==== Front
Exp AnimExp. AnimEXPANIMExperimental Animals1341-13571881-7122Japanese Association for Laboratory Animal Science 2946735217-015610.1538/expanim.17-0156OriginalThioacetamide-induced hepatic fibrosis in the common marmoset Inoue Takashi 1Ishizaka Yukihito 2Sasaki Emi 1Lu Jun 2Mineshige Takayuki 1Yanase Mikio 3Sasaki Erika 14Shimoda Masayuki 51) Department of Marmoset Research, Central Institute for
Experimental Animals, 3-25-12 Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-0821,
Japan2) Department of Intractable Diseases, National Center for
Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan3) Department of Gastroenterology, National Center for Global
Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan4) Keio Advanced Research Center, Keio University, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan5) Islet Cell Transplantation Project, Diabetes Research
Center, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo
162-8655, JapanAddresses corresponding: T. Inoue, Department of Marmoset Research, Central
Institute for Experimental Animals, 3-25-12 Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa
210-0821, Japan. Y. Ishizaka, Department of Intractable Diseases, National Center for Global
Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan22 2 2018 2018 67 3 321 327 11 12 2017 30 1 2018 ©2018 Japanese Association for Laboratory Animal
Science2018This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0:

https://creativecommons.org/licenses/by-nc-nd/4.0/)The common marmoset (Callithrix jacchus) is a nonhuman primate that is
used for preclinical research on stem cell transplantation therapies due to its similarity
to human beings as well as its small size, enabling researchers to perform experiments
without preparing a large number of cells. In this study, we developed a marmoset hepatic
fibrosis model for regenerative medicine research. Six female marmosets aged 4–6 years
were administered thioacetamide (TAA) at a dose of 2.5–40 mg/kg two or three times a week.
Hepatic fibrosis was assessed by liver biopsy when blood chemistry indicated liver damage.
Administration of TAA increased total bile acid, aspartate aminotransferase, and total
bilirubin and decreased serum albumin levels. Following more than 11 weeks of continuous
injection of TAA, histological analyses detected hepatic fibrosis in all animals. Type IV
collagen 7S serum levels in animals with hepatic fibrosis were significantly higher than
in normal animals as a possible marker of hepatic fibrosis in marmosets. Serial liver
biopsies following the last administration of TAA revealed that induced fibrosis remained
up to 11 weeks. The results suggest that continuous TAA administration induces persistent
hepatic fibrosis in the common marmoset and this nonhuman primate hepatic fibrosis model
have the possibility to evaluate the therapeutic effects of test samples to ameliorate
hepatic fibrosis.

liver fibrosismarmosetnonhuman primatethioacetamide
==== Body
Introduction
Hepatic fibrosis has emerged as a global public health problem, and numerous cell therapies
have been developed [5, 6]. For human clinical research of the novel therapies, testing with animal models
is essential and various hepatic fibrosis models using mice and rats have been established
[21]. However, positive results in rodent models
have not always translated through to successful clinical therapies and more realistic
preclinical models need to be developed [6]. Nonhuman
primates have the potential to bridge the gap between rodents and humans due to their
physiological similarity with humans [1, 2].

A common marmoset, a small New World monkey, has recently drawn attention as a preclinical
animal model. Marmosets have several favorable features for biomedical research including
small size (300–500 g), good availability, and relatively rapid generational turnover [17, 18, 22]. They are also highly useful in preclinical studies
in regenerative medicine [3, 9, 11]. One of the advantages of
using marmoset animal model is that it can evaluate the effectiveness and safety of cell
transplantation therapy with a small number of cells. Furthermore, it was reported that
marmosets recapitulate human liver diseases in studies of GB virus B infection, a surrogate
model of hepatitis C virus infection [15], and
spontaneous pathologic findings associated with nonalcoholic fatty liver disease and
nonalcoholic steatohepatitis [12]. However, there are
no reports of experimental hepatic fibrosis models of marmosets. Therefore, we investigated
the induction of a marmoset hepatic fibrosis model by administering TAA, a hepatic
fibrosis-inducing compound used for other animal models [14].

In this study, TAA was administered subcutaneously 2–3 times a week at 2.5–40.0 mg/kg to
common marmosets. Weight measurement, blood biochemical tests, and histological examination
by liver biopsy were performed to evaluate the presence of any liver disorder and the
optimal administration dose and interval were examined.

Materials and Methods
Animals
This study was performed in strict accordance with the Regulation for Animal Experiments
of the Central Institute for Experimental Animals (CIEA) based on the Guidelines for
Proper Conduct of Animal Experiments (Science Council of Japan, 2006). The animal
experiment protocol was approved by the CIEA Institutional Animal Care and Use Committee
(approval no. 16002A).

Seven female common marmosets obtained from CLEA Japan (Tokyo, Japan), were used in this
study. Six animals (A–F) were used for TAA-induced liver fibrosis models, and one (G) was
used as an intact control for serum tests. The animals were 4–7 years old and weighed
303–421 g at the start of TAA administration (A–F) or serum collection (G). They were
housed singly in stainless steel cages (409 × 610 × 728 mm) in a conditioned animal room
at 26–28°C and 40–60% humidity with a 12:12 h light/dark cycle. The cages included wooden
perches and a wooden resting place and positioned facing each other to allow the animals
to communicate visually and vocally. The animals were fed a commercial New World primate
diet (CMS-1M, CLEA Japan) with added vitamins and tap water ad libitum,
and the food was moistened with hot water to vary the texture. The marmosets were also fed
supplemental foods, such as sponge cake, apple jelly, or biscuits.

TAA administration and evaluation of liver fibrosis
TAA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in saline (Otsuka Pharmaceutical,
Tokyo, Japan) was subcutaneously administered to each marmoset 2–3 times a week at a dose
of 2.5–40 mg/kg in a volume of 1–2 ml/kg. Dose and frequency of TAA administration were
changed depending on the blood test and liver biopsy results to examine the optimal
protocol. Blood samples (0.5 ml) were drawn from the femoral vein once a week, and the
collected serum was shipped to a commercial laboratory (Nagahama Institute for Biochemical
Science, Oriental Yeast Co., Ltd., Nagahama, Japan) for albumin (ALB), aspartate
aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total
bilirubin (TBIL), total bile acid (TBA), blood urea nitrogen (BUN), and creatinine (CRE)
measurements. Hepatic fibrosis was assessed by liver biopsy at 3–9-week intervals when
blood chemistry indicated liver disorder. Liver tissue was collected via a small abdominal
incision with the animal under anesthesia with 0.04 mg/kg medetomidine, 0.40 mg/kg
midazolam, 0.40 mg/kg butorphanol, and 1–3% isoflurane. Postoperative analgesia and
infection control following biopsy included 1.2 mg/kg ketoprofen and 15 mg/kg ampicillin
administered once daily for 3 consecutive days.

Liver tissue samples were fixed with 10% neutral buffered formalin and embedded in
paraffin. Sections of the samples were stained with hematoxylin and eosin (HE), Masson
trichrome, and Sirius Red, and the specimens were examined microscopically. Areas stained
with Sirius Red were measured for quantitative analysis of fibrosis using a digital slide
scanner (NanoZoomer S60, Hamamatsu Photonics, Hamamatsu, Japan) and ImageJ software
(National Institute of Health, Bethesda, MD, USA). Blood serum samples were collected for
type IV collagen 7S measurement in five marmosets when liver fibrosis was observed on
liver biopsy in each animal. Measurements in marmosets A, C, D, E, and F were performed
after 33, 24, 18, 22, and 22 weeks of initial TAA administration, respectively. This
measurement was performed 2 weeks after liver biopsy for reducing the effect of surgery.
Serum samples from intact three marmosets (E and F before TAA administration and G) were
also used for the measurement. Type IV collagen 7S assay was performed in a commercial
laboratory (SRL Tokyo Laboratories, Tokyo, Japan).

Statistical analysis
Student unpaired, two-tailed t test was used to compare type IV collagen levels using
Prism 7 (GraphPad Software, San Diego, CA, USA).

Results
Subcutaneous injection of TAA reproducibly induces hepatic fibrosis in
marmosets
Due to the lack of information regarding an effective dose of TAA, we had modified the
doses and frequencies of TAA administration in four trials (Fig. 1Fig. 1. Subcutaneous injection of TAA reproducibly induces hepatic fibrosis in 6 marmosets
(A–E). Upper) Doses and frequencies of TAA administration, hepatic fibrosis observed
in biopsy specimens, and percentages of Sirius Red staining-positive areas in these
specimens; hepatic fibrosis was assessed to evaluate the severity of
histopathological changes as follows: no (−), mild (+), and severe (++). Lower)
Changes of body weight (BW) and blood chemistry: albumin (ALB), aspartate
aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH),
total bilirubin (TBIL), total bile acid (TBA).

). In the first trial, we gradually increased the dose from 2.5 to 10 mg/kg twice a
week in marmoset A (Fig. 1), but neither blood
examinations nor liver biopsies indicated hepatic fibrosis even at 16 weeks after initial
treatment. Next, TAA was increased up to 30 mg/kg three times a week after an interval of
2 weeks. A dose of TAA was further escalated to 40 mg/kg three times a week at the next 2
weeks as liver biopsy did not detected hepatic fibrosis. Then, hepatic fibrosis was
detected at 24 weeks after initial treatment coupled with slight changes in blood
examinations for AST, LDH, TBIL, and TBA levels. Biopsy specimens with Masson trichrome
and Sirius Red stains showed that collagen fiber increased in pericentral regions and
peritoneal areas (Fig. 1).

In the second trial, we started 10 mg/kg of TAA twice a week for 4 weeks, then increased
to 20 mg/kg twice a week and continued for the next 3 weeks in marmoset B (Fig. 1). Next, we escalated TAA up to 40 mg/kg as we
observed no hepatic fibrosis and then detected hepatic fibrosis 6 weeks after increasing
dose to 40 mg/kg (Fig. 1). Hepatic fibrosis was
persistently observed for 9 weeks with continuous TAA injections.

In the third trial, we started 40 mg/kg as an initial dose of TAA in marmosets C and D
(Fig. 1). We observed hepatic fibrosis in both
the marmosets at 11 weeks after the initial treatment (Figs. 1,2Fig. 2. Hepatic fibrosis induced by TAA in a marmoset. Tissue section with Masson trichrome
staining (×2.5) was collected from marmoset D that had been subcutaneously injected
with TAA at 40 mg/kg 2–3 times a week for 16 weeks (Fig. 1). Collagen fiber (blue-colored) increased in the
pericentral and periportal areas with bridging among areas.

 ,3). However, high-dose TAA
administration resulted in temporary withdrawal due to weakening by suspected hepatic
dysfunction. The marmoset D that had been administered TAA (40 mg/kg) three times a week
showed anorexia and nearly 20% loss of body weight 7 weeks after initial administration.
Then, we stopped TAA administration for recovery and resumed TAA injection after an
interval of 2 weeks, reducing the frequency to twice a week.

In the fourth trial, TAA was injected 20, 30, and 40 mg/kg three times a week in
marmosets E and F (Fig. 1). Liver biopsy
detected hepatic fibrosis in the both animals 14 weeks after initial administration.
However, the both animals were weakened during TAA administration period (40 mg/kg three
times a week) and were withdrawn from TAA; the marmoset E showed anorexia, hypoactivity
and marked body weight loss 8 weeks after initial administration, and the marmoset F
showed anorexia, hypoactivity and hypothermia 11 weeks after intimal administration,
respectively.

Quantitative assay of hepatic fibrosis with Sirius Red staining was performed using all
28 liver biopsy specimens in which the average area (± SD) was 6.46 ± 2.66 mm2.
The rate of appearance of Sirius Red-positive area was gradually increased after 40 mg/kg
of TAA administration up to 31% in marmoset B (Fig.
1).

Recovery from hepatic fibrosis after TAA cessation
Serial liver biopsies were performed to examine spontaneous recovery from hepatic
fibrosis. The typical case marmoset C to which 40 mg/kg TAA was administered twice a week
is shown in Fig. 3Fig. 3. Progress and recovery of hepatic fibrosis in the liver tissue of a marmoset
administered TAA. Biopsy specimens with hematoxylin and eosin (HE) and Masson
trichrome stain (MT) of marmoset C (×200) at each time point (w: week) are shown.
Marmoset C was subcutaneously injected with TAA at a dose of 40 mg/kg twice a week
for 23 weeks (Fig. 1). Collagen fiber
increased with the TAA administration period, and fibrosis was observed until 11
weeks after withdrawal.

. Masson trichrome staining detected definite fibrosis at week 21 following the
initiation of TAA. Notably, fibrosis was continuously observed at 6 weeks after the last
injection (Fig. 3). At week 11, mild fibrosis
was still observed, suggesting that TAA-induced hepatic fibrosis persists for
approximately 3 months after discontinuation of TAA treatment.

Blood examinations
Mean values ± SD of blood chemistry in 7 normal marmosets (marmosets A–F before TAA
administration and marmoset G) were ALB 4.6 ± 0.6 g/dl, AST 125 ± 97 U/l, ALT 7.8 ± 4.7
U/l, LDH 184 ± 41 U/l, TBIL 0.09 ± 0.02 mg/dl, TBA 8 ± 7 µmol/l, BUN 30.4
± 5.7 mg/dl and CRE 0.19 ± 0.04 mg/dl. As an early response to TAA administration, AST,
ALT, and LDH were increased (Fig. 1).
Conversely, TBIL and TBA levels increased after relatively long-term TAA administration,
possibly due to altered structure of the hepatic organs. Additionally, serum ALB level
coupled with body weight tends to decrease, which could reflect chronic liver dysfunction.
The marmosets that showed severe clinical signs had either high TBIL (>1 mg/dl), high
TBA (>300 µmol/l) or low ALB (<3.5 g/dl). Following TAA cessation,
TBIL and TBA levels rapidly recovered in most cases. BUN and CRE levels that represent
renal function slightly changed during the experimental period.

Measurement results of blood type IV collagen 7S
We measured the serum level of type IV collagen 7S, which is known as a marker for
hepatic fibrosis, in TAA-treated marmosets. As shown in Fig. 4Fig. 4. Comparison of serum type IV collagen 7S between intact animals and TAA-induced
hepatic fibrosis animals.

, serum levels of type IV collagen 7S in TAA-treated marmosets with hepatic fibrosis
observed on liver biopsy were 7.98 ± 0.50 ng/ml (mean ± SD, n=5) and were significantly
higher than those in the control group (6.30 ± 0.50 ng/ml, n=3).

Discussion
Hepatic fibrosis is defined as the deposition of excess extracellular matrix and is based
on complex interactions between matrix-producing hepatic stellate cells and abundant hepatic
and infiltrating cells. Researching these pathologies and production processes requires
in vitro and in vivo experimental work in animals.
Although hepatic fibrosis in humans can be caused by various stimuli (congenital, metabolic,
inflammatory, parasitic, vascular, toxin, or drug), the molecular mechanisms underlying
fibrosis are basically the same [8]. A highly
regulated program of molecular changes occurs following any kind of liver injury [7]. In fibrosis research, experimental animal models are
currently the gold standard to confirm a proposed disease-associated mechanism, and these
models should at least partially mimic a clinical situation. The most commonly used approach
for inducing toxin-mediated experimental liver fibrosis is the periodic administration of
CCl4 or TAA in mice or rats [4]. However,
there have been few reports of hepatic fibrosis models using nonhuman primates which have
advantages for preclinical translational research. This study succeeded reproducible
induction of hepatic fibrosis using TAA in common marmosets. That is the first report of a
potential liver fibrosis model using the small nonhuman primate species.

As it has been reported that intraperitoneal or oral administration of TAA reproducibly
induced hepatic fibrosis in rodents, we first attempted to administer TAA in drinking water
[10, 20].
However, marmosets did not drink water with TAA administration; hence, we selected
subcutaneous injection as an alternative route of administration. Increased dosages could
reduce modeling time but greatly increase the mortality rate. Our preliminary data
demonstrated that a safe dosage for subcutaneous injection of TAA is<50 mg/kg
(unpublished data). This study adopted the method of administering TAA subcutaneously at 40
mg/kg 2 or 3 times a week to establish a hepatic fibrosis model in common marmosets, which
showed morphological and pathophysiological similarity to human hepatic fibrosis.
Furthermore, fibrosis sustained for up to 11 weeks after withdrawal of TAA. Therefore, 40
mg/kg should be an appropriate initial dose for subcutaneous injection of TAA for hepatic
fibrosis in marmosets. In addition, it is considered appropriate to temporarily stop TAA
administration when hepatic dysfunction occurs, and resume administration following hepatic
function recovery until hepatic fibrosis occurs.

Histological examination by liver biopsy is the most accurate for estimating hepatic
fibrosis but it is invasive. We found that blood levels of TBA, ALB, TBIL, and blood type IV
collagen 7S could be useful markers for evaluating hepatic fibrosis. Simultaneous elevation
of serum TBIL and TBA levels is a sign of acute hepatic failure. Serum TBIL and TBA levels
could be good markers for determining whether we should administer TAA. It is considered
appropriate to temporarily stop TAA administration when these serum markers indicate hepatic
dysfunction occurs, and resume administration following hepatic function recovery until
hepatic fibrosis occurs.

Type IV collagen is a main collagenous component of the basement membrane and the serum
concentration of the 7S fragment of the N-terminal domain of type IV collagen (type IV
collagen 7S) has been identified as a marker for assessing hepatic fibrogenesis and fibrosis
[16, 19].
Serum type IV collagen 7S levels in the TAA-induced hepatic fibrosis marmosets were higher
than control marmosets. This indicates monitoring this marker could aid in the diagnosis of
hepatic fibrosis in marmosets as in humans. The human cutoff value is 6.0 ng/ml [13] but 7.0 ng/ml could be a good cutoff value for
marmosets; however, further evaluation is necessary.

The study suggests that continuous TAA administration induces progressive and persistent
hepatic fibrosis in the common marmoset. A hepatic fibrosis model of common marmosets with
TAA could be widely used, and established models are promising for research on preventing
and treating chronic liver diseases.

This study was mainly supported by the Program for Basic and Clinical Research on
Hepatitis, “Differentiation of Mesenchymal Stem Cells to Hepatic Cells by Artificial
Transcriptional Factors” from the Japan Agency for Medical Research and Development (AMED)
and was partially supported by the Strategic Research Program for Brain Science,
“Maintenance of Systems for Creation and Spread of Primate Model Animals” from AMED. We
wish to thank Dr. Kenji Kawai, Dr. Masahiko Yasuda, and Ms. Mika Yagoto for their helpful
support in preparing sections of this manuscript as well as all members of the CIEA marmoset
research group in Kawasaki, Japan, for the warm support.
==== Refs
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