CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 8, September 2012
452
AFRICA
day, with a 90-s rest period between each set, five times per week
for four weeks.
4
Each rat in the trained group was weighed daily and 120% of
its body weight (approximately 70% of the maximum load that
the rats were able to raise following electrical stimulation) was
used to determine the weight of the piston. The piston movement
for each rat was recorded by a distance sensor which had been
located above the piston and the work performed by each rat
was calculated daily by multiplying the piston weight and piston
movement.
According to the method of Brown
et al
.,
after anaesthetisation
with pentobarbital sodium (35 mg/kg ip injection) the hearts
were excised, placed in ice-cold saline and rapidly hung by
the aorta on the cannula of the Langendorff apparatus.
8
Hearts
were perfused with 37.5°C Krebs buffer (76.5 mmHg perfusion
pressure with 95% O
2
and 5% CO
2
)
containing 117.4 mM NaCl,
4.7
mM KCl, 1.9 mM CaCl
2
, 1.2
mM MgSO
4
, 1.2
mM KH
2
PO
4
,
5
mM pyruvate, 11 mM glucose, 0.5 mM EDTA, 25 mM
NaHCO
3
and 1200 U/l heparin.
A pressure-transducing catheter was placed through the
cannula and aortic valve into the chamber of the left ventricle
(
LV) and the developed pressure was determined with a computer
connected to the transducer (PowerLab, AD Instruments,
Australia). After a 5-min stabilisation period, baseline pressure
was measured, and coronary flow rate was obtained by collection
of the coronary effluent for 1 min.
After baseline records, a suture was threaded through the left
anterior descending coronary artery 3–5 mm distal to the aorta
in 14 rats in each group. Both ends of the suture were inserted
into a small polyethylene tube that was used as a snare, and
ischaemia was induced by tightening the snare so that the artery
was fully compressed. Pressure and coronary flow measurements
were recorded at 5, 15, and 30 min after the onset of ischaemia.
After 40 min, the snare was loosened and reperfusion ensued for
80
min. Coronary flow and pressure data were recorded at 5 min
after the onset of reperfusion and then every 15 min until the end
of the 80-min reperfusion period.
Data were omitted from analysis if the coronary flow did
not decrease at the onset of ischaemia or increase at the onset
of reperfusion (
n
=
3),
or if the hearts did not complete the
IR protocol due to fibrillation or technical difficulty (
n
=
2).
Only 11 hearts in the control group and 12 in the trained group
completed the IR protocol.
In the remaining rats from each group (
n
=
6),
the hearts were
excised, cannulated and perfused as described above but without
the ischaemic period, to observe how the mechanical and flow
measurements changed as a function of time. Pressure and flow
were recorded in these hearts at the same time points as in the
hearts that experienced ischaemia–reperfusion.
Infarct size was measured using methods similar to those
previously described.
8,9
After the reperfusion period, the snare
was re-tightened around the left anterior descending coronary
artery in six hearts from each group, and 100
µ
l of 0.05%
Evans blue solution was injected into the aortic cannula and
perfused through the heart for 3 min. Then the heart was
sliced transversely from base to apex into four slices of equal
width. Each slice was immersed in phosphate buffer and was
photographed with a digital camera.
After both sides of each slice were photographed, each
slice was placed in 100 mM phosphate buffer with 0.1%
triphenyltetrazolium chloride and incubated for 10 min at
37
°C. After incubation, each side of every slice was again
photographed and the slices were weighed. Heart weight was
obtained by summation of the slice weights for each heart.
To avoid experimenter bias, images of the slices were analysed
in a single-blind manner by Scion Image 4.0 software. Total slice
area (TA), zone at risk (ZAR: the area of each slice that did not
turn blue after perfusion with the solution containing Evans blue
dye) and infarct area (IA: the portion of the ZAR that did not
turn red in response to triphenyltetrazolium chloride incubation
and remained white) were measured. ZAR and IA were obtained
from each side of a single slice, and the mean of both sides was
used as the representative ZAR and IA for that slice. Finally, IA
was expressed as a fraction of all ZAR by taking the sum of all
infarcts and was reported as a percentage.
The left ventricle was immersion-fixed in 10% neutral
formalin and embedded in paraffin wax (
n
=
5
for controls,
n
=
6
for exercised rats). Serial sections of 4-
µ
m thicknesses
were prepared. Apoptosis was evaluated via the terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labelling
(
TUNEL) method with the use of an
in situ
Cell Death
Detection Kit, POD (1684817, Roche, Germany) according to
manufacturer’s instructions, with some modifications.
10
Briefly, the tissue sections were dewaxed and rehydrated by
heating at 60°C, followed by washing in xylene and rehydration
through a graded series of ethanol and double distillated water.
Then the sections were incubated for 30 min at 21–37°C with
proteinase K working solution (20
µ
g/ml in 10 mM Tris-Cl,
pH 7.6). The sections were rinsed with PBS and incubated with
the TUNEL reaction mixture for 1 h at 37°C in a humidified
chamber.
As a positive control, sections were treated with DNase I
(1
mg/ml, Sigma) for 10 min to introduce nicks in the genomic
DNA. After converter peroxidase (POD) was added, the sections
were incubated for 30 min at 37°C in a humidified chamber.
Then the 3,3-diaminobenzidine substrate was added for the
visualisation of nuclei with DNA nick-end labelling. The sections
were counter-stained with toluidine blue to show normal nuclei.
The percentage of myocytes with DNA nick-end labelling
was analysed by counting the cells exhibiting brown nuclei at
×
40
magnification in five randomly chosen fields (1 mm
2
)
in
triplicate plates. The number of TUNEL-positive cardiomyocytes
was counted by double-blinded observation.
Statistical analysis
All statistical comparisons were made using SPSS 16.0 software
(
Chicago, IL) and were expressed as means
±
SD. Work
performed, pressures and flow data were analysed using repeated
measures ANOVA. When a significant
p
-
value was obtained, a
post hoc
Bonferroni test was used to determine the differences
between the groups. Between-group comparisons of data of heart
rate, infarct size, body weight, heart weight and apoptosis rate
were made using the Student’s
t
-
test. A
p
-
value of
<
0.05
was
considered statistically significant.
Results
Morphological data from the EXT and Sed rats are presented
in Table 1. The rats in the EXT group had significantly lower
body weights and higher heart weights than the Sed group (
p
