Erosion and rupture of surface layers in atherosclerotic plaque can cause heart attack and stroke; however, changes in luminal surface composition are incompletely defined. Laser-induced fluorescence spectroscopy (LIFS), with limited tissue penetration, was used to investigate the surface of unstable carotid plaque and correlated with microscopy, birefringence and gene expression. Arterial matrix collagens I, III and elastin were assessed in unstable plaques (n = 25) and reference left internal mammary arteries (LIMA, n = 10). LIFS in addition to selective histological staining with picrosirius red, Movat pentachrome and immunostaining revealed decreased elastin and increased collagen I and III (P < 0.05) in carotid plaque when compared with LIMA. Within plaque, collagen I was elevated in the internal carotid region versus the common carotid region. Polarized light microscopy detected layers of aligned collagen and associated mechanical rigidity of the fibrous cap. Microarray analysis of three carotid and three LIMA specimens confirmed up-regulation of collagen I, III and IV, lysyl oxidase and MMP-12. In conclusion, LIFS analysis coupled with microscopy revealed marked regional differences in collagen I, III and elastin in surface layers of carotid plaque; indicative of plaque instability. Birefringence measurements demonstrated mechanical rigidity and weakening of the fibrous cap with complementary changes in ECM gene expression.

Tissue remodeling during maturation, wound healing, and response to vascular stress involves molecular changes of collagen and elastin in the extracellular matrix (ECM). Two optical techniques are effective for investigating these changes--laser-induced fluorescence (LIF) spectroscopy and polarizing microscopy. LIF spectroscopy integrates the signal from both elastin and collagen cross-linked structure, whereas birefringence is a measure of only collagen. Our purpose is (1) to evaluate the rat tail tendon (RTT) spectroscopy against data from purified extracted protein standards and (2) to correlate the two optical techniques in the study of RTT and skin. Spectra from tissue samples from 27 male rats and from extracted elastin and collagen were obtained using LIF spectroscopy (357 nm). Birefringence was measured on 5-mum histological sections of the same tissue. Morphometric analysis reveals that elastin represents approximately 10% of tendon volume and contributes to RTT fluorescence. RTT maximum fluorescence emission intensity (FEI(max)), which includes collagen and elastin, increases with animal weight (R(2)=0.64). Birefringence, when plotted against weight, increases to a plateau (nonlinear correlation: R(2)=0.90), tendon having greater birefringence than skin. LIF spectroscopy and collagen fiber birefringence are shown to provide complementary measurements of molecular structure (tendon birefringence versus FEI(max) at R(2)=0.60).

Saccular aneurysms are balloon-like expansions developing from the branching region of a major brain artery; a small fraction are at risk of rupture with severe clinical consequences. Their wall integrity is related to both collagen fibre strength and orientation. Our approach involved serial section histology and polarized light microscopy, including 3D orientation and birefringence measurements. Results show a marked difference in fibre birefringence across the wall, with strength mainly in the outer layers. Directional and strength measurements on medial gap fibres of major brain artery bifurcations reveal an exceptional structural anisotropy - the apex having a tendon-like backbone.

Background: Arterial bifurcations are commonly the sites of developing atherosclerotic plaque that lead to arterial occlusions and plaque rupture (myocardial infarctions and strokes). Laser induced fluorescence (LIF) spectroscopy provides an effective nondestructive method supplying spectral information on extracellular matrix (ECM) protein composition, specifically collagen and elastin. Purpose: To investigate regional differences in the ECM proteins -- collagen I, III and elastin in unstable plaque by analyzing data from laser-induced fluorescence spectroscopy of human carotid endarterectomy specimens. Methods: Gels of ECM protein extracts (elastin, collagen types I & III) were measured as reference spectra and internal thoracic artery segments (extra tissue from bypass surgery) were used as tissue controls. Arterial segments and the endarterectomy specimens (n=21) were cut into 5mm cross-sectional rings. Ten fluorescence spectra per sampling area were then recorded at 5 sites per ring with argon laser excitation (357nm) with a penetration depth of 200 μm. Spectra were normalized to maximum intensity and analyzed using multiple regression analysis. Tissue rings were fixed in formalin (within 3 hours of surgery), sectioned and stained with H&E or Movat's Pentachrome for histological analysis. Spectroscopy data were correlated with immunohistology (staining for elastin, collagen types I, III and IV). Results: Quantitative fluorescence for the thoracic arteries revealed a dominant elastin component on the luminal side -- confirmed with immunohistology and known artery structure. Carotid endarterectomy specimens by comparison had a significant decrease in elastin signature and increased collagen type I and III. Arterial spectra were markedly different between the thoracic and carotid specimens. There was also a significant elevation (p<0.05) of collagen type I distal to the bifurcation compared to proximal tissue in the carotid specimens. Conclusion: Fluorescence spectroscopy is an effective method for evaluating ECM (collagen and elastin) associated with vascular remodeling despite the considerable variability in the plaque structure. Consistent regional differences were detected in the carotid specimens.

We have demonstrated that in vitro fluorescence spectroscopic analysis of diseased carotid arteries can identify clinically relevant compositional changes in connective tissue (collagen type I, III, IV and elastin) associated with plaque remodeling and instability.

Imbibition analysis, a polarised light microscopy technique, was used to examine the molecular organisation of collagen in normal and diseased mitral valve chordae tendineae. A single strut chorda from each of 23 valves (14 from necropsy specimens and nine from valve replacement surgery) was studied. The degree of molecular organisation of collagen in unstained 7 ,um sections of the chordae was assessed by measuring the retardation of polarised light by the sample. Sections from each tendon were examined, after staining with Movat's pentachrome, for the presence of proteoglycan infiltration and classified as normal or abnormal on that basis. The imbibition analysis results were grouped accordingly. The retardation in the collagen in the seven chordae with proteoglycan infiltration was significantly lower than in the 16 normal chordae, indicating decreased molecular organisation. Five of the seven abnormal chordae with proteoglycan infiltration and decreased retardation were from patients with floppy mitral valves; the other two were from normal necropsy specimens. Although proteoglycan infiltration may not be a specific marker for floppy valve disease, its presence is associated with decreased molecular organisation of collagen in the chordae. Degradation of the ground substance bound to the collagen is the most plausible explanation for the measured optical changes.

BACKGROUND AND PURPOSE:The bifurcation regions of the major human cerebral arteries are vulnerable to the formation of saccular aneurysms. A consistent feature of these bifurcations is a discontinuity of the tunica media at the apex of the flow divider. The objective was to measure the 3-dimensional geometry of these medial gaps or "medial defects."METHODS:Nineteen bifurcations and 2 junctions of human cerebral arteries branches (from 4 male and 2 female subjects) were formalin-fixed at physiological pressure and processed for longitudinal serial sectioning. The apex and adjacent regions were examined and measurements were made from high-magnification photomicrographs, or projection microscope images, of the gap dimensions at multiple levels through the bifurcation.RESULTS:Plots were made of the width of the media as a function of distance from the apex. The media at each edge of the medial gap widened over a short distance, reaching the full width of the media of the contiguous daughter vessel. Medial gap dimensions were compared with the planar angle of the bifurcation, and a strong negative correlation was found, ie, the acute angled branches have the more prominent medial gaps.CONCLUSIONS:A discontinuity of the media at the apex was seen in all the bifurcations examined and was also found in the junction regions of brain arteries. We determined that the gap width is continuous with well-defined dimensions throughout its length and average length-to-width ratio of 6.9. The gaps were generally centered on the prominence of the apical ridge.

Atherosclerosis is the underlying vascular pathology that initiates arterial thromboembolic occlusions (myocardial infarctions, strokes and peripheral vessel blockage). Two imaging modalities, Optical Coherence Tomography (OCT) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), were investigated for detection and compositional analysis of unstable plaque associated with plaque erosion and sudden occlusion. OCT produces high resolution images whereas mass spectrometry images provide information on the spatial distribution of chemical elements. Diseased carotid arteries taken from patients with high-risk lesions were imaged with OCT and ToF-SIMS to give molecular and metabolic information, and matched with histopathology. OCT results show clear indications of vascular remodeling by the presence of fatty acid deposits, fibrous tissue and calcifications. ToF-SIMS further characterized changes based on secondary ion topography analysis where a high 23Na/39K ratio was indicative of arterial tissue degradation and the amount of 40Ca corresponded with late stage atherosclerosis. This pilot experiment has demonstrated that in vitro OCT imaging and ToF-SIMS of diseased carotid arteries have scientific merit for targeting clinically relevant morphology and metabolic changes to compare stable and unstable plaque. These optical techniques provide complimentary metabolic and molecular information on unstable plaque, specifically cell break-down with altered ion ratios of 23Na, 39K and 40Ca.

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.

The 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. © 2000 Biomedical Engineering Society.

The wall of saccular brain aneurysms, lesions that develop at the fork regions of human brain arteries, is a layered multidirectional fabric of fibrous collagen. The wall tissue has the mechanical features of high elastic stiffness and a low tensile strength compared to adjacent arteries. Arterial blood pressure within the sac of an aneurysm stresses the wall in all tangential directions; thus an area of mechanical weakness may be characterized by an area lacking fibre strength, or an area with inadequately aligned fibres. The collagen fibres of the wall are birefringent, with many fibre types, similar to wound healing skin (types I, III, IV, V, VI) The known correlation between fibre birefringence and tissue strength for dermal wound healing3 provided the opportunity in this research to calculate strength maps to compare one lesion from another, and to make regional comparisons around a single lesion.

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 mu m 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 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 al 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. (C) 1997 Published by Elsevier Science Ltd.

Collagen, a naturally birefringent biopolymer and key structural component of the periodontal ligament (PDL), is altered substantially with regard to its molecular cross-linked structure by the dietary lathyrogen, beta-amino-proprionitrile (beta-APN). Our purpose in studying beta-APN-fed animals was to learn if the strength of birefringence of periodontal collagen, measured microscopically by the Senarmont compensator method, correlates with the molecular changes known to occur in these lathyritic animals, and to explore this technique for the periodontal ligament. Five experimental animals were fed a diet containing 0.25% beta-APN for two weeks, and 3 control animals were fed normal rat chow. Tissues were decalcified and the mandibular incisors were cut in cross section at 4 microns and stained with eosin Y. The Senarmont compensator, an attachment for the polarizing microscope, was used to measure phase retardation. Ten measurements were taken from each of the three sides (mesial, lateral and lingual) of the triangularly shaped incisor sections. The collagen fibers of the beta-APN-fed rats had lower values of phase retardation than the controls (p < 0.001), indicating reduced molecular organization. In addition, minor but significant regional differences were revealed, supporting the method for structural studies on the periodontal collagen.

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 μm thickness) of an intact 8 mm aneurysm, a 1.5 mm aneurysm, and a tangential section (3 μm 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° for coherently organized collagen and as high as 40° 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.

We constructed and discussed a mathematical model of intracranial saccular aneurysms based on the static mechanics of hollow vessels and were able to focus on three variables that are fundamental to the process of enlargement and rupture of these lesions. They are blood pressure (P), wall strength (sigma), and total wall substance (VT), which, if assigned values of 150 mm Hg, 10 MPa, and 1.0 mm3, lead to model-predicted values of 8 mm for the diameter and 40 micron for the wall thickness for the critical geometry of aneurysmal rupture. These are quantitatively similar to published measurements. The model is based on the assumption of a uniform thin spherical shell for the saccular aneurysm. The interrelationship of the variables, expressed in the equation for critical size at rupture (dc) (i.e., dc = [4 sigma VT/(pi P)]1/3), draws attention to the need for quantitative studies on aneurysmal geometry and on the stereology of the structural fraction of the aneurysmal wall. We concluded that tissue recruitment from around the initial site or hypertrophy of the wall tissue is commonly involved in the aneurysmal process. We identify the paradox of elastic stiffness and stability, which are characteristic of autopsy specimens in the laboratory, in contrast to plastic behavior and irreversible strain, which are essential to the natural process of enlargement of saccular aneurysms.

The initial step in rouleaux formation, the stacking of two red blood cells, is mechanically a process of three stages, compression, sliding, and creeping. Although this has been thoroughly described for cells in synthetic polymer solutions the present work was undertaken to analyze the process for cells in normal plasma, in order to lay the basis for comparison for ongoing studies of blood from patients with blood disorders. For each experiment a small amount of blood was diluted in its own plasma. A drop from the suspension was then placed on a coverslip, the coverslip covered with paraffin oil in a chamber, and the chamber quickly placed on the stage of an inverted microscope for observation and cinemicroscopy. Twenty-four doublet formations in blood plasma from five subjects were analyzed. After two cells make rim to rim contact, they move closer together by deformation of the contiguous rims to a straight line boundary (compression). Then one of the cells moves up over the other and begins the process of registration which previous work has established as a sliding interaction. The peak sliding velocity (on average 0.35 μm/sec) occurs near halfway to complete overlap. Sliding almost completely stopped just before full overlap, which was achieved very slowly (creeping). The several forces involved during sliding were identified, the force balance was examined, and from the variation of the velocity of sliding it was concluded that the medium between the touching membranes exhibits shear-rate-dependent viscosity.

Others have shown that collagen has a weakly birefringent molecular structure with its optical axis aligned with the longitudinal structural axis. We used a polarizing microscope and universal stage to measure the three-dimensional orientation of individual collagen fibers in sections of cerebral arteries. The arteries were fixed under a transmural pressure of 100 mm Hg, embedded with a separate reference frame of nerve fibers, and cut obliquely and longitudinally. Two dichroic enhancement stains were used—F3BA picro-Sirius red and Gomori's silver impregnation stain. Measurements on stained and unstained sections were reproducible to within approximately 1°. Three hundred measurements from 11 arterial sections revealed that the average direction of alignment was circumferential with a surface component of longitudinal fibers.