Cardiovascular Journal of Africa: Vol 21 No 5 (September/October 2010) - page 16

CARDIOVASCULAR JOURNAL OF AFRICA • Vol 21, No 5, September/October 2010 258 AFRICA
vein and a thoracotomy was immediately performed under sterile
conditions. After sampling, all rabbits were sacrificed using an
overdose of pentobarbital sodium (Euthanase ®) via the ear vein.
The whole aorta was separated, clamped, and then 2 cm was
harvested at the lower end of the aortic arch. The connective
tissue of the outer membrane was removed and part of the aortic
tissue was fixed in 4% paraformaldehyde phosphate buffer for
hematoxylin and eosin (HE) staining. Another 3 cm of aorta was
fixed in 10% paraformaldehyde phosphate buffer for oil red O
staining. Conventional paraffin sections were made and HE stain-
ing for light microscopy was done. The remaining fresh tissue
samples were kept for the detection of endogenous CO content,
NOS activity as well as mRNA expression of HO-1 and ET-1.
After fixation in 10% neutral formalin buffer and conven-
tional dehydration, the aorta was stained with oil red O staining
(plaque shows red). The plaque area and aortic tunica–intima
area were measured with Leica Qwin image analysis software,
and the percentage of the plaque area relative to the aortic
tunica–intima area was calculated.
Assays
Serum lipid levels [total cholesterol (TC), triglycerides (TG),
high-density lipoprotein cholesterol (HDL-C) and low-density
lipoprotein cholesterol (LDL-C)] were determined with an
enzymatic kit from the Shanghai Kehua Bio-engineering Co,
Ltd (Shanghai, China). The oxidised LDL (ox-LDL) level was
determined with the double antibody sandwich method using a
kit provided by Shanghai Rongsheng biological reagents factory
(Shanghai, China).
A homogenate of blood plasma and aortic tissue was prepared
and ET-1 was detected using a radioimmunoassay kit from the
Beijing Huaying Biotechnology Co (Beijing, China).
According to the literature,2 500 μl of serum was centrifuged
at 10 000 rpm at 4°C for 15 min. A 100-μl volume of the super-
natant was removed and 100 μl Griess reagent and 100 μl of 4
mol/l hydrochloric acid were added. The mixture was incubated
at room temperature for 10 min and the optical density was
read at 570 nm with a microplate reader. A standard curve was
prepared using nitrite, and from this curve, serum NO levels
were determined.
Detection of aortic NO and CO contents
The aortic smooth muscle was shaved into 3-mm pieces, rinsed
with 0.01 mol/l phosphate buffer (pH 7.4) and the sample was
added to 2 ml of phosphate buffer for homogenate preparation.
The nitrite content (NO2
–) in the aortic homogenate, representing
NO production was determined with the Greiss method.5 CO
production was determined according to the method of Morita
et al.6
The smooth muscle of the aorta thoracalis was shaved into
3-mm pieces, placed into 2 ml DMEM medium (15 mg/ml)
containing 50 μl/l of haemoglobin (Hb) and incubated at 37°C
in a 95% O2 and 5% CO2 atmosphere for 2 h.
Relative amounts of CO released into the medium were meas-
ured by adding Hb for the last hour of incubation and quantify-
ing carboxyhaemoglobin (HbCO) levels spectrophotometrically
using a CO-oximeter (Coming 270; Ciba Coming Diagnostics,
Medfield, MA). HbCO gives maximal absorbance at a wave-
length of 569 nm and is calculated as percentage total Hb. The
actual amount of Hb was determined from a standard curve
using exogenous CO and varying amounts of Hb over a range of
concentrations where the absorbance was linear.
Aortic NOS activity was detected by calculating the concen-
tration of 3H-citrulline converted from 3H-arginine.7
Protein expression of aortic HO-1 and ET-1 was detected by
immunohistochemistry. Goat anti-rabbit HO-1 and ET-1 poly-
clonal antibodies were purchased from Santa Cruz, USA. An
SP-9001 immunohistochemical staining kit was purchased from
Zhongshan Goldenbridge Biotechnology Co, Ltd. The percent-
age area of positive cells was calculated with Leica Qwin image
analysis software. Brown vessel wall cells were considered posi-
tive for HO-1 or ET-1.
mRNA expression of aortic HO-1 and ET-1
The mRNA expression of aortic HO-1 and ET-1 was determined
by RT-PCR. Total RNA was extracted from the aorta using Trizol
reagent (Roche, Germany). After determination of purity and
concentration, cDNA was generated from total RNA under the
following conditions: 50°C for 30 min, followed by 99°C for 5
min and 5°C for 5 min. Then the PCR reaction was performed
(RT-PCR kit was purchased from TaKaRa, Japan).
Rabbit HO-1 and ET-1 primers and β -actin gene sequence
were obtained according to GenBank(http://www.ncbi.nlm.nih.
gov/). Primers were designed using DNASIS software (Hitachi
Software Engineering Co, Ltd) and synthesised by Shanghai Boya
BiotechnologyCo.TheprimersforHO-1cDNAamplificationwere
as follows: sense: 5′-CAGGTGACTGCCGAGGGTTTTA-3′;
antisense: 5′-GGAAGTAGA GCGGGGCGTAG-3′ . The size of
the target fragment was 118bp.
The primers for ET-1 cDNA amplification were as follows:
sense: 5′-AAGATCCCAGCCAGC ATGGAGAGCG-3′; anti-
sense: 5′-CGTTGCTCCTGCTCCTCC TTGATGG-3′. The size
of target fragment was 543bp.
The primers for β-actin cDNA amplification were as follows:
sense: 5′-CCCATCTACGAGGGCTACGC -3′ ; antisense:
5′ -CAGGAAGGA GG GCTGGAACA-3′. The size of target
fragment was 312bp. β-actin served as the inner control.
The PCR conditions were as follows: HO-1: pre-denaturation
at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at
55°C for 30 s, extension at 72°C for 30 s with 32 cycles, followed
by a final extension at 72°C for 5 min. ET-1: pre-denaturation
at 94°C for 3 min; denaturation at 94°C for 1 min, annealing
at 60°C for min, extension at 72°C for 3 min with 30 cycles,
followed by a final extension at 72°C for 7 min.
A 10-µl volume of the PCR product was separated with
2% agarose gel. A densitometric scan was performed with the
ChemiImagerTM 5500 gel imaging machine (Alpha Innotech,
USA). The mRNA expression was presented as the integral opti-
cal density ratio of HO-1/β-actin and ET-1/β-actin.
Statistical analysis
All data were expressed as mean ± SD. One-way ANOVA was
used for comparison among multiple groups.
Results
At the end of the experiment, compared to the control group,
serum TC, TG, LDL-C and ox-LDL levels in each high-choles-