Ralph G. Kratky

Quantitative measurements of early atherosclerotic lesions on rabbit aortae from vascular casts

We have measured the size and location of fatty atherosclerotic lesions in the cholesterol fed rabbit by scanning electron microscopic analysis of vascular casts. The aortic periorificial lesions examined in this study have been divided into proximal (upstream), distal (downstream) and lateral (to the sides of the branch). Lateral lesions developed after 1 week on diet, followed by distal lesions at 2 weeks, and occasional proximal lesions at 4 weeks. The area covered by the lesions shows a strong linear relationship (P less than 0.001) to the cross-sectional area of the branch after 2 weeks on the cholesterol diet.

The distribution of fibro-fatty atherosclerotic lesions in the aortae of casein- and cholesterol-fed rabbits

Atherosclerotic lesions were induced in the aortas of 50 rabbits by feeding a semi-purified cholesterol-free casein diet or normal rabbit chow with a low level of added cholesterol for 6 or 10 months. Following perfusion fixation, the aortae from these animals were opened along their length, stained with oil red O and photographed en face. Orifice associated lesions were mapped by measuring radial lesion length at 10 degrees intervals circumferentially around ostia. Histology of these lesions revealed abundant smooth muscle cells surrounded by collagen and elastin in the extracellular matrix, typical of fibrous plaques, as well as oil red O staining lipid and some macrophage derived foam cells. These fibro-fatty lesions were found distal and lateral to ostia, at the same locations as fatty streaks seen in rabbits fed a 2% cholesterol diet for 1 week to 2 months in previous studies. The results of this study show that in moderately hypercholesterolemic rabbits fed an atherogenic diet for 6 to 10 months, advanced atherosclerotic plaques develop in the same location as the fatty streaks seen in short term experiments.

Shrinkage of Batson's and its relevance to vascular casting

A number of studies of vascular endothelium using vascular casting have recently been reported in this journal. The most commonly used casting compound is Batson’s (or some modified form of Batson’s) (Polysciences Inc., Paul Valley Industrial Park, Warrington, PA 18976) a methacrylate compound which is pre-polymerized to a specific viscosity [2]. When used, the Batson’s is polymerized to a solid acrylic plastic. Previous users have claimed that Batson’s is ideal in part due to its dimensional stability (shrinkage of less than 1%) [1.4,5]. We would like to report two experiments measuring Batson’s shrinkage of 13% to 20% by volume using Batson’s from 4 different lots. Legg and Gow (1981) report volume shrinkage, similar to ours, of 16% and 20% in rigid containers. Here we report results both in rigid containers and also in distensible arteries both tethered and untethered. In the first experiment, 4 mixtures of Batson’s and methyl methacrylate were placed in 20 l-ml Becton and Dickinson’s syringes (see Table 1). The mixture was set under a hydrostatic pressure 110 mm Hg above atmospheric. Shrinkage increases with the proportion of methyl methacrylate (see Table 2). The setting time reported is the time required for the material to become semisolid Cjello-like). In the second experiment Batson’s (mixture III) was infused into excised sheep femoral arteries and set at 110 mm Hg (see Table 3). Most of the arteries were tethered at the cannulated ends by means of silk suture (# 1). The arteries had internal diameters of 3.5-4.5 mm. Shrinkage was determined from photographs of the arteries. External arterial dimensions were measured by means of these photographs. Arterial wall volume was assumed constant. In vascular casting arteries will be well tethered and shrinkage will be constrained with most shrinkage in diameter and little shrinkage in length. However, forces must be evoked to constrain shrinkage to this mode. These forces are a possible source of artefact.

Quantitative measurement of fixation rate and dimension changes in the aldehyde/pressure-fixed canine carotid artery.

Excised canine carotid arteries held at constant physiologic length were cycled from -13 to +27 kPa with a constant-flow infusion pump, and the pressure-volume curves were recorded. The change in diameter on reducing the pressure from 16 to 0 kPa was determined and the strain of recoil [(diameter16--diameter0)/diameter16] calculated. Diameter recoil was reduced from 51% (fresh tissue) to 5% after 3 h pressure fixation in 4% formaldehyde and to 9% after 15 min in 2% glutaraldehyde with little further change. Lengthwise recoil was reduced from 37 to 10% after 5 min in 2% glutaraldehyde. Photographs were taken to measure outer diameter during pressure fixation. There was no change in diameter in either fixative from the first minute up to 3 days at the constant pressure of 16 kPa.

COLLAGEN QUANTITATION BY VIDEO-MICRODENSITOMETRY IN RABBIT ATHEROSCLEROSIS

A video-microdensitometry method was developed to measure the mass fraction of collagen in portions of tissue sections. A frame grabber and computer are used to capture micrographs and calculate the collagen fraction at each pixel. The method is based on the stoichiometric staining of collagen by picrosirius red stain, and was calibrated for glutaraldehyde fixed tissue; a detailed map of collagen distribution throughout a six-month rabbit atherosclerotic lesion was made. The method is reliable, fast and easily reproduced, since it uses readily available equipment.

Steady flow visualization in a rigid canine aortic cast

Steady flow studies were conducted in a transparent canine aortic cast. The cast segment stretched from the aortic valve to beyond the renal arteries and included all major branches. Flow was visualized by analysis of dye streaklines. Flow rates for basal and exercising cardiovascular states were simulated. The Reynolds numbers in the ascending aorta for basal and exercising conditions were 900 and 1587 respectively. Aortic core flow was laminar in basal simulations. Disturbed flow commenced in the upper descending aorta with exercising flow rates. Separation zones existed along the inner curvature of the aortic arch and the proximal walls of the brachiocephalic, left subclavian, and coeliac arteries. Such zones may exist over a portion of the cardiac cycle. If either renal artery was occluded, then a vortex formed. This vortex is associated with high shear regions which correlate well with sites where sudanophilic lesions have been reported in cholesterol-fed nephrectomized rabbits.

Local changes in collagen content in rabbit aortic atherosclerotic lesions with time

A video-microdensitometry method was used to quantify collagen on a pixel by pixel basis at specific locations within atherosclerotic lesions, induced by feeding New Zealand White rabbits a declining cholesterol diet. Probability mapping (done previously) showed the lesions occurred in predictable locations so the age of any one area of a lesion could be estimated. The oldest part of the lesion was on the lateral lip of the flow divider as lesions always developed there first. Serial sections through three lesions showed the distribution of collagen within the plaque. The measurement of all the lesions showed that early lesions had low collagen levels: 3-6% for the intercostals and 8-11% for the renals. Collagen increased consistently with time on the diet to 11-15%, for the intercostal and approximately 17% for the renal. The aorto-renal lesions were more severe; thicker and with a higher percentage of collagen. The greatest collagen content was on the lateral lip of the flow dividers, where controls had intimal thickening. No fibrous caps were seen. This study shows conclusively that collagen is not evenly distributed in lesions, but develops progressively in specific locations of fatty plaques with the duration of cholesterol feeding. Intimal thickening predisposes to early collagen development.

Taper: An important feature of y-bifurcations in porcine renal arteries and human cerebral arteries

The geometry of arterial bifurcations has been shown to alter fluid flow and the propagation of both pressure and flow waves. Here we provide a more complete description of the renal artery bifurcation geometry and show that the geometry of the bifurcation is more complex than was believed previously. The objective of this study was to quantify changes in cross-sectional luminal area in systemic arterial bifurcations using the method developed by Macfarlane [Ph.D. thesis, University of Western Ontario, Ontario (1985)] to study the geometry of human cerebral bifurcations. Porcine renal arterial bifurcations from seven young (6-14 weeks) and six old (greater than 52 weeks) animals were pressure-fixed (P = 140 mmHg) for 3 h with 10% formalin. Bifurcations were embedded in a block of frozen latex paint. Serial sections were cut at 20 microns (+/- 0.01 microns) using a sledge microtome while the block face was scanned with a video camera and the images were stored on a videotape. The luminal area was measured digitally upon playback. Bifurcations from the two age groups changed in cross-sectional area not only at the flow divider but also along the so-called straight regions. Proximal linear increases in cross-sectional area were observed proximal to the apex of the bifurcation in both young and old vessels, while linear luminal area decreases were measured in the daughter branches of both young and old porcine renals. Taper was defined as the change in luminal area per unit length of parent and daughter branches, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Scanning Electron Microscopic Studies of the Vasa vasorum of Thoracic Aortas

Using hot alkaline solution, the elastic laminae were extracted from aortas and observed with scanning electron microscopy. Vascular structures were found in the elastin layers of the tunica media in descending thoracic aortas of sheep, dogs, and pigs, and these tube-like structures were filled with elastomer which was injected through the heart of the animal in vivo. Sub-intimal microvessels were also found to be filled with the elastomer and it is concluded that vasa vasorum can exist close to the internal elastic lamina in these animals.

A comparison of collagen levels in aorto-renal and aorto-intercostal atherosclerotic lesions in the cholesterol-fed rabbit

Collagen is the most abundant protein found in mammals. Its primary role is to give strength to structures such as bone, tendon and arteries. Collagen develops in atherosclerotic lesions after infiltration of the intima by lipids and monocytes. The authors have developed a video-microdensitometry method to measure, precisely, the collagen mass fraction in sections of atherosclerotic lesion. Four rabbits were fed a declining low level cholesterol diet for 6 months. Lesions were produced primarily to the sides and downstream from branch junctions. Collagen levels were measured in the periorificial lesion of the left aorto-renal and 3rd aorto-intercostal branch sites. Each lesion was sampled in three locations: immediately lateral to both sides of the branch orifice, downstream from the orifice and at the edge of the lesion. The renal values for the downstream, lateral and edge sites were 16.8/spl plusmn/2.1 (SE)%, 16.7/spl plusmn/1.1%, and 17.0/spl plusmn/1.5% respectively, while the corresponding intercostal values were less for all sites, 12.58/spl plusmn/0.85%, 14.5/spl plusmn/1.3% and 10.67/spl plusmn/0.72%. The edge and downstream sites were significantly different between branches (p<0.05 by individual t-tests). The lateral measurements were not shown to be different (p=0.30). These results suggest that collagen is laid down in different amounts according to its position along the artery wall. Physical factors such as local stress/strain distributions may influence collagen production in atherosclerotic lesions.