Three-Dimensional Microvasculature in Rat and Human Hearts Using a Non-injection Ca²⁺-ATPase Method on Thick and Ultra-Thick Sections

Abstract Currently there are no methods available for staining rat and human myocardial microvasculature on thick sections that would allow for specific staining and differentiation of arterioles, venules, and capillaries. A non-injection technique is described that allows for labeling of the microvascular bed (MVB) in formalin-fixed pieces of the myocardium from humans and the white ratRattus norvegicus, as well as human full-mount pericardium. Vessel staining is based on the activity of phosphatases (ATPases) and the precipitation of the released phosphate with calcium ions at high pH (pH 10.5–11.5). The resulting precipitate subsequently is converted to black or brown lead sulfide. The specificity of this reaction to vessels of the MVB allows arterioles, venules, capillaries, and pre- and postcapillaries to be clearly visualized in thick (60–100µm) and ultra-thick (300–500µm) sections against an unstained background of muscle and connective tissue. In addition, smooth muscle cells of arterioles are also stained allowing for differentiation between arteriolar and venular beds. These observations have not been reported in rat or human myocardium using other methods. This procedure should benefit studies of coronary microcirculation in experimental and pathological conditions, as well as in pharmacological investigations.