The document presents a seminar on anti-atherosclerosis screening models conducted at Raghavendra Institute of Pharmaceutical Education and Research. It highlights various in vivo and in vitro models used to study atherosclerosis, including their methodology and outcomes, notably using animal subjects like rats, rabbits, and hamsters. The focus is on understanding the disease mechanisms and evaluating potential therapeutic agents through established experimental protocols.

1. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
Under the guidance of
Dr. K. Somasekhar , M. Pharm, Ph.D.
Associate Professor & Head,
Department of Pharmacology.
Presented by
SHAIK FIRDOUS BANU
(20L81S0105)
PHARMACOLOGY
A Seminar as a part of curricular requirement
for M. Pharmacy, I Year - I semester
Screening Models of Anti-
Atherosclerosis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Atherosclerosis
• Screening models
In vitro models
In vivo models
• References
Contents

3. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Atherosclerosis is a condition in which an artery wall thickens as the
result of a build-up of fatty materials such as cholesterol.
• It is a syndrome affecting arterial blood vessels, a chronic
inflammatory response in the walls of arteries.
• Hyperlipidemia is the most prevalent indicator for susceptibility to
atherosclerotic heart disease.
• It is characterized by abnormally elevated lipid such as triglyceride,
cholesterol and lipoprotein. Increase level of low density lipid and
very low density lipid in the blood.
Atherosclerosis

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
• It is commonly referred to as a hardening of the arteries.
• It is caused by the formation of multiple
plaques within the arteries.

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
Screening models

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
In vivo models
a) Triton Wistar rat induced
hyperlipidaemia
b) Cholesterol diet induced
atherosclerosis in rabbits
c) Hereditary hyperlipidaemia in
rabbits
d) Hypolipidemic activity in Syrian
hamsters
e) Hereditary hypercholesteremia
in rats
f) Transgenic animal model
In vitro models
a) Inhibition of isolated
HMGCOA reductase inhibitors
b) ACAT inhibitory model

7. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
• Purpose
The systemic administration of the surfactant triton to rats
results in a biphasic elevation of plasma cholesterol and
triglycerides.
• Requirement:
Chemical: Surfactant Triton
• Animal:
Wistar strain male albino rats
In vivo models:
A) Triton Wistar Rat Induced
hyperlipidaemia:

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Forty two male wistar rats weighing 190gm to 230 gm
were randomly divided into 7 groups.
In each group contains 6 male rats and kept in their cages for 5 days
prior dosing to allow for acclimation to laboratory condition.
The animals were starved for 18hr and i.p. with 10% aqueous solution
of triton at 400mg /kg body weight.
Procedure :

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
The test drugs employed (or) the solvent for control was
administered simultaneously with triton injection.
Serum analyzed after 24hr and 48 hr after triton injection.
After 24hr, blood was collected by retro orbital puncture under
ether anesthesia and subject to centrifugation to obtain serum.
same procedure is repeated for 48hrs

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• Serum was analysed for serum triglyceride, serum total
cholesterol, serum high density lipoprotein cholesterol,
serum low density lipoprotein cholesterol, serum very low
density lipoprotein cholesterol, serum glucose .
• The result is evaluated by ANOVA test and Dunnet Multiple
comparison test.
Evaluation

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
• Purpose:
Rabbits are known to be susceptible to hypercholesterolemia and
arteriosclerosis after excessive cholesterol feeding. Therefore, this
approach has been chosen by to study the effect of potential anti-
arteriosclerotic drugs.
• Requirements
Animals: White New Zealand male rabbits
Chemical: Dimethyl sulfoxide
Cholesterol-diet induced atherosclerosis
in rabbits

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
• Group of Male rabbits (standard and control) at an age of
8–10 weeks are used and blood is withdrawn from the marginal ear
vein for determination of total cholesterol, total glycerides, and blood
sugar
• The rabbits are switched from commercial food to a diet
supplemented with 0.3–2% cholesterol and kept on this regimen for
a period of 10–12 weeks
• One group is kept on normal diet. During and at the end of the
experiment blood is taken for analysis.
Procedure

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
• The animals are sacrificed and the thoracic aorta is
removed, cleaned of surrounding tissues, and longitudinally cut and
opened for fixation with formaldehyde
• The tissue is stained with oil red. In animals fed a normal diet, the
aorta does not show any staining, whereas in cholesterol-fed rabbits
the aorta shows severe atherogenic lesions

14. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• The percentage of the intimal surface covered by the oil red positive
lesions is calculated with a computerized plan meter.
• Statistical evaluation is performed by Dunnett’s or Scheffe's test.
Evaluation :

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• Purpose
To produce hereditary hyperlipidemia in rabbit. To study the effect
of potential anti-arteriosclerotic drugs
• Requirements
Chemical: Probucol
Animals:
Homozygous Wistar Hereditary Hyperlipidemic rabbits
Hereditary hyperlipemia in rabbits

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
• Homozygous wistar hereditary hyperlipidemic rabbits are raised by
mating heterozygous female wistar hereditary hyperlipidemic
rabbits with homozygous male Wistar hereditary hyperlipidemic
rabbits
• At 2 months of age, eight rabbits are divided into two groups
(group A and group B). Rabbits in group A (two males, two
females) are fed standard rabbit chow for 6 months .
• Rabbits in group B (two males, two females) were raised with
rabbit chow enriched with 1% (wt/wt) probucol for 6 months
procedure

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
• The amount of daily diet for each animal is restricted to 100 g
during the study period
• Six months later (at the age of 8 months), the rabbits are sacrificed
and their blood and aortas are taken for analysis

18. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
• Plasma levels of cholesterol is measured by the enzymatic method.
Evaluation :

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
• Purpose :
The lipoprotein and bile acid metabolism of the hamster is closer
to human. Easy to handle and more human like.
• Requirements :
Animal: Syrian Hamsters
Hypolipidemic activity in Syrian hamsters

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
• Male Syrian hamsters weighing 95–125 g at the start of the
experiment are randomly assigned to form groups of 6 animals each
• After 1 month of cholesterol rich diet they develop sub endothelial
foam cells which are precursors of fatty streaks that develop into
complex plaques
• the animals are anesthetized with diethyl ether, a blood sample is
taken from the superior venacava
Procedure :

21. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
• The plasma is analyzed for total cholesterol using a colorimetric
enzymatic assay.
• The cholesterol content of high density lipoprotein is
determined using a precipitation kit.
Evaluation :

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
• Purpose and rationale
• Transgenic mice lacking apolipoprotein E showed severe
hypercholesterolemia and atherosclerosis.
• Requirement:
Animal: Mice
Transgenic Animal Model

23. RIPER
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Procedure
 The widely used model is Apo E knockout mouse
 This Apo E knockout mice have spontaneously elevated plasma
cholesterol levels, and develop atherosclerosis even on regular chow within
3-4 months
 Walsh and Rubin integrated human apolipoprotein A-1 gene in transgenic
mice causes the increase of high density lipoproteins
 Over production of apolipoprotein E transgenic mice reduced plasma
cholesterol and triglyceride levels, prevented hypercholestrolemia and
inhibited the formation of fatty streak lesions

24. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
• Evaluation
• For evaluation of the anti atherosclerotic effect, the mice were orally
treated with GW501516 for 18 weeks and atherosclerosis at the aortic
valves was determined by cross sectional lesion analysis.

25. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25
• Purpose and rationale
• ACAT stands for Aceyl COA cholesterol acyltransferase.
• In vitro ACAT inhibitory activity can be determined in microsomal
preparations from liver or Intestine of rabbits.
In vitro model:
ACAT inhibitory activity

26. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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 Prior to sacrifice, the animals receive chow supplemented with
2% cholesterol and 10% safflower oil for 6 weeks.
 Each assay contains 0.2 mg of microsomal protein and fatty acid –
poor bovine serum albumin ( 3mg/kg ) in 0.04 M KH2PO4 buffer
ph 7.4, containing 0.05 M KCL, 0.03 M EDTA and 0.3 m sucrose.
 Drug dilutions are made in DMSO.
 The reaction is started by the addition of oleyl CoA ( 50 micro
meters, 7 dpm/pmol )
Procedure

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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 After 3 min the reaction is stopped by the addition of chloroform and
methanol 2:1.
Cholesterol oleate is used as internal standard.
 Lipid extracts are dissolved in chloroform, spotted on TLC plates and developed in
hexane - petroleum ether – acetic acid 80:20:1.
 Unlabelled, carrier cholesterol oleate is added to the internal standard to aid band
visualization with iodine vapour.
 The band corresponding to cholesteryl esters is then scraped into scintillation vials.
 Radioactivity is determined by liquid scintillation spectroscopy

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• Evaluation
• For each compound four concentrations are evaluated in duplicate.
IC50 values are
• determined by performing a nonlinear least-squares fit of the data to
log dose response curve

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• Jose J. Fuster , Ana I. Castillo . Animal models of Atherosclerosis.
Elsevier. 2012;105:1-23.
• Shore B , Shore V. Rabbits as a model for study of
hyperlipoproteinemia and atherosclerosis. Elsevier.1999;172:18-23.
References:

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