Journal of Surgical Research 137, 69–74 (2007)

Edaravone Reduces Ischemia-Reperfusion Injury Mediators in Rat LiverMasanobu Taniguchi, M.D.,1 Masaru Uchinami, M.D., Koji Doi, M.D., Makoto Yoshida, M.D.,

Hisashi Sasaki, M.D., Koji Tamagawa, M.D., Tetsuya Horiuchi, M.D., and Kuniyoshi Tanaka, M.D.Second Department of Surgery, Faculty of Medical Sciences, University of Fukui, Fukui, Japan

Submitted for publication December 22, 2005

doi:10.1016/j.jss.2006.06.033

Background. In hepatic ischemia-reperfusion (I/R)injury, oxidative stress both directly injures the liverand promotes an inflammatory reaction by up-reg-ulating various inflammatory mediators. We inves-tigated whether edaravone, a new hydroxy radicalscavenger, could reduce hepatic I/R injury includingexpression of inflammatory mediators such as cyto-kines and adhesion molecules.

Materials and methods. Male Sprague-Dawley ratswere subjected to 30 min of partial hepatic pedicleclamping (70%) followed by reperfusion. Just after ini-tiation of reperfusion and again 1 h later, edaravone wasadministered intraportally. After reperfusion hepaticlipid peroxidation was measured by thiobarbituric acidassay, and hepatic injury was quantified by measuringhepatic enzymes in plasma. We serially quantified he-patic expression of mRNAs for tumor necrosis factor(TNF)-� and E-selectin, and histologically examinedE-selectin expression and neutrophil accumulation.

Results. In the edaravone group, hepatic lipid peroxi-dation and hepatic enzyme leakage were significantlyless than in the saline group. Hepatic expression ofTNF-� and E-selectin mRNAs was significantly lower inthe edaravone than the saline group, at 2 h after initia-tion of reperfusion. Histologically, E-selectin immunore-activity and neutrophil accumulation were less evidentin hepatic sections from the edaravone group.

Conclusions. Edaravone reduced hepatic I/R injury byminimizing oxidative stress, and inhibited subsequentinjurious inflammation by reducing expression of in-flammatory cytokines and adhesion molecules. © 2007

Elsevier Inc. All rights reserved.

Key Words: edaravone; hepatic ischemia-reperfusioninjury; free radical; tumor necrosis factor (TNF)-�;E-selectin; neutrophils.

1 To whom correspondence and reprint requests should be ad-dressed at Second Department of Surgery, Faculty of Medical Sci-ences, University of Fukui, 23-3 Shimoaizuki, Matsuoka-cho,

Yoshida-gun, Fukui 910-1193, Japan. E-mail: tanibee@hotmail.co.jp.

69

INTRODUCTION

Intraoperative bleeding is a major complication ofliver surgery. Temporary clamping of the hepatic pedi-cle has been carried out as described by Pringle [1] tominimize bleeding from the liver. We previously foundhepatic pedicle clamp time to be related to extent ofhepatic ischemia-reperfusion (I/R) injury [2–4], in whichvarious factors have been implicated [4–6] includingoxygen-derived free radicals [3].

Edaravone has potent free-radical scavenging activ-ity [7, 8], and often is used clinically in the acute phaseof brain infarction. In the present experiments exam-ining whether edaravone could reduce hepatic I/R in-jury, we measured lipid peroxidation in reperfusedischemic rat liver as well as leakage of hepatocellularenzymes into the circulation. We then examined theeffect of edaravone on expression of cytokines and ad-hesion molecules, which exacerbate hepatic I/R injury.

MATERIALS AND METHODS

Animals and Procedures

All animal experiments were performed in compliance with the“Guide for the Care and Use of Laboratory Animals” published by theUS National Institutes of Health. Male Sprague-Dawley rats (CharlesRiver Japan, Kanagawa, Japan) weighing 450 to 550 g were anesthe-tized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Throughout the experiments body temperature was maintainedat 37°C with the aid of a heating pad. The liver was exposed via amidline incision. We induced 70% hepatic ischemia by clamping theportal vein, hepatic artery, and bile duct supplying the median andleft lobes of the liver with a microvascular clip. As blood supply to theomental and right lobes was uninterrupted, no evident vascularcongestion of the alimentary tract was induced. Just after unclamp-ing after 30 min of hepatic ischemia and again 1 h later, edaravone(3 mg/kg) was administered intraportally to rats assigned to anedaravone group, while rats in a saline group received only an equalvolume of saline. Rats were killed at 0, 2, and 4 h after initiation ofreperfusion to collect blood and liver samples, which were frozen at

�80°C until analysis (n � 5 per time point and group).

0022-4804/07 $32.00© 2007 Elsevier Inc. All rights reserved.

70 JOURNAL OF SURGICAL RESEARCH: VOL. 137, NO. 1, JANUARY 2007

Chemicals

Edaravone (MCI-186, 3-methyl-1-phenyl-pyrazolin-5-one), a kindgift from Mitsubishi Welpharma (Osaka, Japan), was dissolved in 1N NaOH, after which the pH was adjusted to 7.0 with 5 N HCl.

Measurement of Lipid Peroxidation and HepaticEnzyme Leakage

Lipid peroxidation was quantified by measuring thiobarbituricacid reactive substances (TBARS) [9, 10]. Briefly, ice-cold 1.15% KClsolution was added to liver samples, which then were homogenizedin a Potter-Elvehjem homogenizer to produce a homogenate contain-ing 30% tissue. In a test tube, 0.10 mL of liver homogenate, 0.20 mLof 8.1% sodium dodecyl sulfate solution, 1.50 mL of 20% acetic acidsolution adjusted to pH 3.5 with 1 N NaOH, 1.50 mL of 0.8% thio-barbituric acid solution, and 0.70 mL of distilled water were added inorder. The mixture was kept at 5°C for 60 min and then heated at100°C for 60 min. After cooling, 1.0 mL of distilled water and 5.0 mLof a mixture of n-butanol and pyridine (15:1, v/v) were added, and themixture was shaken. After centrifugation at 800 g for 10 min, absor-bance was measured in the organic layer at 532 nm. Results areexpressed as molar equivalents of malondialdehyde per milligram ofprotein, using malondialdehyde from tetramethoxypropane as thestandard. Protein content in liver homogenate was determined usingBradford’s method [11].

Blood samples were centrifuged at 800 g for 10 min, and plasmawas obtained. Aspartate aminotransferase (AST) was measured us-ing an automatic analyzer.

Expression of mRNAs for Tumor Necrosis Factor-� andE-Selectin in the Liver

Expression of tumor necrosis factor (TNF)-� and E-selectin mRNAin the liver were examined by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) using a Light Cycler system(Roche Diagnostics, Mannheim, Germany) [12, 13]. Briefly, totalRNA was extracted by an acid guanidium-phenol-chloroform method.One microgram of RNA was treated with deoxyribonuclease I (Sigma,St. Louis, MO), and cDNA was synthesized with a First Strand cDNASynthesis Kit for RT-PCR (AMV; Roche Diagnostics). Primers for rat�-actin were forward, 5=-CCATCATGAAGTGTGACGTTG-3=; and re-verse, 5=-ATCTCCTTCTGCATCCTGTCA-3=. Those for rat TNF-� wereforward, 5=-GCATGATCCGAGATGTGGAA-3=; and reverse, 5=-ACGAGCGGGAATGAGAAGAG-3=. Those for rat E-selectin were forward,5=-TGCAGGGGTACAGTGTTCAA-3=; and reverse, 5=-TTCACAAGTAGGCAGCATCG-3=.

Quantitative PCR in the Light Cycler used QuantiTect SYBRGreen PCR Master Mix (Qiagen, Hilden, Germany). PCR includedinitial denaturation at 95°C for 15 min, followed by 55 cycles ofdenaturation (94°C for 15 s), annealing (57°C for 30 s), and extension(72°C for 15 s). After PCR a melting-curve analysis was carried outfor product identification. Data are shown as a percentage of theexpression detected in a standard liver sample, which was preparedfrom one rat killed 4 h after intravenous injection of lipopolysaccha-ride (7 mg/kg; List Biological Laboratories, Campbell, CA). Amountsof �-actin mRNA were used to normalize sample cDNA content.

Histological Examination

Liver samples were fixed in 10% buffered formalin and embeddedin paraffin. Sections were cut at a thickness of 4 �m, affixed to slides,and stained with hematoxylin and eosin. To investigate accumula-tion of polymorphonuclear leukocytes (PMN), these cells werecounted by light microscopy in 50 high-power fields [4, 14, 15]. Theobserved area of one field was set up for 220 micrometers by 170 �m.

For immunohistochemical staining of E-selectin, portions of the

fresh tissue samples were snap-frozen in liquid nitrogen and stored

at �80°C. Cryostat sections cut at a 5-�m thickness were air-driedand fixed in 0.25% glutaraldehyde for 30 min. After blocking endo-geneous peroxidase, sections were incubated with goat anti-ratE-selectin antibody (R&D Systems, Minneapolis, MN) for 1 h. Thensections were incubated for 30 min with biotinylated rabbit anti-goatimmunoglobulin (DakoCytomation, Kyoto, Japan) as the secondaryantibody and then for 30 min with peroxidase-conjugated streptavi-din (DakoCytomation). Finally, sections were treated with 0.015%hydrogen peroxide-3,3=-diaminobenzidine solution and observed bylight microscopy.

Statistical Analysis

Results are expressed as the mean � SEM. All parameters wereevaluated using the Mann-Whitney U test. Differences were consid-ered significant when the value of P was below 0.05.

RESULTS

Lipid Peroxidation and Hepatic Enzyme Leakage

Time courses of hepatic TBARS are shown in Fig. 1.Hepatic TBARS content increased after initiation ofreperfusion. Edaravone administration significantlydecreased TBARS at 2 and 4 h after initiation of re-perfusion, compared with saline administration (P �0.05). Time courses of AST concentrations in plasmaare shown in Fig. 2. AST also increased after initiationof reperfusion. Similarly to lipid peroxidation, edara-vone administration significantly decreased AST levelsat 2 and 4 h after initiation of reperfusion, comparedwith saline administration (P � 0.05).

TNF-� and E-Selectin mRNA Expression in the Liver

To investigate whether edaravone reduced TNF-�and E-selectin expression at the level of transcription,we quantified mRNA expression in liver by RT-PCR.TNF-� and E-selectin mRNA expression increased at2 h after initiation of reperfusion and then decreased at4 h (Fig. 3), while at 2 h after initiation of reperfusion

FIG. 1. Thiobarbituric acid-reactive substances (TBARS) in liverat various time point, expressed as the mean � SEM for five rats per

group (*P � 0.05).

71TANIGUCHI ET AL.: EDARAVONE REDUCES HEPATIC INJURY MEDIATORS

mRNAs for both were significantly reduced by edara-vone administration (P � 0.05, versus saline adminis-tration).

Histological Examination

We used immunohistochemistry to investigate whetheredaravone decreased E-selectin in sections of liver after

FIG. 2. Plasma AST concentrations at various time point, ex-pressed as the mean � SEM for five rats per group (*P � 0.05).

FIG. 3. Expression of mRNAs for TNF-� (A) and E-selectin (B) inliver at various time points, expressed as the mean � SEM for five

rats per group (*P � 0.05).

I/R. After initiation of reperfusion, E-selectin was ex-pressed along the hepatic microvasculature (brown re-action product in Fig. 4A). This E-selectin expressionwas decreased in the edaravone group (Fig. 4B) com-pared with the saline group (Fig. 4A).

PMN were identified and counted using light micros-copy. The number of PMN in liver sections 4 h afterinitiation of reperfusion is shown in Fig. 5. Infiltrationof liver by PMN was significantly inhibited by edara-vone administration compared with saline administra-tion (P � 0.05).

DISCUSSION

Hepatic I/R has been shown to generate various in-flammatory factors that can lead to hepatic injury[3–6]. Among these, oxygen-derived free radicals suchas superoxide and hydroxy radicals have been reportedto cause cell membrane damage by oxidizing mem-brane phospholipids [3, 16]. This initiates a chain re-action of lipid peroxidation [16] that ultimately com-promises viability of cells, resulting in tissue injury.

FIG. 4. Immunohistochemical staining of E-selectin in salinegroup (A) and edaravone group (B), 4 h after initiation of reperfusion.E-selectin was expressed along the hepatic microvasculature asbrown reaction product.

Furthermore, oxidative stress has been reported to

72 JOURNAL OF SURGICAL RESEARCH: VOL. 137, NO. 1, JANUARY 2007

up-regulate expression of cytokines and adhesion mol-ecules that promote inflammatory responses duringreperfusion [17–19]. Accordingly, restricting free radi-cals has been considered a main treatment strategy inminimizing hepatic I/R injury. In previous investiga-tions various free-radical scavengers such as �-tocopherol[20], ascorbic acid [21], allopurinol [22], coenzyme Q10

[23], and superoxide dismutase [24] have been reportedto prevent hepatic I/R injury. In this study we exam-ined inhibition of hepatic I/R injury by edaravone, anew hydroxy-radical scavenger.

Edaravone has potent hydroxy radical scavengingactivity [7, 8]. In various experimental models, edara-vone has been reported to protect visceral organs suchas brain [25, 26], heart [27], kidney [28], and liver[14, 29, 30] from free-radical mediated injury. Severalreports specifically considered the influence of edara-vone in hepatic I/R injury. In an isolated liver perfusionmodel, Ninomiya et al. [29] reported that edaravoneimproved portal flow and decreased hepatic enzymeleakage into the perfusate, and also decreased malon-dialdehyde concentrations. Okatani et al. [30] reportedthat edaravone decreased oxidative damage to mito-chondria in liver. These results suggested that edara-vone could be useful in treating hepatic I/R injury,although whether edaravone could affect certain in-flammatory mediators important in hepatic I/R injury,such as cytokines, chemokines, and adhesion mole-cules, remained an important unaddressed issue.Therefore, we investigated the protective effect ofedaravone in hepatic I/R injury, including whetheredaravone could reduce the up-regulation of cytokineand adhesion molecule expression that occurs duringreperfusion.

In hepatic I/R injury, free radicals have been re-ported to be generated in the early phase of reperfusion[3]. To obtain an optimal effect from edaravone, thepresent experiments included edaravone administra-

0

60

100

150

pre-reperfusion saline edaravone

Num

ber

of P

MN

(/ 5

0 H

PF)

*

FIG. 5. Numbers of polymorphonuclear neutrophils (PMN) inliver 0 (pre-reperfusion) and 4 h after initiation of reperfusion, ex-pressed as the mean � SEM for five rats per group (*P � 0.05).

tion just before and again of 1 h after initiation of

reperfusion. We administered edaravone intraportallybecause it was considered that intraportal administra-tion was more efficient than intravenous administra-tion for increasing edaravone concentration in liver. Adose of 3 mg/kg edaravone was used because maximaleffect of improvement of survival rates was observed atthis dose in lipopolysaccharide-induced lethal model[14]. Our result indicated that the liver was damagedby oxidative stress during reperfusion, because hepaticlipid peroxidation was increased after the initiation ofreperfusion in association with a marker of liver dam-age, hepatic enzyme leakage into plasma. Further-more, hepatic lipid peroxidation and liver injury weresignificantly decreased in edaravone- compared withsaline-treated rats. Thus, edaravone decreased oxida-tive stress during reperfusion by scavenging free rad-icals generated in the liver, limiting hepatic injury.

Oxidative stress in hepatic I/R injury induces pro-duction of inflammatory mediators by Kupffer cells[31]. Activated Kupffer cells have been reported toproduce oxygen-derived free radicals [32, 33] as well ascytokines [34, 35]. These inflammatory mediators stim-ulate endothelial cells [18, 19, 36], neutrophils [34, 37],and Kupffer cells themselves [38], resulting in an in-tensifying inflammatory reaction. We chose to examinemRNA expression of TNF-� to observe the inflamma-tory reaction after reperfusion because TNF-� is knownto act importantly in hepatic I/R injury by promotingapoptosis of hepatocytes [39] and expression of adhe-sion molecules [18, 36, 40]. We found mRNA expressionof TNF-� to be increased at 2 h after initiation ofreperfusion, showing an inflammatory reaction pro-gressing during reperfusion. Because edaravone signif-icantly inhibited oxidative stress and hepatic injury,we examined whether edaravone could affect inductionof TNF-� in hepatic I/R injury. Edaravone significantlyinhibited mRNA expression of TNF-� at 2 h after ini-tiation of reperfusion, suggesting that edaravone re-duced hepatic I/R injury in part by inhibiting TNF-�expression.

Oxygen-derived free radicals and cytokines gener-ated after reperfusion have been reported to stimulateendothelial cells to up-regulate expression of adhesionmolecules [17, 19, 41]. Adhesion molecules exacerbatehepatic I/R injury, by promoting accumulation of neu-trophils in target tissues [42]. E-selectin, which is ex-pressed on endothelial cells, binds to sialyl groups ofLewisx ligand expressed on the neutrophil surface, aninteraction that contributes to rolling of neutrophilsalong endothelial cell surfaces [42, 43]; this initiates amultistep process leading to neutrophil accumulationand subsequent tissue damage. Although antioxidantssuch as �-tocotrienol [44] and lecithinized superoxidedismutase [45] have been reported to inhibit expres-sion of adhesion molecules and adhesion of inflamma-

tory cells to endothelial cells, few reports have ad-

73TANIGUCHI ET AL.: EDARAVONE REDUCES HEPATIC INJURY MEDIATORS

dressed whether edaravone could reduce expression ofadhesion molecules in hepatic I/R injury. This is thefirst report to examine the effect of edaravone onE-selectin expression in that pathologic state. Wefound that mRNA expression for E-selectin at 2 h afterinitiation of reperfusion was significantly reduced byedaravone administration compared with saline ad-ministration. Immunohistochemical findings showedthat edaravone inhibited E-selectin protein expression,and this drug ultimately reduced neutrophil counts ininjured liver. These observations suggested that neu-trophil accumulation and consequent tissue damagecould be reduced by edaravone administration in he-patic I/R injury.

At present, edaravone is often used clinically as afree radical scavenger in brain infarction. Because nofree radical scavengers other than edaravone are avail-able in clinical use, edaravone has a possibility to beused in various free-radical-related diseases includinghepatic I/R injury, compared with other free radicalscavengers.

In conclusion, edaravone reduced hepatic I/R injuryby minimizing oxidative stress and also by decreasingproduction of inflammatory cytokines and adhesion mol-ecules, inhibiting subsequent inflammation. Edaravonemay prove to be an important therapeutic agent in he-patic I/R injury.

ACKNOWLEDGMENTS

The authors wish to thank Mitsubishi Welpharma (Osaka, Japan)for the kind gift of edaravone.

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