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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 26, No 6, November/December 2015
244
AFRICA
lungs, kidneys and adrenal glands were also excised and weighed.
Haemodynamic parameters, ECG and temperature
measurements were recorded onto the computer using the
PowerLab 4/30 data-acquisition system and the LabChart
7.3.5 software (ADInstruments, Bella Vista, Australia).
Haemodynamic and ECG data were analysed using LabChart
7 Pro BP and ECG analysis modules (ADInstruments, Bella
Vista, Australia). The ECG analysis module was preset to the
rat waveform and Bazett’s formula (QTc
=
QT/√RR) was used to
calculate the QT interval, corrected for heart rate (QTc).
Infarct size quantification
A series of 2-mm-thick ventricular transverse slices of the
frozen heart were cut from apex to base and thawed for
2,3,5-triphenyltetrazolium chloride (TTC) staining. The slices
were incubated in a solution of 1% TTC in phosphate buffer
(pH 7.4) at 37°C for 20 minutes and agitated periodically while
protected from light. The slices were then washed with the buffer
and fixed with 10% formalin to enhance contrast and stored in
the dark at room temperature for 24 hours.
The slices were placed between two glass slides and scanned on
both sides using a flatbed scanner. The ventricular infarct size was
measured as an average of the TTC-negative areas on the slices
from each heart using ImageJ software (Version 1.44p, NIH,
USA) and was expressed relative to the total ventricular area.
Lipid peroxidation assays
Markers of oxidative stress, measured as by-products of lipid
peroxidation, namely conjugated dienes (CD) and thiobarbituric
acid-reactive substances (TBARS), were quantified in the plasma
using spectrophotometric assays. CD assays were carried out
using the methods described by Esterbauer.
36
Briefly, 100
µ
l of plasma was added to 405
µ
l chloroform:
methanol (2:1). After centrifugation at 6 000
g
for 15 minutes,
the top aqueous layer was removed and the organic layer was
isolated and dried under nitrogen. Cyclohexane (250
µ
l) was
added to solubise the dry organic residue and the absorbance
was read at 234 nm on a spectrophotometer (Spectramax Plus
384, Molecular Devices and Labotec, Johannesburg, South
Africa) using Softmax Pro (Version 4.4) software. A molar
extinction coefficient of 2.95
×
10
4
/M/cm was used.
TBARS were measured using the method described by
Jentzsch
et al.
37
Briefly, 6.25
µ
l of 4mMbutylated hydroxytoluene/
ethanol and 50
µ
l of 0.2 M ortho-phosphoric acid were added
to 50
µ
l of plasma samples and vortexed. TBA reagent (6.25
µ
l), dissolved in 0.1 M NaOH, was added and the mixture was
centrifuged at 3 000
g
for two minutes to collect small volumes at
the bottom of the Eppendorf tube. The volumes were heated at
90°C for 45 minutes, placed on ice for two minutes and then left
at room temperature for five minutes before
n
-butanol (500
µ
l)
was added. Phase separation was enhanced by the addition of 50
µ
l of saturated NaCl.
The samples were vortexed and centrifuged at 12 000
g
for
two minutes and 300
µ
l of the top butanol phase was transferred
into wells and read at 532 nm on the spectrophotometer. A
molar extinction coefficient of 1.54
×
10
5
/M/cm was used. The
measurements of CD and TBARS were performed in triplicate
and the mean value was taken as the final result.
Chemicals and reagents
ISO and MgSO
4
were each dissolved in physiological saline.
The TTC buffer was made up of one part 0.1 M monosodium
phosphate (NaH
2
PO
4
) and four parts 0.1 M disodium phosphate
(Na
2
HPO
2
). Sodium pentobarbitone was purchased from Kyron
Laboratories, Johannesburg, South Africa. All other drugs and
chemicals were obtained from Sigma, Johannesburg, South
Africa.
Statistical analysis
Data are expressed as mean
±
standard error of the mean
(SEM), with
n
indicating the number of rats studied under
each condition. Statistical analysis was conducted using Prism
5 (GraphPad, USA). A box-plot analysis was conducted to
exclude outliers. The distribution of data was checked using
the Kolmogorov–Smirnov, D’Agostino and Pearson, and the
Shapiro–Wilk normality tests. Differences among multiple
groups were evaluated using analysis of variance (ANOVA),
followed by a Tukey
post hoc
test. For data not normally
distributed and for normally distributed data that failed the
Bartlett’s test, a Kruskal–Wallis test was conducted followed by
a Dunns
post hoc
test;
p
≤ 0.05 was taken as the threshold for
statistical significance.
Results
Effects of Mg
2+
on ISO-induced infarct size
Fig. 1B shows typical pictures of TTC-stained ventricular slices
cut from four different hearts. Whitish-looking, TTC-negative
areas were more prominent in the ISO-treated hearts, indicating
the presence of irreversible infarction. The infarcted areas were
patchy and more diffusely located on the myocardium, consistent
with a global type of infarction compared to well-demarcated
infarcts due to coronary artery ligation.
In contrast to the effects of ISO, the control and Mg
2+
-only
treated hearts appeared mostly red (TTC positive), suggesting
tissue viability. The quantification of infarct size in whole
ventricles is summarised in Fig. 1C, which confirms that ISO
induced significant increases in infarct size (12.79
±
5.97 vs 6.84
±
1.54% in the controls;
p
<
0.05).
Pre-treatment with Mg
2+
did not prevent or enhance the
ISO-induced infarction compared with ISO-treated rats (infarct
size: 11.67
±
6.63%;
p
>
0.05). Treatment with Mg
2+
alone did
not cause injury to the myocardium compared with the controls
(infarct size: 6.94
±
2.1%;
p
>
0.05).
Effects of ISO and Mg
2+
on body and organ weights
To examine the systemic and organ-specific effects of the various
treatments, the body weight, heart weight and the weights of the
other organs were quantified (Table 1). ISO caused a significant
increase in the heart weight:body weight ratio compared with
the controls (
p
<
0.001). Pre-treatment with Mg
2+
did not prevent
the ISO-induced increase in heart weight:body weight ratio.
Compared with the control, ISO also caused a loss in body
weight (
p
<
0.05), and pre-treatment with Mg
2+
did not rectify
this weight loss.
Compared with ISO‑treated rats, Mg
2+
co-treatment also
did not affect the weights or the gross appearances of the liver,