Male Sprague - Dawly rats, acclimatized in a quarantine room for 2 weeks, and all experiments were approved by the Animal Care and Research Committee of the University of Colorado Denver, and this investigation conforms to the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 1996). The animals divided in to 4 groups, eight rats in each group, control groups (normal saline injected I.P) and group treated with 20 mg/kg doxorubicin in a single injection intraperitoneal i.p. and other two groups one received vitamin E (100 mg/kg)  orally by N/G tube for one week followed by doxorubicin (20 mg/kg) I.P as single dose, and the other one received telmisartan (1 mg/kg)  orally by N/G tube for one week followed by doxorubicin (20 mg/kg) I.P as single dose.
Left ventricular function analysis
Pressure-volume loop and hemodynamic analysis was planned for all animals as described previously by N.Yousif,2011 , in briefly the animals anesthetized with ketamin in dose of 50 mg/kg injected intraperitoneal and neck was opened longitudinally and right common carotid artery exposed and freed, ligated distally and stay suture placed proximally, then small opening was made in artery and size 1 F-micro tipped pressure transducer catheter (Millar Instruments, Houston, TX, USA) was inserted into the LV lumen via the right carotid artery for measurement of LV pressure, volume, function and related parameters. Then after about 20 minutes of data recording, the abdomen is opened by right sub costal incision to reach the inferior vena cava. To acutely change the cardiac preload, caval occlusion was produced over a 3-s period using a nonmetallic occluder applied to the IVC. The data were recorded as a series of pressure-volume loops.
P van software (Conductance Technologies, San Antonio, TX, and Millar, Houston, TX) was used to analyze all pressure-volume loop data. Regression analyses of multiple isochronal pressure-volume loop data were produced by IVC compression. From the baseline and IVC compression loops, comprehensive sets of hemodynamic parameters were calculated. All steady-state and caval occlusion pressure-volume loops were acquired with the computer data acquisition system. From these data we selected the following parameter: LV diameters were measured at end diastole (LVEDD) and end systole (LVESD), ejection fraction (EF), heart rate (HR), LV systolic pressure in the ends of both systole and diastole (LVESP, LVEDP), Cardiac Output (CO) and the maximum elasticity (Emax).
After complete assessed the pressure volume loop, the animal was sacrificed, starting by injection of equal volume of thiopental and heparin intraperotonealy in doses ranging from 300 μl to 600 μl for each one, after giving good time for the animal to go into deep anesthesia, the rat is positioned and taped and the chest is opened in flap like manner revealing the heart then a needle of the syringe is introduced into right ventricle to aspirate around 0.5 ml of blood for plasma analysis.
Immunofluorescent staining was performed as described previously . Myocardial cryosections (5 μm thick) were treated with a mixture of 30% methanol and 70% acetone and fixed in 4% paraformaldehyde. Sections were incubated with blocking solution (10% BSA in PBS) for 45 min. Sections were then incubated with a mixture of rat monoclonal antibody against mouse neutrophil marker protein (Clone 7/4; ABD Serotec, Oxford, UK) and rabbit polyclonal antibody against mouse macrophage marker protein CD68 (Santa Cruz Biotechnology, Santa Cruz, CA), 5 μg/ml each in antibody buffer, for 90 min. Sections were then incubated for 45 min with a mixture of Cy3-labeled goat anti-rat IgG (labeling neutrophils red; 1:250 dilution with antibody buffer) and Alexa488-conjugated goat anti-rabbit IgG (labeling macrophages green; 1:400 dilution with antibody buffer). Sections were counterstained with Alexa Fluor 350-labeled wheat germ agglutinin (for outlining myocardial cells). To assess the staining specificity, adjacent sections were incubated with a mixture of nonimmune rat IgG and nonimmune rabbit IgG (5 μg/ml each in antibody buffer) in replacement of the primary antibodies and then processed in identical conditions. Microscopic observation and photography were performed with a Leica DMRXA digital microscope. Images were analyzed using Slide Book 2.6 software to obtain quantitative estimates of area.
The collected blood from each rat was centrifuged (in 10000 RPM, for 10 minutes at 4 °C) and the yielded plasma samples was obtained before starting drug treatment (at zero time) and after 48 hours of completion treatment (i.e. after 9 days) for determination of serum malondialdehyde (MDA) Level (the byproduct of lipid peroxidation); Serum Catalase Activity (CAT enzyme); Serum Creatine kinase (CK-MB) enzyme level and Serum Lactate dehydrogenase (LDH) enzyme level.
Enzyme-linked immunosorbent assays (ELISAs)
Myocardial MCP-1 and troponin I were measured using commercially available ELISA kits (R and D Systems) according to the manufacturer's instructions.
Western immunoblotting assay
Myocardial tissue was homogenized with a rotor-stator homogenizer and treated in PBS containing 0.5% Triton X-100 and a protease inhibitor cocktail. Size fraction of crude protein (20 μg) was performed by electrophoresis. After transfer, the membrane was incubated in PBS 5% nonfat dry milk to block nonspecific binding. The membrane was then incubated for 60 minutes with an antibody against ICAM-1, at 1:1000 to 1:2000 dilutions with PBS containing 0.05% Tween 20 and 5% dry milk. After thorough washes, the membrane was treated with peroxidase-labeled secondary antibody (1:5000 dilutions with phosphate-buffered saline containing 0.05% Tween 20 and 5% dry milk) for 45 minutes. Protein bands were developed using enhanced chemiluminescence technique. Densitometry was performed using a computerized densitometer (Molecular Dynamics, Sunnyvale, CA).
The data were expressed as mean +/− standard deviation (SD) were calculated and compared using ANOVA test. Significance was set at p < 0.05 for all comparisons unless otherwise stated. Statistical analysis has been done by using paired t-test and ANOVA test. Significant difference was set at P < 0.05 .