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Nitric Oxide Inhibition and Consecutive Aspisol Application Show a Prolonged Survival of Orthotopic Transplanted Livers in a Rat Model
Nitric Oxide Inhibition and Consecutive Aspisol Application Show a Prolonged Survival of Orthotopic Transplanted Livers in a Rat Model E. Matevossian, N. Hüser, H. Kern, V. Assfalg, A. Preissel, I. Sinicina, M. Stangl, and S. Thorban ABSTRACT Background. It is generally accepted that nitric oxide (NO) plays a crucial role in acute rejection caused by inflammatory responses. Therefore, the purpose of this study was to investigate the effect on survival following arterialized orthotopic rat liver transplantations (o-RLTx) of NO inhibition and consequent blockade of platelet aggregation by application of Aspisol. Materials and Methods. Inbred LEWIS-(RT1) rats underwent arterialized o-RLTx under ether anesthesia with DA-(RT1av1) rats as organ donors. After liver transplantation, serum parameters were determined and hepatic biopsy specimens were sampled on postoperative days 5, 8, 10, 30, and 90. Sixty-one rats were divided into 5 groups: syngenic controls (group I, n ⫽ 12); allogenic controls (group II, n ⫽ 11); allogenic with FK506 immunosuppression (group III, n ⫽ 12); allogenic with AGH-treatment (group IV, n ⫽ 13); and allogenic with AGH/low- dose Aspisol treatment for 5 days after liver transplantation (group V, n ⫽ 13) (Bayer, Leverkusen, Germany). Results. Rats of group V with AGH/low-dose Aspisol treatment showed significantly longer graft survival (18.2 days ⫹/⫺ 1.8 days) compared with group II rats with untreated grafts (11.3 days ⫹/⫺ 1.7 days) the allogenic group IV with AGH treatment (11.2 days ⫹/⫺ 1.8 days; P ⬍ .05). Histological examination revealed moderate graft rejection among the AGH-treated group IV; however, marked platelet aggregation in sinusoids was present, which was not observed in the AGH/low-dose Aspisol-treated animals (group V). Conclusion. Our data suggested that simultaneous treatment with AGH/low-dose Aspisol leads to a significant increase in survival and inhibition of platelet aggregation in the graft after orthotopic liver transplantation.
T
HE immunomodulatory role of nitric oxide (NO) in graft rejection is still under discussion. Beside vasodilatatory effects, NO is responsible for immunosuppressive and immunostimulatory functions.1–5 The immunologic influence of Aminoguanidinhydrochloride (AGH), a key enzyme of NO synthesis and a selective inducible NO-synthase (i-NOS) inhibitor, is especially controversial.3–7 In the present study of orthotopic rat liver transplantation (oRLTx), the effects of sole i-NOS inhibition, on the one hand, and simultaneous application of drugs to inhibit platelet aggregation, on the other hand, were investigated. MATERIALS AND METHODS Inbred LEWIS-(RT1) rats underwent orthotopic liver transplantation under ether anesthesia, with DA-(RTlav1) rats as organ donors. The liver transplantation was performed using arterialized o-RLTx. Sixty© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 971–973 (2008)
one rats were divided into 5 groups: syngenic controls (group I, n ⫽ 12); allogenic controls (group II, n ⫽ 11); allogenic with FK506 immunosuppression (group III, n ⫽ 12); allogeneic with AGH treatment (group IV, n ⫽ 13); and allogenic with AGH/low- dose-Aspisol treatment (25 mg intravenous 5 days after liver transplantation; group V, n ⫽ 13). Untreated allogenic transplanted rats as well as allogenic transplanted rats (treated with FK506, 1 mg s.c./kg BW per day) and From the Department of Surgery (E.M., N.H., H.K., V.A., M.S., S.T.) and Department of Preclinical Research (A.P.), Technical University of Munich, and Department of Surgical Research (I.S.), Ludwig Maximilians University of Munich, Munich Germany. Address reprint requests to Edouard Matevossian, MD, Department of Surgery, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany. E-mail: matevossian@ chir.med.tu-muenchen.de 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.041 971
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Fig 1. (a) Group IV, mild rejection signs with infiltration of lymphocytes and multifocal hemorrhagic infarction areas with simultaneous aggregation of platelets inside the arterioles and pre– and post–sinusoid venoles. (b) Group V, mild rejection signs without hemorrhagic infarction areas and platelet aggregation inside the vessels in a largely well-preserved parenchyma. syngenic transplanted rats served as control groups. From the day 7 throughout the whole course until rejection, the recipients (in groups IV and V) were treated orally with AGH (1% in tap water). Postoperatively on days 5, 8, 10, 30, and 90, tissue samples were used for H&E staining. The concentration of stable NO breakdown products (NO2/NO3) in serum was determined using high-performance liquid chromatography (HPLC). To monitor hepatocellular damage after acute graft rejection, we used clinical parameters of liver function and histopathologic findings.
RESULTS
Syngeneic transplants as well as allogeneic FK 506 –treated rats demonstrated long-term survivals (⬎90 days). In group V with AGH/low-dose Aspisol treatment, significantly longer survivals (18.2 days ⫹/⫺ 1.8 days) were observed compared with allogeneic untreated grafts (11.3 days ⫹/⫺ 1.7 days) or allogeneic AGH-treated grafts (11.2 days ⫹/⫺ 1.8 days, group V vs II; P ⬍ .05). NO2/NO3 levels were effectively reduced in both groups by AGH treatment (group IV 30.4 ⫹/⫺ 3.7 mol/L and group V 33.1 ⫹/⫺ 1.8 mol/L, nonsignificant). Similar values were observed in the syngenic group I (88.1 ⫹/⫺ 4.1 mol/L). In the allogenic untreated group II, NO2/NO3 levels increased to 190 mol/L (⫹/⫺ 2.6 mol/L) on day 5 and to 681.1 mol/L (⫹/⫺ 2.6 mol/L) on the day 10. Histological examination revealed moderate graft rejection in group IV; however, marked platelet aggregation was present in sinusoids, which was not observed in group V (Fig 1). On day 5 signs of rejection were observed only in 2 liver specimens of group IV and V, whereas other specimens of this group showed acute rejection (level I and II) on day 10. Compared with group II specimens the histological findings of rejection were mild and identified 2 days later. Eight days after transplantation focal necrosis, hemorrhage, and intravascular thrombosis with mild inflammation were observed in some specimens of group IV, but these findings were not seen among rats of group V under combined AGH/low-dose Aspisol therapy. DISCUSSION
In the present study we investigated the influence of NO after arterialized o-RLTx .8 For selective inhibition of the i-NOS, AGH was used and final products of the endogenous NO production (NO2/NO3) were measured using
HPLC.6,7,9 As shown in heart and lung transplantation models, acute graft rejection was associated with elevation of NO (shown by stable final NO2⫺/NO3 products in serum).3–5 Therefore, a reduction of NO with or without simultaneous inhibition of platelet aggregation to be protective for recipients, resulting in prolonged graft function. Among rats of group V, initial organ protective effects of i-NOS inhibition and Aspisol therapy were observed compared with the allogenic control group II, resulting in prolonged graft survival. However, these findings were not present in control groups I and III. The grafts of group IV revealed severe hemorrhage and multifocal infarction within the liver tissue, consistent with an aggregation of platelets inside the arterioles and pre– and post–sinusoidal venoles. In contrast, no platelet aggregation was found in recipients of group V treated with a combination of AGH and low-dose Aspisol. The reduced production of NO under i-NOS inhibition with AGH decreased the antithrombogenic characteristics of endothelium, increasing the tendency of local platelet aggregation.10,11 Under low NO levels, vascular compensation caused by dilatation of vessels does not take place, the vessels remain narrow with increasing aggregation of platelets and ischemia.12 The loss of NO-mediated functions among animals of groups IV and V could be responsible for the observation of histological changes in the grafts. These histological findings were not observed in group V specimens with simultaneous platelet inhibition. The prolonged graft survival was not identical to the syngenic control group, which could be explained by the low selective i-NOS inhibition by AGH avoiding proinflammatory thresholds.4,5 The combination of selective i-NOS inhibition and inhibition of platelet aggregation resulted in significantly prolonged graft survival as well as in loss of a protective effect against acute organ rejection. i-NOS synthesis in platelets participates in the regulation of the platelet/endothelium and platelet/platelet interactions.12 In conclusion, simultaneous treatment with AGH/lowdose Aspisol resulted in a significantly increased animal survival and an inhibition of platelet aggregation inside graft vessels. ACKNOWLEDGMENTS We sincerely thank Dr. Thomas Brill, Dr. Julia Henke (Department of Preclinical Research, Technical University of Munich), Mr. Christian Bald, and Dr. Sebastian Noe for excellent anaesthesiological and technical assistance.
REFERENCES 1. Kuo PC, Alfrey EJ, Abe KY, et al: Cellular localization and effect of nitric oxide synthesis in a rat model of orthotopic liver transplantation. Transpl 61:305, 1996 2. Novotny AR, Emmauel K, Maier S, et al: Cytochrome P450 activity mirrors nitric oxide levels in postoperative sepsis: predictive indicators of lethal outcome. Surgery 141:376, 2003 3. Worrall NK, Boasquevisque CH, Botney M, et al: Inhibition of inducible nitric oxide synthase ameliorates functional and histological changes of acute lung allograft rejection. Transpl 63:1095, 1997
NO INHIBITION AND ASPISOL APPLICATION 4. Worrall NK, Chang K, Suau GM, et al: Inhibition of inducible nitric oxide synthase prevents myocardial and systemic vascular barrier dysfunction during early cardiac allograft rejection. Circ Res 78:769, 1996 5. Worrall NK, Lazenby WD, Misko TP, et al: Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med 181:63, 1995 6. Corbett JA, Tilton RG, Chang K, et al: Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes 41:552, 1992 7. Corbett JA, McDaniel ML: Selective inhibition of inducible nitric oxide synthase by aminoguanidine. Methods Enzymol 268: 398, 1996
973 8. Lee S, Charters AC, Chandler JG, et al: A technique for orthotopic liver transplantation in the rat. Transpl 16:664, 1973 9. Griess P: Ueber einige Azoverbindungen. Ber Dtsch Chem Ges 1:426, 1879 10. Khandoga A, Biberthaler P, Enders G, et al: P-selectin mediates platelet-endothelial cell interactions and reperfusion injury in the mouse liver in vivo. Shock 18:529, 2002 11. Lindemann S, Krämer B, Seizer P, et al: Paltelets, inflammation and atheroscle-rosis. J Thromb Haemost 5:203, 2007 12. Schmitt-Sody M, Gottschalk O, Metz P, et al: Endothelial iNOS versus platelet iNOS: responsibility for the platelet/leucocyte endothelial cell interaction in murine antigen induced arthritis in vivo. Inflamm Res 56:262, 2007
T
HE immunomodulatory role of nitric oxide (NO) in graft rejection is still under discussion. Beside vasodilatatory effects, NO is responsible for immunosuppressive and immunostimulatory functions.1–5 The immunologic influence of Aminoguanidinhydrochloride (AGH), a key enzyme of NO synthesis and a selective inducible NO-synthase (i-NOS) inhibitor, is especially controversial.3–7 In the present study of orthotopic rat liver transplantation (oRLTx), the effects of sole i-NOS inhibition, on the one hand, and simultaneous application of drugs to inhibit platelet aggregation, on the other hand, were investigated. MATERIALS AND METHODS Inbred LEWIS-(RT1) rats underwent orthotopic liver transplantation under ether anesthesia, with DA-(RTlav1) rats as organ donors. The liver transplantation was performed using arterialized o-RLTx. Sixty© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 971–973 (2008)
one rats were divided into 5 groups: syngenic controls (group I, n ⫽ 12); allogenic controls (group II, n ⫽ 11); allogenic with FK506 immunosuppression (group III, n ⫽ 12); allogeneic with AGH treatment (group IV, n ⫽ 13); and allogenic with AGH/low- dose-Aspisol treatment (25 mg intravenous 5 days after liver transplantation; group V, n ⫽ 13). Untreated allogenic transplanted rats as well as allogenic transplanted rats (treated with FK506, 1 mg s.c./kg BW per day) and From the Department of Surgery (E.M., N.H., H.K., V.A., M.S., S.T.) and Department of Preclinical Research (A.P.), Technical University of Munich, and Department of Surgical Research (I.S.), Ludwig Maximilians University of Munich, Munich Germany. Address reprint requests to Edouard Matevossian, MD, Department of Surgery, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany. E-mail: matevossian@ chir.med.tu-muenchen.de 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.041 971
MATEVOSSIAN, HU¨SER, KERN ET AL
972
Fig 1. (a) Group IV, mild rejection signs with infiltration of lymphocytes and multifocal hemorrhagic infarction areas with simultaneous aggregation of platelets inside the arterioles and pre– and post–sinusoid venoles. (b) Group V, mild rejection signs without hemorrhagic infarction areas and platelet aggregation inside the vessels in a largely well-preserved parenchyma. syngenic transplanted rats served as control groups. From the day 7 throughout the whole course until rejection, the recipients (in groups IV and V) were treated orally with AGH (1% in tap water). Postoperatively on days 5, 8, 10, 30, and 90, tissue samples were used for H&E staining. The concentration of stable NO breakdown products (NO2/NO3) in serum was determined using high-performance liquid chromatography (HPLC). To monitor hepatocellular damage after acute graft rejection, we used clinical parameters of liver function and histopathologic findings.
RESULTS
Syngeneic transplants as well as allogeneic FK 506 –treated rats demonstrated long-term survivals (⬎90 days). In group V with AGH/low-dose Aspisol treatment, significantly longer survivals (18.2 days ⫹/⫺ 1.8 days) were observed compared with allogeneic untreated grafts (11.3 days ⫹/⫺ 1.7 days) or allogeneic AGH-treated grafts (11.2 days ⫹/⫺ 1.8 days, group V vs II; P ⬍ .05). NO2/NO3 levels were effectively reduced in both groups by AGH treatment (group IV 30.4 ⫹/⫺ 3.7 mol/L and group V 33.1 ⫹/⫺ 1.8 mol/L, nonsignificant). Similar values were observed in the syngenic group I (88.1 ⫹/⫺ 4.1 mol/L). In the allogenic untreated group II, NO2/NO3 levels increased to 190 mol/L (⫹/⫺ 2.6 mol/L) on day 5 and to 681.1 mol/L (⫹/⫺ 2.6 mol/L) on the day 10. Histological examination revealed moderate graft rejection in group IV; however, marked platelet aggregation was present in sinusoids, which was not observed in group V (Fig 1). On day 5 signs of rejection were observed only in 2 liver specimens of group IV and V, whereas other specimens of this group showed acute rejection (level I and II) on day 10. Compared with group II specimens the histological findings of rejection were mild and identified 2 days later. Eight days after transplantation focal necrosis, hemorrhage, and intravascular thrombosis with mild inflammation were observed in some specimens of group IV, but these findings were not seen among rats of group V under combined AGH/low-dose Aspisol therapy. DISCUSSION
In the present study we investigated the influence of NO after arterialized o-RLTx .8 For selective inhibition of the i-NOS, AGH was used and final products of the endogenous NO production (NO2/NO3) were measured using
HPLC.6,7,9 As shown in heart and lung transplantation models, acute graft rejection was associated with elevation of NO (shown by stable final NO2⫺/NO3 products in serum).3–5 Therefore, a reduction of NO with or without simultaneous inhibition of platelet aggregation to be protective for recipients, resulting in prolonged graft function. Among rats of group V, initial organ protective effects of i-NOS inhibition and Aspisol therapy were observed compared with the allogenic control group II, resulting in prolonged graft survival. However, these findings were not present in control groups I and III. The grafts of group IV revealed severe hemorrhage and multifocal infarction within the liver tissue, consistent with an aggregation of platelets inside the arterioles and pre– and post–sinusoidal venoles. In contrast, no platelet aggregation was found in recipients of group V treated with a combination of AGH and low-dose Aspisol. The reduced production of NO under i-NOS inhibition with AGH decreased the antithrombogenic characteristics of endothelium, increasing the tendency of local platelet aggregation.10,11 Under low NO levels, vascular compensation caused by dilatation of vessels does not take place, the vessels remain narrow with increasing aggregation of platelets and ischemia.12 The loss of NO-mediated functions among animals of groups IV and V could be responsible for the observation of histological changes in the grafts. These histological findings were not observed in group V specimens with simultaneous platelet inhibition. The prolonged graft survival was not identical to the syngenic control group, which could be explained by the low selective i-NOS inhibition by AGH avoiding proinflammatory thresholds.4,5 The combination of selective i-NOS inhibition and inhibition of platelet aggregation resulted in significantly prolonged graft survival as well as in loss of a protective effect against acute organ rejection. i-NOS synthesis in platelets participates in the regulation of the platelet/endothelium and platelet/platelet interactions.12 In conclusion, simultaneous treatment with AGH/lowdose Aspisol resulted in a significantly increased animal survival and an inhibition of platelet aggregation inside graft vessels. ACKNOWLEDGMENTS We sincerely thank Dr. Thomas Brill, Dr. Julia Henke (Department of Preclinical Research, Technical University of Munich), Mr. Christian Bald, and Dr. Sebastian Noe for excellent anaesthesiological and technical assistance.
REFERENCES 1. Kuo PC, Alfrey EJ, Abe KY, et al: Cellular localization and effect of nitric oxide synthesis in a rat model of orthotopic liver transplantation. Transpl 61:305, 1996 2. Novotny AR, Emmauel K, Maier S, et al: Cytochrome P450 activity mirrors nitric oxide levels in postoperative sepsis: predictive indicators of lethal outcome. Surgery 141:376, 2003 3. Worrall NK, Boasquevisque CH, Botney M, et al: Inhibition of inducible nitric oxide synthase ameliorates functional and histological changes of acute lung allograft rejection. Transpl 63:1095, 1997
NO INHIBITION AND ASPISOL APPLICATION 4. Worrall NK, Chang K, Suau GM, et al: Inhibition of inducible nitric oxide synthase prevents myocardial and systemic vascular barrier dysfunction during early cardiac allograft rejection. Circ Res 78:769, 1996 5. Worrall NK, Lazenby WD, Misko TP, et al: Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med 181:63, 1995 6. Corbett JA, Tilton RG, Chang K, et al: Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes 41:552, 1992 7. Corbett JA, McDaniel ML: Selective inhibition of inducible nitric oxide synthase by aminoguanidine. Methods Enzymol 268: 398, 1996
973 8. Lee S, Charters AC, Chandler JG, et al: A technique for orthotopic liver transplantation in the rat. Transpl 16:664, 1973 9. Griess P: Ueber einige Azoverbindungen. Ber Dtsch Chem Ges 1:426, 1879 10. Khandoga A, Biberthaler P, Enders G, et al: P-selectin mediates platelet-endothelial cell interactions and reperfusion injury in the mouse liver in vivo. Shock 18:529, 2002 11. Lindemann S, Krämer B, Seizer P, et al: Paltelets, inflammation and atheroscle-rosis. J Thromb Haemost 5:203, 2007 12. Schmitt-Sody M, Gottschalk O, Metz P, et al: Endothelial iNOS versus platelet iNOS: responsibility for the platelet/leucocyte endothelial cell interaction in murine antigen induced arthritis in vivo. Inflamm Res 56:262, 2007