the lesions and the areas stained red were obtained by calibrating the software using an image of a micrometer
slide taken at the same magnification.
2
.
9
. Statistical analysis Non-baseline adjusted serum cholesterol and triglyce-
ride, body weight and food consumption data were analysed at weeks 0, 4, 8 and 12 using a mixed-model,
repeated-measures ANOVA. Multiple comparison least significant difference LSD tests were then performed
on the serum triglyceride data. Body weight and food consumption data were natural log transformed prior
to ANOVA to normalise variance. Atherosclerotic le- sion area and oil red O area data were also analysed by
ANOVA followed by a multiple comparison LSD test. Significance was assumed if P 0 0.05. Analyses were
performed using SAS software version 6.11 running on a PC.
The procedures involving animals in this study were subject to both internal review and UK Home Office
regulations.
3. Results
3
.
1
. Body weight and food consumption The body weights of mice in all four groups increased
throughout the study as expected. The weights of C3H apoE − − mice were significantly greater 7 – 24
than those of the C57 apoE − − mice both prior to and 4, 8 and 12 weeks after the start of Western diet
feeding P 0 0.0017 for each timepoint. Male mice of both strains were heavier 18 – 33 than their female
littermates both prior to and throughout the study P = 0.0001. Male mice ate more than females when
fed the normal diet 5.1 vs 4.8 g, P = 0.011. As ex- pected, food consumption fell in all 4 groups when the
mice were fed the Western diet but it did not differ significantly between the sexes and strains throughout
this period 3.3 9 0.2 g, mean 9 S.D..
3
.
2
. Serum cholesterol and triglyceride concentration When fed normal diet, C3H apoE − − mice had
higher serum cholesterol males: 1.9 fold, females: 1.5 fold and very much higher triglyceride males: 5 fold,
females: 4.2 fold concentrations than C57 apoE − − mice P = 0.0001, see Fig. 1. There was no difference
in the serum cholesterol concentrations of male and female mice of either strain or between the serum
triglyceride levels of male and female C57 apoE − − mice. However, male C3H apoE − − mice did have
35 higher serum triglyceride concentrations than their female littermates P = 0.0004.
Total serum cholesterol was increased dramatically in all four groups of mice after 4 weeks of feeding the
Western diet, with C3H apoE − − mice maintaining a 1.5 – 2 fold higher serum cholesterol concentration
than C57 apoE − − mice P = 0.0001, see Fig. 1a. There were no significant differences in the serum
cholesterol concentrations of male and female mice of either strain throughout the study.
Total serum triglyceride concentration decreased rather than increased in all four groups following the
switch to Western diet see Fig. 1b. C3H apoE − − mice had 3 – 6 fold higher serum triglyceride concentra-
Fig. 1. Total serum cholesterol a and triglyceride b concentrations in male and female apoE − − mice on either C3H or C57 back-
grounds fed Western diet for 12 weeks. Total cholesterol and triglyc- eride concentrations mM were measured in blood taken from
non-fasted mice as described in ‘Methods’ mean 9 S.D.. C3H apoE − − mice had significantly higher serum cholesterol and
triglyceride concentrations than C57 apoE − − mice throughout the study a: P = 0.0001, b: P 0 0.0033. Male C3H apoE − −
mice had higher serum triglyceride concentrations than female C3H apoE − − mice throughout the study c: P 0 0.0027. Male C57
apoE − − mice had higher serum triglyceride concentrations than female C57 apoE − − mice at week 8 d: P = 0.024. = Male
C3H apoE − − mice n = 10;
= male C57 apoE − − n = 9; = female C3H apoE − − n = 8;
= female C57 apoE −
−
n = 10.
Table 1 Percentage distribution of serum lipoprotein cholesterol and triglyceride in male and female apoE−− mice on either C3H or C57 backgrounds
before and after 12 weeks of being fed Western diet
a
Cholesterol Triglyceride
Sex Total
VLDLChy VLDLChy
IDLLDL HDL
HDL IDLLDL
Strain Total
Week mM
mM Male
C3H 17.4 9 2.4
80 14
6 9.8 9 2.2
98 2
Female 14.4 9 1.1
72 12
16 7.2 9 1.9
99 1
Male 9.4 9 3.3
59 26
15 2.0 9 1.4
C57 99
1 Female
9.6 9 3.7 73
20 6
1.7 9 0.6 85
10 5
Male 62.7 9 12.8
88 9 2 10 9 1
2 9 0.5 5.9 9 2.5
95 3
12 2
C3H Female
51.9 9 4.4 90 9 1
9 9 1 1 9 0.2
3.6 9 1.8 98
2 0.5
Male 39.2 9 19.4
88 9 0.3 11 9 0.6
2 9 0.5 1.8 9 1.9
C57 95
3 2
Female 37.9 9 8.2
91 9 0.5 8 9 0.3
2 9 0.3 1.3 9 0.3
98 2
a
Equal volumes of serum were pooled from all of the mice in each group at week 0 and 12 of the study. Individual serum samples from five mice in each group were used to produce cholesterol lipoprotein profiles at week 12. Pooled and individual samples were fractionated using a
Superose 6 gel filtration column and the fractions analysed for cholesterol and triglycerides as described in ‘Methods’. Total serum cholesterol and triglyceride concentrations are expressed as mean 9 S.D.
tions than C57 apoE − − mice throughout the study P 0 0.0033 for each timepoint. Male C3H apoE −
− mice had significantly higher 66 – 108 serum
triglyceride concentrations than female C3H apoE − −
mice at weeks 4 – 12 P 0 0.0027 for each time- point. In contrast, male C57 apoE − − mice had
higher triglyceride levels than female C57 apoE − − mice only at week 8 134, P = 0.024.
3
.
3
. Lipoprotein profiles Lipoprotein profiles were acquired from serum sam-
ples taken from normal diet fed mice and again from the mice after 4, 8 and 12 weeks of Western diet
feeding. Equal volumes of serum from all the mice in each group were pooled prior to analysis at all time
points, except week 12 when individual serum samples from five mice from each group were fractionated.
Lipoprotein profile data for weeks 0 and 12 are shown in Table 1. The cholesterol and triglyceride lipoprotein
profiles were very similar in weeks 4, 8 and 12, so only the data from week 12 were included in Table 1.
In male and female C3H apoE − − and female C57 apoE − − mice fed normal diet, the vast major-
ity 72 – 80 of cholesterol was present in the VLDL Chy fraction, whilst in male C57 apoE − − mice fed
chow, a much smaller percentage 59 of the total serum cholesterol was found in this fraction see Table
1. After 12 weeks of feeding Western diet the VLDL Chy fraction represented even more 88 – 91 of the
total serum cholesterol in all mice. The concentrations of HDL cholesterol were small and represented only
1 – 2 of total serum cholesterol in all four groups of mice.
Serum triglyceride was almost exclusively found in the VLDLChy fraction when the mice were fed either
normal or Western diet. Only a negligible amount of triglyceride was present in the IDLLDL and HDL-
sized particles.
3
.
4
. Areas and lipid content of atherosclerotic lesions The total cross-sectional areas of lesions in both male
and female C57 apoE − − mice were more than twice as large as those of C3H apoE − − mice
Fig. 2. Total cross-sectional area of atherosclerotic lesions and cross- sectional area of lesion stained with ORO in male and female
apoE − − mice on either C3H or C57 backgrounds fed Western diet for 12 weeks. Areas mm
2
were measured as described in ‘Methods’ mean 9 SEM. The mean total cross-sectional areas of
lesions in C57 apoE − − mice was more than twice those found in C3H apoE − − mice of the same sex. Filled bars represent cross-
sectional area of lesions stained with ORO and open bars represent total cross-sectional area of lesions. Male C3H apoE − − n = 10,
male C57 apoE − − n = 9, female C3H apoE − − n = 8, female C57 apoE − − n = 10. Significant differences between
groups are shown by the use of the same letter a: P = 0.002; b: P = 0.0088; c: P = 0.0001; d: P = 0.016.
males P = 0.0088, females P = 0.0001, see Fig. 2. The total cross-sectional area of atherosclerotic lesions was
51 greater in female C57 apoE − − than in male C57 apoE − − mice P = 0.016 and although the
area of lesions tended to be larger in female C3H apoE − − than male C3H apoE − − mice, this
difference did not reach significance.
The total area of lesion stained with ORO in male C57 apoE − − mice was higher than in male C3H
apoE − − mice P = 0.002 but there was no signifi- cant difference when these stained areas were expressed
as percentages of the total cross-sectional area of lesion. There was no difference in the ORO stained area
between females of either strain. The percentage of lesion stained by ORO in male C57 apoE − − mice
was twice that in female C57 apoE − − mice 27 vs 14, P = 0.0008. There was no difference in ORO
area between the sexes for C3H apoE − − mice.
4. Discussion
