Rat heart irradiation
All procedures in this study were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. A total of 72 male Sprague-Dawley rats (180–200 g) were obtained from Harlan (Indianapolis, IN). All animals were maintained in our Division of Laboratory Medicine on a 12:12 light-to-dark cycle with free access to food and water.
After two weeks of acclimatization, rats were anesthetized with 2.5% isoflurane and irradiated with a Seifert Isovolt 320 X-ray machine (Seifert X-Ray Corporation, Fairview Village, PA) operated at 250 kV and 15 mA with 3 mm aluminum and 1.85 mm copper added filtration, at a dose rate of 1.17 Gy/min. Dosimetry was performed using an electrometer (Model 206, CNMC, Nashville, TN, calibration factor 1,000 nC/reading) and a cylindrical ionization chamber with volume 0.13 mL (Model N31005, PTW, Hicksville, NY, calibration factor 28.98 R/nC). Calibration of the instruments was performed by the University of Wisconsin Accredited Dosimetry Calibration Laboratory (Madison, WI). The measured Half-Value Layer of the beam used for these experiments was 2.8 mm Cu.
A daily dose of 9 Gy or 0 Gy was administered for 5 consecutive days. This radiation dose corresponds to approximately 21 Gy single dose and was chosen to produce moderate to severe RIHD (14
). Radiation was delivered locally on the heart using parallel opposed fields (anterior-posterior 1:1) with a 19 mm diameter, while the rest of the animal was shielded with lead plates.
Pentoxifylline and α-tocopherol administration
Rats were treated with a combination of PTX (100 mg/kg body weight/day) (Sigma-Aldrich, St Louis MO) and α-tocopherol (in the form of dl-α-tocopheryl acetate, 20 IU/kg body weight/day). These compounds were added to the standard rodent chow TD8640 by Harlan-Teklad. Rats were randomly divided into six treatment groups: 1) sham-irradiation and regular chow; 2) sham-irradiation and customized chow administered from one week before until six months after sham-irradiation; 3) sham-irradiation and customized chow from three months after until six months after sham-irradiation; 4) 5×9 Gy and regular chow; 5) 5×9 Gy and customized chow from one week before until six months after irradiation (“early treatment”); 6) 5×9 Gy and customized chow from three months after until six months after irradiation (“late treatment”).
At six months after irradiation, cardiac function was assessed with echocardiography using the Vevo 770 high-resolution in vivo micro imaging system (VisualSonics, Toronto, Canada). The VisualSonics RMV 716 Scanhead with center frequency 17.5 MHz, frequency band 11.5–23.5 MHz and focal length 17.5 mm was used. Rats were anesthetized with 3% isoflurane and maintained at 1.8% isoflurane delivered through a nose cone during the procedure. All hair was removed from the chest with clippers followed by Nair lotion hair remover (Church and Dwight, Princeton, NJ). Rats were positioned on a heated platform that records both ECG and breathing pattern. The chest was covered with ultrasound transmission gel (Aquasonic, Parker Laboratories, Fairfield, NJ) and the heart was visualized at the parasternal short axis at the mid left ventricular level, verified by the presence of prominent papillary muscles. M-mode recordings were stored for determination of echocardiographic parameters with the Vevo cardiac software package. Care was taken to avoid fragments of M-mode recordings taken during inhalation.
Langendorff perfused rat heart preparation
Langendorff studies were performed as described before (14
). Hearts were isolated from rats in each of the six treatment groups and immediately perfused via the aorta with an oxygenated Krebs-Henseleit solution (37°C) at a flow rate of 8 ml/g heart/min. Both atria were removed and the ventricles were paced with platinum contact electrodes positioned on the interventricular septum to obtain a heart rate of 250 beats/min. A fluid-filled balloon catheter was placed in the left ventricle and left ventricular diastolic pressure, peak systolic pressure, +dP/dtmax
(rate of contraction), and −dP/dtmax
(rate of relaxation) were measured at various preload balloon volumes between 80 μl and 300 μl (a range that elicited maximum contractility in all preparations). Coronary pressure was monitored continuously with a Statham pressure transducer. All data were digitized and analyzed with the use of acquisition and analysis software (CODAS
; DataQ Instruments, Akron, Ohio, USA). After Langendorff studies the hearts were weighed and processed for histology and immunohistochemistry.
According to a method of Radke et al (15
), pressure data obtained from Langendorff preparations were converted into wall stress to correct for possible changes in left ventricular geometry. Left ventricular wall stress was calculated as: Left ventricular pressure/[(left ventricular wall volume/balloon volume +1)2/3 −1]. Left ventricular wall volume was calculated as: heart weight/1.05, assuming that heart weight reflects mainly left ventricular weight.
Histology and immunohistochemistry
Hearts were fixed in methanol Carnoy’s solution (60% methanol, 30% chloroform, 10% acetic acid) and embedded in paraffin to obtain 5 μm cross sections.
For determination of mast cell numbers, sections were stained with 0.5% Toluidine Blue (14
). The total mast cell number in 10 ocular fields with a 10× objective was determined. This area covered almost the entire left ventricular area. Mast cell density was calculated as number of mast cells per area (mm2
Immunohistochemistry was performed with methods established and optimized in our laboratory (16
). Sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked with 1% H2
in methanol. Non-specific antibody binding was reduced by 10% normal rabbit or normal goat serum (Vector Laboratories, Burlingame, CA) in TBS containing 3% dry powdered milk and 0.2% BSA. Sections were incubated with one of the following primary antibodies: goat anti-collagen type I, goat anti-collagen type III (both Southern Biotechnology Associates, Birmingham, AL), each at 1:100, or pan-specific rabbit anti-transforming growth factor beta (TGF-β, R&D, Minneapolis, MN) at 1:300. After incubation with biotinylated goat anti-rabbit IgG or rabbit anti-goat IgG (Vector Laboratories, Burlingame, CA), both at 1:400, sections were incubated with avidin-biotin-peroxidase complex (Vector Laboratories) and visualized with 0.5 mg/ml 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and 0.003% H2
Sections were analyzed with an Axioskop transmitted light microscope (Carl Zeiss, Thornwood, NY) equipped with a color chilled 3CCD camera (Hamamatsu, Hamamatsu City, Japan). Quantitative assessment of areas immunoreactive for collagen type I, collagen type III or extracellular matrix-associated TGF-β was performed with computerized image analysis (Image-Pro Plus, Media Cybernetics, Silver Spring, MD) as validated before (18
). For each heart the average area of 20 fields with a 40× objective was calculated.
Hematoxylin-eosin stained sections were used for assessment of total left ventricular area of myocardial degeneration (myocardial necrosis accompanied by inflammation) with computerized image analysis. Each heart was then given a score for area of myocardial degeneration, as follows: score 0 (no degeneration), score 1 (total area of degeneration 0 –20,000 μm2), score 2 (20,000–40,000 μm2), or score 3 (40,000–60,000 μm2).
Cardiac fibroblast isolation and staining
Hearts were isolated from rats, immediately rinsed and minced in sterile saline, and incubated in HBSS containing 75 U/ml collagenase type II (Worthington Biochemical, Lakewood, NJ) in a trypsinizing flask (Wheaton Science Products, Millville, NJ) under continuous stirring with a star-headed stir-bar (Nalgene Labware, Rochester, NY) at 37°C. The first supernatant obtained after 10 min of incubation was discarded. Subsequent supernatants were collected every 10 min for a total of 40 min and immediately centrifuged at 1,300 g for 4 min. The pellets were resuspended in DMEM containing 10% FBS and antibiotics (Invitrogen, Carlsbad, CA), combined and maintained in a culture flask at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After ten days of incubation colonies of cells were detected with the cellular morphology of fibroblasts. Cells were trypsinized and plated on cover slips. After three days of incubation, cover slips were rinsed with PBS and fixed in 4% methanol free formaldehyde (Pierce, Rockford, IL). After cell permeabilization with 0.1% Triton X-100 for 10 min, cover slips were incubated with FITC-phalloidin (Invitrogen) at 5 U/ml for 20 min to visualize actin filaments. Cells were counterstained with a DAPI nucleus stain and analyzed with an Axioskop 40 fluorescence microscope equipped with an Axiocam MRm camera (Carl Zeiss).
RNA isolation and real-time PCR
Hearts were isolated from three rats in each of the six treatment groups, snap-frozen in liquid nitrogen and subsequently stored at −80°C. Frozen tissue samples from the left ventricle were homogenized in Ultraspec™ RNA reagent (Biotecx Laboratories, Houston, TX), according to the manufacturer’s instructions. After treatment with RQ-DNAse I (Promega, Madison, WI) at 37°C for 30 min, cDNA was synthesized using the High Capacity cDNA Archive Kit™ (Applied Biosystems, Foster City, CA). Steady-state mRNA levels were measured with real-time quantitative PCR (TaqMan™) using the ABI Prism 7700 Sequence Detection System, TaqMan mastermix, TaqMan polymerase, and the pre-designed TaqMan Gene Expression Assay™ for rat TGF-β1 (Rn00572010_m1) or rat connective tissue growth factor (CTGF, Rn00573960_q1) (all Applied Biosystems, Foster City, CA).
Data were evaluated with the software package NCSS 2007 (NCSS, Kaysville, UT). Langendorff data were tested with repeated measures ANOVA. Frequencies of scores for myocardial degeneration were tested with a Chi-Square test. All other data were tested with two-way ANOVA, followed by Newman-Keuls individual comparisons. The criterion for significance was a p<0.05. Data are reported as average ± standard error of the mean (SEM).