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Efficiency of Pentoxifylline in Donor Pretreatment in Rat Liver Transplantation
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
72, 170–176 (1997)
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Efficiency of Pentoxifylline in Donor Pretreatment in Rat Liver Transplantation1 Hiroyasu Nishizawa, M.D.,*,2 Hiroto Egawa, M.D., Yukihiro Inomata, M.D., Shinji Uemoto, M.D., Katsuhiro Asonuma, M.D., Tetsuya Kiuchi, M.D., Yoshio Yamaoka, M.D.,* and Koichi Tanaka, M.D. Department of Transplantation Immunology and *Gastroenterological Surgery, Kyoto University Graduate School of Medicine, Kyoto 606-01, Japan Submitted for publication December 11, 1996
Donor pretreatment is a new concept in organ preservation. Pentoxifylline (PTX) has been reported to suppress the activation of Kupffer cells and to decrease injury to the hepatic graft after rat liver transplantation. We evaluated the efficiency of PTX pretreatment on the donor against hepatic injury following cold ischemia (CI) or warm ischemia (WI) using the rat liver transplantation model. Dose dependency: every rat was injected intraperitoneally with PTX (30, 50, or 80 mg/kg) or saline. One hour later, the portal vein (PV) and the hepatic artery (HA) were clamped for 30 min. Transplantation: the donor rat was injected intraperitoneally with 50 mg/kg PTX or saline, 1 hr before laparotomy. Animals were divided into two groups. In the CI group, grafts were preserved for 12 hr in University of Wisconsin solution at 47C and transplanted. In the WI group, the PV and the HA in the donor were clamped for 30 min before donor surgery, and the grafts were transplanted. Serum levels of tumor necrosis factor-a (TNF-a), glutathione S-transferase-a (GST-a), and aspartate transaminase (AST) were measured at 30 min, 3 hr, and 24 hr after reperfusion of the PV. Compared with those of a control group, the serum levels of TNF-a, GST-a, and AST in the PTXpretreated groups were significantly lower after both CI and WI at 30 min and further suppressed in the WI group at 24 hr. These results indicate that PTX pretreatment on the donor is effective for suppression of hepatic injury after both CI and WI. q 1997 Academic Press
INTRODUCTION
Donor shortage is still one of the greatest problems in organ transplantation. In addition to the increase in organ donations due to public education, innovations 1 This work was supported by a grant from the Scientific Research Fund of the Ministry of Education, Japan. 2 To whom correspondence should be addressed at Kyoto University Graduate School of Medicine, Department of Transplantation Immunology, 54, Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-01, Japan.
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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in the field of preservation solution have played a great role in alleviating this problem. Especially, the advent of University of Wisconsin (UW) solution has contributed to significant progress [1]. This solution inhibits organ injury during cold preservation and allows for a longer preservation time. Transplant organs from suitable donors are protected by UW solution after perfusion during donor surgery. However, following evaluation, these organs may be accidentally exposed to warm ischemia due to hypoxygenemia, hypotension, or cardiac arrest prior to perfusion. Preventing organ injury due to this type of warm ischemia is of great importance in decreasing the incidence of primary graft dysfunction or nonfunction after transplantation. Pretreatment of organ donors was discussed in a meeting of the Society of Organ Sharing in Vancouver in 1993. This concept could also be expanded for the treatment of suboptimal donors. The suboptimal hepatic graft might be susceptible to hypoxygenic injury presumably following oxygen radical injury by activated Kupffer cells. Suppressing the activation of Kupffer cells has been reported to be effective in decreasing ischemia/reperfusion injury [2], and many pharmacological agents have been tried for this purpose [2–5]. Pentoxifylline (PTX) has proven to be one of the most effective of these agents. PTX, which is a methylxanthine phosphodiesterase inhibitor, has been clinically employed for the treatment of peripheral vascular disorders [6, 7] and kidney transplantation [8]. In recent rat experiments, Peng et al. reported that PTX treatment reduced mortality and hepatic injury after normothermic ischemia/ reperfusion [9]; Bachmann et al. reported that PTX treatment on recipients improved graft survival after long storage in rat liver transplantations [10]; and Kozaki et al. reported that an initial PTX flush helped to minimize cold preservation/reperfusion injury in rat liver transplantations [11]. These reports suggest that PTX suppresses the release of tumor necrosis factor-a (TNF-a) from an activated Kupffer cell. Hence, PTX pretreatment of the donor may have the potential to protect the graft liver from (1) warm ischemic injury during procurement, (2) cold ischemic in-
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FIG. 1. Effects of pentoxifylline on TNF-a serum levels during dose-dependent study. TNF-a levels in rats with PTX pretreatment were significantly lower than those of control rats. TNF-a levels in rats with 50 mg/kg PTX pretreatment were significantly lower than those in rats with 20 mg/kg PTX pretreatment at 3 hr. *P õ 0.05, †P õ 0.01 vs control.
jury during preservation, (3) warm ischemic injury during vascular anastomosis, and (4) reperfusion injury. However, the beneficial effects of PTX on hepatic injury involving the release of TNF-a after warm ischemia have not yet been reported when a liver transplantation model is used. Furthermore, in preventive treatment of the donor before surgery in order to suppress procurement/preservation/reperfusion injury, the efficiency of PTX is unknown. Accordingly, the purpose of this study was to evaluate whether PTX pretreatment on the donor can decrease hepatic injury after both cold preservation and warm ischemia. MATERIALS AND METHODS Animals. Male inbred Lewis (RT1l) rats weighing 220 to 270 g were purchased from Charles River Japan, Inc. (Atsugi, Japan). All rats were provided with standard laboratory chow and water ad libitum and housed in accordance with institutional animal care poli-
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cies. Prior to being used in the study, rats were fasted for 1 day and were allowed free access to water. Dose dependency. In this study, a warm ischemia model without liver transplantation was used to determine the appropriate dose of pretreatment in the following liver transplantation study. One milliliter of saline solution containing 20, 50, and 80 mg/kg of PTX (Hoechst Japan Co., Tokyo, Japan) was injected intraperitoneally 1 hr before laparotomy of rats under ether (Wako Pure Chemical Industries, Ltd., Osaka, Japan) anesthesia. One milliliter of saline was injected in the control group (n Å 6, each group). One hour later, the abdomen was opened through a horizontal incision under ether anesthesia. The portal vein and the hepatic artery were clamped with a microvessel clip for 30 min on each animal. Subsequently the abdomen was closed. Liver transplantation. Rat liver transplantation was performed using the technique of Kamada and Calne [12] under ether anesthesia, and the hepatic artery was not reconstructed. UW solution (ViaSpan, Du Pont Merck Pharmaceutical Co., Wilmington, VA) was used for the perfusion [13]. The donor rat was injected intraperitoneally with 50 mg/kg PTX in 1 ml of saline or 1 ml of saline as a control 1 hr before laparotomy. The animals were then divided into a cold ischemic group and a warm ischemic group. In the cold ischemic group, the graft liver was preserved for 12 hr in UW solution at 47C and then transplanted orthotopically. In the warm ischemic group, the portal vein and the hepatic artery were clamped with a microvessel clip for 30 min prior to donor surgery. The graft liver was then transplanted orthotopically (n Å 6, each). The cold preservation time in the warm ischemic group was shorter than 90 min, and the anhepatic time from cross-clamping to reperfusion of the portal vein was less than 15 min. Sampling and assessment. Samples of blood were collected for assay via the jugular vein at 30 min, 3 hr, and 24 hr after reperfusion of the portal vein and the hepatic artery in dose dependency and the portal vein in liver transplantation. During these samplings, the rat remained on ether anesthesia and was given 1 ml of blood from a third rat through the penile vein. The serum was separated immediately and stored at 0707C until analysis. For the assessment, the serum levels of TNF-a, glutathione S-transferase-a (GST-a), lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. TNF-a assay. Serum levels of TNF-a were assayed with a mouse TNF-a ELISA kit from the Genzyme corporation (Cambridge, MA). This was an enzyme-linked immunoadsorbent assay for the quantitation of natural or recombinant rat TNF-a levels as well as that of the mouse TNF-a in the serum. The assay was carried out according to the manufacturer’s instructions. GST-a assay. Serum levels of GST-a were measured with HepkitAlpha for rat GST-a from Biotrin International Ltd. (Dublin, Ireland), which is an enzyme immunoassay providing the quantitative
FIG. 2. Effects of pentoxifylline on GST-a and LDH serum levels during dose-dependent study. Levels in rats with 50 and 80 mg/kg PTX pretreatment were significantly lower than those of control rats at every sampling point. *P õ 0.05, †P õ 0.01 vs control.
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FIG. 3. Effects of pentoxifylline on AST and ALT serum levels during dose-dependent study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min. *P õ 0.05, †P õ 0.01 vs control.
estimation of rat GST-a in serum. The assay was carried out according to the manufacturer’s instructions. GST-a, which is contained in very high concentration in the hepatic cytosol [14], is released into the circulation during hepatocellular damage and is a very sensitive indicator of acute hepatocellular injury [15]. GST-a levels in normal male Lewis rats are 25 ng/ml [16]. GST-a has been shown to have a short half-life (1.5 hr) and stabilizes rapidly after transplant [17]. Statistical analysis. Pretreatment subgroup data, expressed as the mean { standard error (SE), was analyzed by Mann-Whitney’s U test. Differences with P values of less than 0.05 were considered to be significant.
RESULTS
Dose dependency. TNF-a levels of control rats continued to increase gradually, while those in rats with PTX pretreatment remained low. TNF-a levels in rats with PTX pretreatment were significantly lower than those of control rats at every sampling point (P õ 0.01 and P õ 0.05, respectively) (Fig. 1). GST-a levels increased at 30 min and then returned to normal values at 24 hr. GST-a levels in rats with 50 and 80 mg/kg PTX pretreatment were significantly lower than those of the control rats at 30 min and 3 hr (P õ 0.01 and P õ 0.05, respectively) (Fig. 2). LDH levels increased at 30 min, and peaked at 3 hr. However, they decreased rapidly at 24 hr. LDH levels in rats with PTX pretreatment were significantly lower than those of the control rats at every sampling point except 20 mg/kg PTX pretreatment at 24 hr (P õ 0.01 and P õ 0.05, respectively) (Fig. 2). AST and ALT levels peaked at 3 hr. AST and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min (P õ 0.01 and P õ 0.05, respectively), and AST levels in rats with 50 and 80 mg/kg PTX pretreatment were significantly lower than those of control rats at 3 and 24 hr (P õ 0.05 and P õ 0.05, respectively), and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 24 hr (P õ 0.05, each) (Fig. 3). Among rats with 20, 50, and 80 mg/kg PTX pretreatment, no significant differences were seen in serum level parameters except TNF-a. There was a
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significant difference only between TNF-a levels in rats with 20 and 50 mg/kg PTX pretreatment (P õ 0.05) (Fig. 1). Effects of cold ischemia. TNF-a levels in rats with PTX pretreatment increased gradually from 30 min to 24 hr and were significantly lower than those of control rats at 30 min (P õ 0.01) (Fig. 4). GST-a levels increased at 30 min and then remained high. GST-a levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 3 hr (P õ 0.01 and P õ 0.01, respectively) (Fig. 5). LDH levels increased at 24 hr, and LDH levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min (P õ 0.01) (Fig. 5). AST and ALT levels increased at 24 hr. AST and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min (P õ 0.01 and P õ 0.01, respectively) (Fig. 6). Effects of warm ischemia. TNF-a levels of control rats peaked at 3 hr, and TNF-a levels in rats with PTX pretreatment were significantly lower than those
FIG. 4. Effects of pentoxifylline on TNF-a serum levels during cold ischemia (CI) in transplantation study. Levels in rats with PTX pretreatment increased gradually and were significantly lower than those of control rats at 30 mins. †P õ 0.01 vs control.
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FIG. 5. Effects of pentoxifylline on GST-a and LDH serum levels during cold ischemia (CI) in transplantation study. GST-a levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 3 hr. LDH levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min. †P õ 0.01 vs control.
of control rats at 30 min and 3 hr (P õ 0.05 and P õ 0.01, respectively) (Fig. 7). GST-a levels increased in all rats at 30 min, and then in rats with PTX pretreatment they returned to normal values at 24 hr. GSTa levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr (P õ 0.05 and P õ 0.05, respectively) (Fig. 8). LDH levels peaked at 3 hr. LDH levels in rats with PTX pretreatment were significantly lower than those of control rats at every sampling point (P õ 0.05, each) (Fig. 8). AST and ALT levels peaked at 3 hr. AST levels in rats with PTX pretreatment were significantly lower than those of control rats at every sampling point (P õ 0.05, each), and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr (P õ 0.01 and P õ 0.05, respectively) (Fig. 9). Every rat in the dose dependency evaluation and every recipient rat survived more than 100 days. DISCUSSION
In the dose dependency evaluation, the serum levels of TNF-a, GST-a, LDH, AST, and ALT in the three
PTX-pretreated groups were significantly lower than those in the control group at most of the sampling points. The effectiveness of PTX appeared to vary according to the dosage; however, significant differences were not seen among the three PTX-pretreated groups except in serum TNF-a levels at 3 hr. TNF-a levels in the 50 mg/kg PTX-pretreated group were significantly lower than those in the 20 mg/kg PTX-pretreated group at 3 hr, but there was no significant difference in TNFa levels between the 50 mg/kg PTX-pretreated group and the 80 mg/kg PTX-pretreated group. Hence, for transplantation we chose a dose of 50 mg/kg PTX pretreatment on the donor by intraperitoneal injection. In the transplantations, cold preservation injury was greatest at 24 hr after reperfusion and warm ischemic injury peaked at 3 hr according to changes in all hepatic enzymes except GST-a. The target cells of cold ischemic injury are nonparenchymal cells involving Kupffer cells while warm ischemic injury affects parenchymal cells [18, 19]. Indeed, TNF-a levels in the control group with cold ischemia increased to higher levels and remained high longer than the group with warm ischemia. The activation of Kupffer cells was presumably prolonged after cold ischemia. Without GST-a, we might hypothe-
FIG. 6. Effects of pentoxifylline on AST and ALT serum levels during cold ischemia (CI) in transplantation study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min. †P õ 0.01 vs control.
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FIG. 7. Effects of pentoxifylline on TNF-a serum levels during warm ischemia (WI) in transplantation study. Levels in rats with PTX pretreatment remained low, while those of control rats peaked at 3 hr. In rats with PTX pretreatment levels were significantly lower than those of control rats at 30 min and 3 hr. *P õ 0.05, †P õ 0.01 vs control.
size that parenchymal cell damage occurred secondary to nonparenchymal cell injury in grafts with cold ischemia and that parenchymal cell damage occurred concomitant with nonparenchymal cell injury in grafts with warm ischemia. However, the levels of GST-a, which mainly indicate parenchymal cell injury more sensitively than other enzymes [15], increased immediately and remained high longer in the control group with cold ischemia than in the warm ischemia group, seemingly contrary to this theory. Another possible explanation was that the hepatocytes were injured during cold preservation not only by a mechanism related to the activated Kupffer cells but also by the cold ischemia itself. GST-a levels in both control groups increased immediately after reperfusion, although the other hepatic enzyme levels in both groups did not increase at that time. Intracellular concentration of GST-a was high throughout from the centrilobular area to the periportal area in the lobule whereas aminotransferases
were limited to periportal hepatocytes [15]. These results suggested that centrilobular hepatocytes dominated periportal hepatocytes in parenchymal cell injury during the early phase after cold and warm ischemia. PTX pretreatment on the donor significantly decreased hepatic injury during the early phase after cold and warm ischemia and at the 24-hr point after warm ischemia. Although PTX pretreatment on the donor significantly decreased hepatic injury, possibly related to Kupffer cell activation, the effects did not last long in graft with cold ischemia. One possible reason for this may be that PTX activity was lost during preservation. Therefore, concerning clinical usage, it may be more useful to administer PTX to the recipient before and after transplantation as well as in donor pretreatment, in order to maintain optimal effects. PTX seemed to suppress various kinds of cytokines, including TNF-a, IL-1, and IL-6, and toxic oxygen radicals released from activated Kupffer cells [1, 20–25]. Kupffer cell activation was a prominent feature of reperfusion injury after warm and cold ischemia [20–23], and toxic oxygen radicals cause severe injury to the microvascular endothelial cell after reperfusion [26]. In particular, TNF-a plays important roles in the pathogenesis of early graft failure and pulmonary complication after liver transplantation [27]. Although superoxide in the toxic oxygen radicals is released from hepatocytes and leukocytes as well as from Kupffer cells, superoxide from Kupffer cells appears to make a more important contribution to cell damage [11]. The inhibitory effects of PTX on the release of superoxides have already been reported to be effective for liver protection [11, 28]. In this study, PTX significantly suppressed serum TNF-a at the 30 min point in both cold and warm ischemic injuries. PTX also improves microcirculation which has been damaged during preservation and reperfusion. Waxman et al. [29] and Coccia et al. [30] reported that PTX administration to rats with hemorrhage shock increased hepatic surface oxygenation and decreased
FIG. 8. Effects of pentoxifylline on GST-a and LDH serum levels during warm ischemia (WI) in transplantation study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr. GST-a levels in rats with PTX pretreatment returned to normal values at the 24 hr point. *P õ 0.05 vs control.
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FIG. 9. Effects of pentoxifylline on AST and ALT serum levels during warm ischemia (WI) in transplantation study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr. *P õ 0.05, †P õ 0.01 vs control.
whole blood viscosity. Flynn et al. [7] suggested that these facts might be related to the effects of PTX such as improved microvascular blood flow, improved deformability of erythrocytes, inhibition of platelet aggregation, stimulation of prostaglandin production, inhibition of leukocyte adhesion, inhibition of vasoconstriction, and enhancement of fibrinolysis. The hepatocytes in the centrilobular area were susceptible to ischemia, and improvement in microcirculation due to PTX might be the cause of this, as shown by changes in serum GST-a level. This protocol is based on our evaluation of the effects of PTX pretreatment using the serum samples from rats surviving transplantation. Although we did not study the effect on survival following life-threatening injury, our results showed that PTX pretreatment on the donor significantly suppressed hepatic injury after both cold preservation/reperfusion and warm ischemia/ reperfusion in the rat liver transplantation model. In conclusion, PTX pretreatment of the donor displays the potential for protecting the graft liver from procurement/preservation/reperfusion injury in clinical liver transplantation. REFERENCES 1. Clavien, P. A., Harvey, P. R. C., and Strasberg, S. M. Preservation and reperfusion injuries in liver allografts: An overview and synthesis of current studies. Transplantation 53: 957, 1992. 2. Currin, T. R., Reinstein, L. J., Lichtman, S. N., Thurman, R. G., and Lemasters, J. J. Inhibition of tumor necrosis factor release from cultured rat Kupffer cells by agents that reduce graft failure from storage injury. Transplant. Proc. 25: 1631, 1993. 3. Motoyama, K., Kamei, T., Nakafusa, Y., Ueki, M., Hirano, T., Arima, T., Konomi, K., and Tanaka, M. Donor treatment with gadolinium chloride improves survival after transplantation of cold-stored livers reducing Kupffer cell tumor necrosis factor production in rat. Transplant. Proc. 27: 762, 1995. 4. Tilg, H., Eibl, B., Pichl, M., Gachter, A., Herold, M., Brankova, J., Huber, C., and Niederwieser, D. Immune response modulation by pentoxifylline in vitro. Transplantation 56: 196, 1993. 5. Edwards, M. J., Abney D. L., and Miller, F. N. Pentoxifylline inhibits interleukin-2-induced leukocyte-endothelial adherence and reduces systemic toxicity. Surgery 110: 199, 1991.
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6. Ward, A., and Clissold, S. P. Pentoxifylline. A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic efficacy. Drugs 34: 50, 1987. 7. Flynn, W. J., Cryer, H. G., and Garrison, R. N. Pentoxifylline but not Saralasin restores hepatic blood flow after resuscitation from hemorrhagic shock. J. Surg. Res. 50: 616, 1991. 8. Vincenti, F., Danovitch, G. M., Neylan, J. F., Steiner, R. W., Everson, M. P., and Gaston, R. S. Pentoxifylline does not prevent the cytokine-induced first dose reaction following OKT3. A randomized, double-blind placebo-controlled study. Transplantation 61: 573, 1996. 9. Peng, Xing-Xi, Currin, R. T., Thurman, R. G., and Lemasters, J. J. Protection by pentoxifylline against normothermic liver ischemia/reperfusion in rats. Transplantation 59: 1537, 1995. 10. Bachmann, S., Caldwell-Kenkel, J. C., Currin, R. T., Takei, Y., Marzi, I., Thurman, R. G., and Lemasters, J. J. Ultrastructual correlates of liver graft failure from storage injury: Studies of graft protection by Carolina rinse solution and pentoxifylline. Transplant. Proc. 25: 1620, 1993. 11. Kozaki, K., Egawa, H., Bermudez, L., Keefe, E. B., So, S. K., and Esquivel, C. O. Effects of pentoxifylline pretreatment on Kupffer cells in rat liver transplantation. Hepatology 21: 1079, 1995. 12. Kamada, N., and Calne, R. Y. Orthotopic liver transplantation in the rat: Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 28: 47, 1979. 13. Jamieson, N. V., Lindell, S., Sundberg, R., Southard, J. H., and Belzer, F. O. Preservation of the canine liver for 24–48 hours using simple cold storage with UW solution. Transplantation 46: 517, 1988. 14. Hiley, C., Fryer, A., Bell, J., Hume, R., and Strange, R. C. The human glutathione S-transferases: Immunohistochemical studies of the developmental expression of Alpha- and Pi-class isoenzymes in liver. Biochem. J. 254: 255, 1988. 15. Tiainen, P., Hockerstedt, K., and Rosenberg, P. H. Hepatocellular integrity in liver donors and recipients indicated by glutathione transferase alpha. Transplantation 61: 904, 1996. 16. Egawa, H., Martinez, O. M., Quinn, M. B., Villanueva J. C., So, S., Esquivel C. O., and Krams, S. M. Acute liver allograft rejection in the rat: An analysis of the immune response. Transplantation 59: 97, 1995. 17. Bass, N. M., Kirsch, R. E., Tuff, S. A., and Saunders, S. J. Radioimmunoassy of plasma ligandin: A sensitive index of experimental hepatocellular necrosis. Gastroenterology 75: 589, 1978. 18. Caldwell-Kenkel, J. C., Thurman, R. G., and Lemasters, J. J. Selective loss of nonparenchymal cell viability after cold ischemic storage of rat livers. Transplantation 45: 834, 1988.
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19. McKeown, C. M. B., Edwards, V., Phillips, M. J., Harvey, P. R. C., Petrunka, C. N., and Strasberg, S. M. Sinusoidal lining cell damage: The critical injury in cold preservation of liver allografts in the rat. Transplantation 46: 178, 1988. 20. Caldwell-Kenkel, J. C., Currin, R. T., Tanaka, Y., Thurman, R. G., and Lemasters, J. J. Kupffer cell activation and endotherial cell damage after storage of rat livers: Effects of perfusion. Hepatology 13: 83, 1991. 21. Lindert, K. A., Caldwell-Kenkel, J. C., Nukina, S., Lemasters, J. J., and Thurman, R. G. Activation of Kupffer cells on reperfusion following hypoxia: Particle phagocytosis in a low-flow, reflow model. Am. J. Physiol. 262: G345, 1992. 22. Jaeschke, H., and Farhood, A. Neutrophil and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver. Am. J. Physiol. 260: G355, 1991. 23. Lemasters, J. J., and Thurman, R. G. Hypoxia and reperfusion injury to liver. Progr. Liver Dis. 11: 85, 1993. 24. Wake, K., Decker, K., Kirn, A., Knook, D. L., McCuskey, R. S., Bouwens, L., and Wisse, E. Cell biology and the kinetics of Kupffer cells in the liver. Int. Rev. Cytol. 118: 173, 1989. 25. Wang, P., Ba, Z. F., Morrison M. H., Ayala, A., and Chaudry,
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Efficiency of Pentoxifylline in Donor Pretreatment in Rat Liver Transplantation1 Hiroyasu Nishizawa, M.D.,*,2 Hiroto Egawa, M.D., Yukihiro Inomata, M.D., Shinji Uemoto, M.D., Katsuhiro Asonuma, M.D., Tetsuya Kiuchi, M.D., Yoshio Yamaoka, M.D.,* and Koichi Tanaka, M.D. Department of Transplantation Immunology and *Gastroenterological Surgery, Kyoto University Graduate School of Medicine, Kyoto 606-01, Japan Submitted for publication December 11, 1996
Donor pretreatment is a new concept in organ preservation. Pentoxifylline (PTX) has been reported to suppress the activation of Kupffer cells and to decrease injury to the hepatic graft after rat liver transplantation. We evaluated the efficiency of PTX pretreatment on the donor against hepatic injury following cold ischemia (CI) or warm ischemia (WI) using the rat liver transplantation model. Dose dependency: every rat was injected intraperitoneally with PTX (30, 50, or 80 mg/kg) or saline. One hour later, the portal vein (PV) and the hepatic artery (HA) were clamped for 30 min. Transplantation: the donor rat was injected intraperitoneally with 50 mg/kg PTX or saline, 1 hr before laparotomy. Animals were divided into two groups. In the CI group, grafts were preserved for 12 hr in University of Wisconsin solution at 47C and transplanted. In the WI group, the PV and the HA in the donor were clamped for 30 min before donor surgery, and the grafts were transplanted. Serum levels of tumor necrosis factor-a (TNF-a), glutathione S-transferase-a (GST-a), and aspartate transaminase (AST) were measured at 30 min, 3 hr, and 24 hr after reperfusion of the PV. Compared with those of a control group, the serum levels of TNF-a, GST-a, and AST in the PTXpretreated groups were significantly lower after both CI and WI at 30 min and further suppressed in the WI group at 24 hr. These results indicate that PTX pretreatment on the donor is effective for suppression of hepatic injury after both CI and WI. q 1997 Academic Press
INTRODUCTION
Donor shortage is still one of the greatest problems in organ transplantation. In addition to the increase in organ donations due to public education, innovations 1 This work was supported by a grant from the Scientific Research Fund of the Ministry of Education, Japan. 2 To whom correspondence should be addressed at Kyoto University Graduate School of Medicine, Department of Transplantation Immunology, 54, Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-01, Japan.
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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in the field of preservation solution have played a great role in alleviating this problem. Especially, the advent of University of Wisconsin (UW) solution has contributed to significant progress [1]. This solution inhibits organ injury during cold preservation and allows for a longer preservation time. Transplant organs from suitable donors are protected by UW solution after perfusion during donor surgery. However, following evaluation, these organs may be accidentally exposed to warm ischemia due to hypoxygenemia, hypotension, or cardiac arrest prior to perfusion. Preventing organ injury due to this type of warm ischemia is of great importance in decreasing the incidence of primary graft dysfunction or nonfunction after transplantation. Pretreatment of organ donors was discussed in a meeting of the Society of Organ Sharing in Vancouver in 1993. This concept could also be expanded for the treatment of suboptimal donors. The suboptimal hepatic graft might be susceptible to hypoxygenic injury presumably following oxygen radical injury by activated Kupffer cells. Suppressing the activation of Kupffer cells has been reported to be effective in decreasing ischemia/reperfusion injury [2], and many pharmacological agents have been tried for this purpose [2–5]. Pentoxifylline (PTX) has proven to be one of the most effective of these agents. PTX, which is a methylxanthine phosphodiesterase inhibitor, has been clinically employed for the treatment of peripheral vascular disorders [6, 7] and kidney transplantation [8]. In recent rat experiments, Peng et al. reported that PTX treatment reduced mortality and hepatic injury after normothermic ischemia/ reperfusion [9]; Bachmann et al. reported that PTX treatment on recipients improved graft survival after long storage in rat liver transplantations [10]; and Kozaki et al. reported that an initial PTX flush helped to minimize cold preservation/reperfusion injury in rat liver transplantations [11]. These reports suggest that PTX suppresses the release of tumor necrosis factor-a (TNF-a) from an activated Kupffer cell. Hence, PTX pretreatment of the donor may have the potential to protect the graft liver from (1) warm ischemic injury during procurement, (2) cold ischemic in-
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FIG. 1. Effects of pentoxifylline on TNF-a serum levels during dose-dependent study. TNF-a levels in rats with PTX pretreatment were significantly lower than those of control rats. TNF-a levels in rats with 50 mg/kg PTX pretreatment were significantly lower than those in rats with 20 mg/kg PTX pretreatment at 3 hr. *P õ 0.05, †P õ 0.01 vs control.
jury during preservation, (3) warm ischemic injury during vascular anastomosis, and (4) reperfusion injury. However, the beneficial effects of PTX on hepatic injury involving the release of TNF-a after warm ischemia have not yet been reported when a liver transplantation model is used. Furthermore, in preventive treatment of the donor before surgery in order to suppress procurement/preservation/reperfusion injury, the efficiency of PTX is unknown. Accordingly, the purpose of this study was to evaluate whether PTX pretreatment on the donor can decrease hepatic injury after both cold preservation and warm ischemia. MATERIALS AND METHODS Animals. Male inbred Lewis (RT1l) rats weighing 220 to 270 g were purchased from Charles River Japan, Inc. (Atsugi, Japan). All rats were provided with standard laboratory chow and water ad libitum and housed in accordance with institutional animal care poli-
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cies. Prior to being used in the study, rats were fasted for 1 day and were allowed free access to water. Dose dependency. In this study, a warm ischemia model without liver transplantation was used to determine the appropriate dose of pretreatment in the following liver transplantation study. One milliliter of saline solution containing 20, 50, and 80 mg/kg of PTX (Hoechst Japan Co., Tokyo, Japan) was injected intraperitoneally 1 hr before laparotomy of rats under ether (Wako Pure Chemical Industries, Ltd., Osaka, Japan) anesthesia. One milliliter of saline was injected in the control group (n Å 6, each group). One hour later, the abdomen was opened through a horizontal incision under ether anesthesia. The portal vein and the hepatic artery were clamped with a microvessel clip for 30 min on each animal. Subsequently the abdomen was closed. Liver transplantation. Rat liver transplantation was performed using the technique of Kamada and Calne [12] under ether anesthesia, and the hepatic artery was not reconstructed. UW solution (ViaSpan, Du Pont Merck Pharmaceutical Co., Wilmington, VA) was used for the perfusion [13]. The donor rat was injected intraperitoneally with 50 mg/kg PTX in 1 ml of saline or 1 ml of saline as a control 1 hr before laparotomy. The animals were then divided into a cold ischemic group and a warm ischemic group. In the cold ischemic group, the graft liver was preserved for 12 hr in UW solution at 47C and then transplanted orthotopically. In the warm ischemic group, the portal vein and the hepatic artery were clamped with a microvessel clip for 30 min prior to donor surgery. The graft liver was then transplanted orthotopically (n Å 6, each). The cold preservation time in the warm ischemic group was shorter than 90 min, and the anhepatic time from cross-clamping to reperfusion of the portal vein was less than 15 min. Sampling and assessment. Samples of blood were collected for assay via the jugular vein at 30 min, 3 hr, and 24 hr after reperfusion of the portal vein and the hepatic artery in dose dependency and the portal vein in liver transplantation. During these samplings, the rat remained on ether anesthesia and was given 1 ml of blood from a third rat through the penile vein. The serum was separated immediately and stored at 0707C until analysis. For the assessment, the serum levels of TNF-a, glutathione S-transferase-a (GST-a), lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. TNF-a assay. Serum levels of TNF-a were assayed with a mouse TNF-a ELISA kit from the Genzyme corporation (Cambridge, MA). This was an enzyme-linked immunoadsorbent assay for the quantitation of natural or recombinant rat TNF-a levels as well as that of the mouse TNF-a in the serum. The assay was carried out according to the manufacturer’s instructions. GST-a assay. Serum levels of GST-a were measured with HepkitAlpha for rat GST-a from Biotrin International Ltd. (Dublin, Ireland), which is an enzyme immunoassay providing the quantitative
FIG. 2. Effects of pentoxifylline on GST-a and LDH serum levels during dose-dependent study. Levels in rats with 50 and 80 mg/kg PTX pretreatment were significantly lower than those of control rats at every sampling point. *P õ 0.05, †P õ 0.01 vs control.
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FIG. 3. Effects of pentoxifylline on AST and ALT serum levels during dose-dependent study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min. *P õ 0.05, †P õ 0.01 vs control.
estimation of rat GST-a in serum. The assay was carried out according to the manufacturer’s instructions. GST-a, which is contained in very high concentration in the hepatic cytosol [14], is released into the circulation during hepatocellular damage and is a very sensitive indicator of acute hepatocellular injury [15]. GST-a levels in normal male Lewis rats are 25 ng/ml [16]. GST-a has been shown to have a short half-life (1.5 hr) and stabilizes rapidly after transplant [17]. Statistical analysis. Pretreatment subgroup data, expressed as the mean { standard error (SE), was analyzed by Mann-Whitney’s U test. Differences with P values of less than 0.05 were considered to be significant.
RESULTS
Dose dependency. TNF-a levels of control rats continued to increase gradually, while those in rats with PTX pretreatment remained low. TNF-a levels in rats with PTX pretreatment were significantly lower than those of control rats at every sampling point (P õ 0.01 and P õ 0.05, respectively) (Fig. 1). GST-a levels increased at 30 min and then returned to normal values at 24 hr. GST-a levels in rats with 50 and 80 mg/kg PTX pretreatment were significantly lower than those of the control rats at 30 min and 3 hr (P õ 0.01 and P õ 0.05, respectively) (Fig. 2). LDH levels increased at 30 min, and peaked at 3 hr. However, they decreased rapidly at 24 hr. LDH levels in rats with PTX pretreatment were significantly lower than those of the control rats at every sampling point except 20 mg/kg PTX pretreatment at 24 hr (P õ 0.01 and P õ 0.05, respectively) (Fig. 2). AST and ALT levels peaked at 3 hr. AST and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min (P õ 0.01 and P õ 0.05, respectively), and AST levels in rats with 50 and 80 mg/kg PTX pretreatment were significantly lower than those of control rats at 3 and 24 hr (P õ 0.05 and P õ 0.05, respectively), and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 24 hr (P õ 0.05, each) (Fig. 3). Among rats with 20, 50, and 80 mg/kg PTX pretreatment, no significant differences were seen in serum level parameters except TNF-a. There was a
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significant difference only between TNF-a levels in rats with 20 and 50 mg/kg PTX pretreatment (P õ 0.05) (Fig. 1). Effects of cold ischemia. TNF-a levels in rats with PTX pretreatment increased gradually from 30 min to 24 hr and were significantly lower than those of control rats at 30 min (P õ 0.01) (Fig. 4). GST-a levels increased at 30 min and then remained high. GST-a levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 3 hr (P õ 0.01 and P õ 0.01, respectively) (Fig. 5). LDH levels increased at 24 hr, and LDH levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min (P õ 0.01) (Fig. 5). AST and ALT levels increased at 24 hr. AST and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min (P õ 0.01 and P õ 0.01, respectively) (Fig. 6). Effects of warm ischemia. TNF-a levels of control rats peaked at 3 hr, and TNF-a levels in rats with PTX pretreatment were significantly lower than those
FIG. 4. Effects of pentoxifylline on TNF-a serum levels during cold ischemia (CI) in transplantation study. Levels in rats with PTX pretreatment increased gradually and were significantly lower than those of control rats at 30 mins. †P õ 0.01 vs control.
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FIG. 5. Effects of pentoxifylline on GST-a and LDH serum levels during cold ischemia (CI) in transplantation study. GST-a levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 3 hr. LDH levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min. †P õ 0.01 vs control.
of control rats at 30 min and 3 hr (P õ 0.05 and P õ 0.01, respectively) (Fig. 7). GST-a levels increased in all rats at 30 min, and then in rats with PTX pretreatment they returned to normal values at 24 hr. GSTa levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr (P õ 0.05 and P õ 0.05, respectively) (Fig. 8). LDH levels peaked at 3 hr. LDH levels in rats with PTX pretreatment were significantly lower than those of control rats at every sampling point (P õ 0.05, each) (Fig. 8). AST and ALT levels peaked at 3 hr. AST levels in rats with PTX pretreatment were significantly lower than those of control rats at every sampling point (P õ 0.05, each), and ALT levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr (P õ 0.01 and P õ 0.05, respectively) (Fig. 9). Every rat in the dose dependency evaluation and every recipient rat survived more than 100 days. DISCUSSION
In the dose dependency evaluation, the serum levels of TNF-a, GST-a, LDH, AST, and ALT in the three
PTX-pretreated groups were significantly lower than those in the control group at most of the sampling points. The effectiveness of PTX appeared to vary according to the dosage; however, significant differences were not seen among the three PTX-pretreated groups except in serum TNF-a levels at 3 hr. TNF-a levels in the 50 mg/kg PTX-pretreated group were significantly lower than those in the 20 mg/kg PTX-pretreated group at 3 hr, but there was no significant difference in TNFa levels between the 50 mg/kg PTX-pretreated group and the 80 mg/kg PTX-pretreated group. Hence, for transplantation we chose a dose of 50 mg/kg PTX pretreatment on the donor by intraperitoneal injection. In the transplantations, cold preservation injury was greatest at 24 hr after reperfusion and warm ischemic injury peaked at 3 hr according to changes in all hepatic enzymes except GST-a. The target cells of cold ischemic injury are nonparenchymal cells involving Kupffer cells while warm ischemic injury affects parenchymal cells [18, 19]. Indeed, TNF-a levels in the control group with cold ischemia increased to higher levels and remained high longer than the group with warm ischemia. The activation of Kupffer cells was presumably prolonged after cold ischemia. Without GST-a, we might hypothe-
FIG. 6. Effects of pentoxifylline on AST and ALT serum levels during cold ischemia (CI) in transplantation study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min. †P õ 0.01 vs control.
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FIG. 7. Effects of pentoxifylline on TNF-a serum levels during warm ischemia (WI) in transplantation study. Levels in rats with PTX pretreatment remained low, while those of control rats peaked at 3 hr. In rats with PTX pretreatment levels were significantly lower than those of control rats at 30 min and 3 hr. *P õ 0.05, †P õ 0.01 vs control.
size that parenchymal cell damage occurred secondary to nonparenchymal cell injury in grafts with cold ischemia and that parenchymal cell damage occurred concomitant with nonparenchymal cell injury in grafts with warm ischemia. However, the levels of GST-a, which mainly indicate parenchymal cell injury more sensitively than other enzymes [15], increased immediately and remained high longer in the control group with cold ischemia than in the warm ischemia group, seemingly contrary to this theory. Another possible explanation was that the hepatocytes were injured during cold preservation not only by a mechanism related to the activated Kupffer cells but also by the cold ischemia itself. GST-a levels in both control groups increased immediately after reperfusion, although the other hepatic enzyme levels in both groups did not increase at that time. Intracellular concentration of GST-a was high throughout from the centrilobular area to the periportal area in the lobule whereas aminotransferases
were limited to periportal hepatocytes [15]. These results suggested that centrilobular hepatocytes dominated periportal hepatocytes in parenchymal cell injury during the early phase after cold and warm ischemia. PTX pretreatment on the donor significantly decreased hepatic injury during the early phase after cold and warm ischemia and at the 24-hr point after warm ischemia. Although PTX pretreatment on the donor significantly decreased hepatic injury, possibly related to Kupffer cell activation, the effects did not last long in graft with cold ischemia. One possible reason for this may be that PTX activity was lost during preservation. Therefore, concerning clinical usage, it may be more useful to administer PTX to the recipient before and after transplantation as well as in donor pretreatment, in order to maintain optimal effects. PTX seemed to suppress various kinds of cytokines, including TNF-a, IL-1, and IL-6, and toxic oxygen radicals released from activated Kupffer cells [1, 20–25]. Kupffer cell activation was a prominent feature of reperfusion injury after warm and cold ischemia [20–23], and toxic oxygen radicals cause severe injury to the microvascular endothelial cell after reperfusion [26]. In particular, TNF-a plays important roles in the pathogenesis of early graft failure and pulmonary complication after liver transplantation [27]. Although superoxide in the toxic oxygen radicals is released from hepatocytes and leukocytes as well as from Kupffer cells, superoxide from Kupffer cells appears to make a more important contribution to cell damage [11]. The inhibitory effects of PTX on the release of superoxides have already been reported to be effective for liver protection [11, 28]. In this study, PTX significantly suppressed serum TNF-a at the 30 min point in both cold and warm ischemic injuries. PTX also improves microcirculation which has been damaged during preservation and reperfusion. Waxman et al. [29] and Coccia et al. [30] reported that PTX administration to rats with hemorrhage shock increased hepatic surface oxygenation and decreased
FIG. 8. Effects of pentoxifylline on GST-a and LDH serum levels during warm ischemia (WI) in transplantation study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr. GST-a levels in rats with PTX pretreatment returned to normal values at the 24 hr point. *P õ 0.05 vs control.
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FIG. 9. Effects of pentoxifylline on AST and ALT serum levels during warm ischemia (WI) in transplantation study. Levels in rats with PTX pretreatment were significantly lower than those of control rats at 30 min and 24 hr. *P õ 0.05, †P õ 0.01 vs control.
whole blood viscosity. Flynn et al. [7] suggested that these facts might be related to the effects of PTX such as improved microvascular blood flow, improved deformability of erythrocytes, inhibition of platelet aggregation, stimulation of prostaglandin production, inhibition of leukocyte adhesion, inhibition of vasoconstriction, and enhancement of fibrinolysis. The hepatocytes in the centrilobular area were susceptible to ischemia, and improvement in microcirculation due to PTX might be the cause of this, as shown by changes in serum GST-a level. This protocol is based on our evaluation of the effects of PTX pretreatment using the serum samples from rats surviving transplantation. Although we did not study the effect on survival following life-threatening injury, our results showed that PTX pretreatment on the donor significantly suppressed hepatic injury after both cold preservation/reperfusion and warm ischemia/ reperfusion in the rat liver transplantation model. In conclusion, PTX pretreatment of the donor displays the potential for protecting the graft liver from procurement/preservation/reperfusion injury in clinical liver transplantation. REFERENCES 1. Clavien, P. A., Harvey, P. R. C., and Strasberg, S. M. Preservation and reperfusion injuries in liver allografts: An overview and synthesis of current studies. Transplantation 53: 957, 1992. 2. Currin, T. R., Reinstein, L. J., Lichtman, S. N., Thurman, R. G., and Lemasters, J. J. Inhibition of tumor necrosis factor release from cultured rat Kupffer cells by agents that reduce graft failure from storage injury. Transplant. Proc. 25: 1631, 1993. 3. Motoyama, K., Kamei, T., Nakafusa, Y., Ueki, M., Hirano, T., Arima, T., Konomi, K., and Tanaka, M. Donor treatment with gadolinium chloride improves survival after transplantation of cold-stored livers reducing Kupffer cell tumor necrosis factor production in rat. Transplant. Proc. 27: 762, 1995. 4. Tilg, H., Eibl, B., Pichl, M., Gachter, A., Herold, M., Brankova, J., Huber, C., and Niederwieser, D. Immune response modulation by pentoxifylline in vitro. Transplantation 56: 196, 1993. 5. Edwards, M. J., Abney D. L., and Miller, F. N. Pentoxifylline inhibits interleukin-2-induced leukocyte-endothelial adherence and reduces systemic toxicity. Surgery 110: 199, 1991.
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6. Ward, A., and Clissold, S. P. Pentoxifylline. A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic efficacy. Drugs 34: 50, 1987. 7. Flynn, W. J., Cryer, H. G., and Garrison, R. N. Pentoxifylline but not Saralasin restores hepatic blood flow after resuscitation from hemorrhagic shock. J. Surg. Res. 50: 616, 1991. 8. Vincenti, F., Danovitch, G. M., Neylan, J. F., Steiner, R. W., Everson, M. P., and Gaston, R. S. Pentoxifylline does not prevent the cytokine-induced first dose reaction following OKT3. A randomized, double-blind placebo-controlled study. Transplantation 61: 573, 1996. 9. Peng, Xing-Xi, Currin, R. T., Thurman, R. G., and Lemasters, J. J. Protection by pentoxifylline against normothermic liver ischemia/reperfusion in rats. Transplantation 59: 1537, 1995. 10. Bachmann, S., Caldwell-Kenkel, J. C., Currin, R. T., Takei, Y., Marzi, I., Thurman, R. G., and Lemasters, J. J. Ultrastructual correlates of liver graft failure from storage injury: Studies of graft protection by Carolina rinse solution and pentoxifylline. Transplant. Proc. 25: 1620, 1993. 11. Kozaki, K., Egawa, H., Bermudez, L., Keefe, E. B., So, S. K., and Esquivel, C. O. Effects of pentoxifylline pretreatment on Kupffer cells in rat liver transplantation. Hepatology 21: 1079, 1995. 12. Kamada, N., and Calne, R. Y. Orthotopic liver transplantation in the rat: Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 28: 47, 1979. 13. Jamieson, N. V., Lindell, S., Sundberg, R., Southard, J. H., and Belzer, F. O. Preservation of the canine liver for 24–48 hours using simple cold storage with UW solution. Transplantation 46: 517, 1988. 14. Hiley, C., Fryer, A., Bell, J., Hume, R., and Strange, R. C. The human glutathione S-transferases: Immunohistochemical studies of the developmental expression of Alpha- and Pi-class isoenzymes in liver. Biochem. J. 254: 255, 1988. 15. Tiainen, P., Hockerstedt, K., and Rosenberg, P. H. Hepatocellular integrity in liver donors and recipients indicated by glutathione transferase alpha. Transplantation 61: 904, 1996. 16. Egawa, H., Martinez, O. M., Quinn, M. B., Villanueva J. C., So, S., Esquivel C. O., and Krams, S. M. Acute liver allograft rejection in the rat: An analysis of the immune response. Transplantation 59: 97, 1995. 17. Bass, N. M., Kirsch, R. E., Tuff, S. A., and Saunders, S. J. Radioimmunoassy of plasma ligandin: A sensitive index of experimental hepatocellular necrosis. Gastroenterology 75: 589, 1978. 18. Caldwell-Kenkel, J. C., Thurman, R. G., and Lemasters, J. J. Selective loss of nonparenchymal cell viability after cold ischemic storage of rat livers. Transplantation 45: 834, 1988.
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19. McKeown, C. M. B., Edwards, V., Phillips, M. J., Harvey, P. R. C., Petrunka, C. N., and Strasberg, S. M. Sinusoidal lining cell damage: The critical injury in cold preservation of liver allografts in the rat. Transplantation 46: 178, 1988. 20. Caldwell-Kenkel, J. C., Currin, R. T., Tanaka, Y., Thurman, R. G., and Lemasters, J. J. Kupffer cell activation and endotherial cell damage after storage of rat livers: Effects of perfusion. Hepatology 13: 83, 1991. 21. Lindert, K. A., Caldwell-Kenkel, J. C., Nukina, S., Lemasters, J. J., and Thurman, R. G. Activation of Kupffer cells on reperfusion following hypoxia: Particle phagocytosis in a low-flow, reflow model. Am. J. Physiol. 262: G345, 1992. 22. Jaeschke, H., and Farhood, A. Neutrophil and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver. Am. J. Physiol. 260: G355, 1991. 23. Lemasters, J. J., and Thurman, R. G. Hypoxia and reperfusion injury to liver. Progr. Liver Dis. 11: 85, 1993. 24. Wake, K., Decker, K., Kirn, A., Knook, D. L., McCuskey, R. S., Bouwens, L., and Wisse, E. Cell biology and the kinetics of Kupffer cells in the liver. Int. Rev. Cytol. 118: 173, 1989. 25. Wang, P., Ba, Z. F., Morrison M. H., Ayala, A., and Chaudry,
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