Uptake of circulating macromolecules by the aortic wall is greater downstream than upstream of branch sites in immature rabbits, but the opposite pattern is seen at later ages. The mature pattern is nitric oxide dependent; we tested whether it is also flow dependent. Intercostal arteries of anesthetized rabbits were occluded, sham operated, or left alone. Uptake of rhodamine-labeled albumin was assessed by quantitative fluorescence microscopy of the sections through the aorta. In mature animals, uptake was higher upstream than downstream of the control and sham-operated branches, but the pattern was reversed at occluded branches. In young animals, uptake was not significantly different between regions upstream and downstream of control, sham-operated, or occluded branches. The absence of the normal immature pattern may reflect an influence of anesthesia and will assist in the elucidation of mechanisms underlying this pattern. The data for mature animals provide the first direct evidence that flow determines permeability near arterial branches and may account for the inverse spatial correlation between shear stress and disease prevalence at branches of adult human arteries.
SummaryMeasurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ, are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 8C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.
In children, aortic lipid deposition develops in triangular regions of the wall downstream of branch points, whilst in adults these regions are particularly free of disease. Comparable age-related patterns occur in rabbit aortas. They may be explained by patterns of wall permeability to circulating macromolecules: along the longitudinal midline through branches, permeability is greater downstream than upstream in immature rabbits, but is greater upstream at later ages. Here we have mapped permeability in detail around such branches, not just along the midline. Short-term uptake of rhodamine-labeled albumin, measured using digital imaging fluorescence microscopy of serial sections, was greatest in an approximately triangular region downstream of immature branches, but in mature animals it was greater upstream, particularly away from the midline, and in streaks to the side of branches. Hence the maps are consistent with earlier permeability data and closely resemble the patterns of disease.
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