The distribution and morphology of aortic valve cusp lipids.

During dissections of porcine aortic valves, we noted clusters of spherical droplets, roughly 50 micrometers in size, within the central spongiosa of the valve cusps. Oil red-O staining of fresh tissues confirmed that the clusters were lipids, and thin layer chromatography showed that they consisted mainly of free cholesterol and triglycerides, with some cholesterol ester. The distribution of these clusters in 60 cusps from 20 porcine aortic valves was mapped using computerized morphometry. The ventricularis was peeled back to expose the spongiosa, and the spatial distribution of the lipid droplets was analyzed using a stereological point counting method. Computer averaging of lipid density images was done to obtain a mean distribution map of lipid occurrence. This distribution map suggests that porcine aortic valve lipids are found predominantly at the base of the valve cusps, and are conspicuously absent from the coaptation regions and the free edge. Moreover, 19/20 valves had lipids in the left coronary cusp and all 20 valves had lipids in at least two of the three cusps. Endogenous lipids are therefore ubiquitous in porcine aortic valves and may play a role in the calcification of xenograft bioprostheses.

Elasticity and geometry measurements of vascular specimens using a high-resolution laboratory CT scanner.

Vascular diseases are frequently associated with changes in the mechanical properties of the arterial wall. Existing techniques for studying arterial geometry and mechanical properties in vitro are often destructive, since they involve sectioning of the specimen into strips, or provide average measurements of the mechanical properties over the volume of intact specimens. We developed a high-resolution computed tomography (CT) scanner for in vitro studies of arterial geometry and static elastic properties. The x-ray image intensifier based system can acquire single transverse images, or a volume image, with 2 mm-1 resolution. Images were obtained through an intact abdominal aortic aneurysm at five pressures. The incremental circumferential Young's modulus E(inc) was calculated from the internal and external circumferences, and at physiological pressures E(inc) of the aneurysm was found to be 275 times greater than that of the normal aorta proximal to it. A volume image of the specimen provided landmarks that allowed histological sections to be obtained at locations coincident with those where the elasticity was measured. The histological analysis revealed a sixfold decrease in elastin content in the aneurysm, compared to the normal aorta. We have demonstrated that the static mechanical properties and geometry of vascular specimens can be quantified in vitro with the new high-resolution CT scanner and can be compared subsequently with histological analysis to provide further insight into the understanding of atherogenesis.

A digital-radiographic technique for in vitro tissue thickness measurement using iodine displacement.

A novel digital radiographic method has been developed for measuring the thickness of a tissue sample by iodine displacement. This simple, accurate method is useful both in medical research and in the comparison of pathological and clinical findings. Radiographic measurements of tissue samples in air suffer from the limitation that the quantity measured depends on the product of both thickness and radiographic attenuation coefficient. This technique allows one to obtain thickness measurements from a digital radiograph of a tissue sample suspended in a bath of radio-opaque contrast agent. The attenuation of the iodinated contrast agent is much higher than that of tissue or calcium. Thus the resulting image is determined largely by the contour of the surrounding bath, and is only slightly influenced by the composition of the tissue. This technique improves both the accuracy and precision of radiographic thickness determination. In this paper the iodine displacement technique is described and the accuracy and precision of thickness measurements in appropriate phantoms are quantified. This technique has been used with both image intensifier and screen-film based imaging systems to obtain thickness maps of calcified human aorta, with precision better than 4% and spatial resolution of 2.5 mm-1.

Vascular collagen fibril morphology in type IV Ehlers-Danlos syndrome.

Morphometric analysis of transmission electron micrographs of blood vessel, skin and dura mater collagen fibers were performed on postmortem tissues taken from 28-year-old female with Ehlers-Danlos type IV syndrome (EDS IV). Vascular tissue from this patient was compared to 5 age- and sex-matched controls (age range 26-28 years). Our study revealed significant variation in collagen fibril diameter in the walls of almost all the vessels studied. In general, the EDS IV tissue showed a net decrease in average collagen fibril cross sectional area in arterial wall samples. This decrease was significant (p less than .05) across the entire wall of the renal artery, in the media of the carotid artery, and in the media and adventitia of the common iliac artery. Samples from the vena cava show significant increases in collagen fibril cross sectional area across the vessel wall (p less than .005). The only areas studied which did not show significant changes were the intimal and adventitial regions of the common carotid artery. The observed changes may be contributory to the decreased arterial wall strength typical of the syndrome.

The effects of age, vessel size, and Ehlers-Danlos type IV syndrome on the waviness index of arteries.

Ehlers-Danlos Syndrome is a rare hereditary connective tissue disorder produced by collagen abnormalities. The Type IV variant causes vascular rupture in the second and third decades. Definitive diagnosis at present is based on demonstrating a deficiency of Type III collagen or procollagen chemically or in fibroblast cultures. Both require fresh tissue. We have shown in fixed autopsy tissue that the elastin in two EDS IV patients appears accordion-pleated with a high waviness index (WI) of 1.58 +/- 0.028 (n = 90) compared to 1.35 +/- 0.08 (n = 446) for 21 controls aged 9 to 71. The WI is the ratio of the length of elastin in the internal elastic lamina to the circumference. The group were significantly different statistically (p less than 0.01) with a randomized block design analysis. Vessel size and age did not alter the WI in either controls or patients. There was significant overlap of data points between the two groups so that no conclusions can be drawn on individual vessels. However, if the waviness index is greater than 1.8, the vessels are unlikely to be normal. We did not study patients with other connective tissue diseases. The WI may be a useful qualitative guide of abnormality in a retrospective study if the diagnosis of Ehlers-Danlos Type IV is entertained.

A comparison of the size of fenestrations in the internal elastic lamina of young and old porcine aortas as seen with the scanning electron microscope.

The size of the fenestrations (windows) in the internal elastic lamina (IEL) of arteries may be important in the functioning of the blood vessel wall. The fenestrations are filled with collagen, muscle, and (or) ground substance, which must be removed to make the fenestration visible with the scanning electron microscope. All of the nonelastic components are removed with a hot alkali solution. Our experiments were designed to compare the fenestration size in the IEL of the thoracic aorta of young (6-8 weeks) and old (6-9 months) pigs. A protocol for digestion of young pig tissue was developed and showed that fresh young aortas should be digested in 0.1 M NaOH at 75 degrees C for 2 h and fixed tissue should be digested for 5 h. The average area of the fenestrations for young pig thoracic aortas digested for 2 h was 1.8 +/- 0.29 (SE) microns 2 and for the old pig aortas digested for 2 h was 1.7 +/- 0.11 (SE) microns 2. These values were not significantly different (p greater than 0.05), but the IEL from young pigs appeared rougher than the previously reported smooth IEL of the adult pigs.