- Email: [email protected]
Portal Vein Oxygen Supply Through a Liver Extracorporeal Device to Treat Acute Liver Failure in Swine Induced by Subtotal Hepatectomy: Preliminary Data
Portal Vein Oxygen Supply Through a Liver Extracorporeal Device to Treat Acute Liver Failure in Swine Induced by Subtotal Hepatectomy: Preliminary Data B. Nardo, R. Montalti, L. Puviani, V. Pacilè, P. Beltempo, R. Bertelli, M. Licursi, F. Neri, D. Prezzi, M. Tsivian, M. Pariali, and D. Cianciavicchia ABSTRACT Aim. To determine whether the increase of oxygen supply in the portal system by a liver extracorporeal (L.E.O.NARDO) device is effective in treating swine with subtotal hepatectomy leading to acute liver failure (ALF). Methods. Eight swine with ALF induced by 85% to 90% liver resection and 5 minutes of ischemia-reperfusion injury were randomly divided into two groups: four animals received L.E.O.NARDO treatment and four swine were not treated (control group). Blood was withdrawn from the iliac artery and reversed in the portal venous system. An extracorporeal device was interposed between the outflow and the inflow in order to monitor the hemodynamic parameters. Each treatment lasted 6 hours. Serum and liver samples were collected in both groups. The survival was assessed at 1 week. Results. L.E.O.NARDO treatment yielded beneficial effects for subtotal hepatectomyinduced ALF in swine with decreased serum transaminases as compared with the untreated group. International normalized ratio recovered rapidly in the L.E.O.NARDO group, remaining significantly lower than in untreated animals. The 7-day survival of L.E.O.NARDO group swine was significantly higher than that of untreated animals, with a significant difference. Three swine in the L.E.O.NARDO group survived 1 week while none of the swine in the control group were alive at that time. Conclusions. Oxygen supply in the portal vein through the L.E.O.NARDO device is easily applicable, efficacious, and safe and may represent a novel approach for ALF in swine induced by subtotal liver resection.
S
EVERAL ARTIFICIAL AND BIOARTIFICIAL liver support devices have been developed with the aim of temporarily replacing the complex functions of the liver such as synthesis and detoxification and bridging patients with acute liver failure (ALF) until an organ becomes available or to provide liver support for those who have a chance of spontaneous recovery. However, the “bridging systems” currently available for the treatment of ALF aim to support liver function, while a waiting spontaneous regeneration.1,2 Experimental studies have shown that increasing the oxygen supply to the liver through portal vein arterialization (PVA) enhanced liver regeneration after partial hepatectomy.3,4 Recently we reported a patient with massive necrosis due to drug intoxication who was rescued by PVA, thus avoiding liver transplantation.5 This study was performed to assess the efficacy and feasibility of a new device, namely L.E.O.NARDO which
means Liver Extracorporeal Oxygenator NARDO, in a subtotal hepatectomy-induced ALF swine model. Our hypothesis was that increasing the oxygen to the liver through the portal system also with a temporary extracorporeal device might promote hepatocyte regeneration and improve the survival rate of animals subjected to subtotal hepatectomy. METHODS Eight female white swine weighing 28 to 33 kg were obtained from the Montroni Laboratories (Dozza Imolese, Bologna, Italy). They From the Department of Surgery and Transplantations, S. Orsola Hospital, University of Bologna, Bologna, Italy. Address reprint requests to Bruno Nardo, MD, PhD, Department of Surgery and Transplantation, S. Orsola Hospital, Via Massarenti 9, 40138 Bologna, Italy. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.03.057
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 38, 1190 –1192 (2006)
PORTAL VEIN OXYGEN SUPPLY
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were fed and watered ad libitum. Animal care and experimental procedures were conducted according to the guidelines for the care and use of animals approved by our institution. The animals underwent 85% to 90% liver resection as described by Court et al6: all but the caudate lobe was removed. Before surgery the right lateral lobe underwent 5 minutes of ischemia-reperfusion damage. The ALF swine were randomly divided into two groups: the L.E.O.NARDO group (n ⫽ 4) received an extracorporeal oxygenation treatment; the non-L.E.O.NARDO group (n ⫽ 4) were not treated (control group). The operations were well tolerated in both groups. In the L.E.O.NARDO treatment blood was withdrawn from the iliac artery and reinfused into the portal venous trunk at a flow rate of 40 mL/min. An extracorporeal device with a peristaltic pump was interposed between the outflow and inflow in order to monitor the hemodynamic parameters (Bellco S.r.l., Mirandola, Italy). Each treatment lasted 6 hours. The survival of each animal was assessed. Blood samples (5 mL) were collected for biochemical analysis from the inferior vena cava before and after liver resection in both groups, before and after the extracorporeal oxygenation in the L.E.O.NARDO group, and at the time of sacrifice in both groups. We assessed the serum alanine aminotransferase (ALT), total bilirubin, and international normalized ratio (INR) levels. Blood samples (1 mL) were collected from the portal vein and immediately assessed for gas content (OSM3 blood gas analyzer, Radiometer, Copenhagen, Denmark). Liver samples were collected for histological analysis, before and after liver resection in both groups, before and after extracorporeal oxygenation in the L.E.O.NARDO group, and at the time of sacrifice. Liver samples were fixed in either 10% phosphate-buffered formalin or 70% ethanol and embedded in paraffin. Formalin-fixed sections were stained with hematoxylin-eosin. Statistical differences between the groups were determined using analysis of variance and paired Students t test. Statistical significance was considered when P ⬍ .05.
RESULTS
As expected we observed a significant increase in the partial pressure of O2 (64 ⫾ 6 vs 42 ⫾ 3 mm Hg) and oxygen saturation (91% ⫾ 4%, vs 71% ⫾ 2%) with a concomitant decrease in partial pressure of CO2 (41 ⫾ 5 vs 61 ⫾ 3 mm Hg) in the portal blood among the L.E.O.NARDO as compared with an untreated group at the end of treatment. The L.E.O.NARDO treatment yielded beneficial effects for subtotal hepatectomy-induced ALF in swine at 18 hours: there were significantly decreased levels of serum ALT levels as compared with the untreated animals (54 ⫾ 21 vs 123 ⫾
36 U/L). INR recovered rapidly in the L.E.O.NARDO group, remaining significantly lower than in the untreated animals at 18 hours (1.3 ⫾ 0.7 vs 2.8 ⫾ 1.2). No significant differences were seen in either group for the levels of total bilirubin at all experimental times (Table 1). The swine survival time of the L.E.O.NARDO group was significantly higher compared with that of the untreated animals. Three ALF swine in the L.E.O.NARDO group survived 1 week (3/4; 75%). The only animal that did not survive died from gastric hemorrhage. In contrast, all ALF swine in the control group died within 36 hours. At sacrifice, the three animals of the L.E.O.NARDO group that survived 7 days displayed a large volume of the remnant liver with an average weight of 228 ⫾ 22 g. DISCUSSION
Previous experimental studies have shown that increasing the oxygen supply to the liver by arterialization of the portal vein enhanced the regenerative capacity of the liver after extended hepatectomy.3,4 The high oxygen saturation in the portal blood flow seemed to improve oxidative metabolism within the hepatocytes, thereby supporting the energydependent processes of regeneration through increased production of adenosine triphosphate. The present study demonstrates that in a subtotal-induced swine model of ALF, an extracorporeal variant of the portal vein procedure, increased the oxygen concentration in the portal vein without causing hepatic artery deprivation, leading to recovery of hepatic function even when performed after the onset of extended liver resection. This curative effect is probably due to the early, rapid, and massive induction of hepatocyte regeneration. The exact mechanism whereby PVA stimulates liver regeneration is not currently known and further studies are needed. However, it appears reasonable to hypothesize that the promotion of regeneration is mediated by the extra oxygen supplied to the liver through the arterovenous shunt. The L.E.O.NARDO device supplying the arterial blood to the portal system might therefore provide a novel, alternative approach in patients with ALF to enable the native liver to recover. With this in mind, we designed an extracorporeal device to apply in the clinical setting (CPT, IB 2004/001142) to increase the oxygen
Table 1. Biochemical Parameter Values Before (Basal ⴝ 0 Hours) and After 6 and 18 Hours and 7 Days in Swine with SH Treated or Not With the L.E.O.NARDO Device Group
SH
SH ⫹ L.E.O.NARDO
Time
0 6 18 7 0 6 18 7
h h h d h h h d
Ammonia (mol/L)
ALT (U/L)
T.Bil. (mg/dL)
␥-GT (U/L)
INR
142 ⫾ 10 385 ⫾ 53* 334 ⫾ 48* — 137 ⫾ 12 242 ⫾ 39* 185 ⫾ 44* 139 ⫾ 17
42 ⫾ 5 82 ⫾ 24 123 ⫾ 36* — 44 ⫾ 3 63 ⫾ 17 54 ⫾ 21* 48 ⫾ 9
0.9 ⫾ 1 1.3 ⫾ 0.8 4.3 ⫾ 2.5 — 1.0 ⫾ 0.8 1.2 ⫾ 0.7 3.9 ⫾ 2.3 2.2 ⫾ 1.2
52 ⫾ 9 55 ⫾ 10 68 ⫾ 18 — 48 ⫾ 8 49 ⫾ 12 51 ⫾ 14 55 ⫾ 9
1.0 ⫾ 0.4 1.5 ⫾ 0.5 2.8 ⫾ 1.2* — 1.0 ⫾ 0.3 1.2 ⫾ 0.4 1.3 ⫾ 0.7* 1.0 ⫾ 0.4
SH, subtotal hepatectomy; ALT, alanine aminotransferase; T.Bil, total bilirubin; ␥-GT, ␥-glutamyltransferase; INR, international normalized ratio. *P ⬍ .05.
1192
concentration in the portal vein through percutaneous access thus avoiding surgery. REFERENCES 1. Kjaergard LL, Liu J, Nielsen B, et al: Artificial and bioartificial support system for acute and acute-on-chronic liver failure: a sistematic review. JAMA 289:217, 2003 2. O’Obeirne JP, Wendon JA: Liver support. Int J Artif Organs 28:477, 2005
NARDO, MONTALTI, PUVIANI ET AL 3. Shimizu Y, Miyazaki M, Shimizu H, et al: Beneficial effects of arterialization of the portal vein on extended hepatectomy. Br J Surg 87:784, 2000 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. Nardo B, Montalti R, Puviani L, et al: Portal vein arterialization in a patient with acute liver failure. Transplantation 79:851, 2005 6. Court F, Laws P, Morrison C, et al: Subtotal hepatectomy: a porcine model for the study of liver regeneration. J Surg Res 116:181, 2004
S
EVERAL ARTIFICIAL AND BIOARTIFICIAL liver support devices have been developed with the aim of temporarily replacing the complex functions of the liver such as synthesis and detoxification and bridging patients with acute liver failure (ALF) until an organ becomes available or to provide liver support for those who have a chance of spontaneous recovery. However, the “bridging systems” currently available for the treatment of ALF aim to support liver function, while a waiting spontaneous regeneration.1,2 Experimental studies have shown that increasing the oxygen supply to the liver through portal vein arterialization (PVA) enhanced liver regeneration after partial hepatectomy.3,4 Recently we reported a patient with massive necrosis due to drug intoxication who was rescued by PVA, thus avoiding liver transplantation.5 This study was performed to assess the efficacy and feasibility of a new device, namely L.E.O.NARDO which
means Liver Extracorporeal Oxygenator NARDO, in a subtotal hepatectomy-induced ALF swine model. Our hypothesis was that increasing the oxygen to the liver through the portal system also with a temporary extracorporeal device might promote hepatocyte regeneration and improve the survival rate of animals subjected to subtotal hepatectomy. METHODS Eight female white swine weighing 28 to 33 kg were obtained from the Montroni Laboratories (Dozza Imolese, Bologna, Italy). They From the Department of Surgery and Transplantations, S. Orsola Hospital, University of Bologna, Bologna, Italy. Address reprint requests to Bruno Nardo, MD, PhD, Department of Surgery and Transplantation, S. Orsola Hospital, Via Massarenti 9, 40138 Bologna, Italy. E-mail: [email protected]
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.03.057
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1190
Transplantation Proceedings, 38, 1190 –1192 (2006)
PORTAL VEIN OXYGEN SUPPLY
1191
were fed and watered ad libitum. Animal care and experimental procedures were conducted according to the guidelines for the care and use of animals approved by our institution. The animals underwent 85% to 90% liver resection as described by Court et al6: all but the caudate lobe was removed. Before surgery the right lateral lobe underwent 5 minutes of ischemia-reperfusion damage. The ALF swine were randomly divided into two groups: the L.E.O.NARDO group (n ⫽ 4) received an extracorporeal oxygenation treatment; the non-L.E.O.NARDO group (n ⫽ 4) were not treated (control group). The operations were well tolerated in both groups. In the L.E.O.NARDO treatment blood was withdrawn from the iliac artery and reinfused into the portal venous trunk at a flow rate of 40 mL/min. An extracorporeal device with a peristaltic pump was interposed between the outflow and inflow in order to monitor the hemodynamic parameters (Bellco S.r.l., Mirandola, Italy). Each treatment lasted 6 hours. The survival of each animal was assessed. Blood samples (5 mL) were collected for biochemical analysis from the inferior vena cava before and after liver resection in both groups, before and after the extracorporeal oxygenation in the L.E.O.NARDO group, and at the time of sacrifice in both groups. We assessed the serum alanine aminotransferase (ALT), total bilirubin, and international normalized ratio (INR) levels. Blood samples (1 mL) were collected from the portal vein and immediately assessed for gas content (OSM3 blood gas analyzer, Radiometer, Copenhagen, Denmark). Liver samples were collected for histological analysis, before and after liver resection in both groups, before and after extracorporeal oxygenation in the L.E.O.NARDO group, and at the time of sacrifice. Liver samples were fixed in either 10% phosphate-buffered formalin or 70% ethanol and embedded in paraffin. Formalin-fixed sections were stained with hematoxylin-eosin. Statistical differences between the groups were determined using analysis of variance and paired Students t test. Statistical significance was considered when P ⬍ .05.
RESULTS
As expected we observed a significant increase in the partial pressure of O2 (64 ⫾ 6 vs 42 ⫾ 3 mm Hg) and oxygen saturation (91% ⫾ 4%, vs 71% ⫾ 2%) with a concomitant decrease in partial pressure of CO2 (41 ⫾ 5 vs 61 ⫾ 3 mm Hg) in the portal blood among the L.E.O.NARDO as compared with an untreated group at the end of treatment. The L.E.O.NARDO treatment yielded beneficial effects for subtotal hepatectomy-induced ALF in swine at 18 hours: there were significantly decreased levels of serum ALT levels as compared with the untreated animals (54 ⫾ 21 vs 123 ⫾
36 U/L). INR recovered rapidly in the L.E.O.NARDO group, remaining significantly lower than in the untreated animals at 18 hours (1.3 ⫾ 0.7 vs 2.8 ⫾ 1.2). No significant differences were seen in either group for the levels of total bilirubin at all experimental times (Table 1). The swine survival time of the L.E.O.NARDO group was significantly higher compared with that of the untreated animals. Three ALF swine in the L.E.O.NARDO group survived 1 week (3/4; 75%). The only animal that did not survive died from gastric hemorrhage. In contrast, all ALF swine in the control group died within 36 hours. At sacrifice, the three animals of the L.E.O.NARDO group that survived 7 days displayed a large volume of the remnant liver with an average weight of 228 ⫾ 22 g. DISCUSSION
Previous experimental studies have shown that increasing the oxygen supply to the liver by arterialization of the portal vein enhanced the regenerative capacity of the liver after extended hepatectomy.3,4 The high oxygen saturation in the portal blood flow seemed to improve oxidative metabolism within the hepatocytes, thereby supporting the energydependent processes of regeneration through increased production of adenosine triphosphate. The present study demonstrates that in a subtotal-induced swine model of ALF, an extracorporeal variant of the portal vein procedure, increased the oxygen concentration in the portal vein without causing hepatic artery deprivation, leading to recovery of hepatic function even when performed after the onset of extended liver resection. This curative effect is probably due to the early, rapid, and massive induction of hepatocyte regeneration. The exact mechanism whereby PVA stimulates liver regeneration is not currently known and further studies are needed. However, it appears reasonable to hypothesize that the promotion of regeneration is mediated by the extra oxygen supplied to the liver through the arterovenous shunt. The L.E.O.NARDO device supplying the arterial blood to the portal system might therefore provide a novel, alternative approach in patients with ALF to enable the native liver to recover. With this in mind, we designed an extracorporeal device to apply in the clinical setting (CPT, IB 2004/001142) to increase the oxygen
Table 1. Biochemical Parameter Values Before (Basal ⴝ 0 Hours) and After 6 and 18 Hours and 7 Days in Swine with SH Treated or Not With the L.E.O.NARDO Device Group
SH
SH ⫹ L.E.O.NARDO
Time
0 6 18 7 0 6 18 7
h h h d h h h d
Ammonia (mol/L)
ALT (U/L)
T.Bil. (mg/dL)
␥-GT (U/L)
INR
142 ⫾ 10 385 ⫾ 53* 334 ⫾ 48* — 137 ⫾ 12 242 ⫾ 39* 185 ⫾ 44* 139 ⫾ 17
42 ⫾ 5 82 ⫾ 24 123 ⫾ 36* — 44 ⫾ 3 63 ⫾ 17 54 ⫾ 21* 48 ⫾ 9
0.9 ⫾ 1 1.3 ⫾ 0.8 4.3 ⫾ 2.5 — 1.0 ⫾ 0.8 1.2 ⫾ 0.7 3.9 ⫾ 2.3 2.2 ⫾ 1.2
52 ⫾ 9 55 ⫾ 10 68 ⫾ 18 — 48 ⫾ 8 49 ⫾ 12 51 ⫾ 14 55 ⫾ 9
1.0 ⫾ 0.4 1.5 ⫾ 0.5 2.8 ⫾ 1.2* — 1.0 ⫾ 0.3 1.2 ⫾ 0.4 1.3 ⫾ 0.7* 1.0 ⫾ 0.4
SH, subtotal hepatectomy; ALT, alanine aminotransferase; T.Bil, total bilirubin; ␥-GT, ␥-glutamyltransferase; INR, international normalized ratio. *P ⬍ .05.
1192
concentration in the portal vein through percutaneous access thus avoiding surgery. REFERENCES 1. Kjaergard LL, Liu J, Nielsen B, et al: Artificial and bioartificial support system for acute and acute-on-chronic liver failure: a sistematic review. JAMA 289:217, 2003 2. O’Obeirne JP, Wendon JA: Liver support. Int J Artif Organs 28:477, 2005
NARDO, MONTALTI, PUVIANI ET AL 3. Shimizu Y, Miyazaki M, Shimizu H, et al: Beneficial effects of arterialization of the portal vein on extended hepatectomy. Br J Surg 87:784, 2000 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. Nardo B, Montalti R, Puviani L, et al: Portal vein arterialization in a patient with acute liver failure. Transplantation 79:851, 2005 6. Court F, Laws P, Morrison C, et al: Subtotal hepatectomy: a porcine model for the study of liver regeneration. J Surg Res 116:181, 2004