Nitric oxide reduces organ injury and enhances regeneration of re ...

Original article

Nitric oxide reduces organ injury and enhances regeneration of reduced-size livers by increasing hepatic arterial flow D. Cantre´ 1 , H. Schuett2 , A. Hildebrandt1 , S. Dold3 , M. D. Menger3 , B. Vollmar1 and C. Eipel1 1

Institute for Experimental Surgery, University of Rostock, Rostock, 2 Department of Cardiology and Angiology, Hanover Medical School, Hanover, and Institute for Clinical and Experimental Surgery, University of Saarland, Hamburg, Germany Correspondence to: Dr C. Eipel, Institute for Experimental Surgery, University of Rostock, D-18055 Rostock, Germany (e-mail: [email protected])

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Background: Reduced-size livers suffer from portal hyperperfusion, diminished arterial blood flow and

the risk of postoperative liver injury. The aim of this experimental study was to unravel the role of nitric oxide in this setting. Methods: Rats underwent 85 per cent partial hepatectomy and either substitution of nitric oxide with molsidomine or inhibition of nitric oxide synthase (NOS) with N G -nitro-L-arginine methyl ester. Untreated hepatectomized animals served as controls and unresected animals as the sham group. Results: Ultrasonic flowmetry following partial hepatectomy revealed a marked increase in portal venous inflow with a concomitant decrease in hepatic arterial inflow. Nitric oxide substitution counteracted the decline in hepatic arterial inflow and caused a significantly greater increase in cell proliferation after partial hepatectomy compared with control or NOS-inhibited animals. Hepatectomized animals further profited from nitric oxide substitution, as indicated by reduced aminotransferase release and improved liver function. Conclusion: Nitric oxide improves the postoperative course of rats with reduced-size livers by modulating hepatic macrohaemodynamics and mediating regeneration and cytoprotection, but not by reducing hepatic hyperperfusion and the accompanying sinusoidal shear stress. Paper accepted 27 March 2008 Published online 16 April 2008 in Wiley InterScience (www.bjs.co.uk). DOI: 10.1002/bjs.6139

Introduction

Extended liver resection and liver transplantation are often the only reliable strategies for the management of severe liver disease or liver tumours. The main challenge in liver transplantation is the acquisition of adequate donor grafts for patients on waiting lists, who greatly outnumber the number of available donor organs1,2 . Liver surgeons have recently established deceased-donor split and livingdonor graft transplantation as well accepted procedures for helping to meet the high demand for donor livers1 – 3 . The extent of liver resection in the removal of primary or secondary liver tumours is determined by tumour mass and dissemination4 . Modern surgical techniques, such as portal vein embolization before hepatectomy5,6 , are widening the definition of resectability, enabling potentially curative The Editors are satisfied that all authors contributed significantly to this publication

Copyright  2008 British Journal of Surgery Society Ltd Published by John Wiley & Sons Ltd

surgical treatment even in patients with multiple bilobular tumours6,7 . Finding the right balance between secure removal of all malignant tissue, including a microscopically clear margin and sufficient residual liver mass, still is a difficult task8 . All of these surgical procedures are likely to result in reduced-size livers and benefit from the ability of liver tissue to regenerate and regain the missing parenchymal mass9,10 . One of the most common complications in reduced-size livers is the so-called small-for-size syndrome11 – 13 . This is characterized by severe liver dysfunction or even liver failure in the first postoperative week, and is consequently associated with a high mortality rate in the absence of donor organs for (re)transplantation13,14 . Ongoing debate about the causes of small-for-size syndrome has focused mainly on portal hyperperfusion with high intravascular shear stresses13,15 – 17 . Accordingly, surgical techniques that decrease portal venous inflow, such as portosystemic British Journal of Surgery 2008; 95: 785–792

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´ H. Schuett, A. Hildebrandt, S. Dold, M. D. Menger, B. Vollmar and C. Eipel D. Cantre,

shunting or splenic artery ligation, have resulted in an improvement in this syndrome18,19 . As a consequence of portal venous hyperperfusion, hepatic arterial hypoperfusion may occur and contribute to the small-for-size syndrome, as a result of insufficient tissue oxygenation20,21 . In addition, others have demonstrated an imbalance of vasoactive mediators in reduced-size livers, with decreased expression of endothelial nitric oxide synthase (eNOS)22 . The lack of vasodilating nitric oxide with impairment of the hepatic haemodynamics could be of fundamental importance in the development of small-for-size syndrome. Furthermore, in a well defined concentration range nitric oxide is known to act as a trigger for liver regeneration23,24 and is regarded as a cytoprotective agent25 . Thus, lack of nitric oxide may also contribute to the pathogenesis of the small-for-size syndrome as a result of reduced regeneration and diminished cytoprotection. Evaluation of the role of nitric oxide in this setting might enable a therapeutic approach to be identified that would decrease the incidence of the small-for-size syndrome. The 85 per cent liver resection model was thus used to study the influence of nitric oxide on perfusion and regeneration, as well as on function and cell damage, of reduced-size livers by either substitution of nitric oxide with molsidomine or inhibition of NOS with N G -nitro-L-arginine methyl ester (L-NAME). Methods

Reduced-size liver regeneration model Experiments were performed in accordance with German laws on the protection of animals and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Male Wistar rats (bodyweight 230–350 g; Charles River Laboratories, Sulzfeld, Germany) were subjected to 85 per cent hepatectomy. Under isoflurane (1·8 volume per cent) anaesthesia and after midline laparotomy, the liver was freed from its ligaments. The left lateral lobe, median lobe and right superior lobe were then resected by placing 4/0 suture ties most proximally to their origin (left lateral and median lobes) or at a short distance from their origin (right superior lobe), guaranteeing adequate blood flow to the neighbouring residual lobe. The resected lobes were weighed and tissue samples taken. After irrigation, the abdomen was closed with running 5/0 sutures. Animals were allowed to recover from anaesthesia and surgery under a red warming lamp. At 24 h after partial hepatectomy (during maximal DNA synthesis26 ), rats were killed with an overdose of pentobarbital. To study liver cell proliferation, 5bromo-2 -deoxyuridine (BrdU, 50 mg/kg bodyweight) was Copyright  2008 British Journal of Surgery Society Ltd Published by John Wiley & Sons Ltd

administered by intraperitoneal injection 1 h before death. The residual liver was excised and weighed, and liver tissue and blood samples were sent for further analysis.

Experimental groups Four experimental groups of ten animals each were studied. Animals underwent either substitution of nitric oxide with molsidomine (10 mg/kg bodyweight intraperitoneally) or inhibition of NOS with L-NAME (100 mg/kg bodyweight intraperitoneally). Molsidomine is converted enzymatically in the liver to yield the active metabolite 3-morpholinosydnonimine (SIN-1), which subsequently releases nitric oxide27 , whereas L-NAME is a false arginine analogue that competitively inhibits NOS isoenzymes28 . Control animals received equal amounts of physiological saline. The sham group received physiological saline and underwent the surgical protocol but without resection. Pharmacological treatment was given 24 h before and at the time of partial hepatectomy. Molsidomine (Sigma-Aldrich Chemie, Munich, Germany) and L-NAME (Axxora Life ¨ Sciences, Grunberg, Germany) were freshly dissolved in physiological saline on the day of the experiment. Dose and mode of application were chosen in accordance with previously published work29 .

Histology and immunohistochemistry of liver tissue Liver tissue was fixed in 4 per cent phosphate-buffered formalin for 2–3 days and embedded in paraffin. From the paraffin-embedded tissue blocks, 4-µm sections were cut and stained with haematoxylin and eosin for histological analysis. For immunohistochemical demonstration of BrdU incorporation, sections collected on poly-L-lysinecoated glass slides were treated by microwave for antigen unmasking. Mouse monoclonal anti-BrdU (1 : 50; Dako Cytomation, Hamburg, Germany) was used as primary antibody and incubated for 18 h at 4° C. After equilibration at room temperature, sections were incubated with horseradish peroxidase-conjugated goat antimouse IgG (1 : 100; Dako Cytomation) for 30 min; 3,3 -diaminobenzidine was used as chromogen. The sections were then counterstained with haemalaun and examined by light microscopy (Axioskop 40; Zeiss, ¨ Gottingen, Germany). BrdU-positive nuclei were counted (cells/mm2 ) within 30 consecutive high-power fields (×40 objective, numerical aperture 0·65).

Blood analysis The extent of hepatocellular damage was assessed by spectrophotometric determination of plasma aminotransferase www.bjs.co.uk

British Journal of Surgery 2008; 95: 785–792

Nitric oxide and reduced-size livers

and bilirubin levels using commercially available reaction kits (Roche Diagnostics, Mannheim, Germany).

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estimated from VQ and blood viscosity (τ = 4ηVQ/πr3 ). Blood viscosity was assumed according to the findings of Vogel et al.32 .

Intravital fluorescence microscopy Additional animals (four from each group) were used to study red blood cell velocity, diameter, volumetric blood flow and shear stress in hepatic sinusoids and postsinusoidal venules early after partial hepatectomy by intravital fluorescence microscopy. Pharmacological treatment with molsidomine, L-NAME or saline was administered as described above. Spontaneously breathing pentobarbitalanaesthetized (55 mg/kg bodyweight intraperitoneally) rats were prepared for intravital fluorescence microscopy as described previously17,30 . After laparotomy, partial hepatectomy was performed, as described above. The remaining superior caudate lobe was exteriorized on a plasticine stage and the liver surface was covered with a glass slide. At 30 min after partial hepatectomy, sinusoids and postsinusoidal venules were recorded in ten observational fields each. Using a fluorescence microscope equipped with a 100-W mercury lamp (Axiotech vario; Zeiss, Jena, Germany) and a filter for blue light epi-illumination (excitation/emission wavelength 450–490/> 520 nm), microscopic images were taken by a water immersion objective (×20/0·50 W or ×40/0·80 W; Zeiss), recorded by a CCD video camera (FK 6990AIQ; Pieper, Berlin, Germany) and transferred to a video system (S-VHS Panasonic AG 7350-E; Matsushita, Tokyo, Japan). Fluorescein–isothiocyanate dextran (5 per cent, 0·1 ml/100 g bodyweight, molecular weight 150 kDa; Sigma-Aldrich Chemie) was used to enhance tissue contrast, enabling assessment of perfusion within individual microvessels29,30 .

Quantitative video analysis Assessment of hepatic microcirculation parameters was performed off-line by frame-to-frame analysis of the videotaped images at a 424-fold (×20/0·50 W) or 823fold (×40/0·80 W) magnification, using a computerassisted image analysis system with a 19-inch monitor (CapImage; Zeintl, Heidelberg, Germany). For estimation of volumetric blood flow (VQ), red blood cell velocity (VRBC ) and the respective diameter were measured in ten individual sinusoids in mid-zonal regions and in ten postsinusoidal venules per animal. VRBC was assessed by means of the line-shift-diagram method. VQ in individual microvessels was estimated from VRBC and microvascular cross-sectional area (πr 2 ) (VQ = VRBC πr 2 ) and is given in picolitres per second (pl/s)31 . Sinusoidal shear stress was Copyright  2008 British Journal of Surgery Society Ltd Published by John Wiley & Sons Ltd

Parameters of hepatic circulation Additional animals (five per group) were used to investigate portal venous and hepatic arterial inflow, intrahepatic perfusion and tissue oxygenation. Pharmacological treatment with molsidomine, L-NAME and saline was administered as described above. Spontaneously breathing pentobarbital-anaesthetized rats were catheterized and laparotomized, followed by surgical intervention. Intrahepatic perfusion was measured by means of laser Doppler flowmetry (Peri Flux PF3; Perimed, Stockholm, Sweden) with the probe placed on the left lateral liver lobe. The portal vein and hepatic artery were prepared and ultrasonic perivascular flow probes (Transonic Systems, Ithaca, New York, USA) connected to a flowmeter (T402 Animal Research Flowmeter; Transonic Systems) were placed to record hepatic inflow for 30 min. Liver tissue oxygenation were assessed by means of a flexible polyethylene microcatheter Clark-type P O2 probe (diameter 0·5 mm, length 200 mm, Licox Systems; GMS, Kiel-Mielkendorf, Germany) which was positioned inside a liver lobe, penetrating the organ surface33 .

Statistical analysis Data are presented as mean(s.e.m.). After performing a test for normal distribution using the Kolmogorov–Smirnov test, statistical differences between the groups were determined by one-way ANOVA followed by the post hoc Holm–Sidak test for pairwise comparisons. When criteria for parametric tests were not met, Kruskal–Wallis ANOVA on ranks, followed by Dunn’s test, was employed. P < 0·050 was considered statistically significant. Statistical analysis was carried out with the SigmaStat v. 3·5 and SigmaPlot v. 10 · 0 software package (Jandel Scientific, San Rafael, California, USA). Results

Effect of nitric oxide on liver damage and hepatocyte proliferation As indicated by increased aspartate aminotransferase (AST) levels at 24 h after surgical intervention, extended partial hepatectomy resulted in significant liver damage in all groups, compared with unresected sham-operated animals (Fig. 1a). Both inhibition and substitution of nitric oxide resulted in a significant reduction of liver damage compared www.bjs.co.uk



2500

500

2000

400 BrdU-positive cells (mm2)

AST (units/l)

788

1500

1000

0

0

Sham

Control

NO−

AST activity

Control

NO−

NO+

Quantitative analysis of 5-bromo-2 -deoxyuridine (BrdU) immunohistochemistry of liver tissue taken 24 h after 85 per cent hepatectomy and administration of N G -nitro-L-arginine methyl ester (nitric oxide inhibition (NO−) group), molsidomine (nitric oxide substitution (NO+) group) or physiological saline (control group). Animals without hepatectomy served as the sham group. Values are mean(s.e.m.) of at least seven animals per group. * P < 0·050 versus sham, †P < 0·050 versus control, ‡P < 0·050 versus NO− (ANOVA) Fig. 2

30 Bilirubin (µmol/l)

Sham

NO+

40

20

400 BrdU-positive cells/mm2 (Fig. 2). In contrast, inhibition of NOS by L-NAME did not affect the number of proliferating cells in comparison with controls.

10

0 Sham

b

200

100

500

a

300

Control

NO−

NO+

Bilirubin levels

a Plasma aspartate aminotransferase (AST) levels and b systemic bilirubin concentration in rats 24 h after 85 per cent hepatectomy and administration of N G -nitro-L-arginine methyl ester (nitric oxide inhibition (NO−) group), molsidomine (nitric oxide substitution (NO+) group) or physiological saline (control group). Animals without hepatectomy served as the sham group. Values are mean(s.e.m.) of seven to nine animals per group. * P < 0·050 versus sham, †P < 0·050 versus control (ANOVA on ranks) Fig. 1

with that in resected but untreated control animals. This was reflected by significantly lower plasma AST levels and a 20–25 per cent reduction in bilirubin concentration (Fig. 1a, b). BrdU staining revealed a high number of proliferating hepatocytes in untreated control animals, reflecting a marked resection-induced proliferation stimulus. Nitric oxide supplementation further enhanced liver cell proliferation, with an additional twofold increase to about Copyright  2008 British Journal of Surgery Society Ltd Published by John Wiley & Sons Ltd

Effect of nitric oxide on hepatic macrohaemodynamics In relation to liver mass, portal venous inflow after resection was increased about twofold in the control and nitric oxide substitution (molsidomine) groups. In contrast, it was not significantly increased in the NOS-inhibited (L-NAME) group (Fig. 3a). Hepatic arterial inflow decreased slightly after resection. Blockade of nitric oxide further decreased the hepatic arterial inflow by up to twofold when compared with the unresected sham group. The reduction in hepatic arterial inflow was counteracted completely by substitution of nitric oxide, with animals in this group showing arterial blood flow levels similar to those in unresected sham animals (Fig. 3b). The resultant total hepatic inflow was significantly increased in control and molsidomine-treated animals (Fig. 3c), similar to the findings for portal venous inflow.

Effect of nitric oxide on hepatic microhaemodynamics Hepatic tissue perfusion, as assessed by laser Doppler flowmetry, was significantly increased in control and www.bjs.co.uk



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0·25

3 2 1

0·20 0·15 0·10 0·05

0

Sham Control NO−

NO+

b

Portal venous flow

4 3 2 1 0

0 Sham Control NO−

a

5 Total hepatic flow (ml/min·g liver tissue)

Hepatic arterial flow (ml/min·g liver tissue)

Portal venous flow (ml/min·g liver tissue)

4

NO+

Sham Control NO−

c

Hepatic arterial flow

NO+

Total hepatic flow

Ultrasonic flowmetry of a portal venous, b hepatic arterial and c total hepatic flow 1 h after 85 per cent hepatectomy and administration of N G -nitro-L-arginine methyl ester (nitric oxide inhibition (NO−) group), molsidomine (nitric oxide substitution (NO+) group) or physiological saline (control group). Animals without hepatectomy served as the sham group. Values are mean(s.e.m.) of four animals per group. * P < 0·010 versus sham, †P < 0·050 versus control, ‡P < 0.050 versus NO− (ANOVA)

a

300 200 100 0

Sham Control NO−

Intrahepatic perfusion

NO+

5000

30

4000

25

b

Sinusoidal shear stress

Hepatic tissue oxygenation (mmHg)

400

Shear stress in sinusoids (mPa)

Intrahepatic perfusion (units)

Fig. 3

3000 2000 1000 0

Sham Control NO−

20 15 10 5 0

NO+

c

Sham Control NO−

NO+

Hepatic oxygenation

Fig. 4 a Intrahepatic perfusion as assessed by laser Doppler flowmetry, b sinusoidal shear stress and c hepatic tissue oxygenation 1 h after 85 per cent hepatectomy and administration of N G -nitro-L-arginine methyl ester (nitric oxide inhibition (NO−) group), molsidomine (nitric oxide substitution (NO+) group) or physiological saline (control group). Animals without resection served as the sham group. Laser Doppler flowmetry and tissue oxygenation were assessed in one cohort of animals; shear stress was calculated from sinusoidal volumetric blood flow and diameter (see Table 1 for data), assessed from another cohort of animals by intravital microscopy. Values are mean(s.e.m.) of five animals per group. * P < 0·050 versus sham, †P < 0·050 versus control, ‡P < 0·050 versus NO− (ANOVA)

molsidomine-treated animals, whereas it remained unchanged in the L-NAME group when compared with sham animals (Fig. 4a). Quantitative analysis of hepatic microhaemodynamics by in vivo fluorescence microscopy confirmed the marked hyperperfusion of the liver following hepatectomy, as reflected by a twofold rise in VRBC and a two- to threefold rise in VQ in the sinusoids and postsinusoidal venules of control and L-NAMEtreated animals (Table 1). In nitric oxide-substituted animals, hyperperfusion was even more pronounced, with a sevenfold and threefold increase in VQ in hepatic sinusoids and postsinusoidal venules respectively. In addition, the microvascular diameters in these animals were about 20 per cent larger than those of control rats (Table 1). As a consequence, intrahepatic shear stress was markedly increased in all groups after resection, although there were no significant differences between the groups

(Fig. 4b). Hepatic tissue oxygenation was decreased, to approximately 15 mmHg in all groups, after resection (Fig. 4c).


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Discussion

This study has shown that nitric oxide improves the postoperative course of rats with reduced-size livers by modulating hepatic macrohaemodynamics and participating in regeneration and cytoprotection. The theory that hepatic hyperperfusion leads to increased sinusoidal shear stress and results in smallfor-size syndrome in the setting of a reduced-size liver11 – 13,15 – 17 was supported by the present results, which showed that sinusoidal shear stress was more than doubled in association with severe liver damage in animals undergoing 85 per cent hepatectomy. Further British Journal of Surgery 2008; 95: 785–792


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Table 1

Intravital fluorescence microscopic data for hepatic microcirculation following partial hepatectomy in the four groups Sinusoids

Sham Control NO inhibition (L-NAME) NO substitution (molsidomine)

Postsinusoidal venules

VRBC (µm/s)

Diameter (µm)

VQ (pl/s)

VRBC (µm/s)

Diameter (µm)

VQ (pl/s)

199(15) 544(86)* 485(32)* 746(64)*†‡

7·5(0·3) 8·4(0·5) 8·8(0·6)* 9·9(0·2)*†

9·9(1·1) 31·4(5·5)* 30·1(2·5)* 65·1(6·5)*†‡

680(89) 1327(70)* 1205(84)* 1486(59)*

31·6(4·3) 30·8(2·7) 34·3(3·4) 37·4(1·3)

624(184) 1099(147) 1164(186)* 1786(177)*†‡

Values are mean(s.e.m.) (four animals per group). VRBC , red cell blood velocity; VQ, volumetric blood flow; NO, nitric oxide; L-NAME, N G -nitro-L-arginine methyl ester. *P < 0·050 versus sham, †P < 0·050 versus control, ‡P < 0·050 versus NO inhibition (ANOVA).

evidence that increased sinusoidal shear stress is one of the main causes of liver damage in reduced-size livers has been provided by studies showing that surgical reduction in hepatic blood flow can improve outcome after extended hepatectomy18,19 . The present results indicate that this hypothesis needs to be reconsidered. Even though substitution of nitric oxide did influence hepatic circulation, decrease liver damage and improve liver function, this was not achieved by a reduction in sinusoidal shear stress, which is also known to be a strong trigger of early hepatic regeneration24,29,34 . Thus, the positive effects of decreasing blood flow and thus shear stress during surgery might actually be achieved at the expense of regenerative capacity. As imbalance of vasorelaxing and vasoconstricting mediators is considered to be an important pathogenetic feature in reduced-size livers22 , the present authors aimed to establish a counterbalancing strategy by augmenting the vasodilatory response via substitution of nitric oxide. Animals treated with the nitric oxide donor molsidomine had less liver damage together with higher regeneration and improved liver function compared with resected controls. These findings are in line with recent studies of rats undergoing 70 per cent partial hepatectomy24 , suggesting regenerative and protective properties for nitric oxide on the liver. Most of these studies also imply that blockade of nitric oxide leads to further liver damage. In contrast, the present study has shown that inhibiting the release of nitric oxide with L-NAME also resulted in significantly decreased liver damage. This rarely described positive effect of blocking nitric oxide is still a matter for debate. Some groups have ascribed this positive effect to reduced oxidative stress35 or additional inhibition of inducible NOS36 . The present authors further consider that the observed decrease in hepatic inflow after blockade of NOS with L-NAME is a result of systemic vasoconstriction and thus of reduced splanchnic blood flow, which first reduces the portal venous inflow and then the accompanying intrahepatic shear stress. This vasoconstricting effect has a detrimental effect on the supply of oxygen-rich

blood via the hepatic artery, and thus may reduce the positive effects. In contrast to the maintenance of liver cell integrity as a result of decreased intrahepatic shear stress, inhibition of nitric oxide activity did not improve liver regeneration upon extended hepatectomy, as was seen following enhancement of nitric oxide availability by molsidomine. The improved postoperative course following substitution of nitric oxide could be due to one or more of the following possible mechanisms. First, nitric oxide is known to be a cytoprotective and regeneration-triggering agent as a result of its influence on the signalling cascades of both pathways23 – 25 . Second, as shown by the present results, increased portal venous inflow after partial hepatectomy is paralleled by reduced inflow via the hepatic artery. Substitution of nitric oxide restored hepatic arterial flow to supranormal conditions. It can be predicted that the increased oxygen supply due to the nitric oxide-dependent maintenance of hepatic arterial inflow will result in increased energy for regeneration after hepatectomy. In line with this, Shimizu and colleagues37 observed an increase in adenosine 5 -triphosphate activity in rats with extended hepatectomy and arterialized portal venous inflow, although the energy charge (ratio of phosphorylated forms of adenosine) remained unchanged37 . Correspondingly, the partial pressure of oxygen in hepatic tissue was approximately 15 mmHg in all resected groups, despite large differences in the flow of oxygen-rich hepatic arterial blood. This may be related to the fact that the regenerating liver of animals in the nitric oxide substitution group required increased amounts of oxygen, as it has been shown that hepatic venous haemoglobin oxygen saturation predicts the regenerative status of remnant liver after partial hepatectomy in rats38 . Increasing the oxygen supply to the tissue, for instance by portal vein arterialization or by breathing hyperbaric oxygen before or after partial hepatectomy, has already been described as a potential strategy to support regeneration of the reduced-size liver37,39,40 . These findings are supported by the present results, which


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show that the increase in hepatic arterial oxygen supply associated with nitric oxide is important for hepatocellular integrity and regenerative capacity. Acknowledgements

The authors thank Berit Blendow, Doris Butzlaff, Dorothea Frenz and Maren Nerowski (Institute of Experimental Surgery, University of Rostock) for excellent technical assistance, and Dr Ingo Steinke (Institute for Mathematics, University of Rostock) for helpful comments on the statistical analysis. This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Germany (DFG Vo 450/7-3). References 1 Ghobrial RM, Busuttil RW. Challenges of adult living-donor liver transplantation. J Hepatobiliary Pancreat Surg 2006; 13: 139–145. 2 Lopez PM, Martin P. Update on liver transplantation: indications, organ allocation, and long-term care. Mt Sinai J Med 2006; 73: 1056–1066. 3 Fan ST. Live donor liver transplantation in adults. Transplantation 2006; 82: 723–732. 4 Lupinacci R, Penna C, Nordlinger B. Hepatectomy for resectable colorectal cancer metastases – indicators of prognosis, definition of respectability, techniques and outcomes. Surg Oncol Clin N Am 2007; 16: 493–506. 5 Yokoyama Y, Nagino M, Nimura Y. Mechanisms of hepatic regeneration following portal vein embolization and partial hepatectomy: a review. World J Surg 2007; 31: 367–374. 6 Vauthey JN, Zorzi D, Pawlik TM. Making unresectable hepatic colorectal metastases resectable – does it work? Semin Oncol 2005; 32(Suppl 9): S118–S122. 7 Shimada H, Tanaka K, Matsuo K, Togo S. Treatment for multiple bilobar liver metastases of colorectal cancer. Langenbecks Arch Surg 2006; 391: 130–142. 8 Mullin EJ, Metcalfe MS, Maddern GJ. How much liver resection is too much? Am J Surg 2005; 190: 87–97. 9 Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997; 276: 60–66. 10 Fausto N, Campbell JS, Riehle KJ. Liver regeneration. Hepatology 2006; 43(Suppl 1): S45–S53. 11 Pan GD, Yan LN. Problems in adult living donor liver transplantation using the right hepatic lobe. Hepatobiliary Pancreat Dis Int 2006; 5: 345–349. 12 Goldstein MJ, Salame E, Kapur S, Kinkhabwala M, LaPointe-Rudow D, Harren NPP et al. Analysis of failure in living donor liver transplantation: differential outcomes in children and adults. World J Surg 2003; 27: 356–364. 13 Dahm F, Georgiev P, Clavien PA. Small-for-size syndrome after partial liver transplantation: definition, mechanisms of disease and clinical implications. Am J Transplant 2005; 5: 2605–2610.


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