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Ebselen prevents excitotoxicity provoked by glutamate in rat cerebellar granule neurons
Neuroscience Letters 299 (2001) 217±220
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Ebselen prevents excitotoxicity provoked by glutamate in rat cerebellar granule neurons Lisiane O. PorciuÂncula a, JoaÄo Batista T. Rocha b, Carina R. Boeck a, Deusa Vendite a, Diogo O. Souza a,* a
Departamento de BioquõÂmica, ICBS, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil b Departamento de QuõÂmica, CCNE, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Received 10 October 2000; received in revised form 7 December 2000; accepted 2 January 2001
Abstract Ebselen is a selenium compound that have glutathione peroxidase-like activity which is neuroprotective in acute stroke ischemia. The ef®cacy of ebselen to prevent excitotoxicity provoked by glutamate in cerebellar granule neurons was investigated at various time points and concentrations. Simultaneous addition of ebselen with glutamate decreased neuronal death and was completely reversed by 3 mM of ebselen (3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide assays). However, when 1 mM of ebselen was added with glutamate and remained in the culture medium until 24 or 48 h, the neuronal survival increased to the control. The mechanism proposed for neuroprotection was the ability of ebselen to prevent lipoperoxidation provoked by glutamate. The present ®ndings propose to amplify the use of ebselen in others neurodegenerative disorders involving glutamatergic system. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ebselen; Neurotoxicity; Neuroprotection; Glutamate; Cerebellar granule neurons; Lipoperoxidation
Ebselen, 2-phenyl-1,2-benzisoselenazol-3[2H]-one, is a lipid-soluble seleno-organic compound that has glutathione peroxidase-like activity [8,12]. Recently, it has been demonstrated that this compound has a protective effect against brain ischemic insults and decreases the citotoxic effects of 4-hydroxynonenal in spinal cord neurons [9,18,19]. The mechanism underlying the neuroprotection afforded by ebselen is still not completely understood, but it is certainly related to its antioxidant and antiin¯amatory properties [12,15,18]. Recently, clinical trials with humans indicated that ebselen has bene®cial effects on pathological situations where glutamate is involved, such as ischemia and stroke [19]. However, direct evidence supporting that neuroprotective effect of ebselen on the glutamate-induced neurotoxicity is lacking in the literature. Glutamate is the main excitatory neurotransmitter in mammals [13]. However, over stimulation of the glutamatergic neurotransmission may be neurotoxic and can lead to cell death [2]. In fact, excitatory amino acids have been proposed to be involved in the pathophysiology of acute * Corresponding author. Fax: 155-51-316-5540/5535. E-mail address: [email protected] (D.O. Souza).
brain injury and chronic neurodegenerative diseases, such as ischemia, head trauma, stroke, Alzheimer's disease and Parkinson's disease [13]. Reactive oxygen species may be involved in neuronal death caused by alterations in excitatory amino acid neurotransmission [6]. Primary cultures of granule cells derived from cerebella of postnatal rats are endowed with glutamate receptors [3]. Due to their homogenous cellular composition, observations on these cultures re¯ect properties of a single neuronal cell type, making this cellular system one of the most appropriate for the study of glutamate-evoked biological responses. In the present report, the potential neuroprotective effect of ebselen on excitotoxicity induced by glutamate in cerebellar granule neurons culture was investigated. Primary cultures of cerebellar granule cells were prepared from 8-day-old Wistar rats [16]. Brie¯y, freshly dissected cerebella were incubated with 0.025% trypsin solution for 15 min at 378C and disrupted mechanically in the presence of 0.08 mg/ml DNase and 0.05% trypsin inhibitor. For the experiments, the cells were seeded at a density of 2.5 £ 10 5 cell/cm 2 in a 24-well multiwell dish or 6-well dish, coated with 10 mg/ml poly-d-lysine in Eagle's basal medium (BME) supplemented with 10% fetal bovine serum,
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 51 9- 1
218
L.O. PorciuÂncula et al. / Neuroscience Letters 299 (2001) 217±220
50 mg/ml gentamicin and 25 mM KCl. The growth of nonneuronal cells was inhibited by addition of 20 mM cytosine arabinofuranoside 18±20 h after seeding and the medium was maintained without change during the culture period. At 8±9 DIV, cultures were washed twice with Locke's solution (mM): 154 NaCl, 5.6 KCl, 3.6 NaHCO3, 2.3 CaCl2, 5.6 glucose, 5 HEPES, pH 7.4 and were then incubated at 20±228C for 25 min in 1 ml of Locke's solution, with or without 100 mM glutamate, and in the presence or absence of ebselen (Sigma, St. Louis MO) or vehicle. Ebselen was diluted in 50% (v/v) of ethanol. The ®nal concentration of ethanol in control and ebselen-treated samples were 0.25%. After 25 min, the Locke's solution was removed, the cultures were washed twice with fresh solution and then replaced with the previously saved culture-conditioned medium (with or without ebselen) and incubated for 3 h (for lipid peroxidation) or 24 h and 48 h (viability assays) at 378C, 5% CO2. So, the interaction between glutamate and ebselen was evaluated at various time points: cells were exposed (a) to glutamate; (b) to glutamate and ebselen simultaneously; (c) to ebselen after glutamate exposure; (d) to ebselen and glutamate simultaneously and after this incubation just ebselen remained in the culture medium. Cell cultures viability were assessed by MTT, propidium iodide and trypan blue. For MTT, cells (24 and 48 h postexposure) were assessed by the colorimetric MTT 3 (4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [10]. Brie¯y, cultures were incubated for 30 min at 378C with 1 ml Locke's solution with 1.2 MgCl2, containing 0.5 mg/ml of MTT. Only viable cells are able to reduced MTT, into formazan product which was soluble in dimethyl sulfoxide (DMSO) and measured at 490 and 630 nm. For propidium iodide, cells (24 h post-exposure) were stained with 7 mg/ml propidium iodide for 5 min in Locke's Solution with magnesium. Stained cultures were examined and photographed using a ¯uorescence imaging system (Nikon Eclipse TE 300 Diaphot microscope) with standard rhodamine ®lter set (excitation 540 nm; emission 617 nm). For trypan blue, cells (24 h post-exposure) were stained with 0.4 g% of trypan blue in Locke's Solution for 5 min. After, cells were visualized and counted in phase contrast microscopy. Ebselen did not interfere in these viability assays when was incubated alone. Thiobarbituric acid reactive substances (TBARS) were assessed to evaluate the extent of lipid peroxidation in the presence or absence of glutamate (100 mM) and ebselen (3 mM). Brie¯y, after 3 h of glutamate exposure with or without ebselen, the cells were treated with one milliliter of trichloroacetic acid (15% w/v) containing 1 mM ethylenediaminetetra-acetic acid (EDTA). Then, an equal volume of thiobarbituric acid (0.7% w/v) was added and samples heated at 1008C for 20 min. After cooling, the samples were centrifuged at 1000 £ g for 10 min and the supernatant color was monitored at 532 nm. Concentrations of TBARS were calculated using standard curve obtained with malondialdehyde and expressed as nmol of malonilaldehyde formed per well.
All assays were performed from independent cultures. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan's post hoc test. Exposure of cerebellar neurons to 100 mM glutamate for 25 min caused a signi®cant decline (to 40% of control cells) in mitochondrial activity (as observed by MTT reduction to formazan, measured 24 h after glutamate exposure), suggesting neuronal death. Simultaneous addition of ebselen (0.1±3 mM) with glutamate decreased signi®cantly the neuronal death (Fig. 1A). The protection was maximum with the highest concentration of ebselen tested (3 mM). Similar results were obtained by cell counting following trypan blue staining (data not shown) indicating that reduction of MTT coincides with a commensurable reduction of the cell viability. The acute response to glutamate (observed
Fig. 1. (A) Effect of 0.1±3 mM ebselen on glutamate neurotoxicity (24 h after glutamate exposure). Glutamate (100 mM) and ebselen were added simultaneously for 25 min. Values are means ^ SEM (n 4±7); (a) different from all other groups (P , 0.05); (b) different from control and glutamate (P , 0.05). (B) The effect of 1 mM ebselen added in different time points. C, control; G, glutamate 100 mM for 25 min; GE, glutamate and ebselen added simultaneously for 25 min. E, Ebselen added immediately after incubation for 25 min with glutamate and remained in the culture medium until 24 h after the glutamate withdrawal; GEE, glutamate and ebselen added simultaneously for 25 min and ebselen remained in the culture medium until 24 h after the glutamate withdrawal. The mitochondrial activity was tested 24 h after glutamate exposure. Values are means ^ SEM (n 4±5); (a) different from all other groups (P , 0.05); (b) different from control, G and GEE (P , 0.05).
L.O. PorciuÂncula et al. / Neuroscience Letters 299 (2001) 217±220
219
Fig. 2. Fluorescent images of staining with propidium iodide after 24 h of glutamate exposure for 25 min. (A) control; (B) glutamate 100 mM; (C) glutamate 100 mM plus ebselen 3 mM for 25 min. Scale bar, 100 mm.
in the ®rst 5 min of incubation) was characterized by rapid swelling and loss of brightness, as observed by phasecontrast microscopy. Ebselen in all concentrations tested was unable to prevent this acute effect of glutamate (data not shown). Subsequently, we examined whether ebselen confers protection when administered after the glutamate (Fig. 1B). Ebselen (1 mM) added to cerebellar neurons at same time with glutamate or just after exposure to glutamate increased signi®cantly the cell survival from about 40% (glutamate alone) to 60% (P , 0.05). Moreover, the neuronal survival (85%) was not statistically different from control, when ebselen (1 mM) was present simultaneously with glutamate and remained in the culture medium until viability assays after 24 h (Fig. 1B) or 48 h (data not shown). The protection conferred by ebselen (3 mM) against glutamate-induced neuronal death was also observed by staining with propidium iodide (Fig. 2), as observed with the MTT and trypan blue assays. In Fig. 3 we evaluated the ability of ebselen to prevent the lipid peroxidation (used as an index of oxidative damage) provoked by glutamate (100 mM). Ebselen (3 mM) was added simultaneously with glutamate for 25 min and remained in the culture medium until the assay (3 h after
Fig. 3. Effect of ebselen (3 M) in to prevent lipid peroxidation provoked by treatment with glutamate. C, Control; G, glutamate (100 mM) for 25 min; GEE, glutamate (100 mM) for 25 min plus ebselen (3 mM) added simultaneously with glutamate and remained until 3 h in the culture medium. Values are means ^ SEM of ®ve experiments independents. *Different from control and GEE (P , 0.01).
the glutamate exposure). Glutamate treatment increased 135% of lipid peroxidation and ebselen completely prevented the glutamate-triggered effect. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated through NMDA-receptor activation [13]. Overstimulation of NMDA receptors with glutamate results in an excessive in¯ux of Ca 21, that activates neurotoxic mechanisms, including the production of reactive oxygen species, such as superoxide, hydrogen peroxide and nitric oxide [1,4,14,17,20]. Hydrogen peroxide act as a messenger in some synapses [20]. However, one of the breakdown products of this compound is one of the most reactive oxygen specie (OH z), which is an effective neurotoxin, that produces oxidative stress and decreases glutathione levels [5]. Since ebselen demonstrated ef®cacy to prevent lipid peroxidation [11] is plausible to suppose that ebselen protect against glutamate toxicity by enhancing the reduction of hydrogen peroxide to non-reactive products, avoiding the cascade of reactions which may culminate in peroxidation of lipids present in neuronal membranes. Accordingly, ebselen (3 mM) abolished glutamate-triggered lipoperoxidation (Fig. 3). Interestingly, the protection afforded by ebselen greatly depended on the time point and concentration of this compound in relation to glutamate. Ebselen (3 mM) was the most protective concentration when added simultaneously with glutamate for 25 min and low concentrations afforded a partial protection. However, a similar maximum protection was conferred when 1 mM of ebselen was presented together with glutamate, remaining in the culture medium after the glutamate insult for 24 h (Fig. 1B) and for 48 h (data not shown). The delayed addition of 1 mM ebselen after the glutamate exposure and up to assays protected partially, suggesting that ebselen can scavenger reactive intermediates primarily triggered by glutamate or generated by early reactive intermediates in a series of reactions. Ischemia is associated with an excessive activation of the glutamatergic system and membrane lipid peroxidation [7,13]. Many pharmacological tools such glutamate antagonists, have been developed to improve outcome for ischemic patients and to limit their brain damage. However, gluta-
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mate antagonists have not shown the expected ef®cacy, while ebselen was more effective than other proposed neuroprotective agents tested [7]. In conclusion, ebselen is a selenium analogue that protected cerebellar granule neurons from glutamate neurotoxicity. This effect was time and concentration dependent and it seems be involved with ability of ebselen to prevent lipid peroxidation provoked by glutamate. Thus, our ®ndings allow to reinforce the clinical potential use of ebselen for diseases that involve overstimulation of the glutamatergic system. [1] Bindokas, V.P., JordaÂn, J., Lee, C.C. and Miller, R.J., Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine, J. Neurosci., 16 (1996) 1324± 1336. [2] Choi, D.W., Glutamate receptors and the induction of excitotoxic neuronal death, Prog. Brain. Res., 100 (1994) 47±51. [3] Cox, J.A., Felder, C. and Hennebury, R.C., Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons, Neuron, 4 (1990) 941±947. [4] Dugan, L.L., Sensi, S.L., Canzoniero, L.M.T., Handran, S.D., Rothman, S.M., Lin, T.S., Goldberg, M. and Choi, D.W., Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-d-aspartate, J. Neurosci., 15 (1995) 6377±6388. [5] Hoyt, K.R., Gallagher, A.J., Hastings, T. and Reynolds, I.J., Characterization of hydrogen peroxide toxicity in cultured rat forebrain neurons, Neurochem. Res., 22 (1997) 333±340. [6] Lafon-Cazal, M., Pietri, T.G., Culcasi, M. and Bockaert, J., NMDA-dependent superoxide production and neurotoxicity, Nature, 364 (1993) 535±537. [7] Lee, J.M., Zipfel, G. and Choi, D.W., The changing landscape of ischaemic brain injury mechanisms, Nature., 24 (1999) 399. [8] Maiorino, M., Roveri, A., Coassin, M. and Ursini, F., Kinetic mechanism and substrate speci®city of glutathione peroxidase activity of ebselen (PZ51), Biochem. Pharmacol., 37 (1988) 2267±2271. [9] Malecki, A., Garrido, R., Mattson, M.P., Henning, B. and Toborek, M., 4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons, J. Neurochem., 74 (2000) 2278±2287. [10] Mosmann, T., Rapid colorimetric assay for cellular growth
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and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65 (1983) 55±63. MuÈller, A., Gabriel, H. and Sies, H., A novel biologically active selenoorganic compound±IV. Protective glutathione-dependent effect of PZ 51 (ebselen) against ADP-Fe induced lipid peroxidation in isolated hepatocytes, Biochem. Pharmacol., 34 (1985) 1185±1189. MuÈller, A., Cadenas, E., Graf, P. and Sies, H., A novel biologically active seleno-organic compound I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen), Biochem. Pharmacol., 33 (1984) 3235±3239. Ozawa, S., Kamiya, H. and Tsuzuki, K., Glutamate receptors in the mammalian central nervous system, Prog. Neurobiol., 54 (1998) 581±618. Reynolds, I. and Hastings, T.G., Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation, J. Neurosci., 15 (1995) 3318±3327. Saito, I., Asano, T., Sano, K., Takakura, K., Abe, H., Yoshimoto, T., Kikuchi, H., Ohta, T. and Ishibashi, S., Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological de®cits after aneurysmal subarachnoid hemorrhage, Neurosurgery, 42 (1998) 269±278. Sanz, J.M., Vendite, D., FernaÂndez, M., Andres, A. and Ros, M., Adenosine A1 receptors in cultured cerebellar granule cells: role of endogenous adenosine, J. Neurochem., 67 (1996) 1469±1477. Sengpiel, B., Preis, E., Krieglstein, J. and Prehn, J.H., NMDA-induced superoxide production and neurotoxicity in cultured rat hippocampal neurons: role of mitochondria, Eur. J. Neurosci., 10 (1998) 1903±1910. Takasago, T., Peters, E.E., Graham, D.I., Masayasu, H. and Macrae, I.M., Neuroprotective ef®cacy of ebselen an antioxidant with anti-in¯ammatory actions, in a rodent model of permanent middle cerebral artery oclusion, Br. J. Pharmacol., 122 (1997) 1251±1256. Yamaguchi, T., Sano, K., Takakura, K., Saito, I., Shinohara, Y., Asano, T. and Yasuhara, H., Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial, Stroke., 29 (1998) 12±17. Zoccarato, F., Valente, M. and Alexandre, A., Hydrogen peroxide induces a long-lasting inhibition of the Ca(2 1 )dependent glutamate release in cerebrocortical synaptosomes without interfering with cytosolic Ca 21, J. Neurochem., 64 (1995) 2552±2558.
www.elsevier.com/locate/neulet
Ebselen prevents excitotoxicity provoked by glutamate in rat cerebellar granule neurons Lisiane O. PorciuÂncula a, JoaÄo Batista T. Rocha b, Carina R. Boeck a, Deusa Vendite a, Diogo O. Souza a,* a
Departamento de BioquõÂmica, ICBS, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil b Departamento de QuõÂmica, CCNE, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Received 10 October 2000; received in revised form 7 December 2000; accepted 2 January 2001
Abstract Ebselen is a selenium compound that have glutathione peroxidase-like activity which is neuroprotective in acute stroke ischemia. The ef®cacy of ebselen to prevent excitotoxicity provoked by glutamate in cerebellar granule neurons was investigated at various time points and concentrations. Simultaneous addition of ebselen with glutamate decreased neuronal death and was completely reversed by 3 mM of ebselen (3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide assays). However, when 1 mM of ebselen was added with glutamate and remained in the culture medium until 24 or 48 h, the neuronal survival increased to the control. The mechanism proposed for neuroprotection was the ability of ebselen to prevent lipoperoxidation provoked by glutamate. The present ®ndings propose to amplify the use of ebselen in others neurodegenerative disorders involving glutamatergic system. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ebselen; Neurotoxicity; Neuroprotection; Glutamate; Cerebellar granule neurons; Lipoperoxidation
Ebselen, 2-phenyl-1,2-benzisoselenazol-3[2H]-one, is a lipid-soluble seleno-organic compound that has glutathione peroxidase-like activity [8,12]. Recently, it has been demonstrated that this compound has a protective effect against brain ischemic insults and decreases the citotoxic effects of 4-hydroxynonenal in spinal cord neurons [9,18,19]. The mechanism underlying the neuroprotection afforded by ebselen is still not completely understood, but it is certainly related to its antioxidant and antiin¯amatory properties [12,15,18]. Recently, clinical trials with humans indicated that ebselen has bene®cial effects on pathological situations where glutamate is involved, such as ischemia and stroke [19]. However, direct evidence supporting that neuroprotective effect of ebselen on the glutamate-induced neurotoxicity is lacking in the literature. Glutamate is the main excitatory neurotransmitter in mammals [13]. However, over stimulation of the glutamatergic neurotransmission may be neurotoxic and can lead to cell death [2]. In fact, excitatory amino acids have been proposed to be involved in the pathophysiology of acute * Corresponding author. Fax: 155-51-316-5540/5535. E-mail address: [email protected] (D.O. Souza).
brain injury and chronic neurodegenerative diseases, such as ischemia, head trauma, stroke, Alzheimer's disease and Parkinson's disease [13]. Reactive oxygen species may be involved in neuronal death caused by alterations in excitatory amino acid neurotransmission [6]. Primary cultures of granule cells derived from cerebella of postnatal rats are endowed with glutamate receptors [3]. Due to their homogenous cellular composition, observations on these cultures re¯ect properties of a single neuronal cell type, making this cellular system one of the most appropriate for the study of glutamate-evoked biological responses. In the present report, the potential neuroprotective effect of ebselen on excitotoxicity induced by glutamate in cerebellar granule neurons culture was investigated. Primary cultures of cerebellar granule cells were prepared from 8-day-old Wistar rats [16]. Brie¯y, freshly dissected cerebella were incubated with 0.025% trypsin solution for 15 min at 378C and disrupted mechanically in the presence of 0.08 mg/ml DNase and 0.05% trypsin inhibitor. For the experiments, the cells were seeded at a density of 2.5 £ 10 5 cell/cm 2 in a 24-well multiwell dish or 6-well dish, coated with 10 mg/ml poly-d-lysine in Eagle's basal medium (BME) supplemented with 10% fetal bovine serum,
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 51 9- 1
218
L.O. PorciuÂncula et al. / Neuroscience Letters 299 (2001) 217±220
50 mg/ml gentamicin and 25 mM KCl. The growth of nonneuronal cells was inhibited by addition of 20 mM cytosine arabinofuranoside 18±20 h after seeding and the medium was maintained without change during the culture period. At 8±9 DIV, cultures were washed twice with Locke's solution (mM): 154 NaCl, 5.6 KCl, 3.6 NaHCO3, 2.3 CaCl2, 5.6 glucose, 5 HEPES, pH 7.4 and were then incubated at 20±228C for 25 min in 1 ml of Locke's solution, with or without 100 mM glutamate, and in the presence or absence of ebselen (Sigma, St. Louis MO) or vehicle. Ebselen was diluted in 50% (v/v) of ethanol. The ®nal concentration of ethanol in control and ebselen-treated samples were 0.25%. After 25 min, the Locke's solution was removed, the cultures were washed twice with fresh solution and then replaced with the previously saved culture-conditioned medium (with or without ebselen) and incubated for 3 h (for lipid peroxidation) or 24 h and 48 h (viability assays) at 378C, 5% CO2. So, the interaction between glutamate and ebselen was evaluated at various time points: cells were exposed (a) to glutamate; (b) to glutamate and ebselen simultaneously; (c) to ebselen after glutamate exposure; (d) to ebselen and glutamate simultaneously and after this incubation just ebselen remained in the culture medium. Cell cultures viability were assessed by MTT, propidium iodide and trypan blue. For MTT, cells (24 and 48 h postexposure) were assessed by the colorimetric MTT 3 (4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [10]. Brie¯y, cultures were incubated for 30 min at 378C with 1 ml Locke's solution with 1.2 MgCl2, containing 0.5 mg/ml of MTT. Only viable cells are able to reduced MTT, into formazan product which was soluble in dimethyl sulfoxide (DMSO) and measured at 490 and 630 nm. For propidium iodide, cells (24 h post-exposure) were stained with 7 mg/ml propidium iodide for 5 min in Locke's Solution with magnesium. Stained cultures were examined and photographed using a ¯uorescence imaging system (Nikon Eclipse TE 300 Diaphot microscope) with standard rhodamine ®lter set (excitation 540 nm; emission 617 nm). For trypan blue, cells (24 h post-exposure) were stained with 0.4 g% of trypan blue in Locke's Solution for 5 min. After, cells were visualized and counted in phase contrast microscopy. Ebselen did not interfere in these viability assays when was incubated alone. Thiobarbituric acid reactive substances (TBARS) were assessed to evaluate the extent of lipid peroxidation in the presence or absence of glutamate (100 mM) and ebselen (3 mM). Brie¯y, after 3 h of glutamate exposure with or without ebselen, the cells were treated with one milliliter of trichloroacetic acid (15% w/v) containing 1 mM ethylenediaminetetra-acetic acid (EDTA). Then, an equal volume of thiobarbituric acid (0.7% w/v) was added and samples heated at 1008C for 20 min. After cooling, the samples were centrifuged at 1000 £ g for 10 min and the supernatant color was monitored at 532 nm. Concentrations of TBARS were calculated using standard curve obtained with malondialdehyde and expressed as nmol of malonilaldehyde formed per well.
All assays were performed from independent cultures. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan's post hoc test. Exposure of cerebellar neurons to 100 mM glutamate for 25 min caused a signi®cant decline (to 40% of control cells) in mitochondrial activity (as observed by MTT reduction to formazan, measured 24 h after glutamate exposure), suggesting neuronal death. Simultaneous addition of ebselen (0.1±3 mM) with glutamate decreased signi®cantly the neuronal death (Fig. 1A). The protection was maximum with the highest concentration of ebselen tested (3 mM). Similar results were obtained by cell counting following trypan blue staining (data not shown) indicating that reduction of MTT coincides with a commensurable reduction of the cell viability. The acute response to glutamate (observed
Fig. 1. (A) Effect of 0.1±3 mM ebselen on glutamate neurotoxicity (24 h after glutamate exposure). Glutamate (100 mM) and ebselen were added simultaneously for 25 min. Values are means ^ SEM (n 4±7); (a) different from all other groups (P , 0.05); (b) different from control and glutamate (P , 0.05). (B) The effect of 1 mM ebselen added in different time points. C, control; G, glutamate 100 mM for 25 min; GE, glutamate and ebselen added simultaneously for 25 min. E, Ebselen added immediately after incubation for 25 min with glutamate and remained in the culture medium until 24 h after the glutamate withdrawal; GEE, glutamate and ebselen added simultaneously for 25 min and ebselen remained in the culture medium until 24 h after the glutamate withdrawal. The mitochondrial activity was tested 24 h after glutamate exposure. Values are means ^ SEM (n 4±5); (a) different from all other groups (P , 0.05); (b) different from control, G and GEE (P , 0.05).
L.O. PorciuÂncula et al. / Neuroscience Letters 299 (2001) 217±220
219
Fig. 2. Fluorescent images of staining with propidium iodide after 24 h of glutamate exposure for 25 min. (A) control; (B) glutamate 100 mM; (C) glutamate 100 mM plus ebselen 3 mM for 25 min. Scale bar, 100 mm.
in the ®rst 5 min of incubation) was characterized by rapid swelling and loss of brightness, as observed by phasecontrast microscopy. Ebselen in all concentrations tested was unable to prevent this acute effect of glutamate (data not shown). Subsequently, we examined whether ebselen confers protection when administered after the glutamate (Fig. 1B). Ebselen (1 mM) added to cerebellar neurons at same time with glutamate or just after exposure to glutamate increased signi®cantly the cell survival from about 40% (glutamate alone) to 60% (P , 0.05). Moreover, the neuronal survival (85%) was not statistically different from control, when ebselen (1 mM) was present simultaneously with glutamate and remained in the culture medium until viability assays after 24 h (Fig. 1B) or 48 h (data not shown). The protection conferred by ebselen (3 mM) against glutamate-induced neuronal death was also observed by staining with propidium iodide (Fig. 2), as observed with the MTT and trypan blue assays. In Fig. 3 we evaluated the ability of ebselen to prevent the lipid peroxidation (used as an index of oxidative damage) provoked by glutamate (100 mM). Ebselen (3 mM) was added simultaneously with glutamate for 25 min and remained in the culture medium until the assay (3 h after
Fig. 3. Effect of ebselen (3 M) in to prevent lipid peroxidation provoked by treatment with glutamate. C, Control; G, glutamate (100 mM) for 25 min; GEE, glutamate (100 mM) for 25 min plus ebselen (3 mM) added simultaneously with glutamate and remained until 3 h in the culture medium. Values are means ^ SEM of ®ve experiments independents. *Different from control and GEE (P , 0.01).
the glutamate exposure). Glutamate treatment increased 135% of lipid peroxidation and ebselen completely prevented the glutamate-triggered effect. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated through NMDA-receptor activation [13]. Overstimulation of NMDA receptors with glutamate results in an excessive in¯ux of Ca 21, that activates neurotoxic mechanisms, including the production of reactive oxygen species, such as superoxide, hydrogen peroxide and nitric oxide [1,4,14,17,20]. Hydrogen peroxide act as a messenger in some synapses [20]. However, one of the breakdown products of this compound is one of the most reactive oxygen specie (OH z), which is an effective neurotoxin, that produces oxidative stress and decreases glutathione levels [5]. Since ebselen demonstrated ef®cacy to prevent lipid peroxidation [11] is plausible to suppose that ebselen protect against glutamate toxicity by enhancing the reduction of hydrogen peroxide to non-reactive products, avoiding the cascade of reactions which may culminate in peroxidation of lipids present in neuronal membranes. Accordingly, ebselen (3 mM) abolished glutamate-triggered lipoperoxidation (Fig. 3). Interestingly, the protection afforded by ebselen greatly depended on the time point and concentration of this compound in relation to glutamate. Ebselen (3 mM) was the most protective concentration when added simultaneously with glutamate for 25 min and low concentrations afforded a partial protection. However, a similar maximum protection was conferred when 1 mM of ebselen was presented together with glutamate, remaining in the culture medium after the glutamate insult for 24 h (Fig. 1B) and for 48 h (data not shown). The delayed addition of 1 mM ebselen after the glutamate exposure and up to assays protected partially, suggesting that ebselen can scavenger reactive intermediates primarily triggered by glutamate or generated by early reactive intermediates in a series of reactions. Ischemia is associated with an excessive activation of the glutamatergic system and membrane lipid peroxidation [7,13]. Many pharmacological tools such glutamate antagonists, have been developed to improve outcome for ischemic patients and to limit their brain damage. However, gluta-
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