Download Online data supplement Animals – study design

April 3, 2018 | Author: Anonymous | Category: , Science, Health Science, Cardiology
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Animals – study design Male Wistar rats were used in this study (n=63 Harlan, Horst, the Netherlands). Rats were housed in pairs under controlled conditions (22C 12h/12h day/night cycle). Food and water were available ad libitum. PAH was induced in 54 rats by a single injection of MCT dissolved in sterile saline (8 mg/ml in 0.9% NaCl s.c.) at pH 7.4. Control rats (n=9) received saline s.c.. After 14 days, PAH rats were randomly assigned to daily oral treatment with Bosentan (Actelion Pharmaceuticals, Allschwil, Switzerland) (300 mg/kg/day; n=9), Sildenafil (Pfizer Inc. USA10 mg/kg/day; n=9), Fasudil (Mercachem 100 mg/kg/day; n=9), or the combinations Fasudil+Bosentan (same dosages, n=9) or Fasudil+Sildenafil (same dosages, n=9). At day 28, rats were euthanized by excision of the hearts under isoflurane anesthesia (>3.5 %). All experiments were approved by the Institutional Animal Care and Use Committee conform the Helsinki convention for the use and care of animals.

Echocardiography Echocardiography was performed using an Aloka SSD4000 ultrasonographic system (Biomedic, Almere, The Netherlands) equipped with a 13.5 MHz transducer. By means of Doppler imaging of the aorta, heart rate (HR), and stroke volume (SV) were determined(1-3). Subsequently cardiac output (CO) was calculated: CO= SV*HR/1000. In addition, in Mmodus RV end-systolic and end-diastolic diameters (RV EDD and ESD resp.), were measured at mid papillary level as measures for RV dilatation. RV Fractional shortening (FS) was then calculated: FS = ((EDD – ESD) / EDD) x 100%(4;5). In addition, Tricuspid Annular Plane Systolic Excursion (TAPSE) was measured, by measuring the movement form the tricuspid

annulus to the apex, as previously described(1;2). Analyses were performed off-line (ImageArena 2.9.1, TomTec Imaging Systems, Unterschleissheim / Munich, Germany).

Hemodynamic measurements RV systolic pressure (RVSP) was measured using a Millar pressure catheter (Millar, Texas, USA) by direct insertion through the RV wall, after thoracotomy(4). Prior to the procedure rats were intubated with a 16-gauge plastic tube and attached to a mechanical microventilator (UNO, Zevenaar, the Netherlands), assuring a respiratory rate of 75/min with an IPPV/PEEP of 15-5 mmHg. RVSP was measured for 20 seconds and averaged. During the procedure, body temperature was monitored continuously and maintained at 37C with a heating pad. RVSP measurements were discarded when systemic blood pressure decreased more than 15%.

PVR

was

estimated

by

Poiseuille’s

law:

[PVR]≈[mean

PAP]/[cardiac

output]≈(0.61×[systolic RV pressure]+2 mmHg)/[cardiac output] (2;6).

Quantitative histochemistry To study the RV’s capacity to adapt to PAH in more detail, an extensive histochemical analysis was performed on cardiac tissue. Animals were euthanized by excision of the hearts, and hearts were perfused with Tyrode solution (120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2.0 mM Na2HPO4, 27 mM NaHCO3, 1 mM CaCl2, 10 mM glucose and 20 mM 2,3butanedione monoxime, equilibrated with 95% O2 and 5% CO2; pH 7.3–7.4 at 10°C) to remove blood and prevent contraction(7). Hearts were then frozen in liquid nitrogen. Cryosections (5µm) were air-dried for 10 minutes and stained. To determine the degree of individual cardiomyocyte hypertrophy, sections were fixed for 10 min in 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, and stained with Hematoxylin and Eosine. The cross-

sectional area of 40 randomly chosen cardiomyocytes in the RV was measured. In order to be able to compare CSA from different hearts, we normalized CSA to a sarcomere length of 2 μm, by randomly measuring 5 stretches of sarcomeres in both ventricles. In addition, adjacent sections were incubated for succinate dehydrogenase activity (SDHact) as described by des Tombe et al(7) and measured in 40 cardiomyocytes of the RV at 660 nm, as a determinant for the RV’s mitochondrial capacity. We also investigated the RV’s capillary density; sections were stained for 60 min by using primary CD31- (1:35; sc-1506-R, Santa Cruz Biotechnology, Santa Cruz CA) followed by appropriate secondary antibody staining as well as WGA (glycocalyx) and DAPI (nuclei) counterstaining. Image acquisition was performed on a Marianas digital imaging microscopy workstation (Intelligent Imaging Innovations (3i), Denver CO). SlideBook imaging analysis software (SlideBook 4.2, 3i) was used to quantify the number of capillaries per cardiomyocyte in at least three randomly chosen areas per ventricle only selecting transversally sectioned cardiomyocytes(4;7). In addition, sections were myoglobin concentration, which were quantified in 40 random RV cardiomyocytes(7;8). The presence of Cytochrome c release was determined as an indicator of mitochondrial integrity(9).

Results For echocardiographic and haemodynamic results see main manuscript. Body mass at day 28 differed significantly between groups between groups (Fig S1; One way ANOVA P
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