Methods experimental animals



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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 Unfixed frozen samples cut into 20-μm-thick sections were placed on glass 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.

References:

1. Ammarguellat F, Larouche I, Schiffrin EL. Myocardial fibrosis in DOCA-salt hypertensive rats: effect of endothelin ET(A) receptor antagonism. Circulation. Jan 16 2001;103(2):319-324.



2. Fitch RM, Vergona R, Sullivan ME, Wang YX. Nitric oxide synthase inhibition increases aortic stiffness measured by pulse wave velocity in rats. Cardiovasc Res. Aug 1 2001;51(2):351-358.

3. Wu DJ, Xu JZ, Wu YJ, Jean-Charles L, Xiao B, Gao PJ, Zhu DL. Effects of fasudil on early atherosclerotic plaque formation and established lesion progression in apolipoprotein E-knockout mice. Atherosclerosis. Nov 2009;207(1):68-73.

4. Marque V, Kieffer P, Atkinson J, Lartaud-Idjouadiene I. Elastic properties and composition of the aortic wall in old spontaneously hypertensive rats. Hypertension. Sep 1999;34(3):415-422.

5. Carr-White GS, Afoke A, Birks EJ, Hughes S, O'Halloran A, Glennen S, Edwards S, Eastwood M, Yacoub MH. Aortic root characteristics of human pulmonary autografts. Circulation. Nov 7 2000;102(19 Suppl 3):III15-21.

6. Szocs K, Lassegue B, Sorescu D, Hilenski LL, Valppu L, Couse TL, Wilcox JN, Quinn MT, Lambeth JD, Griendling KK. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury. Arterioscler Thromb Vasc Biol. Jan 2002;22(1):21-27.

7. Wind S, Beuerlein K, Armitage ME, Taye A, Kumar AH, Janowitz D, Neff C, Shah AM, Wingler K, Schmidt HH. Oxidative stress and endothelial dysfunction in aortas of aged spontaneously hypertensive rats by NOX1/2 is reversed by NADPH oxidase inhibition. Hypertension. Sep 2010;56(3):490-497.

8. Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J Clin Invest. Jan 1995;95(1):187-194.

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