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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Biofunctional Science, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (H.U., K.Yas., T.S., K.Yam., R.S.); and Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan (T.Y., M.T.)
Received September 3, 2005; accepted December 7, 2005.
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Abstract |
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). NSAIDs cause therapeutic effects by the suppression of prostaglandin biosynthesis via the inhibition of cyclooxygenase (COX) gene expression. This class of agents is also known to cause significant gastrointestinal damage (Vane, 1971). It is known that the COX enzymes have isozymes of three types (Chandrasekharan et al., 2002), and the inhibition of COX-1 in gastric mucosa has been thought to be related with the gastric ulcer formation induced by NSAIDs (Wallace et al., 1998), because COX-1 is involved in maintaining the integrity of gastric mucosa, the increment of mucosal blood flow and mucus secretion (Morris and Wallace, 1981; Kitahora and Guth, 1987). However, ulcer formation did not occur in COX-1 knockout mice (Langenbach et al., 1995) and in mice pretreated with SC-560, which is a COX-1-selective inhibitor, in spite of significant suppression of prostaglandin biosynthesis (Wallace et al., 2000). These findings suggest other factors are involved in the mechanism of NSAID-induced gastric ulcer formation in addition to inhibition of COX-1.
Reactive oxygen species (ROS) are reportedly associated with the pathogenesis in indomethacin-induced gastric ulcer (Takeuchi et al., 1991; Yoshikawa et al., 1993). The formation of gastric ulcer and the increase of thiobarbituric acid-reactive substances in gastric mucosa induced by indomethacin was significantly suppressed by the intravenous infusion of superoxide dismutase (SOD) or dimethylsulfoxide (DMSO) (Takeuchi et al., 1991) and the subcutaneous treatment of the mixture of Cu,Zn-SOD and catalase (Yoshikawa et al., 1993). In these studies, the stable end products of ROS generation were monitored. However, individual free radical reactions or reactive oxygen species were not monitored.
The in vivo electron spin resonance (ESR)/spin probe technique is suitable for the examination of free radical reactions in vivo in experimental diseases, as we (Utsumi et al., 1990; Sano et al., 1998; Phumala et al., 1999; Han et al., 2001; Utsumi et al., 2002; Kasazaki et al., 2003a,b; Matsumoto et al., 2003; Yamato et al., 2003; Takeshita et al., 2004; Yasukawa et al., 2004; Sonta et al., 2005) and others (Miura et al., 1997; Kuppusamy et al., 1998; Leonard et al., 2002) have demonstrated. In this technique, the signal decay of the nitroxyl probes mediated by ROS is monitored by ESR spectroscopy. Recently, we reported, for the first time, the generation of ROS in the stomachs of rats with NH4OH-induced (Kasazaki et al., 2003a,b) and water immersion restraint (WIR)-induced (Yasukawa et al., 2004) gastric lesions using this technique. In the NH4OH-induced gastric damage model, lesion formation induced by NH4OH is acute, occurring within 30 min, and is associated with the enhancement of vascular permeability, neutrophil infiltration into the mucosa, and ROS generation in stomach. This rapidly developing pathology makes it difficult to ascertain whether ROS generation is a cause for and/or a result of ulcer formation. In WIR-induced gastric lesion model, which requires approximately 6 h to produce lesions, the relationships between the enhancement of signal decay and the neutrophil infiltration as well as lesion formation in the WIR model were different from that observed with NH4OH. However, ROS generation in gastric region of WIR-treated rats was similar in extent to that observed in NH4OH-treated rats. These studies demonstrated the usefulness of in vivo ESR/spin probe technique for noninvasive and real-time evaluation of in vivo free radical reaction in rats with gastric ulcers.
The availability of nitroxyl probes with functional groups, which make them compartmentalize in specific cellular/subcellular regions, makes in vivo ESR technique uniquely capable of providing unambiguous information pertaining to the sites of ROS generation noninvasively (Utsumi et al., 2002). The selection of nitroxyl probes with varying membrane permeability enables us to evaluate the site-specific ROS generation in various disease models. The in vitro cellular ESR measurement of three nitroxyl probes having different membrane permeability, carboxy-PROXYL, methoxycarbonyl-PROXYL, and carbamoyl-PROXYL, with liposomes revealed that carboxy-PROXYL, methoxycarbonyl-PROXYL, and carbamoyl-PROXYL locate in aqueous phase, in lipidic phase, and at the interface between aqueous and lipidic phase, respectively (Yamato et al., 2003). In this study, generation sites of ROS in rats with indomethacin-induced gastric ulcer were directly investigated using in vivo 300-MHz ESR spectroscopy and nitroxyl probes with different membrane permeability.
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Materials and Methods |
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). [1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl] acetic acid (indomethacin) and 4,5-dihydroxy-1,3-benzene-disulfonic acid (tiron) were obtained from Sigma Chemical Co. (St. Louis, MO). D-Mannitol (mannitol) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were commercially available reagent grade quality. Animal Treatment. Male Sprague-Dawley rats (5 weeks old; 120–160 g body weight) were purchased from Seac Yoshitomi Co. (Fukuoka, Japan) and were acclimatized for 1 week before experimentation. Diet (MF; Oriental Yeast Co., Tokyo, Japan) and water were provided ad libitum. The animals were fasted for 24 h with free access to water until 1 h before the experiment. Gastric mucosal ulcers were induced by the oral administration of indomethacin (5, 15, or 30 mg/kg). Indomethacin was dissolved in 5% sodium hydrogen carbonate (NaHCO3) solution.
To estimate the effect of antioxidants, either DMTU (0.33 mmol/kg at a time), tiron (0.33 mmol/kg at a time), mannitol (0.33 mmol/kg at a time), or nitroxyl radicals (0.18 mmol/kg at a time) hydroxy-, oxo-, carboxy-, or trimethylammonium-TEMPO or carbamoyl- or methoxycarbonyl-PROXYL were orally administered 5 min before and 1 and 2 h after indomethacin treatment. All procedures and animal care were approved by the Committee on Ethics of Animal Experiments, Graduate School of Pharmaceutical Sciences, Kyushu University, and were conducted according to the Guidelines for Animal Experiments of the Graduate School of Pharmaceutical Sciences, Kyushu University.
ESR Measurement. Rats anesthetized by intramuscular injection of urethane (2 g/kg) were orally given hydroxy-, oxo-, carboxy-, or trimethylammonium-TEMPO or carbamoyl- or methoxycarbonyl-PROXYL (1.0 ml of 10 mM) to individual rats, and the ESR spectra were observed in vivo in the gastric region with a 300-MHz ESR spectrometer (JES-CM-3L; JEOL, Tokyo, Japan). The microwave power was 1.19 mW. The amplitude of the 100-kHz field modulation was 0.125 mT. The external magnetic field was swept at a scan rate of 6.0 mT/min. The signal-decay rate was calculated as described previously (Kasazaki et al., 2003a).
To determine the causes of the enhanced signal decay, DMTU (0.33 mmol/kg), tiron (0.33 mmol/kg), or mannitol (0.33 mmol/kg) was orally administered with the administration of the nitroxyl probe. In each experiment, control rats were treated with distilled water as a vehicle.
Evaluation of Macroscopic and Histologic Gastric Mucosal Injury. The extent of gastric mucosal injury in rats treated with indomethacin was investigated macroscopically. The stomachs were removed, inflated with 10 ml of 1% formaldehyde, and opened along the greater curvature. The ulcer area (millimeters2 per glandular stomach) was determined under a dissecting microscope with a square grid micrometer.
Histologic injury of indomethacin-treated gastric mucosa with hydroxy-TEMPO pretreatment was evaluated by hematoxylin and eosin (H&E) stain. A hydroxy-TEMPO solution (0.18 mmol/kg at a time) was orally administered 5 min before and 1 and 2 h after indomethacin treatment. Three hours after indomethacin treatment, the stomach was collected. All specimens were fixed overnight in 10% neutral-buffered formalin and embedded in paraffin. Sections with thickness of 3 µm were cut, mounted on glass slides, dried overnight, and then stained with H&E.
MRI Measurement. A rat anesthetized by intramuscular injection of urethane (2 g/kg) was turned up on the plate, where two syringes (3 mm inner diameter) containing distilled water were fixed as positional and contrast markers on both sides of the rat. The MR imaging was performed using a 0.2-T MRI system (MRP-20; Hitachi Medical Co., Tokyo, Japan) both before and after the oral administration of distilled water (1.0 ml). The pulse sequence of conventional spin echo proton density-weighted with repetition time (ms)/echo time (ms) = 1600/40 was acquired. All images were acquired using a 200 x 200-mm field of view, two-signal average, coronal plane, and 3.0-mm thickness.
Statistical Analysis. Statistical analyses were carried out using Stat View 5.0. The data were analyzed by Student's t test, by oneway analysis of variance (ANOVA) with the Dunnett's test or the Tukey-Kramer's test as a post hoc test, or by two-way ANOVA with the Tukey-Kramer's test as a post hoc test. All the results are expressed as the mean ± S.D. P < 0.05 was considered statistically significant.
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Results |
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). The studies using the 20-mg/kg (Langenbach et al., 1995), 30-mg/kg (Tanaka et al., 2002), or 48-mg/kg indomethacin doses (Swarnakar et al., 2005) were reported as the indomethacin-induced gastric ulcer model. Thus, the 30-mg/kg indomethacin dose was used to the following experiments. The ulcer area in the mucosa gradually increased with time up to 3 h as shown in Fig. 1C.
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). The continuous infusion via the tail vein of SOD (25,000 U/kg/h) and catalase (25,000 U/kg/h) prevented the gastric damage by approximately 60 and 50%, respectively (Vaananen et al., 1991). The subcutaneous administration of a combination of Cu,Zn-SOD (50,000 U/kg) and catalase (90,000 U/kg) and the intraperitoneal injection of DMSO (550 mg/kg) suppressed the area of erosions by 50 and 55%, respectively (Yoshikawa et al., 1993). However, our previous studies demonstrate that the location of ROS generation observed by in vivo ESR/spin probe technique is not blood vessel but gastric cavity of rats with NH4OH-induced and WIR-induced gastric lesions (Kasazaki et al., 2003a; Yasukawa et al., 2004). To examine the correlation between the in vivo sites of ROS generation evaluated by this technique and the ulcer formation, it is important that the antioxidants are administered in the gastric cavity directly and the protective effects are examined. The suppressive effect of orally administered three antioxidants, both membrane-permeable and impermeable, on the ulcer formation was investigated. When tiron, which is a membrane-permeable superoxide scavenger, was orally administered 5 min before and 1 and 2 h after indomethacin treatment, the gastric ulcer area was suppressed to 35% of that in the vehicle group (Fig. 2). The oral administration of DMTU, which is a membrane-permeable hydroxyl radical scavenger, also depressed the ulcer area to 12% of that in the vehicle group (Fig. 2). However, mannitol, which is a membrane-impermeable hydroxyl radical scavenger, showed little inhibitory effect on the ulcer formation (Fig. 2). In our previous reports, the ulcer formation induced by NH4OH and WIR was suppressed by the oral administration of mannitol by 80 and 50%, respectively (Kasazaki et al., 2003a; Yasukawa et al., 2004). Results from Fig. 2 and results from earlier studies (Kasazaki et al., 2003a; Yasukawa et al., 2004) suggest that the ulcers induced by indomethacin are quite different from the lesions caused by NH4OH or WIR. These findings suggest that only membrane-permeable antioxidants have suppressive effects on the gastric ulcer formation induced by indomethacin, whereas membrane impermeable antioxidants seem to be effective in other models of gastric ulcer induction.
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In vivo ROS generation in the stomach of rats with NH4OH-induced (Kasazaki et al., 2003a,b) and WIR-induced (Yasukawa et al., 2004) gastric lesions were directly detected using a carbamoyl-PROXYL probe and in vivo ESR spectroscopy. In this study, various nitroxyl probes having different Po/w values (Table 1) were orally administered to indomethacin-treated rat and in vivo ESR measurements were carried out. To confirm that indomethacin itself does not promote the excretion from stomach, the MR images at upper abdomen of rats intragastrically injected the water with and without indomethacin were obtained 4 and 14 min after oral administration of water using a clinical 0.2-T MRI system. The oral administration of water gave a clear MR image at the gastric domain, and the intensity of the MR image hardly changed for 15 min as reported previously (Fig. 3, B and C) (Yasukawa et al., 2004). The presence of indomethacin in injected water had no influence on MR image at the gastric domain, and the intensity of the MR image continued to keep for 15 min (Fig. 3, E and F), suggesting that indomethacin itself does not promote the excretion from stomach during the in vivo ESR measurement.
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The signal-decay rate was calculated from the slope of the signal-decay curve, as described previously (Kasazaki et al., 2003b). Because of the protective effect of membrane-permeable antioxidants on the indomethacin-induced gastric mucosal ulcer (Fig. 2), the hydroxy-TEMPO probe with the higher Po/w value [Po/w: 3.6 (Takeshita et al., 1999)] may display greater differences in signal-decay rates between indomethacin-treated and sham groups. The hydroxy-TEMPO probe was orally administered to indomethacin-treated rats, and the ESR spectra were obtained in the gastric region. The semilogarithmic plot of ESR signal intensity of hydroxy-TEMPO against time showed a linear decrease (Fig. 4A). The signal-decay rate, calculated from the slope of the semilogarithmic plot, at 1 h after indomethacin treatment was enhanced as the dose of indomethacin, and the dose response of signal decay was similar to that of ulcer area (Fig. 4B). The signal-decay rate in the 30 mg/kg indomethacin group was significantly enhanced in the 1-h indomethacin group (*, p < 0.05) compared with the sham group, and the enhancing ratio of hydroxy-TEMPO was 3.1 as shown in Fig. 4C. The signal decay was moderately decreased at 3 h compared with that observed at 1 h in spite of the development of ulcer formation (Fig. 4C). This result indicates that the enhanced signal decay of hydroxy-TEMPO occurs at the early phase of the gastric ulcer formation induced by indomethacin.
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)], was orally administered to indomethacin-treated rat, the signal-decay rate was significantly enhanced (*, p < 0.05) compared with the sham group, and the enhancing ratio was 11.7 (Fig. 6). When the oxo-TEMPO probe, which is moderately membrane-permeable [Po/w: 2.0 (Takeshita et al., 1999)], was used to indomethacin-treated rat, the signal-decay rate showed a slight enhancement, and the enhancing ratio was 1.75 (Fig. 6). On the other hand, the signal-decay rate of a carboxy-TEMPO probe, which is membrane-impermeable [Po/w: 0.019 (Eriksson et al., 1986)], showed minimal enhancement, and the enhancing ratio was 1.3. The signal-decay rate of a trimethylammonium-TEMPO probe, which has the lowest membrane permeability of the probes used in the experiments [Po/w: 0.0033 (Takeshita et al., 1999)], did not cause the enhanced signal decay (Fig. 6). These data indicate that the enhanced signal decay increases as partition coefficient of the probe increases. Carbamoyl-PROXYL with a Po/w value of 0.68 (Takeshita et al., 1999) (Table 1) localizes at the interface between aqueous and lipidic phase (Yamato et al., 2003). The ESR signal decay rates of carbamoyl-PROXYL in the group of NaHCO3 and in the group of 1-h indomethacin were 0.0094 ± 0.0037 and 0.0166 ± 0.0067 (mean ± S.D.), respectively (Fig. 6). The carbamoyl-PROXYL signal decay in the 1-h indomethacin group was 1.8 times higher than that of the sham group, which was much smaller compared with that in the 6-h WIR group (Yasukawa et al., 2004) and in the 0.5-h NH4OH group (Kasazaki et al., 2003a,b).
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To assess the relationship between signal-decay rates and protection of the ulcer, nitroxyl probes with different partition coefficients were orally administered to rats 5 min before and 1 and 2 h after indomethacin treatment, and the ulcer formation 3 h after indomethacin treatment was evaluated. The oral administration of hydroxy-TEMPO suppressed the ulcer area by 94.5% compared with the vehicle-treated group, and the suppression was significant (*, p < 0.05). When methoxycarbonyl-PROXYL was used, 75.6% compared with the vehicle inhibited the ulcer area (*, p < 0.05). Oral administration of oxo-TEMPO significantly suppressed the gastric ulcer area by 77.5% of that in the vehicle-treated group (Fig. 7). On the other hand, the oral administration of membrane-impermeable probes carboxy- and trimethylammonium-TEMPO had no effect on the ulcer formation (Fig. 7). These findings suggest that indomethacin treatment induces ROS generation in the hydrophobic environment of gastric mucosa, such as the mucus layer or the interface or the intracellular compartment of epithelial cells.
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Discussion |
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In this study, the use of an in vivo 300-MHz ESR spectrometer and nitroxyl probes with different membrane permeability provided us with, for the first time, the noninvasive and real-time information on the location of ROS generation in stomach of rats with indomethacin-induced gastric ulcer. The association of the disappearance of the nitroxyl probes and their protective effects in probes with high Po/w values in the stomach of rats treated with indomethacin suggests that ROS generation contributing to the ulcer formation occurs at the mucus layer or the interface or the intracellular compartment of epithelial cells.
The enhanced signal decay of administered nitroxyl probe increased as the Po/w value increased and the enhanced signal decay was suppressed by the administration of the hydrophobic antioxidants with the nitroxyl probe. The administered antioxidants or nitroxyl probes should localize in the gastric mucus layer and permeate the mucus layer depending on their Po/w values. It is characteristic that the surface of gastric mucosa is covered with a meshy macromolecule, mucin, maintaining hydrophobic and neutral atmosphere. Thus, the permeability of nitroxyl probes into the gastric mucosa might be different from that into the liposomal membrane.
The signal-decay rates of hydroxy-TEMPO and carbamoyl-PROXYL (280 mM, 100 µl/g) injected intravenously to a normal mouse at the upper abdomen were reported to be 0.71 ± 0.30 (/min) and 0.09 ± 0.02 (/min), respectively (values were expressed as mean ± S.D.) (Utsumi et al., 1990). Hydroxy-TEMPO can easily permeate the membrane of cells and rapidly lose its paramagnetism by bioreduction such as enzymes in electron transport system (Quintanilha and Packer, 1977) and ascorbic acid (Perkins et al., 1980), whereas carbamoyl-PROXYL minimally permeates the cell membrane and is minimally reduced. On the other hand, in this study, the signal-decay rates of all tested probes administered orally to NaHCO3-treated rats at the gastric region were similar. The gastric mucus components in pigs are reported to be 64% proteins, 15% carbohydrates, and 18% lipids, and mucin binds to 20% of the lipids (Sarosiek et al., 1988), and its secretion can not be altered by indomethacin (Nicoloff, 1968; Narumi and Kanno, 1972). The existence of a mucus layer would make the surface of epithelial cells neutral and hydrophobic in spite of indomethacin treatment. However, most of carboxy-TEMPO molecules in gastric liquid at pH 2 are the uncharged form because the previously determined pKa value of carboxy-TEMPO of 4.0, the increment of charged form following the rise of pH in gastric mucus layer, might impede the distribution to epithelial cell layers.
The finding that the enhanced signal decay was significantly suppressed by the coadministration of tiron (77% in 0.05-mmol group) or DMTU (78% in 0.025-mmol group) with the hydroxy-TEMPO probe suggests that the reactive oxygen species are generated in the stomach of indomethacin-treated rats. Another point to be noted from the fact that tiron and DMTU impact the signal-decay rates is that the nitroxyl probes and antioxidants compete for the same reactive species. However, tiron, which is known as a superoxide scavenger, reacts with not only superoxide (1 x 107 M–1 · min–1) but also hydroxyl radical (1 x 109 M–1 · min–1) (Bors et al., 1979) and acts as a chelator of metal such as Fe3+ or Cu2+ (Krishna et al., 1992). Therefore, the suppressive effect of tiron on the enhanced signal decay of hydroxy-TEMPO might be due to the scavenging of several primary species such as hydroxyl radical, superoxide, etc. or secondary species produced in lipid peroxidation chain reactions.
A hydrophobic nitroxyl probe, hydroxy-TEMPO protected the gastric ulcer formation induced by indomethacin, supported by previous report that the oral administration of hydroxy-TEMPO (0.1 g/kg at a time) 5 min before as well as 1 and 2 h after subcutaneous treatment of indomethacin (30 mg/kg) completely prevented the ulcer formation (Rachmilewitz et al., 1994). In this study, not only hydroxy-TEMPO but also other nitroxyl probes with different Po/w values were evaluated, and the evidence that carboxy- and trimethylammonium-TEMPO with relatively low Po/w values had no effect on the ulcer formation shows, for the first time, that the sites of accumulation of antioxidants are critical in protecting against gastric ulcers.
The signal-decay rate of nitroxyl radicals could be influenced by not only their membrane permeability but also the reactivity of nitroxyl radicals with ROS. Using the hydroxyl radical generating system in vitro [Fe(II)/H2O2], the reactivity of nitroxyl probes with hydroxyl radical was reported previously (Kasazaki et al., 2003a). In conclusion, in vivo ESR study with nitroxyl spin probes provides useful information as to the sites of ROS generation and the choice of protective agents to inhibit damage associated with indomethacin-induced gastric ulcers.
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Footnotes |
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ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; ROS, reactive oxygen species; SOD, superoxide dismutase; DMSO, dimethylsulfoxide; ESR, electron spin resonance; WIR, water immersion restraint; carboxy-PROXYL, 3-carboxy-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl; methoxycarbonyl-PROXYL, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl; carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl; hydroxy-TEMPO, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl; oxo-TEMPO, 4-oxo-2,2,6,6-tetramethyl-piperidine-1-oxyl; carboxy-TEMPO, 4-carboxy-2,2,6,6-tetramethyl-piperidine-1-oxyl; DMTU, dimethylthiourea; trimethylammonium-TEMPO, 4-trimethylammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl; MRI, magnetic resonance imaging; MR, magnetic resonance; ANOVA, analysis of variance; Po/w, n-octanol/water partition coefficients.
Address correspondence to: Prof. Hideo Utsumi, Department of Biofunctional Science, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582 Japan. E-mail: utsumi{at}pch.phar.kyushu-u.ac.jp
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) or 1 h after indomethacin (30 mg/kg) group (
), hydroxy-TEMPO (10 mM, 1 ml) was orally administered, and then its ESR spectra were measured in the gastric region with in vivo 300-MHz ESR spectroscopy. The logarithm of peak height at the center field h(0) was plotted against time after administration. The decay rates were calculated from the slope of A in the sham or the indomethacin group. Each value represents mean ± S.D. of five to seven rats indicated in the parentheses. In B, **, p < 0.01 as determined by Dunnett's test compared with the signal-decay rate of the sham group; and ##, p < 0.01 as determined by Dunnett's test compared with the ulcer formation of the sham group. In C, *, p < 0.05 as determined by two-way ANOVA with the Tukey-Kramer's test.
