CARDIOVASCULAR JOURNAL OF AFRICA • Vol 24, No 3, April 2013
AFRICA
59
as the spontaneously hypertensive rat. Interruption of the
RAS pathway, either by preventing the formation of Ang-II
(i.e. angiotensin-converting enzyme inhibitor) or by blocking
its actions at the level of the peptide receptor (AT
1
receptor
antagonists), has been shown to reduced BP and protect against
target-organ injury.
12-15
However, the attenuation or delay of
non-haemodynamic pathophysiological impairments with these
agents does not reduce the risk in hypertensive patients.
9-10
In addition, chronic administration of traditional therapies is
necessary for long-term antihypertensive benefits.
Constriction of the thoracic or abdominal aorta provides an
experimental model of what has previously been described as
pressure-overload cardiac hypertrophy. The increased blood
pressure proximal to the constriction has been postulated to
provide a stimulus for the development of cardiac hypertrophy.
16
This study was designed to examine the effects of AT
1
receptor
antagonists on the non-invasive (indirect) tail-cuff method, using
an automated cuff inflator pulse-detection system to estimate the
endogenous antioxidant enzyme [serum catalase and superoxide
dismutase (SOD)] activity. Histopathological changes in the
target organs (heart, liver, kidneys and thoracic aorta) were
analysed to compare the histopathological changes induced
in untreated abdominal aortic banding-induced hypertension
(AABIH) and cardiac hypertrophy in rats.
Methods
Healthy adult male albino Wistar rats weighing between 150 and
210 g were selected. Animals were maintained under standard
laboratory conditions at 28
±
2°C, relative humidity of 50
±
15%
and normal photo-period (12-h dark and 12-h light). Commercial
pellet diet (Amruth Ltd, India) and water were provided
ad
libitum
.
The experimental protocol was approved by the Institutional
Animal Ethics Committee and by the animal regulatory body of
the government (Al-Ameen College of Pharmacy, India. Reg.
No. 83/1999/CPCSEA). The test drugs losartan, candesartan
and irbesartan were procured from Micro Labs Private Ltd and
Biocon Ltd, India, respectively.
Animals were randomly divided into different groups, each
with eight male Wistar rats and they were treated as follows:
control (normotensive) sham-operated rats; untreated AABIH
rats; AABIH rats treated with losartan (40 mg/kg/day p.o.);
candesartan (10 mg/kg/day p.o.); and irbesartan (10 mg/kg/
day p.o.), respectively. Pressure overload was produced by
abdominal aortic banding (AAB), which has primarily been used
as a model of cardiac hypertrophy.
17
Briefly, animals were anesthetised using a combination of
ketamine (70 mg/kg, i.p) and xylazine (10 mg/kg, i.p.) and the
aorta was exposed through a midline abdominal incision. For the
banding model, a blunt 22-gauge needle was placed adjacent to
the abdominal aorta between the renal arties just below the renal
bifurcations, and a ligature was tightened around the aorta and
adjacent needle. The sham procedure for the control rats included
injection of the same dose of combination anesthesia, an incision
of approximately the same size, and the placement of a loosely
tied ligature at the same position on the abdominal aorta.
18
The
muscular layer was sutured, followed by the abdominal skin,
and the animals were isolated in a cage for recovery. The dead
animals were removed from the cage.
Drug treatment was started on the animals recovering from
surgery, with losartan, candesartan and irbesartan administered
daily for 16 weeks. The three drugs were formulated freshly
using 1% carboxy methyl cellulose (CMC) in distilled water
and were administered orally in a dose volume of 2 ml/kg body
weight; 1% CMC solution was used as vehicle.
After the surgery the animals were placed in their cages
and were observed for general characteristics and mortality.
Non-invasive (indirect) blood pressure (NIBP) was determined
by the tail-cuff method using an automated cuff inflator
pulse-detection system (AD Instruments, NIBP measurement
apparatus).
Non-anaesthetised rats were placed in a restraining holder
from which the tail protruded. Vasodilatation was achieved by
local warming of the tail with an infrared bulb. The cuff and
transducer were placed around the tail, and the cuff was inflated
until the pulse disappeared. When the cuff was deflated, the
point of reappearance of the pulse indicated the value of systolic
blood pressure. The reported values are from a minimum of three
recordings performed on each animal by the same investigator.
The NIBP was measured during weeks 1, 3 and 16. The patency
of the hypertension induced by AAB was ascertained during
week 3.
Endogenous anti-oxidant enzyme activity
After the NIBP measurement, the rats were anaesthetised with
ether and blood was collected in 2-ml Eppendorff tubes from
the retro-orbital plexus, with the help of heparinised capillary
tubes, for the estimation of anti-oxidant enzyme activity. The
collected blood was centrifuged for 15 min at 7 000 rpm and the
supernatant (serum) was used for the estimation of biochemical
parameters, namely catalase and SOD activity.
The catalase activity was determined spectrophotometrically
according to standard protocol as per the Clariborne method.
19
Briefly, to 1.95 ml of 10 mM H
2
O
2
in 60 mM phosphate
buffer (pH 7.0), 0.05 ml of the plasma/serum was added and
degradation of H
2
O
2
was followed at 240 nm per min. The rate
of decomposition of H
2
O
2
was calculated using the formula
k
=
2.303/
Δ
t
×
log (A
1
/A
2
) S
-1
, followed by calculation of catalase
in terms of U/mg of protein. A unit of catalase is defined as
the quantity that decomposes 1.0
μ
mole of H
2
O
2
per min at pH
7.0 and 25°C, while this H
2
O
2
concentration falls from 10.3 to
9.2 mM.
SOD activity was determined based on the ability of SOD
to inhibit the auto-oxidation of epinephrine to adrenochrome at
alkaline pH as per the method of Misra and Fridovich.
20
Briefly,
25
μ
l of the supernatant obtained from the centrifuged blood
was added to the mixture of 0.1 mM adrenaline in carbonate
buffer (pH 10.2) in a total volume of 1 ml, and the formation of
adrenochrome was measured at 295 nm. The SOD activity (U/
mg of protein) was calculated using a standard plot.
Histopathological evaluation of target organs
At the end of 16 weeks, after the NIBP measurement, rats
from each group were anaesthetised with ether and the target
organs (heart, liver, kidneys and thoracic aorta) were collected
and placed in the separate containers containing 10% neutral
buffered formalin, pH 6.8–7.0 (10 ml 40% formaldehyde, 0.35
g anhydrous sodium dihydrogen phosphate, 0.65 g anhydrous
