Functional recovery after liver resection - Chapter 9 Mild steatosis impairs functional recovery after liver resection in an experimental model

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Functional recovery after liver resection

Veteläinen, R.L.

Publication date

2006

Link to publication

Citation for published version (APA):

Veteläinen, R. L. (2006). Functional recovery after liver resection.

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Reetaa Vetelainen

Roeloff J. Bennink

Arlènee K. van Vliet

Thomass M. van Gulik

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INTRODUCTION N

Non-alcoholicc fatty liver disease (NAFLD) is currently the most common parenchymal liver diseasee in the world, affecting 20 per cent of individuals in the general population and up too 95 per cent of obese subjects12. Up to 80 per cent of all patients with steatosis have the mildestt form of hepatic fatty infiltration, defined as uncomplicated mild steatosis, in which fewerr than 30 per cent of hepatocytes show steatosis and no inflammatory or fibrotic

changes3.. Mild steatosis is generally considered to be a reversible and benign condition

withoutt influence on global hepatocelfutar function, whereas moderate-to-severe steatosis

iss a well established risk factor for primary graft dysfunction after transplantation4-6.

Althoughh mild-to-moderate steatosis does not affect graft function after transplantation, patientss with mild steatosis have an increased risk of mortality and morbidity after liver

resection77 9 . This discrepancy suggests that steatosis has more impact on postoperative

recoveryy in a regenerating liver, which is of particular relevance to liver resection and living donorr liver transplantation,

Afterr liver resection, the remaining hepatocytes must maintain a balance between differentiatedd function (protein synthesis and hepatobiliary transport of various substances)

andd increased energy demand because of hepatocyte proliferation10. Existing data

suggestt that hepatic adenosine 5'-triphosphate (ATP) synthesis in steatotic liver is affected

byy increased oxidative stress causing mitochondrial dysfunction11'12. The impact of such

mitochondriall dysfunction on functional recovery during regeneration of livers with mild steatosiss has not been investigated.

Hepatobiliaryy scintigraphy (HB5) is useful in evaluating liver function13-15. 99mTc-labelled mebrofeninn is most frequently used because of its high hepatic uptake and excretion

throughh the biliary transport system together with very low urinary extraction13'16'17 HBS

iss a reliable method for assessment of hepatic metabolic and excretory function in animal

modelss of parenchymal liver diseases18-20, and the reproducibility of HBS in normal and

regeneratingg liver has been demonstrated21, steatotic livers during regeneration after

resection.. Hepatic metabolic uptake rate and excretory function, parenchymal damage andd energy restoration were evaluated in a rat

Thee aim of the present study was to investigate the functional capacity of mild model of mildd hepatic steatosis and 70 per cent liver resection.

MATERIALSS AND METHODS

Liverr steatosis was induced using a standard methione and choline-deficient (MCD) diet as

describedd previously22. Male Wistar rats (250-300 g) (Harlan CPB, Zeist, The Netherlands}

weree acclimatized to laboratory conditions for at least 7 days. They were maintained at

aa constant temperature of C with a 12-h light-dark cycle, and had free access to

standardd rodent chow and water (Harlan, Zeist, The Netherlands). After acclimatization, ratss were fed either a MCD diet (Research Diets, New Brunswick, New Jersey, USA) or standardd chow, containing an adequate amount of choline and methione, ad libitum forr 1 week. Two non-operated groups (standard chow and MCD diet, n = 8/ per group

weree used as baseline controls. During all procedures the animals were treated according too the guidelines of Dutch legislation and international standards for animal care and handling.. The protocol was approved by the Animal Ethical Committee of the University off Amsterdam.

Surgicall procedure

Surgeryy was performed under inhalation anaesthesia (1 : 1 oxygen : nitrous oxide, 1 -22 per cent isoflurane) with intravenous pain medication (3.3 mg/kg buprenorphine).

Liverr regeneration was induced by 70 per cent liver resection23. Rats underwent 99m

Tc-mebrofeninn scintigraphy 1, 2, 3 and 7 days after liver resection. After imaging, animals weree killed by heart puncture, blood was collected and centrifuged, and plasma stored at

- 8 00 . Livers were removed, weighed and thin slices of all liver lobes immersed in 10 per

centt formalin or snap-frozen immediately in liquid nitrogen for further analysis.

Hepatobiliaryy scintigraphy

Ratss were sedated with an intramuscular injection of 40 mg/kg ketamine and 2 mg/kg

xylazine.. They were then injected in the tail vein with 40 MBq 99mTc-mebrofenin (Bridatec

®,, GE-Amersham Health, London, UK) and immediately scanned in the anterior position withh the liver in the field of view. HBS was used to calculate the clearance of mebrofenin

fromm the blood by liver uptake as described in detail elsewhere21-24,25. Regions of interest

(ROIs)) were drawn around the liver, heart and large vessels within the mediastinum (serving ass blood pool) and around the total field of view, indicative of total activity. The rate of

uptakee of mebrofenin by the liver was calculated based on these three parameters21. The

liverr ROI was used to create a time-activity curve for calculation of the time at which maximall hepatic activity occurred (T peak), as well as the time required for the peak

activityy to decrease by 50 per cent (TVi peak). All calculations were repeated twice for

assessmentt of the reproducibility of hepatic uptake calculations.

Liverr regeneration

Thee restitution of liver weight was expressed as a percentage of regenerated liver mass calculatedd using the equation [C - (A - B)]/A, where A was the estimated total liver weightt at the time of liver resection, B the weight of resected liver and C the weight of

regeneratedd liver at final surgery26. MtB-5 is an antibody that detects proliferating cells

off all phases of cell cycle (G1, S, G2 and M phases)27. Briefly, the 4-um sections were

deparaffinizedd and pretreated with citric acid (pH 6.0) at 200 kPa and C for 20 min in

aa pressure cooker. Sections were then incubated for 60 min with MIB-5 antibody (DAKO Cytomation,, Glostrup, Denmark) at a dilution of 1 : 50 for 60 min. After incubation with secondaryy antibody (Poly-HRP ®; Invitrogen, Carlsbad, California, USA) at a dilution off 1 : 1 for 30 min, diaminobenzidine was used to visualize peroxidase complexes and sectionss were counterstained with haematoxyiin. The MIB-5 index, expressed as the rate off positive cells per 1000 hepatocytes, was determined by two blinded investigators who examinedd 30 high-power fields (h.p.f.) per slide at 40 * magnification.

Plasmaa samples were analysed for aspartate aminotransferase (AST), alanine aminotransferasee (ALT) and plasma bilirubin using standard laboratory methods. Plasma hyaluronicc acid concentration was measured as an indicator of sinusoidal endothelial cell function,, using an enzyme-linked immunosorbent assay (Echelon, Salt Lake City, Utah, USA)) according to manufacturer's instructions.

Liverr samples were homogenized in phosphate-buffered saline (pH 7.2) and centrifuged

att 2 000 g for 10 min at . Hepatic lipids were extracted by the chloroform : methanol

extractionn method28, and triglyceride and cholesterol in the tissue extracts were measured

enzymaticallyy using commercial kits (Trig/GB, Roche, Switzerland; chotesterol: Biomerieux, Boxtel,, The Netherlands), The protein concentration in the homogenates was measured, andd hepatic triglyceride and cholesterol levels were expressed in picograms per gram protein. .

Hepaticc ATP was was measured fluorimetrically as described previously29.

Histopathology y

Liverr histopathology was assessed by two blinded investigators who examined 30 h.p.f.

perr slide at 40 x magnification by light microscopy. The degree of steatosis was assessed

ass described previously30. Macrovesicular steatosis (MAS) was classified as 0 (none), 1

(occasionall cells showing MAS), 2 (small clusters of MAS (less 20 cells; 5-10 per cent off field), 3 (larger clusters, >20 cells ; 5-10 per cent of fieldor 4 {more than 10 per cent involved).. Hepatocyte swelling was classified as 0 (absent), 1 (minor) or 2 (marked).

Statisticall analysis

Dataa analysis was performed with GraphPad Prism 4® for Windows (GraphPad Software, Sann Diego, California, USA). The results are presented as mean(s.e.m.). Differences were testedd using Kruskal-Wallls one-way analysis and Mann-Whitney U test. All statistical testss were two tailed and P< 0.050 was considered significant. Scintigraphy data were analysedd by the method of Bland and Altman, which is a supplementary method used to

comparee two different methods when the true value is unknown 31.

RESULTS S

Afterr 1 week of MCD diet, combined MAS and MIS affected approximately 20-30 per centt of all hepatocytes, without inflammatory or fibrotic changes, featuring MIS 1.9(0.1), MASS 0.8(0.2) and swelling 0.2(0.1 (P < 0.050) {Fig. 1a). The increase in amount of steatosiss correlated with a sevenfold increase in hepatic triglyceride content (0.21(0.02) andd 0.03(0.01) mmol per mg protein in steatotic and normal rats respectively; P< 0.050). Noo pathological changes were found in normal rats (Fig. lb)

Liverr regeneration and hepatocellular damage

Theree was no significant difference in regenerating liver weight or number of MIB-5-positivee proliferative hepatocytes between mild steatotic and normal rats at any time pointt (Fig. 2a, b).

Fig.. 1 a Micrograph of liver tissue after 1 week of a methione and choline-deficient (MCD) diet showed mainlyy microvesicular steatosis with occasional macrovesicular foci and no inflammatory cells were observed, bb of control rat showing normal liver parenchyma with no pathological changes (haematoxylin and eosin stain,, original magnification * 10)

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Fig.. 2 Liver regeneration after resection was evaluated by measuring a the increase in postoperative liver mass (regenerationn ratio) and b nepatocyte proliferation index. Hepatocellular damage after liver resection was assessedd by measuring plasma levels of c alanine aminotransferase (AST) and d aspartate aminotransferase (ALT).. Values are mean(s.e.m). *P < 0.050 versus control; ]P < 0 050 versus baseline (Mann-Whitney U test) )

Plasmaa levels of ALT and AST rose in both groups after resection {Fig. 2c, d), reaching peak valuess at 24 h and declining thereafter. The plasma AST level was increased significantly att 24, 4 8 and 72 h ( all time points, P = 0.016), and the ALT level at 24 h, in rats w i t h mild steatosiss compared with controls (P= 0.016. The plasma bilirubin at 24 h was significantly higherr in rats w i t h mild steatotosis than in controls (1.6(0.2 versus 0.5(0.3) u m o l / l ; P < 0.050).. After liver resection, plasma hyaluronic acid levels were increased in both groups, butt had returned t o baseline levels by 72 h after surgery (data not shown).

Hepatobiliaryy transport

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Tc-mebrofenmm uptake rate calculations showed excellent reproducibility (r = 0.999,

PP < 0.0001) as did those for T peak (r = 0.999, P < 0.0001) and T,., peak (r = 0.947, P << 0.0001).. There were good correlations between 99mTc-mebrofenin uptake rate and T peakk (r = - 0.765, P< 0.001) and between T peak and T1/2 peak {r. = 0.807, P< 0.001).

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Tc-mebrofeninn uptake rate decreased, whereas T peak and T,- peak increased in both groupss after liver resection. Values recovered to baseline after 3 days in normal rats (Fig.

3a-c).3a-c). However, in mildly steatotic rats, 9 9 mTc-mebrofenin uptake rate was decreased at alll time points and remained low at 7 days after liver resection (P < 0.050 versus control andd baseline). Tl/? peak in mildly steatotic rats recovered t o baseline level after 72 h after

liverr resection. However, Tpeak remained increased up to 7 days after liver resection (P < 0.0500 versus baseline).

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Fig.. 3 a Uptake rate, b T peak and c T, 7 peak of 99m

Tc-mebrofeninn after liver resection. Values are mean(s.e.m.). *P

<< 0.050 versus control, P < 0.050 versus baseline ( M a n n

-Whitneyy U test)

—©—— Control — o -- Mud steatosis 111 i

Fig.. 4 Hepatic adenosine 5'-tnphosphate (ATP)) after liver resection. Values are mean(s.e.m.).. *P < 0.050 versus control,

]P]P < 0.050 versus baseline ( M a n n

-Whitneyy Utest)

Recoveryy of hepatic ATP

Inn both groups, hepatic ATP was decreased after liver resection (Fig. 6). In mildly steatotic ratss the decrease was more profound and prolonged. The hepatic ATP content returned too baseline level in normal rats after 3 days, but remained low in mildly steatotic rats (P < 0.050). .

DISCUSSION N

Extensivee liver resections are being performed more frequently as resection remains the onlyy curative treatment for patients with secondary hepatic malignancy or primary hepatic

tumourss not eligible for liver transplantation32. Postoperative morbidity and mortality are

closelyy related to the regenerative capacity and functional recovery of the liver33. Patients

withh parenchymal liver disease have impaired regeneration after resection leading to sustainedd postoperative hepatic dysfunction. There are currently no reliable non-invasive methodss by which to classify the extent of parenchymal changes. Biopsy is therefore used too diagnosis steatosis, although accurate assessment is hampered by the heterogenous distributionn of steatosis throughout the liver parenchyma.

Contradictoryy results have been reported concerning the regenerative capacity of steatoticc liver. In leptin-deficient rodents, regenerative capacity is impaired by defective

leptinn signalling and not by the development of steatosis per se34. Mild steatosis induced

byy a MCD diet in the present study did not affect liver regeneration, whereas impaired liverr regeneration after hepatectomy has been reported in the setting of severe steatosis

withh inflammatory components35'36. In accordance with published data, no differences

weree detected in the regenerative capacity of mildly steatotic livers and normal livers

inn the present study 35'36. It should be emphasized that MCD diet-induced steatosis is

consideredd to be a clinically relevant model of steatosis as it parallels human nonalcoholic

fattyy liver disease and non-alcoholic steatohepatitis37. Furthermore, a combination of

MISS and MAS with minimal lobular inflammation was confirmed in steatotic rats after 1

142 2

<< >,

7--'"1 1

weekk on the MCD diet as assessed by histopathological evaluation, which is considered

thee 'gold standard'38. The distribution of steatosis was both pericentral and periportal,

correspondingg to that in hepatic steatosis associated with obesity and diabetes mellitus. Thesee features justify the use of a MCD diet in animals as a model for steatosis.

Thee value of HBS has been evaluated in several experimental models of acute and chronic parenchymall disease, HBS data show an excellent correlation with the magnitude of histopathologicall changes18"20 . Several calculations can be applied in HBS evaluation, but thee hepatic uptake rate of mebrofenin is the only method directly applicable to the clinical situation18'19,39.. Tpeak and ly2 peak are derived from time-activity curves without taking

intoo account the clearance rate of injected mebrofenin. They are therefore considered as

moree descriptive parameters of hepatic 99mTc-mebrofenin transport.

Thee impairment of 99mTc-mebrofenin transport in mild steatotic rats probably reflects

aa combination of decreased hepatocyte uptake and excretion capacity. The sustained functionall impairment probably contributes to the disturbed energy balance observed inn mildly steatotic liver after liver resection and is likely to be caused by pre-existing mitochondriall dysfunction. Such dysfunction is induced by increased oxidative stress causedd by accumulation of reactive oxygen species (ROS) and mitochondrial structural

changes11'122 . Important sources of oxidative stress in the context of steatosis are

intrahepatocellularr fatty acids, via direct hepatocyte toxicity or via induction of cytochrome

p-4500 microsomal lipoxygenase 2E1 (CYP2E1) and peroxisomal "-oxidation40'41. These

furtherr cause mitochondrial dysfunction and together with hepatocellular damage after liverr resection potentially enhanced disturbed ATP synthesis in steatotic livers in the present study40'41'42. .

Thee ATP-dependent uptake (organic anion transporters 1 and 2) and excretion transporters off mebrofenin, bile excretory pump and multidrug resistance protein -2 and 3, might be affectedd by mitochondrial dysfunction and by proinflammatory cytokines (tumor necrosis factorr -alpha, interleukin -6) in mildly steatotic liver after resection 16-17.36<43. Furthermore, inn steatotic livers, the expression of bile excretion transporters is downregulated and bilee secretion impaired owing to lipid accumulation, progressive inflammation and hepatocellularr injury after resection 22'44.

Mildd steatosis impairs functional recovery after liver resection although liver regeneration iss not affected. This is in part explained by increased hepatocellular damage and disturbed energyy balance. These findings provide an explanation for the increased risk of liver resectionn in patients with mild steatosis.

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