With the long term goal of improving our understanding of the mechanisms involved in coronary artery spasm, we have undertaken a two part study of the artery structure. We have made a comparison of the relative proportions of the different layers in proximal and distal regions of the main coronary arteries, and have quantitatively assessed their three dimensional structural fabric. Major coronary arteries from nine hearts were prepared for histological examination after fixation at a transmural pressure of 120 mm Hg. Measurements from 14 proximal and distal pairs of the cross sectioned arteries showed a dominant subendothelial layer, which diminished in thickness distally, with a small fraction of muscle cells interspersed with collagen. Three dimensional orientation measurements of the collagen and muscle components, which are birefringent, were obtained from one pair of segments from each of the left anterior descending, circumflex and right coronary arteries, using the polarising light microscope and Universal stage. Findings showed (1) a single circumferential order of adventitial collagen, with a mean circular standard deviation (CSD) of 22.3 degrees; (2) very highly ordered medial smooth muscle, mean CSD of 5.0 degrees (both findings are quantitatively similar between proximal and distal segments of artery, and between arteries); and (3) a multilayered fabric of collagen in the subendothelium in all vessel segments. The principal contributor to functional differences between proximal and distal regions may be the prominent and structurally varied subendothelial layer of the coronary arteries.

The mechanical properties of collagen as a biopolymer ensures that collagen has a significant influence on the mechanical behavior of the host tissue. Structural organization is a key to that influence. We have assessed this relationship quantitatively in the tunica media of arteries from the heart and brain, using the polarizing light microscope and Universal stage. Arteries from 22 autopsies were isolated, cannulated and fixed with 10% buffered formalin, at a distending pressure spanning normal values in vivo. We prepared the tissue for light microscopy, with paraffin embedding, sectioning at 7 microns, and staining with picrosirius red to enhance the natural birefringence of medial collagen. Individual measurements, 30 to 50 per arterial section, referenced against the central axis of the vessel segment, revealed a coherent organization, with an average orientation which was within 1 to 2 degrees of being perfectly concentric for all artery segments. Analysis was done with Lambert projections and circular statistics. We calculated the circular standard deviation, which was 5.2 degrees for 27 brain arteries (S.D. 1.9 degrees) and 5.6 degrees (S.D. 2.1 degrees), for 5 coronary arteries sectioned at less than 15 degrees. Our interpretation is that medial collagen can be strained even though highly aligned, revealing a mechanical property which contrasts that of type I collagen.

The thickened subendothelium of brain arteries that is characteristic of atherosclerosis was assessed for the directional organization of the two main birefringent components, smooth muscle cells and collagen. Thirty-three arteries from 16 autopsy cases were pressure fixed at 30, 60,110, and 200 mm Hg, sectioned at a thickness of 7 pm, and stained with silver impregnation to enhance tissue birefringence. The intended focus of the study was on muscle organization, but it also included the collagen among the cells because of the coalignment of the two tissues and their similar staining properties for polarized-light microscopy. The birefringent medial fabric at all pressures was circumferentially oriented, with a mean deviation of the 33 sections of 1.4° from circumferential with an average circular standard deviation of 3.5°, thereby showing remarkable coherence. In contrast, the subendothelium showed great variability both in thickness and in organization. Many arteries had no measurable subendothelium, and others had as much as 100%, with some atherosclerotic lesions as much as 300% of the medial width. Measurements from the subendothelium revealed a helical arrangement of tissue, often divided into separate regions, with a balance of left-and right-handed helical components and generally with lower pitch angles in the layers adjacent to the lumen. The average circular standard deviation within individual subendothelial layers was 14.5°. (Arteriosclerosis and Thrombosis 1991;ll:681-690)

We evaluated the effectiveness of the Universal stage, an instrument for measuring three-dimensional orientation of birefringent materials, for studying the collagen fabric in the wall of brain aneurysms. Vessels from autopsy were fixed at normal arterial distending pressure with 10% formalin, and prepared for polarized light microscopy, with paraffin embedding and staining with picrosirius red for birefringent enhancement. Quantitative data were obtained from tangential and oblique sections (7 microns thickness) of an intact 8 mm aneurysm, a 1.5 mm aneurysm, and a tangential section (3 microns thickness) of a cerebral artery. Sections of full-size aneurysms seen through the microscope, adjusted either for plane or circularly polarized light, revealed distinctive layers of collagen across the aneurysmal wall, which at higher magnification were further subdivided. Three-dimensional measurements, numbering 1,082, were made by use of the Universal stage attachment to the polarizing microscope. They were plotted by computer-controlled graphics on Lambert projections and analyzed by circular statistics. When assessed layer by layer, the collagen spanned a full range of orientations relative to the tangential plane. The circular standard deviation, a measure of the spread of alignment about the mean, was as low as 10 degrees for coherently organized collagen and as high as 40 degrees for the least coherently organized collagen, values characteristic of either the organized tunica media, or the least organized tunica adventitia of cerebral arteries. Although there was a marked thinning of the wall of one aneurysm, there was no evidence of structural weakness based only on the directional organization assessed by our measurements.