thiocarbohydrazide and 4-phenylenediamine

Abundant extracellular lipid deposits are associated with cell necrosis and tissue weakening in the core region of human atherosclerotic fibrous plaques. The ultrastructural morphology of the core region, previously undefined because of lipid extraction artifacts, was studied with the aid of new osmium-thiocarbohydrazide-osmium and osmium-tannic acid-paraphenylenediamine sequences for tissue processing. Small droplets of neutral lipid (30 to 400 nm profile diameter) and lipid vesicles with aqueous centers accounted for more than 90% of the area occupied by lipid-rich structures in the core region. No foam cells were present. Cholesterol crystals, lipid droplets of a size similar to those in foam cells (0.4 to 6 mu), and larger neutral lipid deposits (greater than 6 mu) together occupied less than 10% of the total area of lipid structures. Abundant lipid vesicles were associated with the nearby presence of cholesterol crystals, whereas small lipid droplets were predominant in areas without crystals. Many droplets had surface defects in the form of pits and vesicular blebs. These morphologic findings are explained most concisely by postulating direct accumulation of extracellular lipid from interstitial lipoproteins as a major process in core region formation. Moreover, a dynamic state of ongoing physical/metabolic transformation of extracellular lipid deposits is suggested.

Topics: Aorta; Arteriosclerosis; Histological Techniques; Humans; Hydrazines; Hydrolyzable Tannins; Lipid Metabolism; Microscopy, Electron; Osmium; Phenylenediamines

Electron microscopy of atherosclerotic arterial tissue commonly fails to distinguish lipid vesicles from droplets, especially when these are found in the extracellular space. The distinction is important, because vesicular or membranous lipid is composed of phospholipid and unesterified cholesterol, whereas neutral lipid in droplet form implies the presence of cholesteryl ester in atherosclerosis. A new procedure with sequential tannic acid and p-phenylenediamine treatments of osmicated tissue (TA-PDA) allows reliable ultrastructural discrimination of lipid vesicles and droplets. The multilamellar character of many vesicles is revealed. Extracellular droplets are found to possess many surface pits associated with membranous blebs. Pitting of droplets is especially evident after the use of an alternative tissue processing technique, the osmium-thiocarbohydrazide-osmium (OTO) sequence applied en bloc. The two complementary techniques will prove useful for electron microscopic studies of atherosclerotic and other lipid-rich tissues.

Topics: Adult; Aged; Arteries; Arteriosclerosis; Extracellular Space; Humans; Hydrazines; Hydrolyzable Tannins; Indicators and Reagents; Lipids; Male; Membrane Lipids; Microscopy, Electron; Middle Aged; Osmium; Phenylenediamines; Staining and Labeling