- Email: [email protected]
Cerebral penetration injury leads to H2O2 generation in microdialysis samples
Neuroscience Letters 236 (1997) 63–66
Cerebral penetration injury leads to H2O2 generation in microdialysis samples Matthew E. Layton a ,*, Thomas L. Pazdernik b , c, Fred E. Samson c a
Department of Psychiatry and Behavioral Sciences, University of Washington, P.O. Box 356560, Seattle, WA 98195, USA Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA c Smith Mental Retardation Research Center, University of Kansas Medical Center, Kansas City, KS, USA
b
Received 4 September 1997; accepted 6 September 1997
Abstract Delayed tissue damage is proposed to be caused by reactive oxygen species. We investigated the effects of microdialysis probe penetration into rat piriform cortex on hydrogen peroxide (H2O2) in brain extracellular fluid (ECF). H2O2 decreased immediately after probe insertion into the brain, but increased over 300% in samples within minutes after collection. We assessed H2O2 changes in vitro in microdialysis perfusion media containing various ascorbic acid concentrations and confirmed ascorbic acid is a source of H2O2. We conclude that decreased H2O2 concentrations in perfusion media as it passes through the brain reflect an extracellular antioxidant effect, whereas the increase in H2O2 with time after sample collection indicates that H2O2 generating substances are present in ECF. Thus, the potential for producing reactive oxygen species in brain ECF exists following penetration injury, especially if transition metals are released into the neuronal microenvironment. 1997 Elsevier Science Ireland Ltd.
Keywords: Antioxidant; Ascorbic acid; Brain injury; Chemiluminescence; Hydrogen peroxide; Microdialysis; Oxidants; Redox
Delayed brain damage from a variety of diseases or injuries to the central nervous system including trauma, ischemia/reperfusion and seizures is proposed to involve reactive oxygen species [3,4,9]. Hydrogen peroxide (H2O2), ascorbic acid and transition metals have pivotal actions in the formation of these reactive oxygen species [10]. Redox processes are important in brain function as well as in neuropathologies. Intracerebral microdialysis provides a method to detect, quantify and characterize changes in redox-active small molecules, such as ascorbic acid and H2O2, in brain extracellular fluid (ECF) in awake, freely-moving animals [2]. Two groups [13,16] have recently reported on H2O2 levels measured in microdialysates following ischemia/ reperfusion. In addition, we recently confirmed that ascorbic acid levels are high in ECF immediately following probe penetration into the brain [11,15] and demonstrated that
* Corresponding author. Tel.: +1 206 5433750; fax: +1 206 5439520; e-mail: [email protected]
ascorbic acid contributes to a non-linear production of oxidative chemiluminescence [15]. In the current study, we measured the H2O2 component of the oxidative chemiluminescence produced by microdialysates. The microdialysates were analyzed for H2O2 before and immediately after the probe was inserted into the piriform cortex over a period of 2 h. H2O2 was measured in microdialysates both immediately after collection and 8 min post-collection. Since ascorbic acid levels in microdialysates are about 15 mM immediately after probe insertion and decline to about 1 mM by 2 h post probe insertion [15], we also measured H2O2 in vitro in Kreb’s Ringer bicarbonate (KRB) solution containing varying concentrations of ascorbic acid immediately, 8 or 16 min after ascorbic acid was added to the KRB solution. H2O2 (30%), catalase from bovine liver (EC 1.11.1.6), microperoxidase disodium salt from equine heart cytochrome c (MP-11), 6-amino-2,3-dihydro-1,4-phthalazinedione (isoluminol), sodium borate (Na2BO4) and ascorbic acid were all obtained from Sigma Chemical (St. Louis,
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00765- 9
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MO, USA). Dow 50 cellulose microdialysis membranes from a kidney dialysis cartridge (molecular weight cut-off, 5000; o.d., 250 mm) were used in concentric tube microdialysis probes of fused silica hollow tubing (PolymicroTechnology; i.d., 75 mm; o.d., 145 mm) inside Teflon tubing with approximately 4 mm exposed dialysis membrane. All materials that have contact with the perfusate such as glassware, pipets and microdialysis probe construction materials were selected for their minimal production of autoluminescence or chemiluminescence. All glassware was acid washed and rinsed with triple distilled water. Adult male Wistar rats were obtained from Sasco (Omaha, NE, USA). Food and water were provided ad libitum, and a 12:12 h light-dark cycle was maintained. Experiments were conducted in accordance with the guidelines of the National Research Council DHEW publication #(NIH) 80-23 (1980). Animals were anesthetized with pentobarbital (80 mg/kg i.p. injection) prior to aseptic stereotaxic probe implantation. Stereotaxic measurements relative to bregma for the rat piriform cortex are −1.8 mm anteroposterior, −5.7 mm lateral and −10.0 mm vertical [19]. The dura mater was punctured with a 27-gauge needle and epoxy and dental acrylic secured the apparatus to the skull. The microdialysis probes were perfused with modified KRB (pH 7.4; NaCl 144.5 mM; KCl 3.0 mM; CaCl2 1.2 mM; KH2PO4 0.4 mM; MgSO4 1.2 mM; NaHCO3 2.5 mM) at a rate of 2 ml/ min using a Hamilton Microliter 1000 Series Gas tight syringe with a Teflon plunger and syringe attachment hub in a Harvard infusion pump. Microdialysis perfusion was initiated prior to surgery and continued throughout probe implantation; samples were collected at 20 min intervals. A chemiluminescence assay developed for analysis of reactive oxidative species in plasma [7] was adapted for microdialysis samples. This method employs amplified chemiluminescence produced by isoluminol (1 mM) in the presence of microperoxidase (0.01 mM) and sodium borate (100 mM) in 70% water and 30% methanol at pH 10. Chemiluminescence was quantified with an MGM Optocomp I luminometer (Hamden, CT, USA). H2O2 standards and microdialysis samples were placed in a test tube in the sample compartment of the luminometer. The chemiluminescence solution (300 ml) was automatically injected into the sample compartment from a reservoir. Chemiluminescence was quantified with an integrated kinetic photon counting protocol of 60 consecutive 4 s counting intervals for a total counting period of 4 min and expressed as relative light units (RLU). Samples were assayed in the presence and absence of catalase (final concentration 30 U/ml). Catalase activity was tested in the presence and absence of 25 mM ascorbic acid, the upper limit of ascorbic acid concentrations measured in brain microdialysis samples, as well as in the presence and absence of the isoluminol/microperoxidase chemiluminescence solution. A Clark electrode was used to monitor oxygen liberation from a solution containing ascorbic acid, catalase (30 U/ml) in KRB or in the chemiluminescence solution prior to and after addition of
H2O2 (final concentration 4 mM). Catalase activity was unaffected by these conditions [15]. The catalase-sensitive component of chemiluminescence was used as an indirect measure of H2O2-dependent chemiluminescence, which is linear from 15 nM to 1 mM (standard curve; r2 = 0.99). This chemiluminescence analytical procedure was also used to measure H2O2 in KRB samples with known ascorbic acid concentrations. Samples from 0, 1, 2, 5, 10, 25, 50 and 100 mM ascorbic acid solutions were mixed with an equal volume of KRB with or without catalase (final 30 U/ml) prior to chemiluminescence analysis. H2O2 concentrations in the KRB perfusion media at time of preparation were approximately 30 nM, comparable to previous reports of H2O2 concentrations in aqueous media [12]. Simply passing the KRB through the microdialysis system in vitro doubled the H2O2 concentrations in the perfusates to 63.6 ± 10 nM (mean ± SEM, n = 18). Thus, H2O2 is produced in the microdialysis apparatus itself. On the other hand, after probe insertion into the brain, H2O2 concentrations in the first microdialysis samples were significantly lower at 36.3 ± 2.7 nM (P , 0.05, n = 18). Indeed the H2O2 concentrations in samples collected during the first hour after probe insertion remained significantly lower than in perfusates prior to insertion. By 2 h after probe insertion, H2O2 in the perfusates returned to the pre-insertion levels (Fig. 1). The early decrease in H2O2 can be explained by the antioxidant activity of the brain extra-
Fig. 1. H2O2 concentrations in microdialysis samples before and after microdialysis probe insertion into rat brains. Samples were collected at 20 min intervals and analyzed with an isoluminol-amplified chemiluminescence assay in the presence and absence of catalase (30 U/ ml). The catalase-sensitive component of chemiluminescence represents the contribution of H2O2 to total chemiluminescence. H2O2 standards (15 nM–1 mM) produce linear, concentration-dependent increases in chemiluminescence (r2 = 0.99). Samples were reanalyzed 8 min after collection. Values represent the mean ± SEM, n = 18 initial samples and n ≥ 5 repeat sample analyses at each time point. *P , 0.05, t-test versus H2O2 concentrations in samples prior to probe insertion. **P , 0.05, t-test versus H2O2 concentrations in samples measured immediately after collection compared to those measured 8 min after collection.
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cellular microenvironment. However, when samples were reanalyzed for H2O2 only 8 min after collection, the H2O2 concentrations increased 3- to 4-fold to a range of 150–200 nM (n ≥ 5 repeat sample analyses at each time point; Fig. 1). This demonstrates that ECF has H2O2 generating substances (i.e. pro-oxidant substances) that are revealed when the perfusate leaves the antioxidant environment of the brain. To ascertain whether ascorbic acid contributes to H2O2 generation, H2O2 was measured in KRB solutions with various ascorbic acid concentrations (0, 1, 2, 5, 10, 25, 50 and 100 mM). Samples were analyzed for H2O2 concentrations immediately and at 8 and 16 min after sample preparation. The H2O2 concentrations measured immediately were significantly lower in 50 mM than in 1 mM ascorbic acid solutions (P , 0.05; n ≥ 4; Fig. 2), demonstrating the predominately antioxidant properties of ascorbic acid at the high concentrations [15]. H2O2 concentrations at 8 and 16 min increased somewhat over those measured immediately at all ascorbic acid concentrations; however, the increase in H2O2 was the greatest in those samples with the lowest ascorbic acid concentrations (1 and 2 mM; see Fig. 2). When all metals are removed from ascorbic acid solutions with Chelex [4], no H2O2 is formed, so traces of transition metals (i.e. copper or iron), normally occurring in buffers, can support H2O2 formation in oxygenated solutions containing low concentrations of ascorbic acid. Since microdialysates or any oxygenated biological solution with low ascorbic acid and trace amounts of transition metals has pro-oxidant potential, extreme caution must be
Fig. 2. H2O2 concentrations in ascorbic acid solutions. Samples were taken from ascorbic acid solutions of 0, 1, 2, 5, 10, 25, 50 and 100 mM in KRB. Samples were analyzed with an isoluminol-amplified chemiluminescence assay in the presence and absence of catalase (30 U/ml) to quantify H2O2-dependent chemiluminescence. H2O2 (15 nM–1 mM) produces a linear, concentration-dependent increase in chemiluminescence (r2 = 0.99). Values represent the mean ± SEM, n ≥ 4. *P , 0.05, paired t-tests of H2O2 concentrations determined immediately in 1 mM versus 50 mM ascorbic acid solutions and also in solutions measured immediately compared to those measured at 16 min.
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taken when storing and analyzing samples for substances that are easily oxidized, such as catecholamines. This is especially critical because chelating agents are not routinely included in perfusion media used in microdialysis studies [5]. Moreover, use of disodium-EDTA in the perfusion media did not abolish H2O2 production in the microdialysates, because EDTA probably extracted iron from the brain and iron-EDTA can be a stronger pro-oxidant than uncomplexed iron [10]. In fact, dual microdialysis delivery systems are used to deliver iron-EDTA and H2O2 via different probes to create reactive oxygen species to simulate the role of these species in delayed brain damage [17]. Since the extracellular microenvironment contains substances that can either destroy or form H2O2, this must also be taken into consideration when using in vivo or ex vivo techniques which rely on the conversion of a substance to H2O2 for detection and quantitation [1]. In brain ECF, basal ascorbic acid concentrations are estimated to be 200–400 mM [8,18] and at these concentrations antioxidant properties of ascorbic acid should predominate. Moreover, with an injurious insult such as probe insertion, ascorbic acid extracellular levels increase [11,15] and thus should provide additional antioxidant protection in the ECF. This is seen by the reduction of H2O2 in samples collected immediately after probe insertion (Fig. 1). The reduction in H2O2 as the perfusate passes through the brain may be due in part to the antioxidant activities of ascorbic acid or the diffusion of H2O2 from the perfusate into the extracellular space where it is destroyed by peroxidases or catalases. If there is some micro-bleeding around the probe, the catalase in the red blood cells could contribute to the destruction of H2O2. At later times after probe insertion, the accumulation of oxidant producing antiinflammatory cells at the site of the probe/tissue interface can contribute to H2O2 formation [13]. However, since the H2O2 measured in the first sample after probe insertion immediately after collection was decreased from pre-insertion levels, and samples collected 2 h after probe insertion were about the same as pre-insertion levels, there is an inverse relationship with falling ascorbic acid levels in the microdialysates during this same time period. Therefore, it is likely that the high initial ascorbic acid levels in the ECF after probe insertion contribute substantially to the antioxidant effect that occurs as the perfusate passes through the brain. In the microdialysates, ascorbic acid levels are considerably lower than in ECF starting at about 15 mM in the first sample and decreasing to about 1–2 mM by 1 h post-insertion [15]. This is the concentration range that is optimal for ascorbic acid to contribute to H2O2 formation even when only trace amounts of metals are present as in KRB solutions (see Fig. 2). The fact that considerable H2O2 was formed in the first sample collected (see 20 min sample in Fig. 1) when analyzed 8 min post-collection where ascorbic acid concentrations were measured at about 15 mM and there was only a very small increase in H2O2 detected in ascorbic acid KRB solutions above 5 mM (see Fig. 2), sug-
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gests that release of metals from cells injured by insertion of the probe is responsible for the more robust net H2O2 production. Implantation of a microdialysis probe disrupts normal tissue structure, despite the normalization of many cellular and metabolic processes within 24 h of probe implantation [2]. We suggest that site-specific oxidation of macromolecules mediated by ascorbic acid/metal/dioxygen complexes in the cellular microenvironment with the closest proximity to the inserted probe may influence redox changes detected in microdialysates caused by subsequent experimental paradigms such as trauma, ischemia or seizures [9]. Microdialysis has been used to measure changes in extracellular H2O2 in ischemia/reperfusion studies [13,16]. Based on the results presented here, we suggest that the H2O2 detected in microdialysates reported in the above cited studies may have formed ex vivo. The baseline H2O2 levels reported (ECF ca. 25–50 mM [13]; dialysates ca. 1–2 mM [16]) are considerably higher than found in our study or reported to be the upper limits in tissue by Halliwell and Gutteridge [10]. However, what all of these studies do demonstrate is that chariges in H2O2 generating substances occur during brain injury and are revealed when the fluid is removed from the brain. These same substances may participate in site-specific actions in vivo [10] avoiding the norrnal antioxidant defenses and thus, contribute to delayed neuropathologies. This study demonstrates that H2O2 is both formed and destroyed as perfusion media is exchanged with brain ECF, collected and allowed to stand at room temperature. Ascorbic acid can contribute both to the formation and destruction of H2O2. Antioxidant properties should dominate at concentrations of ascorbic acid found in normal ECF and even more so when ascorbic acid ECF concentrations are increased after insults from trauma, ischemia or seizures [11,14,15]. The pro-oxidant actions of ascorbic acid are more important at the lower concentrations which exist in microdialysates, especially if metals are dislocated by the experimental procedure and extracted by the dialysis procedure. Importantly, this pro-oxidant environment can affect the stability of oxidizable substances in microdialysates or the conversion of analytes to H2O2 for detection and quantification. However, a more important and difficult question is the role of ascorbic acid and transition metals in the formation of H2O2 and other reactive oxygen species leading to cell injury associated with disease processes or acute insults such as trauma, ischemia or seizures [10]. Our in vitro studies [6] indicate that ascorbic acid/metal/oxygen complexes could oxidize critical targets in close proximity (i.e. lipids, proteins) and participate in site-specific oxidations, thereby avoiding the normal antioxidant defenses. The results from this study demonstrate that microdialysis can be used to detect rapid fluctuations in redox substances in the brain cell microenvironment after injury and that these redox changes have the potential for H2O2 production, especially if iron is released.
[1] Asai, S., Iribe, Y., Kohno, T. and Ishikawa, K., Real time monitoring of biphasic glutamate release using dialysis electrode in rat acute brain ischemia, NeuroReport, 7 (1996) 1092–1096. [2] Benveniste, H. and Hu¨ttemeier, P.C., Microdialysis-theory and application, Prog. Neurobiol., 35 (1990) 195–215. [3] Bruce, A.J. and Baudry, M., Oxygen free radicals in rat lirnbic structures after kainate-induced seizures, Free Radical Biol. Med., 18 (1995) 993–1002. [4] Buettner, G.H., In the absence of catalytic metals ascorbic acid does not autoxidize at pH 7: test for catalytic metals, J. Biochem. Biophys. Methods, 16 (1988) 27–40. [5] Chen, N.H.N., Lai, Y.J. and Pan, W.H.T., Effects of different perfusion medium on the extracellular basal concentration of dopamine in striatum and medial prefrontal cortex: a zero-net flux microdialysis study, Neurosci. Lett., 225 (1997) 197–200. [6] Emerson, M.R., Samson, F.E. and Pazdernik, T.L., Evidence for an ascorbic acid-copper-peroxide complex in lipid peroxidation, Soc. Neurosci. Abstr., 22 (1996) 562.3. [7] Frei, B., Yamamoto, Y., Niclas, D. and Ames, B.N., Evaluation of an isoluminol chemiluminescence assay for the detection of hydroperoxides in human blood plasma, Anal. Biochem., 175 (1988) 120–130. [8] Grunewald, R.A., Ascorbic acid in the brain, Brain Res. Rev., 18 (1993) 123–133. [9] Halliwell, B., Oxidants and the central nervous system: some fundamental questions. Is oxidant damage relevant to Parkinson’s disease, Alzheimer’s disease, traumatic injury or stroke?, Acta Neurol. Scand., 1265 (1989) 23–33. [10] Halliwell, B. and Gutteridge, J.M.C., Role of free radicals and catalytic metals in human disease: an overview, Methods Enzymol., 186 (1990) 1–85. [11] Hillered, L., Nilsson, P., Ungerstedt, U. and Ponten, U., Trauma-induced increase of extracellular ascorbic acid in rat cerebral cortex, Neurosci. Lett., 113 (1990) 328–332. [12] Hwang, H. and Dasgupta, P.K., Fluorometric flow injection determination of aqueous peroxides at nanomolar level using membrane reactors, Anal. Chem., 58 (1986) 1521–1524. [13] Hyslop, P.A., Zhang, Z., Pearson, D.V. and Phebus, L.A., Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with cytotoxic potential of H2O2 in vitro, Brain Res., 671 (1995) 181–186. [14] Landolt, H., Lutz, T.W., Langemann, H., Stauble, D., Mendelowitsch, A., Gratzl, O. and Honegger, C.G., Extracellular antioxidants and amino acids in the cortex of the rat: monitoring by microdialysis of early ischemic changes, J. Cerebral Blood Flow Metab., 12 (1992) 96–102. [15] Layton, M.E., Wagner, J.K., Samson, F.E. and Pazdernik, T.L., Redox changes in perfusates following intracerebral penetration of microdialysis probes, Neurochem. Res., 22 (1997) 735–741. [16] Lei, B., Adachi, N. and Arai, T., The effect of hypothermia on H2O2 production during ischemia and reperfusion: a microdialysis study in the gerbil hippocampus, Neurosci. Lett., 222 (1997) 91–94. [17] Liu, D., Yang, R., Yan, X. and McAdoo, D.J., Hydroxyl radicals generated in vivo kill neurons in the rat spinal cord: electrophysiological, histological and neurochemical results, J. Neurochem., 62 (1994) 37–44. [18] Milby, K., Oke, A. and Adams, R.N., Detailed mapping of ascorbic acid distribution in rat brain, Neurosci. Lett., 28 (1982) 15– 20. [19] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press Australia, North Ryde, NSW, 1986, Figure 25.
Cerebral penetration injury leads to H2O2 generation in microdialysis samples Matthew E. Layton a ,*, Thomas L. Pazdernik b , c, Fred E. Samson c a
Department of Psychiatry and Behavioral Sciences, University of Washington, P.O. Box 356560, Seattle, WA 98195, USA Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA c Smith Mental Retardation Research Center, University of Kansas Medical Center, Kansas City, KS, USA
b
Received 4 September 1997; accepted 6 September 1997
Abstract Delayed tissue damage is proposed to be caused by reactive oxygen species. We investigated the effects of microdialysis probe penetration into rat piriform cortex on hydrogen peroxide (H2O2) in brain extracellular fluid (ECF). H2O2 decreased immediately after probe insertion into the brain, but increased over 300% in samples within minutes after collection. We assessed H2O2 changes in vitro in microdialysis perfusion media containing various ascorbic acid concentrations and confirmed ascorbic acid is a source of H2O2. We conclude that decreased H2O2 concentrations in perfusion media as it passes through the brain reflect an extracellular antioxidant effect, whereas the increase in H2O2 with time after sample collection indicates that H2O2 generating substances are present in ECF. Thus, the potential for producing reactive oxygen species in brain ECF exists following penetration injury, especially if transition metals are released into the neuronal microenvironment. 1997 Elsevier Science Ireland Ltd.
Keywords: Antioxidant; Ascorbic acid; Brain injury; Chemiluminescence; Hydrogen peroxide; Microdialysis; Oxidants; Redox
Delayed brain damage from a variety of diseases or injuries to the central nervous system including trauma, ischemia/reperfusion and seizures is proposed to involve reactive oxygen species [3,4,9]. Hydrogen peroxide (H2O2), ascorbic acid and transition metals have pivotal actions in the formation of these reactive oxygen species [10]. Redox processes are important in brain function as well as in neuropathologies. Intracerebral microdialysis provides a method to detect, quantify and characterize changes in redox-active small molecules, such as ascorbic acid and H2O2, in brain extracellular fluid (ECF) in awake, freely-moving animals [2]. Two groups [13,16] have recently reported on H2O2 levels measured in microdialysates following ischemia/ reperfusion. In addition, we recently confirmed that ascorbic acid levels are high in ECF immediately following probe penetration into the brain [11,15] and demonstrated that
* Corresponding author. Tel.: +1 206 5433750; fax: +1 206 5439520; e-mail: [email protected]
ascorbic acid contributes to a non-linear production of oxidative chemiluminescence [15]. In the current study, we measured the H2O2 component of the oxidative chemiluminescence produced by microdialysates. The microdialysates were analyzed for H2O2 before and immediately after the probe was inserted into the piriform cortex over a period of 2 h. H2O2 was measured in microdialysates both immediately after collection and 8 min post-collection. Since ascorbic acid levels in microdialysates are about 15 mM immediately after probe insertion and decline to about 1 mM by 2 h post probe insertion [15], we also measured H2O2 in vitro in Kreb’s Ringer bicarbonate (KRB) solution containing varying concentrations of ascorbic acid immediately, 8 or 16 min after ascorbic acid was added to the KRB solution. H2O2 (30%), catalase from bovine liver (EC 1.11.1.6), microperoxidase disodium salt from equine heart cytochrome c (MP-11), 6-amino-2,3-dihydro-1,4-phthalazinedione (isoluminol), sodium borate (Na2BO4) and ascorbic acid were all obtained from Sigma Chemical (St. Louis,
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00765- 9
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M.E. Layton et al. / Neuroscience Letters 236 (1997) 63–66
MO, USA). Dow 50 cellulose microdialysis membranes from a kidney dialysis cartridge (molecular weight cut-off, 5000; o.d., 250 mm) were used in concentric tube microdialysis probes of fused silica hollow tubing (PolymicroTechnology; i.d., 75 mm; o.d., 145 mm) inside Teflon tubing with approximately 4 mm exposed dialysis membrane. All materials that have contact with the perfusate such as glassware, pipets and microdialysis probe construction materials were selected for their minimal production of autoluminescence or chemiluminescence. All glassware was acid washed and rinsed with triple distilled water. Adult male Wistar rats were obtained from Sasco (Omaha, NE, USA). Food and water were provided ad libitum, and a 12:12 h light-dark cycle was maintained. Experiments were conducted in accordance with the guidelines of the National Research Council DHEW publication #(NIH) 80-23 (1980). Animals were anesthetized with pentobarbital (80 mg/kg i.p. injection) prior to aseptic stereotaxic probe implantation. Stereotaxic measurements relative to bregma for the rat piriform cortex are −1.8 mm anteroposterior, −5.7 mm lateral and −10.0 mm vertical [19]. The dura mater was punctured with a 27-gauge needle and epoxy and dental acrylic secured the apparatus to the skull. The microdialysis probes were perfused with modified KRB (pH 7.4; NaCl 144.5 mM; KCl 3.0 mM; CaCl2 1.2 mM; KH2PO4 0.4 mM; MgSO4 1.2 mM; NaHCO3 2.5 mM) at a rate of 2 ml/ min using a Hamilton Microliter 1000 Series Gas tight syringe with a Teflon plunger and syringe attachment hub in a Harvard infusion pump. Microdialysis perfusion was initiated prior to surgery and continued throughout probe implantation; samples were collected at 20 min intervals. A chemiluminescence assay developed for analysis of reactive oxidative species in plasma [7] was adapted for microdialysis samples. This method employs amplified chemiluminescence produced by isoluminol (1 mM) in the presence of microperoxidase (0.01 mM) and sodium borate (100 mM) in 70% water and 30% methanol at pH 10. Chemiluminescence was quantified with an MGM Optocomp I luminometer (Hamden, CT, USA). H2O2 standards and microdialysis samples were placed in a test tube in the sample compartment of the luminometer. The chemiluminescence solution (300 ml) was automatically injected into the sample compartment from a reservoir. Chemiluminescence was quantified with an integrated kinetic photon counting protocol of 60 consecutive 4 s counting intervals for a total counting period of 4 min and expressed as relative light units (RLU). Samples were assayed in the presence and absence of catalase (final concentration 30 U/ml). Catalase activity was tested in the presence and absence of 25 mM ascorbic acid, the upper limit of ascorbic acid concentrations measured in brain microdialysis samples, as well as in the presence and absence of the isoluminol/microperoxidase chemiluminescence solution. A Clark electrode was used to monitor oxygen liberation from a solution containing ascorbic acid, catalase (30 U/ml) in KRB or in the chemiluminescence solution prior to and after addition of
H2O2 (final concentration 4 mM). Catalase activity was unaffected by these conditions [15]. The catalase-sensitive component of chemiluminescence was used as an indirect measure of H2O2-dependent chemiluminescence, which is linear from 15 nM to 1 mM (standard curve; r2 = 0.99). This chemiluminescence analytical procedure was also used to measure H2O2 in KRB samples with known ascorbic acid concentrations. Samples from 0, 1, 2, 5, 10, 25, 50 and 100 mM ascorbic acid solutions were mixed with an equal volume of KRB with or without catalase (final 30 U/ml) prior to chemiluminescence analysis. H2O2 concentrations in the KRB perfusion media at time of preparation were approximately 30 nM, comparable to previous reports of H2O2 concentrations in aqueous media [12]. Simply passing the KRB through the microdialysis system in vitro doubled the H2O2 concentrations in the perfusates to 63.6 ± 10 nM (mean ± SEM, n = 18). Thus, H2O2 is produced in the microdialysis apparatus itself. On the other hand, after probe insertion into the brain, H2O2 concentrations in the first microdialysis samples were significantly lower at 36.3 ± 2.7 nM (P , 0.05, n = 18). Indeed the H2O2 concentrations in samples collected during the first hour after probe insertion remained significantly lower than in perfusates prior to insertion. By 2 h after probe insertion, H2O2 in the perfusates returned to the pre-insertion levels (Fig. 1). The early decrease in H2O2 can be explained by the antioxidant activity of the brain extra-
Fig. 1. H2O2 concentrations in microdialysis samples before and after microdialysis probe insertion into rat brains. Samples were collected at 20 min intervals and analyzed with an isoluminol-amplified chemiluminescence assay in the presence and absence of catalase (30 U/ ml). The catalase-sensitive component of chemiluminescence represents the contribution of H2O2 to total chemiluminescence. H2O2 standards (15 nM–1 mM) produce linear, concentration-dependent increases in chemiluminescence (r2 = 0.99). Samples were reanalyzed 8 min after collection. Values represent the mean ± SEM, n = 18 initial samples and n ≥ 5 repeat sample analyses at each time point. *P , 0.05, t-test versus H2O2 concentrations in samples prior to probe insertion. **P , 0.05, t-test versus H2O2 concentrations in samples measured immediately after collection compared to those measured 8 min after collection.
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cellular microenvironment. However, when samples were reanalyzed for H2O2 only 8 min after collection, the H2O2 concentrations increased 3- to 4-fold to a range of 150–200 nM (n ≥ 5 repeat sample analyses at each time point; Fig. 1). This demonstrates that ECF has H2O2 generating substances (i.e. pro-oxidant substances) that are revealed when the perfusate leaves the antioxidant environment of the brain. To ascertain whether ascorbic acid contributes to H2O2 generation, H2O2 was measured in KRB solutions with various ascorbic acid concentrations (0, 1, 2, 5, 10, 25, 50 and 100 mM). Samples were analyzed for H2O2 concentrations immediately and at 8 and 16 min after sample preparation. The H2O2 concentrations measured immediately were significantly lower in 50 mM than in 1 mM ascorbic acid solutions (P , 0.05; n ≥ 4; Fig. 2), demonstrating the predominately antioxidant properties of ascorbic acid at the high concentrations [15]. H2O2 concentrations at 8 and 16 min increased somewhat over those measured immediately at all ascorbic acid concentrations; however, the increase in H2O2 was the greatest in those samples with the lowest ascorbic acid concentrations (1 and 2 mM; see Fig. 2). When all metals are removed from ascorbic acid solutions with Chelex [4], no H2O2 is formed, so traces of transition metals (i.e. copper or iron), normally occurring in buffers, can support H2O2 formation in oxygenated solutions containing low concentrations of ascorbic acid. Since microdialysates or any oxygenated biological solution with low ascorbic acid and trace amounts of transition metals has pro-oxidant potential, extreme caution must be
Fig. 2. H2O2 concentrations in ascorbic acid solutions. Samples were taken from ascorbic acid solutions of 0, 1, 2, 5, 10, 25, 50 and 100 mM in KRB. Samples were analyzed with an isoluminol-amplified chemiluminescence assay in the presence and absence of catalase (30 U/ml) to quantify H2O2-dependent chemiluminescence. H2O2 (15 nM–1 mM) produces a linear, concentration-dependent increase in chemiluminescence (r2 = 0.99). Values represent the mean ± SEM, n ≥ 4. *P , 0.05, paired t-tests of H2O2 concentrations determined immediately in 1 mM versus 50 mM ascorbic acid solutions and also in solutions measured immediately compared to those measured at 16 min.
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taken when storing and analyzing samples for substances that are easily oxidized, such as catecholamines. This is especially critical because chelating agents are not routinely included in perfusion media used in microdialysis studies [5]. Moreover, use of disodium-EDTA in the perfusion media did not abolish H2O2 production in the microdialysates, because EDTA probably extracted iron from the brain and iron-EDTA can be a stronger pro-oxidant than uncomplexed iron [10]. In fact, dual microdialysis delivery systems are used to deliver iron-EDTA and H2O2 via different probes to create reactive oxygen species to simulate the role of these species in delayed brain damage [17]. Since the extracellular microenvironment contains substances that can either destroy or form H2O2, this must also be taken into consideration when using in vivo or ex vivo techniques which rely on the conversion of a substance to H2O2 for detection and quantitation [1]. In brain ECF, basal ascorbic acid concentrations are estimated to be 200–400 mM [8,18] and at these concentrations antioxidant properties of ascorbic acid should predominate. Moreover, with an injurious insult such as probe insertion, ascorbic acid extracellular levels increase [11,15] and thus should provide additional antioxidant protection in the ECF. This is seen by the reduction of H2O2 in samples collected immediately after probe insertion (Fig. 1). The reduction in H2O2 as the perfusate passes through the brain may be due in part to the antioxidant activities of ascorbic acid or the diffusion of H2O2 from the perfusate into the extracellular space where it is destroyed by peroxidases or catalases. If there is some micro-bleeding around the probe, the catalase in the red blood cells could contribute to the destruction of H2O2. At later times after probe insertion, the accumulation of oxidant producing antiinflammatory cells at the site of the probe/tissue interface can contribute to H2O2 formation [13]. However, since the H2O2 measured in the first sample after probe insertion immediately after collection was decreased from pre-insertion levels, and samples collected 2 h after probe insertion were about the same as pre-insertion levels, there is an inverse relationship with falling ascorbic acid levels in the microdialysates during this same time period. Therefore, it is likely that the high initial ascorbic acid levels in the ECF after probe insertion contribute substantially to the antioxidant effect that occurs as the perfusate passes through the brain. In the microdialysates, ascorbic acid levels are considerably lower than in ECF starting at about 15 mM in the first sample and decreasing to about 1–2 mM by 1 h post-insertion [15]. This is the concentration range that is optimal for ascorbic acid to contribute to H2O2 formation even when only trace amounts of metals are present as in KRB solutions (see Fig. 2). The fact that considerable H2O2 was formed in the first sample collected (see 20 min sample in Fig. 1) when analyzed 8 min post-collection where ascorbic acid concentrations were measured at about 15 mM and there was only a very small increase in H2O2 detected in ascorbic acid KRB solutions above 5 mM (see Fig. 2), sug-
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gests that release of metals from cells injured by insertion of the probe is responsible for the more robust net H2O2 production. Implantation of a microdialysis probe disrupts normal tissue structure, despite the normalization of many cellular and metabolic processes within 24 h of probe implantation [2]. We suggest that site-specific oxidation of macromolecules mediated by ascorbic acid/metal/dioxygen complexes in the cellular microenvironment with the closest proximity to the inserted probe may influence redox changes detected in microdialysates caused by subsequent experimental paradigms such as trauma, ischemia or seizures [9]. Microdialysis has been used to measure changes in extracellular H2O2 in ischemia/reperfusion studies [13,16]. Based on the results presented here, we suggest that the H2O2 detected in microdialysates reported in the above cited studies may have formed ex vivo. The baseline H2O2 levels reported (ECF ca. 25–50 mM [13]; dialysates ca. 1–2 mM [16]) are considerably higher than found in our study or reported to be the upper limits in tissue by Halliwell and Gutteridge [10]. However, what all of these studies do demonstrate is that chariges in H2O2 generating substances occur during brain injury and are revealed when the fluid is removed from the brain. These same substances may participate in site-specific actions in vivo [10] avoiding the norrnal antioxidant defenses and thus, contribute to delayed neuropathologies. This study demonstrates that H2O2 is both formed and destroyed as perfusion media is exchanged with brain ECF, collected and allowed to stand at room temperature. Ascorbic acid can contribute both to the formation and destruction of H2O2. Antioxidant properties should dominate at concentrations of ascorbic acid found in normal ECF and even more so when ascorbic acid ECF concentrations are increased after insults from trauma, ischemia or seizures [11,14,15]. The pro-oxidant actions of ascorbic acid are more important at the lower concentrations which exist in microdialysates, especially if metals are dislocated by the experimental procedure and extracted by the dialysis procedure. Importantly, this pro-oxidant environment can affect the stability of oxidizable substances in microdialysates or the conversion of analytes to H2O2 for detection and quantification. However, a more important and difficult question is the role of ascorbic acid and transition metals in the formation of H2O2 and other reactive oxygen species leading to cell injury associated with disease processes or acute insults such as trauma, ischemia or seizures [10]. Our in vitro studies [6] indicate that ascorbic acid/metal/oxygen complexes could oxidize critical targets in close proximity (i.e. lipids, proteins) and participate in site-specific oxidations, thereby avoiding the normal antioxidant defenses. The results from this study demonstrate that microdialysis can be used to detect rapid fluctuations in redox substances in the brain cell microenvironment after injury and that these redox changes have the potential for H2O2 production, especially if iron is released.
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