Thomas Zand

Quantitation of oil red O staining of the aorta in hypercholesterolemic rats

A technique is described which provides morphologic and quantitative data on the amount of oil red O (ORO) staining in thoracic aortas of rats fed a high cholesterol diet. Samples are stained with ORO, the dye is extracted, and the concentration of ORO in the extract is measured colorimetrically. Wistar rats fed ad libitum either standard chow (control group: n = 15) or chow supplemented with 4% cholesterol, 1% cholic acid, and 0.5% thiouracil (CCT group: n = 23) were maintained on these diets for 1, 3, 6, 9, or 12 months. Plasma cholesterol levels averaged overall 87 and 737 mg/dl for the control and CCT groups, respectively. Animals were killed under anesthesia by perfusion fixation with formalin or glutaraldehyde, and samples of thoracic aorta were stained with ORO. After microscopic study en face and measurement of surface area, the ORO was extracted in chloroform-methanol (2:1). Concentrations of ORO (microM) were determined from a standard curve and expressed as microM/mm2 of aorta. Aortas of CCT animals showed progressive diet- and time-dependent increases in the amount of ORO staining compared to controls. We conclude that this method yields reliable quantitative data applicable to studying atherosclerosis in small animals.

Endothelium and “silver lines”

The significance of endothelial “silver lines” was studied by TEM in rat aortas after perfusion with glutaraldehyde followed by silver nitrate. Standard TEM technique proved unsatisfactory (coarse silver granules, imprecise localization, artefacts). Exposure of the silver-treated aortas to photographic fixer markedly improved the image of the deposits leaving fine, stable, uniform “residual granules” about 100 Å in diameter. Most of these granules were localized along the intercellular junctions; they also tended to pool in the basement membrane beneath each junction. This image suggests that the Ag+ ions pass through the junction, and react with its contents as well as with the basement membrane beyond it. A scheme is proposed to explain the reaction of Ag+ ions with anions and negatively charged radicals within the junction. It is concluded that the “silver lines” represent not only a histochemical effect, but also the visualization of a transendothelial electrolyte pathway.

The significance of endothelial stomata and stigmata in the rat aorta: An electron microscopic study

Perfusion of arteries with dilute silver nitrate produces in the endothelium (a) a pattern of pericellular black lines, which we earlier interpreted as a marker of the physiological electrolyte pathway (Zand et al. 1982), and (b) focal black deposits on or between the cells, either ring-shaped (stomata) or solid (stigmata). The purpose of this study was to clarify the nature and significance of these controversial structures. A glutaraldehyde-fixednormal rat aorta was perfused with silver nitrate; 17 typical stomata and stigmata were photographeden face, then studied on ultrathin serial sections. When seenen face, they fell into three groups: (I) 4 stomata in endothelial cells; (II) 6 stigmata in endothelial cells; (III) 7 stigmata on intercellular junctions. By electron microscopy, (I) all thestomata in endothelial cells corresponded to myoendothelial herniae. (II) Of the 6stigmata in endothelial cells, 4 corresponded again to myoendothelial herniae, 2 corresponded to blebs (it seemed likely that these blebs had existedin vivo, but the possibility of a fixation artefact could not be excluded). (III) Of the 7stigmata on intercellular junctions, one corresponded to the diapedesis of a mononuclear cell; the other 6 did not correspond to visible endothelial changes and are best interpreted as points of normally higher permeability. We conclude that stomata and stigmata (under the conditions of our experiments) can be explained in at least 4 different ways, depending in part on their location (in cells, on junctions). These ancient terms therefore remain useful for descriptive purposes, as long as it is realized that their significance in any given case must be determined by electron microscopic study.

Intimal Responses to Shear Stress, Hypercholesterolemia, and Hypertension

From the perspective of pathology, the three most common challenges to the intima of larger arteries are local disturbances of flow, hypercholesterolemia, and hypertension. We have studied all three in one experimental system, the aorta of the rat. This chapter is a critical summary of these studies. As an introduction, we will examine some relevant features of our experimental model.