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Atherosclerotic lesion formation in rabbits fed on egg yolk-supplemented diet: an inexpensive experimental model
(Portuguese PDF version)

Rodrigo Gibin Jaldin,¹ Hênio Arruda Falcão Filho,¹ Júlio Lopes Sequeira,² Winston Bonetti Yoshida³

1. Medical student, Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (UNESP), Botucatu, SP, Brazil.
2. Physician. Assistant professor, Veterinary Pathology, Faculdade de Medicina Veterinária e Zootecnia (FMVZ), UNESP, Botucatu, SP, Brazil.
3. Associate professor, Vascular Surgery, Department of Surgery and Orthopedics, FMB, UNESP, Botucatu, SP, Brazil.

Correspondence:
Winston Bonetti Yoshida
Departamento de Cirurgia e Ortopedia
Faculdade de Medicina de Botucatu - UNESP
Campus Botucatu - Distrito de Rubião Jr., s/nº
CEP 18618-000 - Botucatu, SP, Brazil
Tel.: 55 14 3811.6230, 55 11 3811.6269
Fax: 55 14 3815.3205
E-mail: winston@fmb.unesp.br

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ABSTRACT

Objective: To observe whether an inexpensive dietary-induced hypercholesterolemia model in rabbits using a chow diet supplemented with egg yolks can be used to develop atherosclerotic lesions in animals.

Material and method: Fourteen rabbits were divided into two groups of seven animals: control group (G1), in which the rabbits were fed standard chow diet; and experimental group (G2), in which the rabbits were fed standard chow supplemented with egg yolks. Both groups were fed for 90 days. The lipid profile of all animals was measured at 0, 30, 60 and 90 days. At the end of the experimental period, euthanasia was performed and the aorta and its direct branches were excised to perform the histopathological examination.

Results: There was a significant increase in plasma total cholesterol and cholesterol fractions only in group G2. The macroscopic examination showed fatty streaks in the aortic arch and abdominal aorta. Histological analyses revealed minimal lesions consisting of macrophages foam cells in the abdominal, renal, carotid and diaphragmatic aorta and femoral arteries. Therefore, the egg yolk diet developed mild atherosclerosis in rabbits and induced the same characteristics and extent of the lesions obtained by low-dosage purified cholesterol. Thus, egg yolk can be used as an inexpensive source of cholesterol in models of experimental atherosclerosis.

Keywords: Atherosclerosis, cholesterol, aorta, rabbits, diet, dyslipidemias.

J Vasc Bras. 2006;5(4):247-56

Article submitted September 12, 2006, accepted December 14, 2006.

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INTRODUCTION

Atherosclerosis is the main cause of death and disability in Western countries.^1,2 It consists of a chronic, progressive and systemic process, characterized by inflammatory and fibroproliferative response of the arterial wall, which is caused by aggressions to the arterial surface.² The disorder of vascular endothelial function is the first step to atherogenesis,^3-5 hypercholesterolemia being one of its main triggering factors.⁶ The atherosclerotic lesion may affect many and important arterial territories, being responsible for 95% of heart diseases, 85% of intermittent claudication of the lower limbs and 75% of strokes,⁷ besides being an important factor in the development of abdominal aortic aneurysm.⁸

Due to the importance of this disease for human beings, researchers have been continuously searching for experimental models to better characterize it. When a cholesterol-rich diet alone was found to be atherogenic, attention was drawn to models based on supplementation with purified cholesterol and there was no interest in researching diets composed by other elements inducing experimental atherosclerosis.^9,10 In a survey on the characteristics of the methods used in experimental atherosclerosis, rabbits were the most studied species and hypercholesterolemia was the most practical approach for inducing it.¹¹

Atherosclerosis has been induced in rabbits through chow diet containing 1% cholesterol for approximately 12 weeks.^12,13 This method allows not only the formation of atherosclerotic lesions in the thoracic and abdominal aorta,^12,13 but also proved to be effective in the study of carotid atherosclerosis in rabbits.¹⁴ Bocan et al. provided rabbits with a diet composed of varied cholesterol levels, showing that the extension and type of atherosclerotic lesion were proportional to the concentration of serum cholesterol, which, in its turn, increased according to the percentage of cholesterol in the diet.¹⁵ However, the purified cholesterol available in the market is expensive, which limits its wide use in the study of experimental atherosclerosis (Table 1).

Table 1 - Compared cost between cholesterol supplement for chow diet and egg yolk (diet at 0.15% cholesterol, for seven rabbits/day, for a 12-week period)

Source of cholesterol	% of cholesterol	Total cholesterol (G) - 3 months	Cost per gram	Cost per day	Cost in US $§	Cost in R$
Cholesterol - 99%, porcine liver, powder, grade I	0.15% diet = 225 mg/150 g of chow diet	141.75 g	1 g = US$ 36.00 5 g = US$ 114.00 25 g = US$ 315.00†	1.575 g/day US$ 19.85 (bottle w/ 25 g)	1,786.50	4,075.00
Egg yolk (each contains 225 mg of cholesterol in average)*	0.15% diet = 1 egg yolk/150 g of chow diet	630 eggs (egg yolks)	30 dozen eggs (81 g chol) = R$ 27.98‡ : 1 g = R$ 0.35	7 eggs/day (egg yolks) R$ 0.56	22.10	50.40

* USDA Nutrient Database for Standard Reference, 2001.
† Catálogo Sigma 2003/2004.
‡ Associação Paulista de Avicultura (APA): mean wholesale price of white egg, on April/2006, box with 30 dozens, São Paulo, Brazil.
§ Reuters (Folha de S. Paulo): commercial dollar exchange rate at R$ 2.281 on 06/06/2006.

 

Atherosclerotic lesions are apparently more evident associating cholesterol, animal saturated fat and protein, which are present at large quantities in egg yolks. In previous studies carried out at our laboratory, there was significant increase in the concentration of serum cholesterol in rabbits fed with chow diet supplemented with egg yolk.¹⁶ Cholesterol overload using egg yolk, although being a pioneering model of experimental atherosclerosis, had no systematic evaluation of distribution and intensity of vascular impairment. As a consequence, we propose to assess whether the diet under discussion induces atherosclerosis efficaciously in the main arteries of the experimental animal (aorta, carotid, renal, common iliac and femoral arteries) and whether it can be used as an inexpensive experimental model.

MATERIAL AND METHODS

Experimental design

The experimental protocol of this study followed the ethical principles for animal experimentation described by the Brazilian College of Animal Experimentation and was approved by the ethic committee in animal experimentation at Faculdade de Medicina de Botucatu-UNESP. The experiment was carried out using 14 male Norfolk rabbits. The animals were received at the experimental laboratory weighing approximately 1,200 g and approximate age of 80 days. For adaptation, weight gain and reaching ideal age to begin the treatment, the animals were maintained in individual cages with water ad libitum and standard chow diet (Nutricoelho, Purina®) for 14 days, at room temperature and light. In addition, the laboratory is equipped with a ventilation and exhaustion system. After this period, the rabbits were randomized into two groups of seven animals: groups G1 (control) and G2 (with diet supplemented with egg yolk). They were then placed in numbered individual cages and tattooed on the left ear with their respective cage numbers, thus minimizing the possibility of loss.

Chow diet preparation

The animals in group G2 were fed with diet containing one egg yolk plus 13.5 ml of corn oil for each 150 g of standard chow diet for rabbits (Nutricoelho, Purina®) for 3 months. This diet was prepared every week, 10 kg at each time, at the experimental laboratories of Faculdade de Medicina de Botucatu. The standard chow diet was ground in a grinder and then mixed with egg yolks, water and corn oil in a mixer (Marconi®). The mass was submitted to pelletization in a meat grinder (CAF®, model 10, cycling 60 Hz) and dried in a drying and sterilizing greenhouse (Fanem®, model 320-SE) at 55 °C for 24 h. The chow diet was maintained at a refrigeration equipment until consumption. It was considered that each egg yolk contained 225 mg cholesterol, 1.8 g saturated fat, 2.16 g monounsaturated fatty acids and 0.72 g polyunsaturated fatty acids.¹⁷ Group G1 was given usual chow diet of the same brand and lot for the same period of time, and water was offered ad libitum.

Weight control and biochemical dosages

The animals were monthly weighed since the day previous to the treatment. After a 14-h fasting,¹⁸ venous blood was collected from the marginal vein of the right ear in 6 ml bottles with anticoagulant for dosage of the lipid profile. Samples were collected at time zero and at the end of each month of treatment. After the collection, the samples were centrifuged at 3,000 rpm for 10 min, and the dosages of serum levels of total cholesterol, HDL-cholesterol (HDL-c) and triglycerides were performed at the Biochemistry Laboratory of Hospital das Clínicas da Faculdade de Medicina de Botucatu-UNESP using dry chemistry in an autoanalyzer device (Johnson & Johnson®, model Vitros 950). The values of LDL-cholesterol (LDL-c) fraction were obtained using the Friedewald formula.¹⁸

Euthanasia

The rabbits were anesthetized with 1-ethyl methyl-butyl-sodium barbiturate (Nembutal®) intravenously. They were then trichotomized from the anterior cervical region to the pubic eminence and heparinized (400 mg/kg, IV). Through a venous approach, they were submitted to euthanasia exceeding the same anesthetic.

Macroscopic evaluation

Thoracic and abdominal aorta were removed after median thoracotomy, median laparotomy and careful dissection of renal arteries and iliac bifurcation of the aorta. The neck was dissected to expose carotid arteries in their cervical tract, and these arteries were stripped since their emergence in the aorta. The femoral trigone was also dissected, isolating femoral arteries. After the excision of the arteries under investigation, the adventitial fat was carefully removed. The aorta was divided into the following segments: arch, thoracic aorta, abdominal aorta and aortoiliac. These segments, along with the carotid and femoral arteries, were opened in their posterior wall, analyzed in their luminal surface and then fixed in a 10% buffered formol solution for 24 h. Later, they were stained with Sudan IV solution (Hexheimer solution), standardized by Holman et al.¹⁹ After staining, arterial luminal surfaces were inspected again, by naked eye and a 10 x microscope (D.F. Vasconcellos S.A.®, São Paulo, Brazil). Presence (sudanophilic areas) or absence (non-sudanophilic areas) of atherosclerotic plaques were investigated, as well as their distribution along the vascular lumen. The kidneys, liver and heart were weighed on an analytic scale (Chyo®, JS-110; max: 110 g/d = 0.0001 g) and longitudinally sectioned for structure analysis. The heart was weighed after opening its chambers to consider only its structure, free from blood or clots.

Histopathologic analysis

Samples of arteries and organs were placed in bottles containing 10% formal solution, for processing in a microtome (Leica®, TP102) and processed in blocks of paraffin. A series of 5 µm sections was performed in a microtome (Leica®, RM 2155) and stained by hematoxylin-eosin (HE) and picrosirius. HE provided the general morphology of the arterial and structure wall of the organs. Picrosirius aimed to identify the intimal fibrous thickening. Samples of all arteries under investigation were collected, independent of plaque formation, and the general changes in the vascular wall were verified (inflammatory process, integrity of elastic blades, proportion of collagen and elastic fibers) and changes on wall layers. The analysis was performed by a pathologist unaware of the groups.

Classification of the lesions

The atherosclerotic lesions found in the macroscopic study were visually classified into a 0-3 scale,^20,21 according to the amount of sudanophilic areas present in the arterial lumen (0 = absence; 1 = discrete, restricted to the ostia; 2 = moderate; 3 = intense). Adding macro- and microscopic findings, we used the classification standardized by the committee on vascular lesion of the American Heart Association.²²

Statistical analysis

Comparisons between groups were performed using the Mann-Whitney test. The data were expressed in median with quartiles 25 and 75, and the results were considered statistically significant if P < 0.05.

RESULTS

Body variables of the animals

The group that was given supplemented diet had body weight greater than the control group (P < 0.001) after a 90-day follow-up. Except for the heart (P < 0,05), the other organs did not present significant difference as to weight (Table 2).

Table 2- Median and quartiles of body weight and visceral organs at the end of the experiment for both groups

Variables	Controls (G1) n = 7	Treated (G2) n = 7	P
Final body weight (g)	3,430 (3,290; 3,535)	3,850 (3,667,5; 3,950)	< 0.001**
Liver weight (g)	117 (112.4; 133.8)	146 (130.8; 164.8)	0.072
Heart weight (g)	7.55 (7.31; 7.92)	8.3 (8.24; 8.96)	< 0.05*
Kidney weight (g)
Right	9.04 (8.72; 9.93)	9.66 (9.11; 10.47)	0.259
Left	9.1 (8.7; 10.14)	9.58 (9.13; 10.68)	0.383

Mann-Whitney test.
g = grams; n = number of animals per group.

 

Biochemical dosages

Serum levels of total cholesterol, LDL-c, HLD-c and triglycerides are described in Figure 1. There was increase in concentration of total cholesterol and fractions in the group receiving diet supplemented with egg yolk, but there was no difference in levels of triglycerides between the groups.

Figure 1 - Means and standard deviations of lipid profile (mg/dl) during the study in both experimental groups. A) total cholesterol; B) LDL-cholesterol; C) HDL-cholesterol; D) triglycerides

o Controls; o Treated (Mann-Whitney test).
* P < 0.05; ** P < 0.001.

Macroscopic evaluation

There was no macroscopic change in the evaluated arterial segments of control group animals, before and after histochemical analysis of fat. On the other hand, the arteries of group G2 animals, after staining, presented sudanophilic areas corresponding to fatty streaks predominantly in the anterior luminal surface of the aortic arch (Figure 2) and abdominal aorta (Figure 3 and Table 3).

Figure 2 - Digital photograph of the anterior luminal surface of the aortic arch longitudinally opened and stained with Sudan IV in animals that were given hypercholesterolemic diet. The areas that are more intensely stained (or arrows) represent atherosclerotic lesions. A) absence of lesions (0); B) discrete lesion (1); C) moderate lesion (2); D) intense lesion (3)

Figure 3 - Digital photographs of the abdominal aorta, stained with Sudan IV, of the animals that were given hypercholesterolemic diet. The areas that are more intensely stained (or arrows) represent atherosclerotic lesions. A) absent (0); B) discrete (1); C) moderate (2); D) intense (3)

Table 3 - Macroscopic evaluation and graduation* of sudanophilic areas in aortic segments and their main branches in all the animals treated with egg yolk

Artery	Frequency according to type of lesion (%)
	0	1	2	3
Carotid	7/7 (100)	-	-	-
Aortic arch	1/7 (14)	1/7 (14)	4/7 (58)	1/7 (14)
Thoracic aorta	6/7 (86)	-	1/7 (14)	-
Abdominal aorta	-	3/7 (43)	3/7 (43)	1/7 (14)
Aortoiliac	5/7 (71)	2/7 (29)	-	-
Femoral	7/7 (100)	-	-	-

* Visually graduated in a scale from 0-3 (0 = absent; 1 = discrete, restricted to the ostia; 2 = moderate; 3 = intense) G2 = group of animals that were given hypercholesterolemic diet; n = number of animals.

 

Histopathologic analysis

There were no architectural changes in the arteries of control group animals. In the animals that received egg yolk, changes were concentrated on the intimal layer and corresponded to discrete lipid stores inside foam cells or forming subendothelial vacuoles (Figure 4). This change was present in samples of abdominal aorta (3/7), renal (2/7), carotid (2/7), thoracoabdominal transition (2/7) and femoral (2/7). In only one animal, there was intimal thickening at a moderate degree, in the thoracoabdominal transition of the aorta.

Figure 4 - Cross-sectional sections of different arterial territories stained with hematoxylin-eosin. There were intimal changes compatible with atherosclerosis. A) artery of a control animal; B) there were foam cells (negative images) in the abdominal aorta; C) common carotid artery; D) there is intimal thickening in the transition area between the thoracic and abdominal aorta

DISCUSSION

Experimental models are important instruments to study atherosclerosis and were responsible for most part of the current knowledge on its etiology, physiopathology and treatment. The first nutritional investigation on atherosclerosis was carried out by Ignatowski, between 1908 and 1909. Assuming that a toxic metabolite of animal protein might lead to atherosclerosis, he fed adult rabbits with meat and weanling rabbits with milk and egg yolk, obtaining atherosclerosis in both. Stuckey, between 1910 and 1911, noted that the egg yolk was atherogenic and, in 1912, concluded that there was a non-protein component in the egg yolk that worked as an atherogenic agent. Wesselkin, in 1913, tested whether cholesterol or lecithin could be the atherogenic agent of Stuckey's experiment. He obtained atherosclerosis only in the rabbits fed with egg yolk and concluded that cholesterol was the atherogenic factor present in the egg yolk.²³

Models based on diet-induced hypercholesterolemia are the most practical and widespread to study experimental atherosclerosis.¹¹ The role of this type of diet in atherogenesis is well determined. Progenitor cells may be seen in only 1 week being exposed to a hypercholesterolemic diet.²⁴ Aikawa et al. showed that diet lipids, causing hypercholesterolemia, were responsible for oxidative stress and endothelial lesion in the aorta of rabbits, so that only lipid reduction in the diet could reduce the mechanisms damaging the arterial surface.²⁵

The atherogenic effects of the diet were studied as naturally as possible, i.e., ad libitum and egg yolk as source of cholesterol were adopted. There was significant weight gain in the animals that were given supplemented chow diet. There was increase in serum concentrations of total cholesterol and fractions in the group receiving diet supplemented with egg yolk in relation to controls. Such increase was expressive over the first 30 days of supplemented chow diet, and in subsequent moments, there was initial decrease followed by stabilization.

The cholesterol levels obtained with diet supplemented with 0.15% cholesterol from egg yolks were lower than those achieved by standard diet containing 1% purified cholesterol.^12,13,15 On the other hand, there were temporal behavior and cholesterolemia values equivalent to those of rabbits receiving between 0.1 and 0.25% purified cholesterol for 60 days.¹⁵ By comparing the increase in cholesterolemia between sources of experimental atherosclerosis, egg yolk was less effective than conjugated linoleic acid.²¹ It also elevated less cholesterolemia in rabbits than casein added to soy oil partially hydrogenated for 6 months,^9,10 but lower exposure time and cost of atherogenic material support the use of egg yolk. On the other hand, hyperhomocysteinemia is also an effective inducer of atherosclerosis, but without interfering the lipid profile of the experimentation animal.²⁶

The serum levels of LDL-c and HDL-c at the end of the experiment were very close to those obtained by the supply of 0.1% purified cholesterol for 60 days,¹⁵ which indicates a certain equivalence in the results obtained. Egg yolk seems to have been inefficient in elevating serum triglycerides, similar to the diet with 1% purified cholesterol.¹³ Casein added to soy oil partially hydrogenated for 6 months⁹ and the conjugated linoleic acid²¹ seem to be more effective in elevating this parameter.

It should be stressed the formation of macroscopic lesions induced only by diet, reaffirming that hypercholesterolemia is sufficient for atherogenesis. Most studies using experimental models in atherosclerosis associate diet and mechanic lesion of the endothelial surface,^15,27,28 causing hypercholesterolemia to be the process intensifier, and not its promoter. That occurs because, even in the absence of hypercholesterolemia, there is formation of atherosclerotic lesions in de-endothelialized arteries.¹⁵

Bocan et al.¹⁵ observed that animal fed with diet of 0.1 and 0.25% purified cholesterol developed minimal lesions in the arteries that did not suffer denudation, and only levels higher than 0.5% diet cholesterol caused fatty streaks, isolated or confluent. However, the method proposed by this study showed that it is possible to develop both minimal microscopic lesions and isolated fatty streaks in arteries with preserved endothelial surface, even with low proportion of diet cholesterol. The lesions caused by this experimental model, due to their macro- and microscopic characteristics, may be classified into types I and II of the American Heart Association,²² i.e., they are compatible with the initial changes in the atherosclerotic process.

This study was proposed to spread the use of experimental atherosclerosis in physiopathological and therapeutic investigations. The diet we tested proved to be effective to induce hypercholesterolemia in experimentation animals, but caused only initial changes in the atherosclerotic process. On the other hand, it can be considered that egg yolk as source of 0.15% diet cholesterol caused changes in the arteries and serum levels of cholesterol equivalent to those caused by commercial purified cholesterol when provided at a low dosage. Therefore, perspectives are open to test whether a higher amount of daily egg yolk would develop more expressive arterial lesions and cholesterolemia, given the low cost and accessibility to this source of diet cholesterol. In conclusion, the egg yolk diet developed mild atherosclerosis in experimentation animals and changes similar to those obtained by low-dosage purified cholesterol. Therefore, egg yolk can be used as an inexpensive source of cholesterol in models of experimental atherosclerosis.

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