23 / 64
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 4, July/August 2019
AFRICA
209
Cocoa powder is rich in antioxidants and several studies
have shown that treating animals with cocoa powder inhibits
the oxidation of LDL,
22
and reduces oxidative stress,
23
inflammation
24
and insulin resistance.
25
Moreover, consumption
of natural cocoa powder reduced hypercholesterolaemia and
atherosclerosis in apolipoprotein E knock-out mice by reducing
the expression of genes related to metabolism, apoptosis and
inflammation.
26
Our study therefore investigated the effect of
maternal hypercholesterolaemia on the vascular morphology
and atherosclerosis in the offspring of hypercholesterolaemic
rabbits. It also sought to validate potential beneficial effects of
consumption of natural cocoa powder (NCP) in the prevention
of foetal onset of atherosclerosis.
Methods
This study was approved by the Ethical and Protocol Review
Committee of the College of Health Sciences, University of
Ghana. It was carried out in accordance with appropriate
institutional regulations on the care and use of laboratory
animals.
Ten New Zealand white female rabbits (age: 6 months,
body weight: 1.5–2.8 kg) were obtained from the Animal
Experimentation Unit of the Noguchi Memorial Institute for
Medical Research (NMIMR), University of Ghana, Accra. The
rabbits were transported to the animal house of the University
of Ghana Medical School, Korle-Bu and kept for two weeks to
acclimatise. The animals were housed under standard conditions
of local temperature (30°C) and relative humidity (80%) and
exposed to a 12-hour light/dark cycle.
Rabbits were then randomly assigned by lottery to three
groups and housed individually in cages. The rabbits were
arbitrarily numbered from one to 10 and the numbers were
written on pieces of paper. After mixing up the pieces of paper,
rabbits were placed in groups when the numbers were drawn.
The first two groups of four rabbits each were fed cholesterol-
enriched feed (CEF). The CEF was prepared (adopted from Sun
et al
.
27
) by mixing standard feed (Kosher Feedmill Ltd, Accra)
with 0.5% (w/w) cholesterol (Hopkin and William Ltd, London)
and 10% (v/w) coconut oil (open market, Accra). The rabbits
were fed CEF for two weeks, and when a routine lipid profile
test confirmed hypercholesterolaemia, they were crossed with
normocholesterolaemic males.
The normal cholesterol range for rabbits is 0.14–1.86 mmol/l
(Olfert
et al
.
28
). Hypercholestrolaemia was defined as a total
plasma cholesterol level higher than twice the upper level of the
normal range.
One group of rabbits on CEF was given 2% (w/v) of NCP
(GoodFood brand, Kakawa Enterprise Ltd, Accra) as an
aqueous suspension instead of drinking water. This group
(HCC) had 24-hour access to the NCP suspension, which they
drank voluntarily, after being mated until they littered (28–30
days). The second group of rabbits on CEF (HC) were given
24-hour access to filtered tap water.
The third group of rabbits (
n
=
2), designated as normal
control (NC), were given standard chow without cholesterol
enrichment and filtered tap water throughout the duration of the
experiment. Animals that were fed CEF were fed with standard
chow after delivery and the cocoa drink was replaced with
drinking water. Twelve pups were delivered by the HCC rabbits,
six by the HC rabbits, and the NC rabbits delivered 12 pups.
Total levels of cholesterol were determined after an overnight
fast by an enzymatic colorimetric test in a laboratory at
the Medical Biochemistry Department (University of Ghana
Medical School), using a semi-automated clinical analyser,
Microlab 300 (Vital Scientific, the Netherlands).
Blood samples were obtained by the bleeding of the marginal
ear vein. The skin over the ear of the rabbits was anaesthetised
using a local anaesthetic cream containing lidocain (Lignocaine
2% Jelly, Purna Pharmaceuticals, Belgium) after the fur over
the ear was shaved and the skin sanitised with alcohol. Blood
samples were then drawn from the marginal ear vein and stored
in sterilised test tubes containing heparin.
Maternal blood samples were collected before treatment with
the cholesterol-enriched diet and after two weeks of feeding with
the cholesterol-enriched diet. Blood samples from the offspring
at the end of the experiment were obtained by cardiac puncture
after chloroform inhalation had anaesthetised them.
The rabbit pups were euthanised by chloroform inhalation
one week after birth and perfusion-fixed using 10% normal
saline, followed by 10% phosphate-buffered formalin at pH
7.3. The aorta was dissected to remove the arch, thoracic and
abdominal segments, which were post-fixed in 10% phosphate-
buffered formalin for two to seven days. The aortae were taken
through a routine histological processing.
Every 10th section of the arch, thoracic and abdominal
segments of the aorta with a thickness of 10 µm was stained
and analysed. Sections were stained with haematoxylin and
eosin (H & E) for assessing intima–media thickness. In order
to assess collagen and elastic fibre deposition in the vascular
walls, Verhoeff–Van Gieson (VVG) staining of the sections was
performed.
To determine the presence or absence of atherosclerotic
lesions, frozen sections of the aortic segments were stained with
Oil red O. Sections were counter-stained in alum haematoxylin.
Five sections each of the aortic arch, thoracic and abdominal
aorta were selected 100 µm apart and examined qualitatively for
the presence or absence of atherosclerotic lesions.
Micrographs of stained sections were obtained using a
digital microscope eyepiece (Premiere MA 88) fitted to a Leica
Galen III light microscope. The digital eyepiece was connected
to a computer and images from the microscope were captured
using Microsoft Publisher software 2003 version. Images were
analysed with Photoshop CS 4 (Adobe Systems, San Jose, CA,
2008).
A stage graticule (Nikon, Japan) was used to calibrate the
ruler tool in Photoshop by mounting it onto the stage of a
microscope and a micrograph, taken using the digital eyepiece,
was connected to the ×10 objective lens. Intima–media thickness
was measured using the ruler tool in Photoshop. Two lines
(DD and EE) perpendicular to each other were drawn across
the image of the artery through the centre, as shown in Fig. 1.
Intima–media thickness readings were taken between points 1/2
and 3/4 on line EE and the average was calculated and recorded.
Statistical analysis
The results of the study were analysed using Graphpad Prism
software (3.0). The
t
-test was used to compare the means of
two groups, while one-way ANOVA was used to compare the