Attenuation of Ischemic Liver Injury by El Analogue, , and Prostaglandin 12 Analogue,OP-41483

Eishi Totsuka, MD, Satoru Todo, MD, FACS, Yue Zhu, MD, Naoki Ishizaki, MD, Yoshiyuki Kawashima, MD, Maeng Bong Jin, MD, Atsushi Urakami, MD, Tsuyoshi Shimamura, MD, and Thomas E Starzl, MD, PhD, FACS

Background: Prostaglandin has been reported to have MP-M, MP-L, and OP animals experienced only tran­ protective effects against liver injury. Use of this agent in sient hypotension. clinical settings, however, is limited because of drug­ Conclusions: These results indicate that misoprostol related side effects. This study investigated whether mi­ and 0 P -41483 prevent ischemic liver damage, although soprostol, prostaglandin El analogue, and OP-41483, careful dose adjustment of misoprostol is required to prostaglandin 12 analogue, which have fewer adverse ef­ obtain the best protection with minimal side effects. (J fects with a longer half-life, attenuate ischemic liver Am ColI Surg 1998;187:276-286. © 1998 by the damage. American College of Surgeons) Study Design: Thirty beagle dogs underwent 2 hours of Since the protective effects of 16, 16-dimethyl pros­ hepatic vascular exclusion using venovenous bypass. Mi­ soprostol was administered intravenously for 30 min­ taglandin E2 on carbon tetrachloride-induced liver utes before ischemia and for 3 hours after reperfusion. necrosis were reported by Stachura and colleagues in o P -41483 was administered intraportally for 30 min­ 1981,1 many investigators have demonstrated the utes before ischemia (2#Lg/kg/min) and for 3 hours after potency of , such as reperfusion (0.5 #Lg/kg/min). Animals were divided into (PGE1) and prostaglandin 12 (PGI2), against liver five groups: untreated control group (n = 10); high-dose injury induced by ischemia,2-7 preservation,8.9 and misoprostol (total 100 #Lg/kg) group (MP-H, n = 5); various toxinslO,11 in experimental studies. On the middle-dose misoprostol (50 #Lg/kg) group (MP-M, other hand, since Abecassis and coworkers showed n = 5); low-dose misoprostol (25 #Lg/kg) group (MP-L, the beneficial effects of PGE1 on acute or subacute n = 5); and OP-41483 group (OP, n = 5). Animal fulminant viral hepatitis in clinical studies for the survival, hepatic tissue blood flow (HTBF), liver func­ first time in 1987,12 PGE1 has been used clinically in tion, and histology were analyzed. cases of liver transplantation, and its potency has been reported by some investigators. 13,14 Results: Two-week animal survival rates were 30% in control, 60% in MP-H, 100% in MP-M, 80% in MP-L, However, the use of prostaglandin in clinical set­ and 100% in OP. The treatments with prostaglandin tings has been limited because of drug-related side analogues improved HTBF, and attenuated liver enzyme effects, such as diarrhea, emesis, and hypoten­ release, adenine nucleotrides degradation, and histo­ sion, 15,16 and its short duration of action. 17,IS Specif­ logic abnormalities. In contrast to the MP-H animals ically, an unstable blood pressure caused by prosta­ that exhibited unstable cardiovascular systems, the glandin could lead to a critical situation at reperfusion after ischemia in liver transplantation. Aided by Research Grants from the Veterans Administration and Project Therefore, it is difficult to administer enough pros­ Grant No. DK-29961 from the Narional Institutes of Health, Bethesda, taglandin to exert its effects. Indeed, some clinical MD. reports showed that PGEI did not have any benefi­ Received Mav 4. 1998; Accepted May 25, 1998. cial effects on the incidence of primary graft nonfunc­ From the Thomas E Starzl Transplantation Institute, University of Pittsburgh, 2 Pittsburgh, PA. tion,19 rejection,20 or renal dysfunction20. ! in recipi­ Correspondence address: Eishi Totsuka, MD, University of Pittsburgh, School ents after liver transplantation. of Medicine, TE Starzl Transplantation Institute, Biomedical Science Tower, E1555, 200 Lothrop St, Pittsburgh, PA 15213-2582. Misoprostol, which is a structurally changed an-

© 1998 by the American College of Surgeons ISSN 1072-7515/98/$19.00 Published by Elsevier Science Inc. 276 PIT S1072-7515(98)00179-3 Vol. 187, No.3, September 1998 Totsuka et a1 Attenuation of Ischemic Liver Injury 277 alogue from natural occurring PGEl' enables us to pass circuit connected the left femoral vein and overcome these problems. In this compound, typical splenic vein to the left jugular vein. The animals were prostaglandin side-effects, such as rhinorrhea, eme­ given heparin (50 U/kg) 5 minutes before the initia­ sis, and diarrhea, are diminished, and undesirable tion of ischemia. At the end ofthe two-hour ischemic actions on the cardiovascular system are also mini­ period, the clamps were removed, bypass was mized.22 Besides, the active duration of misoprostol, stopped, and a splenectomy was performed. Mter whose half life is more than 30 minutes,23 is longer ensuring adequate hemostasis, the abdomen was than that of natural PGE1• Also, the effects of OP- rinsed with warm saline and closed. Cefamandole 41483, a PGI2 analogue, on anti-platelet aggregation (Mandol 1 g; Lilly, Firenze, Italy) was given intraop­ and vasodilation are half and a tenth as potent as eratively and continued daily for 3 postoperative natural occurring PGI2, respectively.24 The aim of days. Animals were allowed to eat and drink the next this study is to determine whether these two kinds of morning, and followed for 2 weeks postoperatively. prostaglandin analogues, misoprostol and OP- Experimental groups 41483, attenuate hepatic warm ischemia and reper­ Misoprostol, a PGE analogue, and OP-41483, a fusion injury, and to investigate usefulness of these 1 PGI analogue, were supplied by Searle (Skokie, IL) drugs in clinical liver transplantation. 2 and Ono Pharmaceutical Co. Ltd. (Osaka, Japan), respectively. Misoprostol was administered by con­ METHODS tinuous intravenous infusion for 30 minutes before Animals ischemia onset (pre) and from 15 minutes before Thirty adult female beagle dogs, weighing 8 to reperfusion for 3 hours (post). Animals were divided 12 kg, were used for this study. The experiment was into three groups based on the dosage of misoprostol; performed with the approval of the Animal Care and the high-dose group (MP-H group, n = 5) receiving Use Committee of the University of Pittsburgh, and 30 JLg/kg (pre) and 70 JLg/kg (post) of misoprostol; was managed in accordance with guidelines issued by the middle-dose group (MP-M group, n = 5) receiv­ the National Institutes of Health and the Public ing 15 JLg/kg (pre) and 35 JLg/kg (post); and the Health Service Policy on the humane use and care of low-dose group (MP-L group, n = 5) receiving 7.5 laboratory animals. The animals were fasted over­ JLg/kg (pre) and 17.5 JLg/kg (post). In an OP-41483 night but had free access to water before the experi­ administered group (OP group; n = 5), animals re­ ments. After intravenous administration of thiopental­ ceived 2 JLg/kg/min OP-41483 for 30 minutes be­ sodium (25 mg/kg; Abbott Laboratories, North fore ischemia and from 30 minutes before reperfu­ Chicago, IL), dogs were intubated and maintained sion 0.5 JLg/kg/min for 3 hours intraportally. with a mixture of oxygen (2 Llmin), nitrous oxide (2 Animals not receiving any study drugs were used as Llmin) and isoflurane (1.0%) by positive pressure controls (control group, n = 10). mechanical ventilation. The right carotid artery and Determinations the right jugular vein were cannulated for monitor­ Two-week animal survival, hepatic tissue blood ing of arterial blood pressure and for serial blood flow (HTBF), liver function tests, tissue biochemis­ sampling. An electrolyte solution (Plasmalyte, Bax­ try, and histologic assessments were used to evaluate ter, Deerfield, IL) was continuously infused into the the efficacy of the treatment in this study. animals at a rate of 20 mLlkg/h. Electrocardiogram, Serial measurements of HTBF were performed arterial blood gas, electrolytes, and esophageal tem­ intraoperatively using a laser Doppler flowmeter perature were monitored throughout the operation. (Advance Laser Flowmeter, ALF21 , Advance Com­ Operative procedure pany, Ltd., Tokyo, Japan). The probe was placed on After midline laparotomy, the liver was com­ three lobes of the liver until a stable flow value was pletely skeletonized by dissecting all of the suspen­ obtained. To avoid a misreading in HTBF measure­ sory ligaments and retrohepatic vena cava. Total he­ ments related to respiratory motion, the ventilation patic vascular exclusion was initiated by cross­ was always stopped briefly. The mean of three values clamping the portal vein, hepatic artery, and infra­ was used as representative value and was expressed as and supra-hepatic inferior vena cava. Pump-driven a percentage of the value of the preischemic level. venovenous bypass (Biomedicus 520D, Minetonka, Peripheral venous blood samples were collected MN) was used to decompress the splanchnic venous serially for determination ofliver enzymes, including bed and infrahepatic inferior vena cava. 25,26 The by- aspartate aminotransferase (AST) , alanine amino- 278 Totsuka et al Attenuation of Ischemic Liver Injury J Am Call Surg transferase (ALT) and lactate dehydrogenase (LDH), RESULTS by a Technicon RA500 autoanalyzer (Bayer, Tarry­ Clinical observation town, NY). Serum total bile acid (TBA) was mea­ Preischemic administration of high dose miso­ sured by the enzymatic fluorimetric method using prostol triggered severe hypotension and bradycar­ the Sterongnost-alpha Flu Kit (Nyegaad, Oslo, Nor­ dia, in contrast to the treatments with middle and way).27 low dose of misoprostal and OP-41483, which Wedge biopsies from the liver were repeated be­ caused only transient and mild hypotension (Fig. 1). fore ischemia, 1 hour and 2 hours after the onset of These hemodynamics in the MP-H group continued ischemia, and 15 and 60 minutes after reperfusion. even after introduction of liver ischemia; systolic ar­ The liver tissue was immediately immersed in liquid terial pressure and heart rate remained below 75 and nitrogen. Adenine nucleotides (adenosine triphos­ 70%, respectively, of the initial level during ischemia. phate, [ATP]; adenosine diphosphate, [ADP]; and After reperfusion, blood pressure markedly decreased adenosine monophosphate, [AMP]), and purine ca­ in the control and MP-H groups, while hypotension tabolites (adenosine; inosine; hypoxanthine, [HX]; in the MP-M, MP-L, and OP groups was slight. and xanthine, [XA]) were assayed with a high­ Heart rate was stable after reperfusion in the control, performance liquid chromatography system (Waters MP-M, MP-L, and OP groups, but in the MP-H Chromatography Division, Millipore Corp., Mil­ group it remained bradycardic even after reperfusion. ford, MA; Model 51 0 pumps, Model 484 absorbance No vasopressor was administered, and atropine was module, and Model 717 WISP system), equipped used for bradycardia of 50 beat/min or less. with a tunable absorbance detector (Water Model Neither complications related to bypass, such as 484).28 The hepatic energy charge (EC), originally systemic thrombosis and intestinal congestion, nor described by Atkinson bleeding tendency caused by misoprostol and OP- 41483 could be found during and after ischemia. Immediately after reperfusion, all livers in the ATP + ~ADP control group and one out of five livers in the MP-L EC = ATP + ADP + AMP group developed swelling, congestion, and an exten­ sive hypo perfused area, which lasted for 30 to 60 was calculated.29 minutes. The livers in the MP-H group, at first, A small portion of each tissue sample taken for showed fair revascularization without swelling after tissue biochemistry was fixed in buffered formalin, reperfusion, but notable congestion appeared after paraffin-embedded and stained with hematoxylin­ 30 minutes. The livers in all MP-M, four MP-L, and eosin. The specimens were examined by a single pa­ all OP animals, by contrast, showed smooth revascu­ thologist without knowledge of the groups and the larization and a remarkably limited hypoperfused timing of tissue sampling. Histologic assessment was area. After operation, a dry or moist rale was auscu­ semiquantitatively performed based on the degree of lated on the lung field of the MP-H dogs. Postoper­ sinusoidal congestion, ischemic hepatocyte injury, ative arterial blood gas analysis showed pulmonary and hepatocyte necrosis, which was expressed as failure only in the high dose misoprostol adminis­ none 0, mild 1, moderate 2, and severe 3. = = = = tered animals (Table 1). At 6 hours after reperfusion, The number of neutrophils that had infiltrated into pressure of arterial carbon dioxide (PaC02) in the the tissue was counted after Leder's staining,30 and MP-H group was significantly higher than that in the expressed as that number per 1,000 hepatocyte other four groups. Likewise, pulmonary oxygenation nuclei. was suppressed only in the MP-H group. Statistical analysis Animal survival The values were expressed as means ± SEM. An­ Survival after surgery is summarized in Fig. 2. imal survival was determined using the Fisher's exact Two hours of warm liver ischemia was poorly toler­ probability test. The comparison of statistical signif­ ated by the untreated control group, in which six icance was performed according to analysis of vari­ dogs died within 24 hours, and one on postoperative ance (ANOYA) analysis for unpaired data; differ­ day 10 from liver failure. Two dogs in the MP-H ences at p < 0.05 were considered to be statistically group and one dog in the MP-L group died within significant. 24 hours of reperfusion. In the MP-M and OP Vol. 187, No.3, September 1998 Totsuka et al Attenuation of Ischemic Liver Injury 279

-...- Control ---0-- MP-fJ 110 ---[}-- 11,11'-\1 'a~ ---r:r-- \11'-1 ~ 100 v ---<)-- 01' ~ C/o <)11 Cf. u.... 0- RO ';::: "" 70 8....

,~"" 60 0 tl 50 >. Vl 40 110

100 .-- c"" 90 ~ 8 ...."" so t u :c"" 70

hO

50 c ,S c c c c c c 'E E E E E E 'E E 0 0 0 0 0 0 0 0 '-D r"\ r"\ '-D 0-. r1 r"\

... ~ .. • .. Pre-ischemia Ischemia Post-reperfusion

Figure 1. Systolic arterial pressure (upper) and heart rate (lower) before, during, and after ischemia (mean ± SEM). Values are expressed as a percentage of the level at 30 min bd()re ischemia. *p < 0.05 versus control value. **p < 0.05 versus control, MP-M, MP-L, and OP values, #p < 0.05 verSllS MP-M, MP-L, and OP values. ##p < 0.05 versus control. MP-M, and OP values. MP-H, high-dose misoprostal; MP-M, medium-dose misoprostal; MP-L, low-dose misoprostal; Ol~ prostaglandin I2 analogue OP-41483. groups, each five animals survived more than 14 days 3). There were significant differences between the (p < 0.05 versus control group). control and study groups in all postischemic Hepatic tissue blood flow measurements. The levels of HTBF after reperfusion were less Liver function test than 25% of initial value in the control group, Serum AST, ALT and LDH increased sharply around 50% in the misoprostol-treated groups, and after reperfusion in all the groups, while the eleva­ around 60% in the OP-41483-treated group (Fig. tions of liver enzymes in the low dose misoprostol group were significantly suppressed during the early Table 1. Arterial Blood Gas Analysis 6 Hours After phase of reperfusion (Fig. 4, Table 2). These values in Reperfusion (Mean ± SEM) ------~------the control and MP-H groups progressively in­ Group Pa02 (mmHg) PaC02 (mmHg) --~------~----~ creased and reached peak levels at 12 hours after Control 92.7 ± 3.56 32.1 ± 1.96 reperfusion. By contrast, treatment with middle and MP-H 69.2 ± 8.77* 43.6 ± 4.03* low dose misoprostol, and OP-41483 significantly MP-M 92.2 ± 3.59 33.6 ± 1.51 MP-L 87.1 ± 4.94 33.6 ± 3.19 inhibited these elevations, although one MP-L ani­ OP 94.3 ± 2.20 31.4 ± 1.1 0 mal that died within 24 hours after surgery showed *p < 0.05 versus control, MP-M. MP-L, and OP values, high levels of liver enzymes. Pa02 , pressure of arterial oxygen; PaC02 • presure of arterial carbon dioxide, Serum TBA values increased in all animals after MP-H. high-dose misoprostal; MP-M. medium-dose misoprostal; MP-L, low­ dose misoprostal; Or, prostaglandin 12 analogue OP-41483. reperfusion (Table 2). In the control group, the value 280 Totsuka et a1 Attenuation ofIschemic Liver Injury ] Am Coll Surg

100

90 MP-L 80

70 -~ MP-H '-' .....

20

10

0 0 2 3 4 5 6 7 8 9 10 II 12 13 14 Days after operation

Figure 2. Survival of dogs after 2 hours of total hepatic vascular exclusion. *p < 0.05 versus control group. MP-M, medium-dose misoprostal; MP-L, low-dose misoprostal; MP-H, high-dose misoprostal.

progressively increased, while those in the MP-H, different from those in control livers. Concerning MP-M, MP-L, and op groups began to decrease 3 changes in hepatic energy charge (EC), no significant hours after reperfusion. There were significant differ­ differences were found among all the groups. ences between the control group and the treated Histopathology groups at 6, 12, and 24 hours after reperfusion. Histopathologic examination showed no differ­ Liver tissue biochemistry ences between control and misoprostol-treated Changes in adenine nucleotides and purine ca­ groups at the end of ischemia. After reperfusion, the tabolites in liver tissue at the end of ischemia and control livers developed sinusoidal congestion and after reperfusion are summarized in Table 3. Two derangement, ballooning, bridging hepatocyte ne­ hours of hepatic vascular exclusion in the control crosis, and endothelial cell detachment. According to animals induced a 54.5% decrease in total adenine the histologic score, it was demonstrated that treat­ nucleotide (TAN) and an 840% increase in purine ment with middle and low dose misoprostol, and catabolites, compared with preischemic values. In OP-41483 significantly alleviated these structural particular, the ischemia caused >90% reduction in abnormalities (Table 4). The high dose-treated liver ATp, and> 1,000% production ofHX and XA. Mi­ showed almost the same sinusoidal congestion and soprostol and OP-41483 treatments retarded the parenchymal injury as those of the control group, degradation of adenine nucleotides to purine catabo­ even though parenchymal necrosis was rarely seen in lites during ischemia, especially in the MP-H and OP the MP-H livers. The numbers of neutrophils infil­ groups. While resynthesis of ATP was suppressed af­ trated into postischemic livers 60 minutes after ter reperfusion in control livers, prompt increases in reperfusion in the MP-H and OP groups were signif­ ATP levels were seen with treated livers, especially in icantly less than that in control group. (Table 4). the MP-L and OP groups. Likewise, the levels of tissue purine catabolites in the treated livers were DISCUSSION significantly lower than those in the control livers In this study, we demonstrated that two kinds of after reperfusion. At 60 minutes after reperfusion, prostaglandin analogues, misoprostol and OP- however, ATP levels in the MP-H livers were not 41483, lessened microcirculatory disturbance, atten- Vol. 187, No.3, September 1998 Totsuka et al Attenuation of Ischemic Liver Injury 281

• Control --0-- MP-H ---0-- MP-M ~ MP-L --0-- OP

,S ,S s: s: s: s: E E 's 's 'S 'S o V) V) V) 0 0 0' 0 M 'D ... :4

Ischemia Post-reperfusion

Figure 3. Tissue blood flow in the liver before, during, and after ischemia (means:!: SEM). Values are expressed as a percentage of the level at 30 min before ischemia. *p < 0.005 versus MP-H, MP-M, MP-L, and OP values. MP-H, high-dose misoprostal; MP-M, medium-dose misoprostal; MP-L, low-dose misoprostal; Or, prostaglandin 12 analogue OP-41483. uated liver injury, and improved animal survival after production of purine catabolites.34 Harvey and asso­ reperfusion following 2 hours of hepatic warm isch­ ciates demonstrated that hepatocyte injury began emia. In addition, these prostaglandins inhibited ad­ during ischemia.35 Their study showed that the level enine nucleotide degradation in ischemic tissues, en­ of tissue malondialdehyde as well as hypoxanthine hanced ATP resynthesis, and improved histopathologic and xanthine significantly increased 2 hours after he­ findings. patic warm preservation. This result indicates that Prostaglandins, including PGEl and PGlz, are some lipid peroxidation by the xanthine oxidase sys­ said to have a wide range of effects against liver injury tem,36 which causes direct cellular injury, occurred by ischemia and reperfusion. This potency is based not only after but also before reperfusion. Likewise, on the ability of prostaglandin to activate adenylate depletion of the tissue stores of ATP results in an cyclase. 31 ,32 Intracellular cyclic adenosine 3', 5' accumulation of intracellular calcium. This accumu­ monophosphate (cyclic AMP), which is increased by lating calcium activates calcium-dependent ATP-ase, adenylate cyclase, acts as a second messenger of pros­ leading to a further depletion of tissue stores of taglandin on hepatocytes, vascular smooth muscles, ATp'37 Intracellular accumulated calcium activates platelets, and neutrophils. proteolytic enzyme systems, which causes additional In hepatocytes, cyclic AMP is well known to pro­ hepatocellular injury at the same time as that caused duce an efflux of calcium.33 This effect leads to an by lipid peroxidation from the xanthine oxidase sys­ attenuation of hepatocyte injury during and after tem.38 Cyclic AMp, which produces an efflux of cal­ liver ischemia. Liver ischemia causes the progressive cium accumulated intracellularly by ischemia and degradation of adenine nucleotides, resulting in the ATP depletion, prevents the intracellular buildup of 282 Totsuka et a1 Attenuation ofIschemic Liver Injury J Am Coli Surg

Figure 4. Changes in serum aspartate aminotransferase (AST) levels after ischemia (means ± SEM). *p < 0.05 versus control value, #p < 0.05 versus control and MP-H values. MP-H, high-dose misoprostal; MP-M, medium-dose misoprostal; MP-L, low-dose misoprostal; Or, prostaglandin 12 analogue OP-41483.

Table 2. Alanine Aminotransferase, Lactate Dehydrogenase, and Total Bile Acid Levels after Ischemia (Mean ± SEM) Preischemia 60 minutes 3 hours 6 hours 12 hours 24 hours ~------Alanine aminotransferase (U/L) Control 32.2±4.2 4,961±798 6,576± 1,007 9,332± 1,128 13,563± 1,379 12,681 ± 1,451 MP-H 26.2±4.0 3,527±686 4,057±736 6,248±696* 9,006± 1,629* 4,414±448* MP-M 26.6±5.5 2,302±1,183* 2,745±1,300* 1,644±465t 1,668±655t 1,315±361 1 MP-L 23.0±2.8 1,444±508* 2,087±749* 2,580±842t 2,544± 1,005t 2,185±1,028t OP 18.2±3.0 2,274±540* 2,873±5lO* 2,082±3331 1,443±272t 723±241 t Lactate dehydrogenase (U/L) Control 80.6±15.7 3,385±474 3,128±179 2,906±559 3,046±634 1,441 ±216 MP-H 40.6±6.3 2,966±480 2,784±291 2,662±505 2,919±532 1,099±491 MP-M 35.2±8.8 1,199±541t 934±358"1 521±153t 358±77.8t 195±53.3t MP-L 42.0±4.9 1,364±527t 1,165±419t 1,290±401* 813±228t 415± 173 t OP 36.4±4.6 1,207±4581 1,109±404t 354±9l.9t 244±36.5t 190±52.1t Total bile acid (f.LmollL) Control 0.3±0.3 48.5±12.4 100.2± 11.6 133.8± 12.5 124.3± 14.0 175.4±42.2 MP-H 0 66.0±17.6 90.0± 14.2 8l.9±22.5* 50.8±35.5* 11.9± 14.5* MP-M 0 13.7±6.3 18.0±8.5t 10.2±lO.Ot 0.74±0.83t 0* MP-L 0 16.3± 11.2 43.9±23.4* 43.9±26.9* 14.6±8.5* 5.4±3.7* OP 0 32.9±30.6 66.2±61.5 45.5±27.0* 23.6±9.1* 16.0± 18.2* *p < 0.05 versus control value. t p < 0.05 versus control and MP-H values. MP-H, high-dose misoprostal; MP-M, medium-dose misoprostal; MP-L, low-dose misoprostal; OP, prostaglandin I2 analogue 01'-41483. Vol. 187, No.3, September 1998 Totsuka et al Attenuation ofIschemic Liver Injury 283

Table 3. Tissue Concentrations of Adenine Nucleotides and Purine Catabolites (Nmol/g tissue) During and after Ischemia (Mean ± SEM) Group EC TAN ATP ADP AMP HX XA Normal 0.82±0.02 3,774±314.1 2,666±311.6 il94.0± 121.9 213.6±29.0 102.7±2l.6 4.20±2.94 The end of ischemia Control 0.23±0.03 1,719±147.8 236.9±45.2 335.0±32.5 1,147± 107.2 460.0±29.7 659.0±54.3 MP-H 0.26±0.05 2,650±285.0* 406.3±120.1 594.3±76.1* 1,649±249.1 * 223.8±58.7* 359.2±88.8* MP-M 0.28±0.03 2,305±127.1 379.3±49.0 498.6±43.3 1426.5± 132.7 254.2±37.2* 491.3±48.9 MP-L 0.30±0.03 2,306±347.2 413.5± 115.0 619.8±12l.5* 1,272± 118.6 183.4±41.1* 362.4± 85.2* OP 0.29±0.O3 2,400±273.5* 405.7±44.2 566.4±56.9* 1,428±230.9 218.0±38.1 * 394.6± 58.8* 15 minutes after reperfusion Control 0.72±0.04 1,417±234.0 846.8±170.2 410.0±73.3 160.0±17.8 13l.6±25.5 24.0±9.3 MP-H 0.66±0.02 2553±337.7* 1,347± 110.2 629.7± 132.0 576.2± 136.0* 38.5±5.9* 6.18±7.14* MP-M 0.65±0.05 2,446±203.5* 1,314±255.4 585.5±52.9 546.4±72.9* 32.9±4.0* l.49±0.81 * MP-L 0.75±0.04 2,884±338.6* 1,909±335.2* 597.2±77.1 378.4±47.5* 36.6±3.7* 0.78±0.55* OP 0.69±0.05 2,738± 193.7* 1,597±205.9* 607.6± 123.7 533.9± 103.1 * 34.8±4.0* l.30±0.92* 60 minutes after reperfusion Control 0.83±0.02 1,669±160.1 1,227±138.3 329.4±37.4 112.2±14.7 105.5±20.5 44.3±9.9 MP-H 0.69±0.04 2,146±307.5 1,225 ± 282.3 574.9±81.7* 345.9±58.9 33.7±7.6* 23.5±21.8 MP-M 0.77±0.04 2,748±276.2* 1,895±304.7 524.4± 16.3* 328.9±41.3 29.5±2.0* O' MP-L 0.78±O.03 2,535 ±294.5* 1,762±250.2 467.9±67.6 305.4±44.9 25.6± 1.1 * 0* OP 0.77±0.10 2,968± 163.7t 1,999 ± 369.2 t 547.9±1l5.7* 42l.0±240.1* 37.2±5.1* 1.03± 1.15* 'p < 0.05 versus control value; tp < 0.05 verSllS control and MI'-H values. ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATI~ adenosine triphosphate; Ee, energy charge; HX, hypoxanthine; MP-H, high-dose miso­ prostal; MP-L, low-dose misoprostal; MP-M, medium-dose misoprostal; Or, proS{aglandin 12 analogue 01'-41483; TAN, total adenine nucleotide; XA, xanthine. calcium and consumption of ATp, resulting in the prostaglandin analogues attenuated liver injury dur­ stabilization of the hepatocellular membrane, and in ing ischemia and immediately after reperfusion. an intracellular adenine nucleotide preservation that After reperfusion, hepatic tissue blood flow was prevents activation of the xanthine oxidase system. In markedly improved in the misoprostol and op- the present study, degradation of adenine nucleotides 41483 treated groups. This observation agrees with and elevation of purine cat abo lites after two hours of previous reports; an improvement was made in the ischemia were retarded by misoprostol and op- hepatic microcirculatory system by vasodilatory ef­ 41483 treatments. These results suggest that these fect and antiplatelet action of prostaglandin after

Table 4. Microscopic Grading of Histologic Changes and Neutrophil Counts (Neutrophils/l,OOO hepatocytes) (Mean ± SEM) Group Sinusoidal congestion Hepatocytes injury Hepatocytes necrosis Neutrophils count ------Normal 0 0 0 16.7 ± 2.4 15 minutes after reperfusion Control 1.47 ± 0.22 1.07 ± 0.12 0.07 ± 0.07 39.4 ± 13.1 MP-H 1.80 ± 0.22 0.80 ± 0.22 0 22.4 ± 8.2 MP-M 1.20 ± 0.22 0.60 ± 0.27 0 35.3 ± 6.7 MP-L 0.80 ± O.22T 1.60 ± 0.27 0.60 ± 0.27 29.3 ± 8.4 OP 1.00 ± a 1.00 ± 0 0 24.0 ± 5.6 60 minutes after reperfusion Control 1.93±0.19 1.87 ± 0.17 1.13±0.17 57.2 ± 19.1 MP-H 2.40 ± 0.27 1.60 ± 0.27 0.20 ± 0.22* 3l.0 ± 9.6§ MP-M 1.00 ± 0.20t 0.60 ± 0.27+ 0* 73.7 ± 6.9 MP-L l.80 ± O.22t 1.00 ± 0.35* 0* 39.0 ± 8.8 OP 1.20 ± 0.20+ 0.60 ± 0.40+ 0* 35.6 ± 1O.5§ *p < 0.05 versus control value. tp < 0.05 versus MP-H value. "p < 0.05 versus control and MP-H values. §p < 0.05 versus control and MP-H values. MP-H, high-dose misoprostal; MP-L, low-dose misoprostal; MP-M, medium-dose misoprostal; Or, prostaglandin 12 analogue 01'-41483. 284 Totsuka et al Attenuation of Ischemic Liver Injury ] Am Coll Surg

reperfusion.3 '),40 Cyclic AMP was reported to pro­ groups. These results suggest that the misoprostol duce an efflux of calcium in vascular smooth muscles and OP-41483 treatments could protect the liver through the activation of the Ca + + -activated K+ from progressive damage by neutrophils after channel. 41 The decline of in tracellular calci urn causes reperfusion. vasodilation as a consequence of vasorelaxation. In this study, we made three misoprostol-treated In platelets, cyclic AMP suppresses the inositol groups depending on the drug dosage, because the phospholipid metabolic turnover that generates both potency and optimal dose of misoprostol against liver inositol I,4,5-triphosphate (IP 3) and I,2-diacyl­ injury has not yet been determined. Dosages of mi­ glycerol (DAG).42,41 This function prevents the mo­ soprostolused were based on a previous report. Using bilization of intracellular calcium by IP 3 and the ac­ a model of 90 minutes partial liver ischemia in rats, tivation of protein kinase C by DAG. In this Lim and associates51 showed that both two-dose mechanism, cyclic AMP inhibits the process of plate­ treatments of 25 mg/kg misoprostal before ischemia let aggregation, such as ADP release and activation of and before reperfusion, and a single dose treatment phospholipase A2 , which produces thromboxan A2 of 25 mg/kg misoprostol before ischemia were effec­ from . These functions produced by tive in attenuating ischemia and reperfusion injury. prostaglandin contribute to an increase in organ We tried three dosages: total IOO, 50, and 25 mg/kg blood flow. misoprostol, and we divided the drug administration Many investigators demonstrated that prosta­ between before and after hepatic ischemia in the ratio glandin has antiinflammatory potency. At the initial of seven to three. To avoid sudden changes in cardio­ phase of reperfusion after liver ischemia, activated vascular system caused by a bolus injection, the drug Kupffer cells release the inflammatory mediators was administered by continuous intravenous infu­ such as tumor necrosis factor or interleukin-I.44 sion. We found that the high dose misoprostol treat­ Neutrophils, which are activated by these mediators ment partially protected the liver against ischemic or activated endothelial cells,45 infiltrate the liver tis­ injury, but produced an unstable cardiovascular sys­ sue and produce reactive oxygen species by means of tem and pulmonary dysfunction that would have an the NADPH oxidase system, resulting in the progres­ adverse influence on liver function. We considered sion of the liver injury.46 This injury urges platelets that this adverse effect was due to not only vasodila­ into an activation, causing aggregation and further tion but also vagal reflexes caused by high-dose mi­ activationY As a result, microcirculatory failure of soprostol administration. Previously, two clinical the liver has occurred throughout these processes. cases were reported in which cardiac arrest occurred On the other hand, cyclic AMp, which is increased by after severe hypotension and bradycardia induced by prostaglandin in neutrophils, inhibits intracellular E-type prostaglandin treatment. 52, 51 In experimental calcium elevation and activation of protein kinase C, study, it was demonstrated that PGI2 stimulated va­ resulting in direct suppression of neutrophil adhe­ gal cardiac c fibers, causing vagal reflexes, such as a sion, lysosomal enzyme release, and NADPH oxidase decrease in cardiac output and bradycardia. 54,55 system.48 Since was reported to in­ Therefore, an excessive dose of misoprostol as well as hibit the release of inflammatory mediators from ac­ PGI2 may produce the vagal reflex, although this tivated Kupffer cells by elevating intracellular cyclic hypothesis has not been proved. In the present study, AMp,49 prostaglandin may block the activation of severe hypotension and bradycardia were exhibited neutrophils induced by the mediators indirectly. In in the MP-H animals, and this cardiovascular insta­ this study, high doses of misoprostol and OP-41483 bility must have an adverse influence on the post­ significantly inhibited the infiltration of neutrophils ischemic condition. In this group, sinusoidal conges­ 60 minutes after reperfusion. Although the numbers tion and hepatocyte injury developed, probably from of neutrophils in the MP-M and MP-L livers were low cardiac function after reperfusion, resulting in not different from those in control livers, the eleva­ suppression of ATP resynthesis, even though the in­ tion ofliver enzyme release was markedly suppressed hibition of adenine nucleotide degradation during in both the MP-M and MP-L groups as well as in the ischemia and the improvement ofHTBF after reper­ OP group compared with the control group for 3 fusion were induced by treatment with high dose hours after reperfusion. The values of TBA, which misoprostol. In addition to the liver findings, the reflect hepatic circulation and hepatocyte function high dose misoprostol treatment led to pulmonary more sensitively than the standard liver function failure after the operation. Although histologic ex­ tests, 50 were sustained at low levels in these three amination of the lung was not performed, we conjec- Vol. 187, No.3, September 1998 Totsuka et al Attenuation of Ischemic Liver Injury 285

tured that pulmonary edema occurred in the MP-H 4. Sikujara 0, Monden M, Toyoshima K, et al. Cytoprotcctive effect of prostaglandin 12 on ischemia-induced hepatic cell injury. Trans- animals by misoprostol-induced heart failure and di­ plantation 1983;36:238-243. . latation of the pulmonary artery. 5. Steininger R, Muhlbacher F, Rauhs R, et al. Protective effect of Regarding two lower dose misoprostol groups, PGI 2 and diltiazem on liver ischemia and reperfusion in pigs. Transplant Proc 1988;20:999-1002. 2-week animal survival and amelioration of histo­ 6. Lambotte L, Hamptinne B, Alvarez-Lopez A, Besse T. Effects of logic abnormalities in the MP-L group were poorer calcium blocking agents and prostaglandin 12 or E, on the toler­ than those in the MP-M group, although inhibitions ance of the rat liver to ischemia. Transplant Proc 1988;20:986. 7. Besse T, Gustin T, Claeys, P, et al. Effect ofPGI2 and of liver enzyme release and bile acid elevation were antagonist on liver ischemic injury. Eur Surg Res 1989;21 :213- significant in both groups. This result suggests that 217. 8. OlthoffKM, WasefE, Seu P, et al. PGEI reduces injury in hepatic the low dose misoprostol treatment had a less desir­ allografts following preservation. J Surg Res 1991 ;50:595-60 1. able effect than the middle dose treatment. 9. Okabe K, Malchesky PS, Nose Y. Protective effect of prostaglan­ din 12 on hepatic mitochondrial function of the preserved rat liver. On the other hand, the dose of OP-41483 was TohokuJ Exp Med 1986;150:373-379. based on our previous study. 56 When PGI2 was ad­ 10. Mizoguchi Y, Ysutsui H, Miyajima K, et al. The protective effects ministered at a rate of 1 or 2 /Lg/kg/min for 60 min­ of prostaglandin E] in an experimental massive hepatic cell necro­ sis model. Hepatology 1987;7: 1184-1188. utes via the portal vein before 3 hours warm ischemia 11. Nakano H, Monden M, Umeshita K, et a1. Cytoprotective effect of prostaglandin 12 analogue on superoxide-induced hepatocyte of the canine liver, 2 /Lg/kg/min-PGI 2 treatment sig­ injury. Surgery 1994;116:883-889. nificantly improved the survival rate and remarkably 12. Abecassis M, Falk R, Blendis L, et al. Treatment of fulminant suppressed the serum liver enzyme elevation, and 1 hepatic failure with a continuous infusion of prostin VR (PGE]). Heparology 1987;7:1104. /Lg/kg/min-PGI2 showed low effects. In addition to 13. Greig PD, Woolf GM, Sinclair SB, et al. Treatment of primary the OP-41483 administration at a rate of2/Lg/g/min liver graft nonfunction with prostaglandin E]. Transplantation for 30 minutes before ischemia, 3 hours infusion of 1989;48:447-453. 14. Takaya S, Doyle H, Todo S, et al. Reduction of primary nonfunc­ 0.5 /Lg/kg/min OP-41483 from 30 minutes before tion with prostaglandin E] after clinical liver transplantation. reperfusion was performed in the present study, be­ Transplant Proc 1995;27:1862-1867. 15. Cattral MS, AltraifI, Grig PD, et al. Toxic effects of intravenous cause it was considered that the halflife ofOP-41483 and oral prostaglandin E therapy in patients with liver disease. is too short to inhibit the actions of platelets and Am J Med 1994;97:369-373. neutrophils after reperfusion. OP-41483 was infused 16. Pickles H, O'Grady]. Side effects occurring during administra­ tion of epoprostenol (, PGI2 ) in man. Br J Clin Phar­ via the portal vein to alleviate the cardiovascular side macoI1982;14:177-185. effects of this drug, because PGI2 including OP- 17. Golub M, Zia P, Matsuno M, Horton R. Metabolism of prosta­ 41483 is not metabolized by the lung but by the glandin Al and E1 in man. J Clin Invest 1975;56: 1404-141 O. 18. Cho MJ, Allen MA. 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