Histological and Ultrastructural Assessment of Myocardial Tissue

Light microscopic study: Myocardial tissue was excised and fixed in 10% buffered formalin. The fixed tissues were embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin (H&E). The specimens were examined under light microscope (Zeiss Axioskop 40) by the experienced pathologists who were blinded to the experimental protocol. Photomicrographs were taken at ×200 magnification. The histological findings were graded using a scoring system which was classified as: (−) no changes; (+) mild (focal myocytes damage or small multifocal degeneration with slight degree of inflammatory process); (++) moderate (extensive myofibrillar degeneration and/or diffuse inflammatory process); (+++) marked (necrosis with diffuse inflammatory process).
Transmission electron microscopy: Small pieces of myocardial tissue were retrimmed into 1mm³ blocks and fixed in 4% glutaraldehyde overnight. The next day, the blocks were washed trice with 0.1 M phosphate buffer and post-fixed for 2 h in 1% osmium tetraoxide in the same buffer at 4 °C. After several washes in 0.1 M phosphate buffer, the specimens were dehydrated using graded acetone solutions and embedded in Spon812. Semi-thin (1 mm) as well as ultrathin sections (50 nm) were cut by an ultramicrotome (LKB-Nova, Sweden). The sections were stained in alcohol uranyl acetate and lead citrate and viewed under JEM-2000EX transmission electron microscope (Hitachi Co., Ltd., Japan).