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HYPEROXIA SUPPRESSES EXCESSIVE SUPEROXIDE ANION RADICAL GENERATION IN BLOOD, OXIDATIVE STRESS, EARLY INFLAMMATION, AND ENDOTHELIAL INJURY IN FOREBRAIN ISCHEMIA/REPERFUSION RATS: LABORATORY STUDY

Fujita, Motoki*; Tsuruta, Ryosuke*; Kaneko, Tadashi*; Otsuka, Yohei*; Kutsuna, Satoshi*; Izumi, Tomonori*; Aoki, Tetsuya; Shitara, Masaki; Kasaoka, Shunji*; Maruyama, Ikuro; Yuasa, Makoto; Maekawa, Tsuyoshi*

doi: 10.1097/SHK.0b013e3181ceeeec
Basic Science Aspects
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This study used an electrochemical O2-· sensor to investigate the effects of hyperoxia on generation of the superoxide radical (O2-·) in the jugular vein during forebrain I/R in rats. Twenty-eight male Wistar rats were allocated to a sham group (n = 7; sham-treated rats with inspired oxygen fraction [FiO2] of 0.4), a hemorrhagic shock and reperfusion (HS/R) group (n = 7; HS without carotid artery occlusion and reperfusion with FiO2 of 0.4), a normoxia group (n = 7; forebrain ischemia produced by bilateral carotid arteries occlusion with HS and reperfusion with FiO2 of 0.4), and a hyperoxia group (n = 7; forebrain ischemia with FiO2 of 0.4 and reperfusion with FiO2 of 1.0). The jugular venous O2-· current was measured for 10 min during forebrain ischemia and for 120 min after reperfusion. The O2-· current increased gradually during forebrain ischemia in the three groups other than the sham group. Immediately after reperfusion, the current showed a marked increase in the normoxia group and a pronounced decrease in the hyperoxia group. Levels of brain and plasma malondialdehyde, high-mobility group box 1 protein, and intercellular adhesion molecule 1 were significantly attenuated in the hyperoxia group relative to those in the normoxia group. In conclusion, hyperoxia suppressed jugular venous O2-· generation and malondialdehyde, high-mobility group box 1, and intercellular adhesion molecule 1 in the brain and plasma in the acute phase of cerebral I/R. Thus, the administration of 100% oxygen immediately after reperfusion suppresses oxidative stress and early inflammation in cerebral I/R.

*Advanced Medical Emergency and Critical Care Center, Yamaguchi University Hospital, Ube; Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, Noda; and Department of Laboratory and Molecular Medicine, Kagoshima University, Kagoshima, Japan

Received 1 Oct 2009; first review completed 6 Nov 2009; accepted in final form 7 Dec 2009

Address reprint requests to Motoki Fujita, MD, PhD, Advanced Medical Emergency and Critical Care Center, Yamaguchi University Hospital, 1-1-1, Minami-Kogushi, Ube, Yamaguchi, Japan. E-mail: motoki-ygc@umin.ac.jp.

This study was mainly supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan (grant no. 19791328) and was partially supported by the Ministry of Health, Labor and Welfare of Japan (grant no. H18-trans-general-003).

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INTRODUCTION

In the pathophysiology of focal and global cerebral I/R, including stroke, head injury, and postresuscitation after cardiac arrest, oxygen is routinely administered to improve tissue hypoxia. The administration of oxygen is thought to prevent the primary and secondary events leading to cell death after cerebral ischemia (1, 2). However, because the superoxide anion radical (O2-·) induces oxidative stress and lipid peroxidation after cerebral I/R (3), oxygen therapy may theoretically exacerbate oxygen-derived oxidative stress as a consequence of increased production of O2-· (1, 2, 4).

It has been reported that oxygen therapy can enhance the generation of free radicals. These radicals induce lipid peroxidation, blood-brain barrier (BBB) disruption, and glutamate-induced cell death, and increase cerebral hemorrhage (1-3, 5, 6). Furthermore, hyperoxia is reported to induce cerebral vasoconstriction and increase brain damage (1, 2, 4, 7-11). Conversely, recent experiments have reported beneficial effects of normobaric hyperoxia (NBO) on the pathophysiology of focal and global cerebral I/R (1, 2, 6, 12-16), and NBO has ameliorated oxidative stress (1, 2, 6, 13). However, the efficacy of oxygen therapy remains controversial, and the dynamics of O2-· during oxygen therapy are unclear.

We recently developed an in vivo real-time quantitative O2-· analysis system with a novel electrochemical O2-· sensor (3, 17-20). The current measured by the sensor correlates strongly with the hitting frequency of the O2-· in the circulating blood on the surface of the sensor (17, 18). Using this method, we have demonstrated in rats that O2-· is increased in the jugular vein during the ischemic and reperfusion periods of forebrain I/R and that one of the major sources of O2-· after reperfusion is xanthine oxidase (3, 21). Furthermore, the excessive production of O2-· after I/R is associated with early inflammation, oxidative stress, and endothelial injury (3, 22). Following these results, we also measured levels of high-mobility group box 1 (HMGB1) protein, an early mediator of neuroinflammation after cerebral I/R (22-24); malondialdehyde (MDA), an endo product of lipid peroxidation; and intercellular adhesion molecule 1 (ICAM-1), a marker of endothelial activation (24).

In this study, we tested the hypothesis that NBO is beneficial to the pathophysiology of cerebral I/R because of its suppressive effect on excessive O2-· generation, oxidative stress, early inflammation, and endothelial activation.

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MATERIALS AND METHODS

This study was approved by the Animal Experiment Committee of Yamaguchi University, Japan, and all animals were handled according to the National Institutes of Health guidelines. Twenty-eight male Wistar rats (230-280 g) were assigned to one of four groups: a sham group (n = 7; sham-treated with an inspired oxygen fraction [FiO2] of 0.4), a hemorrhagic shock and reperfusion (HS/R) group (n = 7; HS without carotid artery occlusion and reperfusion with FiO2 of 0.4), a normoxia group (n = 7; forebrain ischemia produced by bilateral carotid artery occlusion with HS and reperfusion with FiO2 of 0.4), and a hyperoxia group (n = 7; forebrain ischemia induced with FiO2 of 0.4 and reperfusion with FiO2 of 1.0).

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Animal preparation

Transient forebrain ischemia was induced as described in our previous report (3, 21), with minor changes. In brief, under isoflurane anesthesia (3% during surgery), mechanical ventilation was performed through a tracheostomy tube with tidal volume of 10 μL/g body weight (SN-480-7; Shinano Manufacturing Co Ltd, Tokyo, Japan); the respiratory rate was adjusted to maintain normocapnia throughout the experiments. An arterial catheter was inserted to measure blood pressure and to sample blood in the left femoral artery. A venous catheter was inserted into the right atrium through the right external jugular vein to both administer drugs and remove blood, inducing a state of hypotension. The distal side of the right jugular vein was ligated. The tip of the O2-· sensor was inserted from the left anterior facial vein to the external jugular vein. The distal side of the left anterior facial vein and the proximal branches of the external jugular vein from the left anterior facial vein, which included the left posterior external jugular vein and the cephalic vein, were ligated under a surgical microscope to preclude blood flow from the face and neck (3). The isoflurane concentration was reduced to 0.7% with 40% O2, and the physiological parameters were stabilized for at least 20 min.

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Induction of forebrain I/R

After measurement of the baseline O2-· current, forebrain ischemia was induced by bilateral occlusion of the common carotid arteries and removal of blood to achieve a MAP of 40 to 45 mmHg for 10 min in the normoxia and hyperoxia groups. In the HS/R group, the bilateral common carotid arteries were not occluded during the ischemic period after blood had been removed to produce a MAP of 40 to 45 mmHg. In the sham group, identical surgical preparation was made, except that no bilateral common carotid artery occlusion was applied or hemorrhagic hypotension was induced. Forebrain ischemia was confirmed by the complete suppression of electroencephalographic activity. Reperfusion was achieved by releasing the bilateral carotid artery occlusion and returning the previously removed blood. In this FBI/R (bilateral carotid artery occlusion with HS and reperfusion) model, hemorrhagic hypotension was required to precisely establish forebrain ischemia because bilateral occlusion of the common carotid arteries alone was insufficient to reduce the cerebral blood flow to below the ischemic threshold and did not produce sufficient quantifiable cell death.

In the hyperoxia group, FiO2 was increased from 0.4 to 1.0 immediately after reperfusion and was maintained at that level throughout the experiment. In the sham, HS/R, and normoxia groups, FiO2 was maintained at 0.4 throughout the experiment. The generation of O2-· was evaluated using the O2-· sensor as the difference between the detected current and the baseline current during the preischemic, ischemic, and reperfusion periods, as previously described (3, 17). Also measured were arterial oxygen saturation (SaO2), PaO2, PaCO2, pH, and base excess in the arterial blood, MAP, and pharyngeal temperature. The pharyngeal temperature was maintained at 37.0°C throughout the experiment. At 120 min after reperfusion, the blood was sampled and replaced with ice-cold saline. The blood was centrifuged at 900g for 10 min at 4°C, and the plasma was stored at −80°C until analysis. The brain was removed, frozen in liquid nitrogen, and stored at −80°C until analyses of MDA, HMGB1, and ICAM-1.

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Jugular venous O2-· measurement and evaluation

The O2-· generated was measured as the O2-· current (I), as recorded using an O2-· analysis system with an all-synthetic electrochemical O2-· sensor (Actiive Corp, Noda, Japan), as previously described (3, 17-20). The sensor has a carbon working electrode coated with a polymeric iron porphyrin complex, bromo-iron(III) (5,10,15,20-tetra[3-thienyl]porphyrin), ligated to 1-methylimidazole as the axial ligand ([Fe{im}2{tpp}]Br), which mimics cytochrome c, and a stainless steel counter electrode (18-20). It has a high catalytic activity for the oxidation of O2-·, and there is a linear relationship between the current and the O2-· concentration in phosphate-buffered saline or human blood (17). The sensor has been shown to be sensitive and specific for extracellular O2-· and does not respond to NO or H2O2 (17). Furthermore, it was stable for at least 6 h in vivo, as shown in our previous study (17), and its stability in saline was confirmed before use in the present rat experiment.

The current data were recorded at two points per second, and smoothing procedures (i.e., moving average) were applied during the data analysis because the data contained noise and artifacts (17). The current data are presented as ΔI, which represents the difference between the baseline and measured currents, as described in our previous report (3).

The measured O2-· current was evaluated as a quantified partial value of electricity (Q), which correlates with the amount of O2-· generated (17). The baseline current was defined as the stable state before the onset of ischemia. The differences between the baseline and measured O2- currents were integrated during the ischemic period to QI and during the reperfusion period to QR, as described in our previous study (3). Total Q was calculated as the sum of QI and QR, which reflects the amount of O2-· generated throughout forebrain I/R.

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MDA analysis

Brain and plasma MDA levels were measured to evaluate the degree of lipid peroxidation in the brain tissue and the circulating blood, respectively, as previously described (3, 21). The brain tissue of the left frontal lobe was homogenized in ice-cold 50 mM Tris-HCI buffer (pH 7.4) containing 5 mM butylated hydroxytoluene (in acetonitrile), using a Polytron PT-MR3100 homogenizer (Kinematica, Littau, Switzerland). The MDA levels in the brain homogenate and plasma 120 min after reperfusion were analyzed with a Bioxytech MDA-586 kit (OxisResearch, Foster, Calif). The final results are given as picomoles of MDA per milligram of protein in the brain homogenate and as micromolar MDA in the plasma.

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HMGB1 analysis

The cytoplasmic fraction of the brain tissue was prepared as described in our previous report (3, 21). In brief, the brain tissue of the left frontal lobe was gently homogenized in 10 mM N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid/10 mM KCl buffer with 0.08% NP-40, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride; the soluble fraction derived from the cytoplasm was stored at −80°C until further analysis. The HMGB1 levels in the cytoplasm of the brain and plasma 120 min after reperfusion were analyzed with an HMGB1 ELISA Kit II (Shino-Test Corporation, Kanagawa, Japan). The final results are given as nanograms of HMGB1 per milligram of protein in the brain homogenate and as nanograms of HMGB1 per milliliter of plasma.

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ICAM-1 analysis

The ICAM-1 levels in the brain homogenate and the plasma levels of soluble ICAM-1 (sICAM-1) 120 min after reperfusion were analyzed with the Quantikine Rat sICAM-1 (CD54) Immunoassay Kit (R&D Systems Inc, Minneapolis, Minn). The final results are given as picograms of ICAM-1 per milligram of protein in the brain homogenate and as nanograms of sICAM-1 per milliliter of plasma.

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Analyses of arterial blood gas and lactate

Arterial blood gas and lactate were analyzed with the ABL System 555 (Radiometer Medical A/S, Copenhagen, Denmark) during the preischemic and reperfusion periods.

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Statistical analysis

The data were analyzed using the SPSS 10.0 statistical software package (SPSS Inc, Chicago, Ill). The statistical significance of differences in the serial data (ΔI, MAP, SaO2, PaO2, PaCO2, pH, and lactate) was determined with two-way ANOVA. The statistical significance of differences in data measured at a single point (QI, QR, MDA, HMGB1, and ICAM-1) was determined with one-way ANOVA. When the results of ANOVA were significant, the Bonferroni post hoc test was applied to determine specific group differences. Statistical analysis of the correlation between total Q and MDA, HMGB1, or ICAM-1 was performed using Pearson correlation coefficient. All data are expressed as the mean ± SD of seven measurements, except for ΔI, which is expressed as the mean ± SE. A value of P < 0.05 was considered statistically significant.

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RESULTS

Jugular venous O2-· current and Q during forebrain I/R in rats

Figure 1A shows ΔI in the jugular vein during ischemia and after reperfusion. During the ischemic period, the O2-· current increased gradually in the HS/R, normoxia, and hyperoxia groups, but the increase was not significant. Immediately after reperfusion, further elevation of the O2-· current was observed in the normoxia group relative to the sham group (P < 0.01, 4-120 min after reperfusion) and HS/R group (P < 0.05, 4 min after reperfusion; P < 0.01, 6-120 min after reperfusion). In the hyperoxia group, further elevation of the O2-· current was observed, but the current was significantly attenuated compared with that in the normoxia group after reperfusion (P < 0.01, 10-120 min after reperfusion).

Fig. 1

Fig. 1

The ΔI of O2-· was integrated during ischemia as QI and during reperfusion as QR, which reflect the amount of O2-· generated in each period (3). Figure 1, B and C, shows the differences in QI and QR among the four groups. The QI did not differ among the four groups, but QR was significantly higher in the normoxia group than in the sham group (P < 0.01) or the HS/R group (P < 0.01). The QR in the hyperoxia group was significantly attenuated compared with that in the normoxia group (P < 0.01).

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MDA levels in the brain tissue and plasma

The MDA in the forebrain tissue 120 min after reperfusion is shown in Figure 2A. The level of brain MDA in the normoxia and hyperoxia groups was significantly higher than that in the sham group (P < 0.01 vs. the normoxia and hyperoxia groups) or HS/R group (P < 0.01 vs. the normoxia and hyperoxia groups). Brain MDA in the hyperoxia group was significantly attenuated compared with that in the normoxia group (P < 0.01).

Fig. 2

Fig. 2

Plasma MDA at 120 min after reperfusion is shown in Figure 2B. Plasma MDA in the normoxia group was significantly higher than that in the sham group (P < 0.01) or HS/R group (P < 0.01). Plasma MDA in the hyperoxia group was significantly attenuated compared with that in the normoxia group (P < 0.01).

The correlation coefficient was calculated between total Q, which is the sum of QI and QR, and brain MDA (y = 0.7069x + 40.586, r = 0.7587, P < 0.01; Fig. 2C), and between total Q and plasma MDA (y = 0.0241x + 0.7991, r = 0.8888, P < 0.01; Fig. 2D).

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HMGB1 levels in brain tissue and plasma

The HMGB1 levels in the cytoplasm of forebrain tissue 120 min after reperfusion are shown in Figure 3A. Brain HMGB1 in the HS/R, normoxia, and hyperoxia groups was significantly higher than that in the sham group (P < 0.01 vs. the HS/R, normoxia, and hyperoxia groups). Brain HMGB1 in the normoxia group was significantly higher than that in the HS/R group (P < 0.05). Brain HMGB1 in the hyperoxia group was significantly attenuated compared with that in the HS/R group (P < 0.05) or normoxia group (P < 0.01).

Fig. 3

Fig. 3

Plasma HMGB1 levels 120 min after reperfusion are shown in Figure 3B. Plasma HMGB1 was significantly higher in the normoxia group than in the sham group (P < 0.01) or the HS/R group (P < 0.01) and was significantly attenuated in the hyperoxia group compared with the normoxia group (P < 0.01).

The correlation coefficients were calculated between total Q and brain HMGB1 (y = 0.6254x + 221.42, r = 0.6551, P < 0.01; Fig. 3C), and between total Q and plasma HMGB1 (y = 0.6545x + 15.158, r = 0.765, P < 0.01; Fig. 2D).

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ICAM-1 levels in brain tissue and plasma

The ICAM-1 levels in the forebrain tissue 120 min after reperfusion are shown in Figure 4A. Brain ICAM-1 in the HS/R, normoxia, and hyperoxia groups was significantly higher than that in the sham group (P < 0.01 vs. the HS/R, normoxia, and hyperoxia groups). There was no significant difference in brain ICAM-1 between the HS/R group and the normoxia group. Brain ICAM-1 in the hyperoxia group was significantly attenuated compared with that in the HS/R group (P < 0.05).

Fig. 4

Fig. 4

Plasma sICAM-1 levels 120 min after reperfusion are shown in Figure 4B. Plasma sICAM-1 in the normoxia and hyperoxia groups was significantly higher than that in the sham group (P < 0.01 vs. the normoxia and hyperoxia groups) or HS/R group (P < 0.01 vs. the normoxia and hyperoxia groups). Plasma sICAM-1 in the hyperoxia group was significantly attenuated compared with that in the normoxia group (P < 0.01).

The correlation coefficients were calculated between total Q and brain ICAM-1 (y = 0.6546x + 98.597, r = 0.5066, P < 0.01; Fig. 4C), and between total Q and plasma sICAM-1 (y = 0.0184x + 1.413, r = 0.7515, P < 0.01; Fig. 4D).

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Physiological parameters

Physiological parameters are shown in Table 1. The PaO2 in the hyperoxia group was 344 ± 15 torr at 10 min after reperfusion and decreased gradually thereafter. The PaO2 in the hyperoxia group was significantly higher than that in the sham, HS/R, or normoxia groups. The PaO2 in the HS/R and normoxia groups was significantly lower than that in the sham group after reperfusion. The PaCO2 was elevated at 10 min after reperfusion in the HS/R, normoxia, and hyperoxia groups because of systemic hypotension, with or without carotid artery occlusion. Metabolic acidosis was observed in the HS/R, normoxia, and hyperoxia groups after reperfusion relative to the sham group, induced by systemic hypotension with or without carotid artery occlusion. The lactate concentration in the hyperoxia group was the same as that in the HS/R group 30 min after reperfusion and tended to be lower than that in the HS/R and normoxia groups at 60, 90, and 120 min after reperfusion. The MAP in the normoxia and hyperoxia groups was significantly lower than that in the sham group at 90 and 120 min after reperfusion.

Table 1

Table 1

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DISCUSSION

We demonstrated that NBO suppresses the generation of O2-· in the jugular vein (Fig. 1), which is associated with the suppression of MDA, HMGB1, and ICAM-1 in the brain and plasma during forebrain I/R (Figs. 2-4). These findings indicate that the early administration of oxygen suppresses oxidative stress, thereby attenuating lipid peroxidation, early inflammation, and endothelial injury in the pathophysiology of cerebral I/R.

Although it has been reported previously that NBO suppresses oxidative stress in the pathophysiology of focal cerebral I/R (6, 13), the dynamics of O2-· under NBO has not been demonstrated in vivo because of the unavailability of a method of in vivo O2-· monitoring before our development of a suitable O2-· sensor (3, 17). In the present study, NBO initiated immediately after reperfusion attenuated the excessive production of O2-· in the jugular vein (Fig. 1). These results indicate that even after reperfusion, NBO suppresses O2-· generation in the pathophysiology of global cerebral I/R.

Lipid peroxidation is one of the major consequences of free radical-mediated injury to the brain. In the present study, MDA levels in brain tissue reflect oxidative damage to brain lipids. It has also been reported that plasma MDA levels correlate well with infarct size, the severity of stroke, and outcome (25). In the present study, NBO after reperfusion suppressed MDA levels in the brain tissue and plasma (Fig. 2, A and B). Brain and plasma MDA were also associated with the generation of O2-· in the jugular vein (Fig. 2, C and D). These results indicate that early NBO suppresses oxidative stress and prevents cerebral injury by suppressing excessive O2-· generation.

In this study, NBO suppressed oxidative stress immediately after reperfusion by attenuating the generation of O2-·. In the pathophysiology of cerebral ischemia, energy failure occurs in the ischemic tissue and causes the uncontrolled accumulation of intracellular Ca2+ (26). Ca2+ sequentially activates enzymes such as xanthine oxidase and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which accelerate O2-· generation, and neuronal cell death occurs (27). Xanthine oxidase is also one of the major sources of O2-· in the jugular vein in the rat forebrain I/R model (21). Liu et al. (27) reported that NBO inhibits NADPH oxidase activity and protects the BBB. NADPH oxidase-mediated O2-· generation induces lipid peroxidation and membrane disruption, leading to BBB damage (14, 27). Recent studies have reported positive feedback loops involving reactive oxygen species (28-32). Xanthine oxidase activity is reported to be regulated by NADPH oxidase (31) and by H2O2 and calcium (32); consequently, the inhibition of NADPH oxidase by NBO might disrupt the positive feedback loops involving reactive oxygen species. Wada et al. (33) reported that hyperoxic therapy led to increased levels of manganese superoxide dismutase in the gerbil hippocampus; this phenomenon might also contribute to the benefits of hyperoxic therapy.

Another mechanism by which O2-· generation is thought to be attenuated is the salvage of ischemic tissue by NBO after reperfusion. Oxygen is known to cause vasoconstriction and decrease cerebral blood flow in the normal brain (34), but paradoxical increases in blood flow have been documented in ischemic brain regions (13, 35). This effect might explain how NBO salvages ischemic tissue after reperfusion. After the initiation of NBO, blood flow in the ischemic tissue increases and energy failure is ameliorated. Therefore, the activation of xanthine oxidase and NADPH oxidase might be attenuated. In the present study, the early improvement in lactate concentration observed in the hyperoxia group might reflect this effect (Table 1). In addition, MAP was maintained higher than 83 ± 9 mmHg, even in the normoxia group (Table 1), which was higher than the autoregulation level (generally 60 mmHg) of cerebral circulation, although we could not measure hemodynamic parameters other than MAP. Therefore, it is possible that cerebral blood flow was maintained, even until the end of the experiment.

The HMGB1 is one of the early mediators of inflammation in the pathophysiology of focal and global cerebral I/R, as described in our previous report and those of others (3, 22-24). Qiu et al. (24) reported that HMGB1 is translocated from the nucleus to the cytoplasm and is released into the extracellular milieu as early as 1 to 2 h after focal cerebral ischemia, and that neurons are one of the principal sources of the HMGB1 released in the early phase of ischemic injury. Extracellular HMGB1 is reported to function as a proinflammatory cytokine, in the activation of microglia and the endothelium, and in stimulating the release of the other cytokines, thus aggravating brain injury (22-24). In the present study, NBO attenuated brain and plasma HMGB1 (Fig. 3, A and B) and ICAM-1 levels after reperfusion (Fig. 4, A and B). These results indicate that NBO attenuates the inflammatory response in the early phase of reperfusion in association with the suppression of O2-· generation and the attenuation of HMGB1 and ICAM-1. We have previously demonstrated a positive correlation between HMGB1 concentration and O2-· generation, suggesting an O2-· mediated HMGB1 loop (3, 21). The present study revealed significant correlations between total Q and HMGB1 concentrations in the brain and plasma (Fig. 3, C and D). These results confirm our previous data and suggest the existence of an O2-· mediated HMGB1 loop (3, 21).

The timing of initiation and duration of hyperoxia therapy are important in terms of its protective effects (1, 2, 13). It was reported that 60 min of NBO after reperfusion was neuroprotective and did not increase oxidative stress in the pathophysiology of both focal and global cerebral I/R (13, 15, 16). Singhal (1, 2) also suggested that early timing and a short duration of NBO might be beneficial. In the present study, NBO was initiated immediately after reperfusion and was continued for 120 min. The effect of NBO on the generation of O2-· was rapid and continued for 120 min (Fig. 1). These results indicate that the early administration of oxygen after reperfusion has a strong influence on the pathophysiology of cerebral I/R. However, it remains necessary to investigate the effects of much longer oxygen exposure after reperfusion.

In the present study, NBO showed protective effects during cerebral I/R, as also reported in previous studies (1, 2, 6, 12-16). However, other reports have documented the harmful effects of NBO in the pathophysiology of cerebral I/R (1, 2, 4, 7-11). This discrepancy might reflect the settings of the experiments. Hyperoxia was reported to be harmful in neonates probably because of undeveloped antioxidative ability in the immature brain (2, 4). In addition, differences among the studies in terms of species, model type, and background may have affected the results.

The present study involved several limitations. We obtained no data regarding long-term neurological outcome because the FBI/R model used (two-vessel occlusion and hemorrhagic hypotension) was too severe for the rats to survive. Our results reflect only the acute phase (120 min) of the pathophysiology of forebrain I/R, with or without NBO. Further studies are required with a much longer duration and with different timing of the induction of NBO. Furthermore, we were unable to obtain data regarding infarct size, histology, immune histochemistry, and antioxidative parameters, as the entire sample in each case was used to measure MDA, HMGB1, and ICAM-1. Another limitation of this study was that our in vivo real-time quantitative O2-· analysis system cannot directly measure O2-· generation in brain tissue. However, the Q value for O2-· in the jugular vein correlated well with brain MDA (Fig. 2C), brain HMGB1 (Fig. 3C), and brain ICAM-1 (Fig. 4C). These results indicate that the value of O2-· in the jugular vein reflects the oxidative status in both the cerebral circulation and the brain.

In conclusion, NBO after reperfusion suppressed jugular venous O2-· generation and levels of MDA, HMGB1, and ICAM-1 in the brain and plasma in the acute phase of cerebral I/R. Thus, the early-phase administration of 100% oxygen suppresses oxidative stress, early inflammation, and endothelial injury in the brain and plasma during the acute phase of cerebral I/R.

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ACKNOWLEDGMENTS

The authors thank Ms Chihiro Kobayashi and Mr Masahiro Nanba (Tokyo University of Science) for their assistance with the O2-· sensor, Mrs Hitomi Ikemoto and Dr Takahiro Yamamoto for their valuable technical assistance, and Ms Masako Ueda for her patience in preparing the original manuscript.

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Keywords:

Reactive oxygen species; high-mobility group box 1; malondialdehyde; intercellular adhesion molecule 1; electrochemical sensor

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