Shukei Sugita

A Novel Method for Measuring Tension Generated in Stress Fibers by Applying External Forces

The distribution of contractile forces generated in cytoskeletal stress fibers (SFs) contributes to cellular dynamic functions such as migration and mechanotransduction. Here we describe a novel (to our knowledge) method for measuring local tensions in SFs based on the following procedure: 1), known forces of different magnitudes are applied to an SF in the direction perpendicular to its longitudinal axis; 2), force balance equations are used to calculate the resulting tensions in the SF from changes in the SF angle; and 3), the relationship between tension and applied force thus established is extrapolated to an applied force of zero to determine the preexisting tension in the SF. In this study, we measured tensions in SFs by attaching magnetic particles to them and applying known forces with an electromagnetic needle. Fluorescence microscopy was used to capture images of SFs fluorescently labeled with myosin II antibodies, and analysis of these images allowed the tension in the SFs to be measured. The average tension measured in this study was comparable to previous reports, which indicates that this method may become a powerful tool for elucidating the mechanisms by which cytoskeletal tensions affect cellular functions.

Quantitative measurement of the distribution and alignment of collagen fibers in unfixed aortic tissues

Determination of the local amount and direction of collagen fibers during deformation is crucial for an understanding of the mechanical behavior of aortic tissues. Since most conventional methods cannot be used for this purpose, we propose a method to quantify the local amount and direction of fibers by simply measuring the optical properties of the specimen. After confirming the linear correlation between the retardance and thickness of sections of porcine thoracic aortas (PTAs) ranging from 15 to 300 μm, we investigated the effects of their structural components, i.e., smooth muscle cells (SMCs), elastin and collagen, on the retardance of whole tissues. Decellularization of SMCs did not change the retardance of PTA sections significantly. Patterns in autofluorescent and immunofluorescent images of elastin purified from bovine nuchal ligaments did not match those in retardance images. Images of collagen in PTA sections stained with picrosirius red were similar to corresponding retardance images. The slow axis azimuth corresponded to the circumferential direction of the aorta. Results indicate that collagen in aortas can be quantified by measuring the retardance and slow axis azimuth of whole aortic tissues. Application of this technique to PTAs showed that retardance was higher in dorsal and distal regions than ventral and proximal regions, respectively, indicating that the aortas contain more collagen in distal and dorsal regions than proximal and ventral regions, respectively. Both results were in accordance with previous findings. Measurement of retardance is useful to quantify the amount of collagen in unfixed aortas.

Multiphoton microscopy observations of 3D elastin and collagen fiber microstructure changes during pressurization in aortic media

Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal–circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.

Novel biaxial tensile test for studying aortic failure phenomena at a microscopic level

BackgroundAn aortic aneurysm is a local dilation of the aorta, which tends to expand and often results in a fatal rupture. Although larger aneurysms have a greater risk of rupture, some small aneurysms also rupture. Since the mechanism of aortic rupture is not well understood, clarification of the microstructure influencing the failure to rupture is important. Since aortic tissues are stretched biaxially in vivo, we developed a technique to microscopically observe the failure of an aortic rupture during biaxial stretch.MethodsA thinly sliced porcine thoracic aortic specimen was adhered to a circular frame and pushed onto a cylinder with a smaller diameter to stretch the specimen biaxially. To induce failure to rupture at the center, the specimen was thinned at the center of the hole as follows: the specimen was frozen while being compressed with metal plates having holes, which were 3 mm in diameter at their centers; the specimen was then sliced at 50-μm intervals and thawed.ResultsThe ratio of the thickness at the center to the peripheral area was 99.5% for uncompressed specimens. The ratio decreased with an increase in the compression ratio εc and was 47.3% for specimens with εc = 40%. All specimens could be stretched until failure to rupture. The probability for crack initiation within the cylinder was <30% and 100% for specimens with εc <10% and εc >30%, respectively. Among specimens ruptured within the cylinder, 93% of those obtained from the mid-media showed crack initiation at the thin center area.ConclusionsAortic tissues were successfully stretched biaxially until failure, and their crack initiation points were successfully observed under a microscope. This could be a very useful and powerful method for clarifying the mechanism of aortic rupture. We are planning to use this technique for a detailed investigation of events occurring at the point of failure when the crack initiates in the aortic aneurysm wall.

Heterogeneity of deformation of aortic wall at the microscopic level: contribution of heterogeneous distribution of collagen fibers in the wall.

There is growing evidence of heterogeneous distribution of collagen fibers in the aortic wall. To investigate the effects of collagen microstructure on local aortic wall deformation, porcine thoracic aortas were sliced into 100 μm-thick sections perpendicular to their radial direction and stretched biaxially with a laboratory-made tensile tester under a microscope equipped with a birefringence imaging system. Strain tensor components were calculated from fluorescent images of the cell nuclei for each 50 × 50 μm² area. Retardance Ret and slow axis azimuth θ were measured as indices of collagen density and fiber direction, respectively. Aortic wall deformation was highly heterogeneous: standard deviations of strains were significantly larger, by 3-5 times, in aortic slices than in homogeneous silicone sheets. A significant negative correlation was found between maximum principal strain and Ret (R=-0.077), and a positive correlation between minimum principal strain direction and θ (R=0.345). These indicate that the aorta is less distensible in areas with higher collagen density and in the direction of collagen fiber alignment. Microscopic heterogeneity may induce heterogeneous responses of smooth muscle cells and have crucial effects on mechanical homeostasis in the aortic wall.

Dynamic Control of Sliding Directions of Kinesin-Driven Microtubules With Rotating Electric Fields

Kinesins, biomolecular motors that move along microtubules (MTs) can potentially be utilized as an actuator in nanoscale transporting systems. Recent studies have reported inverted geometry in vitro, in which MTs randomly moved on kinesins fixed to substrates. To develop the transporting systems, one of key elements includes precise control of the direction of sliding MTs. One possible method is to utilize electric field (EF) to direct the MTs because MTs are negatively charged in neutral solutions [1,2]. For example, MTs have been shown to orient to the direction of uniaxially or biaxially applied EFs [3,4]. However, for a reliable transporting system, further studies are still required to control the direction of sliding MTs dynamically and effectively. In our previous study [5], we applied EF to MTs in random direction and showed that the rate of change in angle (angular velocity) was proportional to the sin of the angle between the directions of MTs and the generated electrophoretic force. The result indicates that it is most efficient to continuously apply EF perpendicular to the direction of MTs. In this study, the direction of sliding MTs was dynamically controlled with EF, particularly demonstrating a circular movement of MTs.Copyright

Local distribution of collagen fibers determines crack initiation site and its propagation direction during aortic rupture

Although elucidation of the mechanism of aortic aneurysm rupture is important, the characteristics of crack initiation and propagation sites remain unknown. To determine the microscopic properties of these sites, the characteristics of local strains and constituents at crack initiation and propagation sites were investigated during biaxial stretching of porcine thoracic aortas (PTAs). PTAs were sliced into approximately 50-

Tensile Properties of Smooth Muscle Cells, Elastin, and Collagen Fibers

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