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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 3, May/June 2016
AFRICA
135
found in hospitals, to investigate cardiac changes in sham rats
or rats with ACC-induced myocardial hypertrophy by using an
extensive analysis, including echocardiography, heart and left
ventricular mass, cardiomyocyte area, plasma B-type natriuretic
peptide (BNP) concentration, and interstitial and perivascular
fibrosis.
BNP is a circulating hormone produced in the heart, primarily
by ventricular cells. This hormone is mainly secreted in response
to increases in ventricular wall stress, such as ventricular
hypertrophy, and it is detectable at high concentrations in a
number of circumstances, including cardiac ischaemia and severe
heart failure.
8
In addition, the expression of BNP is significantly
increased in animal models of chronic haemodynamic overload.
Therefore, we measured blood BNP concentrations to enhance
our understanding of BNP secretion in both AAC and sham
rats sequentially, as a marker to evaluate the extent of cardiac
hypertrophy.
The primary goals of this study were to modify the AAC
procedure in order to establish a safer and more stable LVH
model in rodents, evaluate the utility of standard human
ultrasound probes to detect structural and functional changes
in rats with cardiac hypertrophy, and provide a theoretical
and experimental foundation for the application of novel drug
interventions aimed at interfering with clinical LVH.
Methods
Fifty male Wistar rats (80–100 g) were used in all experiments.
They were allowed standard laboratory chow and tap water
ad libitum
and housed in stable conditions at 22°C with a
12-hour light/dark cycle for one week prior to AAC surgery.
All procedures were performed in accordance with institutional
guidelines for animal research.
After a one-week acclimatisation, the AAC surgery was
performed. All experimental rats were weighed prior to surgery,
and at three, four and six weeks post surgery. Echocardiographic
studies were conducted at three, four and six weeks post surgery.
For LVweight and histological measurements, rats were sacrificed
following echocardiography, and blood samples were collected
from the right carotid artery for enzyme-linked immunosorbent
assay (ELISA) analyses of plasma BNP concentrations.
AAC was induced in outbred male rats, as previously
described.
9
In brief, the animals were anesthetised with sodium
pentobarbital (45 mg/kg, i.p.). The abdominal aorta proximal to
the left renal artery was exposed and separated from the vena
cava. A 2-0 silk suture was tied, using a blunt 24-G probe (the
external diameter was 0.55 mm), beside the aorta between the
branches of the coeliac and anterior mesenteric arteries. The
probe was removed, leaving the vessel constricted to a diameter
of 0.55 mm. Saline (1 ml) was administered into the peritoneal
cavity in order to replenish any fluid loss, and the abdominal wall
and skin were sutured closed. All rats were allowed to recover on
a warming pad. The same procedure was performed in the sham
animals except that the silk suture around the aorta was pulled
through and not tied.
Following surgery, 50 mg/kg, i.m. ampicillin was administered
once daily for three days after surgery to prevent infection.
The day following surgery, the sham and AAC animals were
randomly divided into six groups as follows: (1) sham for three
weeks (
n
=
8); (2) sham for four weeks (
n
=
8); (3) sham for six
weeks (
n
=
8); (4) AAC for three weeks (
n
=
8); (5) AAC for four
weeks (
n
=
8); (6) AAC for six weeks (
n
=
10).
Echocardiographic studies
Echocardiographic studies were performed between 16:00 and
20:00, with the animals in the left lateral decubitus position,
using sodium pentobarbital (45 mg/kg, i.p.) for anaesthesia.
The IE 33 echocardiographic system (Philips Medical Systems,
Nederland BV) was used to perform two-dimensional (2D)
guided M-mode echocardiography and pulse-wave Doppler
echocardiography with a linear-phase array probe (L15-7io;
frequency range 7–15 MHz), which was placed on the shaved
left hemithorax. 2D images of the heart were obtained in the
parasternal long-axis view, followed by the short-axis and apical
four-chamber views.
M-mode echocardiography is useful for assessing LVH,
allowing accurate measurements of wall thickness and LV
dimensions during systole and diastole. For M-mode recordings,
the parasternal long-axis view was used to image the heart in
2D, with a depth setting of 2 cm. M-mode recordings were then
analysed at a sweep speed of 66 mm/s, with the axis of the probe
aligned near the posterior leaf mitral valve.
The following parameters were measured: LV posterior
wall (LVPW) dimensions during both diastole and systole
(LVPWd and LVPWs, respectively), interventricular septal
(IVS) dimensions during both diastole and systole (IVSd and
IVSs, respectively), LV internal dimensions (LVID) during
both diastole and systole (LVIDd and LVIDs, respectively), LV
end-diastolic volume (EDV), LV end-systolic volume (ESV),
percentage LV fractional shortening (FS), LV ejection fraction
(EF), cardiac output (CO), and heart rate (HR). LV mass (LVm)
was obtained from echocardiography and derived from the cubic
equation at the end of diastole:
LVm
=
1.04
×
[(LVIDd
+
LVPWd
+
IVSd)
3
– LVIDd
3
]
×
0.8
+
0.14.
10
All data are means of three consecutive cardiac cycles. 2D
guided M-mode recordings were obtained from the parasternal
short-axis view of the left ventricle at the level of the papillary
muscles. The angle of the M-mode beam was focused on the
middle of the LV, and aligned at the anteroposterior axis,
perpendicular to the LV walls. Parameters and methods were
the same as in the parasternal long-axis view, except that the
thickness of the LV anterior wall (LVAW) was obtained instead
of IVS. To assess inflow and outflow of the left ventricle,
Doppler recordings were acquired in the apical four-chamber
view to obtain inflow values parallel to the sample volume.
Pulse-wave Doppler (PWD) recordings were acquired with the
sample volume placed midway between the mitral and aortic valves
to determine the velocity of mitral inflow and aortic outflow. PWD
spectra of mitral inflow were recorded with the sample volume
placed at the tips of the mitral valve leaflets and adjusted to the
position at which velocity was maximal, with the sample volume
set to the smallest size available (1 mm). Due to the high heart rates
of rodents, which caused fusion of the E and A waves, diastolic
function was not evaluated using Doppler imaging.
LV outflow velocity was recorded from the apical long-axis
view, with the sample volume positioned just below the aortic