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reperfusion in rat
Free Radical Biology & Medicine 38 (2005) 369 – 374 www.elsevier.com/locate/freeradbiomed
Original Contribution
Edaravone protects against lung injury induced by intestinal ischemia/reperfusion in rat Koji Itoa,b, Hisashi Ozasac, Saburo Horikawaa,* a
Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan b Department of Surgery, Tsuchiura Kyodo Hospital, Tsuchiura 300-0053, Japan c Minami-Ikebukuro Clinic, Tokyo 171-0022, Japan Received 8 June 2004; revised 7 October 2004; accepted 22 October 2004 Available online 28 November 2004
Abstract Intestinal ischemia/reperfusion (I/R) is a critical and triggering event in the development of distal organ dysfunction, frequently involving the lungs. Respiratory failure is a common cause of death and complications after intestinal I/R. In this study we investigated the effects of edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) on the prevention of lung injury induced by intestinal I/R in rats. Edaravone has been used for protection against I/R injury in patients with cerebral infarction. When rats were subjected to 180 min of intestinal ischemia, a high incidence of mortality was observed within 24 h. In this situation, intravenous administration of edaravone just before the start of reperfusion reduced the mortality in a dose-dependent manner. To examine the efficacy of edaravone on the lung injury induced by intestinal I/R in more detail, we performed 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone treatment decreased the neutrophil infiltration, the lipid membrane peroxidation, and the expression of proinflammatory cytokine interleukin-6 mRNA in the lungs after intestinal I/R compared to the I/R-treated rat lungs without edaravone treatment. Histopathological analysis also indicated the effectiveness of edaravone. In conclusion, edaravone ameliorated the lung injury induced by intestinal I/R, resulting in a reduction in mortality. D 2004 Elsevier Inc. All rights reserved. Keywords: Oxidative stress; Free radical scavenger; Lung injury; Intestinal ischemia; Edaravone; Free radicals
Intestinal ischemia/reperfusion (I/R) is considered to be a critical and triggering event in the development of organ dysfunction [1,2]. Respiratory failure is a common cause of death and complications after intestinal I/R [3,4]. Intestinal I/R occurs often in clinical situations, including intestinal surgical operation and intestine transplantation. A number of chemical and cellular mediators, including reactive oxygen species [5], cytokines (tumor necrosis factor-a (TNF-a) and interleukin (IL)-6) [6–8], endotoxins [7,9], platelet-activating factor [10,11], and neutrophils [12,13], have been implicated in the pathogenesis of intestinal I/R. Clinical and experimental studies suggest that oxidative stress induced by reactive oxygen species is one of the most important mediators in this process * Corresponding author. Fax: +81 3 5280 8075. E-mail address: [email protected] (S. Horikawa). 0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.10.029
[14–16]. Reactive oxygen species can cause direct oxidative damage to DNA, proteins, and lipids [17–19]. It has been known that neutrophils attached to the injured tissues produce a large amount of reactive oxygen species and cause tissue injury [20,21]. It has been reported that increased expression of pulmonary inducible nitric oxide synthase protects the lung from injury after intestinal I/R [22,23]. We have also recently shown that pharmacological preconditioning with doxorubicin, an anticancer drug, protects the acute lung injury induced by intestinal I/R [24]. In addition, it has been shown that the free radical scavenger methylene blue prevents the development of polymorphonuclear cell-related lung injury after intestinal I/R in rat [25]. Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) is a potent free radical scavenger and has the antioxidant ability to inhibit lipid peroxidation [26]. It is therefore speculated
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that the tissue damage induced by reactive oxygen species may be improved by administration of this drug. Edaravone has protective effects on both hemispheric embolization and transient cerebral ischemia in rats [27,28]. Edaravone has been used clinically to treat acute brain infarction in Japan [27,29]. Therefore, the aim of this study was to elucidate its possible therapeutic effects on lung injury induced by intestinal I/R in rats.
Materials and methods Animals All animal experiments were performed following the institution’s criteria for the care and use of laboratory animals. Male Wistar rats weighing 200–250 g were used. They were housed in a temperature-controlled room (23 F 18C) with 12-h light/dark cycles. All rats were fed water and rodent chows ad libitum. Each experimental group consisted of 4–6 animals, but in the survival experiment each group consisted of 18–20 animals. Intestinal ischemia/reperfusion Rats were fasted and allowed free access to water for 16–18 h before the experiments. Rats were anesthetized with sodium pentobarbital (50 mg/kg body wt, ip). Via a midline laparotomy, the superior mesenteric artery was carefully isolated at its origin from the abdominal aorta and occluded with a small surgical clamp. During this period the abdomen was covered with saline-moistened gauze. After occlusion for 120 or 180 min, reperfusion was initiated by removing the clamp and the midline incision sutured. Sham-operated control animals underwent the same surgical procedures without clamping. During the period of ischemia, most rats required an additional bolus of sodium pentobarbital (20 mg/kg, ip) to ensure stable anesthesia. Either edaravone, a kind gift from Mitsubishi Pharma Corp. (Tokyo, Japan), or the same volume of vehicle was intravenously injected into rats via the tail vein just before the start of reperfusion. Edaravone was dissolved in 1 N NaOH, and the pH was adjusted to 7.0 with 1 N HCl. At the indicated times (see below) the small intestines and lungs were removed and then stored at 808C until use. Rats were given food and water ad libitum throughout the study. Histopathological assessment For histopathological analyses, 120 min of intestinal ischemia followed by 120 min of reperfusion was conducted. At the indicated times rats were sacrificed by removing the blood from the heart. Isolated intestines and lungs were fixed in 10% formalin. The samples were
dehydrated and embedded in paraffin. Sections (4 Am thickness) were cut and stained with hematoxylin and eosin. Lung myeloperoxidase activity Activity of myeloperoxidase, an enzyme present in neutrophils, was used as a marker of neutrophil infiltration. Lung myeloperoxidase activity was determined as described [30]. Lung tissue (100 mg wet wt) was homogenized in 2 ml of 10 mM phosphate buffer (pH 7.4). After centrifugation at 15,000 g for 20 min, the pellet was resuspended in 1 ml of 10 mM phosphate buffer (pH 7.0) containing 0.5% hexadecyltrimethylammonium and sonicated for 20 s. After being heated at 608C for 2 h, the samples were centrifuged at 8000 g for 10 min. The reaction mixture for myeloperoxidase activity consisted of an aliquot of the supernatant, 1.6 mM tetramethylbenzidine, 0.3 mM H2O2, 80 mM sodium phosphate (pH 5.4), 8% N,N-dimethylformamide, and 40% phosphate-buffered-saline in a total volume of 500 Al. The mixture was incubated for 3 min at 378C and then immersed into an ice bath. The reaction was terminated by addition of 2 ml of 0.2 M sodium acetate buffer (pH 3.0). Myeloperoxidase product was measured by spectrophotometry at 650 nm. Myeloperoxidase activity (1 unit defined as change in absorbance of 1/min) was expressed as units per milligram protein. Lung malondialdehyde levels Levels of malondialdehyde, as an index of membrane lipid peroxidation, were determined [31]. The tissues were homogenized in 10 vol of 1.15% KCl solution containing 0.85% NaCl and then centrifuged at 1500 g for 15 min. In brief, the reaction mixture consisted of 40 Al of 8.1% sodium dodecyl sulfate, 300 Al of 20% acetic acid solution adjusted to pH 3.5 with NaOH, 300 Al of 0.8% aqueous solution of thiobarbituric acid, and 40 Al of the tissue supernatant. The mixture was heated at 958C for 60 min in a 1.5-ml tube with a tight cap. After cooling on ice, 200 Al of distilled water was added and the sample was centrifuged at 15,000 g for 20 min. The supernatant was taken out and its absorbance was measured at 532 nm. The amount of red pigment reflecting malondialdehyde was calculated using a molecular extinction coefficient of red pigment of 156,000. Interleukin-6 mRNA levels Total RNA was extracted from rat lungs using RNeasy Mini Kits (Qiagen) according to the manufacturer’s instructions. The expression of IL-6 mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA. Two micrograms of total RNA was reverse transcribed using random primers and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). For PCR, 2 Al of first-strand synthesis
K. Ito et al. / Free Radical Biology & Medicine 38 (2005) 369–374
Fig. 1. Effects of edaravone on intestinal I/R-induced mortality. Survival was assessed to 72 h after reperfusion after 180 min of intestinal ischemia. Edaravone (1 or 6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Intestinal I/R-treated rats n = 20; edaravone-treated rats with I/R, each group, n = 18; sham-operated control rats n = 18.
reaction was used as template and each fragment for IL-6 mRNA or GAPDH mRNA was amplified using a FastStart Taq DNA polymerase Kit (Roche Diagnostic GmbH, Mannheim, Germany) in 10 Al of reaction volume. The primers for IL-6 were 5V primer, 5VACAGCGATGATGCACTGTCAG-3V, and 3Vprimer, 5VATGGTCTTGGTCCTTAGCCAC-3V, and for GAPDH were 5Vprimer, 5V-TGATGGGTGTGAACCACGAG-3V, and 3Vprimer, 5V-AACGGATACATTGGGGGTAG-3V. For each primer couple, the following PCR conditions were used. For IL-6, after a bhot startQ for 10 min at 948C, 32 cycles were used for amplification, with a melting temperature of 948C and an annealing temperature of 658C and an extending temperature of 728C, each for 1 min, followed by a final extension at 728C for 7 min; for GAPDH, after a hot start for 10 min at 948C, 32 cycles were used for amplification, with a melting temperature of 948C and an annealing temperature of 608C and an extending temperature of 728C, each for 1 min, followed by a final extension at 728C for 7 min. The
Fig. 2. Pulmonary myeloperoxidase levels. Lung tissues were removed from rats after 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone (6 mg/kg) was intravenously injected into rats just before the start of reperfusion. Myeloperoxidase (MPO) activity was determined as described under Materials and methods. Each experimental group was comprised of four to six rats. C denotes the sham-operated control rats. Data represent means F SEM. *p b 0.05.
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RT-PCR product was confirmed by electrophoresis of samples in a 2% agarose gel. To rule out contaminating DNA as being responsible for results, controls for samples were performed in which RT-PCR was performed similarly, except for the absence of reverse transcriptase. These controls showed no detectable band for IL-6 and GAPDH, respectively (data not shown). The amount of amplified product was estimated by densitometry of ethidium bromide-stained 2% agarose gels using a CCD camera. Densitometric analysis of the captured image was performed on a Macintosh computer using NIH Image 1.62 analysis software. The intensity of each IL-6 band was normalized to GAPDH content. Statistical analysis Data are expressed as means F SEM for each group. The differences among experimental groups were detected by one-way analysis of variance using a multiple comparison test (Bonferroni’s multiple t test). A p value of less than 0.05 was considered to be significant.
Results Effects of edaravone on survival in rats with lung injury induced by intestinal I/R We examined the effects of edaravone on the survival rate in rats after intestinal ischemia (Fig. 1). Rats underwent 180 min of intestinal ischemia followed by 72 h of reperfusion. Of the rats with intestinal I/R, 12 of 20 died. When edaravone (6 mg/kg body wt) was intravenously administered to rats just before the start of reperfusion, 14 rats of 18 survived. In contrast, of edaravone (1 mg/kg body wt)-treated rats with intestinal I/R, 9 of 18 survived. All rats that died after the start of reperfusion died within 24 h and all rats that survived for
Fig. 3. Pulmonary malondialdehyde levels. Lung tissues were removed from rats after 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone (6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Malondialdehyde (MDA) levels were determined as described under Materials and methods. Each experimental group was comprised of four to six rats. C denotes the sham-operated control rats. Data represent means F SEM. *p b 0.05.
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Fig. 4. Expression of IL-6 mRNA. Lung tissues were removed from rats after 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone (6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Levels of IL-6 mRNA and GAPDH mRNA were determined as described under Materials and methods. Representative data of RT-PCR for IL-6 and GAPDH are shown. These experiments were performed three times independently and similar results were obtained.
24 h also survived for 72 h. Sham-operated control rats were all alive during the experimental period. Neutrophil infiltration into the lungs after intestinal I/R We examined the levels of myeloperoxidase activity, a marker of neutrophil infiltration, in rat lungs after intestinal I/R (Fig. 2). To examine the effects of edaravone on lung injury for biochemical analyses, we adopted a more moderate ischemic condition. Rats underwent 120 min of intestinal ischemia followed by 120 min of reperfusion. In the rats with intestinal I/R, high levels of myeloperoxidase activity were observed compared to the sham-operated control rats. On the other hand, edaravone treatment significantly reduced the levels of myeloperoxidase activity in rat lungs with intestinal I/R. Malondialdehyde levels in the lungs after intestinal I/R We examined the levels of malondialdehyde, a marker of membrane lipid peroxidation, in rat lungs after intestinal I/R (Fig. 3). Rats underwent 120 min of intestinal ischemia
followed by 120 min of reperfusion. In rat lungs with intestinal I/R, malondialdehyde levels slightly but significantly increased compared to the sham-operated control rats. On the other hand, edaravone treatment significantly decreased the levels of malondialdehyde in rat lungs with intestinal I/R, comparable to those of sham-operated control rat lungs. IL-6 mRNA levels in the lungs after intestinal I/R We examined the effects of edaravone on the expression of proinflammatory cytokine IL-6 mRNA in the lungs after intestinal I/R using the RT-PCR technique (Fig. 4). Rats underwent 120 min of intestinal ischemia followed by 120 min of reperfusion. In the sham-operated rat lungs, IL-6 mRNA was not detected. On the other hand, IL-6 mRNA levels increased in rat lungs with intestinal I/R. Edaravone treatment reduced the levels of IL-6 mRNA compared to those of the I/R rats without edaravone. The relative ratio of the levels of IL-6 mRNA in edaravone-treated rat lungs with intestinal I/R to those in rat lungs with I/R was 0.23 F 0.04 ( p b 0.05, n = 6). Histopathology of the lungs and intestines Next, we examined histopathologically the effects of edaravone treatment on rat lungs with intestinal I/R (Fig. 5). Rats underwent 120 min of intestinal ischemia followed by 120 min of reperfusion. Routine hematoxylin and eosin staining revealed that widespread edema, neutrophil infiltration, atelectasis, disruption of alveolar and bronchiolar epithelial cells, and hemorrhage were present compared to the sham-operated control rat lungs (Fig. 5, top). On the other hand, edaravone treatment obviously suppressed these morphological changes. A similar pattern of response was seen in three independent experiments.
Fig. 5. Histopathology of rat intestines and lungs with 120 min of intestinal ischemia followed by 120 min of reperfusion. After 120 min of reperfusion after 120 min of intestinal ischemia, the intestines and lungs were removed for histological examination using hematoxylin/eosin staining as described under Materials and methods. Edaravone (6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Representative data are shown. Original magnification, 100.
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Under these circumstances, we also assessed the histopathological changes in rat intestines (Fig. 5, bottom). In rat intestines with I/R, hematoxylin–eosin staining showed that the intestinal villi presented with an irregularity in their architecture with edema and inflammatory infiltrates in the villi and submucosa compared to the sham-operated control rat intestines. In contrast to the lungs, when edaravone was administered into rats intravenously just before the start of reperfusion, severe morphological changes observed in intestines with I/R were not improved by the drug. A similar pattern of response was seen in three independent experiments.
Discussion Intestinal I/R is an unavoidable process in intestine transplantation and in intestinal surgical operations. Acute lung injury is a common cause of organ failure accompanying intestinal I/R [3]. Thus, it is clinically important to prevent the lung injury induced by intestinal I/R. Intestinal ischemia induces disruption of the intestinal mucosal barrier, allowing translocation of bacteria and endotoxin into the circulation, which may trigger a systemic inflammatory response and lung injury. In addition, the postischemic intestine releases proinflammatory molecules such as reactive oxygen species and cytokines (e.g., TNF-a, IL-6, IL-8) into the portal and systemic circulation. Actually, the development of remote pulmonary dysfunction was observed only after reperfusion [32]. Although the molecular mechanisms regulating pulmonary injury after intestinal I/R are not fully elucidated, clinical and experimental studies suggest oxidant species, complement activation, cytokine/chemokine generation, and activated neutrophils as the agents responsible for lung injury after intestinal I/R [33–37]. I/R is initiated by production of reactive oxygen species, which initially seem to be responsible for the generation of chemotactic activity for neutrophils. In reperfusion injury, a variety of cytokines and mediators may be responsible for priming neutrophils. These proinflammatory molecules can induce direct tissue damage and are also potent activators of leukocytes and thereby promote their sequestration in organs susceptible to leukocytemediating injury, such as the lung alveolar capillary bed, leading to endothelial cell injury, increased vascular permeability, and the development of pulmonary hypertension [38,39]. Later, once adherent to endothelium, neutrophils mediate damage by secretion of additional reactive oxygen species. Therefore, the therapeutic options for limiting I/R injury include inhibition of oxygen radical formation and pharmacological prevention of neutrophil activation. It was previously reported that administration of methylene blue attenuated the lung injury induced by intestinal I/R [40]. This finding indicated that reactive oxidant species participated in this injury. In addition, the lung injury seems to be mediated, at least in part, by TNF-a [41], platelet-
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activating factor [42], nitric oxide [22], and neutrophil sequestration [43,44]. Terada et al. have also shown that endogenous lung nitric oxide production diminishes the lung injury by decreasing neutrophil sequestration [45]. In our present results, edaravone significantly decreased the expression of proinflammatory cytokine IL-6 mRNA and the neutrophil infiltration into the lungs with intestinal I/R. In this study, we demonstrated that administration of edaravone ameliorated acute lung injury induced by intestinal I/R. In fact, edaravone treatment reduced the malondialdehyde levels in rat lungs with intestinal I/R. These data showed the important role of reactive oxygen species in acute lung injury induced by intestinal I/R. In addition, histopathological analysis indicated that 120 min of intestinal ischemia followed by 120 min of reperfusion induced severe injury in both intestine and lung. It is important to note that the efficacy of edaravone administration is observed in the lungs, but not in the intestines. These findings strongly suggest that edaravone may protect the lung directly against acute lung injury and not by mediating the improvement of intestinal I/R injury. Recently, Tomatsuri et al. reported that edaravone treatment protected against I/R injury of the small intestine in rats [46]. In their report, a short period of 30 min intestinal ischemia was conducted for intestinal I/R injury. On the other hand, our experimental protocol of 120 or 180 min of intestinal ischemia might induce severe damage in the intestines, because the intestinal mucosa was extremely sensitive to I/R. Therefore, in contrast to the lungs the serious intestinal injury induced by prolonged ischemia might not be improved by edaravone treatment in our experiments. Our report is the first findings that edaravone has a protective effect against lung injury induced by intestinal I/R. Application of edaravone shows promising results in animal models of lung injury induced by intestinal I/R and may become powerful tools in the treatment of acute lung injury that occurs in intestine transplantation and intestinal surgery.
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Original Contribution
Edaravone protects against lung injury induced by intestinal ischemia/reperfusion in rat Koji Itoa,b, Hisashi Ozasac, Saburo Horikawaa,* a
Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan b Department of Surgery, Tsuchiura Kyodo Hospital, Tsuchiura 300-0053, Japan c Minami-Ikebukuro Clinic, Tokyo 171-0022, Japan Received 8 June 2004; revised 7 October 2004; accepted 22 October 2004 Available online 28 November 2004
Abstract Intestinal ischemia/reperfusion (I/R) is a critical and triggering event in the development of distal organ dysfunction, frequently involving the lungs. Respiratory failure is a common cause of death and complications after intestinal I/R. In this study we investigated the effects of edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) on the prevention of lung injury induced by intestinal I/R in rats. Edaravone has been used for protection against I/R injury in patients with cerebral infarction. When rats were subjected to 180 min of intestinal ischemia, a high incidence of mortality was observed within 24 h. In this situation, intravenous administration of edaravone just before the start of reperfusion reduced the mortality in a dose-dependent manner. To examine the efficacy of edaravone on the lung injury induced by intestinal I/R in more detail, we performed 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone treatment decreased the neutrophil infiltration, the lipid membrane peroxidation, and the expression of proinflammatory cytokine interleukin-6 mRNA in the lungs after intestinal I/R compared to the I/R-treated rat lungs without edaravone treatment. Histopathological analysis also indicated the effectiveness of edaravone. In conclusion, edaravone ameliorated the lung injury induced by intestinal I/R, resulting in a reduction in mortality. D 2004 Elsevier Inc. All rights reserved. Keywords: Oxidative stress; Free radical scavenger; Lung injury; Intestinal ischemia; Edaravone; Free radicals
Intestinal ischemia/reperfusion (I/R) is considered to be a critical and triggering event in the development of organ dysfunction [1,2]. Respiratory failure is a common cause of death and complications after intestinal I/R [3,4]. Intestinal I/R occurs often in clinical situations, including intestinal surgical operation and intestine transplantation. A number of chemical and cellular mediators, including reactive oxygen species [5], cytokines (tumor necrosis factor-a (TNF-a) and interleukin (IL)-6) [6–8], endotoxins [7,9], platelet-activating factor [10,11], and neutrophils [12,13], have been implicated in the pathogenesis of intestinal I/R. Clinical and experimental studies suggest that oxidative stress induced by reactive oxygen species is one of the most important mediators in this process * Corresponding author. Fax: +81 3 5280 8075. E-mail address: [email protected] (S. Horikawa). 0891-5849/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2004.10.029
[14–16]. Reactive oxygen species can cause direct oxidative damage to DNA, proteins, and lipids [17–19]. It has been known that neutrophils attached to the injured tissues produce a large amount of reactive oxygen species and cause tissue injury [20,21]. It has been reported that increased expression of pulmonary inducible nitric oxide synthase protects the lung from injury after intestinal I/R [22,23]. We have also recently shown that pharmacological preconditioning with doxorubicin, an anticancer drug, protects the acute lung injury induced by intestinal I/R [24]. In addition, it has been shown that the free radical scavenger methylene blue prevents the development of polymorphonuclear cell-related lung injury after intestinal I/R in rat [25]. Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) is a potent free radical scavenger and has the antioxidant ability to inhibit lipid peroxidation [26]. It is therefore speculated
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that the tissue damage induced by reactive oxygen species may be improved by administration of this drug. Edaravone has protective effects on both hemispheric embolization and transient cerebral ischemia in rats [27,28]. Edaravone has been used clinically to treat acute brain infarction in Japan [27,29]. Therefore, the aim of this study was to elucidate its possible therapeutic effects on lung injury induced by intestinal I/R in rats.
Materials and methods Animals All animal experiments were performed following the institution’s criteria for the care and use of laboratory animals. Male Wistar rats weighing 200–250 g were used. They were housed in a temperature-controlled room (23 F 18C) with 12-h light/dark cycles. All rats were fed water and rodent chows ad libitum. Each experimental group consisted of 4–6 animals, but in the survival experiment each group consisted of 18–20 animals. Intestinal ischemia/reperfusion Rats were fasted and allowed free access to water for 16–18 h before the experiments. Rats were anesthetized with sodium pentobarbital (50 mg/kg body wt, ip). Via a midline laparotomy, the superior mesenteric artery was carefully isolated at its origin from the abdominal aorta and occluded with a small surgical clamp. During this period the abdomen was covered with saline-moistened gauze. After occlusion for 120 or 180 min, reperfusion was initiated by removing the clamp and the midline incision sutured. Sham-operated control animals underwent the same surgical procedures without clamping. During the period of ischemia, most rats required an additional bolus of sodium pentobarbital (20 mg/kg, ip) to ensure stable anesthesia. Either edaravone, a kind gift from Mitsubishi Pharma Corp. (Tokyo, Japan), or the same volume of vehicle was intravenously injected into rats via the tail vein just before the start of reperfusion. Edaravone was dissolved in 1 N NaOH, and the pH was adjusted to 7.0 with 1 N HCl. At the indicated times (see below) the small intestines and lungs were removed and then stored at 808C until use. Rats were given food and water ad libitum throughout the study. Histopathological assessment For histopathological analyses, 120 min of intestinal ischemia followed by 120 min of reperfusion was conducted. At the indicated times rats were sacrificed by removing the blood from the heart. Isolated intestines and lungs were fixed in 10% formalin. The samples were
dehydrated and embedded in paraffin. Sections (4 Am thickness) were cut and stained with hematoxylin and eosin. Lung myeloperoxidase activity Activity of myeloperoxidase, an enzyme present in neutrophils, was used as a marker of neutrophil infiltration. Lung myeloperoxidase activity was determined as described [30]. Lung tissue (100 mg wet wt) was homogenized in 2 ml of 10 mM phosphate buffer (pH 7.4). After centrifugation at 15,000 g for 20 min, the pellet was resuspended in 1 ml of 10 mM phosphate buffer (pH 7.0) containing 0.5% hexadecyltrimethylammonium and sonicated for 20 s. After being heated at 608C for 2 h, the samples were centrifuged at 8000 g for 10 min. The reaction mixture for myeloperoxidase activity consisted of an aliquot of the supernatant, 1.6 mM tetramethylbenzidine, 0.3 mM H2O2, 80 mM sodium phosphate (pH 5.4), 8% N,N-dimethylformamide, and 40% phosphate-buffered-saline in a total volume of 500 Al. The mixture was incubated for 3 min at 378C and then immersed into an ice bath. The reaction was terminated by addition of 2 ml of 0.2 M sodium acetate buffer (pH 3.0). Myeloperoxidase product was measured by spectrophotometry at 650 nm. Myeloperoxidase activity (1 unit defined as change in absorbance of 1/min) was expressed as units per milligram protein. Lung malondialdehyde levels Levels of malondialdehyde, as an index of membrane lipid peroxidation, were determined [31]. The tissues were homogenized in 10 vol of 1.15% KCl solution containing 0.85% NaCl and then centrifuged at 1500 g for 15 min. In brief, the reaction mixture consisted of 40 Al of 8.1% sodium dodecyl sulfate, 300 Al of 20% acetic acid solution adjusted to pH 3.5 with NaOH, 300 Al of 0.8% aqueous solution of thiobarbituric acid, and 40 Al of the tissue supernatant. The mixture was heated at 958C for 60 min in a 1.5-ml tube with a tight cap. After cooling on ice, 200 Al of distilled water was added and the sample was centrifuged at 15,000 g for 20 min. The supernatant was taken out and its absorbance was measured at 532 nm. The amount of red pigment reflecting malondialdehyde was calculated using a molecular extinction coefficient of red pigment of 156,000. Interleukin-6 mRNA levels Total RNA was extracted from rat lungs using RNeasy Mini Kits (Qiagen) according to the manufacturer’s instructions. The expression of IL-6 mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA. Two micrograms of total RNA was reverse transcribed using random primers and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). For PCR, 2 Al of first-strand synthesis
K. Ito et al. / Free Radical Biology & Medicine 38 (2005) 369–374
Fig. 1. Effects of edaravone on intestinal I/R-induced mortality. Survival was assessed to 72 h after reperfusion after 180 min of intestinal ischemia. Edaravone (1 or 6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Intestinal I/R-treated rats n = 20; edaravone-treated rats with I/R, each group, n = 18; sham-operated control rats n = 18.
reaction was used as template and each fragment for IL-6 mRNA or GAPDH mRNA was amplified using a FastStart Taq DNA polymerase Kit (Roche Diagnostic GmbH, Mannheim, Germany) in 10 Al of reaction volume. The primers for IL-6 were 5V primer, 5VACAGCGATGATGCACTGTCAG-3V, and 3Vprimer, 5VATGGTCTTGGTCCTTAGCCAC-3V, and for GAPDH were 5Vprimer, 5V-TGATGGGTGTGAACCACGAG-3V, and 3Vprimer, 5V-AACGGATACATTGGGGGTAG-3V. For each primer couple, the following PCR conditions were used. For IL-6, after a bhot startQ for 10 min at 948C, 32 cycles were used for amplification, with a melting temperature of 948C and an annealing temperature of 658C and an extending temperature of 728C, each for 1 min, followed by a final extension at 728C for 7 min; for GAPDH, after a hot start for 10 min at 948C, 32 cycles were used for amplification, with a melting temperature of 948C and an annealing temperature of 608C and an extending temperature of 728C, each for 1 min, followed by a final extension at 728C for 7 min. The
Fig. 2. Pulmonary myeloperoxidase levels. Lung tissues were removed from rats after 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone (6 mg/kg) was intravenously injected into rats just before the start of reperfusion. Myeloperoxidase (MPO) activity was determined as described under Materials and methods. Each experimental group was comprised of four to six rats. C denotes the sham-operated control rats. Data represent means F SEM. *p b 0.05.
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RT-PCR product was confirmed by electrophoresis of samples in a 2% agarose gel. To rule out contaminating DNA as being responsible for results, controls for samples were performed in which RT-PCR was performed similarly, except for the absence of reverse transcriptase. These controls showed no detectable band for IL-6 and GAPDH, respectively (data not shown). The amount of amplified product was estimated by densitometry of ethidium bromide-stained 2% agarose gels using a CCD camera. Densitometric analysis of the captured image was performed on a Macintosh computer using NIH Image 1.62 analysis software. The intensity of each IL-6 band was normalized to GAPDH content. Statistical analysis Data are expressed as means F SEM for each group. The differences among experimental groups were detected by one-way analysis of variance using a multiple comparison test (Bonferroni’s multiple t test). A p value of less than 0.05 was considered to be significant.
Results Effects of edaravone on survival in rats with lung injury induced by intestinal I/R We examined the effects of edaravone on the survival rate in rats after intestinal ischemia (Fig. 1). Rats underwent 180 min of intestinal ischemia followed by 72 h of reperfusion. Of the rats with intestinal I/R, 12 of 20 died. When edaravone (6 mg/kg body wt) was intravenously administered to rats just before the start of reperfusion, 14 rats of 18 survived. In contrast, of edaravone (1 mg/kg body wt)-treated rats with intestinal I/R, 9 of 18 survived. All rats that died after the start of reperfusion died within 24 h and all rats that survived for
Fig. 3. Pulmonary malondialdehyde levels. Lung tissues were removed from rats after 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone (6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Malondialdehyde (MDA) levels were determined as described under Materials and methods. Each experimental group was comprised of four to six rats. C denotes the sham-operated control rats. Data represent means F SEM. *p b 0.05.
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Fig. 4. Expression of IL-6 mRNA. Lung tissues were removed from rats after 120 min of intestinal ischemia followed by 120 min of reperfusion. Edaravone (6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Levels of IL-6 mRNA and GAPDH mRNA were determined as described under Materials and methods. Representative data of RT-PCR for IL-6 and GAPDH are shown. These experiments were performed three times independently and similar results were obtained.
24 h also survived for 72 h. Sham-operated control rats were all alive during the experimental period. Neutrophil infiltration into the lungs after intestinal I/R We examined the levels of myeloperoxidase activity, a marker of neutrophil infiltration, in rat lungs after intestinal I/R (Fig. 2). To examine the effects of edaravone on lung injury for biochemical analyses, we adopted a more moderate ischemic condition. Rats underwent 120 min of intestinal ischemia followed by 120 min of reperfusion. In the rats with intestinal I/R, high levels of myeloperoxidase activity were observed compared to the sham-operated control rats. On the other hand, edaravone treatment significantly reduced the levels of myeloperoxidase activity in rat lungs with intestinal I/R. Malondialdehyde levels in the lungs after intestinal I/R We examined the levels of malondialdehyde, a marker of membrane lipid peroxidation, in rat lungs after intestinal I/R (Fig. 3). Rats underwent 120 min of intestinal ischemia
followed by 120 min of reperfusion. In rat lungs with intestinal I/R, malondialdehyde levels slightly but significantly increased compared to the sham-operated control rats. On the other hand, edaravone treatment significantly decreased the levels of malondialdehyde in rat lungs with intestinal I/R, comparable to those of sham-operated control rat lungs. IL-6 mRNA levels in the lungs after intestinal I/R We examined the effects of edaravone on the expression of proinflammatory cytokine IL-6 mRNA in the lungs after intestinal I/R using the RT-PCR technique (Fig. 4). Rats underwent 120 min of intestinal ischemia followed by 120 min of reperfusion. In the sham-operated rat lungs, IL-6 mRNA was not detected. On the other hand, IL-6 mRNA levels increased in rat lungs with intestinal I/R. Edaravone treatment reduced the levels of IL-6 mRNA compared to those of the I/R rats without edaravone. The relative ratio of the levels of IL-6 mRNA in edaravone-treated rat lungs with intestinal I/R to those in rat lungs with I/R was 0.23 F 0.04 ( p b 0.05, n = 6). Histopathology of the lungs and intestines Next, we examined histopathologically the effects of edaravone treatment on rat lungs with intestinal I/R (Fig. 5). Rats underwent 120 min of intestinal ischemia followed by 120 min of reperfusion. Routine hematoxylin and eosin staining revealed that widespread edema, neutrophil infiltration, atelectasis, disruption of alveolar and bronchiolar epithelial cells, and hemorrhage were present compared to the sham-operated control rat lungs (Fig. 5, top). On the other hand, edaravone treatment obviously suppressed these morphological changes. A similar pattern of response was seen in three independent experiments.
Fig. 5. Histopathology of rat intestines and lungs with 120 min of intestinal ischemia followed by 120 min of reperfusion. After 120 min of reperfusion after 120 min of intestinal ischemia, the intestines and lungs were removed for histological examination using hematoxylin/eosin staining as described under Materials and methods. Edaravone (6 mg/kg body wt) was intravenously injected into rats just before the start of reperfusion. Representative data are shown. Original magnification, 100.
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Under these circumstances, we also assessed the histopathological changes in rat intestines (Fig. 5, bottom). In rat intestines with I/R, hematoxylin–eosin staining showed that the intestinal villi presented with an irregularity in their architecture with edema and inflammatory infiltrates in the villi and submucosa compared to the sham-operated control rat intestines. In contrast to the lungs, when edaravone was administered into rats intravenously just before the start of reperfusion, severe morphological changes observed in intestines with I/R were not improved by the drug. A similar pattern of response was seen in three independent experiments.
Discussion Intestinal I/R is an unavoidable process in intestine transplantation and in intestinal surgical operations. Acute lung injury is a common cause of organ failure accompanying intestinal I/R [3]. Thus, it is clinically important to prevent the lung injury induced by intestinal I/R. Intestinal ischemia induces disruption of the intestinal mucosal barrier, allowing translocation of bacteria and endotoxin into the circulation, which may trigger a systemic inflammatory response and lung injury. In addition, the postischemic intestine releases proinflammatory molecules such as reactive oxygen species and cytokines (e.g., TNF-a, IL-6, IL-8) into the portal and systemic circulation. Actually, the development of remote pulmonary dysfunction was observed only after reperfusion [32]. Although the molecular mechanisms regulating pulmonary injury after intestinal I/R are not fully elucidated, clinical and experimental studies suggest oxidant species, complement activation, cytokine/chemokine generation, and activated neutrophils as the agents responsible for lung injury after intestinal I/R [33–37]. I/R is initiated by production of reactive oxygen species, which initially seem to be responsible for the generation of chemotactic activity for neutrophils. In reperfusion injury, a variety of cytokines and mediators may be responsible for priming neutrophils. These proinflammatory molecules can induce direct tissue damage and are also potent activators of leukocytes and thereby promote their sequestration in organs susceptible to leukocytemediating injury, such as the lung alveolar capillary bed, leading to endothelial cell injury, increased vascular permeability, and the development of pulmonary hypertension [38,39]. Later, once adherent to endothelium, neutrophils mediate damage by secretion of additional reactive oxygen species. Therefore, the therapeutic options for limiting I/R injury include inhibition of oxygen radical formation and pharmacological prevention of neutrophil activation. It was previously reported that administration of methylene blue attenuated the lung injury induced by intestinal I/R [40]. This finding indicated that reactive oxidant species participated in this injury. In addition, the lung injury seems to be mediated, at least in part, by TNF-a [41], platelet-
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activating factor [42], nitric oxide [22], and neutrophil sequestration [43,44]. Terada et al. have also shown that endogenous lung nitric oxide production diminishes the lung injury by decreasing neutrophil sequestration [45]. In our present results, edaravone significantly decreased the expression of proinflammatory cytokine IL-6 mRNA and the neutrophil infiltration into the lungs with intestinal I/R. In this study, we demonstrated that administration of edaravone ameliorated acute lung injury induced by intestinal I/R. In fact, edaravone treatment reduced the malondialdehyde levels in rat lungs with intestinal I/R. These data showed the important role of reactive oxygen species in acute lung injury induced by intestinal I/R. In addition, histopathological analysis indicated that 120 min of intestinal ischemia followed by 120 min of reperfusion induced severe injury in both intestine and lung. It is important to note that the efficacy of edaravone administration is observed in the lungs, but not in the intestines. These findings strongly suggest that edaravone may protect the lung directly against acute lung injury and not by mediating the improvement of intestinal I/R injury. Recently, Tomatsuri et al. reported that edaravone treatment protected against I/R injury of the small intestine in rats [46]. In their report, a short period of 30 min intestinal ischemia was conducted for intestinal I/R injury. On the other hand, our experimental protocol of 120 or 180 min of intestinal ischemia might induce severe damage in the intestines, because the intestinal mucosa was extremely sensitive to I/R. Therefore, in contrast to the lungs the serious intestinal injury induced by prolonged ischemia might not be improved by edaravone treatment in our experiments. Our report is the first findings that edaravone has a protective effect against lung injury induced by intestinal I/R. Application of edaravone shows promising results in animal models of lung injury induced by intestinal I/R and may become powerful tools in the treatment of acute lung injury that occurs in intestine transplantation and intestinal surgery.
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