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ORIGINAL ARTICLE

Decreased accumulation of ultrasound contrast in the liver

of nonalcoholic steatohepatitis rat model

Yuki Miyata, Takeo Miyahara, Fuminori Moriyasu

Yuki Miyata, Takeo Miyahara, Fuminori Moriyasu, Depart-ment of Gastroenterology and Hepatology, Tokyo Medical Uni-

versity, 6-7-1, Nishi-Shinjuku, Shinjukuku-ku, Tokyo 160-0023,

Japan

Author contributions: Miyata Y and Miyahara T contributedequally to this work; Moriyasu F designed research; Miyata Y

and Miyahara T performed research; Miyata Y and Miyahara T

contributed new reagents/analytical tools; Miyata Y wrote the

paper.

Correspondence to: Fuminori Moriyasu, MDFuminori Moriyasu, MD, Department ofGastroenterology and Hepatology, Tokyo Medical University,

6-7-1, Nishi-Shinjuku, Shinjukuku-ku, Tokyo 160-0023,

Japan. [email protected]

Telephone: +81-3-53256838 Fax: +81-3-53256840Received: February 11, 2011 Revised: April 7, 2011

Accepted:April 14, 2011

Published online:October 7, 2011

Abstract

AIM: To investigate the diagnosis of nonalcoholic ste-atohepatitis (NASH) using contrast ultrasonography inthe NASH rat model.

METHODS: The liver in methionine choline-deficientdiet (MCDD) rats, a NASH model constructed by feedingan MCDD, was examined by contrast ultrasonographyat weeks 2, 4, 8, 12 and 16, with late phase images ofcontrast ultrasonography (Kupffer imaging) in whichcontrast enhancement was achieved by incorporationof a contrast agent by Kupffer cells (KCs), and imageswere compared to those in rats taking a regular chow.

RESULTS: Decrease in contrast enhancement was ob-served frst in MCDD rats at week 2. KCs were counted

based on immunohistochemistry, but their numberswere not reduced and it was assumed that attenuation

of contrast enhancement was attributable to reducedphagocytic activity of the KCs.

CONCLUSION: It is suggested that clinical application

of contrast ultrasonography may be valuable for non-invasive diagnosis of NASH.

2011 Baishideng. All rights reserved.

Key words: Nonalcoholic steatohepatitis; Leptin; Kupffercell; Methionine choline-deficient diet; Contrast ultra-sound

Peer reviewer: Sren Rafaelsen, MD, Consultant Radiologist,Associate Professor, Department of Radiology, Vejle Hospital,

Vejle 7100, Denmark

Miyata Y, Miyahara T, Moriyasu F. Decreased accumulation of

ultrasound contrast in the liver of nonalcoholic steatohepatitis rat

model. World J Gastroenterol 2011; 17(37): 4191-4198 Avail-

able from: URL: http://www.wjgnet.com/1007-9327/full/v17/

i37/4191.htm DOI: http://dx.doi.org/10.3748/wjg.v17.i37.4191

INTRODUCTION

Nonalcoholic steatohepatitis (NASH) is a relatively newdisease entity, proposed by Ludwig in 1980 [1], which

exhibits a clinical manifestation similar to that of alco-holic steatohepatitis in terms of liver histology, despitea lack of history of alcohol abuse causing hepatitis. Thediagnosis of fatty liver without a history of drinking al-cohol is generally termed nonalcoholic fatty liver disease(NAFLD), which is most simply fatty liver with an excel-lent prognosis, while NASH is a subtype of NAFLD

[2].

It is important to differentiate NASH from NAFLDat an early stage to start relevant treatment. Althoughthere have been a number of reports aimed at differ-entiating NASH patients[2], histological diagnosis basedon liver biopsy is the only diagnostic method at present.

However, liver biopsy is an invasive method, with a smallrisk of complications, and is too invasive for diagnosis ofa benign disease, such as NASH.

It has been previously reported that liver-specific

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World J Gastroenterol 2011 October 7; 17(37): 4191-4198ISSN 1007-9327 (print) ISSN 2219-2840 (online)

2011 Baishideng. All rights reserved.

Online Submissions: http://www.wjgnet.com/[email protected]

doi:10.3748/wjg.v17.i37.4191

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Miyata Y et al. Microbubbles of MCDD in NASH rat model

Kupffer images of contrast ultrasound can be used fordifferential diagnosis between NASH and NAFLD [3]. Inthis study, using a rat model of NASH, we carried outcontrast ultrasonography to compare contrast enhance-ment between the NASH model and control rats that

were fed a regular chow. Levovist

TM

(Schering AG, Berlin,Germany) and SonazoidTM (GE Healthcare, Oslo, Nor-way) were used as contrast ultrasonography agents. Mi-crobubbles of Levovist

TM and Sonazoid

TMare phagocy-

tosed and incorporated by Kupffer cells (KCs) in the latecontrast phase (Kupffer imaging)[4]. There is a report thatcontrast enhancement by the contrast agent in the latecontrast phase was decreased in patients diagnosed withNASH, compared to NAFLD patients[3,5]. The reason fordecreased microbubble accumulation in the NASH liveris presumed to be because phagocytosis of microbubblesby KCs is decreased.

We constructed a NASH model by feeding a methio-nine choline-decient diet (MCDD, Oriental Yeast Co.,Tokyo, Japan) to rats

[6-8]. Then, examining the liver using

contrast ultrasonography, we compared the phagocyticactivity by KCs in rats eating an MCDD and those givenregular chow, using contrast enhancement in the latecontrast phase.

MATERIALS AND METHODS

Animals and construction of a NASH modelMale Wistar rats weighing about 50 g at the time of pur-chase were used and kept in a room at 21 2 with

a 12-h cycle of light and darkness. The NASH modelwas constructed by feeding an MCDD to 15 rats, andthe group was designated the MCDD group. Conversely,a diet of regular chow was fed to 5 rats and they weredesignated the control group. Rats were subjected toexperiments at 2, 4, 8, 12 and 16 wk after beginning thediet, and three rats in the MCDD group and one in thecontrol group were used for experiments at each timepoint. During the follow-up period, water and regularchow were given ad libitum. Body weight was measured inall rats in both groups at the designated time points. Inaddition, to observe the histological changes in liver tis-

sue in MCDD rats at the designated time points, part ofthe resected liver was xed in 10% formalin after the ex-periments, and parafn slices were prepared and stainedwith hematoxylin and eosin for histological examination.All animals received humane care and the experimentalprotocols were approved by the Animal Ethical Commit-tee of Tokyo Medical University.

Incorporation of the contrast ultrasonography agentAfter rats were anesthetized at the designated time pointby inhalation of diethyl ether (Wako Pure Chemical In-dustries, LTD., Tokyo, Japan), the abdomen and thighswere shaved and the fascia exposed by an excision of ab-

dominal skin. At the same time, the thigh was incised toexpose the femoral vein and a 24-gauge indwelling needle(TERUMO Corp., Tokyo, Japan) was inserted to secure

the vessel. From this site, a total of 500 L of LevovistTM

solution (10 L/g body weight) with physiological sa-/g body weight) with physiological sa-line was infused as a bolus. In both groups, the liver wasscanned through the exposed fascia with an echo probe10 min later. Images were scanned at mechanical index

(MI) of 1.5 and then any Levovist

TM

remaining in the liverwas destroyed by sweep scanning over the whole liver.Next, as for Levovist

TM, a total of 500 L of Sonazoid

TM

10 times diluted solution (1 L/g of Sonazoid/g of SonazoidTM

solu-tion/body weight) with physiological saline was infusedintravenously as a bolus and, 10 min later, images werescanned and recorded at Advanced Dynamic FlowTM(ADF) ultrasound mode at MI of 1.5. An AplioTM (ToshibaMedical Systems, Tokyo, Japan) was used in this study.The frequency used was 7.5 MHz and the type of thetransducer was linear. ADF was harmonic power Dop-pler mode from Toshiba Medical Systems.

A region of interest (ROI) was determined arbitrarilyin the constant area of ultrasound images of the liverparenchyma obtained at 10 min, and contrast enhance-ment was quantied in the MCDD and control groupsby intensity analysis software Image J (http://rsb.info.nih.gov/ij/). Quantified values in the MCDD groupwere expressed as relative values compared to those inthe control group at each designated time point. An ROIwas set in the region covering a wide area of the liver pa-renchyma, excluding large vessels as far as possible. Theaverage intensity in the ROI was measured and relativevalues were compared.

In vivo administration of FITC-latex beads and isolationof KCsAfter experiments using contrast ultrasound, 50 L ofofFITC-latex beads (2 m in diameter, Polyscience, War-rington, PA, United States) were mixed with 500 L ofofphysiological saline and infused from the tail vein to ex-amine the phagocytic activity of KCs in rats in the MCDDand control groups. One hour later, KCs were isolated ac-cording to the following methods: Rats were anesthetizedby inhalation of diethyl ether and a midline abdominalincision was made. A 20-G indwelling needle was insertedinto the portal vein and after perfusion with Hanks Bal-

anced Salt Solution (HBSS, Sigma, St. Louis, MO, UnitedStates), the portal vein was perfused with 100 mL of Dul-L of Dul-of Dul-beccos Modied Eagles Medium (DMEM/F-12, Sigma)containing 200 mg pronase (Roche Diagnostics Corp.,Indianapolis, IN, United States), followed by 100 mL ofL ofofDMEM/F-12 containing 25 mg collagenase (Nitta Gela-tin Inc., Osaka, Japan). When collagenase leaked out ofthe blood vessel because of rupture during perfusion,and it was judged that the liver was not perfused, theprocedure was stopped. After completion of perfusion,the digested liver was extracted carefully and incubated inDMEM/F-12 supplemented with 0.035% pronase and62.5 units/mL of DNase (Sigma) for 20 min at 37on

a shaker incubator.Next, the suspension was ltered through gauze and

undigested liver tissue was discarded. The liver cell sus-

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pension containing KCs was centrifuged for 1 min at50g/min and the supernatant collected. Then, the super-natant was centrifuged again at 50g/min and parenchymal

liver cells were removed as far as possible. Subsequently,the supernatant was centrifuged at 500 g/min for 8 minand a pellet of non-parenchymal cells was obtained.The supernatant was discarded and the pellet was resus-pended with a small amount of washing buffer. The non-parenchymal liver cell suspension containing KCs wascentrifuged at 900 g/min for 15 min with 50% and 25%Percoll (Pharmacia, Uppsala, Sweden) for density-gradi-ent centrifugation, and the first layer from the top wascollected. Next, the suspension was rinsed with 40 mLof HBSS and percentages of KCs that had phagocytosedFITC-latex beads were measured by ow cytometry. The

purity of the KCs was examined by incorporation of la-tex beads 2 m in size and conrmed to be always 96%or greater. Viability was examined by the trypan blue dyeexclusion test and unstained cells accounted for 90% ormore.

Changes in phagocytic activity of KCsPhagocytosis by KCs isolated from each group wasexamined using a BD LSR- flow cytometer (BectonDickenson, Franklin Lakes, NJ, United States). First, ina preliminary experiment, it was determined whetherFITC-latex beads phagocytosed by KCs were measurableby the ow cytometer. The ow cytometry histogram of

KCs in the control group is shown in Figure 1. The up-per panel shows the ow cytometer histogram of KCsin the control rat without adding FITC-latex beads and

the lower panel shows the flow cytometer histogramof KCs in the control rat given FITC-latex beads. Theamount of FITC-latex beads added was the same as inthe subsequent experiments. There were peaks represent-ing the number of FITC-latex beads, but the number of

KCs that phagocytosed ve beads or more was not de-termined. Nevertheless, it was possible to detect phago-cytosed FITC-latex beads. KCs isolated in the controlgroup were used to determine the position of gating andthe gates were set a little wider. After the ow of 10 000counts of KCs, the percentage of cells positive for uo-rescence of FITC-latex beads in all gated cells was calcu-lated in the MCDD and control groups and taken as thephagocytic activity.

Changes in KCsBecause there was a possibility that microbubble accumu-lation in the liver was dependent on the number of KCsin the liver, the liver was removed from one rat in eachgroup, embedded in O.C.T. compound (Sakura Finetech-nical Co. LTD, Tokyo, Japan) and snap-frozen immedi-ately in liquid nitrogen. Later, 5 m frozen sections werecut with a cryostat. Sections were xed with acetone andsubjected to immunohistochemistry for KCs with anti-rat macrophage antibody (ED2, SEROTEC Ltd, Oxford,United Kingdom) to count positive cells under the samemagnication in the MCDD and control groups. In addi-tion, 5 m frozen liver specimens were cut with the cryo-stat and immunouorescent latex beads were observed.

Statistical analysisUnpaired Students t test was employed for all statisticalanalyses and a Pvalue less than 0.01 was considered sta-tistically signicant.

RESULTS

Body weight changes and liver histology in the MCDD

and control groupsBody weight initially was about 50 g and increased in theMCDD group to 270 g at week 9, but decreased slightlythereafter. Although body weight reduced, general con-

ditions were stable. In the control group, body weightincreased steadily to about 440 g at week 16. Generalconditions also were stable (data not shown). In terms ofhistological changes in the liver, large-droplet fat deposi-tion was recognized in the entire liver lobules at week 2and later. At week 8, necrotic inammatory changes wereobserved around the central vein and inammatory nd-ings were evident. At week 16, brosis was observed ex-tending from the central vein and the MCDD group wasjudged to be a NASH model. In contrast, no fatty liver,inammation, or brosis was observed up to week 16 inthe control group (Figure 2).

Changes in contrast ultrasonographic fndingsSignal intensity of the liver obtained by contrast ultrasoundwith LevovistTMwas compared between the two groups.


200

150

100

50

0

Count

102

103

104

105

FITC-A

1

4~3

2

FITC labeled-latex beads (+)

KC phagocytic index = [FITC (+) cells/total KC (gated cells)] 100%

200

150

100

50

0

Count

102

103

104

105

FITC-A

FITC labeled-latex beads (-)

Figure 1 Measurement of Kupffer cells phagocytic activity by flow cy-

tometry.A: The negative control, into which uoresceinisothiocyanate (FITC)-

labeled latex beads were not injected; B: The positive control to which FITC-

labeled latex beads were administered. Note the several peaks of the FITC

from the beads in the positive control. The numbers on the peak shows the

number of latex beads phagocytosed. KC: Kupffer cell.

A

B


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In the MCDD group, the intensity showed a significantdecrease at weeks 2, 8 and 12, and a decrease, although

not signicant, at weeks 4 and 16(Figures 3A and 4A). OnA and 4A). Onand 4A). On4A). On). Onthe other hand, signal intensity of the liver obtained by

contrast ultrasound with SonazoidTM

showed a signicantdecrease at all weekly time points in the MCDD group,

compared to the control group(Figures 3B and 4B).3B and 4B).and 4B).4B).).Phagocytic activity of KCs in each groupPhagocytic activities (control vsMCDD) of KCs exam-ined with uorescent latex beads were 44.3% 12.6%vs18.5% 9.8% (P < 0.01) at week 2, 55.1% 0.1% vs


A B C

D E F

Figure 2 Histological changes of methionine choline-decient diet-fed rat liver (hematoxylin eosin x 200).Extremely large vesicle fat deposits in almost

the entire lobules were detected even as early as two weeks after the start of the methionine choline-decient diet. By 8 wk, spotty necrosis was dispersed and

by 16 wk, there was brosis extending from the central vein. A: Control; B: Two weeks methionine choline-decient (MCD); C: Four weeks MCD; D: Eight weeks

MCD; E: Twelve weeks MCD; F: Sixteen weeks MCD.

A Levovist

Control 2 wk MCD

4 wk MCD 8 wk MCD

12 wk MCD 16 wk MCD

Control 2 wk MCD

4 wk MCD 8 wk MCD

12 wk MCD 16 wk MCD

B Sonazoid

Figure 3 Ultrasound images of the liver 10 min after evovistafter evovistfter evovistTM

(A) and onaoidonaoidTM

injection ().(). Note signal intensity of the liver was lower in all methionineNote signal intensity of the liver was lower in all methionineNote signal intensity of the liver was lower in all methionine

choline-decient diet rats than in the controls. MCD: Methionine choline-decient.


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18.5% 9.8% (P < 0.01) at week 8, and 57.4% 3.4%vs30.3% 0.6% (30.3% 0.6% (P < 0.01) at week 12, and the activi-ties in the MCDD group were about half those in thecontrol group. At week 4, only one rat was examined butthe activities were 61.9% in the control and 7.6% in theMCDD group, and again they tended to be lower in theMCDD group (Figure 5).5).).

Counting of KCs and images of phagocytosed

uorescent latex beadsBecause the decrease in the accumulation of the ultra-sound contrast agent in the MCDD group potentially was

attributable to a decrease in the number of KCs com-pared to the control group, KCs were counted histologi-cally based on immunohistochemistry with anti-rat mac-rophage antibody. As a result, there was no significantdifference between the MCDD and control groups inthe number of KCs (Table 1). In addition, uorescencemicroscopy revealed a decrease in the accumulation ofuorescent latex beads in the MCDD group compared tothe control group (Figure 6).6).).

Serum leptin concentrationsSerum leptin concentrations (control vs MCDD) were120.6 ng/L 39.4 ng/L vs31.5 ng/L 77.6 ng/L (L ((P

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