The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging

Abstract

Changes in arterial wall composition and function underlie all forms of vascular disease. The fundamental structural and functional unit of the aortic wall is the medial lamellar unit (MLU). While the basic composition and organization of the MLU is known, three-dimensional (3D) microstructural details are tenuous, due (in part) to lack of three-dimensional data at micro- and nano-scales. We applied novel electron and confocal microscopy techniques to obtain 3D volumetric information of aortic medial microstructure at micro- and nano-scales with all constituents present. For the rat abdominal aorta, we show that medial elastin has three primary forms: with approximately 71% of total elastin as thick, continuous lamellar sheets, 27% as thin, protruding interlamellar elastin fibers (IEFs), and 2% as thick radial struts. Elastin pores are not simply holes in lamellar sheets, but are indented and gusseted openings in lamellae. Smooth muscle cells (SMCs) weave throughout the interlamellar elastin framework, with cytoplasmic extensions abutting IEFs, resulting in approximately 20 degrees radial tilt (relative to the lumen surface) of elliptical SMC nuclei. Collagen fibers are organized as large, parallel bundles tightly enveloping SMC nuclei. Quantification of the orientation of collagen bundles, SMC nuclei, and IEFs reveal that all three primary medial constituents have predominantly circumferential orientation, correlating with reported circumferentially dominant values of physiological stress, collagen fiber recruitment, and tissue stiffness. This high resolution three-dimensional view of the aortic media reveals MLU microstructure details that suggest a highly complex and integrated mural organization that correlates with aortic mechanical properties.

Figure 1. Sample Location and Size

Represents scale of imaging. Images presented are from (left to right) geometric model of rat aorta, light microscopy of aortic cross section, confocal laser scanning microscopy (CLSM), and serial block face scanning electron microscopy (SBFSEM). Note the branching lamellae in the CLSM image (yellow arrow).

Figure 2. 3D CLSM Images Show In Vivo Arrangement of Combined and Individual Constituents

3D confocal images showing in vivo microstructure with green representing elastin, red collagen, and blue SMC nuclei. (a) Combined, (b) Elastin only, (c) SMC nuclei, and (d) Collagen only. Lumen surface located at top, circumferential direction noted. Volumes are 92μm × 92μm × 60μm. (r indicates radial direction, z axial, and θcircumferential).

Figure 3. 3D SBFSEM Images Showing Elastin Features

SBFSEM images show how SMC nuclei (SMC) reside within the complex 3D architecture of elastin. (a. Full view, b. Magnified section). Thickness of elastic lamellae (EL) varies. 3 primary forms of elastin are evident, with continuous sheets of circumferential elastin forming elastic lamellae (EL), a dense network of interlamellar elastin fibers (IEFSs) protruding from top & bottom lamellar surfaces in oblique (circumferential & radial) directions, and a thick radial elastin strut (ES) connecting adjacent lamellae. (Small volume dimensions (θ × Z × r) are 31μm × 36μm × 50μm, and magnified volume 22μm × 16μm × 25μm. (r indicates radial direction, z axial, and θ circumferential).

Figure 4. 3D SBFSEM Volume Showing Smooth Muscle Cell Configuration

Nuclei align between lamellae in staggered arrangement. The shape of healthy aortic smooth muscle cell nuclei is elliptical (Long axis/ radius = 6.2±1.4). The nuclei’s long axis is oriented circumferentially with a 19°radial tilt that directs ends toward top & bottom lamellae. The cytoplasm weaves throughout interlamellar space, extending numerous offshoots. (N = Nuclei, Cyt = Cytoplasm, EL = Elastic Lamella)

Figure 5. Sequential 2D CLSM Images Show Collagen Arrangement

Sequential confocal images of the media of one representative healthy rat abdominal aorta (Collagen = Red, SMC nuclei = Blue). The first image in the series is taken just below the internal elastic lamella, and each subsequent image is 2.2μm deeper. Collagen fiber bundles do not form a helical mesh within each layer, rather bundles are parallel within each layer, closely enveloping SMC nuclei. Fiber orientation for each layer differs slightly from adjacent layers, achieving a variation of orientations throughout mural thickness.

Figure 6. 2D SBFSEM Images Showing Helical Bundles of Collagen Fibers

2D SBFSEM image shows collagen fibers in bundles of 40-50 fibers (arrows). Collagen bundles do not appear taut, despite fixation of specimens at mean intraluminal pressure. Fiber bundles spiral in & out of image plane, suggesting helical rather than crimped structure. IEFs are seen extending circumferentially and radially throughout interlamellar space, with cytoplasm weaving in close proximity. (E = Elastic Lamella, IEF = Interlamellar Elastin Fiber, Arrow = Collagen fiber bundle, Cyt = SMC Cytoplasm, N = Nucleus).

Figure 7. Distributed Orientation of Smooth Muscle Cell Nuclei, Elastin, & Collagen

Normalized distribution of orientation indicators (OIs) of mural constituents shows that smooth muscle cell nuclei, interlamellar elastin fibers, and collagen preferentially align in the circumferential direction (90°).

Figure 8. Revised 3D Medial Aortic Microstructure

Artistic rendering of aortic medial microstructure from SBFSEM volume. Aortic media shows complex, interconnected structure. ELASTIN features include elastic lamellae (EL), dense network of interlamellar elastin fibers (IEFs shown with black arrows), elastin struts (ES), & reinforced elastin pores (EP). SMOOTH MUSCLE CELL (SMC) features include staggered elliptical nuclei (N) oriented circumferentially with radial tilt, & cytoplasm (Cyt) abuts IEFs & overlaps adjacent SMCs. COLLAGEN features include large & small fiber bundles (white arrows) adjacent to lamellar surfaces, arranged in layers of parallel bundles oriented predominantly circumferentially. Image dimensions (θ × Z × r) are 80μm × 60μm × 45μm, with lumen surface at top. (r indicates radial direction, z axial, and θcircumferential).