The pH dependence of borohydride as an aldehyde reductant.

The aldehyde-reducing capacity of borohydride has been investigated in the sequence periodic acid-borohydride-periodic acid-Schiff and variants. Densitometric studies on rat colonic mucins show that borohydride incompletely blocks periodate-engendered aldehydes unless the pH is above 8.2. Below this value, some aldehydes are not reduced and continue to be Schiff-stainable, while others are subsequently gerenated by the second exposure to periodic acid. The effect is more pronounced in paraffin than in cryostat sections, but does not apply to human colonic mucins.

Permeability in atherosclerosis: fluorescence test in green light with trypan blue.

A new microscopic fluorescence method for trypan blue at 570 nm has been used to follow the entry of albumin into the atheromatous rabbit aorta. Permeability into the inner aortic wall increases before the onset of gross lesions and seems just to precede intraendothelial deposition of lipid. Thereafter, permeability of the inner wall progressively increases until streaks or small plaques develop. These raised lesions stain and fluoresce variably, some intensely so while others are almost unreactive. This variability might reflect the difference between progressive and quiescent lesions. However, a zone of increased permeability surrounds many raised lesions, suggesting that the edge is a major site of growth and progression.

Permeability of inner and outer layers of rat and rabbit aortic wall. Two new microscopic test with trypan blue.

Two new permeability tests are described for use with intravenously injected trypan blue. One depends on the demonstration of trypan blue by its specific red fluorescence in green light at 570 nm, while the other is a surface microscopy technique at low magnification, using illumination from a fibre-optics light source. The routes of entry of the trypan blue-albumin complex into the rat and rabbit aorta appear to be from both the inner and outer surfaces. Over the period 1/4-24 h after injection of the dye, more entered from the outer surface than the inner surface in the rat aorta and rather more in the rabbit thoracic aorta. The arch of the rabbit aorta showed in general rather greater entry from the inner surface. Trypan blue that has entered the aortic wall is partly taken up by the elastic lamellae. Elastic competes successfully for the dye and captures it from the trypan blue-albumin complex; this uptake is blocked by deamination with nitrous acid.

The giant cell in cholesterol resorption.

Giant cells in the experimental cholesterol granuloma provided a convenient, recognizable cellular model in which to follow the transformation of crystalline cholesterol to liquid lipid. Crystals that were too large to be ingested by individual macrophages were incorporated into a multinucleate cell by a process of circumfusion, and were progressively converted to cholesterol esters within the giant cell. The liquid ester droplets seemed then to be mobilized by mononuclear cells until the entire implant had been resorbed.

Detection of macrophages in atherosclerotic lesions with cytochrome oxidase.

The anaerobic metabolism of the arterial wall allows macrophages to be demonstrated therein by the cytochrome oxidase histochemical method. Monocytes (macrophages) in human fatty streaks (WHO grade I) or fibrofatty (WHO grade II) human atherosclerotic lesions are normally confined to subendothelial regions. Lesions complicated by ulceration, mural thrombosis or intimal haemorrhage (WHO grade III) showed numerous monocytes (macrophages) around newly-formed capillaries in focal areas of organization. By contrast with grades I and II atherosclerotic lesions, macrophages in lipid granulomas induced by subcutaneous injection of cholesterol oleate are more numerous and distributed throughout the lesion. The slow or absent resorption of lipid from atheromatous lesions may in part result from the paucity of macrophages therein.

Crystals in atherosclerotic lesions: real or artefact?

Atherosclerotic lesions were obtained from man during surgery and from cholesterol-fed rabbits. They were maintained at about 37 degrees C during handling. Smears were prepared on glass slides and these were examined microscopically at 37 degrees C. Solid rhomboidal or thick needle-like crystals were present at 37 degrees C but increased in numbers or in size or both on cooling. Staining studies and measurement of melting point (133-153 degrees C) suggested that such crystals are composed largely of free cholesterol or a related sterol. Liquid crystals exhibiting conic focal (Maltese cross) anisotropism were present at 37 degrees C and did not appreciably increase in either size or numbers on cooling. Staining studies and their resistance to digitonin suggested that these Maltese cross crystals are largely esterified cholesterol. Thin needle-like crystals arranged like feathers or in rosettes were seen in smears of adipose tissue and were attributed to triglycerides.

Phagocytes, lipid-removal and regression of atheroma.

Reticuloendothelial (RE) phagocytes (macrophages and histiocytes) can be distinguished from locally-derived lipid-containing cells (e.g., arterial smooth muscle) or locally derived phagocytes (e.g., Schwann cells and microglia) by the demonstration of a diffuse catalase reaction in a proportion of these RE cells with a short incubation modification of the Novikoff-Golfischer diaminobenzidine histochemical methods. Even though only a proportion of an RE population is catalase-positive, the results accord with the majority of current opinion that most of the cells in atherosclerotic lesions are derived locally, whereas the phagocytes in lipid implants and xanthomas are of RE origin. The phagocytes in the peripheral nerve undergoing Wallerian degeneration appear to be of mixed RE and endogenous origin, whereas microglia around multiple sclerosis plaques seem to be derived locally. Lipid in lesions with RE phagocytes (subcutaneous lipid implants and xanthomas) is relatively rapidly resorbed, whereas lipid in lesions with few RE phagocytes (atherosclerosis) or phagocytes of endogenous origin (CNS degeneration) is more slowly resorbed or partly retained within the tissue. Wallerian degeneration in the peripheral nerve, with its mixed population of RE and endogenous phagocytes, occupies an intermediate position in the speed of lipid removal.