rotic lesions in the aortic root after only 7 weeks, whereas lesions are absent in C3H mice fed the same
diet for up to 1 year, although total serum cholesterol concentrations in C3H mice are usually higher than
those in C57 mice [1 – 3]. Differences in the susceptibil- ity of these strains to developing atherosclerosis has
been attributed to at least eight genes Ath1 – Ath8, for which the approximate gene loci have been iden-
tified [4].
The recent development of technology for gene si- lencing and gene transfer has generated many novel
strains of mice with much greater susceptibility to developing atherosclerosis. Among these the knockout
of the apoE gene has been shown to give one of the most severe atherogenic phenotypes. ApoE is a major
constituent of very low density lipoproteins VLDL and chylomicrons and is an important ligand for the
receptor mediated uptake of these lipoproteins from the blood into the liver, as reviewed by Hofker et al.,
[5].
Furthermore, apoE
is implicated
in HDL
metabolism and
reverse cholesterol
transport. ApoE − − mice were initially produced by two
groups [6,7]. The apoE − − mice were subsequently bred into the C57 background and have now been
studied extensively. ApoE − − mice have impaired clearance of cholesterol-ester enriched VLDL and chy-
lomicrons from the blood which results in hyperlipi- daemia and the development of atherosclerotic lesions
[5]. It is reported that apoE − − mice develop spontaneous lesions when fed normal chow diet and
this process is further accelerated by feeding a high fathigh cholesterol diet [6,8,9]. Complex lesions that
include a fibrous cap can be seen in mice as young as 15-weeks-old and these are not just confined to the
aortic sinus but are found in the aortic arch, the major branches of the aorta and the pulmonary and carotid
arteries. The atherosclerotic lesions of apoE − − mice exhibit a similar distribution, microscopic ap-
pearance and cellular composition to those found in humans [9].
As mentioned above, wild-type C57 mice are rela- tively susceptible to diet-induced atherosclerosis. For
the present study we have bred the apoE − − mu- tation into the C3H strain, which has a low suscepti-
bility for atherosclerosis, to address the question as to whether the apoE knockout is so dominant in its
effect on atherosclerosis that the difference in suscepti- bility between the C57 and C3H strains is lost. Here
we report the results of a study in which we compared plasma lipid and lipoprotein concentrations and the
amount of atherosclerosis that developed in the aortic root of C57- and C3H apoE − − mice fed a West-
ern-type diet. As female mice of the C57 strain are reported to be more susceptible to diet-induced
atherosclerosis than males, these studies were per- formed using both sexes [10].
2. Materials and methods
2
.
1
. Materials Wako cholesterol CII kit no. 270-5439954499 was
purchased from Wako Chemicals GmbH, Germany. Boehringer-Mannheim Peridochrom triglyceride kit
no. 701904 and EDTA sodium salt were from Boehringer-Mannheim GmbH, Germany. Pyruvic acid
sodium salt, cholesterol standard solutions, glycerol gelatine for slide coverslipping and fumaric acid were
from Sigma, USA. Isopropyl alcohol,
D
-glucose, NaHCO
3
, KCl and NaCl were from Fisher, UK. Oil Red O certistain grade, for lipid staining, dextrin,
gelatine for slide coating, Mayer’s haematoxylin,
L
- glutamic acid, glycerol for triglyceride assay stan-
dard, NaN
3
, CaCl
2
, MgSO
4
.7H
2
O and Na
2
HPO
4
were obtained from MerckBDH, UK. All chemicals were
of analytical grade. Buffered formal saline 4 ww formaldehyde solution was from Pioneer Research
Chemicals, UK. Butterfly-25 cannulae were from Venisystems, Ireland, Microvette CB-300 blood tubes
from Sarstedt, Germany, 96-well tissue culture plates from Costar, USA and OCT compound from Bayer
Diagnostics, Germany.
2
.
2
. Diet Normal mouse diet RM1 was purchased from Spe-
cial Diet Services, UK. Western diet was from Hope Farms, The Netherlands. Western diet was essentially
the same as that used by Nishina et al. [11] and contained the following constituents ww: cocoa but-
ter 15, cholesterol 0.25, sucrose 40.5, corn- starch 10, corn oil 1, cellulose 5.95, casein
20, 50 choline chloride 2, methionine 0.2 and mineral mixture 5.1.
2
.
3
. Mice ApoE − −
tm1Unc
mice [7] were purchased from the Jackson laboratory, having been backcrossed for
six generations into a C57BL6J background and un- dergone three generations of brothersister mating.
The colony underwent a further eight generations of breeding and expansion before being used in the
present study. C57BL6J apoE − − mice were also backcrossed for six generations into C3HHeNHsd
mice. The C3HHeNHsd apoE − − mice were then bred for two generations before being used in the
present study.
2
.
4
. Methods Male and female apoE − − mice on C3H
HeNHsd or C57BL6J backgrounds n = 10sexstrain
were randomised to eight cages five per cage with regard to age C3H: age = 8.1 weeks, C57: age = 7.7 9
0.3 weeks, mean 9 S.D. and body weight. Body weights for male and female C3H apoE − − and
C57 apoE − − mice were respectively: 25.7 9 1.5, 20.9 9 1.8, 23.3 9 1.8 and 19.4 9 1.7 g mean 9 S.D..
Mice received food and water ad libitum and were housed in a room that was lit from 06:00 h to 18:00 h
and maintained at 21°C. The mice were fed powdered normal diet for 2 weeks prior to receiving the Western
diet. All of the mice were weaned onto Western diet over 4 days and were fed the diet for 12 weeks. Body
weights were measured at weekly intervals both before and during the study. Food consumption per cage was
measured both prior to and throughout the study.
2
.
5
. Serum lipid determination Blood samples were taken from the tails of non-
fasted mice at least 4 h after the start of the light phase when food consumption would be expected to be mini-
mal. Blood samples were taken just prior to treatment and at 4 weekly intervals throughout the study. Serum
was prepared and aliquots were either frozen − 20°C awaiting analysis for cholesterol and triglycerides or
stored at 4°C for lipoprotein profile analysis. Total cholesterol and total triglyceride concentrations were
measured enzymatically, using commercial kits from Wako Chemicals and Boehringer-Mannheim, respec-
tively, and a 96-well plate reader Thermomax, Molecu- lar Devices, UK. In weeks 0, 4, 8 and 12 of the study,
equal volumes of serum were pooled from all of the mice in each group for lipoprotein analysis. Lipoprotein
profiles were analysed by size-exclusion chromatogra- phy using a SMART™ micro-FPLC system Pharma-
cia, Sweden and a method similar to that described by Hennes et al. [12]. Briefly, pooled serum samples were
filtered 0.2 mm filter, Anotop 10, Whatman, UK and 30 ml of serum was applied to a Superose 6, PC 3.230
column. The column was eluted with a solution con- taining NaCl 150 mM, EDTA 1 mM and sodium
azide 7.7 mM at pH 8.0. The first 920 ml of column eluent represented the void volume. The lipoproteins
were contained in the next 42 fractions of 20 ml each. The flow rate of the mobile phase was 65 mlmin. The
cholesterol and triglyceride content of the fractions was measured as described above. VLDLchylomicrons
VLDLChy, intermediatelow density lipoproteins IDLLDL and high density lipoproteins HDL were
present in fractions 1 – 16 0.92 – 1.24 ml, 17 – 29 1.24 – 1.5 ml and 30 – 42 1.5 – 1.76 ml, respectively. In addi-
tion, individual terminal serum samples from five of the mice in each of the four groups were separated into
20-ml fractions and their cholesterol concentration mea- sured as described above.
2
.
6
. Perfusion fixing of hearts and aortae The mice were killed by cervical dislocation. The
hearts and aortae were immediately perfused in situ with oxygenated Krebs – Henseleit buffer at 37°C under
a pressure of approximately 110 cm of water via a cannula inserted in the left ventricle and an outlet
created by cutting the right atrium. After 30 min the buffer was replaced with buffered formal saline at 37°C
and the perfusion was continued for a further 30 min. The hearts and aortae were then removed, cleaned of
extravascular fat and stored in formalin until they were processed.
2
.
7
. Sectioning and staining of the aortic sinus Cross-sections of the aortic root were taken as de-
scribed previously [13,14]. Briefly, the hearts were bi- sected just below the level of the atria and the base of
the heart plus aortic root were taken for analysis. After equilibrating the tissue in OCT compound overnight
the hearts were immersed in OCT compound on a cryostat chuck Bright Instrument Company Ltd, UK
with the aorta facing the chuck. The tissue was frozen by surrounding the chuck with dry ice. The hearts were
then sectioned perpendicular to the axis of the aorta, starting within the heart and proceeding in the direction
of the aorta. After the aortic root had been identified by the appearance of the three-valve leaflets, alternate
10-mm sections were taken and mounted on gelatinised slides. Sections were air dried for 1 h and subsequently
rinsed briefly in 60 isopropyl alcohol. The sections were stained with Oil Red O, counterstained with
Mayer’s haematoxylin, cover slipped using glycerol gelatine and sealed with nail varnish.
2
.
8
. Quantification of atherosclerosis in the aortic root Ten sections of the aortic root per mouse were
imaged using an Olympus BH-2 microscope equipped with a × 4 objective total magnification = × 40, three
neutral density 2 × ND-6 and 1 × ND-25 filters and a video camera Hitachi, HV-C10. Twenty-four bit
colour images were acquired and analysed using a PC Datacell Pentium P5-133, Datacell, Berks, UK fitted
with a framegrabbing board IC-PCI, Imaging Tech- nologies, MA, USA and running Optimas software
version 6.1, Optimas Corp., WA, USA. The images were captured under identical lighting, microscope,
camera and PC conditions. Some of the sections were torn or folded and the images of these were discarded.
Quantification of the atherosclerotic lesion areas was performed by drawing around the lesions by hand using
the Optimas software. Colour thresholds were set that quantified the areas that were stained red within the
lesions. Absolute values for the cross-sectional areas of
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
