the cells with 10 mM VINP in these conditions signi®cantly increased the amount of apoptotic nuclei in comparison with control (Fig. 2F). The microscopic
Exposure of astrocyte cultures to hypoxia signi®cantly increased caspase-3 activity. Treatment with 0.01 mM or 1 mM PIR as well as 0.1 mM VINP did not change the enzyme proteolytic activity in comparison to normoxia. However, simultaneous exposure of astrocytes to hypoxia and 10 mM VINP substantially increased the caspase-3 activity (Fig. 4A). During a 24 h reoxygenation period and after PIR (0.01 or 1 mM) administration a similar activity of the enzyme to that in normoxia was observed. In these conditions, treatment of astrocytes with VINP caused the increase of the intracellular caspase-3 activity (Fig. 4B). Effect of PIR and VINP on Mitochondrial Function Mitochondrial function was measured by the MTT reduction method, which may also serve as a general indicator of cell viability. Fig. 5. shows the results of the in¯uence of PIR and VINP on MTT conversion into formasan in the cultures. A substantial increase of MTT conversion in astrocytes in normoxic conditions stimulated both by PIR and VINP indicated a signi®cant increase in mitochondrial activity (Fig. 5A). A 24 h hypoxia period attenuated the conversion of MTT. A similar effect was observed after an additional exposition of astrocytes in these conditions to 0.01 mM PIR or 10 mM VINP. However, treatment with 1 mM PIR or 0.01 mM VINP induced the MTT conversion process during 24 h hypoxia, although the process intensity was weaker in comparison with the cells in normoxia (Fig. 5B). The 24 h reoxygenation as well as 1 mM PIR administration during this period weakened mitochondrial functions. The exposition of astrocytes in these conditions to 0.01 mM PIR, 1 mM PIR or 0.1 mM VINP increased the MTT conversion (Fig. 5C).
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Fig. 2. Effect of piracetam or vinpocetine on astrocyte apoptosis induced by hypoxia. Cell nuclei were stained with the Hoechst 33342 dye. Microphotographs show experimental groups of astrocyte cultures treated with drugs according to protocols described in Section 2. Microphotographs were taken with the use of fluorescent microscope and 20 objective. Thin arrows point at apoptotic cell nuclei and the thick arrows point at live cell nuclei. (A) normoxia; (B) hypoxia; (C) 0.1 mM piracetam; (D) 1 mM piracetam; (E) 0.1 mM vinpocetine; (F) 10 mM vinpocetine.
Influence of PIR and VINP on Intracellular ATP and PCr Concentrations Tables 1 and 2 show changes in the intracellular amount of ATP and PCr in astrocytes treated with PIR and VINP in normoxia, hypoxia and reoxygenation. Increased ATP and PCr levels in cells treated with 1 mM PIR or 0.1 mM VINP was observed in normoxia. The exposure of astrocytes to 24 h hypoxia caused a signi®cant reduction in the amount of both high-energy phosphates. In this experimental option only 1 mM PIR produced increase of the ATP concentration in cells. The most signi®cant decrease in value of this parameter was observed in hypoxia after treatment with 10 mM VINP. VINP at the concentration 0.1 mM maintained the amount of ATP and PCr at the level similar to normoxia control. After 24 h reoxygenation the concentration of both ATP and PCr in astrocytes increased signi®cantly. The increase in amount of both high-
energy phosphates was also observed as a result of astrocytes treatment with 0.01 or 1 mM PIR as well as 0.1 mM VINP. At the same time no changes were observed in intracellular concentrations of ATP and PCr when the cells were exposed during reoxygenation to 10 mM VINP. Influence of PIR and VINP on LDH Release The LDH level in the medium of normoxic culture was very low (Fig. 6A). After 24 h hypoxia, an increase in LDH activity in the medium occurred as a result of cell membrane integrity loss and an enzyme leakage. (Fig. 6B). Both drugs added in normoxia reduce the LDH activity. The amount of released LDH increased in cultures exposed simultaneously to hypoxia as well as to 0.01, 1 mM PIR or 10 mM VINP. In these conditions only 0.1 mM VINP did not cause the increase of LDH activity in the medium (Fig. 6B). A signi®cant
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Fig. 3. Effect of piracetam or vinpocetine on astrocyte apoptosis induced by hypoxia (A) and reoxygenation (B). The results are shown as a percentage relation of the apoptotic nuclei to the total amount of nuclei in the field and are a mean S:D. of the six randomly selected areas from three coverslips. At least 1000 cells were counted in each experiment. Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment; The symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
reduction of the LDH release was observed 24 h after hypoxia both in cultures subjected only to reoxygenation as well as treated with the tested drugs (Fig. 6C). Influence of PIR and VINP on [3H]thymidine Incorporation [3 H]thymidine incorporation into astrocytes treated with PIR and VINP was studied as an indicator of DNA synthesis and astrocytic proliferation.
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Fig. 4. Effect of piracetam or vinpocetine on caspase-3 activity in cultured rat astrocytes exposed to hypoxia (A) and reoxygenation (B). The results are shown as a percentages of the control value in the normoxia conditions. Data were analyzed with the use of oneway ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. Each value is mean of eight dishes S:D. (n 8); the symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
An insigni®cant increase in [3 H]thymidine incorporation occurred after administration of 0.01 mM PIR and 0.1 mM VINP into the culture medium in normoxia (Fig. 7A). It has been observed that 24 h hypoxia slowed [3 H]thymidine incorporation in comparison with the cells maintained in normoxia conditions. Similarly, exposure of hypoxic astrocytes to the tested concentrations of PIR and VINP abated [3 H]thymidine incorporation into their DNA (Fig. 7B). However, the increase in proliferation of astrocytes was
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Fig. 5. Effect of piracetam and vinpocetine on mitochondrial function of cultured rat astrocytes exposed to normoxia (A), hypoxia (B) and reoxygenation (C) measured by MTT conversion assay. Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman± Keuls test was performed in order to compare effects of studied drugs treatment. The results are shown as a percentage relation of the control value in the normoxia conditions. Data are S.D. (n 8); the symbol () P < 0:05 vs. normoxia; (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
Fig. 6. Effect of piracetam and vinpocetine on LDH release in cultured rat astrocytes exposed to normoxia (A), hypoxia (B) and reoxygenation (C). Data were analysed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. The results are shown as a percentage relation of the control value in the normoxia conditions. Data are S.D. (n 8); () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
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Table 1 Effect of piracetam and vinpocetine on ATP levels in cultured rat astrocytesa Conditions
24-h Normoxia
24-h Hypoxia
24-h Reoxygenation
Protein (nM/mg) Untreated Piracetam (0.01 mM) Piracetam (1 mM) Vinpocetine (0.1 mM) Vinpocetine (10 mM)
33.4 47.7 48.7 54.2 36.8
8.7 12b 14.2b 5.1b 13.9b
14.0 23.4 43.6 29.4 4.4
2.1b 12b,c 8.3b,c 7.6c 2.2b,c
99.8 57.8 106.6 125.6 43.7
10.2b 16.7b,d 10.4b 24.3b 8.8b,d
a
Data were analysed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. Each value is mean obtained from eight dishes S.D. (n 8). b P < 0:01 vs. normoxia. c P < 0:01 vs. hypoxia. d P < 0:01 vs. reoxygenation.
Table 2 Effect of piracetam and vinpocetine on intracelluar PCr levels in cultured astrocytesa Conditions Untreated Piracetam (0.01 mM) Piracetam (1 mM) Vinpocetine (0.1 mM) Vinpocetine (10 mM)
24-h Normoxia 52.6 76.1 108.3 120.0 63.4
11.3 22.4b 20b 16.2b 13.4b
24-h Hypoxia 30.1 29.5 29.7 45.4 25.8
b
6.1 5.7b 4.8b 12.5c 6.2b,c
24-h Reoxygenation 61.9 136.2 133.8 78.9 65.1
9.9b 34.5b,d 16.9b,d 12.5b,d 13.9b
a Data were analysed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. Each value is mean obtained from eight dishes S.D. (n 8). b P < 0:01 vs. normoxia. c P < 0:01 vs. hypoxia. d P < 0:01 vs. reoxygenation.
observed during 24 h reoxygenation. Yet, intensity of the [3 H]thymidine incorporation differed among groups and depended both on the kind of the drug examined and on the concentration used (Fig. 7C). The biggest increase in the astrocytes' DNA synthesis was induced by treatment with 0.1 mM VINP. A weaker mitogenic effect was observed during reoxygenation after 10 mM VINP administration to the cultures. The results obtained indicated that PIR and VINP prevented the proliferation of astrocytes during 24 h hypoxia, but stimulated their multiplication during reoxygenation. The most intensive reaction was caused by exposure of astrocytes to 0.1 mM VINP both in normoxia and reoxygenation conditions. DISCUSSION In the cultures subjected to 24 h hypoxia we observed a signi®cant increase in the number of both necrotic and apoptotic astrocytes in comparison to the control. The changes were accompanied by signi®cant energy depletion, cell membrane desintegra-
tion, impairment of mitochondrial function, caspase-3 activation and inhibition of cell proliferation. There are authors suggesting that nootropic drugs including PIR and VINPare effective only in cells injured by various factors, e.g. hypoxia or trauma (Giurgea and Salama, 1977; King, 1987). However, in the experiments we conducted the exposure of astrocytes to the drugs in normoxia signi®cantly intensi®ed the MTT reduction and decreased the amount of LDH released into the medium. The phenomenon was accompanied by elevated intracellular ATP and PCr concentration and increased intensity of cell proliferation re¯ected by the amount of incorporated [3 H]thymidine. Therefore, the results obtained did not indicate the toxic activity of any of the drugs in selected concentrations towards astrocytes in normoxia conditions. Perineuronal glial cells response to hypoxic brain damage by transforming themselves into so called reactive astrocytes. They are characterized by stellate morphology, increased GFAP expression and increased antioxidant activity (Petito, 1986; Liu et al., 1993; Takizawa et al., 1994). However, prolonged hypoxia, causes disturbances in the astrocytes functions, such
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Fig. 7. Effect of piracetam or vinpocetine on [3 H]thymidine incorporation into cultured rat astrocytes astrocytes exposed to normoxia (A), hypoxia (B) and reoxygenation (C). Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. The results are shown as a percentage relation of the control value in the normoxia conditions. Each value is mean activity obtained form eight dishes S:D. (n 8); the symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
as: loss of cell volume control resulting in swelling of brain parenchyma and constriction of blood vessels, leading consequently to termination of brain circulation (Hossmann, 1985). Hypoxia induces cell death possessing features characteristic for both necrosis and apoptosis (Shimizu et al., 1996). It is currently believed that mitochondria are the intracellular organelles which constitute a kind of switch between necrosis and apoptosis. The factors determining the type of cell death are the change in their membrane permeability as a result of the occurrence of mitochondrial permeability transition pore (MTP) (Zoratti and Szabo', 1995) and intracellular level of ATP (Leist et al., 1997; Eguchi et al., 1997). The opening of MTP is accompanied by inhibition of oxidative phosphorylation and activation of the mitochondrial ATP-ase which increases the consumption of ATP (Nieminen et al., 1994; Cai and Jones, 1998). When glycolysis or the use of energy stores accumulated in PCr are able to partly compensate the ATP de®ciency, the apoptosis program is commenced. However, if sudden and parallel mitochondrial transmembrane potential (Dc) break down occurs in the majority of mitochondria, the intracellular energy de®ciency is so severe that the live functions are stopped leading to necrosis (Kroemer et al., 1998). The energy stored in ATP is necessarily used in major apoptotic pathways leading from cytochrome c (Li et al., 1997), through apoptosis inducing factor (AIF), cell death receptors to caspases activity (Susin et al., 1999). Likewise, typical for apoptosis morphological changes of the cell nuclei are the result of the signal transduction from cytoplasm to the nuclei through active transport requiring ATP hydrolysis (Melchior and Gerace, 1995; Pante and Aebi, 1996). The extent of astrocyte damage in hypoxia was radically limited when they were simultaneously exposed to 1 mM PIR or 0.1 mM VINP. In our study, PIR added during hypoxia prevented necrosis as well as apoptosis of the astrocytes. The drug prevented ATP loss probably through partial use of PCr as an energy store which also enabled maintenance of mitochondria capacity. Reports demonstrating the normalization of the pathological platelet aggregation (Grotemeyer et al., 1986) and the increase in erythrocyte deformation (Nalbandian et al., 1983) suggest that PIR may also in¯uence the variety of cell types other than neurons. PIR ability to restore the disturbed cell membrane ¯uidity seems particularly interesting in the context of the loss of membrane asymmetry in the initial stage of apoptosis as a results of the phosphatidylserine externalization (Leist et al., 1997). The conceivable mechanism of PIR antiapoptic activity
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is also supported by its recently reported antioxidant properties (Quian et al., 1992; Rauca et al., 2000a,b). In case of VINP, both during hypoxia and reoxygenation, we observed signi®cant differences in its activity depending on the concentration. The obtained results indicate the protective effect of VINP on astrocytes in hypoxia and reoxygenation only at concentration 0.1 mM. It is intriguing due to the lack of undesirable activity of 10 mM VINP in normoxia. It is commonly accepted that VINP intensi®es stimulation of postsynaptic adenosine receptors (A1) as a result of inhibition of neuronal adenosine re-uptake (Krieglstein and Rischke, 1991). This mechanism leads to hyperpolarization of neuronal membranes and reduction of the Ca2 in¯ux through NMDA receptors (Choi, 1988). Moreover, stimulation of the presynaptic A1 receptors inhibits the release of glutaminian (Goldberg et al., 1988). With regard to our experiment the VINP activity pro®le is particularly interesting due to a considerable number of adenosine receptors on astrocytes (Murphy et al., 1991; Peakman and Hill, 1994) and the stimulating effect of adenosine on their proliferation (Abbarachio et al., 1994; Christjanson et al., 1993). In our study, VINP had a remarkable mitogenic in¯uence on astrocytes after 24 h reoxygenation. We also showed that the modulation of caspase-3 activity may be involved in the mechanism of PIR and VINP cytoprotective action. Caspase-3 activity is one of the fundamental events of the so called apoptosis execution phase. This enzyme decomposes various proteins, i.e. cell cycle regulatory proteins, poly(ADP-rybose)polymerase and activates other caspase family members, such as caspase-2, -6 and -7 (Nicholson, 1999). Recently, a caspase-3-activated deoxyribonuclease termed CAD (caspase activated domain) has been identi®ed which causes the degradation of chromosomal DNA into nucleosomal unitsÐlandmarks of apoptosis (Enari et al., 1998). We were encouraged to explore PIR and VINP in¯uence upon astrocytes exposed to 24 h hypoxia with subsequent 24 h reoxygention by the fact that during brain ischemia leading to serious neuron damage glial cells are activated in the form of gliosis with hypertrophy and increased astrocyte proliferation (Eng et al., 1987; Norton et al., 1992; Kimelberg and Norenberg, 1989). The factor stimulating mitogenesis is ATP released from damaged cells and acting synergistically with polypeptide growth factors, e.g. FGF (Rozengurt, 1986; Burnstock, 1993; Edwards et al., 1992). The results of both in vivo (Rathbone et al., 1992) and in vitro (Neary et al., 1994) studies indicating the GFAP immuno-reactivity, con®rm the
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participation of ATP in the development of reactive astrocytes. Twenty-four hour after the end of hypoxia we observed a signi®cant stimulation of astrocyte viability in parallel with the decrease of apoptotic nuclei number and caspase-3 activity to the level observed in normoxia as well as the increase of intracellular ATP and PCr concentration and intensi®cation of proliferation. Opinions, whether reactive gliosis contributes to escalated neuronal death or, on the contrary, exerts a neuroprotective effect, vary. Some authors claim that reactive astrocytes constitute a barrier for regrowing neurons, while others maintains that astrocytes may promote regeneration processes (Hatten et al., 1991; Kimelberg and Norenberg, 1989). The key enzyme mediating the proliferation of astrocytes in reactive post-injury gliosis is protein kinase C (PKC) (Yong, 1992). Astrocytes have been found to have signi®cant PKC activity (Bhat, 1989). However, of the many isoforms of PKC known to exist, astrocytes appear to have only the a- and b-isotypes (Todo et al., 1990; Masliash et al., 1991). The activation of PKC is believed to result from translocation of inactive cytosolic enzyme to the lipid environment of the membranes. Translocation of PKCa occurs during recirculation after brain ischemia (Cardell et al., 1990). Moreover, it has been proved that the PKCa isoenzyme is an apoptotic inhibitor and that particular effect is caused by the prevention of ceramide engagement in the apoptotic process. The analysis of some antioxidant enzymes (e.g. superoxide dismutaseÐ SOD) sequence revealed the existence of the site which might be phosphorylated by PKC. Therefore, it is likely that PKC participates in their activation. It seems to be particularly interesting due to a very effective system of ROS scavenging in astrocytes, which is based on marked activities of antioxidant enzymes, such as catalase, SOD, glutathione peroxidase (GPx) and glutathione reductase (GR) (Blaauwgeers et al., 1996; Aschner, 1998) and high level of reduced glutathione (GSH). Thus, the possible common mitogenic and antiapoptotic effect of reoxygenation and drugs caused by their possible in¯uence on the PKC activity in astrocytes should be considered. Therefore, the studies on PIR and VINP effects on the PKC activity of in astrocytes during hypoxia are warranted. Summing up, the glioprotective effects of PIR and VINP, we observed are the result of the drugs interference in a number of factors determining cell apoptosis and viability. Thus, the hereby studies presents new mechanisms of PIR and VINP antiischemic activity indicating their potential protective
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in¯uence towards astrocytes in hypoxia/reoxygenation condition. ACKNOWLEDGEMENTS The work was supported by grant of State Committee for Scienti®c Research 4 PO5D 00516. REFERENCES Abbarachio MP, Saffrey MJ, Hopker V, Burnstock G. Modulation of astroglial cell proliferation by analogues of adenosine and ATP in primary cultures of rat striatum. Neuroscience 1994;59: 67±76. Aschner M. Astrocytic functions and physiological reactions to injury: the potential to induce and/or exacerbate neuronal dysfunctionÐa forum position paper. NeuroToxicology 1998; 19:7±18. Bereczki D, Fekete I. A systematic review of vinpocetine therapy in acute ischaemic stroke. Eur J Clin Pharmacol 1999;55: 349±52. Bhat NR. Role of protein kinase C in glial cell proliferation. J Neurosci Res 1989;22:20±7. Blaauwgeers HGT, De Jong JMBV, Verspaget HW, Van den Berg FM, Troost D. Enhanced superoxide dismutase-2 immunoreactivity of astrocytes and occasional neurons in amyotrophic lateral sclerosis. J Neurol Sci 1996;140:21±9. Burnstock G. Hypoxia, endothelium, and purines. Drug Dev Res 1993;28:301±5. Cai J, Jones DP. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrom c loss. J Biol Chem 1998; 273:11401±4. Cardell M, Bingren H, Wieloch T, Zwin J, Saitoh T. Protein kinase C is translocated to cell membranes during cerebral ischemia. Neurosci Lett 1990;119:228±32. Choi DW. Glutamate neurotoxicity and diseases of the nervous systemÐreview. Neuron 1988;1:623±34. Christjanson LJ, Middlemiss PJ, Rathbone MP. Stimulation of astrocyte proliferation by purine nad pyrimidine nucleotides and nucleosides. Glia 1993;7:176±82. Coq J-C, Xerri Ch. Acute reorganization of the forepaw representation in the rat SI cortex after focal cortical injury: neuroprotective effects of piracetam treatment. Eur J Neurosci 2000;11:2597±608. Edwards FA, Gibb AJ, Colquhoun D. ATP receptor-mediated synaptic currents in the central nervous system. Nature 1992;359:144±7. Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell fated by apoptosis or necrosis. Cancer Res 1997;57:1835±40. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. A caspase-activated DNAse that degrates DNA during apoptosis, and its inhibitor ICAD. Nature 1998;391:43±50. Eng LR, Reier PJ, Houle JD. Astrocyte activation and fibrous gliosis: glial fibrillary acidic protein immunostaining of astrocytes following intraspinal cord grafting of fetal CNS tissue. Prog Brain Res 1987;71:439±55.
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Peuvot J, Schanck A, Deleers M, Brasseur R. Piracetam-induced changes to the membrane physical properties: a combined approach by 31 P nuclear magnetic resonance and conformation analysis. Biochem Pharmacol 1995;50:1129±34. Poeck K. Piracetam treatment in post-stroke aphasia. CNS Drugs 1998;9:51±6. Quian ZL, Jin LQ, Xie ML, Chen BQ. Effects of piracetam on Na()-K()-ATPase and monoamine oxidase in rat brain and its antioxidation effect. Chung Kuo Yao Li Hsueh Pao 1992;13:48±50. Rathbone MP, Middlemiss PJ, Kim JL, Gysbers JW, DeForge SP, Smith RW, Hughes DW. Adenosine and its nucleosides stimulate proliferation of chick astrocytes and human astrocytoma cells. Neurosci Res 1992;13:1±17. Rauca C, Zerbe R, Jantze H, Krug M. The importance of free hydroxyl radicals to hypoxia preconditioning. Brain Res 2000a;868:147±9. Rauca C, Jantze H, Krug M. Does fucose or piracetam modify the effect of hypoxia preconditioning against pentylenetetrazolinduced seizures? Brain Res 2000;880:187±90. Rozengurt E. Early signals in the mitogenic response. Science 1986;234:161±6. Sauer D, Rischke R, Beck T, Rossberg C, Mennel HD, Bielenberg GW, Krieglstein J. Vinpocetine prevents ischemic cell damage in rat hippocampus. Life Sci 1988;43:1733±9. Shearman MS, Hawtin SR, Tailor VJ. The intracellular component of cellular 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) reduction is specifically inhibited by b-amyloid peptides. J Neurochem 1995;65:218±27. Shimizu S, Eguchi Y, Kamiike W, Itoh Y, Hasegawa J, Yamabe K, Otsuki Y, Matsuda H, Tsujimoto Y. Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-XL. Cancer Res 1996;56:2161±6. Stockmans F, Deberdt W, Nystrom A, Nystrom E, Stassen JM, Vermylen J, Deckmijn H. Inhibitory effect of piracetam on plated-rich thrombus formation in an animal model. Throm Hoemost 1998;7:222±7. Susin SA, Lorenzo HK, Zamzani N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Constantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999;397:441±6. Takizawa S, Matsushima K, Shinohara Y, Komatsu N, Utsunomiya H, Watanabe K. Immunohistochemical localization of glutathione peroxidase in infracted human brain. J Neurol Sci 1994;122:66±73. Todo T, Shitara N, Nakamura H, Takakura K, Tomonaga M, Ikeda K. Astrocytic localization of the immunoreactivity for protein kinase C isozyme (type III) in human brain. Brain Res 1990;517:351±3. Zoratti M, Szabo' I. The mitochondrial permeability transition. Biochim Biophys Acta 1995;1241:139±76. Yong VW. Proliferation of human and mouse astrocytes in vitro: signaling through the protein kinase C pathway. J Neurol Sci 1992;111:92±103.
Piracetam and Vinpocetine Exert Cytoprotective Activity and Prevent Apoptosis of Astrocytes In Vitro in Hypoxia and Reoxygenation BozÇena Gabryel1,*, Mariusz Adamek1,2, Anna Pudeøko1, Andrzej Maøecki1, Henryk I. Trzeciak1 1
Department of Pharmacology, Silesian Medical University, 18 MedykoÂw St., PL 40752 Katowice, Poland Department and Clinic of Internal Diseases and Physical Medicine, Center for Laser Diagnostics and Therapy, Silesian Medical University, 15 Batory St., PL 41902 Bytom, Poland 2
Received 23 July 2001; accepted 7 November 2001
Abstract 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 under ¯uorescence 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 mM) 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 mM VINP to the cultures during hypoxia signi®cantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also con®rmed 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 mM) was detrimental in hypoxic conditions. Our experiment proved the signi®cant cytoprotective effect of 1 mM PIR and 0.1 mM VINP on astrocytes in vitro. # 2002 Published by Elsevier Science Inc.
Keywords: Piracetam; Vinpocetine; Astrocytes; Hypoxia; Reoxygenation; Apoptosis
INTRODUCTION Nootropic drugs, including piracetam (2-pyrrolidonN-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP), are used in the treatment of brain injures, cognitive dysfunction caused by hypoxia, hypogycaemia and heavy metal intoxication as well as in senile dementia of the Alzheimer's type (Nicholson, 1990; Gabryel and Trzeciak, 1994). * Corresponding author. Tel.: 48-3225-23835; fax: 48-3225-23835. E-mail address: [email protected] (B. Gabryel).
Results of studies using various models in vivo and in vitro of experimental brain ischemia as well as of clinical trials indicate neuroprotective properties of PIR and VINP (Krieglstein and Rischke, 1991; Erdo et al., 1990; Sauer et al., 1988). Yet, mechanisms through which the drugs exert the protective effects are different and not fully known. The ef®cacy of PIR in ischemia prevention is connected mainly with the drug positive in¯uence on neuronal cell membrane ¯uidity. It has been shown that PIR acts on the cell membrane, to which it binds, being inserted at the level of the polar heads of phospholipids (Muller et al., 1994, 1997; Peuvot et al., 1995). Moreover, PIR exerts
0161-813X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 1 6 1 - 8 1 3 X ( 0 2 ) 0 0 0 0 4 - 9
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antithrombotic as well as haemorrheological effects (Moriau et al., 1993; Stockmans et al., 1998). VINP, on the other hand, mainly inhibits intraneuronal adenosine re-uptake and also has vasodilatory effect (Krieglstein and Rischke, 1991; Gabryel and Trzeciak, 1994). Despite a number of experimental data concerning the protective effects of PIR (Poeck, 1998; Coq and Xerri, 2000; Rauca et al., 2000a,b) and VINP (Kiss et al., 1991; Bereczki and Fekete, 1999) on damaged neurons in vivo and in vitro it remains unknown whether they also have the glioprotective activity. It seems particularly important in the context of coexistence of necrotic and apoptotic death of both neurons and astrocytes as a result of ischemic brain damage. Studies on the pathomechanism of brain ischemia indicate that a decrease in neuronal survival during and after ischemia is also associated with astrocytic dysfunction (Aschner, 1998; Gabryel and Trzeciak, 2001). Therefore, using the cell culture model, we decided to investigate if and in what way drugs of potentially different neuroprotective mechanisms protect astrocytes from rat cerebral cortex in primary cell culture during in vitro simulated hypoxia and reoxygenation. For this purpose we decided to examine the effect of PIR and VINP on astrocyte apoptosis by determining the amount of apoptotic nuclei, the percentage of lactate dehydrogenase (LDH) released into the culture medium, caspase-3 activity, mitochondrial function, and by determining intracellular high-energy phosphates concentration (ATP and PCr). Additionally, we evaluated the proliferation of astrocytes treated in vitro with selected drugs in normoxia, hypoxia and reoxygenation. MATERIALS AND METHODS Materials Chemicals and materials were obtained from the following sources: PIR (Polfa, Poland), VINP (Cavinton1, Gedeon Richter, Hungary), Dulbecco's modi®ed Eagle's medium (DMEM), foetal bovine serum (FBS), phosphate buffered saline (PBS), antibioticantimycotic mixture (all from GIBCO Laboratories, USA), DAKO PAP kit system glial ®brillary acidic protein (GFAP) test (DAKO Co., Denmark), Live/Dead Kit (Molecular Probes Inc., USA), Caspase-3 Colorimetric Assay (R&D System, UK), ATP monitoring kit (Labsystems, Finland), creatine kinase (Boehringen Mannheim, Germany), adenosine 50 -pentaphosphate, ADP, poly-D-lysine, 3-(4,5-dimethylthazol-2-yl)-2,5-
diphenyltetrazolinum bromide (MTT), Hoechst 33342, etylenediaminetetraacetic acid disodium salt (EDTA 2 Na), trichloracetic acid (TCA), trypan blue solution (0.4%), NADH, sodium pyruvate and other chemicals all from Sigma (USA), [methyl-3 H] thymidine (2.00 Ci/ mM) (Dupont, NEN, USA), plastic tissue culture Petri dishes (Becton Dickinson, USA). Primary Culture of Rat Astrocytes Astrocytes were isolated and cultured essentially according to the method of Hertz et al. (1985). Brie¯y, hemispheres from one-day old Wistar rat pups were removed aseptically from skulls, freed of the meninges, minced and mechanically disrupted by vortexing in DMEM containing penicillin (100 U/ml) and streptomycin (100 mg/ml). This suspension was ®ltered through sterile nylon screening cloth with pore sizes 70 mm (®rst sieving) and 10 mm (second sieving). Subsequently, cultures were incubated at 37 8C in 95% air and 5% CO2 with relative humidity 95% (CO2-Incubator, Kebo±Assab, Sweden). Before the material was plated onto the plastic dishes the cells were counted in a Coulter Z1 counter (Coulter Counter, UK). Cells destined for bioluminescent and isotopic studies were plated at a density of 3 105 cells per dish (35 mm in diameter). For MTT conversion measurements cells were seeded at 1 104 per well on 96-well plates. Astrocytes for ¯uorescent studies (Live/Dead Kit and Hoechst 33342 staining) were cultured on coverslips covered with poly-D-lysine (100 mg/ml). The cells destined for determination of caspase-3 activity with the colorimetric assay were seeded onto plastic dishes of 100 mm in diameter at the density of 1 106 cells per dish. The culture medium initially contained 20% of FBS and after 4 days was replaced with medium containing 10% FBS. The whole volume of culture medium was changed twice a week. In order to remove contaminating non-astroglial cells, con¯uent cultures were shaken overnight on a rotary platform at 200 rpm (37 8C) (McCarthy and De Vellis', 1980). Cell culture medium was replaced after overnight shaking. To identify astrocytes, cultures (21 days old) were stained immunocytochemically for GFAP. Analysis of the cultures has shown that 90±95% of cells were GFAP-positive. All experiments were performed on 21-day cultures. Experimental Design Control astrocytes (Normoxia) were cultured in DMEM medium with 5.5 mM glucose and 10% FBS
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in 95% air/5% CO2. In order to mimic the in vivo hypoxia, cultures were placed for 24 h in 37 8C in 3% O2/5% CO2/92% N2 (CO2 incubator, Heraeus, Germany) in DMEM without glucose and serum. Osmolarity of the medium was measured and adjusted to 319 mOsm with mannitol. Some astrocyte cultures were exposed for 24 h to hypoxia (Hypoxia) or 24 h hypoxia followed by 24 h reoxygenation (Reoxygenation). Cells were treated with PIR (0.01 and 1 mM) and VINP (0.1 and 10 mM) according to three distinct protocols: (i) for 24 h in normoxia; (ii) for 24 h of hypoxia; (iii) for 24 h of reoxygenation. Treatment of astrocytes with PIR and VINP in normoxia and reoxygenation was conducted in a medium containing glucose and serum at 37 8C in 95% air/5% CO2. Cell Death Cell death was determined by a ¯uorescent method with the use of a Live/Dead Kit (Molecular Probes Inc., USA) containing calcein/AM and ethidium homodimer-1 (EthD-1). Calcein/AM is absorbed by living cells and subsequently converted by cytosolic esterases into a green ¯uorescent product (ex/em 495 nm/ 530 nm), whereas EthD-1 is known to enter only the cell with compromised cell membrane permeability and after being attached to nucleic acids yields red ¯uorescence (ex/em 495/635 nm). After twice washing with PBS both reagents diluted in PBS were applied to the cell cultures immediately after exposure to hypoxia. Astrocytes were incubated with 2 mM of calcein/AM and 4 mM of EthD-1 for 30 min at 37 8C. The number of dead and live cells was determined on at least six randomly selected areas from three coverslips of every experimental group, each containing approximately 200 cells. Cells were examined by ¯uorescence imaging MiraCal Pro III workstation (Life Science Resources Ltd., UK) comprising of inverted microscope Eclipse TE200 (Nikon, Japan) and high-resolution cooled CCD camera (Photonic Science Ltd., UK). The results were expressed as a percentage of dead cells relative to the total number of cells. Assessment of Apoptosis Cell nuclei staining by Hoechst 33342 (Molecular Probes Inc., USA) enables visualization of fragmented and condensed DNA, a characteristic feature of apoptosis. After washing with PBS astrocytes cultured on coverslips were ®xed for 10 min with a 4% paraformaldehyde
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at room temperature. Subsequently, after being washed twice with PBS the samples were dehydrated ®rst in 70% ethanol and then in absolute ethanol. The samples were kept in 20 8C until they were stained with Hoechst 33342 (5 mg/ml in PBS) for 5 min at room temperature. Then the cells were washed again with PBS. Cell nuclei analysis was conducted with the above-described imaging system (ex/em 340/510 nm). A 20 objective was used. The number of apoptotic nuclei was determined on at least six randomly selected areas from three coverslips of every experimental group, each containing approximately 200 cells. The results were expressed as a percentage of apoptotic cells relative to the total number of cells. Measurement of Caspase-3 Activity Enzymatic activity of caspase-3 was determined by Caspase-3 Colorimetric Assay (R&D System, UK), according to the manufacturer's instructions. This assay is based on the release of the ¯uorochrome p-nitroaniline (p-NA) combined with a caspase-3 speci®c peptide substrate (DEVD±p-NA). Peptide cleavage through active caspase-3 releases chromophor p-NA, which can be quanti®ed using colorimetric plate reader (405 nm). Brie¯y, cultured astocytes were lysed and cell extracts were centrifuged to eliminate cellular debris. Aliquots (20 ml of the cell extract) were incubated for 2 h at 37 8C in the presence of the substrate. The level of caspase-3 activity, proportional to the color reaction intensity was expressed as a percentage of control. Mitochondrial Function Mitochondrial function of astrocytes treated with PIR and VINP was evaluated with 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion method (Mosman, 1983). The cells ability to convert MTT indicates mitochondrial integrity and activity, which might in turn indicate cell viability. The cleavage of tetrazoline ring in MTT takes place mainly with the participation of the mitochondrial succinate dehydrogenase and depends on the activity of the respiratory chain and the redox state of the mitochondria (Mosman, 1983; Shearman et al., 1995). MTT (®nal concentrationÐ0.25 mg/ml) was added to the medium three hours before the scheduled end of the experiment and then the cultures were incubated at 37 8C in proper conditions. At the end of the experiment, after being washed twice with PBS, cells were lysed in 100 ml dimethyl sulphoxide which
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enabled the release of the blue reaction productÐ formasan. Absorbance at the wavelength of 570 nm was read on a microplate reader and results were expressed as a percentage of absorbance measured in control cells. Measurement of ATP and PCr Concentrations The ATP and PCr concentrations were determined by high-speci®c ®re¯y luciferin±luciferase bioluminescence assay system using a Fluoroscan Ascent plate reader (Labsystems, Finland). The extraction of ATP and PCr was performed according to procedure described by Fitzpatrick et al. (1988). The conversion of PCr into ATP catalyzed by creatine kinase was performed according to the method of Lowry and Passonneau (1972). ATP concentrations were measured by bioluminescence quanti®cation according to the method of Lust et al. (1981). Brie¯y, 100 ml samples of disrupted cells were added to equal volumes of ice-cold 5% TCA solution with 2 mM EDTA, vortexed rapidly for 5 s and incubated for 30 min at room temperature. Then the samples were 20-fold diluted with Tris±acetate buffer pH 7.75 and 10 ml solution of known ATP concentration was added to each well as internal standard and the total amount of ATP was determined. The results were calculated as nM of ATP or PCr per mg of protein (nM/mg). Lactate Dehydrogenase (LDH) Activity LDH activity was estimated by measuring the decrease in absorbance at 340 nm due to the conversion of enzyme co-factor NADH to NAD (one enzyme activity unit 0:001 DA/min). Brie¯y, 100 ml aliquots of culture media were collected and dissolved in 0.1 M pyruvate buffer (0.25 mg sodium pyruvate in PBS). NADH solution (0.25 mg in PBS) was then added to the samples. Cells were lysed by repeated freezing and thawing and total released LDH was measured. Absorbance was measured immediately after the addition of NADH and 60 s after the initial reading. The data are presented as the percentage of the total releasable LDH. [3H]Thymidine Incorporation The amount of incorporated [3 H]thymidine was measured according to the method of Neary et al. (1994) with slight modi®cations. In brief, astrocytes treated with PIR and VINP in the above-mentioned
experimental conditions were incubated with [3 H]thymidine (20 Ci/mM) 0.5 mCi/ dish (2 ml) for 18 h in DMEM containing 0.5% FBS. At the end of incubation, cells were washed twice with 1 ml of ice-cold phosphate-buffered saline (PBS). Then cells were scraped with a plastic policeman (Costar Co, USA) and centrifuged for 10 min (2000 rpm/min). A total of 1 ml 10% TCA was added to the sediment and samples were incubated at 4 8C for 30 min. The samples were centrifuged again for 10 min (2000 rpm/min). A total of 1 ml of 0.1 N NaOH was then added to each tube and samples were lysed overnight at 4 8C. Next day the samples were neutralized by 10% formic acid and transferred to scintillation vials. After addition of 8 ml of Bray scintillating solution, radioactivity was determined in a Beckman LS 6000 IC counter (Beckman Instruments Inc., USA). The results were calculated in dpm per mg of protein and expressed as percentage of control. Protein Determination Protein content in astrocytes was measured according to the method of Lowry et al. (1951). Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) followed by the Newman±Keuls test. A P-value of 0.05 was considered as statistically signi®cant. All data were expressed as mean S:D. RESULTS Effect of PIR and VINP on Cell Death The number of dead astrocytes in cell cultures treated with PIR and VINP in hypoxia was determined by ¯uorescent staining with the use of the Live/Dead Kit. Nuclei of cells undergoing necrosis stain red whereas calcein identi®es viable cells. In the astrocyte cultures exposed to normoxia or reoxygenation few dead cells were observed, therefore, the Live/Dead kit was only used for determining the increase in necrosis in cultures exposed to hypoxia. The percentage of dead cells stained with EthD-1 in comparison with the cells stained with calcein in particular groups is presented in Fig. 1. Hypoxia signi®cantly increased the number of dead astrocytes in the cultures as compared to the normoxia. Interestingly, adding 1 mM PIR into the culture medium in
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pictures were analyzed in order to determine the percentage of apoptotic cells in particular groups. The results are shown in Fig. 3A. After the 24 h reoxygenation period, we observed a similar amount of apoptotic nuclei as in normoxic conditions (Fig. 3B). The presence of 0.01 mM PIR or 10 mM VINP in the culture medium in these conditions signi®cantly increased the amount of apoptotic nuclei. Exposing astrocytes to 0.1 mM VINP during 24 h reoxygenation period turned out to be the most effective method in the prevention of morphological changes of nuclei induced by hypoxia. Influence of PIR and VINP on Caspase-3 Activity Fig. 1. Effect of piracetam or vinpocetine on astrocyte death induced by hypoxia. The results are shown as a percentage relation of dead cells to the total amount of cells in the field and are a mean S:D. of six randomly selected areas from three coverslips. At least 1000 cells were counted in each experiment. Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. The symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia.
hypoxia signi®cantly reduces the number of dead cells. 0.01 mM PIR or 0.1 mM VINP did not cause statistically signi®cant changes in the amount of dead astrocytes in hypoxic conditions. On the other hand, 10 mM VINP signi®cantly increased the number of dead cells which might indicate the drug toxic activity at this concentration. Influence of PIR and VINP on Apoptosis (Hoechst 33342 Staining) In order to determine the in¯uence of PIR and VINP on the process of apoptosis the astrocyte cell nuclei were visualized by DNA-speci®c ¯uorescent dye Hoechst 33342. Fig. 2 shows microphotographs representative for experimental groups. Most cells from the control group (Normoxia) had big, regular nuclei and few had apoptotic nuclei with condensated chromatin (Fig. 2A). In the cultures of astrocytes exposed to hypoxia many more picnotic nuclei occured (Fig. 2B). Treatment of astrocytes with 0.01, 1 mM PIR or 0.1 mM VINP during hypoxia prevented the process of apoptosis (Fig. 2C, D, E). However, exposure of the cells with 10 mM VINP in these conditions signi®cantly increased the amount of apoptotic nuclei in comparison with control (Fig. 2F). The microscopic
Exposure of astrocyte cultures to hypoxia signi®cantly increased caspase-3 activity. Treatment with 0.01 mM or 1 mM PIR as well as 0.1 mM VINP did not change the enzyme proteolytic activity in comparison to normoxia. However, simultaneous exposure of astrocytes to hypoxia and 10 mM VINP substantially increased the caspase-3 activity (Fig. 4A). During a 24 h reoxygenation period and after PIR (0.01 or 1 mM) administration a similar activity of the enzyme to that in normoxia was observed. In these conditions, treatment of astrocytes with VINP caused the increase of the intracellular caspase-3 activity (Fig. 4B). Effect of PIR and VINP on Mitochondrial Function Mitochondrial function was measured by the MTT reduction method, which may also serve as a general indicator of cell viability. Fig. 5. shows the results of the in¯uence of PIR and VINP on MTT conversion into formasan in the cultures. A substantial increase of MTT conversion in astrocytes in normoxic conditions stimulated both by PIR and VINP indicated a signi®cant increase in mitochondrial activity (Fig. 5A). A 24 h hypoxia period attenuated the conversion of MTT. A similar effect was observed after an additional exposition of astrocytes in these conditions to 0.01 mM PIR or 10 mM VINP. However, treatment with 1 mM PIR or 0.01 mM VINP induced the MTT conversion process during 24 h hypoxia, although the process intensity was weaker in comparison with the cells in normoxia (Fig. 5B). The 24 h reoxygenation as well as 1 mM PIR administration during this period weakened mitochondrial functions. The exposition of astrocytes in these conditions to 0.01 mM PIR, 1 mM PIR or 0.1 mM VINP increased the MTT conversion (Fig. 5C).
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Fig. 2. Effect of piracetam or vinpocetine on astrocyte apoptosis induced by hypoxia. Cell nuclei were stained with the Hoechst 33342 dye. Microphotographs show experimental groups of astrocyte cultures treated with drugs according to protocols described in Section 2. Microphotographs were taken with the use of fluorescent microscope and 20 objective. Thin arrows point at apoptotic cell nuclei and the thick arrows point at live cell nuclei. (A) normoxia; (B) hypoxia; (C) 0.1 mM piracetam; (D) 1 mM piracetam; (E) 0.1 mM vinpocetine; (F) 10 mM vinpocetine.
Influence of PIR and VINP on Intracellular ATP and PCr Concentrations Tables 1 and 2 show changes in the intracellular amount of ATP and PCr in astrocytes treated with PIR and VINP in normoxia, hypoxia and reoxygenation. Increased ATP and PCr levels in cells treated with 1 mM PIR or 0.1 mM VINP was observed in normoxia. The exposure of astrocytes to 24 h hypoxia caused a signi®cant reduction in the amount of both high-energy phosphates. In this experimental option only 1 mM PIR produced increase of the ATP concentration in cells. The most signi®cant decrease in value of this parameter was observed in hypoxia after treatment with 10 mM VINP. VINP at the concentration 0.1 mM maintained the amount of ATP and PCr at the level similar to normoxia control. After 24 h reoxygenation the concentration of both ATP and PCr in astrocytes increased signi®cantly. The increase in amount of both high-
energy phosphates was also observed as a result of astrocytes treatment with 0.01 or 1 mM PIR as well as 0.1 mM VINP. At the same time no changes were observed in intracellular concentrations of ATP and PCr when the cells were exposed during reoxygenation to 10 mM VINP. Influence of PIR and VINP on LDH Release The LDH level in the medium of normoxic culture was very low (Fig. 6A). After 24 h hypoxia, an increase in LDH activity in the medium occurred as a result of cell membrane integrity loss and an enzyme leakage. (Fig. 6B). Both drugs added in normoxia reduce the LDH activity. The amount of released LDH increased in cultures exposed simultaneously to hypoxia as well as to 0.01, 1 mM PIR or 10 mM VINP. In these conditions only 0.1 mM VINP did not cause the increase of LDH activity in the medium (Fig. 6B). A signi®cant
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Fig. 3. Effect of piracetam or vinpocetine on astrocyte apoptosis induced by hypoxia (A) and reoxygenation (B). The results are shown as a percentage relation of the apoptotic nuclei to the total amount of nuclei in the field and are a mean S:D. of the six randomly selected areas from three coverslips. At least 1000 cells were counted in each experiment. Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment; The symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
reduction of the LDH release was observed 24 h after hypoxia both in cultures subjected only to reoxygenation as well as treated with the tested drugs (Fig. 6C). Influence of PIR and VINP on [3H]thymidine Incorporation [3 H]thymidine incorporation into astrocytes treated with PIR and VINP was studied as an indicator of DNA synthesis and astrocytic proliferation.
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Fig. 4. Effect of piracetam or vinpocetine on caspase-3 activity in cultured rat astrocytes exposed to hypoxia (A) and reoxygenation (B). The results are shown as a percentages of the control value in the normoxia conditions. Data were analyzed with the use of oneway ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. Each value is mean of eight dishes S:D. (n 8); the symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
An insigni®cant increase in [3 H]thymidine incorporation occurred after administration of 0.01 mM PIR and 0.1 mM VINP into the culture medium in normoxia (Fig. 7A). It has been observed that 24 h hypoxia slowed [3 H]thymidine incorporation in comparison with the cells maintained in normoxia conditions. Similarly, exposure of hypoxic astrocytes to the tested concentrations of PIR and VINP abated [3 H]thymidine incorporation into their DNA (Fig. 7B). However, the increase in proliferation of astrocytes was
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Fig. 5. Effect of piracetam and vinpocetine on mitochondrial function of cultured rat astrocytes exposed to normoxia (A), hypoxia (B) and reoxygenation (C) measured by MTT conversion assay. Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman± Keuls test was performed in order to compare effects of studied drugs treatment. The results are shown as a percentage relation of the control value in the normoxia conditions. Data are S.D. (n 8); the symbol () P < 0:05 vs. normoxia; (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
Fig. 6. Effect of piracetam and vinpocetine on LDH release in cultured rat astrocytes exposed to normoxia (A), hypoxia (B) and reoxygenation (C). Data were analysed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. The results are shown as a percentage relation of the control value in the normoxia conditions. Data are S.D. (n 8); () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
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Table 1 Effect of piracetam and vinpocetine on ATP levels in cultured rat astrocytesa Conditions
24-h Normoxia
24-h Hypoxia
24-h Reoxygenation
Protein (nM/mg) Untreated Piracetam (0.01 mM) Piracetam (1 mM) Vinpocetine (0.1 mM) Vinpocetine (10 mM)
33.4 47.7 48.7 54.2 36.8
8.7 12b 14.2b 5.1b 13.9b
14.0 23.4 43.6 29.4 4.4
2.1b 12b,c 8.3b,c 7.6c 2.2b,c
99.8 57.8 106.6 125.6 43.7
10.2b 16.7b,d 10.4b 24.3b 8.8b,d
a
Data were analysed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. Each value is mean obtained from eight dishes S.D. (n 8). b P < 0:01 vs. normoxia. c P < 0:01 vs. hypoxia. d P < 0:01 vs. reoxygenation.
Table 2 Effect of piracetam and vinpocetine on intracelluar PCr levels in cultured astrocytesa Conditions Untreated Piracetam (0.01 mM) Piracetam (1 mM) Vinpocetine (0.1 mM) Vinpocetine (10 mM)
24-h Normoxia 52.6 76.1 108.3 120.0 63.4
11.3 22.4b 20b 16.2b 13.4b
24-h Hypoxia 30.1 29.5 29.7 45.4 25.8
b
6.1 5.7b 4.8b 12.5c 6.2b,c
24-h Reoxygenation 61.9 136.2 133.8 78.9 65.1
9.9b 34.5b,d 16.9b,d 12.5b,d 13.9b
a Data were analysed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. Each value is mean obtained from eight dishes S.D. (n 8). b P < 0:01 vs. normoxia. c P < 0:01 vs. hypoxia. d P < 0:01 vs. reoxygenation.
observed during 24 h reoxygenation. Yet, intensity of the [3 H]thymidine incorporation differed among groups and depended both on the kind of the drug examined and on the concentration used (Fig. 7C). The biggest increase in the astrocytes' DNA synthesis was induced by treatment with 0.1 mM VINP. A weaker mitogenic effect was observed during reoxygenation after 10 mM VINP administration to the cultures. The results obtained indicated that PIR and VINP prevented the proliferation of astrocytes during 24 h hypoxia, but stimulated their multiplication during reoxygenation. The most intensive reaction was caused by exposure of astrocytes to 0.1 mM VINP both in normoxia and reoxygenation conditions. DISCUSSION In the cultures subjected to 24 h hypoxia we observed a signi®cant increase in the number of both necrotic and apoptotic astrocytes in comparison to the control. The changes were accompanied by signi®cant energy depletion, cell membrane desintegra-
tion, impairment of mitochondrial function, caspase-3 activation and inhibition of cell proliferation. There are authors suggesting that nootropic drugs including PIR and VINPare effective only in cells injured by various factors, e.g. hypoxia or trauma (Giurgea and Salama, 1977; King, 1987). However, in the experiments we conducted the exposure of astrocytes to the drugs in normoxia signi®cantly intensi®ed the MTT reduction and decreased the amount of LDH released into the medium. The phenomenon was accompanied by elevated intracellular ATP and PCr concentration and increased intensity of cell proliferation re¯ected by the amount of incorporated [3 H]thymidine. Therefore, the results obtained did not indicate the toxic activity of any of the drugs in selected concentrations towards astrocytes in normoxia conditions. Perineuronal glial cells response to hypoxic brain damage by transforming themselves into so called reactive astrocytes. They are characterized by stellate morphology, increased GFAP expression and increased antioxidant activity (Petito, 1986; Liu et al., 1993; Takizawa et al., 1994). However, prolonged hypoxia, causes disturbances in the astrocytes functions, such
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Fig. 7. Effect of piracetam or vinpocetine on [3 H]thymidine incorporation into cultured rat astrocytes astrocytes exposed to normoxia (A), hypoxia (B) and reoxygenation (C). Data were analyzed with the use of one-way ANOVA in order to compare mean value among different groups. Newman±Keuls test was performed in order to compare effects of studied drugs treatment. The results are shown as a percentage relation of the control value in the normoxia conditions. Each value is mean activity obtained form eight dishes S:D. (n 8); the symbol () P < 0:05 vs. normoxia; the symbol (#) P < 0:05 vs. hypoxia; the symbol ( ) P < 0:05 vs. reoxygenation.
as: loss of cell volume control resulting in swelling of brain parenchyma and constriction of blood vessels, leading consequently to termination of brain circulation (Hossmann, 1985). Hypoxia induces cell death possessing features characteristic for both necrosis and apoptosis (Shimizu et al., 1996). It is currently believed that mitochondria are the intracellular organelles which constitute a kind of switch between necrosis and apoptosis. The factors determining the type of cell death are the change in their membrane permeability as a result of the occurrence of mitochondrial permeability transition pore (MTP) (Zoratti and Szabo', 1995) and intracellular level of ATP (Leist et al., 1997; Eguchi et al., 1997). The opening of MTP is accompanied by inhibition of oxidative phosphorylation and activation of the mitochondrial ATP-ase which increases the consumption of ATP (Nieminen et al., 1994; Cai and Jones, 1998). When glycolysis or the use of energy stores accumulated in PCr are able to partly compensate the ATP de®ciency, the apoptosis program is commenced. However, if sudden and parallel mitochondrial transmembrane potential (Dc) break down occurs in the majority of mitochondria, the intracellular energy de®ciency is so severe that the live functions are stopped leading to necrosis (Kroemer et al., 1998). The energy stored in ATP is necessarily used in major apoptotic pathways leading from cytochrome c (Li et al., 1997), through apoptosis inducing factor (AIF), cell death receptors to caspases activity (Susin et al., 1999). Likewise, typical for apoptosis morphological changes of the cell nuclei are the result of the signal transduction from cytoplasm to the nuclei through active transport requiring ATP hydrolysis (Melchior and Gerace, 1995; Pante and Aebi, 1996). The extent of astrocyte damage in hypoxia was radically limited when they were simultaneously exposed to 1 mM PIR or 0.1 mM VINP. In our study, PIR added during hypoxia prevented necrosis as well as apoptosis of the astrocytes. The drug prevented ATP loss probably through partial use of PCr as an energy store which also enabled maintenance of mitochondria capacity. Reports demonstrating the normalization of the pathological platelet aggregation (Grotemeyer et al., 1986) and the increase in erythrocyte deformation (Nalbandian et al., 1983) suggest that PIR may also in¯uence the variety of cell types other than neurons. PIR ability to restore the disturbed cell membrane ¯uidity seems particularly interesting in the context of the loss of membrane asymmetry in the initial stage of apoptosis as a results of the phosphatidylserine externalization (Leist et al., 1997). The conceivable mechanism of PIR antiapoptic activity
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is also supported by its recently reported antioxidant properties (Quian et al., 1992; Rauca et al., 2000a,b). In case of VINP, both during hypoxia and reoxygenation, we observed signi®cant differences in its activity depending on the concentration. The obtained results indicate the protective effect of VINP on astrocytes in hypoxia and reoxygenation only at concentration 0.1 mM. It is intriguing due to the lack of undesirable activity of 10 mM VINP in normoxia. It is commonly accepted that VINP intensi®es stimulation of postsynaptic adenosine receptors (A1) as a result of inhibition of neuronal adenosine re-uptake (Krieglstein and Rischke, 1991). This mechanism leads to hyperpolarization of neuronal membranes and reduction of the Ca2 in¯ux through NMDA receptors (Choi, 1988). Moreover, stimulation of the presynaptic A1 receptors inhibits the release of glutaminian (Goldberg et al., 1988). With regard to our experiment the VINP activity pro®le is particularly interesting due to a considerable number of adenosine receptors on astrocytes (Murphy et al., 1991; Peakman and Hill, 1994) and the stimulating effect of adenosine on their proliferation (Abbarachio et al., 1994; Christjanson et al., 1993). In our study, VINP had a remarkable mitogenic in¯uence on astrocytes after 24 h reoxygenation. We also showed that the modulation of caspase-3 activity may be involved in the mechanism of PIR and VINP cytoprotective action. Caspase-3 activity is one of the fundamental events of the so called apoptosis execution phase. This enzyme decomposes various proteins, i.e. cell cycle regulatory proteins, poly(ADP-rybose)polymerase and activates other caspase family members, such as caspase-2, -6 and -7 (Nicholson, 1999). Recently, a caspase-3-activated deoxyribonuclease termed CAD (caspase activated domain) has been identi®ed which causes the degradation of chromosomal DNA into nucleosomal unitsÐlandmarks of apoptosis (Enari et al., 1998). We were encouraged to explore PIR and VINP in¯uence upon astrocytes exposed to 24 h hypoxia with subsequent 24 h reoxygention by the fact that during brain ischemia leading to serious neuron damage glial cells are activated in the form of gliosis with hypertrophy and increased astrocyte proliferation (Eng et al., 1987; Norton et al., 1992; Kimelberg and Norenberg, 1989). The factor stimulating mitogenesis is ATP released from damaged cells and acting synergistically with polypeptide growth factors, e.g. FGF (Rozengurt, 1986; Burnstock, 1993; Edwards et al., 1992). The results of both in vivo (Rathbone et al., 1992) and in vitro (Neary et al., 1994) studies indicating the GFAP immuno-reactivity, con®rm the
29
participation of ATP in the development of reactive astrocytes. Twenty-four hour after the end of hypoxia we observed a signi®cant stimulation of astrocyte viability in parallel with the decrease of apoptotic nuclei number and caspase-3 activity to the level observed in normoxia as well as the increase of intracellular ATP and PCr concentration and intensi®cation of proliferation. Opinions, whether reactive gliosis contributes to escalated neuronal death or, on the contrary, exerts a neuroprotective effect, vary. Some authors claim that reactive astrocytes constitute a barrier for regrowing neurons, while others maintains that astrocytes may promote regeneration processes (Hatten et al., 1991; Kimelberg and Norenberg, 1989). The key enzyme mediating the proliferation of astrocytes in reactive post-injury gliosis is protein kinase C (PKC) (Yong, 1992). Astrocytes have been found to have signi®cant PKC activity (Bhat, 1989). However, of the many isoforms of PKC known to exist, astrocytes appear to have only the a- and b-isotypes (Todo et al., 1990; Masliash et al., 1991). The activation of PKC is believed to result from translocation of inactive cytosolic enzyme to the lipid environment of the membranes. Translocation of PKCa occurs during recirculation after brain ischemia (Cardell et al., 1990). Moreover, it has been proved that the PKCa isoenzyme is an apoptotic inhibitor and that particular effect is caused by the prevention of ceramide engagement in the apoptotic process. The analysis of some antioxidant enzymes (e.g. superoxide dismutaseÐ SOD) sequence revealed the existence of the site which might be phosphorylated by PKC. Therefore, it is likely that PKC participates in their activation. It seems to be particularly interesting due to a very effective system of ROS scavenging in astrocytes, which is based on marked activities of antioxidant enzymes, such as catalase, SOD, glutathione peroxidase (GPx) and glutathione reductase (GR) (Blaauwgeers et al., 1996; Aschner, 1998) and high level of reduced glutathione (GSH). Thus, the possible common mitogenic and antiapoptotic effect of reoxygenation and drugs caused by their possible in¯uence on the PKC activity in astrocytes should be considered. Therefore, the studies on PIR and VINP effects on the PKC activity of in astrocytes during hypoxia are warranted. Summing up, the glioprotective effects of PIR and VINP, we observed are the result of the drugs interference in a number of factors determining cell apoptosis and viability. Thus, the hereby studies presents new mechanisms of PIR and VINP antiischemic activity indicating their potential protective
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