Photochemical fluorochrome staining for detection of prenecrotic changes in cardiomyocyte myofibrils

Abstract

Ischemic and metabolic damages to cardiomyocytes induce immediate ultrastructural changes in their myofibrils. Photochemical fluorochromize staining of prenecrotic cardiomyocytes allows to determine with high sensitivity local redistribution of sarcomere mass. It is shown that different types of destructive processes in cardiomyocyte myofibrils underlie similar polarization microscopic picture.Key Words: necrobiosis; cardiomyocytes; myofibrils; luminescence and polarization microscopy Double refraction of myosin fibrils of sarcomeres allowed to use polarization microscopy for detection of the most early reactions of cardiomyocytes (CMC) to damage [3][4][5][6]10,11,15]. These results provide the basis for classification of CMC alterations. We proposed a method of photochemical fluorochromize staining for detection of early necrobiotic changes in CMC based on the formation of luminescent products in CMC after short wave UV (SUV) irradiation [12][13][14].Polarized light microscopy of CMC is based on changes in anisotropy and topography of myosin filaments in altered cell. Here we used photochemical fluorochromize staining in polarization microscopy of myofibrils during CMC alterations. MATERIALS AND METHODSOcclusion myocardial infarction was modeled in 39 male C57BI mice weighing 17-20 g. The left carotid artery was ligated at the level of the middle third of the left ventricle under Nembutal narcosis (0,5%, 1.25Laboratory of General Pathological Anatomy, Institute of Regional Pathology and Pathomorphologyl Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Medical Sciences, Novosibirsk ml/100 g body weight). The animals were sacrificed 5 min and 1 day after coronary occlusion. Catecholamine-induced damage to the myocardium was modeled in 34 male Wistar rats weighing 160-270 g. Isopropylnoradrenaline (8-10 mg/100 g) was injected intraperitoneally 0.5-18 h before decapitation.After decapitation the heart was isolated and incubated at 0~ to termination of contraction and muscle relaxation. The atria were separated and the heart was cut along through the ventricles and septum. The tissue was washed in a cold neutral buffer. Some samples were frozen in liquid nitrogen and 10-g sections were prepared on an MK-25 cryostate at -18~ mounted on warm glass slides, dried on air, and exposed to phosphorus pentoxide. Fixed and paraffin-embedded tissue samples were cut into 7-~t sections. Before fluorochromize staining the sections were treated with xylene, alcohol, ammonia, and water to ensure complete removal of paraffin and formaldehyde [2].Luminescent, polarization, interference, and brightfield microscopy was used in the study. The preparations were stained by the following techniques: hematoxylin and eosin staining combined with Perls reaction, PAS-reaction combined with colloid iron, according to Hale, and hematoxylin orange, PAS-reaction after amylase treatment and hematoxylin-picrofuchsin 0007-4888/99/0009-0967522.00 9Kluwer Academic/Plenum Publishers