Perfusion and superfusion fixation effects on rat mesentery microvascular beds. Intravital and electron microscope...

The effects of glutaraldehyde on dimensions and ultrastructure of microvascular beds in rat mesentery were studied in two kinds of experiment, administering the fixative by intra-arterial perfusion at a pressure of 80 mm Hg and by superfusion of the exteriorized mesenteric membrane. The microvascular segments were observed by means of intravital microscopy and recorded on videotape before, during, and after glutaraldehyde reached the microvascular segment being observed. Vascular outer diameters were measured at exactly the same points before and after fixation; in Epon embedded whole-mounts; and in sections analyzed by light and transmission electron microscopy, confirming positively the various segments of the microvascular bed and yielding information concerning the preservation of cellular components. Both experiments confirmed that neither perfusion nor superfusion of glutaraldehyde changes the outer diameter of any segment of the microvascular bed compared to the dimensions 5-10 sec before the blood vessels are reached by the fixative. They remain unaltered also after embedding in epoxy resin. During superfusion, there is a 20-50 sec delay until the blood flow comes to a complete stop. This delay is assumed to give rise to the recorded small undulations of luminal endothelial cell membranes and slight buckling of the entire endothelial layer, probably due to a gradual fall in intravascular pressure. Occasionally, the ultrastructure of some endothelial cells is less well preserved after superfusion fixation. This study demonstrates that intraarterial perfusion of glutaraldehyde renders an instantaneous fixation of mesenteric microvessels, preserving the prefixation dimensions of the various segments and the ultrastructure of the cells. Superfusion of glutaraldehyde is slower in reaching the microvessels and may change slightly the appearance of the vascular wall, and cause some impairment of microvascular functions, such as increased postcapillary leukocyte margination and extravasation.