Mechanical Properties Of Blood Vessels Research Articles

Measurement of the mechanical properties of blood vessels using a vascular simulation rig

Measurement of the mechanical properties of blood vessels using a vascular simulation rig

Dynamic indentation and microstructural analysis of artery under pre-strain in different directions

Strain from mechanical overload is a critical factor in vascular wall rupture associated with vascular diseases. Therefore, investigating the mechanical properties of blood vessels under strain is essential. Previous studies have predominantly focused on strain-free tests, and lacking microstructural analysis under load conditions. In this study, a self-made indentation device was used to combine with a pre-strain apparatus. Dynamic indentation tests were conducted on porcine arteries under pre-strain. Hematoxylin & Eosin staining and Masson staining were employed to observe microstructural changes in the arteries under strain. The results indicate that under pre-strain, the shear modulus of the arteries is positively correlated with the strain ratio. Radial strain increase the arterial relaxation ratio, while axial strain decrease it. Pre-strain enhance the arterial storage and loss modulus, which peaks at resonance frequencies of approximately 0.04 Hz and 1 Hz. Under pre-strain, elastic fibers experience a decrease in axial diameter and a reduction in spacing. The distribution of collagen is abnormal under radial strain. In the design of artery stents, a rationally designed stent structure should be considered so that the localized strain of the vessel wall is more decomposed into the axial direction.

Assessing friction and damage of cell monolayers on soft substrates in vitro.

In the area of surgical applications, understanding the interaction between medical device materials and tissue is important since this interaction may cause complications. The interaction often consists of a cell monolayer touching the medical device that can be mimicked in vitro. Prominent examples of this are contact lenses, where epithelial cells interact with the contact lens, or stents and catheters, which are in contact with endothelial cells. To investigate those interactions, in previous studies, expensive microtribometers were used to avoid pressures in the contact area far beyond physiologically relevant levels. Here, we aim to present a new methodology that is cost- and time-efficient, more accessible than those used previously and allows for the application of more realistic pressures, while permitting a quantification of the damage caused to the monolayer. For this, a soft polydimethylsiloxane is employed that better mimics the mechanical properties of blood vessels than materials used in other studies. Furthermore, a technique to account for misalignments within the experiment set-up is presented. This is carried out using the raw spatial and force data recorded by the tribometer and adjusting for misalignments. The methodology is demonstrated using an endothelial cell (human umbilical vein endothelial cells) monolayer.

Experimental Investigation of the Anisotropic Mechanical Response of the Porcine Thoracic Aorta.

Knowledge of the mechanical properties of blood vessels and determining appropriate constitutive relations are essential in developing methodologies for accurate prognosis of vascular diseases. We examine the directional variation of the mechanical properties of the porcine thoracic aorta by performing uniaxial extension tests on dumbbell-shaped specimens cut at five different orientations with respect to the circumferential direction of the aorta. Specimens in all the orientations considered exhibit a nonlinear constitutive response that is typical of collagenous soft tissues. Shear strain under uniaxial extension demonstrates clearly discernible anisotropy of the mechanical response of the porcine aorta, and samples oriented at 45[Formula: see text] and 60[Formula: see text] with respect to the circumferential direction show a peculiar crescent-shaped shear strain-nominal stretch response not displayed by axial and circumferential specimens. Failure stress indicates decreasing tensile strength of the porcine aortic wall from the circumferential direction to the longitudinal direction. Furthermore, we determine the material parameters for the four-fiber-family and Gasser-Holzapfel-Ogden models from the mechanical response data of the circumferential and longitudinal specimens. It is shown how the material parameters derived from the uniaxial tests on circumferential and longitudinal specimens are insufficient to characterize the response of off-axis specimens.

Numerical Investigation of the Dynamical Behavior of a Fluid-Filled Microparticle Suspended in Human Arteriole.

The study of artificial microparticles (capsules and vesicles) has gained a growing interest with the emergence of bio-engineering. One of their promoting applications is their use as therapeutic vectors for drug delivery, when capsules and vesicles release their capacity in a targeted environment. The dynamic behavior of capsules and vesicles in confined or unbounded flows was widely studied in the literature and their mechanical response was truthfully described using constitutive laws with good agreement with experiences. However, in a context of biological application, to our knowledge, none of published studies investigating the mechanical response of deformable microparticle took into account the real physiological conditions: the rheological properties of blood such as carrying fluid and the mechanical properties of blood vessels. In this paper, we consider a hyperelastic microparticle suspended in human arteriole. We investigate the deformation of the microparticle resulting from its interaction with blood flow and the arteriolar wall using various capillary numbers and respecting physiological properties of blood and arterial wall. The influence of the blood viscosity model (Newtonian versus shear thinning) is investigated and a comparison with a rigid microchannel and a muscle-embedded arteriole is carried out. The fluid structure interaction (FSI) problem is solved using arbitrary Lagrangian Eulerian (ALE) method. Our simulations have revealed that the arteriolar wall distensibility deeply influences both the deformation and velocity of the microparticle: the deformation strongly increases while the velocity decreases in comparison to an infinitely rigid wall. In the context of therapeutic procedure of targeted drug-delivery, a particular attention should be addressed to these observations, in particular for their implication in the burst mechanism.

Effect of fluid dynamics on decellularization efficacy and mechanical properties of blood vessels.

Decellularization of blood vessels is a promising approach to generate native biomaterials for replacement of diseased vessels. The decellularization process affects the mechanical properties of the vascular graft and thus can have a negative impact for in vivo functionality. The aim of this study was to determine how detergents under different fluid dynamics affects decellularization efficacy and mechanical properties of the vascular graft. We applied a protocol utilizing 1% TritonX, 1% Tributyl phosphate (TnBP) and DNase on porcine vena cava. The detergents were applied to the vessels under different conditions; static, agitation and perfusion with 3 different perfusion rates (25, 100 and 400 mL/min). The decellularized grafts were analyzed with histological, immunohistochemical and mechanical tests. We found that decellularization efficacy was equal in all groups, however the luminal ultrastructure of the static group showed remnant cell debris and the 400 mL/min perfusion group showed local damage and tearing of the luminal surface. The mechanical stiffness and maximum tensile strength were not influenced by the detergent application method. In conclusion, our results indicate that agitation or low-velocity perfusion with detergents are preferable methods for blood vessel decellularization.

Micromechanical property analyses of decellularized vessels by atomic force microscopy

Vascular transplantation involves embedding vascular grafts between normal blood vessels to replace damaged blood vessels and allow blood to flow normally. There is an increasing need for small-diameter vascular transplantation in clinical environments. However, the implementation of small-diameter vascular grafts (SDVGs) still has some drawbacks, such as thrombosis. Decellularization is the most common method of constructing SDVGs, and fully understanding the mechanical properties of decellularized vessels is crucial. Thus, when determining the scale-dependent mechanical properties of blood vessels, exclusively analyzing the macroscopic mechanical properties is not comprehensive. In this study, we evaluated the mechanical properties at the microscale and nanoscale. Atomic force microscopy was used as the basic technique to characterize the mechanical properties of specific layers (lumen, tunica media and tunica adventitia) of various samples (decellularized, native, and frozen aortic vessels). Moreover, the finite element method (FEM) was applied to analyze the influences of probes at different scales. The results showed that the mechanical properties of the various layers were significantly different. Decellularization significantly enhanced the mechanical properties of the tunica media and tunica adventitia, and reduced the mechanical properties of the lumen. The freezing treatment also increased the stiffnesses of the tunica media and tunica adventitia. However, the ratios of the elastic modulus of the tunica media to that of the tunica adventitia were similar before and after the freezing treatment at the nanoscale, and the elastic modulus of the lumen was not significantly different after freezing. FEM simulations illustrated that the measurements at the nanoscale were significantly larger than those at the microscale. The aims of this study were to emphasize the value of microscale mechanical properties and provide a more comprehensive data reference for modeling multilayer vessels and constructing bionic decellularized blood vessels.

Fluid Structure Interactions in Ascending Thoracic Aortic Aneurysms

The fluid mechanics community has been interested for many years in hemodynamics. More recently, significant endeavours of the solid mechanics community have permitted to establish constitutive equations and to achieve stress analyses in arterial lesions (atheromatous plaque in coronary or carotid arteries, aneurysms of the aorta). The mechanical properties of blood vessels have often been characterized ex vivo, but medical imaging, including MRI, now allows non-intrusive identifications in vivo. The spatial heterogeneity of these mechanical properties, even at the macroscopic scale, remains poorly explored despite its undeniable interest in understanding the mechanisms of remodeling and degeneration of the tissue. We are interested in the problem of identifying the fields of mechanical properties of aneurysms of the aorta. Scientific barriers are related to the complex geometry, the nonlinear and anisotropic behavior of tissues, the multiaxial loading conditions, and to the measurement of a local response in these tissues. Our identification approaches, based on digital image correlation field measurements and inverse methods, have demonstrated the link between the heterogeneity of mechanical properties and the existence of localized failure modes. A micromechanical approach has also made it possible to develop a mechanobiological model to reproduce the behavior of the aorta in surgical situations and a simulation software is being developed for assistance to personalized surgery in the cardiovascular field.

Arterial pulse attenuation prediction using the decaying rate of a pressure wave in a viscoelastic material model

The present study examines the possibility of attenuating blood pulses by means of introducing prosthetic viscoelastic materials able to absorb energy and damp such pulses. Vascular prostheses made of polymeric materials modify the mechanical properties of blood vessels. The effect of these materials on the blood pulse propagation remains to be fully understood. Several materials for medical applications, such as medical polydimethylsiloxane or polytetrafluoroethylene, show viscoelastic behavior, modifying the original vessel stiffness and affecting the propagation of blood pulses. This study focuses on the propagation of pressure waves along a pipe with viscoelastic materials using the Maxwell and the Zener models. An expression of exponential decay has been obtained for the Maxwell material model and also for low viscous coefficient values in the Zener model. For relatively high values of the viscous term in the Zener model, the steepest part of the pulse can be damped quickly, leaving a smooth, slowly decaying wave. These mathematical models are critical to tailor those materials used in cardiovascular implants to the mechanical environment they are confronted with to repair or improve blood vessel function.

Microstructure-based constitutive model of coronary artery with active smooth muscle contraction

Currently, there is no full three-dimensional (3D) microstructural mechanical model of coronary artery based on measured microstructure including elastin, collagen and smooth muscle cells. Many structural models employ mean values of vessel microstructure, rather than continuous distributions of microstructure, to predict the mechanical properties of blood vessels. Although some models show good agreements on macroscopic vessel responses, they result in a lower elastin stiffness and earlier collagen recruitment. Hence, a full microstructural constitutive model is required for better understanding vascular biomechanics in health and disease. Here, a 3D microstructural model that accounts for all constituent microstructure is proposed to predict macroscopic and microscopic responses of coronary arteries. Coronary artery microstructural parameters were determined based on previous statistical measurements while mechanical testing of arteries (n = 5) were performed in this study to validate the computational predictions. The proposed model not only provides predictions of active and passive stress distributions of vessel wall, but also enables reliable estimations of material parameters of individual fibers and cells and thus predicts microstructural stresses. The validated microstructural model of coronary artery sheds light on vascular biomechanics and can be extend to diseased vessels for better understanding of initiation, progression and clinical treatment of vascular disease.

Simulation of one-dimensional blood flow in networks of human vessels using a novel TVD scheme.

An extension of a total variation diminishing (TVD) scheme to solve one-dimensional (1D) blood flow for human circulation is proposed. This method is simple as it involves only a few modifications to existing shock-capturing TVD schemes. We have applied the method to a wide range of test cases including a complete simulation of the human vascular network. Excellent solutions have been demonstrated for problems involving varying and discontinuous mechanical properties of blood vessels. For 1D network simulations, the method has been shown to agree well with the reported computational results. Finally, the method has been demonstrated to compare favorably with in vivo experiments set up to study the impact of circle of Willis anomalies on flow patterns in the cerebral arterial system.

Biaxial vasoactivity of porcine coronary artery

The passive mechanical properties of blood vessel mainly stem from the interaction of collagen and elastin fibers, but vessel constriction is attributed to smooth muscle cell (SMC) contraction. Although the passive properties of coronary arteries have been well characterized, the active biaxial stress-strain relationship is not known. Here, we carry out biaxial (inflation and axial extension) mechanical tests in right coronary arteries that provide the active coronary stress-strain relationship in circumferential and axial directions. Based on the measurements, a biaxial active strain energy function is proposed to quantify the constitutive stress-strain relationship in the physiological range of loading. The strain energy is expressed as a Gauss error function in the physiological pressure range. In K(+)-induced vasoconstriction, the mean ± SE values of outer diameters at transmural pressure of 80 mmHg were 3.41 ± 0.17 and 3.28 ± 0.24 mm at axial stretch ratios of 1.3 and 1.5, respectively, which were significantly smaller than those in Ca(2+)-free-induced vasodilated state (i.e., 4.01 ± 0.16 and 3.75 ± 0.20 mm, respectively). The mean ± SE values of the inner and outer diameters in no-load state and the opening angles in zero-stress state were 1.69 ± 0.04 mm and 2.25 ± 0.08 mm and 126 ± 22°, respectively. The active stresses have a maximal value at the passive pressure of 80-100 mmHg and at the active pressure of 140-160 mmHg. Moreover, a mechanical analysis shows a significant reduction of mean stress and strain (averaged through the vessel wall). These findings have important implications for understanding SMC mechanics.

Hydrogel–Electrospun Mesh Composites for Coronary Artery Bypass Grafts

The aim of the present study was to investigate the potential of hydrogel-electrospun mesh hybrid scaffolds as coronary artery bypass grafts. The circumferential mechanical properties of blood vessels modulate a broad range of phenomena, including vessel stress and mass transport, which, in turn, have a critical impact on cardiovascular function. Thus, coronary artery bypass grafts should mimic key features of the nonlinear stress-strain behavior characteristic of coronary arteries. In native arteries, this J-shaped circumferential stress-strain curve arises primarily from initial load transfer to low stiffness elastic fibers followed by progressive recruitment and tensing of higher stiffness arterial collagen fibers. This nonlinear mechanical response is difficult to achieve with a single-component scaffold while simultaneously meeting the suture retention strength and tensile strength requirements of an implantable graft. For instance, although electrospun scaffolds have a number of advantages for arterial tissue engineering, including relatively high tensile strengths, tubular mesh constructs formed by conventional electrospinning methods do not generally display biphasic stress-strain curves. In the present work, we demonstrate that a multicomponent scaffold comprised of polyurethane electrospun mesh layers (intended to mimic the role of arterial collagen fibers) bonded together by a fibrin hydrogel matrix (designed to mimic the role of arterial elastic fibers) results in a composite construct which retains the high tensile strength and suture retention strength of electrospun mesh but which displays a J-shaped mechanical response similar to that of native coronary artery. Moreover, we show that these hybrid constructs support cell infiltration and extracellular matrix accumulation following 12-day exposure to continuous cyclic distension.

Rosiglitazone reverses endothelial dysfunction but not remodeling of femoral artery in Zucker diabetic fatty rats.

ObjectivesEndothelial dysfunction precedes atherogenesis and clinical complications in type 2 diabetes. The vascular dysfunction in Zucker diabetic fatty (ZDF) rats was evaluated at different ages along with the effect of treatment with rosiglitazone (Rosi) on endothelial function and mechanical remodeling.MethodsThe Rosi treatment was given to ZDF rats for 3 weeks. The endothelium-dependent vasodilation and α-adrenoceptor-dependent vasoconstriction of femoral arteries were studied using an ex-vivo isovolumic myograph. The biomechanical passive property of the arteries was studied in Ca2+-free condition. The expressions of endothelial nitric oxide synthase (eNOS), α-adrenoceptor, matrix metalloproteinase 9 (MMP9), and elastase were evaluated.ResultsEndothelium-dependent vasorelaxation of the femoral artery was blunted at low doses in ZDF rats at 11 weeks of age and attenuated at all doses in ZDF rats at 19 weeks of age. The expression of eNOS was consistent with the endothelium-dependent vasorelaxation. The α-adrenoceptor was activated and the mechanical elastic modulus was increased in ZDF rats at 19 weeks of age. The expressions of α-adrenoceptor, MMP9, and elastase were up regulated in ZDF rats at 19 weeks of age. Rosi treatment for 3 weeks restored endothelium-dependent vasorelaxation and the expression of eNOS and the adrenoceptor activation at the doses below 10-6 mole/L in ZDF rats at 19 weeks of age. Rosi treatment for 3 weeks did not, however, improve the mechanical properties of blood vessel, the expressions of α-adrenoceptor, MMP9, and elastase in ZDF rats.ConclusionThe endothelial dysfunction and mechanical remodeling are observed as early as 19 weeks of age in ZDF rat. Rosi treatment for 3 weeks improves endothelial function but not mechanical properties.

Copper nanoparticle cues for biomimetic cellular assembly of crosslinked elastin fibers

Elastin, a structural protein distributed in the extracellular matrix of vascular tissues, is critical to maintaining the elastic stability and mechanical properties of blood vessels, as well as regulating cell-signaling pathways involved in vascular injury response and morphogenesis. Pathological degradation of vascular elastin or its malformation within native vessels and the poor ability to tissue-engineer elastin-rich vascular replacements due to innately poor elastin synthesis by adult vascular cells can compromise vascular homeostasis, and must thus be addressed. Our recent studies attest to the utility of hyaluronan (HA) oligomers for elastin synthesis and organization by adult vascular smooth muscle cells (SMCs), though the elastin matrix yields in these cases were quite low relative to total elastin produced. Thus, in this study, we investigated the utility of copper (Cu2+) ions to enhance cellular elastin deposition, crosslinking and maturation into structural fibers. Copper nanoparticles (CuNPs; 80–100nm) in the dose range of 1–100ngml−1 were tested for Cu2+ ion release, and based on mathematical modeling of their release profiles, CuNPs (1, 10, and 400ng ml−1) were chosen for supplementation to adult SMC cultures. The 400ngml−1 dose of CuNPs cumulatively delivered Cu2+ doses in the range of 0.1M, over the 21day culture period. It was observed that while exogenous CuNP supplements do not up-regulate tropoelastin production by vascular SMCs, they promoted formation of crosslinked elastin matrices. The deposition of crosslinked matrix elastin was further improved by the additional presence of HA oligomers in these cultures. Immunofluorescence imaging and structural analysis of the isolated elastin matrices indicate that amorphous elastin clumps were formed within non-additive control cultures, while aggregating elastin fibrils were observed within SMC cultures treated with CuNPs (1–10ngml−1) alone or together with HA oligomers. The presence of 400ngml−1 of CuNPs concurrent with HA oligomers furthered aggregation of these elastin fibrils into mature fibers with diameters ranging from 200 to 500nm. Ultrastructural analysis of elastin matrix within cultures treated with HA oligomers and 400ngml−1 of CuNPs suggest that elastin matrix deposition as stimulated by Cu2+ ions proceeds via a fibrillin-mediated assembly process, with enhanced crosslinking occurring via stimulation of lysyl oxidase. Overall, the data suggest that CuNPs and HA oligomers are highly useful for regenerating crosslinked, fibrillar elastin matrices by adult vascular SMCs. These results have immense utility in tissue-engineering vascular replacements.

Measurements of Dynamic Viscoelasticity of Poly (vinyl alcohol) Hydrogel for the Development of Blood Vessel Biomodeling

In vitro blood vessel biomodeling with realistic mechanical properties and geometrical structures is helpful for training in surgical procedures, especial those used in endovascular treatment. Poly (vinyl alcohol) hydrogel (PVA-H), which is made of Poly (vinyl alcohol) (PVA) and water, may be useful as a material for blood vessel biomodeling due to its low surface friction resistance and good transparency. In order to simulate the mechanical properties of blood vessels, measurements of mechanical properties of PVA-H were carried out with a dynamic mechanical analyzer, and the storage modulus (G’) and loss modulus (G”) of PVA-H were obtained. PVA-Hs were prepared by the low-temperature crystallization method. They were made of PVA with various concentrations (C) and degrees of polymerization (DP), and made by blending two kinds of PVA having different DP or saponification values (SV). The G’ and G” of PVA-H increased, as the C or DP of PVA increased, or as the proportion of PVA with higher DP or SV increased. These results indicate that it is possible to obtain PVA-H with desirable dynamic viscoelasticity. Furthermore, it is suggested that PVA-H is stable in the temperature range of 0°C to 40°C, indicating that biomodeling made of PVA-H should be available at 37°C, the physiological temperature. The dynamic viscoelasticity of PVA-H obtained was similar to that of the dog blood vessel measured in previous reports. In conclusion, PVA-H is suggested to be useful as a material of blood vessel biomodeling.

Proposal of a Mechanical Index for Arteries

Arteriosclerosis is such a phenomena hardening of arteries, with thickening and loss of elasticity. Previous indexes are included both geometric factor and mechanical factor as the radius, the wall thickness and mechanical properties of blood vessel. Furthermore, those indexes are formulated based on the static relationship between a pressure and a radial change. In this study, we suggested an evaluation index formulated by thin cylindrical shell theory estimated dynamic strain, and this index was independent of wall thickness and radius of arterial vessel. To confirm the validity of the evaluation index, we evaluated the parameters of viscosity and elasticity using the latex tube with different wall thickness of blood vessel model. We measured a radius of the latex tube and an inner pressure maintained a pulsatile pump in a mock circuit filled with the water. Estimating the parameters of viscosity and elasticity using these measurement values, we concluded that a proposal index was independent of the wall thickness and the radius of the arterial vessel.

A bilinear stress–strain relationship for arteries

A comprehensive understanding of the mechanical properties of blood vessels is essential for vascular physiology, pathophysiology and tissue engineering. A well-known approach to study the elasticity of blood vessels is to postulate a strain energy function such as the exponential or polynomial forms. It is typically difficult to fit experimental data to derive material parameters for blood vessels, however, due to the highly nonlinear nature of the stress–strain relation. In this work, we generalize the strain definition to absorb the elastic nonlinearity and then propose a two-dimensional bilinear stress–strain relation between second Piola-Kirchhoff stress and the new strain measure. The model is found to represent the Fung's exponential model very well. The novel linearized constitutive relation simplifies the determination of material constants by reducing the nonlinearity and provides a clearer physical interpretation of the model parameters. The limitations of the constitutive model and its implications for vascular mechanics are discussed.

A Mechanical Model of Porcine Vascular Tissues-Part II: Stress–Strain and Mechanical Properties of Juvenile Porcine Blood Vessels

Our laboratory is studying the influence of the volume fractions and the organization of various macromolecules and cells in vascular tissue on the physical properties of vessel wall. The current investigation reports various mechanical properties of aorta, vena cava, carotid artery and iliac artery of the swine including: low-strain and high-strain moduli, transition from low-strain to high-strain behavior, the ultimate tensile stress, and the ultimate strain. Tissues were tested using a uniaxial incremental stress–strain technique, which allowed us to capture viscous, elastic, and total data simultaneously. Vessels were strained throughout their entire strain range until failure was achieved. Low-strain modulus showed sporadic differences between blood vessels and some dependence on strain-rate. The high-strain modulus was consistently greater in vena cava (2.2 MPa for viscous stress–strain—9.6 MPa for total stress–strain) than other vessels. The ultimate tensile strength of vena cava (0.46 MPa for viscous and 2.5 MPa for total) was again greater than in other vessels and there was some strain-rate dependence. The ultimate strain of carotid artery (180%) was greater in the transverse direction than in other vessels. Viscosity measurements indicated that blood vessels are thixotropic (viscosity decreased 2 orders of magnitude for 2 orders of magnitude increase in strain-rate). Our data suggests that differences in mechanical properties of blood vessels occur depending on where in the vascular tree the tissue is located. These differences may be required by the physiological and mechanical demands on individual vessels.

Imaging Coronary Artery Microstructure Using Second-Harmonic and Two-Photon Fluorescence Microscopy

The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.