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Table of Contents
Vol. 40, No. 4, 2003
Issue release date: July–August 2003
Section title: Research Paper
J Vasc Res 2003;40:406–415
(DOI:10.1159/000072831)

Collagen Biomechanics in Cerebral Arteries and Bifurcations Assessed by Polarizing Microscopy

Rowe A.J. · Finlay H.M. · Canham P.B.
Department of Medical Biophysics, University of Western Ontario, London, Ont., Canada
email Corresponding Author

Abstract

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.

© 2003 S. Karger AG, Basel


  

Key Words

  • Bifurcations
  • Collagen
  • Extracellular matrix
  • Tunica adventitia
  • Tunica media
  • Polarized light

 Introduction

The bifurcation regions of human brain arteries deserve scientific attention because of their vulnerability to forming saccular aneurysms [1, 2]. Most bifurcations of the cerebral vasculature are structurally stable, but a small number develop a weakness that causes the wall to expand outwardly in the region near the flow divider of the branching artery. Once the small ‘blister-like’ lesion starts, the distending forces of blood pressure contribute to the processes of enlargement, shape changes and eventual rupture. These lesions lack the organized layers of elastin and smooth muscle cells that are part of the structure of the parent vessel wall. Collagen remains the dominant structural protein and is thus critically involved in the gradual remodeling and weakening of the artery and the aneurysmal wall.

Brain arteries are muscular arteries with a composite structure of smooth muscle cells and extracellular matrix fibers of collagen and elastin. The outer layer, the adventitia, is made up of bundles of wavy collagen fibers mostly oriented in a longitudinal direction, but also some with helical and circumferential alignments [3]. Cerebral arteries lack the external elastic lamina found in systemic arteries, and the boundary between the adventitia and the media is generally taken as the edge of the smooth muscle layer [1, 4]. The birefringent optical properties of collagen permit a better determination of the boundary between media and adventitia in brain arteries, and polarized light microscopy of tangentially cut sections reveals a thin outer layer of closely packed collagen fibers belonging to the tunica media but external to the medial smooth muscle cells. The tunica media in its entirety, including layers of smooth muscle cells, is the dominant layer of the brain artery. Smooth muscle cells, comprising approximately two thirds by volume (monkey brain arteries studied by electron microscopy [5] and human brain arteries by light microscopy [6]), are highly aligned circumferentially and are parallel to the extracellular matrix of collagen fibers [1, 6, 7]. The tunica intima consists of a layer of endothelial cells, and a generally thin subendothelium separated from the media by a well-defined internal elastic lamina [1, 8].

The region where blood vessels branch, or come together, is mechanically complicated and more varied structurally. Flaring or tapered luminal caliber, widely varying branch angles, curving vessels and varied daughter vessel size constitute an array of factors that must be accommodated in the structure and mechanics for withstanding blood pressure. Within the general region of the bifurcation, but away from the flow divider, the structure is similar to and continuous with the three layers of the nearby arteries [9, 10]. There is a transition region of the trunk vessel just proximal to the apex which has multidirectional layered muscle cells in the tunica media, and extracellular matrix collagen fibers assumed to be parallel and similarly aligned to the cells (fig. 1) [9]. The flow divider region is very differently structured with a discontinuity of the muscle cells of the tunica media, identified by Stehbens [1] and Hassler [11] as a medial gap or defect. As a result, the adventitial fibers are ‘filling in’ up to the internal elastic lamina. In this region, running in the direction of the ridge of the flow divider, there is a narrow band of highly aligned tendon-like collagen. This collagen band is strongly birefringent and is observed best under polarized light [10]. The question arises whether this region of the medial gap may be associated with collagen fibers from the outer media or aligned fibers of collagen from the adventitia.

FIG01

Fig. 1. Schematic of an artery bifurcation showing the three sectioning planes used in the study. Attention is drawn to the apical ridge visible in the longitudinal perpendicular plane of sectioning.

The primary stress as a result of blood pressure in the straight section of blood vessels is circumferential, or hoop stress, and the organization of the extracellular matrix fibers and cellular network of smooth muscle reflects the mechanics of bearing that pressure load. In addition there are longitudinal and obliquely arranged fibers aligned to resist the longitudinal stress, which is half the circumferential stress [12]. The integrity of the artery wall depends in part on both the strength and organization of the extracellular matrix fibers, properties that may be examined using polarizing microscopy. The birefringence of collagen is related to mechanical strength through the molecular cross-linked structure and the diameter of its fibers [13, 14]. In this study we have undertaken to investigate the birefringence of the collagen fabric in the bifurcation region to look for structural inadequacies related to mechanical stress.

 

 Methods

For the main part of the study involving branch sites, 16 artery bifurcations were obtained from 15 autopsies (9 male, 6 female) between the ages of 30 and 85 years. Causes of death were cardiac arrest in 3, myocardial infarction in 3, dementia in 2, liver cirrhosis in 2, and 1 each of pneumonia, post-heart transplant, congestive heart failure, Alzheimer’s disease and leukemia. Eight of the bifurcations were from the middle cerebral artery, which is the most common site of aneurysm formation. Of the remaining half, 7 were from the basilar artery bifurcation and 1 was from the bifurcation of the posterior cerebral artery and the posterior communicating artery. Segments of the arteries were ligated, cannulated and tested for leaks with saline. They were then pressure distended and fixed in 10% neutral buffered formalin. Ten were fixed at physiological pressure between 110 and 120 mm Hg, 5 were fixed at 30 mm Hg and 1 was fixed at 60 mm Hg (the lower pressure samples drawn from another study). The bifurcation regions were paraffin embedded and sectioned at 4-μm thickness, which is the preferred sectioning thickness for polarized light microscopy [3, 15]. The sections were stained with sirius red F3B in 0.05% saturated picric acid to enhance the natural birefringence of collagen. Picrosirius red has been shown to increase considerably the sensitivity and resolution of polarized light microscopy [16]. Three orthogonal cutting planes were used to enable observation of the apical ridge from different directions (fig. 1). Seven bifurcations were sectioned in the longitudinal perpendicular plane, which slices through the apex to give a profile of the flow divider. Five were cut in cross section and 4 were sectioned in the longitudinal planar direction to reveal the width of the medial gap. Micrographs were taken of each bifurcation to include the region of the apex as well as a tangential section of adjacent artery wall in which the fibers were oriented in the plane of the section.

The method of measuring phase retardation is well established [17, 18, 19]. Collagen has two indices of refraction, no and ne, the difference indicating the strength of birefringence. When linearly polarized light passes through a birefringent tissue, it is resolved into two rays, an ordinary and an extraordinary ray. These pass through the tissue at different velocities, resulting in a phase difference, or retardation of one ray relative to the other. We measured retardation on a Nikon Optiphot-Pol polarized light microscope with a Sénarmont compensator and a narrow-band interference filter, which produces near monochromatic light (λ = 546 nm). The Sénarmont compensator is a λ/4 plate inserted between the specimen and the analyzer that retards one of the emerging rays by a quarter wavelength, converting the elliptically polarized light emerging from the fibers back to linearly polarized light at a rotated angle. This angle, θ, is proportional to the phase retardation, and is measured by rotating the analyzer to obtain an extinction position. Retardation, B (nm), is calculated from B = (λ/180) × θ, where λ is the wavelength of the incoming light. The retardation can also be expressed in terms of the thickness of birefringent material by B = t(ne – no), where t is the thickness of the section or the fiber and ne – no is the difference in the refractive indices. Each measurement of birefringence is made from a small region of a single collagen fiber or group of fibers, and is independent of its waviness.

From each of the longitudinal perpendicular sections (fig. 1, a) we sampled four to ten areas in the region of the apex for the retardation measurements, including each end of the medial gap and regions along the apical ridge. In order to compare the apex region with the straight vessel wall, we included a corresponding region of the artery in which the section plane was approximately tangential. Tangential and near tangential sections include those in which the outer part of the artery wall is sectioned (fig. 2), and may be planar or oblique to the surface. In some cases the asymmetry of the bifurcation provides the opportunity to get these data from a region of adjacent vessel from the same section, but in others a different section was used, stained and processed at the same time. We took ten measurements from each layer across the wall within each of the designated regions and carefully delineated those regions on micrographs.

FIG02

Fig. 2. Circularly polarized light micrographs of two middle cerebral arteries from different autopsies. The cutting plane (shown schematically) for both arteries is close to tangential. a Curved artery and intersects with the lumen. Individual layers are identified – a thin subendothelium (s.e.), cellular media, outer media and tunica adventitia. a, b The high birefringence of the outer media shows clearly, with the collagen fibers continuous with the fine fibers of the cellular media. b Many less coherent fine collagen fibers (0.5–2 μm) that border the adventitial fibers.

A complementary part of the study involved the determination of the three-dimensional orientation of collagen fibers. These orientation measurements were made using the universal stage attachment to a Zeiss polarizing microscope (detailed methods provided in other studies [8, 15]). The universal stage permits the microscope section to be rotated about three orthogonal axes in such a way that the azimuth angle and the elevation angle of an individual birefringent fiber can be measured precisely. These two angles define the alignment of the fiber in three dimensions relative to the plane of the section. Since measurements of retardation are dependent on the inclination of the fibers out of the section plane (i.e. the elevation angle), we measured the three-dimensional alignment of the collagen in the region of the apex in sections cut in the longitudinal perpendicular plane (fig. 1, a). Measurements were made from collagen of the media and the adventitia from either the same or adjacent sections to those used for the retardation measurements. Readings from the adventitia were made through the apical ridge, and since there is an absence of media right at the apex, the media measurements were made from each side of the medial gap. Fifty orientation measurements were made from each layer, and the results were analyzed using circular statistics [15, 20]. The measurement of retardation is relatively unaffected by the elevation angle for angles below 10°, but the underestimation becomes significant beyond an elevation angle of 30° (an angle at which the reduction is approximately 15% [21]).

A further part of the methods was to determine the influence of the section thickness. Since birefringence is dependent on the thickness of the birefringent material, both the fiber thickness and the section thickness are factors for consideration. When the collagen fiber is less than the section thickness, it is the fiber thickness that determines birefringence. However, when the fibers are tightly packed and parallel the light travels through a series of coherent collagen fibers that comprise the full thickness of the section. The retardation measurements of artery wall show a much greater birefringence in the outer media and the adventitia than within the media. We investigated the possible correlation of this birefringence with section thickness for each of the layers of the artery wall. Six arteries (two vertebral, one basilar and three middle cerebral) from 6 autopsies (2 male and 4 female, ages 41–84) were used. Tangential sections were cut from two of the vessels at three different thicknesses and cut from the other four vessels at two thicknesses, ranging from 3 to 7 μm. Twelve measurements of birefringence were taken from the media, the outer media and the adventitia from each of these 14 sections.

 

 Results

Examination of 12 bifurcations sectioned in the longitudinal perpendicular plane (fig. 1, a) and in cross section (fig. 1, c) revealed that the collagen of the tendon-like band through the apical ridge appeared to be continuous with the adventitial layer. The media tapered abruptly to an end just adjacent to the medial gap. In some of the bifurcations there was a visible layer of highly birefringent collagen on the outer surface of the media, coaligned with the smooth muscle and collagen fibers of the media. This outer media layer is particularly distinctive with polarized light when the sectioning plane is slightly oblique from tangential or perfectly tangential (i.e. parallel to the vessel axis), and was seen in all the brain arteries that we examined. Examples of this layer in tangential sections of two middle cerebral arteries are shown in figure 2, obtained by circularly polarized light optics under which all orientations of birefringent fibers appear bright. The parallel alignment of the collagen of the cellular media and outer media is clearly visible, in contrast to the thick wavy fibers aligned mainly longitudinally in the adventitia. Within the tunica media, the curvature of the muscle cells and groups of cells at their boundary can be seen as a ‘scalloped’ outer edge to the cellular zone of the media.

For the 7 bifurcations sectioned in the longitudinal perpendicular plane (fig. 1, a), we compared the birefringence in the region of the medial gap to that of a nearby tangential section from the same bifurcation (table 1). The mean values of collagen birefringence of the adventitia and outer media layers are presented relative to that of the cellular media in the tangential sections because this collagen showed the lowest coefficient of variation among bifurcations. The outer media was measurable in four of the tangential sections where it was generally found to be more birefringent than the neighboring adventitia (table 2).

TAB01

Table 1. Birefringence of the adventitial fibers and of the nearby media in the medial gap region of bifurcations, sectioned in the longitudinal perpendicular direction

TAB02

Table 2. Birefringence of the adventitia and outer media, relative to the cellular media, in tangential sections of bifurcations cut in the longitudinal perpendicular direction

Measurements from cross sections (fig. 1, c) revealed that the collagen in the medial gap was more birefringent than the adjacent adventitia as well as the adventitia of a distal region of the bifurcation (table 3). However, we also noted the higher coherence of the collagen in the region of the gap and believe that this may have contributed to its higher birefringence. The collagen of the tunica media in these sections had increased birefringence and coherence when closer to the apex (table 3). The very few sections at the start of the sectioning into the ridge tend to be influenced by the asymmetrical geometry of most of the bifurcations, and are sensitive to histological artifact. In some of the sections studied, the geometry was such that it did not reveal the discontinuity of the tunica media through the apex. Instead, there was a thin layer of medial collagen on either side of the adventitial band in the apex, which was more highly aligned and more birefringent than the majority of the artery wall in the balance of the two daughter vessels.

TAB03

Table 3. Birefringence of the adventitia and cellular media of the bifurcations and distal regions of vessels cut in cross section (see fig. 1, c)

Longitudinal planar sections (fig. 1, b) were also examined to determine the shape and width of the medial gap. With this sectioning geometry, the collagen band of the apical ridge was perpendicular to the section plane, and therefore birefringence measurements could not be made. However, these sections did reveal that there was a wide range of bifurcation angles within the brain arteries, and that the medial gap was evident in all of them. The width of the gap, from four arteries sectioned in this plane as well as from five arteries from a previous study, varied widely from one of 25 μm to one having an extreme width of 1,100 μm, possibly the start of an aneurysm. Six of the gaps were between 50 and 200 μm and one was 350 μm, with a mean value of 244 μm. From the longitudinal perpendicular sections, the length of the medial gap varied from 88 to 1,750 μm (fig. 3) with a mean value of 532 μm, and the breadth was between 20 and 55 μm with a mean of 34 μm. It is noted that these measurements of medial gap length are influenced partly by the choice of midline section, and on its angle of sectioning. The long apical ridge shown in figure 3 reveals the highly aligned collagen band that extends between the two regions above and below the apex center where the medial layer is seen to end abruptly. In summary, the normal medial gap (i.e. smooth muscle gap) in three dimensions is a long narrow saddle of collagen following the curving apical ridge, which reveals its length on perpendicular sections (fig. 1, a, 3) and its width on longitudinal planar sections (fig. 1, b).

FIG03

Fig. 3. Circularly polarized light micrographs of a longitudinal perpendicular section of a middle cerebral bifurcation, and of a cross-sectioned basilar fenestration bifurcation (cutting planes shown schematically). a Apical ridge with a long continuum of the tendon-like fibers. b Cross section. Very strongly birefringent collagen of the ridge is only evident at the junction of the two daughter vessels. A region of intimal thickening is seen in the vessel to the left.

Results from the three-dimensional orientation measurements showed that in five of the seven longitudinal perpendicular sections examined, the mean inclination angles of the collagen of both the media and adventitia were less than 10°, in one section both were less than 20°, and in the other they were less than 30°. This translates to an underestimate of the birefringence of the collagen by a maximum of 15% in the case of a 30° angle.

When we examined the birefringence of the various regions of the vessel walls with arteries cut at different section thickness, we compared regions of the media, the strongly birefringent outer media and the adventitia. The defining relationship is B = t(ne – no) for birefringence, B, thickness, t, and the two indices of refraction, ne and no. For regions of densely packed parallel fibers, the phase retardation can be expected to depend more directly on the section thickness than on individual fiber size. Results for each layer were plotted as the ratio of birefringence measurements (Bratio) as a function of the ratio of the section thicknesses (Tratio) of each pair of measurements, i.e. B1/B2 versus T1/T2 where Bi and Ti are the birefringence and thickness measurements for section i. Statistical analysis revealed a regression slope of near unity (Bratio = 0.988 Tratio –0.01) for the outer medial fibers. The adventitia had a lower slope (Bratio = 0.709 Tratio +0.24). There is also a much lower slope for the muscle cell region of the media (Bratio = 0.404 Tratio +0.55) indicating that the fiber diameter is the determining factor in the birefringence of that part of the tunica media. The implication of this analysis is that the collagen of the outer media occupies the full thickness of the section, while in the main part of the media, where the extracellular matrix is approximately 35% by volume, the measurements of birefringence are taken from single collagen fibers that have a diameter less than the section thickness.

Since variations in birefringence and the directional anisotropy of the four layers of the brain artery wall are known, it is possible to make an estimate of ultimate tensile strengths, both circumferentially and longitudinally, in the straight part of the artery wall. Calculations were based on the measurements of birefringence and the data from Doillon et al. [13] on dermal wound healing with the assumption that these wound-healing results are applicable to the artery wall. Results are shown in table 4 to which we have added new data on the subendothelial layer [3]. The four layers are treated separately, and for each we incorporated (1) tissue strength correlating with birefringence [13, 22], (2) percentage of the matrix occupied by collagen, (3) wall dimensions measured from the histological sections, and (4) previously published results on collagen alignment [3]. Elastin is not included in the calculations. It is a high-strain, low-strength matrix protein that is a major component of elastic arteries, but much less a component in muscular arteries. Waviness has not been considered as a factor in calculating the integrated wall strength since it primarily affects the tolerance of strain before failure. The tunica media, including the outer media, has fibers precisely aligned circumferentially (thus contributing only to circumferential strength) and the components of tissue in the adventitia and the subendothelium are resolved from the helical angle, Φ, into the circumferential and longitudinal directions using cos2(Φ) [22, 23]. Combining all four layers, the integrated wall strength is 3.6 MPa for longitudinal strength, and 2.4 MPa for circumferential strength. An assumption is made for the volume fraction of the collagen within each region of the wall. For the purposes of the strength calculation we used the same birefringence for individual collagen fibers of the outer media as that measured in the cellular media, but we assumed a packing density of 150% relative to that of skin. In this way the higher birefringence measured in this layer is accounted for in our calculations. The volume fraction of collagen in the different layers is an assumed quantity. To illustrate the sensitivity to this value, we repeated the calculations using a different assumed volume fraction (75 instead of 100%) for the adventitia – the layer with the greatest influence. The result is a reduction of the total longitudinal strength component by 23% and the circumferential component by 9%.

TAB04

Table 4. Assessment of composite strength of the cerebral artery wall

 

 Discussion

Variations in geometry of the branching region of arteries make it difficult to generalize about overall structure. However, a consistent finding outside the proximity of the flow divider is the three-layered wall with the dominant circumferentially organized muscle cells of the tunica media. The tunica adventitia continues with its characteristic organizational structure of strongly birefringent wavy collagen fibers with a wide distribution of alignment, from longitudinal to circumferential, while the tunica intima is usually very thin. We focused attention on the cell-free thin outer media, exploring its nature as coherently aligned in the circumferential direction and strongly birefringent, and whether it might be continuous with the fibers of the medial gap.

The identification of a coherent and strongly birefringent outer media of densely packed fibers was consistent among the brain arteries of the study. However, examination of similarly sectioned and stained coronary and internal thoracic human arteries, also muscular arteries, did not reveal the same structure. The birefringence of the outer media fibers is much higher than the extracellular matrix fibers of the cellular media [i.e. 130 ± (SD) 46 nm compared to 63 ± 18 nm], and on average the outer media birefringence is higher than that of the adventitia. An explanation of the high birefringence of the outer media was explored. In general the birefringence of collagen fibers depends, to a large measure, on fiber size [13] but also on the section thickness [17, 21]. Results from measurements of birefringence of the various layers of artery wall were taken from sections of different thicknesses. It was only for the outer media fibers that the birefringence was nearly directly proportional to section thickness. Our interpretation is that the outer medial fibers are a continuation of the fibers among the cells of the media, augmented in birefringence because of the continued coherence of fibers and increased matrix volume fraction (approaching 100 instead of 35%).

The true width of the coherent outer media is difficult to measure directly from cross sections, and was obtained from obliquely cut sections on which the measurement could be made directly with the polarizing microscope. When cross-sectioned vessels are viewed with polarized light, the longitudinally aligned adventitial fibers are not visible, but those that are aligned either helically or circumferentially will be bright, and their predominant orientation appears to be around the perimeter of the vessel. This masks the much thinner circumferential outer media that is similarly aligned. Data were obtained from several sections of six obliquely cut arteries, and converted to radial distances using the vessel radius, projection width of the layer on the microscope, and the assumption of uniformity of layer thickness and cylindrical symmetry of the artery. The average value of 11 μm (SD 5.3 μm) and its slight dependence on vessel size indicate that its anatomical role may be mainly to close off the cellular zone of the tunica media. Brain arteries are distinct from other arteries in that they do not have an external elastic lamina, hence this outer coherent zone of media collagen acts to separate the cellular media from the differently structured adventitia. We note that when making geometric measurements from these brain arteries we have not taken into account any distortion caused by shrinkage due to paraffin embedding. This shrinkage has been measured for brain arteries at 8.7% radially and 14.5% longitudinally [6]. The collagen fibers of the media in tangential sections (fig. 2) remain straight despite any shrinkage, although there is histological distortion in the thin apical ridge in the longitudinal perpendicular section in figure 3. We assume that our strength calculations based on the work of Doillon et al. [13] will not be influenced since that study employed the same tissue preparation and staining methods as ours.

The high degree of alignment makes untenable the conclusion that these outer medial fibers are solely type I fibers because such a layer would be a mechanical ‘straight jacket’ allowing at most 3–5% strain before major tearing of the fibers (well-known mechanics of type I collagen in tendon [24]). The general interpretation of the typing of the fibrillary collagen in the media of muscular arteries is that of type III fibers, or a complex of type I and III [25]. The work on brain arteries by Austin et al. [26] supports, in our view, the interpretation that the medial collagen is a type I/III complex. Their published micrographs of the fluorescently identified type I collagen show clearly the thin band of labeled fibers of the outer edge of the tunica media (enhanced by the obliqueness of the sectioning plane, as in our study). That study did not include the complementary labeling of type III fibers; however, the fibronectin, which is primarily associated with the cellular component of the media, did not have any increased concentration at the outer edge of the tunica media. In a comparison of saccular aneurysms with the internal carotid artery studied by immunofluorescence [27] type I collagen was seen in all layers, although less strongly in the media, type III in all layers, type IV in the subendothelium and the media, type V in all the layers but predominantly in the adventitia, and type VI only in the adventitia.

From the pressure cycling studies of Macfarlane et al. [28] we have strong evidence that the branching region of brain arteries is relatively stiff mechanically in comparison to nearby unbranching segments. The tendon-like ridge of the flow divider would contribute to that stiffness, particularly if it were primarily type I fibers with a high degree of alignment. The strength of the birefringence along the ridge was slightly higher than the nearby adventitial fibers on the same tissue section, 2.96 compared to 2.28 (both expressed as a ratio of birefringence as shown in table 3). Our interpretation of the collagen fibers along the ridge (and thus in the medial gap or defect) is that they are primarily type I fibers, partly because of the much greater breadth of the gap and apical ridge, 20–55 compared to 11 μm for the full width of the outer media, and also that the coherence and parallel alignment of these fibers are continuous with the adventitia of the two daughter branches distal to the apex.

Strength of birefringence has been shown to correlate strongly with tissue mechanical strength and size of the collagen fibers (experiments on dermal wound healing in the guinea pig by Doillon et al. [13]). Because of the closely matched histological and measurement methods between our studies on vascular tissue and those of Doillon et al. on wound healing we assumed we could calculate relative strength values for the components of the blood vessel wall. From their published data we derived the relationship of tensile tissue strength, σmax, versus birefringence, B: σmax = 0.000304 B2.33, where the units of σmax are MPa, and of B are nm. We have done similar mathematical modeling for predicting vascular wall strength for brain aneurysms [22].

The waviness of the matrix proteins is acknowledged to be a contributing factor in the mechanics of layered composite tissues, and would affect the degree of fiber recruitment in the direction of tension as it gradually is loaded to failure. However, in our theoretical calculation of ultimate strength we have assumed that each layered component contributes proportionately to its individual birefringence and alignment, and thus is not dependent on fiber waviness. The adventitia is the only layer contributing significant strength in both the circumferential and longitudinal directions (table 4), and since its fibers have relatively high birefringence, the adventitia dominates the anisotropic strength characteristics of the composite brain artery. It is of interest that despite its thin radial dimension the outer media, with its densely packed coaligned fibers, does contribute to the wall strength in the circumferential direction. Our estimates of the circumferential and longitudinal tensile strength in the straight portion of brain artery are 2.4 and 3.6 MPa, values that are higher than the mechanical measurements for muscular arteries reported by Yamada [29] (circumferential σmax = 1.0 MPa, longitudinal σmax = 1.3 MPa); however, they likewise indicate greater strength for the longitudinal compared to the circumferential direction. When the same calculations are applied to the flow divider with a birefringence of 129 nm (table 1), the strength along the ridge is 25.1 MPa. This estimate of strength is consistent with the theme of a tendon-like structure with very high strength and high stiffness in the direction of the apical ridge fibers.

The nonuniform collagen framework of the branching region of brain arteries invites further consideration of why aneurysms develop. A parallel is drawn with manufactured fabrics strengthened by reinforced seams or inserted stiffeners (e.g. tent fabrics and clothing). The immediately adjacent material is commonly exposed to higher strains and local stresses which predispose these sites to wear and failure. Likewise, the unidirectional strength and stiffness of the apical ridge may expose the adjacent wall tissue to stresses from cyclic blood pressure that the vessel wall is unsuited to withstand. Researchers studying brain aneurysm development in a rat model demonstrated the formation of early aneurysms adjacent to an ‘intimal pad’ (interpreted as apical ridge in our study) [30, 31]. Future experiments including cyclic high-pressure loading of brain artery branches might reveal zones of nonuniform elastic (or inelastic) deformation alongside the apical ridge.

 

 Acknowledgments

The authors thank Linda Jackson and Dr. John Kiernan for assistance with histology, and summer student Binh Nguyen for help with measurements. We acknowledge, too, Dr. Rob Hammond, Department of Pathology, for advice and support in obtaining artery specimens. The work was supported by the Canadian Institutes of Health Research (grant No. MOP38026) and an NIH grant to Dr. Jay Humphrey (HL54957) that helped fund the summer student A. Rowe.


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  17. Bennett HS: The microscopical investigation of biological materials with polarized light; in McClung-Jones R (ed): McClung’s Handbook of Microscopical Technique. New York, Hafner, 1950, pp 591–677.
  18. Whittaker P, Schwab ME, Canham PB: The molecular organization of collagen in saccular aneurysms assessed by polarized light microscopy. Connect Tissue Res 1988;17:43–54.
  19. Canham PB, Finlay HM, Kiernan JA, Ferguson GG: Layered structure of saccular aneurysms assessed by collagen birefringence. Neurol Res 1999;21:618–626.
  20. Fisher NI, Lewis T, Embleton BJJ: Statistical Analysis of Spherical Data. New York, Cambridge University Press, 1987, pp 101–151.
  21. Whittaker P: Structural Analysis of Cardiovascular Tissue Using Quantitative Polarised Light Microscopy; PhD thesis University of Western Ontario, 1986, pp 33–34.
  22. MacDonald DJ, Finlay HM, Canham PB: Directional wall strength in saccular brain aneurysms from polarized light microscopy. Ann Biomed Eng 1999;28:533–542.

    External Resources

  23. Lanir Y, Lichtenstein O, Imanuel O: Optimal design of biaxial tests for structural material characterization of flat tissues. J Biomech Eng 1996;118:41–47.
  24. Alexander RM: Animal Mechanics, ed 2. Oxford, Blackwell Scientific, 1983.
  25. Fleischmajer R, Perlish JS, Burgeson RE, Shaikh-Bahai F, Timpl R: Type I and type III collagen interactions during fibrillogenesis. Ann NY Acad Sci 1990;580:161–175.
  26. Austin G, Fisher S, Dickson D, Anderson D, Richardson S: The significance of the extracellular matrix in intracranial aneurysms. Ann Clin Lab Sci 1993;23:97–105.
  27. Mimata C, Kitaoka M, Nagahiro S, Iyama K, Hori H, Yoshioka H, Ushio Y: Differential distribution and expressions of collagens in the cerebral aneurysmal wall. Acta Neuropathol 1997;94:197–206.
  28. Macfarlane TWR, Canham PB, Roach MR: Shape changes at the apex of isolated human cerebral bifurcations with changes in transmural pressure. Stroke 1983;14:70–76.
  29. Yamada H: Strength of Biological Materials. Baltimore, Williams & Wilkins, 1970, pp 114–130.
  30. Kojima M, Handa H, Hashimoto N, Kim C, Hazama F: Early changes of experimentally induced cerebral aneurysms in rats: Scanning electron microscope study. Stroke 1986;17:835–841.
  31. Kim C, Kikuchi H, Hashimoto N, Kojima M, Kang Y, Hazama F: Involvement of internal elastic lamina in development of induced cerebral aneurysms in rats. Stroke 1988;19:507–511.

  

Author Contacts

Dr. Peter B. Canham
Department of Medical Biophysics, Faculty of Medicine and Dentistry
University of Western Ontario
London, Ont. N6A 5C1 (Canada)
Tel. +1 519 661 3053, Fax +1 519 661 2123, E-Mail pcanham@uwo.ca

  

Article Information

Received: January 31, 2002
Accepted after revision: May 22, 2003
Published online: August 8, 2003
Number of Print Pages : 10
Number of Figures : 3, Number of Tables : 4, Number of References : 31

  

Publication Details

Journal of Vascular Research (Incorporating International Journal of Microcirculation)
Founded 1964 as Angiologica by M. Comèl and L. Laszt (1964–1973) continued as Blood Vessels by J.A. Bevan (1974–1991)
Official Journal of the European Society for Microcirculation

Vol. 40, No. 4, Year 2003 (Cover Date: July-August 2003)

Journal Editor: U. Pohl, Munich
ISSN: 1018–1172 (print), 1423–0135 (Online)

For additional information: http://www.karger.com/jvr


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

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.

© 2003 S. Karger AG, Basel


  

Author Contacts

Dr. Peter B. Canham
Department of Medical Biophysics, Faculty of Medicine and Dentistry
University of Western Ontario
London, Ont. N6A 5C1 (Canada)
Tel. +1 519 661 3053, Fax +1 519 661 2123, E-Mail pcanham@uwo.ca

  

Article Information

Received: January 31, 2002
Accepted after revision: May 22, 2003
Published online: August 8, 2003
Number of Print Pages : 10
Number of Figures : 3, Number of Tables : 4, Number of References : 31

  

Publication Details

Journal of Vascular Research (Incorporating International Journal of Microcirculation)
Founded 1964 as Angiologica by M. Comèl and L. Laszt (1964–1973) continued as Blood Vessels by J.A. Bevan (1974–1991)
Official Journal of the European Society for Microcirculation

Vol. 40, No. 4, Year 2003 (Cover Date: July-August 2003)

Journal Editor: U. Pohl, Munich
ISSN: 1018–1172 (print), 1423–0135 (Online)

For additional information: http://www.karger.com/jvr


Article / Publication Details

First-Page Preview
Abstract of Research Paper

Received: 1/31/2002
Accepted: 5/22/2003
Published online: 9/26/2003
Issue release date: July–August 2003

Number of Print Pages: 10
Number of Figures: 3
Number of Tables: 4

ISSN: 1018-1172 (Print)
eISSN: 1423-0135 (Online)

For additional information: http://www.karger.com/JVR


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

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  2. Ferguson GG: Intracranial arterial aneurysms; A surgical perspective; in Toole JF (ed): Handbook of Clinical Neurology, vol 2: Vascular Diseases. Part 3. New York, Elsevier, 1989.
  3. Finlay HM, McCullough L, Canham PB: Three-dimensional collagen organization of human brain arteries at different transmural pressures. J Vasc Res 1995;32:301–312.
  4. Ross MH, Romrell LJ, Kaye GI: Histology; a Text and Atlas, ed 3. Baltimore, Williams & Wilkins, 1995, pp 306–308.
  5. Canham PB, Henderson RM, Peters MW: Coalignment of the muscle cell and nucleus, cell geometry and Vv in the tunica media of monkey cerebral arteries, by electron microscopy. J Microsc 1982;127:311–319.
  6. Walmsley JG: Vascular smooth muscle orientation in straight portions of human cerebral arteries. J Microsc 1983;131:361–375.
  7. Canham PB, Talman EA, Finlay HM, Dixon JG: Medial collagen organization in human arteries of the heart and brain by polarized light microscopy. Connect Tissue Res 1991;26:121–134.
  8. Finlay HM, Whittaker P, Hicks JG, Taylor CP, Park YW, Canham PB: Spatial orientation of arterial sections determined from aligned vascular smooth muscle. J Microsc 1989;155:213–226.
  9. Walmsley JG, Campling MR, Chertkow HM: Interrelationships among wall structure, smooth muscle orientation, and contraction in human major cerebral arteries. Stroke 1983;14:781–790.
  10. Finlay HM, Whittaker P, Canham PB: Collagen organization in the branching region of human brain arteries. Stroke 1998;29:1595–1601.
  11. Hassler O: Morphological Studies of the Large Cerebral Arteries, with Reference to the Aetiology of Subarachnoid Haemorrhage. Acta Psychiatr Neurol Scand 1961;154:1–145.
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  14. Wolman M, Gillman TA: Polarized light study of collagen in dermal wound healing. Br J Exp Pathol 1972;53:85–89.
  15. Canham PB, Finlay HM, Tong SY: Stereological analysis of the layered collagen of human intracranial aneurysms. J Microsc 1996;183:170–180.

    External Resources

  16. Junqueira LCU, Bignolas G, Brentani RR: Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 1979;11:447–455.
  17. Bennett HS: The microscopical investigation of biological materials with polarized light; in McClung-Jones R (ed): McClung’s Handbook of Microscopical Technique. New York, Hafner, 1950, pp 591–677.
  18. Whittaker P, Schwab ME, Canham PB: The molecular organization of collagen in saccular aneurysms assessed by polarized light microscopy. Connect Tissue Res 1988;17:43–54.
  19. Canham PB, Finlay HM, Kiernan JA, Ferguson GG: Layered structure of saccular aneurysms assessed by collagen birefringence. Neurol Res 1999;21:618–626.
  20. Fisher NI, Lewis T, Embleton BJJ: Statistical Analysis of Spherical Data. New York, Cambridge University Press, 1987, pp 101–151.
  21. Whittaker P: Structural Analysis of Cardiovascular Tissue Using Quantitative Polarised Light Microscopy; PhD thesis University of Western Ontario, 1986, pp 33–34.
  22. MacDonald DJ, Finlay HM, Canham PB: Directional wall strength in saccular brain aneurysms from polarized light microscopy. Ann Biomed Eng 1999;28:533–542.

    External Resources

  23. Lanir Y, Lichtenstein O, Imanuel O: Optimal design of biaxial tests for structural material characterization of flat tissues. J Biomech Eng 1996;118:41–47.
  24. Alexander RM: Animal Mechanics, ed 2. Oxford, Blackwell Scientific, 1983.
  25. Fleischmajer R, Perlish JS, Burgeson RE, Shaikh-Bahai F, Timpl R: Type I and type III collagen interactions during fibrillogenesis. Ann NY Acad Sci 1990;580:161–175.
  26. Austin G, Fisher S, Dickson D, Anderson D, Richardson S: The significance of the extracellular matrix in intracranial aneurysms. Ann Clin Lab Sci 1993;23:97–105.
  27. Mimata C, Kitaoka M, Nagahiro S, Iyama K, Hori H, Yoshioka H, Ushio Y: Differential distribution and expressions of collagens in the cerebral aneurysmal wall. Acta Neuropathol 1997;94:197–206.
  28. Macfarlane TWR, Canham PB, Roach MR: Shape changes at the apex of isolated human cerebral bifurcations with changes in transmural pressure. Stroke 1983;14:70–76.
  29. Yamada H: Strength of Biological Materials. Baltimore, Williams & Wilkins, 1970, pp 114–130.
  30. Kojima M, Handa H, Hashimoto N, Kim C, Hazama F: Early changes of experimentally induced cerebral aneurysms in rats: Scanning electron microscope study. Stroke 1986;17:835–841.
  31. Kim C, Kikuchi H, Hashimoto N, Kojima M, Kang Y, Hazama F: Involvement of internal elastic lamina in development of induced cerebral aneurysms in rats. Stroke 1988;19:507–511.