Abstract
Dear Editor
The purpose of this letter is to communicate some key factors, which have to be taken into consideration when experimental protocols using cholesterol-fed rabbit as an animal model are designed. The following issues are not referred to in original articles in detail; however they should be considered for discussion and conclusions, as this may be useful for researchers in the field.
Cholesterol-fed rabbit is an animal model which has been used for the study of atherosclerosis. The initiating events of the disease are similar to those in humans.1,2 In a large number of studies rabbits are used for testing the effectiveness of new drugs, food constituents and foodstuff and their impact on the progression and/or regression of early vascular lesions. Recently transgenic mice have been extensively used for atherosclerosis research – on a large scale. However, the traditional animal model of cholesterol-fed rabbit exerts a series of advantages such as the fact that a simple high-cholesterol diet can trigger vascular lesions without a requirement for complementary techniques, plus the availability of larger vessels, higher blood volumes and tissue amounts, all of which make it preferable in many research protocols.
The cholesterol percentage in the diet, the amount of food provided to the animals – restricted or ad libitum – and the duration of the diet are some of the main points that have to be considered when the dietary pattern is designed. When a foodstuff is to be tested, the threshold amount, which can be supplemented to the animals’ chow, triggers further discussion.
The selection of the percentage of cholesterol added in the diet protocol depends mainly on the extent of lesions, and this is determined by the research protocol. In brief, bibliographic data review demonstrates that a diet enriched with 0.2% (w/w) cholesterol causes the adhesion of leukocytes to the endothelium of thoracic and upper abdominal aortas within three weeks, a process which is detected by using scanning electron microscopy. When 0.25% (w/w) cholesterol is supplemented, for a duration of four weeks, monocyte adhesion and foam cell accumulation in the subendothelial space can be observed with specific dyes. 3 However, the lesions are not macroscopically visible. When the amount of cholesterol rises to 0.5% (w/w), for a duration of eight weeks, fatty streaks are formed which mainly originate from macrophages. The lesions are macroscopically visible; this percentage of cholesterol is well tolerated by the animals without any occurring deaths. Furthermore, histopathological evaluation through determination of the ratio between the thickness of the intima and the media is feasible. 4 A diet enriched with 1% (w/w) cholesterol for a period of 12 weeks can cause lesions rich in foam cells, originating from macrophages and smooth muscle cells. Large fatty streaks are formed; and a number of animals will not survive to the end of the dietary intervention 3 period. Longer periods where 1–2% of cholesterol is supplemented usually lead to high mortality rates. The induced hepatotoxicity reduces the survival rate of the animals. However, 2% can be provided for a short duration, such as four weeks. 5 Since all these types of lesions are classified as being at the stage of fatty streaks, the questions remain of the percentage which has to be selected and the criteria it has to be based on. The answers may be found in the importance of the parameters that can be measured at each step of lesion formation, and even in the same type of lesions for each protocol.
In most protocols, young animals of a body weight of approximately 2.5 kg are used. The rabbit’s daily food intake normally constitutes almost 5% of its body weight. When restricted food is provided, 125 g can be given to the animals at the beginning of the study. 6 However, this amount should not remain constant since the animal grows older and nutritional needs increase while the atherogenic diet will not cause the aimed degree of lesions. The animal will be stressed and will lose weight. The restricted food levels must be gradually elevated after the first week of the dietary intervention, so as not to affect the animals’ weight, behavior and overall health. On the other hand, when food is provided ad libitum, different net amounts of cholesterol are consumed, which leads to a great variation in serum cholesterol concentrations and lesion size between animals following the same diet. In this case a more in-depth statistical analysis must be performed in order to compensate for unpredictable factors.
Another issue that should be considered is the maximum amount/percentage of the studied foodstuff that can be supplemented to the animals’ chow. If the amount is high the consumed cholesterol will be lower compared with the group receiving only the cholesterol diet (without supplemented substances). In the literature, this amount is shown to exert a broad range of effects, depending on the food supplement.6–8 In addition, the caloric differences between diets provided to the groups of animals and their effects on body weight present another consideration that must be further studied and elucidated. Otherwise, experimental protocols must include more animal groups, and data must be evaluated with more complicated statistical analysis. However, researchers always need to compare similar properties or similar characteristics, while avoiding the use of an extensive number of animals, in order to achieve more consistency in their scientific results.
The points highlighted above should provide researchers in the field with food for thought, ultimately leading to fruitful discussion and development of guidelines for the establishment of a standardized model for cholesterol-fed rabbit in atherosclerosis research.