Hartwig Bunzendahl

Transplantation einer Spenderleber auf zwei Empfänger (Splitting-Transplantation) - Eine neue Methode in der Weiterentwicklung der Lebersegmenttransplantation

Tumor surgery in this field is no longer such a high risk as previously. Prolonged survival can be achieved by resection of hepatocellular carcinomas in non-cirrhotic livers (3-year survival 58%, n = 54 patients) and for colorectal liver metastases (3-year survival 44%, n = 124 patients). But surgery is rarely successful for the most frequent type of liver malignancy, the hepatocellular carcinoma in cirrhosis. Central bile duct carcinomas are now resected more frequently than in the past. Liver grafting seems indicated in special cases of liver and bile duct tumors. The future developments of operating on the in situ-perfused liver was discussed and the first operation on an ex situ-liver was demonstrated.SummaryA donor liver was divided in such a way that the left part (segment 11 and III without caval vein) could be transplanted into a child, the right part (segment I, IV, V to VIII) into an adult successfully. Common bile duct and common hepatic artery remained with the left part of the liver, portal vein with the right one. In the recipient of the left part of the liver the own caval vein was preserved and anastomosed with the left hepatic vein; the other anastomoses were carried out in the typical way. In the recipient of the right part of the liver the right hepatic artery of the graft was anastomosed with the recipients common hepatic artery using a saphenous interponate. Two separate intrahepatic bile ducts were anastomosed with a Roux-en-Y loop of the jejunum. The other anastomoses were carried out in the typical way. Thus the possibility of using one donor liver for two recipients (splitting transplantation) has been demonstrated.ZusammenfassungEin Spenderleberorgan wurde so getrennt, daß der linke Teil (Segment II und III ohne Vena cava) auf ein Kind, der rechte Teil (Segment 1, IV, V bis VIII) auf einen Erwachsenen erfolgreich transplantiert werden konnte. Choledochus und Arteria hepatica communis bzw. propria blieben beim linken Leberteil, Vena portae beim rechten. Beim Empfänger der linken Seite blieb die eigene Vena cava erhalten; in sie wurde die linke Vena hepatica anastomosiert; die übrigen Anastomosen wurden in üblicher Weise durchgeführt. Beim Empfänger des rechten Leberteiles wurde die spenderseitige Arteria hepatica dextra mit einem Saphenainterponat verlängert und mit der Arteria hepatica communis des Empfängers anastomosiert; zwei getrennte Hepaticusäste wurden mit einer Jejunumschlinge anastomosiert. Die übrigen Anastomosen wurden in typischer Weise ausgeführt. Die Möglichkeit der Verwendung einer Spenderleber für zwei Empfänger (Splitting-Transplantation) ist damit gezeigt.

Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation: Role of Kupffer cells

BACKGROUND The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here. METHODS Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared. RESULTS In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied. CONCLUSION These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.

Intravenous glycine improves survival in rat liver transplantation

In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-α after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.

Liver regeneration is suppressed in small-for-size liver grafts after transplantation: involvement of c-Jun N-terminal kinase, cyclin D1, and defective energy supply.

Background. Small-for-size liver grafts have decreased survival compared to full-size grafts. This study investigated mechanisms of suppression of liver regeneration in small-for-size grafts. Methods. Rat liver explants were reduced in size to 50% and implanted into recipients of different body weights, resulting in graft weight/standard liver weights of ∼50% (half-size) and ∼25% (quarter-size). Results. Hepatic cellular 5-bromo-2′-deoxyuridine (BrdU) incorporation increased from 0.2% after sham operation to 2%, 18%, and 1.2% in full-size, half-size, and quarter-size grafts, respectively. Graft weight did not increase in full- and quarter-size grafts but increased 40% in half-size grafts. By contrast, apoptosis remained low (≤0.7%) and stem cells did not increase in all conditions. Phospho-c-Jun increased 27-fold in half-size grafts but only sevenfold in quarter-size grafts. Activating protein-1 activation increased 14-fold in half-size grafts but only fivefold in quarter-size grafts. Cyclin D1 (CyD1), which was barely detectable in full- and quarter-size grafts, increased 8.3-fold in half-size grafts. Adenosine 5′-triphosphate (ATP) per gram tissue decreased 70% in quarter-size grafts. Treatment of quarter-size grafts with radical scavenging C. sinenesis polyphenols (20 μg/ml) increased BrdU labeling and weight gain to 35% and 56%, respectively, reversed inhibition of CyD1 expression, c-Jun phosphorylation, and AP-1 activation in quarter-size grafts compared to half-size grafts, and restored ATP levels to 75%. Conclusions. Liver regeneration is stimulated in half-size grafts but suppressed in quarter-size grafts. Defective liver regeneration in small grafts is associated with an inhibition of the c-Jun N-terminal kinase/c-Jun and CyD1 pathways and compromised energy production.

Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat.

Background. Oxygen‐derived free radicals play a central role in ischemia/reperfusion injury after organ transplantation and are degraded by endogenous radical scavengers such as superoxide dismutase (SOD). Overexpression of SOD by delivery of the cytosolic SOD gene with an adenovirus (Ad.SOD1) decreases organ injury and increases survival in a rat model of liver transplantation. However, it is unclear which of the three isoforms of SOD provides the most protective effect. The purpose of this study was to identify the isoform with the highest effectiveness against ischemia/reperfusion injury after transplantation of fatty livers, which are particularly susceptible. Methods. Donor rats were given ethanol by gavage before harvest to induce steatotic livers. Some of the donors were infected with adenoviruses expressing either the gene lacZ encoding bacterial &bgr;‐galactosidase (Ad.lacZ), Ad.SOD1, Ad.SOD2 (mitochondrial isoform), or Ad.SOD3 (extracellular isoform). After transplantation, SOD activity in liver, survival, histopathology, transaminases, and activation of nuclear factor (NF)‐&kgr;B, I&kgr;B kinase, Jun‐N‐terminal kinase (JNK), and tumor necrosis factor (TNF)‐&agr; were evaluated. Results. Ad.SOD1 treatment increased survival, blunted transaminase release, and reduced necrosis, whereas Ad.SOD3 had no protective effect. Ad.SOD2 was not as protective as Ad.SOD1. Ad.SOD1 reduced the activation of NF‐&kgr;B, blunted JNK activity, and reduced TNF‐&agr; activity. Ad.SOD2 treatment resulted in lower kinase, TNF‐&agr;, and NF‐&kgr;B activities but was not as effective as Ad.SOD1. I&kgr;B kinase activity was not affected. Conclusion. This study demonstrates that cytosolic SOD represents the most effective isoform of SOD to protect transplanted livers from failure; this may be related to lowered NF‐&kgr;B and JNK activities because of reduced oxygen‐derived radical production.

Delivery of Cu/Zn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat.

BACKGROUND Oxygen-derived free radicals play a central role in pathomechanisms of reperfusion injury after organ transplantation. Endogenous radical scavenger systems such as superoxide dismutase (SOD) degrade toxic radicals; however, SOD is degraded rapidly when given exogenously. Therefore, the hypothesis that treatment of the donor liver with an adenoviral vector encoding the Cu/Zn-SOD gene (Ad-SOD1) would lead to permanent gene expression and therefore protect the organ against injury and increase survival in a rat model of liver transplantation was tested. METHODS Some donors were infected with Ad-SOD1, whereas untreated grafts and livers infected with the indicator gene lacZ encoding bacterial beta-galactosidase (Ad-lacZ) served as controls. After orthotopic liver transplantation, survival, serum transaminases, and histopathology were evaluated. RESULTS Approximately 80% of hepatocytes expressed beta-galactosidase 72 hr after injection of Ad-lacZ. Moreover, SOD1 gene expression and activity were increased 3- and 10-fold in the Ad-SOD1 group, respectively. After transplantation, 20-25% of rats treated with Ad-lacZ survived. In contrast, all SOD1-treated animals survived. Transaminases measured 8 hr after transplantation in Ad-SOD1 rats were only 40% of those in controls, which increased 40-fold above normal values. Approximately 20% of hepatocytes in untreated and Ad-lacZ-infected organs were necrotic 8 hr after reperfusion, whereas necrosis was nearly undetectable in grafts from rats treated with Ad-SOD1. CONCLUSIONS This study provides clear evidence for the first time that gene therapy with Ad-SOD1 increases survival and decreases hepatic injury after liver transplantation. Genetic modification of the liver represents a future approach to protect organs against injury where oxygen-derived free radicals are involved.

Prevention of cyclosporine-induced nephrotoxicity with dietary glycine

BACKGROUND The nonessential amino acid glycine has been used previously to prevent hypoxic and ischemic injury to kidney tissue in vitro. Furthermore, it was recently shown that glycine prevents activation of macrophages and neutrophils in vitro. Because there is some evidence that the immunosuppressant cyclosporine causes nephrotoxicity through a hypoxia-reoxygenation mechanism that could involve infiltration and activation of macrophages and neutrophils, we hypothesized that dietary glycine could prevent this injury. METHODS Rats were fed a diet containing glycine (5%) or a control diet for 3 days before cyclosporine treatment. To produce nephrotoxicity, cyclosporine (25 mg/kg daily by gavage) was administered for 28 days while animals were maintained on glycine or control diets. Serum creatinine, urea, glomerular filtration rates, and kidney histology were evaluated in different treatment groups. RESULTS All rats gained weight; however, overall weight gain in the cyclosporine, glycine, and cyclosporine+glycine groups was significantly less by about 40% compared with the control group. Diet consumption was not statistically different between the groups. As expected, cyclosporine caused kidney damage in the rats fed control diet, reflected in significantly elevated serum urea and creatinine. In addition, cyclosporine treatment decreased glomerular filtration rate by nearly 70%, caused proximal tubular dilation and necrosis as well as increased macrophage and neutrophil infiltration into the kidney. Dietary glycine prevented or minimized kidney damage due to cyclosporine in all parameters studied nearly completely. Furthermore, feeding glycine for up to 1 month had no detrimental effect on kidney function. CONCLUSIONS Dietary glycine is a safe and effective treatment to reduce the nephrotoxicity of cyclosporine.

Renal transplant recipients - Selection and management of risk factors

SummaryDecision making in renal transplantation is largely based on considerations concerning risk factors in the treatment of end-stage renal disease. The attempt to identify minimize individual risks has proven to be a key to success in renal transplantation.

Evaluation of the liver graft before procurement : Significance of arterial ketone body ratio in brain-dead patients

Hepatic energy metabolism was assessed by measuring the blood ketone body ratio (KBR), that is, the ratio of acetoacetate to β-hydroxybutyrate in the arterial blood, in 31 brain-dead patients in an intensive care unit (ICU) in Japan and in 25 donors just before procurement of the liver for transplantation in Germany. In the study in Japan, 7 of the 12 brain-dead patients treated with highdose catecholamine showed significantly decreased KBRs, revealing the detrimental effect of catecholamine on livermmetabolism. In contrast, 8 of the 9 untreated patients with blood pressure below 80 mm Hg showed almost normal KBRs. In the 25 donors in Germany, KBR was maintained within the normal range. Based upon conventional criteria, 21 livers were selected for use and the other 4 were discarded. Nineteen of the grafts were able to normalize KBR within 24 h after reperfusion, while 2 failed to function and required a second transplantation. It was suggested that a KBR in the normal range in donors is a prerequisite to immediate recovery of metabolic function of the liver graft after transplantation, and that hypotensive donors as a potential source of liver grafts may warrant further study.

Graft survival is improved by hepatic denervation before organ harvesting.

BACKGROUND In a recent study, disturbances of hepatic microcirculation at harvesting caused by in situ organ manipulation dramatically reduced survival after a liver transplant. Because hepatic innervation is involved in the regulation of liver hemodynamics, the effect of denervation before harvesting was assessed here. METHODS The livers were harvested from female Lewis rats (200-230 g) within 25 min. Briefly, after minimal dissection during the first 12 min, the livers were either manipulated gently or left alone for 13 min. Subsequently, an orthotopic liver transplant was performed after 1 hr of storage in cold UW solution. Some donors livers underwent microsurgical denervation before harvesting or rats were given hexamethonium (10 mg/kg, i.v.), a ganglionic blocking agent. RESULTS In the nonmanipulated group, survival was 100% after the transplant; however, gentle manipulation decreased survival by about 50%. Furthermore, manipulation elevated serum transaminases and bilirubin 6- to 8-fold 8 hr after the transplant and caused necrosis of about 25% of hepatocytes. After organ harvesting, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased 2- to 4-fold, and the maximal increase of surface fluorescence was blunted about 2-fold. Pimonidazole binding, which reflects tissue hypoxia, was increased 2-fold by manipulation. Denervation of the liver before organ harvesting or treatment with hexamethonium prevented the effects of organ manipulation on all parameters studied. CONCLUSION These data indicate for the first time that hepatic denervation before organ harvesting prevents detrimental effects of brief, gentle manipulation of the liver during harvesting on survival after the transplant. This is consistent with the hypothesis that organ manipulation disturbs the hepatic microcirculation and causes hypoxia at harvesting using mechanisms dependent on innervation.