- Email: [email protected]
Protein S100B release from rat brain slices during and after ischemia: Comparison with lactate dehydrogenase leakage
Neurochemistry International 47 (2005) 580–588 www.elsevier.com/locate/neuint
Protein S100B release from rat brain slices during and after ischemia: Comparison with lactate dehydrogenase leakage Rifat Levent Bu¨yu¨kuysal Uludag˘ University, Medical School, Department of Pharmacology and Clinical Pharmacology, 16059 Bursa, Turkey Received 1 March 2005; received in revised form 20 June 2005; accepted 22 June 2005 Available online 27 September 2005
Abstract One hour of ischemia significantly increased protein S100B release from rat brain slices without altering lactate dehydrogenase leakage. Reoxygenation of the ischemic slices, however, increased the levels of these biochemical markers in the medium. Although removal of extracellular Ca+2 ions from the medium did not alter the basal lactate dehydrogenase leakage from cortical slices, an excessive increase in basal protein S100B release was seen under this condition. Ischemia and/or reoxygenation induced enhancements in these markers were attenuated by removal of Ca+2 ions from the medium. Ischemia significantly increased glutamate release, but neither ischemia nor reoxygenation induced rises in protein S100B and lactate dehydrogenase levels were altered by glutamate receptor antagonists. Rising the glutamate levels in the medium by each ouabain or exogenous glutamate, moreover, failed in exerting an ischemia like effect on protein S100B and LDH outputs. In contrast, exogenous glutamate added into the medium protected the slices against reoxygenation induced increments in protein S100B and lactate dehydrogenase levels. These results indicate that protein S100B has a greater sensitivity against ischemia than lactate dehydrogenase in in vitro brain slice preparations. Since neither exogenous glutamate nor enhancements of the extracellular glutamate levels by ouabain had an ischemia like effect, and since glutamate receptor antagonists were also unsuccessful, it seems unlikely that ischemia-induced increase in glutamate release is directly involved in protein S100B release or lactate dehydrogenase leakage determined in the present study. # 2005 Elsevier Ltd. All rights reserved. Keywords: Protein S100B; Ischemia; Lactate dehydrogenase leakage
1. Introduction Several methods have been used for determination of ischemia and/or reoxygenation (REO) induced tissue damage in in vitro studies. One of these methods is densitometric evaluation after staining of the brain tissue with 2,3,5-triphenyltetrazolium (TTC) (Mathwes et al., 2000). Lactate dehydrogenase (LDH), a cytoplasmic enzyme, is most commonly used biochemical marker in ischemic studies. Neuron-specific enolase (NSE), a neuronal form of the intracytoplasmic glycolytic enzyme enolase, on the other hand, has also been used as a biochemical marker in brain slice studies (Moro et al., 2000). It must be noted that significant alterations in each TTC-staining or LDH and NSE outputs have been observed after a transient exposure E-mail address: [email protected]. 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.06.009
of brain slices to oxygen/glucose deprivation followed by REO (Tatsumi et al., 1998; Mathwes et al., 2000; Moro et al., 2000; De La Cruz et al., 2002; Bu¨yu¨kuysal, 2004; Mog˘ol et al., 2005), indicating that neuronal damage that becomes after a delay is an active process and blocking the postischemic events seems to be a more promising strategy against ischemia-induced neuronal damage. Increasing body of evidence indicates that acute ischemic stroke or traumatic brain damage also increases protein S100B levels in serum or CSF (Rothermundt et al., 2003). This protein is a member of the S-100 family of acidic, calcium binding proteins and mainly expressed in astrocytes and constitutively released from these cells (Donato, 2003). Although extracellular S100B exerts a dual effect on neurons depending on its concentration, i.e. pro-survival effect on neurons at nM and a toxic effect at m M concentrations (Donato, 2003; Rothermundt et al., 2003), mechanisms
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
involved in its release are not clear. While stimulation of 5HT1 serotonin, A1 adenosine or mGlu3 metabotropic glutamate receptors releases protein S100B from cultured astrocytes (Whitaker-Azmitia et al., 1990; Ciccarelli et al., 1999; Pinto et al., 2000), it has been reported that high concentration of glutamate has a decreasing effect on protein S100B secretion induced by serum deprivation in astrocytes (Goncalves et al., 2002). As increased S100B concentration induced by brain trauma and ischemia is believed to provide a biochemical information about the extent of the brain damage, either clarifying its release mechanism under ischemic conditions or understanding its advantages over other biochemical markers seems to be important for developing new therapeutic approaches against ischemia-induced neuronal damage. Thus, this study was undertaken to investigate protein S100B release from rat brain slices during and after ischemia. Each release and involvement of extracellular calcium ions and glutamate in protein S100B and LDH outputs were also compared in the present study.
2. Experimental procedures 2.1. Materials
581
medium. Following 90 min of equilibration period, slices were incubated in either control or ischemic medium for 1 h at 37 8C. Ischemic medium was not containing glucose and oxygen, but gassed with 95% N2 and 5% CO2. When glucose or calcium was omitted, osmolarity of the medium was maintained by increasing the NaCl concentration. Calcium-free medium was also containing EGTA (1 mM). At the end of ischemic incubation period, slices were transferred into the glucose and oxygen-containing medium (REO period). In order to determine time dependency of protein S100B release and LDH leakage, REO of the slices was extended up to 3 h and incubation medium was replaced with fresh medium at 1 h interval during this period. As both LDH and protein S100B outputs were highest during first 1 h of the REO period and then declined gradually (Fig. 1), in the rest of the studies, incubation of the slices was terminated at the end of first 1 h of the REO period. Half of the incubation medium (1 ml) collected at the end of ischemia was acidified with HClO4 (final concentration 0.4 M) and stored at 20 8C for quantification of excitatory amino acids released from the slices. Another half of the samples and others obtained after REO period were used for determination of protein S100B and LDH. Glutamate receptor antagonists were added into the medium 10 min
MK-801(+),CNQX, ouabain and glutamate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). aMethyl-4-carboxyphenylglycine (()-MCPG) was obtained from Research Biochemicals International (Natick, MA, USA). Other chemicals were at pure analytical grade and obtained from Merck KGaA (Darmstadt, Germany) or from Sigma Chemical Co. 2.2. Preparation and incubation of brain slices Male Sprague–Dawley rats (weighing 250–300 g and obtained from Experimental Animals Breeding and Research Center, Bursa, Turkey) were used. All experimental protocols were approved by Uludag˘ University Medical Center Institutional Review Board for animal research, and all efforts were made to minimize the number of animals used and their suffering. Rats were decapitated, their brains were removed quickly and were placed in cold oxygenated physiological medium with 95% O2 and 5% CO2 (in mmol/L: 120 NaCl, 1.3 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 3.5 KCl, 25 NaHCO3 and 10 glucose). After dissection, cortical, corpus striatal and hippocampal slices (0.3 mm of thickness) were prepared with a McIlwain tissue chopper (Brinkmann Instruments; Westbury, NY, USA). Slices were washed with oxygenated cold physiological medium and then transferred to 2 ml of incubation tubes. Each tube was divided into four separate chambers and contained three slices that did not touch each other. Slices were incubated in a water bath at 37 8C and the medium was changed every 10 min with fresh oxygenated
Fig. 1. Effects of ischemia and REO on protein S100B and LDH release from rat cortical slices. After 90 min of equilibration period, cortical slices were incubated in control or ischemic medium for 1 h. At the end of this incubation, all slices were transferred into oxygen and glucose containing medium (REO) for another 3 h. During this period, incubation medium was changed at 1 h intervals to fresh medium and samples collected at the end of each period were studied for determination of protein S100B and LDH levels. Data are given as means S.E.M. of six–seven determinations. * p < 0.05, ***p < 0.001, significantly different from their corresponding control levels.
582
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
before the ischemia and they were present in the medium until the end of the REO period. The effects of glutamate (2 mM) and ouabain (100 mM) on each amino acid, protein S100B and LDH outputs were studied on cortical slices incubated under normoxic conditions. In another part of the study, glutamate (2 mM) was also added into the medium during REO period of the ischemic cortical slices.
2–2.5 with 2 M of LiOH. A portion of the supernatant (20 ml) was then injected onto HPLC system without further purification. Glutamate and aspartic acid levels were calculated by comparing peak heights of the samples with their standards. Amino acid standards were prepared in physiological medium, acidified with HClO4 and processed together with samples.
2.3. LDH assay
2.6. Total protein assay
LDH activity in the incubation medium was assayed by using a commercial kit from Merck-Biotrol Diagnostics (France). Namely, 50 ml of incubation medium was mixed with 0.7 ml of reaction medium in a temperature controlled cuvette (30 8C). Changes in the absorbance were read at 340 nm after 30 s then every minute during 2 min. LDH activity was calculated according to average absorbance change and then corrected with protein levels of the slices.
Incubation of the slices was terminated by transferring them in 2 ml of 0.4 M HClO4. After homogenization of the slices, tissue protein levels were measured in 50 ml of homogenate according to the procedure of Lowry et al. (1951). Protein standards were prepared in 0.4 M HClO4 and processed together with the tissue samples. All results were corrected according to total protein levels of the slices.
2.4. Protein S100B assay
All the results in the text are expressed as mean S.E.M. The differences between the results were tested by Tukey– Kramer multiple comparison test or Student’s t-test. A probability of p < 0.05 was considered significant.
Protein S100B released into the medium was determined by an enzyme immunoassay test kit (Nexus Dx S100, from Synx Pharma Inc. Toronto, Canada), which was specific for the b-subunit of the S100 protein and measured the bsubunit concentration in both bb and ab isoforms of the protein. Samples were first diluted with distilled water (dilution ratio 1/20) and 25 m l of diluted samples was used for protein S100B assay. 2.5. Quantification of glutamate and aspartic acid Glutamate and aspartic acid levels in the medium were determined by a HPLC system (HP 1100 series, HewlettPackard, Palo Alto, CA, USA) coupled to a post-column derivatization unit (Pickering Laboratories, Mountain View, CA, USA). This system was combined with a quaternary pump (HP, G1311A), a fluorometric detector (HP, G1321 A) and an autosampler (HP, G1329 A). The amino acids which were separated on lithium exchange column (Pickering Labs., series number 5338) with Li280 and Li750 eluents (Pickering Labs.) were reacted with OPA in a post-column derivatization unit (both from the Pickering Labs.). The flow rates of the quaternary pump and post-column derivatization unit were 0.3 ml/min. Column and post-column reaction temperatures were adjusted to 40 and 45 8C, respectively. Other chromatographic conditions, such as the gradient program of the Li280, Li750 and lithium regenerant eluents, were similar with the conditions reported by Grunau and Swaider (1992). OPA reactive compounds were detected at excitation 330 nm, emission 465 nm wavelengths, and chromatograms were analyzed with a software (HP Chemstation, revision A. 08. 03. 847). Acidified samples were centrifuged for 5 min in a Beckman microfuge and pH of the samples was adjusted to
2.7. Data analysis
3. Results 3.1. Ischemia and REO-induced protein S100B and LDH outputs from rat cortical slices Basal protein S100B release from cortical slices incubated under normoxic conditions was 2.65 0.6 ng/ ml/mg protein/1 h. Omission of both glucose and oxygen from the medium (ischemia) increased protein S100B release more than 50%. When ischemic slices were transferred into the normoxic medium (REO period), a greater increase in protein S100B level was determined (Fig. 1). In contrast to protein S100B, 1 h of ischemia did not alter the LDH leakage from cortical slices. REO of the ischemic slices, on the other hand, enhanced LDH output to 331 21 DmOD/mg protein from its control value of 131 18 DmOD/mg protein (Fig. 1). It was also determined that both protein S100B and LDH outputs were highest during first 1 h of the REO period. As lengthening of REO up to 3 h did not cause a further increment in the release of these biochemical markers (Fig. 1), in the rest of the studies, REO of the ischemic slices was terminated at the end of 1 h of the REO period. 3.2. Comparison of the ischemia and REO-induced protein S100B and LDH outputs in cortical, striatal and hippocampal slices Basal levels of protein S100B released from cortical, striatal and hippocampal slices incubated under control
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
583
Table 1 Ischemia and REO-induced protein S100B and LDH outputs from rat cortical, striatal and hippocampal slices Brain region
Control
Ischemia
Control-REO
Ischemia-REO
(A) Protein S100 B (ng/ml/mg protein) Cortex 2.65 0.6 Corpus striatum 1.83 0.3 Hippocampus 1.79 0.6
4.56 0.5 * 3.57 0.3 ** 7.40 1.7 *
2.35 0.3 1.10 0.4 2.68 0.5
10.11 1.1 *** 4.86 0.6 *** 21.3 2.7***,a
(B) LDH (DmOD/mg protein) Cortex Corpus Striatum Hippocampus
177 40 156 20 321 19
131 18 106 14 262 18
331 21 *** 243 47 * 747 72***,a
168 19 152 30 309 27
After 90 min of equilibration period, brain slices were incubated in control or ischemic medium for 1 h followed by 1 h of REO period. At the end of each period, incubation medium was collected and studied for determination of protein S100B and LDH levels. Data are given as means S.E.M. of six–seven determinations. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from their corresponding control levels. ap < 0.001, significantly different from the value determined in cortical or striatal slices.
(normoxic) conditions were similar ( p > 0.05). Basal LDH leakage, on the other hand, was found higher in hippocampal slices ( p < 0.01). As obtained in cortical slices, ischemia did not alter the LDH leakage from striatal and hippocampal slices, but REO caused significant increases in the levels of this enzyme in the medium (Table 1). Although extents of the ischemia-induced LDH leakage were similar in each brain area (144 49%, 152 16% and 185 28% for striatal, cortical and hippocampal slices, respectively, p > 0.05), net amount of the LDH leakage into the medium was higher in hippocampal slices ( p < 0.001). Ischemia, as obtained in cortical slices, significantly increased protein S100B release from striatal and hippocampal slices. REO of the ischemic brain slices, on the other hand, further enhanced protein S100B outputs. When ischemia and REO-induced increments in protein S100B outputs from different brain regions were compared, greater increases were seen in hippocampal slices (Table 1). Like cortical slices, protein S100B and LDH outputs from striatal and hippocampal slices were highest during first 1 h of REO period, increases in their levels then declined gradually (data not shown).
in REO-induced LDH leakage ( p < 0.001). If calcium ions were removed during only REO period, a significant decline in REO-induced LDH leakage was also present ( p < 0.01). In agreement with these observations, REOinduced LDH leakage further declined when omitting the calcium ions from incubation medium during both ischemia and REO periods ( p < 0.001; Fig. 3).
3.3. Calcium dependency of protein S100B and LDH outputs from rat cortical slices Removal of extracellular calcium ions from normoxic incubation medium caused 455% increase in protein S100B output from cortical slices. Similar amount of increases were also seen when calcium ions were omitted during ischemia and REO periods (Fig. 2). Although chasing calcium-free ischemia with a calcium containing REO period brought the protein S100B levels back below what would have been generated from calcium-free ischemia alone, its level was not different than the value obtained from ischemia (+Ca+2)-REO (+Ca+2) group (Fig. 2). In contrast to protein S100B, basal LDH leakage did not alter in the absence of calcium ions in the medium (data not shown). When calcium ions were omitted during ischemic period, a significant attenuation was determined
Fig. 2. Effect of extracellular Ca+2 omission on basal, ischemia and REOinduced protein S100B outputs from cortical slices. After equilibration period, cortical slices were incubated either in Ca+2-free or Ca+2-containing control or ischemic medium for 1 h followed by 1 h REO period in the absence or presence of extracellular Ca+2 ions. Data are given as means S.E.M. of three determinations for Ca+2-containing control and Ca+2containing ischemia groups, and eight determinations for the remaining groups. ***p < 0.001, significantly different from the corresponding value determined in the presence of Ca+2 ions, ap > 0.05, not significantly different from the ischemia (+Ca+2)-REO (+Ca+2) group.
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
584
Fig. 3. Effect of extracellular Ca+2 omission on REO-induced LDH leakage from cortical slices. After equilibration period, cortical slices were incubated either in Ca+2-free or Ca+2-containing ischemic medium for 1 h followed by 1 h REO period in the absence or presence of extracellular Ca+2 ions. Data are given as means S.E.M. of eight determinations for each group. **p < 0.01, ***p < 0.001, significantly different from the value determined in ischemia (+Ca+2)-REO (+Ca+2) group.
3.4. Involvement of glutamate in ischemia and REOinduced protein S100B and LDH outputs from rat cortical slices Since ischemia causes an excessive increase in extracellular glutamate level, it is one possibility that this increase contributes to ischemia and/or REO-induced protein S100B and LDH outputs. This possibility was tested by increasing the glutamate levels in normoxic medium by different pharmacological manipulations, or by adding the glutamate receptor antagonists into the medium before ischemia. As seen in Table 2, 1 h of ischemia or incubation of cortical slices in ouabain (100 mM) containing normoxic medium for 1 h significantly increased glutamate and aspartic outputs. Incubation of cortical slices with high concentration of glutamate (2 mM), on the other hand, significantly increased aspartic acid level in the medium (Table 2). Despite these excessive increases in excitatory amino acid levels, neither exogenous glutamate nor ouabain increased protein S100B and LDH outputs. In contrast, basal protein Table 2 Excitatory amino acid release from rat cortical slices: effects of ischemia, ouabain and glutamate
Control Ischemia Ouabain Glutamate
Aspartic acid
Glutamate
37 8 1991 193*** 1370 81 *** 6651 928***
29 6 12757 1672*** 6307 872*** –
After 90 min of equilibration period, cortical slices were incubated in control or in ischemic medium, or ouabain (100 mM) or glutamate (2 mM) containing normoxic medium for 1 h. Incubation medium collected at the end of the incubation period was used for determination of aspartic acid and glutamate levels. Data are given as means S.E.M. of five–six determinations. ***p < 0.001, significantly different from their corresponding control levels.
S100B release was declined significantly by 2 mM of glutamate (Table 3). Similarly, 1 h of drug-free period followed by glutamate or ouabain incubation also failed to show a REO-like effect. When added into the medium during REO period, on the other hand, high concentration of glutamate protected the slices against ischemia and/or REOinduced increments in protein S100B and LDH leakages (Table 3). Presence of MK-801 (100 mM), CNQX (50 mM) and MPCG (50 mM) in the medium during both ischemia and REO periods, which are known to block NMDA, kainat/ AMPA and metabotropic glutamate receptors, respectively, were unsuccessful to inhibit ischemia and REO-induced increases in protein S100B outputs from cortical slices (Fig. 4). Similarly, LDH leakage enhanced by REO of the ischemic cortical slices was not altered significantly by glutamate receptor antagonists (Fig. 5).
4. Discussion Since protein S100B has been shown to be increased in human blood and CSF after traumatic brain damage, many studies have focused on protein S100B under various ischemic conditions. Although results obtained from these studies demonstrated a close correlation between the protein S100B concentration and infarct volume as well as clinical outcome (Rothermundt et al., 2003), neither ischemia and/or REO-induced alterations in protein S100B release nor its release mechanism have been studied in brain slice preparations. Now I am reporting here that ischemia increases protein S100B release from brain slices and this increase is further enhanced by REO. In contrast, LDH levels in the medium, another biochemical marker which is widely used to evaluate and quantify cellular damage, was not altered by ischemia. It has been suggested that LDH assay is not suitable for acute brain preparations, because its release is slow relative to deterioration of cellular morphology (Izumi et al., 2001). It is known that, however, not only LDH leakage, but NSE release or TTC staining in brain slices does not response to ischemia (Mathwes et al., 2000; Moro et al., 2000). REO of the ischemic slices, on the other hand causes significant alterations in the levels of these biological markers (Tatsumi et al., 1998; Mathwes et al., 2000; Moro et al., 2000; De La Cruz et al., 2002; Bu¨yu¨kuysal, 2004; Mog˘ol et al., 2005) supports the conclusion that ischemia-induced neuronal damage actually becomes after a delay, and REO seems to be a critical period for resultant tissue damage. Because ischemia increased protein S100B release in the absence of signs of brain cell death determined as LDH leakage, this increase probably reflects a specific cell response to ischemia. It is noteworthy that ischemia and REO caused greater increases in protein S100B release from hippocampal slices than the slices prepared from other two brain regions. Similarly, basal or REO-induced LDH leakage was also higher in hippocampal
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
585
Table 3 Effects of ouabain and glutamate on protein S100B and LDH release from rat cortical slices
(A) Normoxic medium Control Ouabain Glutamate (B) After ischemia REO REO + glutamate
Protein S100B (ng/ml/mg protein)
LDH (DmOD/mg protein)
First incubation (drug added)
Second incubation (drug free)
First incubation (drug added)
Second incubation (drug free)
2.93 0.4 2.18 0.4 3.10 0.6
116 25 101 30 82 9
90 5 75 5 82 8
3.54 0.7 4.12 0.4 2.63 0.2* 10. 36 1 4.95 0.2 ***
424 40 151 10***
After equilibration period, cortical slices were incubated in control, ouabain (100 mM) or glutamate (2 mM) containing normoxic medium for 1 h followed by drug-free period for another 1 h. When the effect of exogenous glutamate on REO-induced protein S100B and LDH outputs was tested, cortical slices were incubated first in ischemic condition for 1 h followed by 1 h REO period in the absence or presence of 2 mM glutamate. At the end of each incubation period, medium was collected and used for determination of protein S100B and LDH levels. Data are given as means S.E.M. of three–seven determinations. * p < 0.05, significantly different from their corresponding control values. ***p < 0.001, significantly different from the value obtained in the absence of glutamate.
slices. Either regional difference of neuronal vulnerability to ischemia (Yanagihara et al., 1985) or differences in cell densities between the brain regions, as determined previously by number of the S100B positive astrocytes (Savchenko et al., 2000), may result in an unequal release or leakage of these biochemical markers from the slices. As increased levels of intracellular free Ca+2 are implicated in ischemia and/or REO-induced alterations (Kristian and Siesjo¨, 1998; Zhang and Lipton, 1999; Sattler and Tymianski, 2000), Ca+2 dependency of protein S100B release was also compared with LDH leakage in the present study. As seen in Fig. 3, removal of Ca+2 ions from the medium during ischemia significantly attenuated REO-induced LDH leakage from
Fig. 4. Effects of glutamate receptor antagonists on ischemia and REOinduced protein S100B outputs from rat cortical slices. After 90 min of equilibration period, cortical slices were incubated in control or ischemic medium for 1 h followed by 1 h REO period. At the end of ischemia and REO periods, incubation medium was used for determination of protein S100B levels. MK-801 (100 mM), CNQX (50 mM) and MCPG (50 mM) were added to the medium 10 min before ischemia and were present in the medium until the end of REO period. Data are given as means S.E.M. of four determinations. Ischemia or REO-induced enhancements determined in the presence of glutamate receptor antagonists are not different from the value obtained in the absence of these drugs ( p > 0.05).
Fig. 5. Effects of glutamate receptor antagonists on REO-induced LDH leakage from rat cortical slices. After 90 min of equilibration period, cortical slices were incubated in each control or ischemic medium for 1 h followed by 1 h REO period. At the end of REO period, incubation medium was used for determination of LDH levels. MK-801 (100 mM), CNQX (50 mM) and MCPG (50 mM) were added to the medium 10 min before ischemia and were present in the medium until the end of REO period. Data are given as means S.E.M. of seven–eight determinations. REO-induced enhancements determined in the presence of glutamate receptor antagonists are not different than the value obtained in the absence of these drugs ( p > 0.05).
586
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
cortical slices ( p < 0.001). Similar but slightly less improvement ( p < 0.01) was also seen when Ca+2 ions were omitted during REO period. If Ca+2 ions were omitted during both ischemia and REO periods, on the other hand, REO-induced LDH leakage was further attenuated, indicating that removal of extracellular Ca+2 ions, as reported previously (Tatsumi et al., 1998; Bu¨yu¨kuysal, 2004), would protect the slices against REO-induced LDH leakage. In contrast, basal protein S100B release significantly augmented when extracellular Ca+2 ions were omitted from the normoxic medium. Neither ischemia nor REO, however, caused an additional increase in protein S100B release in Ca+2 free medium. Although these findings indicate that removal of extracellular Ca+2 ions, as observed in LDH leakage, protects the slices against ischemia and/or REO-induced protein S100B outputs, it is one possibility that removal of Ca+2 ions from the medium completely depletes releasable pool of the protein S100B from the slices. Thus, neither ischemia nor REO could cause an additional increase in the absence of Ca+2 ions. However, it was determined that REO still increases protein S100B release from the slices that were incubated first in Ca+2-free ischemic medium (see Fig. 2), suggesting that even after Ca+2free medium-induced depletion, a releasable pool of protein S100B probably remains in the slices. Mechanism(s) involved in Ca+2-free medium-induced protein S100B release was not investigated the in present study. As removal of Mg+2 ions from the medium (but 1 mM of EGTAwas also present) failed in enhancing the control protein S100B level (data not shown), neither an alteration in the membrane charges (Piccolino and Pignatelli, 1996), nor an unspecific effect of EGTA seems involved. It is known that protein S100B, like some other S100 proteins, undergoes Ca+2-induced conformational changes and interacts with its target proteins in a Ca+2-dependent manner (Donato, 2003). Thus, it is likely that possible influences of Ca+2 removal on these parameters causes an abundant increase in protein S100B release. Since ischemia decreases extracellular Ca+2 concentration by translocating almost all extracellular Ca+2 ions into cells (Kristian and Siesjo¨, 1998), it is one possibility that declining in extracellular Ca+2 levels during ischemia also contributes, at least partly, to ischemia-induced protein S100B release as determined in the present study. It has been repeatedly shown that extracellular accumulation of excitatory amino acids plays a major role in ischemia and/or REO-induced tissue damage (Martin et al., 1994; Szatkowski and Attwell, 1994; Calabresi et al., 1999; Nishizawa, 2001) leading to glutamate–calcium hypothesis in the pathology of ischemia (Kristian and Siesjo¨, 1998; Sattler and Tymianski, 2000). According to this hypothesis, energy failure results in loss of Na+, K+-ATPase activity and causes membrane depolarization (anoxic depolarization) which triggers glutamate release. Thus, elevation glutamate level in the extracellular space and subsequent activation of glutamate receptors are implicated in neuronal damage. In a good agreement with previous reports (Pellegrini-Giampietro et al., 1990; Nishizawa, 2001; Nelson et al., 2003;
Bu¨yu¨kuysal, 2004; Mog˘ol et al., 2005), ischemia significantly increased glutamate release from cortical slices. The presence of high concentration of glutamate receptor antagonists MK-801, CNQX and MCPG, those block the NMDA, kainat/AMPA and metabotropic glutamate receptors, respectively, however, did not alter the ischemia and REO-induced increments in protein S100B release. Similarly, REO-induced increase in LDH leakage from cortical slices was not attenuated by glutamate receptor antagonists. Although these findings seem to be in opposite to glutamate hypothesis mentioned above, failure or limited effects of glutamate receptor antagonists under in vitro conditions (Oka et al., 2000; Joshi and Andrew, 2001; Bu¨yu¨kuysal, 2004) and in clinical trials (De Keyser et al., 1999; Plum, 2001) have also been reported. Additionally, collapse of the Na+ and K+ gradients by 100 mM of ouabain, a Na+–K+ATPase inhibitor, caused an ischemia-like effect on glutamate release, but neither protein S100B nor LDH leakage was increased by ouabain. If 100 mM of ouabain, as observed in mouse hemi-brain slices (Joshi and Andrew, 2001), elicits a neuronal depolarization similar to that induced by oxygen-glucose deprivation, then failure of the ouabain on protein S100B and LDH leakages also excludes a possible involvement of ischemic depolarization in the release of these biochemical markers. In the present study, it was also determined that, glutamate added into the normoxic medium at high concentration also failed in increasing the protein S100B and LDH outputs, strengthening the conclusion that excessive accumulation of glutamate in extracellular space is not directly or solely involved in ischemia and/or REOinduced protein S100B and LDH outputs. Interestingly, high concentration of glutamate added into the medium during REO period completely protected the slices against REOinduced protein S100B and LDH leakages. A similar attenuation of S100B secretion by high glutamate has also been reported in serum deprived hippocampal astrocytes (Goncalves et al., 2002). In patients with acute ischemic stroke, moreover, ZK200775, an AMPA antagonist, has worsened neurological outcome that was associated with a higher serum S100B levels than ischemia alone (Elting et al., 2002). The protective mechanism of glutamate on REOinduced LDH and protein S100B outputs are still under progress, but involvement of metabotropic glutamate receptors that are linked to protein kinase C activation is likely (Adamchik and Baskys, 1999; Schro¨der et al., 2000). Since glutamate is an essential element for glutathione synthesis in astroglia and ischemia decreases glutathione levels dramatically (Huster et al., 2000), addition of excessive glutamate into the medium during REO may increase the astrocyte antioxidant defense system and thus may lead to a decreased vulnerability to free radical damage. Although high concentration of exogenous glutamate added into the normoxic medium or inhibition of Na+, K+ATPase by ouabain did not show an ischemia-like effect on protein S100B and LDH outputs, many events such as altered Ca+2 homeostasis, activation of Ca+2-dependent enzymes,
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
mitochondrial permeability transition, excessive production of either reactive oxygen species or proinflamatory cytokines also occur during ischemia and/or REO period (Neumar, 2000). Thus, putative roles of these pathways in combination with glutamate release may not be ruled out. As its release mechanism, functional roles of S100B released during and/or after ischemia are also unclear. Data collected from S100B studies suggest that it has a dual effect on neurons depending on its concentration, i.e. it stimulates neurite outgrowth and enhances neuronal survival at nanomolar, but has deleterious effects at micromolar concentrations (Rothermundt et al., 2003). In rat hippocampal neurons, tissue damage induced by glucose deprivation has been protected by protein S100B, and it has been suggested that its elevation in the medium may be a compensatory response (Barger et al., 1995). Similarly, stretch injury has caused an immediate and prolonged release of protein S100B from neuronal plus glial cells, and S100B added to culture medium has reduced delayed neuronal injury (Willoughby et al., 2004). One of the most relevant findings in the present study is that protein S100B release increases during ischemia in the absence of signs of brain cell death and its levels in the extracellular medium increase further during REO. Although this finding seems to be important in the context of the putative role of protein S100B as a neurotropic factor during early phase of brain insults and a neurotoxic factor during late phase of insults, detailed studies are required in order to clarify whether protein S100B released during and after ischemia has similar and/or opposite effects on neuronal survival. In summary, results presented in this paper clearly indicate that ischemia causes significant increases in protein S100B release from brain slices without altering the LDH leakage. REO of the ischemic slices, on the other hand, enhances the levels of these biochemical markers in the medium by a mechanism seems to be not directly or solely related with the ischemia-induced glutamate output. Since ischemia-induced increase in protein S100B occurs in the absence of signs of brain cell death and probably reflects a specific cell response to ischemia, it is likely that protein S100B is a more valuable biological marker than LDH, at least, for in vitro brain slice studies.
Acknowledgement This study was supported by a grant from Uludag˘ University Research Council (2002/65).
References Adamchik, Y., Baskys, A., 1999. Glutamate-mediated neuroprotection against N-methyl-D-aspartate toxicity: a role for metabotropic glutamate receptors. Neuroscience 99, 731–736. Barger, S.W., Van Eldik, L.J., Mattson, M.P., 1995. S100 beta protects hippocampal neurons from damage induced by glucose deprivation. Brain Res. 677, 167–170.
587
Bu¨yu¨kuysal, R.L., 2004. Ischemia and reoxygenation-induced amino acid release and tissue damage in the slices of rat corpus striatum. Amino Acids 27, 57–67. Calabresi, P., Marfia, G.A., Centonze, D., Pisani, A., Bernardi, G., 1999. Sodium influx plays a major role in the membrane depolarization induced by oxygen and glucose deprivation in rat striatal spiny neurons. Stroke 30, 171–179. Ciccarelli, R., Di Lorio, P., Bruno, V., Battaglia, G., D’Alimonte, I., D’Onofrio, M., Nicoletti, F., Caciagli, F., 1999. Activation of A1 adenosine or mGlu3 metabotropic receptors enhances the release of Nerve Growth Factor and S100b protein from cultured astrocytes. Glia 27, 275–281. De Keyser, J., Sulter, G., Luiten, P.G., 1999. Clinical trials with neuroprotective drugs in acute ischemic stroke: are we doing the right thing? Trends Neurosci. 22, 535–540. De La Cruz, J.P., Villalobos, M.A., Cuerda, M.A., Guerrero, A., GonzalesCorrea, J.A., De La Cuesta, F.S., 2002. Effects of S-adenosyl-methionine on lipid peroxidation and glutathione levels in rat brain slices exposed to reoxygenation after oxygen–glucose deprivation. Neurosci. Lett. 318, 103–107. Donato, R., 2003. Intracellular and extracellular roles of S100 proteins. Microsc. Res. Techn. 60, 540–551. Elting, J.-W., Sulter, G.A., Kaste, M., Lees, K.R., Diener, H.C., Hommel, M., Versavel, M., Teelken, A.W., De Keyser, J., 2002. AMPA antagonist ZK200775 in patients with acute ischemic stroke. Possible glial cell toxicity determined by monitoring of S-100B serum levels. Stroke 33, 2813–2818. Goncalves, D., Karl, J., Leite, M., Rotta, L., Salbego, C., Rocha, E., Wofchuk, S., Goncalves, C.A., 2002. High glutamate decreases S100B secretion stimulated by serum deprivation in astrocytes. Neuroreport 27, 1533–1535. Grunau, J.A., Swaider, J.M., 1992. Chromatography of 99 amino acids and other ninhydrin-reactive compounds in Pickering lithium gradient system. J. Chromatogr. 594, 165–171. Huster, D., reichenbach, A., Reichelt, W., 2000. The glutathione content of retinal Muller (glial) cells: effect of pathological conditions. Neurochem. Int. 36, 461–469. Izumi, Y., Izumi, M., Benz, A.M., Zorumski, C.F., 2001. Lactate dehydrogenase release is facilitated by brief sonication of rat hippocampal slices and isolated retinas following acute neuronal damage. J. Neurosci. Methods 108, 49–55. Joshi, I., Andrew, R.D., 2001. Imaging anoxic depolarization during ischemia-like conditions in the mouse hemi-brain. J. Neurophys. 85, 414–424. Kristian, T., Siesjo¨, B.K., 1998. Calcium in ischemic cell death. Stroke 29, 705–718. Lowry, O.H., Rosebrough, N.J., Farr, A.L., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Martin, R.L., Lloyd, H.G.E., Cowan, A.I., 1994. The early events of oxygen and glucose deprivation: setting the scene for neuronal death? Trends Neurosci. 17, 251–257. Mathwes, K.S., McLaughlin, D.P., Ziabari, L.H., Toner, C.C., Street, P.C., Hisgrove, E., Bezzina, E.L., Stamford, J.A., 2000. Rapid quantification of ischaemic injury and cerebroprotection in brain slices using densitometric assessment of 2,3,4-triphenyltetrazolium chloride staining. J. Neurosci. Methods 102, 43–51. Mog˘ol, E.B., Bu¨yu¨kuysal, R.L., Korfalı, G., 2005. Effects of ketamine and thiopental on ischemia-reoxygenation induced LDH leakage and amino acid release from rat striatal slices. J. Neurosurg. Anesthesiol. 17, 20– 27. Moro, M.A., De Alba, J., Cardenas, A., De Cristobal, J., Leza, J.C., Lizasoain, I., Diaz-Guerra, M.J.M., Bosca, L., Lorenzo, P., 2000. Mechanism of neuroprotective effect of aspirin after oxygen and glucose deprivation in rat forebrain slices. Neuropharmacology 39, 1309–1318. Nelson, R.M., Lambert, D.G., Green, A.R., Hainsworth, A.H., 2003. Pharmacology of ischemia-induced glutamate efflux from rat cerebral cortex in vitro. Brain Res. 964, 1–8.
588
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
Neumar, R.W., 2000. Molecular mechanisms of ischemic neuronal injury. Ann. Emerg. Med. 36, 483–506. Nishizawa, Y., 2001. Glutamate release and neuronal damage in ischemia. Life Sci. 69, 369–381. Oka, M., Itoh, Y., Ukai, Y., 2000. Preferential inhibition by a novel Na+/Ca2+ channel blocker NS-7 of severe to mild hypoxic injury in rat cerebrocortical slices: a possible involvement of a highly voltage-dependent blockade of Ca2+ channel. J. Pharmacol. Exp. Therap. 293, 522–529. Pellegrini-Giampietro, D.E., Cherici, G., Alesiani, M., Carla, V., Moroni, F., 1990. Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage. J. Neurosci. 10, 1035–1041. Piccolino, M., Pignatelli, A., 1996. Calcium-independent synaptic transmission: artifact or fact. Trends Neurosci. 19, 120–125. Pinto, S.S., Gottfried, C., Mendez, A., Goncalves, D., Karl, J., Goncalves, C.A., Wofchuk, S., Rodnight, R., 2000. Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology. FEBS Lett. 486, 203–207. Plum, F., 2001. Neuroprotection in acute ischemic stroke. J. Am. Med. Assoc. 13, 1760–1761. Rothermundt, M., Peters, M., Prehn, J.H.M., Arolt, V., 2003. S100B in brain damage and neurodegeneration. Microsc. Res. Techn. 60, 614–632. Sattler, R., Tymianski, M., 2000. Molecular mechanisms of calciumdependent excitotoxicity. J. Mol. Med. 78, 3–13. Savchenko, V.L., Mckanna, J.A., Nikonenko, I.R., Skibo, G.G., 2000. Microglia and astrocytes in the adult rat brain: comparative immuno-
cytochemical analysis demonstrates the efficacy of lipocotin I immunoreactivity. Neurosciences 96, 195–203. Schro¨der, U.H., Opitz, T., Jager, T., Sabelhaus, C.F., Breder, J., Reymann, K.G., 2000. Protective effects of group I metabotropic glutamate receptor activation against hypoxic/hypoglycemic injury in rat hippocampal slices: timing and involvement of protein kinase C. Neuropharmacology 38, 209–216. Szatkowski, M., Attwell, D., 1994. Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms. Trends Neurosci. 17, 359–365. Tatsumi, S., Itoh, Y., Ukai, Y., Kimura, K., 1998. A novel Na+/Ca+2 channel blocker, NS-7, suppresses hypoxic injury in rat cerebrocortical slices. Naunyn-Schmiedeberg’s Arch. Pharmacol. 358, 191–196. Whitaker-Azmitia, P.M., Murphy, R., Azmitia, E.C., 1990. S100 proteins is released from astroglial cells by stimulation of 5-HT1A receptors. Brain Res. 528, 155–158. Willoughby, K.A., Kleindienst, A., Mu¨ller, C., Chen, T., Muir, J.K., Ellis, E.F., 2004. S100B protein is released by in vitro trauma and reduces delayed neuronal injury. J. Neurochem. 91, 1284–1291. Yanagihara, T., Yoshimine, T., Morimoto, K., Yamamoto, K., Homburger, H.A., 1985. Immunohistochemical investigation of cerebral ischemia in gerbils. J. Neuropathol. Exp. Neurol. 44, 204–215. Zhang, Y., Lipton, P., 1999. Cytosolic Ca+2 changes during in vitro ischemia in rat hippocampal slices: major roles of glutamate and Na+-dependent Ca+2 release from mitochondria. J. Neurosci. 19, 3307–3315.
Protein S100B release from rat brain slices during and after ischemia: Comparison with lactate dehydrogenase leakage Rifat Levent Bu¨yu¨kuysal Uludag˘ University, Medical School, Department of Pharmacology and Clinical Pharmacology, 16059 Bursa, Turkey Received 1 March 2005; received in revised form 20 June 2005; accepted 22 June 2005 Available online 27 September 2005
Abstract One hour of ischemia significantly increased protein S100B release from rat brain slices without altering lactate dehydrogenase leakage. Reoxygenation of the ischemic slices, however, increased the levels of these biochemical markers in the medium. Although removal of extracellular Ca+2 ions from the medium did not alter the basal lactate dehydrogenase leakage from cortical slices, an excessive increase in basal protein S100B release was seen under this condition. Ischemia and/or reoxygenation induced enhancements in these markers were attenuated by removal of Ca+2 ions from the medium. Ischemia significantly increased glutamate release, but neither ischemia nor reoxygenation induced rises in protein S100B and lactate dehydrogenase levels were altered by glutamate receptor antagonists. Rising the glutamate levels in the medium by each ouabain or exogenous glutamate, moreover, failed in exerting an ischemia like effect on protein S100B and LDH outputs. In contrast, exogenous glutamate added into the medium protected the slices against reoxygenation induced increments in protein S100B and lactate dehydrogenase levels. These results indicate that protein S100B has a greater sensitivity against ischemia than lactate dehydrogenase in in vitro brain slice preparations. Since neither exogenous glutamate nor enhancements of the extracellular glutamate levels by ouabain had an ischemia like effect, and since glutamate receptor antagonists were also unsuccessful, it seems unlikely that ischemia-induced increase in glutamate release is directly involved in protein S100B release or lactate dehydrogenase leakage determined in the present study. # 2005 Elsevier Ltd. All rights reserved. Keywords: Protein S100B; Ischemia; Lactate dehydrogenase leakage
1. Introduction Several methods have been used for determination of ischemia and/or reoxygenation (REO) induced tissue damage in in vitro studies. One of these methods is densitometric evaluation after staining of the brain tissue with 2,3,5-triphenyltetrazolium (TTC) (Mathwes et al., 2000). Lactate dehydrogenase (LDH), a cytoplasmic enzyme, is most commonly used biochemical marker in ischemic studies. Neuron-specific enolase (NSE), a neuronal form of the intracytoplasmic glycolytic enzyme enolase, on the other hand, has also been used as a biochemical marker in brain slice studies (Moro et al., 2000). It must be noted that significant alterations in each TTC-staining or LDH and NSE outputs have been observed after a transient exposure E-mail address: [email protected]. 0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.06.009
of brain slices to oxygen/glucose deprivation followed by REO (Tatsumi et al., 1998; Mathwes et al., 2000; Moro et al., 2000; De La Cruz et al., 2002; Bu¨yu¨kuysal, 2004; Mog˘ol et al., 2005), indicating that neuronal damage that becomes after a delay is an active process and blocking the postischemic events seems to be a more promising strategy against ischemia-induced neuronal damage. Increasing body of evidence indicates that acute ischemic stroke or traumatic brain damage also increases protein S100B levels in serum or CSF (Rothermundt et al., 2003). This protein is a member of the S-100 family of acidic, calcium binding proteins and mainly expressed in astrocytes and constitutively released from these cells (Donato, 2003). Although extracellular S100B exerts a dual effect on neurons depending on its concentration, i.e. pro-survival effect on neurons at nM and a toxic effect at m M concentrations (Donato, 2003; Rothermundt et al., 2003), mechanisms
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
involved in its release are not clear. While stimulation of 5HT1 serotonin, A1 adenosine or mGlu3 metabotropic glutamate receptors releases protein S100B from cultured astrocytes (Whitaker-Azmitia et al., 1990; Ciccarelli et al., 1999; Pinto et al., 2000), it has been reported that high concentration of glutamate has a decreasing effect on protein S100B secretion induced by serum deprivation in astrocytes (Goncalves et al., 2002). As increased S100B concentration induced by brain trauma and ischemia is believed to provide a biochemical information about the extent of the brain damage, either clarifying its release mechanism under ischemic conditions or understanding its advantages over other biochemical markers seems to be important for developing new therapeutic approaches against ischemia-induced neuronal damage. Thus, this study was undertaken to investigate protein S100B release from rat brain slices during and after ischemia. Each release and involvement of extracellular calcium ions and glutamate in protein S100B and LDH outputs were also compared in the present study.
2. Experimental procedures 2.1. Materials
581
medium. Following 90 min of equilibration period, slices were incubated in either control or ischemic medium for 1 h at 37 8C. Ischemic medium was not containing glucose and oxygen, but gassed with 95% N2 and 5% CO2. When glucose or calcium was omitted, osmolarity of the medium was maintained by increasing the NaCl concentration. Calcium-free medium was also containing EGTA (1 mM). At the end of ischemic incubation period, slices were transferred into the glucose and oxygen-containing medium (REO period). In order to determine time dependency of protein S100B release and LDH leakage, REO of the slices was extended up to 3 h and incubation medium was replaced with fresh medium at 1 h interval during this period. As both LDH and protein S100B outputs were highest during first 1 h of the REO period and then declined gradually (Fig. 1), in the rest of the studies, incubation of the slices was terminated at the end of first 1 h of the REO period. Half of the incubation medium (1 ml) collected at the end of ischemia was acidified with HClO4 (final concentration 0.4 M) and stored at 20 8C for quantification of excitatory amino acids released from the slices. Another half of the samples and others obtained after REO period were used for determination of protein S100B and LDH. Glutamate receptor antagonists were added into the medium 10 min
MK-801(+),CNQX, ouabain and glutamate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). aMethyl-4-carboxyphenylglycine (()-MCPG) was obtained from Research Biochemicals International (Natick, MA, USA). Other chemicals were at pure analytical grade and obtained from Merck KGaA (Darmstadt, Germany) or from Sigma Chemical Co. 2.2. Preparation and incubation of brain slices Male Sprague–Dawley rats (weighing 250–300 g and obtained from Experimental Animals Breeding and Research Center, Bursa, Turkey) were used. All experimental protocols were approved by Uludag˘ University Medical Center Institutional Review Board for animal research, and all efforts were made to minimize the number of animals used and their suffering. Rats were decapitated, their brains were removed quickly and were placed in cold oxygenated physiological medium with 95% O2 and 5% CO2 (in mmol/L: 120 NaCl, 1.3 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 3.5 KCl, 25 NaHCO3 and 10 glucose). After dissection, cortical, corpus striatal and hippocampal slices (0.3 mm of thickness) were prepared with a McIlwain tissue chopper (Brinkmann Instruments; Westbury, NY, USA). Slices were washed with oxygenated cold physiological medium and then transferred to 2 ml of incubation tubes. Each tube was divided into four separate chambers and contained three slices that did not touch each other. Slices were incubated in a water bath at 37 8C and the medium was changed every 10 min with fresh oxygenated
Fig. 1. Effects of ischemia and REO on protein S100B and LDH release from rat cortical slices. After 90 min of equilibration period, cortical slices were incubated in control or ischemic medium for 1 h. At the end of this incubation, all slices were transferred into oxygen and glucose containing medium (REO) for another 3 h. During this period, incubation medium was changed at 1 h intervals to fresh medium and samples collected at the end of each period were studied for determination of protein S100B and LDH levels. Data are given as means S.E.M. of six–seven determinations. * p < 0.05, ***p < 0.001, significantly different from their corresponding control levels.
582
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
before the ischemia and they were present in the medium until the end of the REO period. The effects of glutamate (2 mM) and ouabain (100 mM) on each amino acid, protein S100B and LDH outputs were studied on cortical slices incubated under normoxic conditions. In another part of the study, glutamate (2 mM) was also added into the medium during REO period of the ischemic cortical slices.
2–2.5 with 2 M of LiOH. A portion of the supernatant (20 ml) was then injected onto HPLC system without further purification. Glutamate and aspartic acid levels were calculated by comparing peak heights of the samples with their standards. Amino acid standards were prepared in physiological medium, acidified with HClO4 and processed together with samples.
2.3. LDH assay
2.6. Total protein assay
LDH activity in the incubation medium was assayed by using a commercial kit from Merck-Biotrol Diagnostics (France). Namely, 50 ml of incubation medium was mixed with 0.7 ml of reaction medium in a temperature controlled cuvette (30 8C). Changes in the absorbance were read at 340 nm after 30 s then every minute during 2 min. LDH activity was calculated according to average absorbance change and then corrected with protein levels of the slices.
Incubation of the slices was terminated by transferring them in 2 ml of 0.4 M HClO4. After homogenization of the slices, tissue protein levels were measured in 50 ml of homogenate according to the procedure of Lowry et al. (1951). Protein standards were prepared in 0.4 M HClO4 and processed together with the tissue samples. All results were corrected according to total protein levels of the slices.
2.4. Protein S100B assay
All the results in the text are expressed as mean S.E.M. The differences between the results were tested by Tukey– Kramer multiple comparison test or Student’s t-test. A probability of p < 0.05 was considered significant.
Protein S100B released into the medium was determined by an enzyme immunoassay test kit (Nexus Dx S100, from Synx Pharma Inc. Toronto, Canada), which was specific for the b-subunit of the S100 protein and measured the bsubunit concentration in both bb and ab isoforms of the protein. Samples were first diluted with distilled water (dilution ratio 1/20) and 25 m l of diluted samples was used for protein S100B assay. 2.5. Quantification of glutamate and aspartic acid Glutamate and aspartic acid levels in the medium were determined by a HPLC system (HP 1100 series, HewlettPackard, Palo Alto, CA, USA) coupled to a post-column derivatization unit (Pickering Laboratories, Mountain View, CA, USA). This system was combined with a quaternary pump (HP, G1311A), a fluorometric detector (HP, G1321 A) and an autosampler (HP, G1329 A). The amino acids which were separated on lithium exchange column (Pickering Labs., series number 5338) with Li280 and Li750 eluents (Pickering Labs.) were reacted with OPA in a post-column derivatization unit (both from the Pickering Labs.). The flow rates of the quaternary pump and post-column derivatization unit were 0.3 ml/min. Column and post-column reaction temperatures were adjusted to 40 and 45 8C, respectively. Other chromatographic conditions, such as the gradient program of the Li280, Li750 and lithium regenerant eluents, were similar with the conditions reported by Grunau and Swaider (1992). OPA reactive compounds were detected at excitation 330 nm, emission 465 nm wavelengths, and chromatograms were analyzed with a software (HP Chemstation, revision A. 08. 03. 847). Acidified samples were centrifuged for 5 min in a Beckman microfuge and pH of the samples was adjusted to
2.7. Data analysis
3. Results 3.1. Ischemia and REO-induced protein S100B and LDH outputs from rat cortical slices Basal protein S100B release from cortical slices incubated under normoxic conditions was 2.65 0.6 ng/ ml/mg protein/1 h. Omission of both glucose and oxygen from the medium (ischemia) increased protein S100B release more than 50%. When ischemic slices were transferred into the normoxic medium (REO period), a greater increase in protein S100B level was determined (Fig. 1). In contrast to protein S100B, 1 h of ischemia did not alter the LDH leakage from cortical slices. REO of the ischemic slices, on the other hand, enhanced LDH output to 331 21 DmOD/mg protein from its control value of 131 18 DmOD/mg protein (Fig. 1). It was also determined that both protein S100B and LDH outputs were highest during first 1 h of the REO period. As lengthening of REO up to 3 h did not cause a further increment in the release of these biochemical markers (Fig. 1), in the rest of the studies, REO of the ischemic slices was terminated at the end of 1 h of the REO period. 3.2. Comparison of the ischemia and REO-induced protein S100B and LDH outputs in cortical, striatal and hippocampal slices Basal levels of protein S100B released from cortical, striatal and hippocampal slices incubated under control
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
583
Table 1 Ischemia and REO-induced protein S100B and LDH outputs from rat cortical, striatal and hippocampal slices Brain region
Control
Ischemia
Control-REO
Ischemia-REO
(A) Protein S100 B (ng/ml/mg protein) Cortex 2.65 0.6 Corpus striatum 1.83 0.3 Hippocampus 1.79 0.6
4.56 0.5 * 3.57 0.3 ** 7.40 1.7 *
2.35 0.3 1.10 0.4 2.68 0.5
10.11 1.1 *** 4.86 0.6 *** 21.3 2.7***,a
(B) LDH (DmOD/mg protein) Cortex Corpus Striatum Hippocampus
177 40 156 20 321 19
131 18 106 14 262 18
331 21 *** 243 47 * 747 72***,a
168 19 152 30 309 27
After 90 min of equilibration period, brain slices were incubated in control or ischemic medium for 1 h followed by 1 h of REO period. At the end of each period, incubation medium was collected and studied for determination of protein S100B and LDH levels. Data are given as means S.E.M. of six–seven determinations. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from their corresponding control levels. ap < 0.001, significantly different from the value determined in cortical or striatal slices.
(normoxic) conditions were similar ( p > 0.05). Basal LDH leakage, on the other hand, was found higher in hippocampal slices ( p < 0.01). As obtained in cortical slices, ischemia did not alter the LDH leakage from striatal and hippocampal slices, but REO caused significant increases in the levels of this enzyme in the medium (Table 1). Although extents of the ischemia-induced LDH leakage were similar in each brain area (144 49%, 152 16% and 185 28% for striatal, cortical and hippocampal slices, respectively, p > 0.05), net amount of the LDH leakage into the medium was higher in hippocampal slices ( p < 0.001). Ischemia, as obtained in cortical slices, significantly increased protein S100B release from striatal and hippocampal slices. REO of the ischemic brain slices, on the other hand, further enhanced protein S100B outputs. When ischemia and REO-induced increments in protein S100B outputs from different brain regions were compared, greater increases were seen in hippocampal slices (Table 1). Like cortical slices, protein S100B and LDH outputs from striatal and hippocampal slices were highest during first 1 h of REO period, increases in their levels then declined gradually (data not shown).
in REO-induced LDH leakage ( p < 0.001). If calcium ions were removed during only REO period, a significant decline in REO-induced LDH leakage was also present ( p < 0.01). In agreement with these observations, REOinduced LDH leakage further declined when omitting the calcium ions from incubation medium during both ischemia and REO periods ( p < 0.001; Fig. 3).
3.3. Calcium dependency of protein S100B and LDH outputs from rat cortical slices Removal of extracellular calcium ions from normoxic incubation medium caused 455% increase in protein S100B output from cortical slices. Similar amount of increases were also seen when calcium ions were omitted during ischemia and REO periods (Fig. 2). Although chasing calcium-free ischemia with a calcium containing REO period brought the protein S100B levels back below what would have been generated from calcium-free ischemia alone, its level was not different than the value obtained from ischemia (+Ca+2)-REO (+Ca+2) group (Fig. 2). In contrast to protein S100B, basal LDH leakage did not alter in the absence of calcium ions in the medium (data not shown). When calcium ions were omitted during ischemic period, a significant attenuation was determined
Fig. 2. Effect of extracellular Ca+2 omission on basal, ischemia and REOinduced protein S100B outputs from cortical slices. After equilibration period, cortical slices were incubated either in Ca+2-free or Ca+2-containing control or ischemic medium for 1 h followed by 1 h REO period in the absence or presence of extracellular Ca+2 ions. Data are given as means S.E.M. of three determinations for Ca+2-containing control and Ca+2containing ischemia groups, and eight determinations for the remaining groups. ***p < 0.001, significantly different from the corresponding value determined in the presence of Ca+2 ions, ap > 0.05, not significantly different from the ischemia (+Ca+2)-REO (+Ca+2) group.
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
584
Fig. 3. Effect of extracellular Ca+2 omission on REO-induced LDH leakage from cortical slices. After equilibration period, cortical slices were incubated either in Ca+2-free or Ca+2-containing ischemic medium for 1 h followed by 1 h REO period in the absence or presence of extracellular Ca+2 ions. Data are given as means S.E.M. of eight determinations for each group. **p < 0.01, ***p < 0.001, significantly different from the value determined in ischemia (+Ca+2)-REO (+Ca+2) group.
3.4. Involvement of glutamate in ischemia and REOinduced protein S100B and LDH outputs from rat cortical slices Since ischemia causes an excessive increase in extracellular glutamate level, it is one possibility that this increase contributes to ischemia and/or REO-induced protein S100B and LDH outputs. This possibility was tested by increasing the glutamate levels in normoxic medium by different pharmacological manipulations, or by adding the glutamate receptor antagonists into the medium before ischemia. As seen in Table 2, 1 h of ischemia or incubation of cortical slices in ouabain (100 mM) containing normoxic medium for 1 h significantly increased glutamate and aspartic outputs. Incubation of cortical slices with high concentration of glutamate (2 mM), on the other hand, significantly increased aspartic acid level in the medium (Table 2). Despite these excessive increases in excitatory amino acid levels, neither exogenous glutamate nor ouabain increased protein S100B and LDH outputs. In contrast, basal protein Table 2 Excitatory amino acid release from rat cortical slices: effects of ischemia, ouabain and glutamate
Control Ischemia Ouabain Glutamate
Aspartic acid
Glutamate
37 8 1991 193*** 1370 81 *** 6651 928***
29 6 12757 1672*** 6307 872*** –
After 90 min of equilibration period, cortical slices were incubated in control or in ischemic medium, or ouabain (100 mM) or glutamate (2 mM) containing normoxic medium for 1 h. Incubation medium collected at the end of the incubation period was used for determination of aspartic acid and glutamate levels. Data are given as means S.E.M. of five–six determinations. ***p < 0.001, significantly different from their corresponding control levels.
S100B release was declined significantly by 2 mM of glutamate (Table 3). Similarly, 1 h of drug-free period followed by glutamate or ouabain incubation also failed to show a REO-like effect. When added into the medium during REO period, on the other hand, high concentration of glutamate protected the slices against ischemia and/or REOinduced increments in protein S100B and LDH leakages (Table 3). Presence of MK-801 (100 mM), CNQX (50 mM) and MPCG (50 mM) in the medium during both ischemia and REO periods, which are known to block NMDA, kainat/ AMPA and metabotropic glutamate receptors, respectively, were unsuccessful to inhibit ischemia and REO-induced increases in protein S100B outputs from cortical slices (Fig. 4). Similarly, LDH leakage enhanced by REO of the ischemic cortical slices was not altered significantly by glutamate receptor antagonists (Fig. 5).
4. Discussion Since protein S100B has been shown to be increased in human blood and CSF after traumatic brain damage, many studies have focused on protein S100B under various ischemic conditions. Although results obtained from these studies demonstrated a close correlation between the protein S100B concentration and infarct volume as well as clinical outcome (Rothermundt et al., 2003), neither ischemia and/or REO-induced alterations in protein S100B release nor its release mechanism have been studied in brain slice preparations. Now I am reporting here that ischemia increases protein S100B release from brain slices and this increase is further enhanced by REO. In contrast, LDH levels in the medium, another biochemical marker which is widely used to evaluate and quantify cellular damage, was not altered by ischemia. It has been suggested that LDH assay is not suitable for acute brain preparations, because its release is slow relative to deterioration of cellular morphology (Izumi et al., 2001). It is known that, however, not only LDH leakage, but NSE release or TTC staining in brain slices does not response to ischemia (Mathwes et al., 2000; Moro et al., 2000). REO of the ischemic slices, on the other hand causes significant alterations in the levels of these biological markers (Tatsumi et al., 1998; Mathwes et al., 2000; Moro et al., 2000; De La Cruz et al., 2002; Bu¨yu¨kuysal, 2004; Mog˘ol et al., 2005) supports the conclusion that ischemia-induced neuronal damage actually becomes after a delay, and REO seems to be a critical period for resultant tissue damage. Because ischemia increased protein S100B release in the absence of signs of brain cell death determined as LDH leakage, this increase probably reflects a specific cell response to ischemia. It is noteworthy that ischemia and REO caused greater increases in protein S100B release from hippocampal slices than the slices prepared from other two brain regions. Similarly, basal or REO-induced LDH leakage was also higher in hippocampal
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
585
Table 3 Effects of ouabain and glutamate on protein S100B and LDH release from rat cortical slices
(A) Normoxic medium Control Ouabain Glutamate (B) After ischemia REO REO + glutamate
Protein S100B (ng/ml/mg protein)
LDH (DmOD/mg protein)
First incubation (drug added)
Second incubation (drug free)
First incubation (drug added)
Second incubation (drug free)
2.93 0.4 2.18 0.4 3.10 0.6
116 25 101 30 82 9
90 5 75 5 82 8
3.54 0.7 4.12 0.4 2.63 0.2* 10. 36 1 4.95 0.2 ***
424 40 151 10***
After equilibration period, cortical slices were incubated in control, ouabain (100 mM) or glutamate (2 mM) containing normoxic medium for 1 h followed by drug-free period for another 1 h. When the effect of exogenous glutamate on REO-induced protein S100B and LDH outputs was tested, cortical slices were incubated first in ischemic condition for 1 h followed by 1 h REO period in the absence or presence of 2 mM glutamate. At the end of each incubation period, medium was collected and used for determination of protein S100B and LDH levels. Data are given as means S.E.M. of three–seven determinations. * p < 0.05, significantly different from their corresponding control values. ***p < 0.001, significantly different from the value obtained in the absence of glutamate.
slices. Either regional difference of neuronal vulnerability to ischemia (Yanagihara et al., 1985) or differences in cell densities between the brain regions, as determined previously by number of the S100B positive astrocytes (Savchenko et al., 2000), may result in an unequal release or leakage of these biochemical markers from the slices. As increased levels of intracellular free Ca+2 are implicated in ischemia and/or REO-induced alterations (Kristian and Siesjo¨, 1998; Zhang and Lipton, 1999; Sattler and Tymianski, 2000), Ca+2 dependency of protein S100B release was also compared with LDH leakage in the present study. As seen in Fig. 3, removal of Ca+2 ions from the medium during ischemia significantly attenuated REO-induced LDH leakage from
Fig. 4. Effects of glutamate receptor antagonists on ischemia and REOinduced protein S100B outputs from rat cortical slices. After 90 min of equilibration period, cortical slices were incubated in control or ischemic medium for 1 h followed by 1 h REO period. At the end of ischemia and REO periods, incubation medium was used for determination of protein S100B levels. MK-801 (100 mM), CNQX (50 mM) and MCPG (50 mM) were added to the medium 10 min before ischemia and were present in the medium until the end of REO period. Data are given as means S.E.M. of four determinations. Ischemia or REO-induced enhancements determined in the presence of glutamate receptor antagonists are not different from the value obtained in the absence of these drugs ( p > 0.05).
Fig. 5. Effects of glutamate receptor antagonists on REO-induced LDH leakage from rat cortical slices. After 90 min of equilibration period, cortical slices were incubated in each control or ischemic medium for 1 h followed by 1 h REO period. At the end of REO period, incubation medium was used for determination of LDH levels. MK-801 (100 mM), CNQX (50 mM) and MCPG (50 mM) were added to the medium 10 min before ischemia and were present in the medium until the end of REO period. Data are given as means S.E.M. of seven–eight determinations. REO-induced enhancements determined in the presence of glutamate receptor antagonists are not different than the value obtained in the absence of these drugs ( p > 0.05).
586
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
cortical slices ( p < 0.001). Similar but slightly less improvement ( p < 0.01) was also seen when Ca+2 ions were omitted during REO period. If Ca+2 ions were omitted during both ischemia and REO periods, on the other hand, REO-induced LDH leakage was further attenuated, indicating that removal of extracellular Ca+2 ions, as reported previously (Tatsumi et al., 1998; Bu¨yu¨kuysal, 2004), would protect the slices against REO-induced LDH leakage. In contrast, basal protein S100B release significantly augmented when extracellular Ca+2 ions were omitted from the normoxic medium. Neither ischemia nor REO, however, caused an additional increase in protein S100B release in Ca+2 free medium. Although these findings indicate that removal of extracellular Ca+2 ions, as observed in LDH leakage, protects the slices against ischemia and/or REO-induced protein S100B outputs, it is one possibility that removal of Ca+2 ions from the medium completely depletes releasable pool of the protein S100B from the slices. Thus, neither ischemia nor REO could cause an additional increase in the absence of Ca+2 ions. However, it was determined that REO still increases protein S100B release from the slices that were incubated first in Ca+2-free ischemic medium (see Fig. 2), suggesting that even after Ca+2free medium-induced depletion, a releasable pool of protein S100B probably remains in the slices. Mechanism(s) involved in Ca+2-free medium-induced protein S100B release was not investigated the in present study. As removal of Mg+2 ions from the medium (but 1 mM of EGTAwas also present) failed in enhancing the control protein S100B level (data not shown), neither an alteration in the membrane charges (Piccolino and Pignatelli, 1996), nor an unspecific effect of EGTA seems involved. It is known that protein S100B, like some other S100 proteins, undergoes Ca+2-induced conformational changes and interacts with its target proteins in a Ca+2-dependent manner (Donato, 2003). Thus, it is likely that possible influences of Ca+2 removal on these parameters causes an abundant increase in protein S100B release. Since ischemia decreases extracellular Ca+2 concentration by translocating almost all extracellular Ca+2 ions into cells (Kristian and Siesjo¨, 1998), it is one possibility that declining in extracellular Ca+2 levels during ischemia also contributes, at least partly, to ischemia-induced protein S100B release as determined in the present study. It has been repeatedly shown that extracellular accumulation of excitatory amino acids plays a major role in ischemia and/or REO-induced tissue damage (Martin et al., 1994; Szatkowski and Attwell, 1994; Calabresi et al., 1999; Nishizawa, 2001) leading to glutamate–calcium hypothesis in the pathology of ischemia (Kristian and Siesjo¨, 1998; Sattler and Tymianski, 2000). According to this hypothesis, energy failure results in loss of Na+, K+-ATPase activity and causes membrane depolarization (anoxic depolarization) which triggers glutamate release. Thus, elevation glutamate level in the extracellular space and subsequent activation of glutamate receptors are implicated in neuronal damage. In a good agreement with previous reports (Pellegrini-Giampietro et al., 1990; Nishizawa, 2001; Nelson et al., 2003;
Bu¨yu¨kuysal, 2004; Mog˘ol et al., 2005), ischemia significantly increased glutamate release from cortical slices. The presence of high concentration of glutamate receptor antagonists MK-801, CNQX and MCPG, those block the NMDA, kainat/AMPA and metabotropic glutamate receptors, respectively, however, did not alter the ischemia and REO-induced increments in protein S100B release. Similarly, REO-induced increase in LDH leakage from cortical slices was not attenuated by glutamate receptor antagonists. Although these findings seem to be in opposite to glutamate hypothesis mentioned above, failure or limited effects of glutamate receptor antagonists under in vitro conditions (Oka et al., 2000; Joshi and Andrew, 2001; Bu¨yu¨kuysal, 2004) and in clinical trials (De Keyser et al., 1999; Plum, 2001) have also been reported. Additionally, collapse of the Na+ and K+ gradients by 100 mM of ouabain, a Na+–K+ATPase inhibitor, caused an ischemia-like effect on glutamate release, but neither protein S100B nor LDH leakage was increased by ouabain. If 100 mM of ouabain, as observed in mouse hemi-brain slices (Joshi and Andrew, 2001), elicits a neuronal depolarization similar to that induced by oxygen-glucose deprivation, then failure of the ouabain on protein S100B and LDH leakages also excludes a possible involvement of ischemic depolarization in the release of these biochemical markers. In the present study, it was also determined that, glutamate added into the normoxic medium at high concentration also failed in increasing the protein S100B and LDH outputs, strengthening the conclusion that excessive accumulation of glutamate in extracellular space is not directly or solely involved in ischemia and/or REOinduced protein S100B and LDH outputs. Interestingly, high concentration of glutamate added into the medium during REO period completely protected the slices against REOinduced protein S100B and LDH leakages. A similar attenuation of S100B secretion by high glutamate has also been reported in serum deprived hippocampal astrocytes (Goncalves et al., 2002). In patients with acute ischemic stroke, moreover, ZK200775, an AMPA antagonist, has worsened neurological outcome that was associated with a higher serum S100B levels than ischemia alone (Elting et al., 2002). The protective mechanism of glutamate on REOinduced LDH and protein S100B outputs are still under progress, but involvement of metabotropic glutamate receptors that are linked to protein kinase C activation is likely (Adamchik and Baskys, 1999; Schro¨der et al., 2000). Since glutamate is an essential element for glutathione synthesis in astroglia and ischemia decreases glutathione levels dramatically (Huster et al., 2000), addition of excessive glutamate into the medium during REO may increase the astrocyte antioxidant defense system and thus may lead to a decreased vulnerability to free radical damage. Although high concentration of exogenous glutamate added into the normoxic medium or inhibition of Na+, K+ATPase by ouabain did not show an ischemia-like effect on protein S100B and LDH outputs, many events such as altered Ca+2 homeostasis, activation of Ca+2-dependent enzymes,
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
mitochondrial permeability transition, excessive production of either reactive oxygen species or proinflamatory cytokines also occur during ischemia and/or REO period (Neumar, 2000). Thus, putative roles of these pathways in combination with glutamate release may not be ruled out. As its release mechanism, functional roles of S100B released during and/or after ischemia are also unclear. Data collected from S100B studies suggest that it has a dual effect on neurons depending on its concentration, i.e. it stimulates neurite outgrowth and enhances neuronal survival at nanomolar, but has deleterious effects at micromolar concentrations (Rothermundt et al., 2003). In rat hippocampal neurons, tissue damage induced by glucose deprivation has been protected by protein S100B, and it has been suggested that its elevation in the medium may be a compensatory response (Barger et al., 1995). Similarly, stretch injury has caused an immediate and prolonged release of protein S100B from neuronal plus glial cells, and S100B added to culture medium has reduced delayed neuronal injury (Willoughby et al., 2004). One of the most relevant findings in the present study is that protein S100B release increases during ischemia in the absence of signs of brain cell death and its levels in the extracellular medium increase further during REO. Although this finding seems to be important in the context of the putative role of protein S100B as a neurotropic factor during early phase of brain insults and a neurotoxic factor during late phase of insults, detailed studies are required in order to clarify whether protein S100B released during and after ischemia has similar and/or opposite effects on neuronal survival. In summary, results presented in this paper clearly indicate that ischemia causes significant increases in protein S100B release from brain slices without altering the LDH leakage. REO of the ischemic slices, on the other hand, enhances the levels of these biochemical markers in the medium by a mechanism seems to be not directly or solely related with the ischemia-induced glutamate output. Since ischemia-induced increase in protein S100B occurs in the absence of signs of brain cell death and probably reflects a specific cell response to ischemia, it is likely that protein S100B is a more valuable biological marker than LDH, at least, for in vitro brain slice studies.
Acknowledgement This study was supported by a grant from Uludag˘ University Research Council (2002/65).
References Adamchik, Y., Baskys, A., 1999. Glutamate-mediated neuroprotection against N-methyl-D-aspartate toxicity: a role for metabotropic glutamate receptors. Neuroscience 99, 731–736. Barger, S.W., Van Eldik, L.J., Mattson, M.P., 1995. S100 beta protects hippocampal neurons from damage induced by glucose deprivation. Brain Res. 677, 167–170.
587
Bu¨yu¨kuysal, R.L., 2004. Ischemia and reoxygenation-induced amino acid release and tissue damage in the slices of rat corpus striatum. Amino Acids 27, 57–67. Calabresi, P., Marfia, G.A., Centonze, D., Pisani, A., Bernardi, G., 1999. Sodium influx plays a major role in the membrane depolarization induced by oxygen and glucose deprivation in rat striatal spiny neurons. Stroke 30, 171–179. Ciccarelli, R., Di Lorio, P., Bruno, V., Battaglia, G., D’Alimonte, I., D’Onofrio, M., Nicoletti, F., Caciagli, F., 1999. Activation of A1 adenosine or mGlu3 metabotropic receptors enhances the release of Nerve Growth Factor and S100b protein from cultured astrocytes. Glia 27, 275–281. De Keyser, J., Sulter, G., Luiten, P.G., 1999. Clinical trials with neuroprotective drugs in acute ischemic stroke: are we doing the right thing? Trends Neurosci. 22, 535–540. De La Cruz, J.P., Villalobos, M.A., Cuerda, M.A., Guerrero, A., GonzalesCorrea, J.A., De La Cuesta, F.S., 2002. Effects of S-adenosyl-methionine on lipid peroxidation and glutathione levels in rat brain slices exposed to reoxygenation after oxygen–glucose deprivation. Neurosci. Lett. 318, 103–107. Donato, R., 2003. Intracellular and extracellular roles of S100 proteins. Microsc. Res. Techn. 60, 540–551. Elting, J.-W., Sulter, G.A., Kaste, M., Lees, K.R., Diener, H.C., Hommel, M., Versavel, M., Teelken, A.W., De Keyser, J., 2002. AMPA antagonist ZK200775 in patients with acute ischemic stroke. Possible glial cell toxicity determined by monitoring of S-100B serum levels. Stroke 33, 2813–2818. Goncalves, D., Karl, J., Leite, M., Rotta, L., Salbego, C., Rocha, E., Wofchuk, S., Goncalves, C.A., 2002. High glutamate decreases S100B secretion stimulated by serum deprivation in astrocytes. Neuroreport 27, 1533–1535. Grunau, J.A., Swaider, J.M., 1992. Chromatography of 99 amino acids and other ninhydrin-reactive compounds in Pickering lithium gradient system. J. Chromatogr. 594, 165–171. Huster, D., reichenbach, A., Reichelt, W., 2000. The glutathione content of retinal Muller (glial) cells: effect of pathological conditions. Neurochem. Int. 36, 461–469. Izumi, Y., Izumi, M., Benz, A.M., Zorumski, C.F., 2001. Lactate dehydrogenase release is facilitated by brief sonication of rat hippocampal slices and isolated retinas following acute neuronal damage. J. Neurosci. Methods 108, 49–55. Joshi, I., Andrew, R.D., 2001. Imaging anoxic depolarization during ischemia-like conditions in the mouse hemi-brain. J. Neurophys. 85, 414–424. Kristian, T., Siesjo¨, B.K., 1998. Calcium in ischemic cell death. Stroke 29, 705–718. Lowry, O.H., Rosebrough, N.J., Farr, A.L., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Martin, R.L., Lloyd, H.G.E., Cowan, A.I., 1994. The early events of oxygen and glucose deprivation: setting the scene for neuronal death? Trends Neurosci. 17, 251–257. Mathwes, K.S., McLaughlin, D.P., Ziabari, L.H., Toner, C.C., Street, P.C., Hisgrove, E., Bezzina, E.L., Stamford, J.A., 2000. Rapid quantification of ischaemic injury and cerebroprotection in brain slices using densitometric assessment of 2,3,4-triphenyltetrazolium chloride staining. J. Neurosci. Methods 102, 43–51. Mog˘ol, E.B., Bu¨yu¨kuysal, R.L., Korfalı, G., 2005. Effects of ketamine and thiopental on ischemia-reoxygenation induced LDH leakage and amino acid release from rat striatal slices. J. Neurosurg. Anesthesiol. 17, 20– 27. Moro, M.A., De Alba, J., Cardenas, A., De Cristobal, J., Leza, J.C., Lizasoain, I., Diaz-Guerra, M.J.M., Bosca, L., Lorenzo, P., 2000. Mechanism of neuroprotective effect of aspirin after oxygen and glucose deprivation in rat forebrain slices. Neuropharmacology 39, 1309–1318. Nelson, R.M., Lambert, D.G., Green, A.R., Hainsworth, A.H., 2003. Pharmacology of ischemia-induced glutamate efflux from rat cerebral cortex in vitro. Brain Res. 964, 1–8.
588
R.L. Bu¨yu¨kuysal / Neurochemistry International 47 (2005) 580–588
Neumar, R.W., 2000. Molecular mechanisms of ischemic neuronal injury. Ann. Emerg. Med. 36, 483–506. Nishizawa, Y., 2001. Glutamate release and neuronal damage in ischemia. Life Sci. 69, 369–381. Oka, M., Itoh, Y., Ukai, Y., 2000. Preferential inhibition by a novel Na+/Ca2+ channel blocker NS-7 of severe to mild hypoxic injury in rat cerebrocortical slices: a possible involvement of a highly voltage-dependent blockade of Ca2+ channel. J. Pharmacol. Exp. Therap. 293, 522–529. Pellegrini-Giampietro, D.E., Cherici, G., Alesiani, M., Carla, V., Moroni, F., 1990. Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage. J. Neurosci. 10, 1035–1041. Piccolino, M., Pignatelli, A., 1996. Calcium-independent synaptic transmission: artifact or fact. Trends Neurosci. 19, 120–125. Pinto, S.S., Gottfried, C., Mendez, A., Goncalves, D., Karl, J., Goncalves, C.A., Wofchuk, S., Rodnight, R., 2000. Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology. FEBS Lett. 486, 203–207. Plum, F., 2001. Neuroprotection in acute ischemic stroke. J. Am. Med. Assoc. 13, 1760–1761. Rothermundt, M., Peters, M., Prehn, J.H.M., Arolt, V., 2003. S100B in brain damage and neurodegeneration. Microsc. Res. Techn. 60, 614–632. Sattler, R., Tymianski, M., 2000. Molecular mechanisms of calciumdependent excitotoxicity. J. Mol. Med. 78, 3–13. Savchenko, V.L., Mckanna, J.A., Nikonenko, I.R., Skibo, G.G., 2000. Microglia and astrocytes in the adult rat brain: comparative immuno-
cytochemical analysis demonstrates the efficacy of lipocotin I immunoreactivity. Neurosciences 96, 195–203. Schro¨der, U.H., Opitz, T., Jager, T., Sabelhaus, C.F., Breder, J., Reymann, K.G., 2000. Protective effects of group I metabotropic glutamate receptor activation against hypoxic/hypoglycemic injury in rat hippocampal slices: timing and involvement of protein kinase C. Neuropharmacology 38, 209–216. Szatkowski, M., Attwell, D., 1994. Triggering and execution of neuronal death in brain ischemia: two phases of glutamate release by different mechanisms. Trends Neurosci. 17, 359–365. Tatsumi, S., Itoh, Y., Ukai, Y., Kimura, K., 1998. A novel Na+/Ca+2 channel blocker, NS-7, suppresses hypoxic injury in rat cerebrocortical slices. Naunyn-Schmiedeberg’s Arch. Pharmacol. 358, 191–196. Whitaker-Azmitia, P.M., Murphy, R., Azmitia, E.C., 1990. S100 proteins is released from astroglial cells by stimulation of 5-HT1A receptors. Brain Res. 528, 155–158. Willoughby, K.A., Kleindienst, A., Mu¨ller, C., Chen, T., Muir, J.K., Ellis, E.F., 2004. S100B protein is released by in vitro trauma and reduces delayed neuronal injury. J. Neurochem. 91, 1284–1291. Yanagihara, T., Yoshimine, T., Morimoto, K., Yamamoto, K., Homburger, H.A., 1985. Immunohistochemical investigation of cerebral ischemia in gerbils. J. Neuropathol. Exp. Neurol. 44, 204–215. Zhang, Y., Lipton, P., 1999. Cytosolic Ca+2 changes during in vitro ischemia in rat hippocampal slices: major roles of glutamate and Na+-dependent Ca+2 release from mitochondria. J. Neurosci. 19, 3307–3315.