Download METHODS Experimental animals Experiments were performed in 12

METHODS
Experimental animals
Experiments were performed in 12-week-old male Sprague-Dawley rats
weighing 250-280 g (SLAX Laboratories, Shanghai, China). Animals were kept in
a climate-controlled room maintained at 24°C with a 12:12-h light-dark cycle with
access to food and water ad libitum. All experimental procedures were approved
by the Animal Care and Use Committee, and were conducted under the
guidelines for the care and use of laboratory animals as established by the
Shanghai Institute of Hypertension (permit number: 2008125). DSH rats were
generated as previously described. 1 Briefly, DSH animals were anesthetized and
uninephrectomized. After 7 days of recovery, rats were administered DOCA
(Sigma, D7000) subcutaneously at a dose of 12.5 mg/rat/week for 5 weeks and
1% NaCl was added to drinking water. The sham group received sham operation
without kidney removal and given normal water. For the apocynin treatment
groups, three concentrations of apocynin (0.5 mM, 1.0 mM, and 1.5 mM, Sigma)
were administered orally in a tap water solution.
Measurement of blood pressure and PWV
Systolic blood pressure (SBP) was measured every 2 weeks after surgery
using a non-invasive computerized tail cuff system (Blood Pressure Analysis
System BP- 98AW monitor, Softron Co., Ltd., Tokyo, Japan). Six weeks after
surgery, animals were anesthetized and placed on a heated water pad at 37°C to
maintain body temperature for measurement of central blood pressure and PWV.
Two 1.4 Fr. Millar Mikro-tip pressure transducers (Millar, Houston, TX) were
implanted: one in the aortic arch via the left carotid artery, and one in the
abdominal aorta just proximal to the iliac bifurcation via the left femoral artery.
Pressure waves from the two Millar transducers were simultaneously imported to
a recording system (Shanghai Alcott Biotech, Shanghai, China) and displayed on
a data acquisition system (PowerLab, 16/s, AD Instruments, Australia) at a
sampling rate of 1000 Hz. After the experiment was completed, the animal was
euthanized with chloral hydrate (200 mg/kg), and the aorta exposed. A silk thread
was placed along the contour of the aorta and marked at the tips of the two
pressure transducers. The distance between the two marks, (L) was then
measured. PWV was calculated by dividing the distance (L) by the propagation
time (t). The propagation time was determined using the manual foot-to-foot
method described previously, in which the propagation time for the pulse wave
moving from the aortic arch to the abdominal aorta is equal to the time delay
between the upstrokes (foot) of each pressure wave front.
2
At least 10 normal
consecutive cardiac cycles were measured individually and averaged.
Ultrasound biomicroscopy
Ultrasound biomicroscopy (UBM) was performed, as previously described 3,
at the end of studies in all rats. Briefly, rats were anesthetized with chloral hydrate
(30 mg/kg, Butler, Union City, CA), and a UBM system (Vevo 770, Visualsonics,
Toronto, Canada) equipped with a 40 MHz mechanical transducer used for all
examinations. Intima-media thickness (IMT) of the left common carotid artery was
measured using a long axis view. IMT was measured from the leading echo edge
(intima) closest to the blood stream to the next leading echo edge representing
the layer of adventitia as described earlier. All measurements were repeated three
times at the same site. All images were analyzed by another operator blinded to
the identities of the animals.
Aortic wall structure and composition
After delipidation and recording of dry weight, the thoracic aorta was
subjected to hydrolysis in 6 M HCl for 16 h, and the resulting hydrolyzate was
used in assays for hydroxyproline, desmosine, and isodesmosine content. Briefly,
hydroxyproline content was determined by the chloramine T and the
paradimethylaminobenzaldehyde method.
4
Collagen content was calculated as
hydroxyproline content × 7.46. The content of the elastin-specific cross-linking
amino acids desmosine and isodesmosine was determined by capillary zone
electrophoresis and ultraviolet detection. Elastin content was calculated as
(desmosine + isodesmosine) × 200. Elastin (or collagen) content is expressed as
mg/cm of the aorta.
Histological examination and immunocytochemistry
Morphometric studies were performed in the thoracic aorta. Sections (5 μm)
taken from the descending aorta were stained with hematoxylin and eosin (HE) to
estimate the thickness of the arterial wall using a valid method as previously
described.
5
In addition, Masson trichrome staining of fixed samples was used to
show collagen in the artery, and Weigert iron hematoxylin was used to detect
elastin fibers. For immunocytochemistry, sections were blocked with 1% bovine
serum albumin and incubated with p47phox primary antibody (1:100, sc-376614,
Santa
Cruz
Biotechnology,
CA),
then
incubated
with
horseradish
peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, CA,
USA). Every parameter was quantified using computer-assisted morphometry
(Leica Qwin, Heidelberg, Germany) and performed by one investigator blinded to
the treatment.
Evaluation of ROS in aorta by dihydroethidium and lucigenin
Dihydroethidium oxidative fluorescence dye was used to evaluate the in situ
production of ROS, as previously described.
6
20-μm-thick
glass
sections
were
placed
on
Unfixed frozen samples cut into
slides,
augmented
with
dihydroethidium (10 mmol/l), and then capped with coverslips. The slides were
incubated in a light-protected humidified chamber at 37℃ for 30 min. The
dihydroethidium image was obtained by a laser scanning confocal imaging
system (MRC-1024) equipped with a 585 nm long-pass filter. 7
Superoxide production from aortic tissue was measured using lucigenin
chemiluminescence according to a method modified from Munzel et al. Briefly,
specimens of thoracic aorta were incubated in vials containing 5 μM lucigenin
(Sigma).
8
The light reaction between superoxide and lucigenin was detected
using a chemiluminescence reader (BLR-201, Aloka Co., Tokyo, Japan). The
chemiluminescent signal was expressed as average counts/min/mg dry tissue
over a 20-min interval. 7
Statistical analysis
Results are expressed as mean ± SEM. Statistical comparisons between
groups were made using one-way ANOVA. Statistical differences were
considered significant at P<0.05.
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