Histological quantification of myocardial fibrosis and collagen deposition in RV

The procedure and protocol were described in detail in our previous report²⁵. In detail, Masson's trichrome staining was used for identifying the fibrosis of RV myocardium. Three 4-μm-thick serial sections from the same levels of the RV myocardium from each animal were prepared using a Cryostat (Leica CM3050S). The integrated area (μm²) of infarct area and fibrosis on each section were calculated using the Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA). Three randomly selected high-power fields (HPFs) (×100) were analyzed in each section. After determining the number of pixels in each infarct and the fibrotic area per HPF, the numbers of pixels obtained from three HPFs were summated. The procedure was repeated in two other sections for each animal. The mean pixel number per HPF for each animal was then determined by summating all pixel numbers and dividing by 9. The mean integrated area (μm²) of fibrosis in RV myocardium per HPF was obtained using a conversion factor of 19.24 (1 μm² represented 19.24 pixels).

To analyze the extent of collagen synthesis and deposition, cardiac paraffin sections (6 μm) were stained with picrosirius red (1% Sirius red in saturated picric acid solution) for one hour at room temperature using standard methods. The sections were then washed twice with 0.5% acetic acid. The water was physically removed from the slides by vigorous shaking. After dehydration in 100% ethanol three times, the sections were cleaned with xylene and mounted in a resinous medium. High power fields (×100) of each section were used to identify the Sirius red-positive area on each section. Analyses of collagen deposition area in RV myocardium were identical to the description for the calculations of the infarct and fibrotic areas.