Imageready 7

Fresh heart ventricles from the sham-operated rats (n=8) and the H-HF rats (n=8) were fixed using 4% paraformaldehyde, dehydrated with alcohol, embedded in paraffin and cut into 5-mm-thick slices using a rotary microtome (RM2016; Leica Microsystems, Wetzlar, Germany). The slices were then stained with hematoxylin and eosin or Masson’s trichrome, and observed at x200 magnification using an inverted microscope (IX71; Olympus, Tokyo, Japan).The area of cardiac and perivascular fibrosis was scored in a blinded manner in 6 randomly selected areas per sample and analyzed using Adobe ImageReady 7.0.1 software (Adobe Systems, San Jose, CA, USA).

Fresh heart ventricles were fixed using 4% paraformaldehyde, dehydrated with alcohol, embedded in paraffin, and cut into 5 mm slices (RM2016, Leica, Wetzlar, Germany). All hearts were embedded in a cross section orientation, and all slices were taken from the cross section at the papillary muscle level of the left ventricles. Three cross sections of each heart were analyzed. From each section, eight areas of hematoxylin eosin and Masson’s trichrome stained slices were randomly selected across the wall thickness to assess the relative fibrosis area (expressed as a percentage of total heart area). Each field was taken at 200 magnification (IX71, Olympus, Tokyo, Japan) and then analyzed using the Adobe ImageReady 7.0.1 software (Adobe Systems, San Jose, CA).
Apoptosis assays were carried out using the terminal deoxynucleotidyl transferase dUTP nick- end labeling (TUNEL) kit (ZSGB-BIO, Beijing, China) and the protocol recommended by the manufacturer. The TUNEL index (%) was calculated as the ratio of the number of TUNEL- positive cells divided by the total number of cells. At least eight representative fields were evaluated for each experimental group, from which an average value was calculated.