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Departments of 1Physiology and 2Anesthesiology and 3Cognitive and Molecular Research Institute of Brain Disease, Kurume University School of Medicine, Kurume 830-0011 Japan
Submitted 3 June 2003; accepted in final form 7 August 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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5 min after the onset of the superfusion. Even when oxygen and glucose were reintroduced immediately after rapid depolarization, the membrane depolarized further (persistent depolarization) and reached 0 mV (irreversible depolarization) after 5 min from the reintroduction. The pretreatment of the slice preparation with a phospholipase A2 (PLA2) inhibitor, para-bromophenacyl bromide, or a cytochrome P-450 inhibitor, 17-octadecynoic acid, significantly restored the membrane to the preexposure potential level after the reintroduction of oxygen and glucose. The administration of 14,15-epoxyeicosatrienoic acid or 20-hydroxyeicosatetraenoic acid did not change the latency of the rapid depolarization and did not allow the membrane potential to recover after the ischemic exposure. In contrast, after pretreatment with cyclooxygenase or lipoxygenase inhibitors, such as indomethacin, resveratrol, Dup-697, nordihydroguaiaretic acid, and 3,4-dihydrophenyl ethanol, a minority of neurons tested showed postischemic recovery from the persistent depolarization. Improved recovery was also seen after treatment with the free radical scavengers, edaravone and 
-tocopherol. These results suggest that the activation of the arachidonic acid cascade via PLA2 and the free radicals produced by arachidonic acid metabolism contribute to the irreversible depolarization produced by in vitro ischemia. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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; Sapirstein and Bonventre 2000). Lysophospholipids and arachidonic acid are therefore mainly produced by the hydrolysis of the ester bond at the sn-2 position of glycerophospholipids with phospholipase A2 (PLA2). PLA2 isozymes are classified into four subtypes and type VI PLA2 isozymes are cytosolic. The type VI PLA2 isozymes are further classified into two groups, either Ca2+-dependent or -independent. The initiation of cerebral anoxia or ischemia immediately accumulates free fatty acids including arachidonic acid in the brain in a time-dependent manner (Bazan 1970; Siesjö and Katsura 1992). Arachidonic acid produced by the activation of PLA2 can be metabolized by lipoxygenase (LOX) isozymes, cyclooxygenase (COX) isozymes, and cytochrome P-450 isozymes to leukotrienes, prostaglandins, and other eicosanoids, respectively (Sapirstein and Bonventre 2000). The cytochrome P-450 isozymes are divided into two groups by their functions, 
-hydroxylases and epoxygenases (Roman 2002). In the rat brain, -hydroxylases produce mainly 20-hydroxyeicosatetraenoic acid (20-HETE), whereas epoxygenases produce mainly 14,15-epoxyeicosatrienoic acid (14,15-EET) (Alkayed et al. 1996; Amruthesh et al. 1993; Roman 2002). In vivo experiments show the accumulation of leukotrienes and prostanoids in the mammalian brain during and after complete cerebral ischemia (Moskowitz et al. 1984; Stevens and Yaksh 1988). In humans, both the leukotriene C4-and prostaglandin-E2-like activity increase in acute cerebral ischemia patients (Aktan et al. 1991). The enzymes that metabolize arachidonic acid also produce oxygen radicals (superoxyl and hydroxyl radicals) (Paller and Jacob 1994; Siesjo and Katsura 1992). Oxygen radicals produce wide-ranging cellular effects, such as lipid peroxidation, inactivation of enzyme, damage to cytoskeleton, and DNA damage (Kontos 2001). It is therefore possible that activation of arachidonic acid metabolism induce severe neuronal damage or death during ischemia and after reperfusion.
Recently, several lines of evidence support the proposal that the activation of PLA2 and the subsequent arachidonic acid metabolites are closely related to the neuronal cell death produced by brain ischemia. Administration of an inhibitor of COX isozymes before exposure to ischemia reduces the area of infarction produced by either occlusion of common carotid arteries or a middle cerebral artery in gerbils or mice (Sasaki et al. 1988). The administration of an inhibitor for LOX isozymes also attenuates the neuronal death produced by occlusion of carotid arteries in gerbils (Rao et al. 1999). In rat hippocampal slice cultures, LOX inhibitors also attenuate the injury caused by oxygen and glucose deprivation (Arai et al. 2001b). Moreover, PLA2 or COX-2 knockout mice show a reduction in the infarcted area produced by occlusion of the middle cerebral artery in comparison with wild-type mice (Bonventre et al. 1997; Iadecola et al. 2001).
In CA1 pyramidal neurons of the rat hippocampal slices, superfusion with oxygen- and glucose-deprived medium (in vitro ischemia) produce a rapid depolarization 5 min after the onset of the ischemic exposure (Tanaka et al. 1997). When oxygen and glucose are reintroduced immediately after rapid depolarization, the membrane depolarizes further (persistent depolarization) and reaches 0 mV (irreversible depolarization) after 5 min from the onset of the reintroduction: as a result, the neurons show no functional recovery. The persistent depolarization is a Ca2+-dependent process that is mediated by the activation of ionotropic glutamate (Glu) receptors and the Ca2+-induced Ca2+ release from intracellular Ca2+ store sites (Yamamoto et al. 1997). Moreover, blebs appear on the cell body of the CA1 pyramidal neuron 1 min after starting the reintroduction of oxygen and glucose, and the cell body becomes swollen 3 min after (Tanaka et al. 1999). It is therefore possible that during the persistent depolarization, the Ca2+-dependent PLA2 may be activated and thereby induce the neuronal damage. Nevertheless, the critical metabolic pathway of arachidonic acid cascade for generating the irreversible depolarization is still unclear.
In the present study, we examined whether or not PLA2 and the subsequent arachidonic acid metabolites contribute to the generation of the irreversible depolarization induced by in vitro ischemia. Inhibitors for PLA2 have been administered to the rat hippocampal slices, and the potential changes in CA1 neurons during and after ischemic exposure have been compared between the slices with no administration (control) and the slices administered these drugs. In addition, to clarify the metabolic pathway of arachidonic acid cascade, which leads to the irreversible change, we have examined effects of an inhibitor for LOX, COX, or cytochrome P-450 isozymes on the potential changes after in vitro ischemia.
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METHODS |
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Intracellular recordings from CA1 pyramidal cells were made with glass micropipettes filled with K acetate (2 M). The electrode resistance was 50-90 M
. In conventional intracellular recordings, the apparent input resistance in CA1 neurons was monitored by passing small hyperpolarizing pulses (0.2-0.4 nA, 200 ms) through the recording electrode every 3 s.
Slices were made "ischemic" by superfusion with medium equilibrated with 95% N2-5% CO2 and deprived of glucose, which was replaced with NaCl isoosmotically (ischemia-simulating medium). When switching the superfusing media, there was a delay of 15-20 s before the new medium reached the chamber due to the volume of the connecting tubing. As a result, the chamber was filled with the test solution 30 s after switching the solution. We used one slice for one experiment because the responses to in vitro ischemia could not be reproduced after the first ischemic exposure.
The drugs used were (from Sigma) para-bromophenacyl bromide (para-BPB), aristolochic acid, 17-octadecynoic acid (17-ODA), -tocopherol, allopurinol; (from Funakoshi) nordihydroguaiaretic acid (NDGA), 3,4-dihydroxyphenyl ethanol, indomethacin, resveratrol, Dup-697, 20-hydroxyeicosatetraenoic acid (20-HETE), 14,15-epoxyeicosatrienoic acid (14,15-EET); and edaravone (gift from Mitsubishi Welpharma). All drugs were dissolved in the perfusate and applied by bath application. The slices were pretreated with media containing test compounds for 20 min before ischemic exposure.
The latency of the rapid depolarization was measured from the onset of superfusion to onset of the rapid depolarization estimated by extrapolating the slope of the rapid depolarization to the slope of the slow depolarization (Tanaka et al. 1997). Recovery after the reintroduction of oxygen and glucose is defined as follows: no recovery, 30-60 min after reintroduction the membrane potential lay between 0 and -19 mV; complete recovery, the membrane potential was more negative than -60 mV; partial recovery, membrane potential repolarized to a value between -20 and -59 mV (Yamamoto et al. 1997).
All quantitative results were expressed as the means ± SD. The number of slices examined is given in parentheses. A one-way ANOVA with the Scheffé post hoc test was used to compare the data. Statistical significance was determined at the level of P < 0.05 unless otherwise indicated.
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RESULTS |
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Hippocampal CA1 pyramidal neurons with stable membrane potentials more negative than -60 mV were used for the following studies. The resting membrane potential and the apparent input resistance in CA1 neurons were -70 ± 5 mV (n = 297) and 38 ± 10 M (n = 297), respectively. To elucidate the effects of PLA2 on the potential change after in vitro ischemia, broad-spectrum inhibitors of PLA2, para-BPB, and aristolochic acid were administered to the slice preparations. The administration of aristolochic acid at concentrations >100 µM induced a hyperpolarization of a few millivolts, whereas para-BPB (3-100 µM) or aristolochic acid (50-100 µM) did not induce any potential change before ischemic exposure. Figure 1A shows the typical potential changes in CA1 neurons during and after in vitro ischemia in the presence and absence (control) of para-BPB (10 µM) or aristolochic acid (100 µM). In the control condition, in vitro ischemia produced a sequence of potential changes consisting of an initial hyperpolarization, a slow depolarization, and a rapid depolarization (Fig. 1A top). When oxygen and glucose were reintroduced immediately after generating the rapid depolarization, the membrane depolarized further (persistent depolarization) and reached 0 mV. The membrane never showed a restoration to the potential level before exposure to in vitro ischemia (irreversible depolarization). The latency and the maximal slope of the rapid depolarization were 5.2 ± 1.0 min and 9.6 ± 3.7 mV/s (n = 96), respectively. The pretreatment of the slices with para-BPB (100 µM) or aristolochic acid (100 µM) did not significantly change the latency and the maximal slope of rapid depolarization. After the reintroduction of oxygen and glucose, however, both para-BPB and aristolochic acid restored the membrane potential either partially or completely to the preexposure level in a concentration-dependent manner (Fig. 1, A and B, and Table 1). The membrane 30 min after the reintroduction significantly repolarized in the presence of para-BPB (10-100 µM) or aristolochic acid (100 µM) in comparison with the absence of the drugs (Fig. 1C and Table 1). It is therefore possible that the arachidonic acid produced by PLA2 activation thus contributed to the generation of the irreversible depolarization (cf. Fig. 6).
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Effects of inhibitors for LOX, COX, and cytochrome P-450 on the irreversible depolarization produced by in vitro ischemia
We next examined effects of enzymes for arachidonic acid metabolism on the irreversible depolarization produced by in vitro ischemia. Solutions containing the cytochrome P-450 inhibitor, 17-ODA, at concentrations >10 µM produced a fine precipitate; we therefore administered 17-ODA at concentrations 
10 µM. The administration of 17-ODA (1-10 µM) did not change the membrane potential. Figure 2A shows the typical potential changes in CA1 neurons during and after in vitro ischemia in the absence and presence of 17-ODA (10 µM). The pretreatment of the slices with 17-ODA did not significantly change the latency and the maximal slope of rapid depolarization. After the reintroduction, 17-ODA restored the membrane potential either partially (8 of 16 neurons) or completely (2 neurons) to the preexposure level in a concentration-dependent manner (Fig. 2B). The membrane significantly repolarized toward the preexposure level in the presence of 17-ODA (10 µM) 30 min after reintroduction in comparison to the control condition (Fig. 2C, Table 1). On the other hand, LOX or COX inhibitors showed a tendency to restore the membrane potential toward the preexposure level. As shown in Fig. 3A, three of nine neurons showed either a partial or complete recovery in the presence of a broad LOX inhibitor, NDGA (100 µM). Four of nine neurons showed a partial or complete recovery in the presence of a 5- and 12-LOX inhibitor, 3,4-dihydroxyphenyl ethanol (20 µM). Three of 10 neurons showed either a partial or complete recovery in the presence of COX2 inhibitor, Dup-697 (5 µM). Two of 11 neurons showed a partial recovery in the presence of nonselective COX inhibitor, indomethacin (100 µM). One of eight neurons showed a partial recovery in the presence of COX1 inhibitor, resveratrol (100 µM). Two of eight neurons showed a partial recovery in the presence of a xanthine oxidase inhibitor, allopurinol (300 µM). Nevertheless, the membrane potential 30 min after the reintroduction of oxygen and glucose did not show a significant restoration to the preexposure level in the presence of NDGA (100 µM), 3,4-dihydroxyphenyl ethanol (20 µM), Dup-697 (5 µM), indomethacin (100 µM), resveratrol (100 µM), or allopurinol (300 µM; Fig. 3B, Table 1). The pretreatment of the slices with LOX, COX, or xanthine oxidase inhibitors did not significantly change the latency and the maximal slope of rapid depolarization. In addition, the administration of LOX, COX, or xanthine oxidase inhibitors did not induce any voltage change in the presence of oxygen and glucose. It is therefore possible that cytochrome P-450 isozymes contribute to the irreversible depolarization produced by in vitro ischemia (cf. Fig. 6).
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Effects of eicosanoids on the irreversible depolarization produced by in vitro ischemia
We therefore examined the effects of 14,15-EET and 20-HETE, which are the main products of the activation of cytochrome P-450 isozymes in the rat brain. 14,15-EET or 20-HETE (1 µM) did not produce any potential change. In addition, the pretreatment of the slices with 14,15-EET (1 µM), or 20-HETE (1 µM) did not significantly change the latency of the rapid depolarization: the latency was 5.3 ± 0.2 min (n = 6) and 5.0 ± 0.3 min (n = 6), respectively, in the presence of 14,15-EET and 20-HETE. After the reintroduction of oxygen and glucose, 14,15-EET restored the membrane potential partially toward the preexposure level in only one neuron (Fig. 4A). The membrane did not repolarize at all in the presence of 14,15-EET or 20-HETE 30 min after the reintroduction (Fig. 4B). The membrane potential 30 min after the reintroduction was -4.6 ± 7.4 mV (n = 6) and -5.4 ± 4.8 mV (n = 6), respectively, in the presence of 14,15-EET (1 µM) and 20-HETE (1 µM; Table 1). These results suggest that the arachidonic acid metabolites produced by cytochrome P-450 isozymes do not contribute to the generation of the irreversible depolarization induced by in vitro ischemia.
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Effects of free radical scavengers on the irreversible depolarization produced by in vitro ischemia
Cytochrome P-450, LOX, and COX produce oxygen radicals during the arachidonic acid metabolism. To elucidate the effects of oxygen radicals on the irreversible depolarization, free radical scavengers, edaravone and -tocopherol, were administered. Figure 5A shows the typical potential changes in CA1 neurons during and after in vitro ischemia in the presence and absence of edaravone or -tocopherol. The pretreatment of the slices with either edaravone (300 µM) or -tocopherol (50 µM) did not significantly change the latency and the maximal slope of rapid depolarization. After the reintroduction, edaravone and -tocopherol restored the membrane potential either partially or completely to the preexposure level in a concentration-dependent manner (Fig. 5, A and B). The membrane 30 min after the reintroduction was significantly repolarized in the presence of edaravone or -tocopherol in comparison with the control condition (Fig. 5C, Table 1). From these results, it is concluded that the oxygen radicals generated from arachidonic acid metabolism via cytochrome P-450 isozymes produce the irreversible depolarization following in vitro ischemia (cf. Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-tocopherol and edaravone, significantly restored the membrane to the preexposure level. This result indicates that the oxygen radicals produced by metabolism of arachidonic acid via cytochrome P-450 isozymes are responsible for the generation of the irreversible depolarization induced by in vitro ischemia. Effects of PLA2 inhibitors on the irreversible depolarization after the reintroduction
The present results show that PLA2 inhibitors, such as para-BPB and aristolochic acid, provide protection against the generation of the irreversible depolarization, and their action is comparable to previous reports that reported that in a hippocampal slice culture, para-BPB reduces the propidium iodide uptake after oxygen and glucose deprivation (Arai et al. 2001a). The aristolochic acid partially restored the membrane toward the preexposure level after the reintroduction of oxygen and glucose. Because IC50 value of the aristolochic acid for PLA2 activity in human neutrophil is 40 µM (Rosenthal et al. 1989), the concentration (100 µM) used for the hippocampal slices in the present study seems to be reasonable. A previous report showed the intracellular Ca2+ concentration to be markedly elevated at the generation of the rapid depolarization (Tanaka et al. 1997). Both the ionotropic-Glu-receptor-mediated Ca2+ influx and the Ca2+-induced Ca2+ release result in the generation of the persistent depolarization (Yamamoto et al. 1997). Moreover, the cell membrane of the CA1 neuron may be irreversibly damaged during the persistent depolarization because blebs appear on the cell body of the CA1 pyramidal neuron 1 min after starting the reintroduction of oxygen and glucose, and the cell body becomes swollen 3 min after that (Tanaka et al. 1999). Arai et al. (2001a) has also shown that arachidonyl trifluoromethyl keton, a Ca2+-dependent PLA2 inhibitor, reduces neuronal cell death in a hippocampal slice culture after oxygen and glucose deprivation. It is therefore possible that the Ca2+-dependent PLA2 is involved in the generation of the irreversible depolarization after the reintroduction. The result that the broad-spectrum inhibitors of PLA2, para-BPB and aristolochic acid, protected against the irreversible depolarization produced by in vitro ischemia in a concentration-dependent manner suggests that the activation of PLA2 is thus involved to some extent in the induction of the irreversible depolarization (Fig. 6).
Arachidonic acid produced by the activation of PLA2 via an increase in [Ca2+]i may be redistributed to the interstitial space because in rat-cultured cerebellar neurons, the intraand extracellular content of arachidonic acid have been reported to increase after the administration of Glu (Aronica et al. 1990; Lazarewicz et al. 1988). Moreover, the administration of arachidonic acid induces a concentration-dependent release of Glu in the rat hippocampal mossy fiber synaptosomes (Dorman et al. 1992; Freeman et al. 1990). In both synaptosomes and cultured astrocytes from rat cerebral cortex, the administration of arachidonic acid also produces reversible reduction in the uptake of Glu (Volterra et al. 1992a,b). It is therefore possible that the redistributed arachidonic acid induces the accumulation of Glu in the interstitial space, which may produce a further increase in [Ca2+]i and augment the neuronal damage. In fact, we previously reported that in rat hippocampal CA1 neurons, the ionotropic Glu receptor antagonists, 2-amino-5-phosphonopentanoic acid (AP5) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), restore the membrane to the preexposure level after ischemic exposure (Yamamoto et al. 1997).
Effects of cytochrome P-450 isozymes on the irreversible depolarization after the reintroduction of oxygen and glucose
The administration of 14,15-EET or 20-HETE (1 µM) did not alter the membrane potential before the ischemic exposure and the latency of the rapid depolarization during the ischemic exposure, thus suggesting that these products catalyzed by cytochrome P-450 isozymes are not cytotoxic and did not accelerate the generation of the rapid depolarization. On the other hand, a cytochrome P-450 isozyme inhibitor, 17-ODA (10 µM), significantly restored the membrane potential toward the preexposure level. In the brain or liver, the metabolism of various substrates via cytochrome P-450 isozymes leads to the formation of oxygen radicals during the production of metabolites, such as 14,15-EET or 20-HETE (Barth et al. 1998; Mattia et al. 1993; Montoliu et al. 1994; Thompson et al. 2000). These results indicate that the oxygen radicals produced by arachidonic acid metabolism via cytochrome P-450 isozymes contribute to the generation of the irreversible depolarization after in vitro ischemia. Cytochrome P-450 isozymes are found in astrocytes and neurons, including hippocampal pyramidal neurons, in the rat, mouse, and human brains (Bylund et al. 2002; Roman 2002). It is therefore possible that the oxygen radicals produced in both astrocytes and CA1 neurons after in vitro ischemia result in the generation of the irreversible depolarization of the CA1 neurons. The 17-ODA concentration (10 µM) used in this study is comparable with the IC50 for -hydroxylation (0.1-7 µM) and epoxidation (0.1-5 µM) via cytochrome P-450 isozymes on the rat renal function (Wang et al. 1998; Zou et al. 1994).
Effects of free radical scavengers on the irreversible depolarization after the reintroduction
There is ample evidence that the arachidonic acid product catalyzed by cytochrome P-450 isozymes leads to the formation of superoxyl radical in the liver and coronal artery (Bondy and Naderi 1994; Fleming et al. 2001; Puntarulo and Cederbaum 1998). Arachidonic acid is also metabolized by COX (and LOX) and thus yields eicosanoids, and a superoxyl radical is thus formed during this process (Kukreja et al. 1986). -Tocopherol has been shown to be a scavenger of lipoperoxyl radical, superoxide, and hydroxyl radical in many mammalian organs (Burton and Ingold 1986; Fukazawa and Gebicki 1983; Nishikimi et al. 1980; Zalkin and Tappel 1960). The present study showed that the free radical scavengers, -tocopherol (50 µM) and edaravone (300 µM), restored the membrane after the reintroduction toward the preexposure level in a concentration-dependent manner. In the rat brain homogenate, oxygen radicals produce lipid peroxidation 30 min after incubation at 37°C; the peroxidation is blocked by edaravone with IC50 of 15 µM (Tanaka 2002). It is therefore possible that oxygen radicals, such as superoxyl and hydroxyl radical, mediate the irreversible depolarization after the reintroduction. Our previous report showed that in rat hippocampal CA1 neurons, the inhibitor for nitric oxide (NO) synthase and the NO scavenger restore the membrane toward the preexposure potential level after in vitro ischemia (Onitsuka et al. 1998). Because NO can react with superoxyl radical to yield peroxynitrate, which decomposes and produces a highly toxic free radical, hydroxyl radical, it is possible that NO and oxygen radicals are responsible for the generation of the irreversible depolarization after the reintroduction. It is also possible that the free radicals, including peroxynitrate and hydroxyl radical, may damage the cytoskeleton and/or induce peroxidation of the membrane lipid because large blebs and cell swelling are observed in rat CA1 pyramidal neurons during the persistent depolarization (Tanaka et al. 1999).
Effects of LOX and COX inhibitors on the irreversible depolarization after the reintroduction
In vivo study shows that in all forebrain regions of the gerbil, neurons exhibit dense 5-LOX immunoreactivity and that the transient occlusion of common carotid arteries induces an increase in production of leukotriene C4, the metabolite from arachidonic acid by 5-LOX, after the reperfusion (Ohtsuki et al. 1995). A 5-LOX inhibitor reduces the neuronal death of gerbil hippocampal CA1 region after 6 days from the transient carotid arteries occlusion (Rao et al. 1999). The present results demonstrated that potent LOX isozyme inhibitors, NDGA (100 µM) and 3,4-dihydroxyphenyl ethanol (20 µM), showed a complete recovery of the membrane potential after the reintroduction in the minority of neurons tested. In human leukocytes and platelets, the IC50 value of NDGA for 5-LOX is 0.2 µM and that of 3,4-dihydroxyphenyl ethanol for 12-LOX is 4.2 µM (Kohyama et al. 1997; Salari et al. 1984). NDGA inhibits 12- and 15-LOXs at the high concentration; IC50 values for 12- and 15-LOXs are 30 µM. NDGA also inhibits the COX activity and the IC50 is 100 µM (Salari et al. 1984). The neuroprotective action of NDGA against the ischemic insult in this study may thus result from the inhibition of LOX and COX isozymes. On the other hand, 3,4-dihydroxyphenyl ethanol also inhibits 5-LOX at a high concentration (IC50 = 13 µM) (Kohyama et al. 1997). The neuroprotective effect of 3,4-dihydroxyphenyl ethanol may therefore mainly result from the inhibition of 12-LOX. The present finding seems to be comparable with the previous result in the hippocampal slice culture; the blockade of 12-LOX by baicalein or cinnamyl-3,4-dihydroxy--cyanocinnamate inhibits the neuronal death induced by oxygen and glucose deprivation for 35 min and a 5-LOX inhibitor, AA861, and thus reduces the neuronal death induced by the 40-min deprivation (Arai et al. 2001b). It is therefore possible that 5- and 12-LOX isozymes contribute to the neuronal death in CA1 region induced by in vitro and in vivo ischemia.
Rat cortical and hippocampal neurons are relatively rich in the COX-2 mRNA and immunoreactivity (Yamagata et al. 1993). The transient occlusion of both common carotid arteries in gerbil and a middle cerebral artery in rat increases in the COX-2 mRNA and immunoreactivity (Miettinen et al. 1997; Ohtsuki et al. 1996). In the present study, Dup-697 (5 µM), a relatively selective COX-2 inhibitor, restored completely the membrane to preexposure level after the reintroduction in the minority of neurons tested. DuP-697 is 
50-fold more selective for COX-2 than for COX-1: the IC50 value for COX-2 is 7 nM and that for COX-1 is 260-500 nM in inflamed kidney (Kargman et al. 1996; Seibert et al. 1996). It is therefore possible that the activation of COX-2 may contribute in some way to the irreversible depolarization induced by in vitro ischemia. On the other hand, potent COX-1 inhibitors, resveratrol (100 µM) and indomethacin (100 µM), only partially restored the membrane toward the preexposure level. The ED50 value of resveratrol for COX-1 (ability to inhibit the COX-1 activity) is 15 µM on cancer chemopreventive activity (Jang et al. 1997). The IC50 value of indomethacin for COX-1 is 13 µM, whereas that for COX-2 is >1 mM on human COXs expressed in COS-1 cells (Laneuville et al. 1994). These previous findings support the idea that the activation of COX-2 rather than COX-1 may be involved in the generation of the irreversible depolarization induced by in vitro ischemia. The present results are comparable to those described in previous reports in which several COX-2 inhibitors where shown to protect the neurons against neuronal death after the transient in vivo ischemia in gerbil or rat (Candelario-Jalil et al. 2002; Govoni et al. 2001; Hara et al. 1998; Nogawa et al. 1997).
In the present study, a xanthine oxidase inhibitor, allopurinol (300 µM), only partially restored the membrane toward the preexposure level in a few neurons. The Ki value of allopurinol for xanthine oxidase is 0.2-0.7 µM on the mouse liver (Elion 1966). Therefore the allopurinol concentration used in this study is high enough to inhibit xanthine oxidase. The contribution of xanthine oxidase to the generation of the irreversible depolarization induced by in vitro ischemia may, if present, be minimal. Based on the findings of all the above-mentioned results, it is likely that the LOX isozymes and the COX-2 may contribute in some extent to the generation of the irreversible depolarization induced by in vitro ischemia (Fig. 6).
Pathophysiological relevance
In our previous reports, changes in the membrane potential produced in CA1 neurons by in vitro ischemia are a mirror image of the DC potential changes produced by in situ ischemia (e.g., cardiac arrest) or asphyxia in the rat brain cortex (Hansen 1978, 1985; Tanaka et al. 1997, 1999; Yamamoto et al. 1997). The rapid depolarization induced by in vitro ischemia corresponds to the terminal depolarization (or phase II depolarization) produced by in situ ischemia or asphyxia. Simultaneous recordings of both intra- and extracellular recordings from rat hippocampal CA1 region have revealed that the rapid depolarization simultaneously occurs with the negative-going DC potential, which resembles the spreading depression in the cerebral cortex observed during in vivo ischemia (Hansen 1985; Leão 1947; Rader and Lanthorn 1989; Uchikado et al. 2000). Moreover, the cell swelling and no functional recovery of the membrane in rat hippocampal CA1 neurons 3 min after the reintroduction of oxygen and glucose indicate that the process leading to the irreversible depolarization after ischemic exposure is a necrotic event (Tanaka et al. 1999). These morphological changes are similar to so-called severe cell change, which has long been recognized as the response of the neuron to hypoxia (Brierley and Graham 1984; Brown and Brierley 1968). However, 10-30 min ischemia of forebrain in vivo produced by four-vessel occlusion (permanent occlusion of vertebral arteries and transient occlusion of common carotid arteries) induces ischemic neuronal damage in only a small number of hippocampal neurons 3 h after reperfusion and in most of the neurons 72 h after (delayed neuronal damage) (Pulsinelli and Brierley 1979; Pulsinelli et al. 1982). Therefore in vitro ischemia in the present study would be a relatively strong ischemic condition in comparison to the four-vessel occlusion model in vivo.
After this strong ischemic condition, the activation of PLA2 contributes to the generation of the irreversible depolarization. The activation of PLA2 may produce arachidonic acid, which would be mainly metabolized by cytochrome P-450 isozymes. The production of oxygen radicals from arachidonic acid via cytochrome P-450 isozymes may thus contribute to the generation of the irreversible depolarization after the reintroduction of oxygen and glucose. In addition, LOX isozymes and the COX-2 may also play some role in the onset of the irreversible depolarization. Because the expression of COX-2 is induced in the rat cortex and hippocampus after spreading depression and transient focal ischemia in vivo (Miettinen et al. 1997), the COX-2 may play an important role in the delayed neuronal damage observed after in vivo ischemia.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Tanaka, Dept. of Physiology, Kurume Univ. School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan (E-mail: eacht{at}med.kurume-u.ac.jp).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Alkayed NJ, Narayanan J, Gebremedhim D, Medhora M, Roman RJ, and Harder DR. Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke 27: 971-979, 1996.
Amruthesh SC, Boershel MF, McKinney JS, Willoughby KA, and Ellis EF. Metabolism of arachidonic acid to epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and prostaglandins in cultured rat hippocampal astrocytes. J Neurochem 61: 150-159, 1993.[Web of Science][Medline]
Arai K, Ikegaya Y, Nakatani Y, Kudo I, Nishiyama N, and Matsuki N. Phospholipase A2 mediates ischemic injury in the hippocampus: a regional difference of neuronal vulnerability. Eur J Neurosci 13: 2319-2323, 2001a.[Web of Science][Medline]
Arai K, Nishiyama N, Matsuki N, and Ikegaya Y. Neuroprotective effects of lipoxygenase inhibitors against ischemic injury in rat hippocampal slice cultures. Brain Res 904: 167-172, 2001b.[Web of Science][Medline]
Aronica E, Nicoletti F, and Canonico PL. Activation of excitatory amino acid receptors increases intracellular free [3H]arachidonate levels in cultured cerebellar neurons. Funct Neurol 5: 15-20, 1990.[Medline]
Barth A, Bauer R, Gedrange T, Walter B, Linss C, and Klinger W. Influence of hypoxia and hyperthermia upon peroxidative and glutathione status in growth-restricted newborn piglets. Exp Toxicol Pathol 50: 31-33, 1998.[Web of Science][Medline]
Bazan NGJ. Effects of ischemia and electroconvulsive shock on free fatty acid pool in the brain. Biochem Biophys Acta 218: 1-10, 1970.[Medline]
Bondy SC and Naderi S. Contribution of hepatic cytochrome P-450 systems to the generation of reactive oxygen species. Biochem Pharmacol 48: 155-159, 1994.[Web of Science][Medline]
Bonventre JV, Huang Z, Taheri MR, O'Leary E, Li E, Moskowitz MA, and Sapirstein A. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390: 622-625, 1997.[Medline]
Brierley JB and Graham DI. Hypoxia and vascular disorders of the central nervous system. In: Greenfield's Neuropathology (4th ed.), edited by Adams JH, Corsellis JAN, and Duchen LW. London, UK: Edward Arnold, 1984, p. 125-207.
Brown AW and Brierley JB. The nature, distribution and earliest stages of anoxic-ischaemic nerve cell damage in the rat brain as defined by the optical microscope. Br J Exp Pathol 49: 87-106, 1968.[Web of Science][Medline]
Burton GW and Ingold KU. Vitamin E: application of the principles of physical organic chemistry to the exploration of its structure and function. Acc Chem Res 19: 194-201, 1986.[Web of Science]
Bylund J, Zhang C, and Harder DR. Identification of a novel cytochrome P450, CYP4X1, with unique localization specific to the brain. Biochem Biophys Res Commun 296: 677-684, 2002.[Web of Science][Medline]
Candelario-Jalil E, Alvarez D, Castaneda JM, Al-Dalain SM, Martinez-Sanchez G, Merino N, and Leon OS. The highly selective cyclooxygenase-2 inhibitor DFU is neuroprotective when given several hours after transient cerebral ischemia in gerbils. Brain Res 927: 212-215, 2002.[Web of Science][Medline]
Dorman RV, Hamm TF, Damron DS, and Freeman EJ. Modulation of glutamate release from hippocamapl mossy fiber nerve endings by arachidonic acid and eicosanoids. Adv Exp Med Biol 318: 121-136, 1992.[Medline]
Elion GB. Enzymatic and metabolic studies with allopurinol. Ann Rheum Dis 25: 608-614, 1966.[Web of Science][Medline]
Farooqui AA, Yang H-C, Rosenberger TA, and Horrocks LA. Phospholipase A2 and its role in brain tissue. J Neurochem 69: 889-901, 1997.[Web of Science][Medline]
Fleming I, Michaelis UR, Bredenkotter D, Fisslthaler B, Dehghani F, Brandes RP, and Busse R. Endothelium-derived hyperpolarizing factor synthase (cytochrome P-450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries. Circ Res 88: 44-51, 2001.
Freeman EJ, Terrian DM, and Dorman RV. Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid. Neurochem Res 15: 743-750, 1990.[Web of Science][Medline]
Fukazawa K and Gebicki JM. Oxidation of -tocopherol in micelles and liposomes by the hydroxyl, perhydroxyl, and superoxide free radicals. Arch Biochem Biophys 226: 242-251, 1983.[Web of Science][Medline]
Govoni S, Masoero E, Favalli L, Rozza A, Scelsi R, Viappiani S, Buccellati C, Sala A, and Folco G. The cycloxygenase-2 inhibitor SC58236 is neuroprotective in an in vivo model of focal ischemia in the rat. Neurosci Lett 303: 91-94, 2001.[Web of Science][Medline]
Hansen AJ. The extracellular potassium concentration in brain cortex following ischemia in hypo- and hyperglycemic rats. Acta Physiol Scand 102: 324-329, 1978.[Web of Science][Medline]
Hansen AJ. Effect of anoxia on ion distribution in the brain. Physiol Rev 65: 101-148, 1985.
Hara K, Kong DL, Sharp FR, and Weinstein PR. Effect of selective inhibition of cyclooxygenase 2 on temporary focal cerebral ischemia in rats. Neurosci Lett 256: 53-56, 1998.[Web of Science][Medline]
Iadecola C, Niwa K, Nogawa S, Zhao X, Nagayama M, Araki E, Morham S, and Ross ME. Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc Natl Acad Sci USA 98: 1294-1299, 2001.
Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CWW, Fong HHS, Farnsworth NR, Kinghorn AD, Mehta RG, Moon RC, and Pezzuto JM. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275: 218-220, 1997.
Kargman S, Wong E, Greig GM, Falgueyret J-P, Cromlish W, Ethier D, Yergey JA, Riendeau D, Evans JF, Kennedy B, Tagari P, Francis DA, and O'Neill GP. Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells. Biochem Pharmacol 52: 1113-1125, 1996.[Web of Science][Medline]
Kohyama N, Nagata T, Fujimoto S, and Sekiya K. Inhibition of arachidonate lipoxygenase activities by 2-(3,4-dihydroxyphenyl)ethanol, a phenolic compound from olives. Biosci Biotech Biochem 61: 347-350, 1997.[Medline]
Kontos HA. Oxygen radicals in cerebral ischemia: the 2001 Willis lecture. Stroke 32: 2712-2716, 2001.
Kukreja RC, Kontos HA, Hess ML, and Ellis EF. PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH. Circ Res 59: 612-619, 1986.
Laneuville O, Breuer DK, Dewitt DL, Hla T, Funk CD, and Smith WL. Differential inhibition of human prostaglandin endoperoxide H synthases-1 and -2 by nonsterodal anti-inflammatory drugs. J Pharmacol Exp Ther 271: 927-934, 1994.
Lazarewicz JW, Wroblewski JT, Palmer ME, and Costa E. Activation of N-methyl-D-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells. Neuropharmacology 27: 765-769, 1988.[Web of Science][Medline]
Leão AAP. Further observations on spreading depression of activity in the cerebral cortex. J Neurophysiol 10: 409-414, 1947.
Mattia CJ, Adams JD, and Bondy SC. Free radical induction in the brain and liver by products of toluene catabolism. Biochem Pharmacol 46: 103-110, 1993.[Web of Science][Medline]
Miettinen S, Fusco FR, Yrjänheikki J, Keinänen R, Hirvonen T, Roivainen R, Närhi M, Hökfelt T, and Koistinaho J. Spreading depression and focal brain ischemia induce cyclooxygenase-2 in cortical neurons through N-methyl-D-aspartic acid-receptors and phospholipase A2. Proc Natl Acad Sci USA 94: 6500-6505, 1997.
Montoliu C, Valles S, Renau-Piqueras J, and Guerri C. Ethanol-induced oxygen radical formation and lipid peroxidation in rat brain: effect of chronic alchol consumption. J Neurochem 63: 1855-1862, 1994.[Web of Science][Medline]
Moskowitz MA, Kiwak KJ, Hekimian L, and Levine L. Synthesis of compounds with properties of leukotrienes C4 and D4 in gerbil brains after ischemia and reperfusion. Science 224: 886-889, 1984.
Nishikimi M, Yamada H, and Yagi K. Oxidation by superoxide of tocopherols dispersed in aqueous media with deoxycholate. Biochim Biophys Acta 627: 101-108, 1980.[Medline]
Nogawa S, Zhang F, Ross ME, and Iadecola C. Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage. J Neurosci 17: 2746-2755, 1997.
Ohtsuki T, Kitagawa K, Yamagata K, Mandai K, Mabuchi T, Matsushita K, Yanagihara T, and Matsumoto M. Induction of cyclooxygenase-2 mRNA in gerbil hippocampal neurons after transient forebrain ischemia. Brain Res 736: 353-356, 1996.[Web of Science][Medline]
Ohtsuki T, Matsumoto M, Hayashi Y, Yamamoto K, Kitagawa K, Ogawa S, Yamamoto S, and Kamada T. Reperfusion induces 5-lipoxygenase translocation and leukotriene C4 production in ischemic brain. Am J Physiol Heart CircPhysiol 268: H1249-H1257, 1995.
Onitsuka M, Mihara S, Yamamoto S, Shigemori M, and Higashi H. Nitric oxide contributes to irreversible membrane dysfunction caused by experimental ischemia in rat hippocampal CA1 neurons. Neurosci Res 30: 7-12, 1998.[Web of Science][Medline]
Paller MS and Jacob HS. Cytochrome P-450 mediates tissue-damaging hydroxyl radical formation during reoxygenation of the kidney. Proc Natl Acad Sci USA 91: 7002-7006, 1994.
Pulsinelli WA and Brierley JB. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10: 267-272, 1979.
Pulsinelli WA, Brierley JB, and Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 11: 491-498, 1982.[Web of Science][Medline]
Puntarulo S and Cederbaum AI. Production of reactive oxygen speices by microsomes enriched in specific human cytochrome P-450 enzymes. Free Radical Biol Med 24: 1324-1330, 1998.[Web of Science][Medline]
Rader RK and Lanthorn TH. Experimental ischemia induces a persistent depolarization blocked by decreased calcium and NMDA antagonists. Neurosci Lett 99: 125-130, 1989.[Web of Science][Medline]
Rao AM, Hatcher JF, Kindy MS, and Dempsey RJ. Arachidonic acid and leukotriene C4: role in transient cerebral ischemia of gerbils. Neurochem Res 24: 1225-1232, 1999.[Web of Science][Medline]
Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131-185, 2002.
Rosenthal MD, Vishwanath BS, and Franson RC. Effects of aristolochic acid on phospholipase A2 activity and arachidonate metabolism of human neutrophils. Biochem Biophys Acta 1001: 1-8, 1989.[Medline]
Salari H, Braquet P, and Borgeat P. Comparative effects of indomethacin, acetylenic acids, 15-HETE, nordihydroguaiaretic acid and BW755C on the metabolism of arachidonic acid in human leukocytes and platelets. Prostaglandins Leuk Med 13: 53-60, 1984.
Sapirstein A and Bonventre JV. Phospholipase A2 in ischemic and toxic brain injury. Neurochem Res 25: 745-753, 2000.[Web of Science][Medline]
Sasaki T, Nakagomi T, Kirino T, Tamura A, Noguchi M, Saito I, and Takakura K. Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA1 sector. Stroke 19: 1399-1403, 1988.
Seibert K, Masferrer JL, Needleman P, and Salvemini D. Pharmacological manipulation of cyclo-oxygenase-2 in the inflamed hydronephrotic kidney. Br J Pharmacol 117: 1016-1020, 1996.[Web of Science][Medline]
Siesjö BK and Katsura K. Ischemic brain damage: focus on lipids and lipid mediators. In: Neurobiology of Essential Fatty Acids, edited by Bazan NG, Murphy MG, and Toffano G. New York: Plenum, 1992, p. 41-56.
Stevens MK and Yaksh TL. Time course of release in vivo of PGE2, PGF2, 6-keto-PGF1, and TXB2 into the brain extracellular space after 15 min of complete global ischemia in the presence and absence of cyclooxygenase inhibition. J Cereb Blood Flow Metab 8: 790-798, 1988.[Web of Science][Medline]
Tanaka E, Yamamoto S, Inokuchi H, Isagai T, and Higashi H. Membrane dysfunction induced by in vitro ischemia in rat hippocampal CA1 pyramidal neurons. J Neurophysiol 81: 1872-1880, 1999.
Tanaka E, Yamamoto S, Kudo Y, Mihara S, and Higashi H. Mechanisms underlying the rapid depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J Neurophysiol 78: 891-902, 1997.
Tanaka M. Pharmacological and clinical profile of the free radical scavemger edaravone as a neuroprotective agent. Nippon Yakurigaku Zasshi 119: 301-308, 2002.[Medline]
Thompson CM, Capdevila JH, and Strobel HW. Recombinant cytochrome P450 2D18 metabolism of dopamine and arachidonic acid. J Pharmacol Exp Ther 294: 1120-1130, 2000.
Uchikado H, Tanaka E, Yamamoto S, Isagai T, Shigemori M, and Higashi H. Na+/Ca2+ exchanger activity induces a slow DC potential after in vitro ischemia in rat hippocampal CA1 region. Neurosci Res 36: 129-140, 2000.[Web of Science][Medline]
Volterra A, Trotti D, Cassutti P, Tromba C, Galimberti R, Lecchi P, and Racagni G. A role for the arachidonic acid cascade in fast synaptic modulation: ion channels and transmitter uptake systems as target proteins. Adv Exp Med Biol 318: 147-158, 1992a.[Medline]
Volterra A, Trotti D, Cassutti P, Tromba C, Salvaggio A, Melcangi RC, and Racagni G. High sensitivity of glutamate uptake to extracellular free arachidonic acid levels in rat cortical synaptosomes and astrocytes. J Neurochem 59: 600-606, 1992b.[Web of Science][Medline]
Wang M-H, Brand-Schieber E, Zand BA, Nguyen X, Falck JR, Balu N, and Schwartzman ML. Cytochrome P450-derived arachidonic acid metabolism in the rat kidney: characterization of selective inhibitors. J Pharmacol Exp Ther 284: 966-973, 1998.
Yamagata K, Andreasson KI, Kaufman WE, Barnes CA, and Worley PF. Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids. Neuron 11: 371-386, 1993.[Web of Science][Medline]
Yamamoto S, Tanaka E, Shoji Y, Kudo Y, Inokuchi H, and Higashi H. Factors that reverse the persistent depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J Neurophysiol 78: 903-911, 1997.
Zalkin H and Tappel AL. Studies of the mechanism of vitamin E action. IV. Lipid peroxydation in the vitamin E-deficient rabbit. Arch Biochem Biophys 88: 113-117, 1960.[Web of Science][Medline]
Zou A-P, Ma Y-H, Sui Z-H, Ortiz de Montellano PR, Clark JE, Masters BS, and Roman RJ. Effects of 17-octadecynoic acid, a suicide-substrate inhibitor of cytochrome P450 fatty acid -hydroxylase, on renal function in rats. J Pharmacol Exp Ther 268: 474-481, 1994.
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and 
. · · ·, the preexposure level in this and subsequent figures. A: typical changes in the membrane potential during and after in vitro ischemia in the absence of the drugs (top), and in the presence of para-BPB (middle), or aristolochic acid (bottom). Downward deflections are hyperpolarizing electrotonic potentials elicited by anodal current pulses (in range: 0.2-0.4 nA for 200 ms every 3 s) in this and subsequent figures. The resting potentials were -72, -75, and -73 mV from top to bottom. B: the effects of PLA2 inhibitors on the percent of neurons exhibiting recovery. 
, 
, and 
, no, partial, and complete recovery, respectively. C: the effects of PLA2 inhibitors on the potential recovery 30 min after the reintroduction of oxygen and glucose. Each column shows the mean ± SD in this and subsequent figures. In the presence of para-BPB or aristolochic acid, the membrane potential repolarized toward the preischemic exposure level in a concentration-dependent manner after the reintroduction of oxygen and glucose. *, P < 0.05; **, P < 0.01; one-way ANOVA with Scheffé post hoc comparisons.
