1. Introduction & Pathophysiology of Liver fibrosis 2. Experimental Models of Hepatic fibrosis 3. Timeline of development of Fibrotic models 4. Surgically developed models for Fibrosis 5. Chemically Induced Models for Fibrosis 6. Diet Induced Models for Fibrosis 7. Infection based models 8. Extra points 9. Conclusion 10. References

1. EXPERIMENTAL MODELS
OF LIVER DAMAGE
Priyansha Singh
B.Pharm- UIPS, Panjab University
MS (Pharm). Pharmacology & Toxicology- NIPER, Guwahati

2. CONTENTS
Introduction & Pathophysiology of Liver fibrosis
Experimental Models of Hepatic fibrosis
Timeline of development of Fibrotic models
Surgically developed models for Fibrosis
Chemically Induced Models for Fibrosis
Diet Induced Models for Fibrosis
Infection based models
Extra points
Conclusion
References

3. INTRODUCTION
• Models of liver damage can provide useful tools for the study of hepatic histopathology. Almost all of
the known acute and chronic lesions of the liver can be induced experimentally.
• Liver diseases are strongly associated with oxidative and nitrosative stress; thus, models of experimental
damage described herein share the characteristic that are caused, at least in part, by free radicals.
• Therefore, these experimental models constitute essential tools for basic researchers to study
biochemical and molecular mechanisms of injury and to investigate rationally the efficacy of new
treatments before utilizing them in humans.
• Use of whole animals is imperative to show that an agent has an adverse effect on the liver in a setting
of physiologic significance.
• Whole animals also must be employed if the effects of various factors and manipulations on disease are
to have meaning for the mechanism of injury and for the pathophysiologic impact of the hepatic injury

4. PATHOPHYSIOLOGY OF HEPATIC FIBROSIS

5. Timeline of animal and organoid liver fibrosis mode

6. SURGICALLY INDUCED MODELS FOR
FIBROSIS
cBDL induced hepatic fibrosis

7. SURGICAL MODELS FOR LIVER CIRRHOSIS- cBDL
• Common bile duct ligation (cBDL) is a model of secondary biliary cirrhosis that can be performed both in rats and mice. The surgical
obstruction of the common bile duct causes bile to accumulate in the liver, leading to hepatic injury, inflammation and, ultimately, fibrosis and
cirrhosis.
• Cholestatic animal models such as partial or total bile duct ligation (BDL) can approximately mimic clinical obstructive cholangiopathies, for
example, biliary strictures and biliary atresia. Hepatic apoptosis is a routine manifestation of hepatobiliary injury in BDL models.

9. • Activation of the TNF-related apoptosis-inducing ligand receptor (TRAILR) and Fas death receptor signaling
pathway is an important pathway for hepatocyte apoptosis induced by bile acid.
• Bile acids activate Fas-related death signals in a ligand-dependent and - dependent hepatocyte apoptosis manner.
• Bile acid stimulates intracellular vesicles associated with the Golgi complex and the trans-Golgi network, and
transfers Fas-containing vesicles to hepatocyte membranes, initiating a ligand-dependent death signaling
pathway, while increasing Fas density on the surface of hepatocytes to making it more sensitive to Fas
agonists.
• Bile acid-mediated apoptosis of hepatocytes not only activates ligand-independent death receptor oligomerization,
but also regulates the sensitivity of death receptor-related signaling pathways.

10. • Death receptor-mediated apoptosis of hepatocytes is regulated by different apoptotic signals. On the death-inducing signaling
complex (DISC), bile acid stimulates the phosphorylation of cFLIP to reduce the binding of two different isoforms of cFLIP long
(cFLIP-L) and cFLIP short (cFLIP-S) to Fas-associated death domain (FADD) in DISC, and then reduce the recruitment of cFLIP
to DISC, promoting the activation of caspases 8 and 10
• Activated caspases 8 and 10 cleave bid into tBid and enter it mitochondria with Bax to induce mitochondrial dysfunction and
promote the release of cytochrome c. The released cytochrome c binds to apoptosis-activating factor-1 (Apaf-1) to promote the
activation of Caspase 9.
• And caspase 9 further activates Caspase3/6/7, which eventually leads to liver cell death. Besides, bile acids can also directly cause
Bax translocation into mitochondria, which can also lead to the release of cytochrome c and the downstream effectors of caspases
signaling pathway.
• Bile acids can also stimulate mitochondrial respiratory chain to stimulate the production of reactive oxygen species (ROS) and
cause mitochondrial membrane permeability transition (MPT), and release cytochrome C.

11. CHEMICALLY INDUCED LIVER FIBROSIS
1. CCl4 induced liver fibrosis
2. TAA induced liver fibrosis
3. DMN and Diethylnitrosamine (DEN) induced fibrosis
4. Ethanol induced fibrosis

12. CARBON TETRACHLORIDE IS A USEFUL MODEL TO INDUCE ACUTE
AND CHRONIC LIVER DAMAGE
• Carbon tetrachloride (CCl4) is one of the most widely used toxicant for inducing experimental liver injury in
animals. It consistently produces liver injury in many species, including nonhuman primates.
• CCl4 directly damages liver cells (mainly endothelial cells and hepatic parenchyma cells in the hepatic portal vein
region) by altering the permeability of lysosomes and mitochondrial membranes.
• The oxidase system in liver cells can also form highly active free radical metabolites through CYP2E1, leading to
severe central lobular necrosis.
• The damage mechanism of CCl4 is mainly oxidative damage caused by lipid peroxidation. Cytochrome P450
enzyme, especially CYP2E1, converts CCl4 into highly toxic trichloromethyl radical (⋅CCl3) and trichloromethyl
peroxide (⋅CCl3O2). This model has been widely used to study the pathogenesis of liver fibrosis and cirrhosis.

13. CELLULAR MECHANISMS OF CCl4 CAUSING
HEPATIC DAMAGE

14. FREE RADICAL MECHANISMS FOR HEPATIC CIRRHOSIS

15. SOPs FOR THE CCl4 MOUSE MODEL BY SCHOLTEN
• The toxicological mechanism of liver fibrosis induced by CCl4 may be related to multiple biological processes, pathways
and targets. CD133 was significantly up-regulated after CCl4 treatment, and the levels of desmin and glial fibrillary acidic
protein, the representative markers of HSC, were also significantly increased.
• The EGF expression was significantly reduced, contrary to what has been observed in humans. In A/J mice, chronic liver
injury induced by CCl4 differs from HCC induced by human cirrhosis.
• The collagen expression was found to be significantly increased after CCl4 injury, and the number of cells expressing
cytoglobin was also increased. Cytoglobin may be an early biomarker of liver fibrosis. In addition to intraperitoneal
injection, CCl4 can also be inhaled to establish a liver fibrosis model.
• Compared with intraperitoneal injection, inhalation route is a complex process, with great individual differences, and can
cause multiple organ damage. An intraperitoneal injection can reach the liver directly from the hepatic portal vein.
• The animal model of liver fibrosis induced by CCl4 is relatively low-cost to develop, and the implementation method is
relatively simple.

16. SOPs FOR THE CCl4 MOUSE MODEL BY SCHOLTEN
Rats were exposed to CCl4 vapor
twice a week for 30 s each time,
while phenobarbital (0.3 g/L) was
added to drinking water
The duration of inhalation was
increased by 30 s after the first three
sessions and by 1 min after every
three sessions until a steady state
was reached for 5 min.
After 9 weeks, it can lead to liver
fibrosis
• BALB/c mice manifest more liver fibrosis upon CCl4 administration compared to C57BL/6 and DBA/2 counterparts.
• In the most routinely followed strategy, CCl4 is injected intraperitoneally 2 to 3 times per week during 4 to 6 weeks at a dose range of 300
to 1000 μl/kg.
• Recently, a C57BL/6 mouse model was standardized relying on intraperitoneal administration of CCl4 in a concentration range between
0.5 to 0.7 μl/g body weight 2 times per week for 6 weeks or 3 times per week for 4 weeks.
• Alternatively, CCl4 can be administered orally, subcutaneously or through inhalation 2 times per week 10 weeks, between 4 and 8 weeks
or between 2 and 6 weeks, respectively.

17. TAA INDUCED FIBROSIS MODEL

18. Thioacetamide
CYP2E1
Reactive radicals
Oxidative stress
• Hepatocytes damage
• Activation of Kupffer cells & monocytes
Release of cytokines
Quiescent HSC
Activated HSC
(activated myofibroblasts)
FIBROSIS
MECHANISM OF TAA INDUCED FIBROSIS MODEL

19. TAA INDUCED FIBROSIS MODEL
• TAA itself is not hepatotoxic, and its active metabolites covalently bind to proteins and lipids, causing oxidative stress
leading to central lobular necrosis of the liver.
• Compared with CCl4, TAA resulted in more periportal inflammatory cell infiltration and more pronounced ductal
hyperplasia.
• Although both CCl4 and TAA-induced liver injury and fibrosis are dependent on CYP2E1, in some cases, CYP2A5 may
have a protective effect against TAA-induced liver injury and fibrosis but has no effect on the hepatotoxicity of CCl4.
• Although both CCl4 and TAA-induced liver injury and fibrosis are dependent on CYP2E1, in some cases, CYP2A5 may
have a protective effect against TAA-induced liver injury and fibrosis but has no effect on the hepatotoxicity of CCl4.
• After 12-week oral administration of TAA in rats, bile duct fibrosis was induced, characterized by tubular hyperplasia
surrounded by fibrous tissue.

20. DMN(Dimethylnitrosamine) & DEN(Diethylnitrosamine) INDUCED FIBROSIS
MODEL
DMN-induced hepatic fibrosis in rats. Rats were injected with DMN (10 mg/kg body weight, i.p.) every other day for 4 weeks. (A) Staining
with H&E (upper panels) or Sirius Red (lower panels) of liver samples from treated or not treated with DMN. (B) The collagenous fibres (A,
lower panels) were quantified by imaging analysis. (C) Serum levels of ALT and AST of rats treated or not treated with DMN.

21. Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) INDUCED FIBROSIS
MODEL
• Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) are carcinogenic compounds that are frequently used to
experimentally induce liver fibrosis in animals. As a consequence of their biotransformation, ROS are abundantly produced, all
which react with nucleic acids, proteins and lipids, causing cell malfunction and triggering the development of centrilobular
necrosis.
The susceptibility
of mice to
develop HCC due
to DEN
administration is
determined, at
least in part, by
the strain.
In this respect,
C3H and B6C3F1
mice are most
likely to develop
tumors compared
to C57BL mice.
In rats, the
R16 strain is
most
susceptible to
carcinogenic
chemicals.
DEN is routinely
administered
orally to mice at a
dose of 100 μl/kg
body weight for 12
weeks.
DEN is administered to rats
with weekly oral gavage of 5
ml of 1.5 %/kg DEN during 3
to 11 weeks or
intraperitoneally once per
week for 2 weeks, applying
doses between 40 and 100
mg/kg.
DMN is
administered
intraperitoneally to
mice 10 μg/g 3
times per week
during 3 weeks

22. PATHOLOGIES OF DMN CAUSING FIBROSIS

23. DMN is a potent liver specific toxin.
Its metabolism, tissue distribution,
and ability to cause injury to livers of
rats. Intermittent administration of
this compound was reported to
induce liver fibrosis in dogs and rats.
The toxicity of various nitrosamines
in animals and humans is well
established, and trace amounts of
DEN or DMN can cause severe liver
injury in either the enteric or oral
form.
The most prominent manifestations
are extensive neutrophilic
infiltration, extensive central lobular
hemorrhaging and necrosis, bile duct
hyperplasia, fibrosis, bridging
necrosis and ultimately HCC.
Due to the stability of DMN- and
DEN-induced liver changes, the
administration of these agents to
rodents has become a commonly
used experimental model.
Iron deposition and fat accumulation
were shown to play an important
role in the pathological changes of
DMN-induced liver fibrosis in rats.
Rats were intraperitoneally injected
with DMN 3 days a week for 3
weeks. Severe central lobular
congestion and hemorrhaging and
necrosis were observed on day 7.
On day 14, central lobular necrosis
and numerous neutrophils
infiltration were observed.
Collagenous fibrous deposition was
seen on day 21, along with severe
central lobular necrosis, focal fatty
changes, bile duct hyperplasia and
bridging necrosis and fibrosis around
the central vein.
DMN- induced liver injury in rats
seems to be an animal model similar
to early human cirrhosis.
The model shows significantly
increased liver collagen fibraldehyde
content due to DMN administration,
and the cross-linking of liver fibrosis
collagen induced by DMN is greater
than that in normal liver.
In the comparative study of
dimethylnitrosamine (DMN), CCl4
and TAA rat liver fibrosis models,
lipid peroxidation was highest in the
CCl4 model, and the serum liver
enzyme levels increased with
severity.

24. CELLULAR & MOLECULAR MECHANISMS FOR DMN
INDUCED FIBROSIS

25. PATHOLOGICAL FINDINGS OF DEN/ DMN IN LIVER
• DMN treated rats lose weight and become less vigorous with ruffled hair coat. There is significant loss
in average body weights of DMN treated rats; first detectable after 2 weeks of DMN treatment, and this
difference remains through weeks 3 and 4 after DMN treatment.
• As the rats receive DMN over successive weeks, damage to the liver causes it to become smaller. The
liver index; which is the percent of liver weight at final body weight was significantly lower for the
DMN treated rats
• At sacrifice, after 4 weeks of DMN treatment, the liver is smaller and harder compared to those from
aged matched control animals. Fibrin may be present on the liver surface and adjacent liver lobes are
adhered. About 20% of rats have ascites.

26. • Injury to the liver causes increased permeability of the hepatocyte cell membrane. Increased serum ALT and AST are indicators of
hepatocyte damage. Serum ALT and AST of the DMN treated group are significantly higher than the control group after weeks 2 and 4 of
DMN injection. Serum ALT and AST levels typically increase after each week of DMN treatment.
• Histological examination of livers from DMN treated rats show that there is progressive increase and expansion of fibrous septa, with loss
of hepatocytes, over time compared with control rats. Masson's Trichome stain is commonly used to highlight collagen deposits in liver
tissue
a) liver section from a normal control rat; b) liver section from a rat after receiving 1 week of
dimethylnitrosamine (DMN); c) liver section from a rat after receiving 2 weeks of DMN. There is fibrous
expansion of most portal areas with occasional portal to portal bridging. d) Liver section from a rat after
receiving 3 weeks of DMN. Note the fibrous expansion of portal areas with marked portal to portal as well
as portal to central bridging. e) Liver section from a rat after receiving 4 weeks of DMN. There is cirrhosis
with nodule formation. The control liver is from the group sacrificed together with rats after 4 weeks of
DMN injection. There is a pattern of progressive increase of fibrosis score from 0 in (a); 2 in (b); 3 in (c) 4
in (d) to 5/6 in (e)

27. ETHANOL INDUCED FIBROSIS
• ALD usually starts with hepatic steatosis that may progress into fibrosis and subsequent cirrhosis. In the liver,
ethanol is mainly metabolized by alcohol dehydrogenases and CYP450 enzymes. This process is associated
with several deleterious events, such as the production of ROS, glutathione depletion, lipid peroxidation and
increased collagen synthesis.
• Collectively, these mechanisms induce hepatocyte apoptosis, inflammation and the activation of HSCs.
• Although rodents have a natural aversion for alcohol consumption, with the exception of HAP-2 and
C57BL/6 mice, they remain the most routinely used model in the study of ALD. Mice are more prone to
alcohol-induced ALD than rats, with female mice being most susceptible.
• A combination of ethanol administration with a second stimulus, including specific diets, pharmacological
agents, CYP450 inducers, hormones, Toll-like receptor ligands, genetic manipulation or viral infection are
used to stimulate ALD

28. MECHANISMS OF ALD
• Ethanol metabolism and hepatic oxidant stress. Ethanol is oxidized
principally in liver hepatocytes by ADH, CYP2E1, and catalase to
acetaldehyde (Ach).
• Ach is a highly reactive intermediate that, itself, covalently binds to
protein or can undergo secondary reactions to form MAA.
• CYP2E1 is induced by ethanol and produces radicals, including
superoxide and hydroxyl radicals, which by themselves are reactive and
can undergo secondary reactions with PUFA, producing ROS and RNS
as defined in this figure and in the text. The latter reactive molecules
can also form adducts with proteins.

30. HOW DOES ALCOHOL LEAD TO FATTY LIVER DISEASE
AND FIBROSIS
• Three pathways are involved in alcohol metabolism and all of them converge on the oxidation of ethanol to
acetaldehyde.
• Acetaldehyde is further converted to acetate by aldehyde dehydrogenase in the mitochondria.
• Acetate can be rapidly oxidized into CO2 and H2O by peripheral tissues, or can be diverted to the tri-
carboxylic acid (TCA) pathway.
• The oxidation of ethanol to acetaldehyde by microsomal ethanol oxidation system (MEOS) occurs in the
smooth endoplasmic reticulum and changes the NADPH/NADP ratio which in turn influences the
regeneration of glutathione thereby increasing cellular oxidative stress.
• The alcohol dehydrogenase pathway is the major pathway and occurs in the cytosol, generating large amounts
of NADH.
• NADH in turn inhibits TCA cycle enzymes and leads to accumulation of acetyl CoA and increase in ketone
body generation and acidosis.
• NADH also inhibits fatty acid oxidation leading to accumulation of fats and causing “fatty liver.” A
combination of the above factors leads to tissue injury and activation of the fibrogenic pathway.

31. DIET INDUCED LIVER DISEASE MODEL
• A number of specific diets can be used to induce progression of NAFLD to non-alcoholic steatohepatitis
(NASH) in experimental animals.
• It seems that the rodent strain is the major determinant of liver fibrosis caused by dietary ingredients.
• Overall, C57BL/6 mice are more susceptible to develop diet-induced fibrosis compared to the BALB/c strain.
• Nevertheless, these diet-based models fail to mimic the typical characteristics of the human pathology, thus
restricting interspecies extrapolation of results

33. METHIONINE-DEFICIENT AND CHOLINE-DEFICIENT
DIET
• Mice fed a methionine-deficient and choline-deficient (MCD) diet constitute a frequently addressed model to study NASH.
However, this dietary model lacks some of the major human pathological features, including obesity and pronounced peripheral
insulin resistance. MCD diets mimic the hepatic stress caused by the fatty acid flux from adipose tissue to the liver as well as
increased production of triglycerides, resulting in liver steatosis and lipotoxicity.
• Kupffer cells may play a role in the initiation and progression of MCD diet-induced liver steatosis, as they are the firsts to respond
to hepatocyte injury. Activated Kupffer cells increase the production of TNFα, the recruitment of monocytes and may control
collagen deposition by secreting high levels of MMP-13.
• In addition, the infiltration of these macrophages can also promote the upregulation of pro-inflammatory pathways and mediators,
including nuclear factor kappa-light-chain-enhancer of activated B-cells, intracellular adhesion molecule 1, cyclooxygenase 2,
monocyte chemo-attractant protein-1 and IL6.
• In a following next step, HSCs become activated, which directs the pathology into a more fibrotic stage. Mice fed a MCD diet
present steatohepatitis after 8 weeks, whereas the more fibrotic stage, in particular affecting the portal and bridging areas, is only
observed after 16 weeks.

34. CHOLINE-DEFICIENT L-AMINO ACID DEFINED DIET
• The choline-deficient L-amino acid defined diet causes a similar phenotype as the MCD diet,
though animals also gain weight and develop peripheral insulin resistance.
• Choline-deficient L-amino acid-fed rats and C57BL/6J mice frequently produce liver tumors
associated with fibrosis, rendering these models eligible to study the progression from NAFLD to
NASH and further to HCC.
• Mice fed this diet develop evident liver fibrosis after 22 weeks and HCC after 84 weeks

35. HIGH-FAT DIET
• High-fat (HF) diets overcome the shortcomings of the MCD diet, since animals gain body weight
and develop peripheral insulin resistance. Although this model has phenotypic hallmarks similar to
human NASH, it requires 50 weeks to develop steatohepatitis with merely mild fibrosis in mice.
• Male inbred C57BL/6 mice are the most suitable rodents to develop NASH using a HF diet. This is
in contrast to rats, which are not responsive to HF diets. Because of this flaw, an alternative high-
cholesterol diet has been proposed for rats.
• This high-cholesterol diet induces fibrotic NASH in 9 weeks, whereby the rats occasionally
develop cirrhosis, reminiscent of human NASH. Nonetheless, the main disadvantage of this high-
cholesterol diet model is the lack of both obesity and insulin resistance.

36. INFECTION-BASED MODELS

37. INFECTION-BASED MODELS
• Infection-based models have aided researchers in the elucidation of the mechanisms mediated by the immune
system, which occur during liver fibrosis and that can not be reproduced in other models.
• Hepatitis virus infection induces liver fibrosis in humans, but not in rodents. Therefore, genetically engineered
animals able to express the HBV envelope coding region under the constitutive transcriptional control of the
mouse albumin promoter are typically used.
• These mice do not spontaneously develop liver hepatitis unless their immune system is compromised and
replaced by non-transgenic bone marrow cells and spleen cells previously immunized with the HBV antigen.
This model has shown the importance of immune reactions in the progression of the disease to HCC.
• An alternative to this model is the use of immuno-deficient mice transfected with a HBV plasmid.
Schistosoma mansoni infection is readily established in mice due to high resemblance to human infection and
high reproducibility. Nevertheless, different mouse strains can show great variations in hepatic fibrosis levels,
with the C3H/ HeN strain being the most prone to develop higher levels of fibrosis.

38. INFECTION-BASED MODELS
• Alternatively, animals can be infected by percutaneous administration of 35 cercarias through the tail or by
intravenous administration of 10.000 viable eggs.
• The cercarias evolve into adults and can produce more than 100 eggs per day, which can be trapped in the
liver. This forms the main cause for the development of granulomas associated with liver fibrosis.
• Development of the latter is mediated by the action of T-helper 2 cytokines, especially IL13 in a Schistosoma
mansoni model and IL17A in a Schistosoma japonicum infection, which highlights the role of cytokines in the
development of this chronic liver disease.
• Moreover, the presence of activated HSCs in the periphery of the egg granulomas from Schistosoma
japonicum has been observed in rodents and humans. Collectively, the role of the cytokines in these infection
models contributes to the activation of the HSCs and thus to the progression of liver fibrosis.

39. DRUG INDUCED LIVER DISEASE MODELS

40. Drug Modeling methods Pathology features Molecular mechanisms
Acetaminophen Dose: 300-500 mg/kg
Administration: Single i.p., observe 4
hours later
Sinusoidal congestion and
hemorrhage, dilated central vein,
inflammatory cell infiltration,
degenerated hepatocytes showing
perinuclear vacuolization
1. GSH depleted by NAPQI
2. Mitochondrial dysfunction
3. Oxidative stress
4. Activating the protein kinase JNK
5. Hepatocyte apoptosis
6. ER stress and UPR
Isoniazid 1. Dose: 200 or 400 mg/kg/day
Administration: Gavage daily for one
week
2. Dose: INH 75 mg/kg/day and RIF
150 mg/kg/day
Administration: Gavage daily for one
week
Hepatocyte steatosis and edema, the
sinus almost disappears, part of the
mitochondrial cristae disappeared,
and the endoplasmic reticulum was
vesicular
1. Mitochondrial injury and dysfunction
2. Hydrazine, the toxic metabolite
3. Apoptosis
4. Oxidative stress
5. Co- administration of RIF induces CYP 450
enzymes and promotes hepatotoxicity
6. Free radical lipid peroxidation
Cyclosporine A Dose: 20 mg/kg, (Sandimmun infusion
dissolved in olive oil, 25mg/ml)
Administration: Subcutaneous
injection daily for 21 days
Hepatocyte steatosis, apoptosis,
vacuolar degeneration hepatocytes,
lipid droplets, reduced mitochondrial
cristae, and rough endoplasmic
reticulum cystic expansion
1. Imbalance between production of oxygen free
radicals and the endogenous antioxidant defense
system
2. Substantial increase in caspase 3 activity that
induces apoptosis
Tetracycline 1. Dose: 50 mg/kg
Administration: Single i.p., observe 6
hours later
2. Dose: 200 mg/kg in saline
Administration: Single i.p., observe
after 36-hour free diets and 12-hour
diet deprivations
Hepatic parenchymal cells micro-
vesicular steatosis, hydropic
degeneration around the pericentral
zone
1. Affecting cellular lipid metabolism
2. Apoptosis
3. ER stress
4. Oxidative stress

41. Drug Modeling methods Pathology features Molecular mechanisms
Tripterygium wilfordii
multiglycoside
1. Dose: 300 mg/kg TP
Administration: Gavage, observe after 18
hours
2. Dose: 600 μg/kg TP
Administration: Intragastric gavage daily for
5 days
3. Dose: 120 mg/kg GTW
Administration: Gavage daily for 28 days
Extensive hepatocyte turbidity, focal
hepatocyte ballooning in the central
vein and peripheral areas, scattered
eosinophilic changes in hepatocytes
Tendencies toward augmented focal
necrosis, inflammatory cell infiltration,
and bile duct hyperplasia Partial
necrosis with inflammatory cell
infiltration in hepatocytes
1. Cells apoptosis
2. Mitochondria lesions
3. Immune response
4. Lipid peroxidation
5. Inflammation
6. Oxidative stress
Polygonum
multiflorum
1. Dose: 2.8 mg/kg LPS with uncertain doses
of EA extract of PM
Administration: Tail vein injection of LPS and
intragastrically administer EA
extract of PM
2. Dose: 5.4 g/kg water extract of processed
PM
Administration: i.p. for 7 days
Hepatocyte focal necrosis, loss of central
vein intima and a large number of
inflammatory cell infiltration
1. Disruption of energy
metabolism, amino acid and lipid
metabolism
2. Inflammatory response
3. Steatosis
4. CYP1A2 and CYP2E1 mRNA
expression levels were
significantly inhibited
Polygonum
multiflorum
Dose: 500 mg/kg/d
Administration: Gavage for 2 weeks
Massive necrosis, liver steatosis and
increase of lipid accumulation
1. Oxidative stress
2. Hepatotoxic metabolites
Carbamazepine Dose: 400 mg/kg for 4 days and 800 mg/kg
on the 5th day
Administration: Oral gavage
Prominent hepatic necrosis and loss of
hepatocytes,
especially around the central vein
Hepatocytes showed hemorrhage,
centrilobular and sinusoidal congestion
1. The neutralization of IL-17
2. Metabolite(s) indirectly
activates TLR4 and RAGE,
resulting in inflammation

42. Drug/inflammation
interaction models
Dose Pathological changes Mechanisms
Methimazole or
propylthiouracil with LPS
Dose: MMI, 10-50 mg/kg, PTU, 10-
50 mg/kg, and LPS, 100 µg/kg
Administration: MMI&PTU: oral, and
LPS: i.p.
Inflammatory cells infiltration,
intracanalicular cholestasis,
fatty changes
1. Drug reactive metabolite formation and
inflammation induction
2. Immunological reactions
3. Oxidative stress
Trovafloxacin with LPS Dose: TVX: 150 mg/kg, LPS: 67×106 EU/
kg
Administration: TVX: oral, and LPS: i.p.
Inflammatory cell infiltration;
coagulative necrosis located
predominantly midzonally and
in centrilobular region
1. Enhanced TNF release
2. Activation of the hemostatic system
3. Neutrophils accumulation
Sulindac with LPS Dose: SLD: 50 mg/kg, LPS: 8.25 × 105
EU/kg
Administration: SLD: oral; two
administrations with a 16-hour interval.
LPS: i.v.; half an hour before the second
administration of SLD
Midzonal hepatic necrosis
Diclofenac with LPS Dose: DCLF: 20 mg/kg, LPS: 29 × 106
EU/kg
Administration: DCLF: i.p., and LPS: i.v., 2
hours before DCLF
Parenchymal edema
(multifocal); parenchymal
hemorrhage (multifocal);
apoptosis (random); leukocyte
infiltration
Ranitidine with LPS Dose: RAN: 30mg/kg, LPS: 44.4 × 106
EU/kg
Administration: RAN: i.v., and LPS: i.v., 2
hours before RAN
Acute, multifocal, midzonal
hepatic necrosis developed as
early as 3 h; hepatocellular
cytoplasmic eosinophilia and
nuclear pyknosis; variable
numbers of infiltrating PMNs

43. IN VIVO MODELS OF LIVER FIBROSIS
Model Mechanistic basis WHY DO WE USE IT
Ethanol • CYP450-mediated biotransformation to reactive metabolites
• Enhanced immune response
• Increased collagen synthesis
Carbon tetrachloride • CYP2E1-mediated biotransformation to reactive metabolites • High reproducibility
• Close to human liver fibrosis
Thiocetamide • CYP450-mediated biotransformation to reactive metabolites
• Immunological response
• Can be used to confirm results obtained from
other models
Dimethylnitrosamine and
diethylnitrosamine
• CYP2E1-mediated biotransformation to reactive metabolites • Good model to study HCC
Methionine choline-
deficient diet
• Lipotoxicity
• Kupffer cells activation and monocytes recruitment
• HSC activation
• Hepatocyte apoptosis and release of danger signals
• Close to human NASH
Common bile duct
ligation
• Increased biliary pressure
• Infiltration of inflammatory cells
• ROS generation
• Portal fibroblast activation
• Reversibility after relief of the obstruction
• Close to human cholestatic injury
High fat diet • In the liver, excess calories lead to Kupffer cell (hepatic macrophage)
activation, which promotes inflammation and increased hepatocyte
fatty acid synthesis, leading to hepatic steatosis (abnormal retention
of lipids within the hepatocytes) and eventual fibrosis or cirrhosis
• Obesity and peripheral insulin resistance

44. MODEL MECHANISTIC BASIS ADVANTAGES
Multidrug resistance-
associated protein 2-deficient
mice
• Lack of phospholipid secretion into the bile
• Hepatocyte necrosis
• HSCs activation
• Canalicular and small bile ductular destruction
• Inflammatory cells infiltration
• Similar to human chronic biliary disease
Schistosoma spp. • Cytokines production • Similar to human parasitic infections
Hepatitis virus models • Immune response • Similar to human viral infections
Alms1 Fat ausi mutant mice • Lipotoxicity
• Inflammatory cells infiltration
• Ballooned hepatocytes
• HSC activation
• Close to human NASH
High cholesterol diet • Oxidative stress
• Activated stellate cells
• Increased TNF-a, IL-1B, TLR4
• NAFLD  NASH
• Induces NASH and in some cases cirrhosis.
Choline deficient L-amino acid
defined diet
• Mitochondrial dysfunction
• Decreased mitochondrial number
• Decreased antioxidants
• Lipid peroxidation
• Mimics the human main characteristics,
namely obesity and peripheral insulin
resistance
IN VIVO MODELS OF LIVER FIBROSIS

45. IN VITRO MODELS FOR LIVER FIBROSIS

46. IN VITRO MODELS FOR HEPATIC FIBROSIS
MODEL MODEL ORIGIN CHARACTERISTICS ADVANTAGE DISADVANTAGES
Primary
stellate cells
Primary
stellate cells
• Rodent
• Human
• Cells derived from healthy
liver: quiescent HSCs
• Cells derived from injured
liver: myofibroblasts
Close link with the in vivo
situation
• Activation occurs when seeded on
plastic culture dishes
• Limited life span
• Cell culture heterogeneity
• Restricted human material
Co-cultures Co- cultures Rodent and human
primary cells and cell
lines
Combination of liver cell types Establishment of cell to cell
interactions
Restricted to HSCs and hepatocytes
Precision-cut
liver slices
Precision-cut
liver slices
Liver explants from
rodents and humans
Liver explants with different
cell types
Establishment of cell to cell
interactions.
Limited human supply
Limited viability
CELL LINES GRX • C3H/ HeN mice
infected with
Shistosoma mansoni
• 2 phenotypes:
myofibroblasts and
lipocyte-like cells
Myofibroblasts resemble
activated HSCs
Of use for the study of
lipid-related changes and
anti-fibrotic molecules.
No difference between both
phenotypes at the expression level as
occur in vivo
A640-IS HSCs from ICR mice
transfected with TSV40
33 °C: myofibroblastic
phenotype
39 °C: HSC-like phenotype
Myofibroblasts resemble
activated HSCs
No difference between both
phenotypes at the expression level as
occur in vivo

47. IN VITRO MODELS FOR HEPATIC FIBROSIS
MODEL MODEL ORIGIN CHARACTERISTICS ADVANTAGE DISADVANTAGES
CELL LINES SV68c-IS HSCs from ICR mice transfected
with TSV40
Myofibroblastic phenotype - Lack of correlation with
activated HSCs in vivo
M1-4HSC HSCs from male p19ARF null
mice
• Absence of TNF-β1: HSC-
like phenotype
• Presence of TNF-β1:
Myofibroblastic phenotype
Myofibroblast resembles
activated HSCs
Lack of correlation with the
in vivo situation
JS1 HSCs from C57BL/6 transfected
with TSV40
Myofibroblast • Easily transfected
• Of use in studies of
apoptotic
mechanisms
Required characterization
Col-GFP HSCs from transgenic mice
expressing GFP under the
control of collagen I gene
promoter and transfected with
TSV40 and the hygromycin
resistance gene
Myofibroblast phenotype Of use for drug screening Lack of correlation with
activated HSCs in vivo

48. EXTRA POINTS TO BE NOTED
Myofibroblast Phenotype of HSC
Summary of pre clinical models for liver fibrosis

49. MYOFIBROBLAST PHENOTYPE

51. REFERENCES
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