Protocol Considerations

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Many of the experimental design decisions have to be based on empirical information. When initiating a study on the development of atherosclerosis, the following questions need to be asked.

1.2.1. What Is the Most Appropriate Type of Mouse?

As noted above, none of the currently available mouse models of atherosclerosis develop the full spectrum of lesions present in humans (1). Many mouse models develop lesions that resemble American Heart Association (AHA) type I and II; a more limited number have lesions that evolve into AHA type IV. No mouse models are currently available that develop the ruptured and eroded lesions that precipitate acute cardiovascular events in humans. Therefore, investigations that study lipid deposition and monocyte adhesions may be

Selected Genetically Manipulated Mice That Have Been Used in Atherosclerosis Research

Mouse

Characteristics of atherosclerosis

Area of lesion Modified characterization diet required

Commercial availability

CD ros i s

O CD

Targeted Deletions

Trangenics

Cholesterol ester transfer

ApoE(arg112,Cys142) (29)

ApoE3Leiden (30)

apoC-III (31) Compound

Progress from predominantly foam cell lesion to lesions with necrotic cores and fibrous caps

Lesions composed predominantly of lipid-laden foam cells

Lesions composed predominantly of lipid-laden foam cells

Lesions composed predominantly of lipid-laden foam cells

Lesions composed predominantly of lipid-laden foam cells

Lipid-rich lesions of undefined cellularity protein (28)

Lesions composed predominantly of lipid-laden foam cells

Lesions composed predominantly of lipid-laden foamcells

Not defined

A range of lesions from lipid-laden foam cells to those containing fibrosis and smooth muscle hyperplasia

Aortic root, aorta, No carotid

Aortic root Yes

Aortic root, aorta Yes

Aortic root

Aortic root

Aortic root

Aortic root

Aortic root

Aortic root

Aortic root, aorta No

Jackson Taconic

Jackson

Taconic

Jackson

Oi performed in mice that develop lesions of simple morphology. However, the study of mechanisms of more complex tissue remodeling may require models that have mature aspects of the disease process (see details in Table 1).

Early studies used inbred mice, primarily of the C57BL/6 strain. However, as mentioned above, these mice develop small lesions, even when fed diets that have been modified to induce hyperlipidemia. Therefore, most contemporary studies have used a genetically modified variant, of which the most commonly used have been low-density lipoprotein (LDL) receptor-- and apoE-/- mice. LDL receptor-- mice require a modified diet to generate lesions. These lesions tend to have a simple morphology, in which lipid-laden macrophages are the predominant cell type throughout development. LDL receptor-- mice are preferred by some investigators because the distribution of cholesterol within plasma lipoproteins bears more resemblance to humans in that most of the sterol is present in LDL. Lesions in apoE-/- mice start with a simple morphology of lipid-laden contained macrophages. As they mature, they will acquire a more complex morphology that includes acellular lipid cores containing cholesterol clefts and fibrous caps (8,9). Much of the cholesterol in apoE-/-mice is transported in the very low-density lipoprotein (VLDL) fraction. A characteristic of the LDL fraction is that it contains a more complex array of apolipoproteins other than apoB.

Therefore, deciding which mouse model to use will depend in part on the specific pathogenic process under investigation that is most accurately mimicked compared with humans.

1.2.2. Should a Normal or Modified Diet Be Used?

Early studies using inbred mice required the use of a modified diet to generate a hyperlipidemic state, with subsequent development of atherosclerotic lesions. The most common modification was to have mice consume a diet highly enriched in saturated fat, cholesterol, and cholate (10). This diet is often referred to as the Paigen diet, after the investigator who popularized it. However, in the era in which genetically modified mice are available, such pronounced dietary manipulations are not needed. The apoE-/- mouse develops hypercholesterolemia and atherosclerosis when maintained on a normal diet. However, many studies have been performed on these mice during feeding of a diet that is primarily enriched with saturated fat and cholesterol to a level present in the diet of western industrial nations. This diet contains 40% of calories from fat (21% by weight) and is colloquially referred to as the western diet. It has been demonstrated that feeding this diet accelerates the development of atherosclerosis in apoE-/- mice without promoting gross changes in morphologic characteristics of lesions (8). However, feeding a saturated fat diet may influence the mechanism of the disease process. This is illustrated by the study of total lymphocyte deficiency in apoE-/- mice. Total lymphocyte deficiency was produced by the deletion of either recombinant activator genes 1 or 2, which are required to produce mature B- and T-lymphocytes. Total lymphocyte deficiency had no effect on the extent of atherosclerosis development in mice that were fed a western diet. In contrast, there was a 42% decrease in lesion size in the aortic root when mice were fed a normal diet (11,12). Therefore, it is possible that the regulation of atherosclerosis by some mechanisms may be overridden by the production of severe hypercholesterolemia.

1.2.3. What Duration of Study Is Needed?

There are evolving technologies in the noninvasive quantification of atherosclerosis in mice by modalities such as magnetic resonance imaging (13). However, such techniques are in a developmental phase, and lesion quantification presently requires termination of the mouse to acquire the vascular tissue. Therefore, decisions have to be made on the interval of tissue acquisitions. These decisions are facilitated by knowledge of the extent of atherosclerosis under specific circumstances. However, there appears to be substantial variation in the extent of atherosclerosis generated between investigators, even using the same strain of mice and the same diet. Therefore, each individual laboratory needs to define the extent of lesion formation in that environment.

The duration of the study is partially dictated by the underlying hypothesis being studied and the mode of analysis. If an intervention is being studied that inhibits the development of atherosclerosis, then a robust response needs to be generated in the control mice. Lesions form more rapidly in the aortic root, and therefore studies of shorter duration are permissible in this region compared with en face analysis of the entire aortic intima.

In the vast majority of studies, the extent of atherosclerosis is quantified at a single interval. There are many reasons for constraining studies to one interval, including the expense of the studies and the considerable work needed to complete the analysis. However, for some studies it may be important to define the effect of an intervention on the temporal characteristics of atherosclerosis.

1.2.4. How Many Mice Should Be Used per Group?

The number of mice needed per group can be determined from power calculations. This requires prior knowledge of the variance within a control group. Given the differences in variance that are noted between investigators' laboratories, it is not possible to provide these estimates from literature values. Therefore, this information needs to be derived for each environment.

Studies that quantify atherosclerotic lesions are frequently characterized by wide variances in the data. These wide variances occur even in mouse studies performed with inbred strains, which adds genetic equivalence to a standardized environment of such variables as feeding and housing. The wide variance, combined with a common lack of normal distribution for the data, frequently leads to an inability to perform the most commonly used parametric statistical tests. Although parametric tests have a considerably enhanced sensitivity over nonparametric tests, their inappropriate use can lead to a type I statistical error. In our studies, we use the SigmaStat (SPSS) statistical package, which provides information on the appropriate use of a specific statistical test. (See Note 1.)

1.2.5. Which Arteries Should Be Used for Quantifying Atherosclerosis?

The majority of atherosclerosis determinations in mice are performed in the aortic root or in the entire aortic tree. This emphasis is due to the early experience with wild-type C57BL/6 mice fed modified diets in which lesions only formed in these regions. Subsequent experiments used en face quantification of the entire aorta (14), in a process that had been used in several other species, particularly rabbits (15,16). Use of this technique requires genetically modified mice since these are the only strains in which significant disease occurs throughout the aorta. In our hands, the quantification of atherosclerosis by the en face technique is considerably more rapid than using the aortic root.

The carotid artery has been used extensively in vascular damage and transplant studies (17,18), but has had scant use in atherosclerosis studies. The coronary bed is of obvious interest, although there is no publication demonstrating a formal quantification process in this region.

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