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Protective Effect of Portal Vein Arterialization in Acute Liver Failure Induced by Hepatectomy in Normal and Fatty Liver Rat
Protective Effect of Portal Vein Arterialization in Acute Liver Failure Induced by Hepatectomy in Normal and Fatty Liver Rat B. Nardo, L. Puviani, D. Prezzi, F. Neri, M. Tsivian, B. Mattioli, M. Pariali, A.M. Pertosa, P. Caraceni, M. Bernardi, and A.D. Pinna ABSTRACT Aim. We sought to determine whether an additional supply of oxygenated blood achieved by partial portal vein arterialization (PPVA) was protective on normal or fatty liver (FL) in rats with acute liver failure (ALF) induced by hepatectomy. Methods. Sprague-Dawley rats with normal or FL were segregated either to receive or not to undergo PPVA after hepatectomy. FL was induced by feeding a choline-deficient diet (5 days). PPVA was performed by anactamasing the left renal artery to the splenic vein with a stent following a left nephrectomy and splenectomy; the control rats underwent left nephrectomy and splenectomy only. Liver injury was evaluated by the serum alanine aminotransferase (ALT) level. The animals were sacrificed at 24 hours, 48 hours, and 7 days to collect blood and liver tissue samples for biochemical analysis. The 7-day survival was assessed in separate experimental groups. Results. PPVA significantly increased PO2 and oxygen saturation in the portal blood compared to non PPVA rats. PPVA significantly improved the 7-day survival compared with controls in both groups: hepatectomy of normal liver (90% vs 30%) and hepatectomy of FL (75% vs 25%). Serum ALT levels were slightly lower in the PPVA groups compared with the non-PPVA groups without a significant difference. Prothrombin activity decreased soon after hepatectomy in the normal and the FL liver groups but recovered rapidly thereafter without differences between the PPVA and non-PPVA treated animals. Conclusion. An additional supply of arterial oxygenated blood through a PPVA promotes rapid resolution of ALF after partial hepatectomy in rats with normal or fatty livers, significantly improving 7-day survivals compared to hepatectomy controls.
A
FATTY LIVER is a common problem in Western countries, affecting about 25% of liver transplant donors and 20% of patients undergoing liver resection. It is associated with primary dysfunction or non-function after transplantation, increasing the risk of postoperative morbidity and mortality after liver resection.1 Hyperoxygenation of the liver through portal vein arterialization has been applied by our group to improve the regenerative capacity of the liver in two clinical cases of acute liver failure (ALF), one owing to hepatic artery thrombosis after liver transplantation with a normal portal venous system2 and the other owing to drug intoxication.3 On the basis of our successful clinical experience, we decided to test the hypothesis that a technical variant of partial portal vein arterialization (PPVA), which increases the oxygen concentration in the portal vein without causing hepatic artery
deprivation, is an effective approach to treat ALF in rats with normal or fatty livers subjected to extended hepatectomy. MATERIALS AND METHODS Male Sprague-Dawley rats of average weight 250 g (range, 230 –280 g) were fed a rodent chow diet and water ad libitum. Animal care From the Department of Surgery and Transplantation (B.N., L.P., D.P., F.N., M.T., A.D.P.), Biomedical Research Center (B.M., M.P.), and the Department of Cardiology, Hepatology and Internal Medicine (A.M.P., P.C., M.B.), University of Bologna, Bologna, Italy. Address reprint requests to Prof Bruno Nardo, Department of Surgery and Transplantation, University of Bologna, Massarenti 9, Bologna 40138, Italy. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.139
Transplantation Proceedings, 38, 3249 –3250 (2006)
3249
3250 and experimental procedures were approved by the institutional ethical committee and conducted according to the guidelines for care and use of laboratory animals approved by our institution. Extended 85% hepatectomy was performed in rats with a normal liver (n ⫽ 30) and a standard 70% partial hepatectomy (PH) in FL (n ⫽ 30), with a PPVA procedure (in 15 or a left nephrectomy and splenectomy only in 15 the control group). A standard 70% partial hepatectomy, used as a liver regeneration model, has no effect on host survival of animals with a normal liver, which is the reason we chose to perform an extended hepatectomy in this group. Briefly, under enflurane anesthesia, the abdomen of the rat was opened to perform a left nephrectomy. Then, 24-G polyethylene tubing was interposed between the left renal artery and the distal part of the splenic vein and closed with a nontraumatic clamp. Afterward, the spleen was removed and a partial hepatectomy performed. Finally, the shunt was opened by removing the clamp and the peritoneal cavity closed in two layers. At the end of the operation, 1.0 mL of heparinized physiologic saline solution was transfused intravenously. To evaluate liver function, the PPVA rats and control rats were sacrificed at 24 hours, 48 hours, and 7 days after surgery to collect blood and liver tissue samples for biochemical analyses. At the time of sacrifice, blood samples (1 mL) collected from the portal vein were immediately assessed for gas content (OSM3 blood gas analyzer; Radiometer, Copenhagen, Denmark). Blood samples (1 mL) were collected from the inferior vena cava at the time of sacrifice for the measurement of serum alanine aminotransferase (ALT), prothrombin time, and serum creatinine. Statistical differences between groups were analyzed by means of a two-way analysis of variance. Survival was analyzed using Kaplan-Meier curves with differences evaluated using the log-rank test. The analysis was performed with SPSS 8.0 statistical package on a personal computer. Data are reported as mean values ⫾ standard errors (SE). Two-tailed P values ⬍ .05 were regarded as significant.
RESULTS
As expected, PPVA induced a significant increase in O2 partial pressure among the normal liver group (at 48 hours, PH ⫹ PPVA ⫽ 70 ⫾ 1.8 mmHg versus PH ⫽ 43 ⫾ 5 mmHg; P ⬍ .05) and oxygen saturation (at 48 hours, PH ⫹ PPVA ⫽ 88.4 ⫾ 3.6% versus PH ⫽ 72 ⫾ 1.4%; P ⬍ .05) with a concomitant decrease of CO2 partial pressure in the portal blood (at 48 hours, PH ⫹ PPVA ⫽ 39.4 ⫾ 0.9 mmHg versus PH ⫽ 61 ⫾ 5.4 mmHg; P ⬍ .05). As to the FL group, an increased O2 partial pressure at 48 hours (PH ⫹ PPVA ⫽ 50.4 ⫾ 5.74 mmHg versus PH ⫽ 33.8 mmHg; P ⬍.05) and oxygen saturation (at 48 hours, PH ⫹ PPVA ⫽ 87.5 ⫾ 3% versus PH ⫽ 67.8%; P ⬍ .05) with a concomitant decrease of CO2 partial pressure in the portal blood (at 48 hours, PH ⫹ PPVA ⫽ 36.8 ⫾ 3.76 mmHg versus PH ⫽ 65 mmHg; P ⬍ .05). These changes were maintained up to 7 days, suggesting that the arteriovenous shunt remained functional for the experiment. As expected, PH in a normal or fatty liver associated or not with PPVA was followed by significant transaminase release, which peaked after 24 hours, namely normal liver, PH ⫹ PPVA ALT ⫽ 775 U/L versus PH, ALT ⫽ 753 U/L. In the fatty liver model, the results were: PH ⫹ PPVA, ALT 860 U/L versus PH, ALT
NARDO, PUVIANI, PREZZI ET AL
⫽ 910 U/L. The values decline thereafter. No significant differences were observed between arterialized and nonarterialized rats. Prothrombin activity decreased soon after PH in both groups: normal liver at 24 hours showed PH ⫹ PPVA 72% versus PH 69%, whereas the displayed fatty liver group at 24 hours PH ⫹ PPVA 68% versus PH 64%. However, the changes recovered rapidly thereafter without differences between the two experimental groups: namely, normal liver at 48 hours: PH ⫹ PPVA 98% versus PH 96% in contrast to fatty liver at 48 hours PH ⫹ PPVA 94.5% versus PH 89%. The most significant results were the survivals: PPVA significantly improved 7-day survivals compared with controls in both groups: extended hepatectomy of normal liver (90% versus 30%) and standard hepatectomy in FL (75% versus 25%). DISCUSSION
As the poorly oxygenated portal vein blood represents approximately 80% of the total liver flow, the PPVA procedure, which brings the oxygen saturation up to a level close to that seen in arterial blood, greatly increases the total amount of oxygen supplied to the liver, thereby probably matching the increased metabolic demand of regenerating cells. It is tempting to speculate that the improved microcirculatory blood flow and tissue oxygenation obtained with PPVA favors oxidative metabolism within hepatocytes, thereby supporting energydependent processes of regeneration.4,5 Ischemic preconditioning and intermittent clamping are currently the only clinical available strategies to decrease liver injury in fatty organs after liver resection.1 In conclusion, arterialization of the portal vein lead to a resolution of ALF induced by extended hepatectomy in the rat with a normal or a fatty liver. This effect was probably mediated by activation of rapid and extensive hepatocyte regeneration; however, further studies are needed to clarify this mechanism. PPVA may represent an additional procedure to improve the outcome of resections or transplantations involving fatty livers. REFERENCES 1. Selzner M, Clavien PA: Fatty liver in liver transplantation and surgery. Semin Liver Dis 21:105, 2001 2. Cavallari A, Nardo B, Caraceni P: Arterialization of the portal vein in a patient with a dearterialized liver graft and massive necrosis. N Engl J Med 345:1352, 2001 3. Nardo B, Montalti R, Puviani L, et al: Portal vein arterialization in a patient with acute liver failure. Transplantation 79:851, 2005 4. Fan Y-D, Praet M, Van Huysse J, et al: Effects of portal vein arterialization on liver regeneration after partial hepatectomy in the rat. Liver Transpl 8:146, 2002 5. Yoshioka S, Miyazaki M, Shimizu H, et al: Hepatic venous hemoglobin oxygen saturation predicts regenerative status of remnant liver after partial hepatectomy in rats. Hepatology 27:1349, 1998
A
FATTY LIVER is a common problem in Western countries, affecting about 25% of liver transplant donors and 20% of patients undergoing liver resection. It is associated with primary dysfunction or non-function after transplantation, increasing the risk of postoperative morbidity and mortality after liver resection.1 Hyperoxygenation of the liver through portal vein arterialization has been applied by our group to improve the regenerative capacity of the liver in two clinical cases of acute liver failure (ALF), one owing to hepatic artery thrombosis after liver transplantation with a normal portal venous system2 and the other owing to drug intoxication.3 On the basis of our successful clinical experience, we decided to test the hypothesis that a technical variant of partial portal vein arterialization (PPVA), which increases the oxygen concentration in the portal vein without causing hepatic artery
deprivation, is an effective approach to treat ALF in rats with normal or fatty livers subjected to extended hepatectomy. MATERIALS AND METHODS Male Sprague-Dawley rats of average weight 250 g (range, 230 –280 g) were fed a rodent chow diet and water ad libitum. Animal care From the Department of Surgery and Transplantation (B.N., L.P., D.P., F.N., M.T., A.D.P.), Biomedical Research Center (B.M., M.P.), and the Department of Cardiology, Hepatology and Internal Medicine (A.M.P., P.C., M.B.), University of Bologna, Bologna, Italy. Address reprint requests to Prof Bruno Nardo, Department of Surgery and Transplantation, University of Bologna, Massarenti 9, Bologna 40138, Italy. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.139
Transplantation Proceedings, 38, 3249 –3250 (2006)
3249
3250 and experimental procedures were approved by the institutional ethical committee and conducted according to the guidelines for care and use of laboratory animals approved by our institution. Extended 85% hepatectomy was performed in rats with a normal liver (n ⫽ 30) and a standard 70% partial hepatectomy (PH) in FL (n ⫽ 30), with a PPVA procedure (in 15 or a left nephrectomy and splenectomy only in 15 the control group). A standard 70% partial hepatectomy, used as a liver regeneration model, has no effect on host survival of animals with a normal liver, which is the reason we chose to perform an extended hepatectomy in this group. Briefly, under enflurane anesthesia, the abdomen of the rat was opened to perform a left nephrectomy. Then, 24-G polyethylene tubing was interposed between the left renal artery and the distal part of the splenic vein and closed with a nontraumatic clamp. Afterward, the spleen was removed and a partial hepatectomy performed. Finally, the shunt was opened by removing the clamp and the peritoneal cavity closed in two layers. At the end of the operation, 1.0 mL of heparinized physiologic saline solution was transfused intravenously. To evaluate liver function, the PPVA rats and control rats were sacrificed at 24 hours, 48 hours, and 7 days after surgery to collect blood and liver tissue samples for biochemical analyses. At the time of sacrifice, blood samples (1 mL) collected from the portal vein were immediately assessed for gas content (OSM3 blood gas analyzer; Radiometer, Copenhagen, Denmark). Blood samples (1 mL) were collected from the inferior vena cava at the time of sacrifice for the measurement of serum alanine aminotransferase (ALT), prothrombin time, and serum creatinine. Statistical differences between groups were analyzed by means of a two-way analysis of variance. Survival was analyzed using Kaplan-Meier curves with differences evaluated using the log-rank test. The analysis was performed with SPSS 8.0 statistical package on a personal computer. Data are reported as mean values ⫾ standard errors (SE). Two-tailed P values ⬍ .05 were regarded as significant.
RESULTS
As expected, PPVA induced a significant increase in O2 partial pressure among the normal liver group (at 48 hours, PH ⫹ PPVA ⫽ 70 ⫾ 1.8 mmHg versus PH ⫽ 43 ⫾ 5 mmHg; P ⬍ .05) and oxygen saturation (at 48 hours, PH ⫹ PPVA ⫽ 88.4 ⫾ 3.6% versus PH ⫽ 72 ⫾ 1.4%; P ⬍ .05) with a concomitant decrease of CO2 partial pressure in the portal blood (at 48 hours, PH ⫹ PPVA ⫽ 39.4 ⫾ 0.9 mmHg versus PH ⫽ 61 ⫾ 5.4 mmHg; P ⬍ .05). As to the FL group, an increased O2 partial pressure at 48 hours (PH ⫹ PPVA ⫽ 50.4 ⫾ 5.74 mmHg versus PH ⫽ 33.8 mmHg; P ⬍.05) and oxygen saturation (at 48 hours, PH ⫹ PPVA ⫽ 87.5 ⫾ 3% versus PH ⫽ 67.8%; P ⬍ .05) with a concomitant decrease of CO2 partial pressure in the portal blood (at 48 hours, PH ⫹ PPVA ⫽ 36.8 ⫾ 3.76 mmHg versus PH ⫽ 65 mmHg; P ⬍ .05). These changes were maintained up to 7 days, suggesting that the arteriovenous shunt remained functional for the experiment. As expected, PH in a normal or fatty liver associated or not with PPVA was followed by significant transaminase release, which peaked after 24 hours, namely normal liver, PH ⫹ PPVA ALT ⫽ 775 U/L versus PH, ALT ⫽ 753 U/L. In the fatty liver model, the results were: PH ⫹ PPVA, ALT 860 U/L versus PH, ALT
NARDO, PUVIANI, PREZZI ET AL
⫽ 910 U/L. The values decline thereafter. No significant differences were observed between arterialized and nonarterialized rats. Prothrombin activity decreased soon after PH in both groups: normal liver at 24 hours showed PH ⫹ PPVA 72% versus PH 69%, whereas the displayed fatty liver group at 24 hours PH ⫹ PPVA 68% versus PH 64%. However, the changes recovered rapidly thereafter without differences between the two experimental groups: namely, normal liver at 48 hours: PH ⫹ PPVA 98% versus PH 96% in contrast to fatty liver at 48 hours PH ⫹ PPVA 94.5% versus PH 89%. The most significant results were the survivals: PPVA significantly improved 7-day survivals compared with controls in both groups: extended hepatectomy of normal liver (90% versus 30%) and standard hepatectomy in FL (75% versus 25%). DISCUSSION
As the poorly oxygenated portal vein blood represents approximately 80% of the total liver flow, the PPVA procedure, which brings the oxygen saturation up to a level close to that seen in arterial blood, greatly increases the total amount of oxygen supplied to the liver, thereby probably matching the increased metabolic demand of regenerating cells. It is tempting to speculate that the improved microcirculatory blood flow and tissue oxygenation obtained with PPVA favors oxidative metabolism within hepatocytes, thereby supporting energydependent processes of regeneration.4,5 Ischemic preconditioning and intermittent clamping are currently the only clinical available strategies to decrease liver injury in fatty organs after liver resection.1 In conclusion, arterialization of the portal vein lead to a resolution of ALF induced by extended hepatectomy in the rat with a normal or a fatty liver. This effect was probably mediated by activation of rapid and extensive hepatocyte regeneration; however, further studies are needed to clarify this mechanism. PPVA may represent an additional procedure to improve the outcome of resections or transplantations involving fatty livers. REFERENCES 1. Selzner M, Clavien PA: Fatty liver in liver transplantation and surgery. Semin Liver Dis 21:105, 2001 2. Cavallari A, Nardo B, Caraceni P: Arterialization of the portal vein in a patient with a dearterialized liver graft and massive necrosis. N Engl J Med 345:1352, 2001 3. Nardo B, Montalti R, Puviani L, et al: Portal vein arterialization in a patient with acute liver failure. Transplantation 79:851, 2005 4. Fan Y-D, Praet M, Van Huysse J, et al: Effects of portal vein arterialization on liver regeneration after partial hepatectomy in the rat. Liver Transpl 8:146, 2002 5. Yoshioka S, Miyazaki M, Shimizu H, et al: Hepatic venous hemoglobin oxygen saturation predicts regenerative status of remnant liver after partial hepatectomy in rats. Hepatology 27:1349, 1998