Helen M. Finlay

Three-Dimensional Collagen Organization of Human Brain Arteries at Different Transmural Pressures

Measurements on the directional organization of collagen are necessary for relating the structure and mechanical function of blood vessels. The birefringent optical property of collagen has enabled us to assess the collagen architecture for brain arteries, which are prone to spasm and aneurysm formation. Using the universal stage and polarizing microscope, we measured the three-dimensional organization of the collagen of the main layers of the artery wall, and examined the effect of distending pressure on that organization. Adult arteries obtained from autopsy were fixed at one of three distending pressures, 30, 120 and 200 mm Hg; they were embedded in paraffin and sectioned parallel to the vessel axis at 4 microns thickness. Sections were stained with picrosirius red, a birefringent enhancement stain specific for collagen. Orientation data were obtained from tangential sections from thirteen arteries. We chose to use tangential sections that graze the curving surface of individual layers, to permit measurements that are equally sensitive to fibres in the mechanically meaningful range of directions including longitudinal, helical and circumferential. Each measurement was from a single fibre or group of fibres at a specific location; the mean direction and its variation of alignment within each artery layer were calculated. In some arteries, the adventitia and subendothelium measurements were separated into sublayers, distinguishable by the birefringent optical appearance. Main findings included a substantial component of longitudinal fibres in the adventitia and subendothelium, highly varied in coherence and mean direction, and a thin collagen layer of the adventitia, radially outside the medial muscle cells, that was highly organized circumferentially (circular standard deviation of 9 degrees). At higher pressures, the collagen fabric of all the layers was increasingly coherent and more circumferential in direction.

Collagen Organization in the Branching Region of Human Brain Arteries

BACKGROUND AND PURPOSE Unruptured saccular aneurysms are relatively common, occurring in 4% to 9% of autopsies. Their development at the apex region of brain artery bifurcations is attributed to a combination of structural factors and the effect of blood pressure. Collagen is a primary tension-bearing fabric of the vessel wall, and our purpose was to examine its 3-dimensional alignment at arterial branches. METHODS Sixteen segments of arteries from the circle of Willis, including bifurcations, were pressure distended, fixed, and sectioned in 1 of 3 orthogonal planes. We measured the 3-dimensional organization of collagen at the flow divider by using the polarized light microscope. An electron microscopy study performed in tandem provided measurements on the collagen fibril diameters and packing density. RESULTS Orientation data of the collagen fabric were obtained from sections from 3 different cutting planes. The tunica media of all bifurcations had an alignment that was primarily circumferential, and the medial gap (medial defect) was distinguishable at the apex of all bifurcations. The subendothelial layer was thin at the apex but thicker and more disorganized distally. Adventitial collagen showed little organization except for a high degree of alignment along the apex. Results from the electron microscopy study showed densely packed collagen fibrils of uniform diameter at the apex, compared with slightly smaller and less densely packed fibrils nearby. CONCLUSIONS In the region of the medial gap, a narrow band of highly aligned tendonlike collagen running in the direction of the ridge of the flow divider was a consistent finding. This structure would provide strength and stability to the vessel and is inconsistent with the concept of an inherent defect in the structure of bifurcations.

The behaviour of collagen fibres in stress relaxation and stress distribution in the jaw-joint disc of rabbits.

The physical properties of the temporomandibular joint disc are largely attributable to its collagen fibre and proteoglycan composition and organization. Structural and stress relaxation data were obtained from the discs of six rabbits. Two stainless-steel balls, 4.8 mm dia, were used to load the disc surfaces in compression. Stress relaxation tests were performed at loads of 0.8-1.4 kg, and the disc was then placed in fixative while still in the loading apparatus in order to preserve its deformed state at equilibrium stress. After overnight fixation the discs were sectioned and assessed by means of a polarizing microscope with a rotating universal stage. This allowed measurement of three-dimensional changes in collagen fibre waviness and alignment as the result of loading. The data showed that despite significant stress relaxation and strains, only minor changes in fibre waviness and alignment occurred within the disc, reflecting its effectiveness as a tough but compliant structure, well suited to distribute load in the temporomandibular joint.

Contrasting structure of the saphenous vein and internal mammary artery used as coronary bypass vessels

OBJECTIVES To report quantitatively on the three-dimensional layered organization of the collagen and smooth muscle component of the two most successful vessels for coronary bypass-the internal mammary artery (IMA) and the long saphenous vein (SV). Our aim was to provide an explanation for the differential structural stiffness of these two vessels (both functioning at arterial pressures in their new environment), and how they might be susceptible to endothelial thickening. METHODS Eleven human saphenous veins and 23 internal mammary arteries were fixed at arterial distending pressure of 110 mmHg, and were sectioned in cross-section at 7 microns thickness. A subset of these was also sectioned tangentially. Measurements of the three-dimensional alignment of collagen and smooth muscle fibers within the vessel wall were made using polarized light microscopy and the universal stage attachment. Data were plotted and analysed using circular statistics. RESULTS The IMA, structured like an elastic artery, is dominated by a media with discrete lamellae of wavy collagen and smooth muscle, aligned nearly circumferentially, with a low variability of alignment (mean circular SD 12 degrees). The SV is more variable in its size and structure, characteristically with a narrow circumferential media comprised mostly of collagen which is straightened and highly aligned at arterial pressures (mean circular SD 9 degrees). Circumferential collagen in the vein was often adjacent to longitudinal bundles of smooth muscle and collagen. CONCLUSIONS The strikingly aligned structure of the SV complements the known high mechanical stiffness of this vessel when at arterial distending pressure. The high fraction of longitudinal muscle, in addition to the circumferential muscle cells in the SV make it vulnerable to any pre-implant surgical preparation, and to the cyclical luminal pressures and longitudinal strains characteristic for epicardial arteries.

The layered fabric of cerebral artery fenestrations.

Intravascular bridges, resulting from developmental anomalies of brain arteries, are now better known as arterial fenestrations. Their tendency to develop aneurysms, similar to arterial bifurcations, makes their anatomy and microstructure important for study. Methods Six segments of artery, each including a fenestration (five from the vertebrobasilar junction and one from the middle cerebral artery), were pressure distended, fixed, and sectioned. We made three-dimensional orientation measurements of smooth muscle and collagen, stained to enhance their birefringence, using the polarized light microscope. Results The general contour of the fenestrations is streamlined, with a thickened layered subendothelium at the trailing or distal edge, structurally similar to the region of convergence of major brain arteries. Defects of the medial layer were found at both proximal and distal edges of all the fenestrations. Results included regional mean orientations of individual layers, with circular SDs. The medial layer was found to be coherently aligned perpendicular to the direction of blood flow, with a mean circular SD of 12°. The adventitia was less coherent (mean circular SD, 16°) with the same average orientation, and the multilayered subendothelium had layers of obliquely oriented fibers with a wide range of coherence for individual fiber groups. Layers of the side regions were analogous to those in segments of brain artery and differed significantly from the proximal and distal edges of the fenestration structure. Conclusions The plasticity of form of the fenestrations at both the proximal and distal edges is in response to hemodynamic forces and is analogous to branching regions of brain arteries. Medial defects, a common feature in both brain arteries and fenestrations, may predispose the arterial fenestration to aneurysm formation.

Directional Wall Strength in Saccular Brain Aneurysms from Polarized Light Microscopy

AbstractThe aneurysm wall, which must withstand arterial blood pressure, is composed of layered collagen. Wall strength is related to both collagen fiber strength and orientation. When the aneurysm enlarges, the amount and organization of the collagen fibers change, potentially increasing the risk of rupture. We studied the directional organization and molecular strength of the collagen fibers layer by layer across the walls of four aneurysms in order to measure their mechanical integrity. The technique incorporates the birefringent properties of collagen, enabling us to use linearly polarized light for measuring the orientation of the fibers, and the Sénarmont compensator to measure the birefringence and thus mechanical strength. Intact aneurysms were obtained at autopsy, fixed at physiological pressure, sectioned at 4 μm, and stained with 0.05% picrosirius red. By combining birefringence and orientation data we estimated tensile strength as a function of direction on the aneurysmal wall. The average breaking strength of the wall ranged from 0.73 to 1.9 MPa. Comparing the weakest to the strongest direction, the breaking strength varied by a factor of up to 2×, implying a significant degree of mechanical anisotropy.

Collagen Biomechanics in Cerebral Arteries and Bifurcations Assessed by Polarizing Microscopy

Collagen is the main matrix protein of the artery wall. We have used the known correlation between collagen birefringence and its mechanical properties to assess the wall structural integrity in brain arteries and their bifurcation regions, which are the sites of formation of saccular aneurysms. Segments of 28 brain arteries, including bifurcations, were pressure fixed and sectioned in one of three orthogonal planes. Measurements were taken by polarizing microscopy of the birefringence of collagen fibers at the apex of bifurcations and in the main layers of the artery wall – adventitia, media and intima. Dimensional data were obtained of the layers in order to estimate wall properties. Along the apex of the flow divider we measured a narrow band of collagen (birefringence 30% higher than the adjacent adventitia) providing strength and stiffness in that region. There is a thin cell-free outer layer of the tunica media (mean thickness 11 µm) comprised of densely packed coaligned collagen with high birefringence. From the fiber birefringence and directional alignment of the individual layers we calculated that the adventitia contributes about one third of circumferential and almost all of longitudinal strength of intracranial arteries.

Medial collagen organization in human arteries of the heart and brain by polarized light microscopy.

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.

Layered structure of saccular aneurysms assessed by collagen birefringence

Cerebral aneurysms are composed principally of collagen, a birefringent protein which is responsible for withstanding the forces of blood pressure. The known correlation between collagen birefringence and its mechanics provides the basis for using polarizing microscopy to evaluate the strength of collagen, layer by layer across the aneurysmal wall. In order to obtain better quantitative measurements, several birefringent enhancement stains were investigated. We concluded that sirius red F3B, at a concentration of 0.05% in saturated picric acid, is an excellent stain to enable measurement of both birefringence and directional organization on the same tissue sections. Six aneurysms from autopsy, fixed at 120 mmHg, and one surgical specimen were cut at 4 microns to provide sets of tangential sections. The polarizing optics emphasizes the multi-layered structure of the aneurysmal wall with the mean fiber alignments distinguishing one layer from another. Birefringence measurements showed that the outer third of the wall had mainly higher strength collagen, although not as high as nearby artery adventitia. The inner layers of the aneurysms had intermediate values, similar to the artery media and subendothelium. Our results are consistent with a model of aneurysmal enlargement that requires the reorganization of higher strength outer fibers while new collagen is added to the inner layers.

Stereological analysis of the layered collagen of human intracranial aneurysms

Intracranial saccular aneurysms are balloon‐like distensions of the arterial wall; they increase in size gradually, a few to the point of bleeding or catastrophic rupture. Collagen is the primary structural component of the aneurysmal wall, and because only a small fraction of aneurysms fail, the collagen fabric must effectively reorganize in order to maintain mechanical integrity as an aneurysm changes size. Data were obtained from four human aneurysms, fixed at 110 mmHg of distending pressure with 10% buffered formalin, and sectioned completely through at 4 μm thickness. Each set of measurements included groups of data taken layer by layer from a radial corridor across the aneurysm wall. Each three‐dimensional orientation measurement, for which we used a Zeiss polarizing microscope with a universal stage attachment, is defined by an azimuth and elevation angle relative to the section plane. We compared the interdependence of these measured angles with a mathematical model based on fibres following great circle trajectories, and related the measured azimuth and elevation angles to the relative depth of the section into the aneurysm. Data were plotted on Lambert equal‐area projections, along with the theoretical relation between azimuth and elevation, that included wall thickness and depth of sectioning. The graphical relationship between measured azimuth and elevation for collagen fibres across the layered fabric of the aneurysmal wall is consistent with the theoretical great circle trajectories for collagen fibre alignment. Analysis was based on statistics for spherical data to give values for the mean orientation and the circular standard deviations (CSD) about that mean. The results indicate that any given region on the aneurysm wall is made up of many, very thin sublayers, most of which have a relatively coherent organization (mean CSD 8°). These measurements agree well with the mathematical model and, when considered collectively, the layers provide a balanced distribution for bearing the biaxial tensile stress of the wall.