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Functional recovery after liver resection

Veteläinen, R.L.

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Citation for published version (APA):Veteläinen, R. L. (2006). Functional recovery after liver resection.

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Download date: 11 Aug 2020

Reetaa L. Vetelainen

Arlènee van Vliet

Thomass M. van Gulik

Severee steatosi s increase s larr injur y and impair s live r inn a rat mode l of partia l h

atoceilu --generatio n n

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Liverr resections have become safer in the past ten years, largely owing to improvements O inn preoperative diagnosis, surgical techniques and postoperative care. ]-2'3 Consequently, "> overalll postoperative mortality has decreased to below 5% correlating directly with 3 preoperativee liver function and resected liver volume. Function of the remnant liver rapidly recoverss in patients with normal liver parenchyma as hepatocytes proliferate to restore O thee loss of volume. However, in the presence of parenchymal liver disease, hepatocellular £ proliferationn is impaired, exposing patients to liver dysfunction and associated -^ complications,, culminating into posthepatectomy liver failure which has a high mortality rt (60-90%).. S Currently,, hepatic fat accumulation i.e. steatosis, is the most common parenchymal liver O diseasee in the Western world, affecting 20% of individuals in a general population and up < too 95% among obese.4 As steatosis is closely related to obesity, dyslipidemia and diabetes, fj thee prevalence will increase as a consequence of the epidemic obesity in the Western Q population.. Steatosis is classified by histopathology into three categories according to the Q extentt of fat infiltration: mild/uncomplicated steatosis (<30% of hepatocytes affected, < withoutt inflammatory component), moderate (30-60% of hepatocytes affected) and severee steatosis (>60% of hepatocytes affected).5 Furthermore, progressive inflammation andd eventually fibrogenesis is observed throughout the development of steatosis. Evenn though there are only few clinical studies evaluating the impact of steatosis on postoperativee recovery after liver resection, any grade of steatosis is associated with increasedd postoperative morbidity. The steatosis severity grade has been correlated with increasedd infectious complications and increased postoperative mortality. 6-7 Although thee mechanisms of impaired liver recovery in steatotic patients are largely unclear; the underlyingg pathological derangements together with disturbed liver regeneration have beenn implicated as potential factors.7'8 In fatty hepatocytes, intracellular fatty acids promotee the generation of reactive oxygen species (ROS) increasing mitochondrial oxidativee stress and lipid peroxidation. 9 In addition, steatotic livers have impaired antioxidantt scavenging of free radicals causing ROS accumulation and consequently, thee activation of hepatic macrophages, i.e. the Kupffer cells (KC). KC's produce proinflammatoryy cytokines such as tumor necrosis factor (TNF)-", interleukin (IL) -6 and IL-166 further exacerbating hepatocellular damage in steatotic livers. 5- 9 Specific knowledge off the mechanisms involved in the hepatocellular damage incurred in steatotic livers would enablee development of interventional strategies to improve postoperative outcome of patientss with liver steatosis. Wee hypothesize that preoperative^ increased lipid peroxidation and impaired antioxidant responsee aggravate KC-mediated inflammatory responses and hepatocellular injury inn steatotic livers after partial hepatectomy, and that liver regeneration under these circumstancess is impaired (PH). We therefore, assessed the effects of mild and severe steatosiss on hepatocellular recovery after PH in a rat model of diet-induced liver steatosis.

Material ss and Method s

Hepaticc steatosis was induced using a standard MCD diet (Harlan Teklad, Madison Wl, US).. Male Wistar rats (250-300g) (Harlan CPB, Zeist, The Netherlands) were acclimatized att least for 7 days to laboratory conditions, maintained at constant temperature of 24 C withh 12 h light-dark cycle and fed a standard rodent chow (Harlan, Zeist, The Netherlands) andd water ad libitum. After acclimatization, rats (n=30) were fed the MCD diet for 1 or 55 weeks ad libitum. The control group (n=15) received a standard chow with adequate methionee levels ad libitum. Two groups without surgery were used as baseline controls: aa group (n=5) fed with standard chow and a group (n=5) fed with MCD diet. During all procedures,, the animals were treated according to the guidelines of the Dutch legislation andd international standards for animal care and handling. The protocol was approved by thee Animal Ethics Committee of the University of Amsterdam, The Netherlands. Afterr a period of 1 or 5 weeks of diet, laparotomy was performed under inhalation anesthesiaa using a mixture of 02/N20 (1:1) and isoflurane (1-2 % Florene, Abbott laboratoriess Ltd, Queensborough, UK) and pain medication (buprenorphine, Temgesic i.v.. 0,033 mg/ 0,1 kg). 70 % partial hepatectomy consisting of resection the median andd left lateral liver lobes was performed according to the method described by Higgins andd Anderson.10 After 24h, 48h and 72h, respectively, rats were sacrificed. Blood was collectedd by vena cava puncture, centrifuged (10 min, 3,000x g, ) and plasma wass stored at -80 . Livers were removed, weighed and thin slices of all lobes were immersedd in 10% formalin and embedded in paraffin. 4 urn sections were routinely stainedd with haematoxylin-eosin (H&E) for assessment of morphology and with Sirius redd (0,1% Fast red in picric acid, Immunotech, Netherlands) for collagen deposition.

Assessmen tt of hepati c steatosi s Liverr samples were homogenized in phosphate buffered saline (pH 7.2) and centrifuged (4,000xx g, 10 min, 4"C). Hepatic lipids were extracted by chlorofornvmethanol extraction methodd and lipid levels were measured enzymatically using commercial kits (Trig/GB, Roche, Switzerlandd and Cholesterol, Biomerieux, Boxtel, Netherlands). Protein concentration wass measured using BCA Protein Assay kit (Pierce, Rockford, US) and lipid levels were expressedd as mg/g total protein. Histopathological features of hepatic steatosis were evaluatedd using a semi-quantitative, histopathological score adapted from the recently acceptedd AASLD criteria for steatosis staging in H&E and for detection of fibrosis in Sirius redd stained sections by light microscopy in 30 high power field (HPF)'s (magnification 40 X)) by two blinded investigators. 11

Assessmen tt of live r regeneratio n Threee independent markers for hepatic regeneration were used as follows: restitution of liverr weight was expressed as percentage of regenerated liver mass relative to total liver weightt as previously described; % = [C- (A-B)] / A X 100%, where A= estimated total liver weightt at the time of partial hepatectomy, B=resected liver weight and C= the weight off the regenerated liver at sacrifice.12 For assessment of hepatic proliferation, MIB-5, a ratt equivalent of Ki-67 antibody was used which detects all active parts of the cell cycle.

Thee MIB-5 index has a strong positive correlation with proliferating antigen expression, £ bromodeoxyuridinee incorporation and thymidine incorporation.13 Briefly, sections were 8| deparaffinizedd and pretreated (citric acid pH 6.0, 2 bar, , 20 min). For primary § andd secondary antibody, MIB-5 antibody (dilution 1:50, 60 min, DAKO Cytomation, <" Copenhagen,, Denmark) and Poly-HRP (dilution 1:1, 30 min, Invitrogen, Carlsbad, US), 3 weree used, respectively. The mitotic index was determined in HE stained sections using previouslyy reported criteria for mitosis as follows: complete absence of cell membrane, O slightt eosinophilic staining of nucleus, nuclear spindle matrix formation, absence of a j -nucleoluss and slight increase in cell size.14 All indexes were determined by two blinded -^ investigatorss (40X magnification, 30 HPF's) and expressed as the rate of positive cells per r*

10000 hepatocytes/HPF. n

O O

Hepatocellula rr necrosi s and apoptosi s *<

Plasmaa samples were analyzed for aspartate aminotransferase (AST), alanine fD aminotransferasee (ALT) and for albumin (the major hepatic plasma protein product) in the O Departmentt of Clinical Chemistry (AMC) using standard laboratory methods. 2> Histopathologicall injury was evaluated in HE stained sections in 10 HPF's at magnification 40xx by two blinded investigators and by a point-counting method using an ordinal scale ass reported previously: grade 0, minimal or no evidence of injury; grade 1, mild injury consistingg of cytoplasmic vacuolization and focal nuclear pyknosis; grade 2, moderate to severee injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, and loss of intercellularr borders; and grade 3, severe necrosis with disintegration of hepatic cords, hemorrhage,, and neutrophil infiltration.1515 Also, for determination of apoptotic cells, cleavedd caspase- 3 antibody detecting endogenous large fragment of activated caspase-3 wass used. Briefly, 4 urn sections were deparaffinized and pretreated (citric acid, pH 6.0, 22 bar, , 10 min). For primary and secondary antibody, a cleaved caspase- 3 (dilution 1:200,, 60 min, Celt Signaling Technology, Frankfurt, Germany) and Poly-HRP (dilution 1:1,, 30 min, Invitrogen, Carlsbad, US), respectively, were used and positive cells were determinedd in 20 HPF's at magnification of 80x by two blinded investigators.

Hepati cc and systemi c proinflammator y cytokin e respons e

Sectionss were incubated with an ED-1 antibody against the lysosomal membrane glycoproteinn on resident KC's and circulatory macrophages (dilution 1:100, 60 min, Serotec,, Humbeek, Belgium). After incubation with a secondary antibody (1:100, 60 min, GAM-lgG1,, Southern Biotechnologies, Birmingham, AB, US), Fast DAB (Sigma chemical, Munich,, Germany) was used to visualize the peroxidase complexes. Macrophages were countedd (30 HPF's, magnificationn of 25) by two blinded investigators. AA bioplex kit for determination of plasma and hepatic levels of KC-mediated cytokine's TNF-a,, IL-1 p1, IL-6 and IL-10 was used (Biorad, Hercules, US). Samples of all regenerating liverr lobes were homogenized (NaPi buffer 5mM, pH 6.0), centrifuged (10,000x g, , 10 min)) and the supernatants were used in the cytokine assay. The samples were analyzed accordingg to manufacturer's instructions and concentrations were expressed as pg/mg protein. .

3 3

Assessmen tt of hepati c lipi d peroxidatio n and antioxidan t respons e Antioxidantt response was assessed by measurement of hepatic total glutathione (tGSH)) concentrations as follows: samples were homogenized in buffer (MPA, pH 6.0), centrifugedd (4,000x g, 10 min, ) and supernatants were analyzed for tGSH, Lipid peroxidationn was assessed by hepatic malondialdehyde (MDA) levels. MDA was measured ass free thiobarbituric acid reactive substances (TBARS) in the presence of the antioxidant butylatedd hydrotoluene that limits the generation of new TBARS during the assay. The hepaticc tGSH and TBARS concentrations were expressed as mmol/mg total protein.

Statistica ll analysi s Dataa analysis was performed with GraphPad Prism 3.02 for Windows (GraphPad Software Inc.,, San Diego, US)The results are presented as means SEM. Significant differences betweenn groups were tested using ANOVA one-way analysis and Mann-Whitney's U-test followedd by Bonferroni post-test. P values less than 0.05 were considered significant.

Result s s

Inductio nn of steatosi s Inn control rats no pathological changes were seen (Fig.lA). After one week of MCD diet, steatosiss mainly consisted of mild microvesicular steatosis with scattered macrovesicular hepatocytess (Fig.lB). After 5 weeks of MCD diet, steatosis consisted of extensive macrovesicularr steatosis (>60 % hepatocytes affected) with foci of inflammatory cells alongg with increased fibrosis in the portal area's (Fig.lC). Corresponding with these histopathologicall features of steatosis, a 10-fold increase in hepatic triglyceride content wass seen after one week and a 20-fold increase after 5 weeks of MCD diet (vs. controls, p<0.01,, Fig.lD).

Liverr regeneratio n is impaire d in the presenc e of severe steatosi ss after 70% PH Afterr 70% PH, the increase in regenerating liver mass was lowest in the severe steatosis groupp at 24h, 48h and 72 h, as compared to the mild steatosis and control groups (p<0.05) (Fig.2A).. Also, hepatocyte proliferation (MIB-5 index) was lower at all time points in the severee steatosis group, as compared to the mild steatosis and control groups (p<0.05) (Fig.2B).. The mitotic index results followed the pattern of MIB-5 index in all groups at alll time points (data not shown). Between the control and mild steatosis groups, no differencess were observed at all time points. There were no differences in preoperative mitoticc or MIB-5 indexes between the groups (data not shown).

Hepatocellula rr damag e is increase d in severe steatosi s after 70% PH Plasmaa ALT and AST were significantly increased preoperative^ in the severe steatosis groupp (p<0.05), indicating increased hepatocellular damage. After PH, AST and ALT weree significantly elevated in the severe steatosis group at all time points as compared to

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Figur ee 1 Micrograph of liver tissue of control rat (A) showing no pathological changes. After one week MCD diett (B), mainly microvesicular steatosis with occasional macrovesicular foci and no inflammatory cells were observed.. After five weeks of MCD diet (C), mainly macrovesicular steatosis and prominent inflammation wass observed. All slides stained with H&E, 20x magnification.

Thee histopathological changes corresponded with increased hepatic triglycerides content (D). Values are expressedd as mean SEM. (* = p<0.05 vs controls, * *= p<.05 vs the mild steatosis group).

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Figur ee 2. Liver regeneration after PH was evaluated byy measuring increase in postoperative liver mass (A) andd hepatocyte proliferation index (B). A decreased responsee was seen in the severe steatotic rats withh respect to both parameters in contrast to no differencess between the mild steatotic and control rats.. Values are expressed as mean SEM. (* = p<0.055 vs controls, **= p<.05 vs the mild steatosis group,). .

127 7

thee mild steatosis and control groups (p<0.05) (Fig.3A, B). Concerning the mild steatosis group,, AST and ALT were increased at 24h as compared to the controls (p<0.05) but thereafter,, no differences were observed.

Thee histopathology scores corresponded with the plasma markers and was increased at alll time points in the severe steatosis group compared to the mild steatosis and control groupss (p<0.01) (Fig.4A). The initial hepatocellular damage in the severe steatosis group wass mainly due to necrosis as no increase in the apoptotic cells was seen at 24h after PH (Fig.4B).. However, the rate of apoptotic hepatocytes remained increased at 48h and 72h inn the severe steatosis group compared to the control and mild steatosis groups (p<0.01). Inn the mild steatosis group, apoptosis was slightly increased at 48h compared to the controlss (p<0.05).

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Figuree 3. Hepatocellular damage after PH assessed Figure 4 Histopathology scores in HE stained liver byy plasma ALT (A) and AST (B) was most increased tissue (A) after PH was significantly increased in severe inn the severe steatotic rats. Values are expressed as steatotic rats as compared to no changes between meann SEM (* = p<0.05 vs controls, * * = p<0.05 the control and mild steatotic rats (p<0.01). The rate vss the mild steatosis group). of apoptotic cells (B) remained increased in the severe

steatoticc rats compared to decreasing rates in the controll and mild steatotic rats after PH. Values are expressedd as mean SEM. (* = p<0.05 vs controls, * * == p<0.05 vs the mild steatosis group).

Severee steatosis increases lipid peroxidation and decreases

antioxidantt response after 70% PH

Hepaticc TBARS was significantly increased preoperatively in the severe steatosis groupp compared to the mild steatosis and control groups and remained increased postoperativelyy as compared to the control group. When compared to the mild steatosis

128 8

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Figuree 5 Effect of severe and mild steatosis on hepaticc lipid peroxidation after PH as measured byy free TBARS (A) and hepatic total GSH response (B).. Hepatic TBARS was increased in the severe steatoticc rats after PH and hepatic GSH was increasedd in the mild steatotic rats as compared too severe steatotic and control rats. Values are expressedd as mean SEM (* = p<0.05 vs controls, *** = p<0.05 vs the mild steatotic group).

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group,, hepatic TBARS was significantly higher at 24h and 48h in the severe steatosis

groupp (p<0.01)(Fig.5A). Hepatic levels of antioxidant, total glutathione, were significantly

increasedd in the mild steatosis group as compared to the control and severe steatosis

groupss at 24h and 48h postoperatively (p<0.01). In the control group, no significant

changess were detected postoperatively (Fig.5B).

1L-66 IL-10 TNF-Plasmaa cytokine levels 24h after 70% PHX

postoperativee hours

Figuree 6 Kupffer cell-mediated inflammatory response was increased in severe steatotic rats as assessed by plasmaa (A) and hepatic cytokines (B) and by the amount of hepatic resident and circulatory macrophages (C-E,, ED-1 positive macrophages in the control, mild steatosis and severe steatosis groups, respectively). Values aree expressed as mean SEM (* = p<0.05 vs controls, * * = p<0.05 vs the mild steatotic group).

29 9

Severee steatosis increases Kupffer cell release of proinflammatory cytokines after 70% PH Plasmaa IL-6, IL-10 and TNF-a were significantly higher in the severe steatosis group at 24h afterr PH compared to the control and mild steatosis groups (p<0.01) (Fig.6A) whereas plasmaa IL-10 levels were increased in the mild steatosis group compared to controls (p<0.05).. Also at 24h, plasma IL-1B was significantly increased in the severe steatosis groupp 5 ng/ml, p<0.01) compared to undetectable levels in the mild steatosis andd control groups (p<0.01, detection limit 5 ng/mlr data not shown). There were no differencess in preoperative plasma cytokine levels between the groups (data not shown). Alsoo hepatic 1MB, IL-6, 11-10 and TNF-a were significantly increased the severe steatosis groupp compared to the mild and control groups at all time points (p<0.001, Fig.6B, IL-100 and IL-1 6 data not shown). The number of hepatic macrophages was significantly increasedd at all time points in the severe steatosis group compared to mild and control groupss (macrophages per HPF: in the severe at 24h, 48h and 72 h respectively; 90 8, 218+511 and 162+53, in mild , 5 and , p<0.05) (Fig.6C-E).

Discussion n

Severee steatosis had a negative impact on hepatocyte proliferation and on regeneration off remnant liver mass after 70% PH. In rats with norma! liver parenchyma or mild steatosis withoutt inflammation, no differences in the regenerative capacity of the remnant liver weree detected. In contrast, rats with severe steatosis showed an excessive proinflammatory cytokinee response and insufficient antioxidant response affecting liver regeneration and increasingg hepatocellular damage after PH.

Inn experimental models of parenchymal liver injury and KGmediated inflammation, liver regenerationn is impaired after resection. 17<18.19 However, the influence of chronic KC-activationn in combination with steatosis is unclear. To date, experimental studies have mainlyy applied steatosis models based either on genetic leptin mutation {Zucker rats, ob/ obb mice) or on choline deficiency in which the development of prominent and progressive inflammationn as seen in human steatosis lacks. 20'21 A period of one week MCD diet wass chosen to induce mild steatosis without inflammatory components since activation off hepatic inflammation is already observed after two weeks of this diet. 22 A five week diett period was applied to induce severe steatosis with prominent inflammation, however withoutt extensive fibrosis. 21 We speculate that these changes would resolute if the rats wouldd be returned to normal chow. However, that might not be the case if irreversible changes,, i.e. severe fibrosis or cirrosis have already developed. This model because of its inflammatoryy component might be also useful in investigating the uprising major clinical problem,, chemotherapy induced steatohepatitis. However, still only little is known about thiss subject and more studies are urgently needed.23

Mildd steatosis had no effect on liver regeneration as has been reported in previous studies. 22,233 However, in severely steatotic rats, both the restoration of liver mass and hepatocyte proliferationn were impaired. 24> 25 In contrast to our results, Rao et al described an unaffectedd regenerative response in steatotic rats with inflammation. 22 However, in their

130 0

studyy only histopathology was used to assess hepatic changes and the steatosis model rt appliedd in their study was based on a choline deficient diet which is considered mainly W aa model of uncomplicated steatosis. In similar studies using MCD diet, a delayed but § nott impaired liver regeneration in the presence of mode rate-to-severe steatosis has been <* reported.. This discrepancy is most likely explained by the shorter diet period of 4 weeks 3

a. . •a a

3--

appliedd in these studies in contrast to the 5 weeks in our study.22, 23 During the MCD diet,, hepatic inflammation progresses rapidly as a result of increased lipid peroxidation O triggeringg hepatocellular necroinflammation. 23-24

Afterr PH in the presence of severe steatosis and inflammation, increased oxidative stress, ass measured indirectly by the amount of lipid peroxidation (T8ARS) and by decreased levels off the hepatocellular antioxidant, total GSH, was observed. The intracellular lipids induce rt

cytochromee p-450 microsomal lipoxygenases and generate ROS oxidants that promote § celll membrane damage via activation of lipid peroxidation and production of reactive N< lipidd aldehyde, MDA, 26-2^28 in addition, the decreased antioxidant scavenger capacity ^ inn severe steatotic rats contribute to the amplified hepatic ROS levels and predispose 8 hepatocytess to extensive necrosis. In contrast, in mild steatotic rats, sufficient antioxidant o responsee together with attenuated hepatocellular necrosis was observed. The main type *< off cell death in livers with severe steatosis was necrosis in contrast to apoptosis as seen in normall and mildly steatotic livers. In steatotic livers, both the direct inhibition of caspase-3 andd the inhibition of Jnk kinase family that activate caspase-3 are observed in the presence off mod e rate-to-severe steatosis. 29 These protective anti-apoptotic mechanisms help to explainn why the severely steatotic hepatocytes in our study did not undergo massive apoptosiss but showed necrosis when progression of the cell cycle was disrupted. Increasedd ROS formation after PH in rats with severe steatosis triggered the release of proinflammatoryy cytokines by KC's, such as TNF-a, tL-1 p and IL-6. Increased TNF-ct and IL-66 levels were observed preoperative^ in steatotic rats indicating chronic KC activation as hass also been reported in other studies.30'31 Extensive studies by a number of groups have provenn that liver injury after PH in normal rodents, is mediated by TNF-a.32,33 In addition, KC'ss also produce cytotoxins such as hydrogen peroxidase which further activate TNF-a, subsequentlyy creating a vicious circle.34 Therefore, KC dysfunction can be considered one off the key features contributing to the increased hepatocellular damage seen after PH in severee steatosis. This mechanism of injury can be speculated to play also role in orthotopic andd living donor liver transplantations.

Thee role of KC's is controversial, as after PH, KC's reduce endotoxin-induced hepatocyte damagee and are also essential triggers inducing liver regeneration.35 Conversely, factors enhancingg TNF-a production generally exacerbate liver injury, whereas TNF-a inhibitors suchh as!L-10are hepatoprotective.36 Although a significant increase in IL-10 levels in severe steatoticc rats was observed, it was not sufficient enough to attenuate hepatocellular injury.. Increased IL-10 levels together with an increased antioxidant response in the mildly steatoticc rats, potentially attenuated hepatocellular damage as no increased damage was observedd compared to controls. It is noteworthy that control animals had no significant increasee in hepatic glutathione levels and had unchanged plasma IL-10 levels. It seems that inn normal livers after PH, the actual hepatocellular injury is so limited that the protective mechanismss that become active in steatotic livers are unnecessary, —

131 1

InIn conclusion, steatosis wi th prominent inflammation impaired liver regeneration wi th a

significantt role of increased lipid peroxidation and KC-mediated hepatocellular injury after

PH.. These results suggest an increased risk when performing extensive liver resection in

thee presence of severe steatosis.

Acknowledgments s

Thee authors want to thank Adrie Maas for his assistance in the animal experiments, Chris

vann der Loos (Department of Pathology) for his assistance in the immunohistochemical

assayss and Nikol Snoeren for her assistance in the histopathology assessment.

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