Oxygen content of the fixative is important in the interpretation of the ultrastructure of ischaemic myocardium

Abstract

Isolated rat hearts were subjected to 15, 45, or 60 minutes of global ischaemia and then fixed by perfusion at 37 degrees C with glutaraldehyde containing various amounts of oxygen. This either had been bubbled with 100% oxygen (PO2 620 mm Hg) or with 100% nitrogen (PO2 40 mm Hg) immediately before use, or it had been routinely prepared and stored exposed to atmospheric oxygen (PO2 245 mm Hg). The ultrastructure of myocytes and endothelial cells subjected to 15 minutes of ischaemia was not affected by the treatment of the fixative. However, when the tissue subjected to longer periods of ischaemia was fixed with routinely prepared or oxygen-bubbled glutaraldehyde, ultrastructural changes characteristic of reoxygenation damage were uniformly evident in both the microvasculature and myocytes. These qualitatively distinct changes included mitochondrial swelling, cell swelling, endothelial bleb formation, and narrowing of capillary lumina. These abnormalities were not observed in tissue fixed with nitrogen-bubbled glutaraldehyde. These findings indicate that deliberate steps should be taken to reduce or eliminate dissolved oxygen from the fixatives used to study ischaemic tissues. Otherwise artefactual reoxygenation damage in vitro may occur and make valid ultrastructural interpretation difficult or impossible.