Modelling hypercholesterolaemia in rats using high cholesterol diet

Abstract

Hypercholesterolaemia is a complex condition with multiple causes, including both lifestyle and genetic aspects. It is also a risk factor for cardiovascular diseases (CVDs), which are responsible for 172 million deaths/year. Although the reasons for hypercholesterolaemia are known, there are many critical questions that remain to be answered so that new therapeutics can be developed. In order to elucidate the pathobiology of this condition, animal models can mimic the pathology of human hypercholesterolaemia. One example of an animal model is induced by the hypercholesterolaemic diet in Wistar rats. The present review first summarizes the current understanding of the metabolic profile involved in hypercholesterolaemia in humans. Next it comments about the lack of consensus as to which hypercholesterolaemia induction protocol should be used. The present work aimed to review experimental studies that induced hypercholesterolaemia in Wistar rats it was not intended to judge the “best” model, since they all achieved the goal of inducing an increase in serum cholesterol.

1. INTRODUCTION

Cardiovascular diseases (CVDs) are responsible for 17.2 million deaths/year all over the world. ¹ There is a constant search for long term alternative dietary treatments, since this kind of disease starts appearing at early age. ² CVDs are included in the group of chronic non‐communicable diseases, which also include obesity, systemic arterial hypertension (SAH), diabetes mellitus type 2 (DM2), dyslipidaemia and cancer. ³

There is a strong relation between CVSs and genetic, environmental and lifestyle factors. When these conditions are aligned with other risk factors for CVD, the possibility of coronary artery disease increases exponentially. ⁴ The main risk factors for CVD are high total cholesterol, SAH, reduced HDL cholesterol, DM2 and age. ⁵

Increased serum total cholesterol and low LDL cholesterol are components of hypercholesterolaemia. Besides being a risk factor for cardiovascular diseases, these alterations can also be harmful to the organism in other ways. Hypercholesterolaemia can be classified as primary when the lipid disorder has genetic influence, or secondary, caused by inadequate diet and lifestyle, as well as medications. ⁶

The decrease in serum cholesterol levels can be achieved by the consumption of appropriate food, specific foods and/or drugs. Foods and drugs which have hypocholesterolaemic effects should be tested in vitro and in vivo before being consumed by humans in order to verify their efficacy and safety. ⁷ , ⁸

Hypercholesterolaemia, as one of the main risk factors for CVD, has been studied extensively in animal models, especially as part of the search for new therapeutic approaches. However, there is no consensus about which hypercholesterolaemic induction protocol should be used. Therefore the present work aimed to review experimental studies that induced hypercholesterolaemia in Wistar rats.

2. METABOLIC PROFILE OF HYPERCHOLESTEROLAEMIC HUMANS

Before studying the effects of hypercholesterolaemic diet in animals, it is important to understand the cholesterol metabolism in humans; thus, thereafter, the similarity with between animals and humans can be verified.

Cholesterol is a vital compound, essential in cell membrane formation, sex hormone production and bile salt formation, among other functions of the organism. ⁹ It can be found in animal fats, basically in its free form. Its main food sources are egg yolk, milk and milk derivates, shrimp, beef, bird skin and viscera. Some classic studies have shown a strong association between high cholesterol consumption and increased incidence of atherosclerosis. ¹⁰ , ¹¹ Despite that, it is known that 56% of dietetic cholesterol is absorbed, and other fatty acids also have influence on plasmatic cholesterol concentration. ⁹

Cholesterol circulates in the bloodstream in structures called lipoproteins, formed by lipids and proteins. These are classified according to their composition (chylomicrons and VLDL—rich in triglycerides; LDL—rich in cholesterol; and HDL—rich in cholesterol and proteins). ⁶

Atherosclerosis can be classified as a chronic inflammatory disease of multifactorial origin, which mainly affects medium‐ and large‐calibre arteries reducing blood flow. The initial injuries are originated in childhood and are characterized by cholesterol accumulation in macrophages. The deposition of lipoproteins in the arterial wall, a key process at the beginning of atherogenesis, occurs in proportion with the plasma lipoprotein concentration. The severity of atherosclerosis is related to classic risk factors, such as dyslipidaemia, diabetes, tobacco addiction, hypertension and others. At the cellular level, cholesterol crystals, microfilaments released by neutrophils, ischaemia and changes in haemodynamic drag pressure have been implicated in the activation of the inflammatory process, which is associated with atherosclerotic plaque rupture or endothelial erosion. ¹²

The implications of this are that the tissue factor of the vascular intima interacts with circulating VIIa factor, leading to thrombin generation, platelet activation and thrombus formation, thereby determining the main complications of atherosclerosis, acute myocardial infarction and cerebrovascular accident (stroke). ⁶

Taking into account both physiology and metabolism, Wistar rats have been shown to be a very useful animal model to evaluate both hypercholesterolaemia and the comorbidities of metabolic syndrome due to their similarity to humans. ¹³

3. DATA COLLECTION

Articles were found using the keywords “Hypercholesterolemia OR Hyperlipidemias AND Diet AND Rats, Wistar” in PubMed and “Hypercholesterolemia OR Hyperlipidemias AND Diet AND Rats Wistar” in BVS (Biblioteca Virtual de Saúde, a Brazilian database). We included data from experimental studies that efficiently induced hypercholesterolaemia through diet in Wistar rats from 2013 until 2017. We excluded articles that induced hypercholesterolaemia by injection, drug tests and reviews. Studies that did not have a control group were also excluded as in that case we could not prove and quantify the induction. The data collection consisted of three phases: title evaluation, abstract evaluation and full‐text evaluation. Two researchers analysed the manuscripts. The findings are summarized in Table 1.

TABLE 1.

Summary of hypercholesterolaemic diet–fed rat studies

Author	Year	Initial age/ body weight	Diet composition	Diet duration	Serum cholesterol diet group—comparison with the control group
38	2013	4 wk ± 100 g	5 g/kg cholesterol, 2.5 g/kg sodium chloride	14 d	11.2 ± 0.6 mmol/L a —increased
23	2013	8 wk 250‐300 g	2% cholesterol, 0.2% colic acid	60 d	127 ± 6.5 mg/dL—increased
39	2013	±175g	1, 0.5% cholesterol, 0.2% NaTDC, 5% sugar, 0.05% propylthiouracil	28 d	5.98 ± 1.07 mmol/L a —increased
14	2013	140‐160 g	1% cholesterol	8 wk	114.26 ± 1.67 mg/dL—increased
40	2013	90 g	2.43% cholesterol, 0.49% cholic acid	1 wk	2.23 ± 0.19 mmol/L a —increased
48	2013	180‐200 g	3% cholesterol, 0.2% cholic acid, 0.5% propylthiouracil, 10% lard	4 wk	19 ± 0.11 mmol/L a —increased
24	2013	8 wk 250 g	33.5% lard, 1.5% soya bean oil	7 wk	53 ± 3.4 mg/dL—increased
33	2013	6‐8 wk 180‐210 g	1% cholesterol, 2% coconut oil	60 d	168.55 ± 14.69 mg/dL—increased
27	2013	180‐210 g	10 g lard, 4 g corn oil, 0.15 g margarine	15 d	223.91 ± 20.4 mg/dL—increased
41	2013	6 wk 170‐200 g	2% cholesterol, 0.25% cholic acid	8 wk	Values not informed—serum cholesterol significantly increased
42	2014	±140 g	4% cholesterol, 1% cholic acid, 0.5% thiouracil	17 d	153.63 ± 11.72 mg/dL—increased
26	2014	8 wk 150‐200 g	2% cholesterol, 0.2% cholic acid. 5% lard	3 mo	Values not informed—serum cholesterol significantly increased
28	2014	190‐230 g	1% cholesterol, 0.2% bile salts, 10% lard, 10% yolk powder	42 d	2.65 ± 0.17 mmol/L a —increased
43	2015	4 wk 80‐100 g	12.5% palm oil, 12.5% lard, 5% cholesterol, 2% cholic acid	30 d	150.29 ± 20.51 mg/dL—increased
37	2015	1 y 500 g	16.3 g/kg cholesterol	8 wk	3.3 ± 0.34 mmol/L a —increased
17	2015	±200 g	1% cholesterol	28 d	Values not informed—serum cholesterol significantly increased
44	2015	200‐300 g	365 g/kg cholesterol, 310 g/kg yard	6 wk	252.17 ± 14.72 mg/dL—increased
45	2015	150‐200 g	100 g/kg corn oil	7 wk	131 ± 9.6 mg/dL—increased
19	2016	4‐6 wk	2% cholesterol	60 d	194.2 ± 35.6 mg/dL—increased
18	2016	190‐210 g	2% cholesterol	9 wk	88.4 (74.5‐100.3) mg/dl—increased
30	2016	170‐190 g	60% lard	90 d	194.2 ± 35.6 mg/dL—increased
47	2016	8 wk 250 g	33.5% lard, 1.5% soya bean oil	5 wk	46.0 ± 3.2 mg/dL—increased
25	2016	8 wk 180‐220 g	10% lard, 20% sucrose, 2% cholesterol, 1% bile salt	8 wk	Values not informed—serum cholesterol significantly increased
16	2016	±114 g	1% cholesterol	42 d	2.25 ± 1.07 mmol/L a —increased
36	2017	±110 g	1% cholesterol, 10% yolk powder, 5% lard	10 wk	Values not informed—serum cholesterol significantly increased
22	2017	8 wk 200‐300 g	6% cholesterol	6 wk	151.00 ± 10.74 mg/dL—increased
20	2017	8 wk 245 ± 5 g	2% cholesterol, 0.5% cholic acid	7 wk	198.25 ± 10.61 mg/dL—increased
15	2017	±178 g	1% cholesterol	4 wk	156.09 ± 0.92 mg/dL—increased
29	2017	1 y ±500 g	1.26% cholesterol, 0.25% cholic acid	8 wk	126.9 ± 13.2 mg/dL—increased
20	2017	250 g	2% cholesterol, 0.5% cholic acid	7 wk	Values not informed—serum cholesterol significantly increased
34	2017	±180 g	10% sheep's fat, 0.1% cholic acid	6 wks	1.14 ± 0.03 mmol/L a —increased
35	2017	±190 g	10 g/kg cholesterol, 1g/kg cholic acid	2 mo	3.21 ± 0.12 mmol/L a —increased
32	2017	±180 g	400 g/kg beef tallow	12 wk	53.26 ± 1.90 mg/dL—increased
46	2017	4 wk 120‐150 g	15% lard, 10% custard powder, 1.2% cholesterol, 0.3% sodium taurocholate	28 d	Values not informed—serum cholesterol significantly increased

^aConversion factor: 1 mmol/L = 18.018 mg/dL.

4. PATTERNS OF HYPERCHOLESTEROLAEMIC INDUCTION

Of the 208 articles initially analysed, 33 met the inclusion criteria and were included in the present study. It is noticable that the most commonly used protocol for inducing hypercholesterolaemia was the addition of 1% cholesterol to animal diets which appears in 12.1% of the analysed articles, ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ followed by the addition of 2% cholesterol ¹⁸ , ¹⁹ and 2% cholesterol + 0.5% cholic acid, ²⁰ , ²¹ Both of the latter protocols were used by 6% of the analysed articles. Only one study used each of the other listed protocols. Eight of the studies analysed did not add cholesterol to the diet and still managed to obtain induction.

Concerning the study time, 5 articles used the 8‐week protocol. ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ The shortest study time was 15 days, and it used 10 g lard, 4 g corn oil and 0.15 g margarine added to the diet. ²⁷

Regarding the age of the animals, most studies used animals aged 6‐8 weeks at the beginning of the study, though some articles provided only the weight of the animals and not the initial age, therefore not allowing a complete analysis.

All articles claim to have been able to induce hypercholesterolaemia in animals and consequently were included in the present study. However, some of them did not provide the total serum cholesterol values at the beginning and end of the study, making it impossible to verify and quantify the induction.

5. IMPACT OF THE HYPERCHOLESTEROLAEMIC DIET

The regulation of bile acids in the body depends on cholesterol, and this causes cholesterol to modify the composition of bile salts in various parts of the organism, including faecal excretion. ¹⁸ , ²¹ Wang et al demonstrated in their study a smaller faecal bolus in the high cholesterol diet group, ²⁸ although no evidence of this has been found in other studies.

The induction of hypercholesterolaemia can lead to obesity, even when the amount of lipids in control and hypercholesterolaemic groups is the same. ¹⁵ , ²¹ , ²⁸ Santos‐Lopez et al observed that animals fed with a high cholesterol diet developed weight loss, even without significant difference in energy consumption when compared to the control group animals. ²⁹

Hypercholesterolaemia induced by diet also affects the animal's liver, increasing its weight and causing damage. These alterations are probably caused by the higher fat content in the liver. ³⁰ , ³¹ , ³² Furthermore, some studies correlate the liver damage with the high fatty acid content in the diet. However, other studies, in which the fat content was not modified, have also observed hepatic damage in the animals. ¹⁶ , ¹⁹ , ²⁸ , ³³ Some studies have found increased weight of heart and kidneys as well. ⁷ , ¹⁹

The induction of hypercholesterolaemia additionally decreases the activity of some superoxide dismutase (SOD) and catalase (CAT) (important antioxidant enzymes) in the liver, which also decreases the antioxidant defence. ³² , ³⁴ , ³⁵ In addition, hypercholesterolaemic diet can lower vitamin C levels in the animal's body. ³⁵

In animals with hypercholesterolaemia there is a decrease in myocardium function, increasing the chance of ischaemia and reperfusion and enlarging the size of infarction. However, the mechanism involved in these alterations is still not known. ⁷ , ¹⁸ , ³⁶

The animal's age may influence diet consumption, and this directly affects weight gain throughout the experiment. ³⁷

The major limitation of our study is that there is not much information about the impact of the hypercholesterolaemic diet itself. This diet is generally used as a positive control, and the studies do not show significant results exclusively in the hypercholesterolaemic group.

6. LIMITATIONS

The main limitation of this study is the fact that the articles analysed were not intended to examine the model, but the cholesterol reduction parameters instead. Thus, many of the papers do not illustrate cholesterol data accurately.

7. CONCLUSION

The current study reviewed hypercholesterolaemic models induced by diet and analysed many protocols that induce hypercholesterolaemia in animals with different ages and diets. The aim was to collect information, rather than judge the best model, considering that they all achieved their primary goal of inducing an increase in serum cholesterol. Even so, it was noted that the most commonly used protocol for hypercholesterolaemic induction is the addition of 1% cholesterol to animal diets.

Cunha LF, Ongaratto MA, Endres M, Barschak AG. Modelling hypercholesterolaemia in rats using high cholesterol diet. Int J Exp Path. 2021;102:74–79. 10.1111/iep.12387