Local Tissue Stiffness Research Articles

Differential Effects of Confinement on the Dynamics of Normal and Tumor-Derived Pancreatic Ductal Organoids.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the epithelia comprising the ductal network of the pancreas. During disease progression, PDAC tumors recruit fibroblasts that promote fibrosis, increasing local tissue stiffness and subjecting epithelial cells to increased compressive forces. Previous in vitro studies have documented cytoskeletal and nuclear adaptation following compressive stresses in two-dimensional (2D) and three-dimensional (3D) environments. However, a comparison of the responses of normal and tumor-derived ductal epithelia to physiologically relevant confinement remains underexplored, especially in 3D organoids. Here we control confinement with an engineered 3D microenvironment composed of Matrigel mixed with a low yield stress granular microgel. Normal and tumor-derived murine pancreas organoids (normal and tumor) were cultured for 48 h within this composite 3D environment or in pure Matrigel to investigate the effects of confinement on morphogenesis and lumen expansion. In confinement, tumor organoids (mT) formed a lumen that expanded rapidly, whereas normal organoids (mN) expanded more slowly. Moreover, a majority of normal organoids in more-confined conditions exhibited an inverted apicobasal polarity compared to those in less-confined conditions. Tumor organoids exhibited a collective "pulsing" behavior that increased in confinement. These pulses generated forces sufficient to locally overcome the yield stress of the microgels in the direction of organoid expansion. Normal organoids more commonly exhibit unidirectional rotation. Our in vitro microgel confinement platform enabled the discovery of two distinct modes of collective force generation in organoids that may shed light on the mutual interactions between tumors and the microenvironment. These insights into in vitro dynamics may deepen our understanding of how the confinement of healthy cells within a fibrotic tumor niche disrupts tissue organization and function in vivo.

Insights into the Mechanical Characterization of Mouse Brain Tissue Using Microindentation Testing

AbstractIndentation testing is the most common approach to quantify mechanical brain tissue properties. Despite a myriad of studies conducted already, reported stiffness values vary extensively and continue to be subject of study. Moreover, the growing interest in the relationship between the brain's spatially heterogeneous microstructure and local tissue stiffness warrants the development of standardized measurement protocols to enable comparability between studies and assess repeatability of reported data. Here, we present three individual protocols that outline (1) sample preparation of a 1000‐µm thick coronal slice, (2) a comprehensive list of experimental parameters associated with the FemtoTools FT‐MTA03 Micromechanical Testing System for spherical indentation, and (3) two different approaches to derive the elastic modulus from raw force‐displacement data. Lastly, we demonstrate that our protocols deliver a robust experimental framework that enables us to determine the spatially heterogeneous microstructural properties of (mouse) brain tissue. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Mouse brain sample preparationBasic Protocol 2: Indentation testing of mouse brain tissue using the FemtoTools FT‐MTA03 Micromechanical Testing and Assembly SystemBasic Protocol 3: Tissue stiffness identification from force‐displacement data

Characterization of transient and progressive pulmonary fibrosis by spatially correlated phase contrast microCT, classical histopathology and atomic force microscopy

Pulmonary fibrosis (PF) is a severe and progressive condition in which the lung becomes scarred over time resulting in pulmonary function impairment. Classical histopathology remains an important tool for micro-structural tissue assessment in the diagnosis of PF. A novel workflow based on spatial correlated propagation-based phase-contrast micro computed tomography (PBI-microCT), atomic force microscopy (AFM) and histopathology was developed and applied to two different preclinical mouse models of PF - the commonly used and well characterized Bleomycin-induced PF and a novel mouse model for progressive PF caused by conditional Nedd4-2 KO. The aim was to integrate structural and mechanical features from hallmarks of fibrotic lung tissue remodeling. PBI-microCT was used to assess structural alteration in whole fixed and paraffin embedded lungs, allowing for identification of fibrotic foci within the 3D context of the entire organ and facilitating targeted microtome sectioning of planes of interest for subsequent histopathology. Subsequently, these sections of interest were subjected to AFM to assess changes in the local tissue stiffness of previously identified structures of interest. 3D whole organ analysis showed clear morphological differences in 3D tissue porosity between transient and progressive PF and control lungs. By integrating the results obtained from targeted AFM analysis, it was possible to discriminate between the Bleomycin model and the novel conditional Nedd4-2 KO model using agglomerative cluster analysis. As our workflow for 3D spatial correlation of PBI, targeted histopathology and subsequent AFM is tailored around the standard procedure of formalin-fixed paraffin-embedded (FFPE) tissue specimens, it may be a powerful tool for the comprehensive tissue assessment beyond the scope of PF and preclinical research.

Mechano-biological and bio-mechanical pathways in cutaneous wound healing.

Injuries to the skin heal through coordinated action of fibroblast-mediated extracellular matrix (ECM) deposition, ECM remodeling, and wound contraction. Defects involving the dermis result in fibrotic scars featuring increased stiffness and altered collagen content and organization. Although computational models are crucial to unravel the underlying biochemical and biophysical mechanisms, simulations of the evolving wound biomechanics are seldom benchmarked against measurements. Here, we leverage recent quantifications of local tissue stiffness in murine wounds to refine a previously-proposed systems-mechanobiological finite-element model. Fibroblasts are considered as the main cell type involved in ECM remodeling and wound contraction. Tissue rebuilding is coordinated by the release and diffusion of a cytokine wave, e.g. TGF-β, itself developed in response to an earlier inflammatory signal triggered by platelet aggregation. We calibrate a model of the evolving wound biomechanics through a custom-developed hierarchical Bayesian inverse analysis procedure. Further calibration is based on published biochemical and morphological murine wound healing data over a 21-day healing period. The calibrated model recapitulates the temporal evolution of: inflammatory signal, fibroblast infiltration, collagen buildup, and wound contraction. Moreover, it enables in silico hypothesis testing, which we explore by: (i) quantifying the alteration of wound contraction profiles corresponding to the measured variability in local wound stiffness; (ii) proposing alternative constitutive links connecting the dynamics of the biochemical fields to the evolving mechanical properties; (iii) discussing the plausibility of a stretch- vs. stiffness-mediated mechanobiological coupling. Ultimately, our model challenges the current understanding of wound biomechanics and mechanobiology, beside offering a versatile tool to explore and eventually control scar fibrosis after injury.

Breaking down the barrier to medulloblastoma treatment: Piezo2 knockout disrupts the BTB and increases vascular permeability

In this issue, Chen etal.1 show that genetic knockout of Piezo2 in SOX2+ medulloblastoma cells decreased local tissue stiffness, increased drug delivery across the blood-tumor barrier, and improved survival, uncovering a mechanosensitivity-dependent mechanism of action in a mouse model of pediatric brain tumors.

Acute Effects of Manual Lymphatic Drainage and Compression with Exercise Therapy on Breast Lymphedema Following Breast-Conserving Surgery and Radiotherapy.

Background: Breast lymphedema is a common complication of breast cancer treatments but there are limited studies about the treatment of breast lymphedema. The aim of this study was to investigate the acute effects of manual lymphatic drainage (MLD), compression with exercise on the local tissue water percentage, pain, and stiffness following breast-conserving surgery and radiotherapy. Materials and Methods: Twenty-two patients (52.54 ± 12.18 years, 28.55 ± 5.11 kg/m2) were included. The sociodemographic and clinical information was recorded. The pain and stiffness severity were measured with Visual Analog Scale. Measurements of water percentages in local tissue were performed in all quadrants of the breast with the Moisture Meter D Compact device. All measurements were performed baseline, after MLD, and after compression with exercise. Results: There was a significant difference in local tissue water percentages between the affected and unaffected sides before treatment. The percentage of water only in the lower outer quadrant of the affected breast increased significantly after acute treatment (p: 0.002). In addition, pain (p: 0.001) and stiffness (p: 0.001) scores decreased. Conclusions: Local tissue water percentages increased with MLD and decreased with compression with exercise. In the treatment of breast lymphedema, MLD and compression bandage with exercise may be beneficial in the management of the symptoms of swelling, pain, and stiffness.

182 Evaluation of skin stiffness in cutaneous fibrosing disorders: A novel constructive shear wave interference ultrasound technology

There is a critical need for objective and reproducible methods to evaluate skin sclerosis in individuals with fibrotic skin conditions. Constructive Shearwave Interference (CSI) is a novel ultrasonic method for noninvasive characterization of skin elasticity; the tool uses a cylindrical source to generate constructively interfering shear waves in the target medium. The speed at which the shear waves propagate is directly proportional to local tissue stiffness, with stiffer areas of tissue exhibiting faster shear wave speed (SWS).

Remodeling of the Aged and Emphysematous Lungs: Roles of Microenvironmental Cues.

Aging is a slow process that affects all organs, and the lung is no exception. At the alveolar level, aging increases the airspace size with thicker and stiffer septal walls and straighter and thickened collagen and elastic fibers. This creates a microenvironment that interferes with the ability of cells in the parenchyma to maintain normal homeostasis and respond to injury. These changes also make the lung more susceptible to disease such as emphysema. Emphysema is characterized by slow but progressive remodeling of the deep alveolar regions that leads to airspace enlargement and increased but disorganized elastin and collagen deposition. This remodeling has been attributed to ongoing inflammation that involves inflammatory cells and the cytokines they produce. Cellular senescence, another consequence of aging, weakens the ability of cells to properly respond to injury, something that also occurs in emphysema. These factors conspire to make alveolar walls more prone to mechanical failure, which can set emphysema in motion by driving inflammation through immune stimulation by protein fragments. Both aging and emphysema are influenced by microenvironmental conditions such as local inflammation, chemical makeup, tissue stiffness, and mechanical stresses. Although aging and emphysema are not equivalent, they have the potential to influence each other in synergistic ways; aging sets up the conditions for emphysema to develop, while emphysema may accelerate cellular senescence and thus aging itself. This article focuses on the similarities and differences between the remodeled microenvironment of the aging and emphysematous lung, with special emphasis on the alveolar septal wall. © 2022 American Physiological Society. Compr Physiol 12:3559-3574, 2022.

Assessment of alternative techniques to quantify the effect of injury on soft tissue in closed ankle and pilon fractures.

IntroductionLocal soft tissue status (STS) guides the timing for definitive surgical treatment strategies of fracture fixation around the ankle joint. The aim of this study was to assess different types of new technical devices in relation to the surgical treatment in closed ankle and pilon fractures.MethodsThis study was designed as a cohort study. Adult patients admitted between February 1, 2019 and December 31, 2020 presenting with closed ankle fracture requiring surgical treatment were eligible. The exclusion criteria were previous injuries to the lower extremity, acute deep venous thrombosis, skin diseases, and delayed presentation (admission >24 hours after injury). Moderate-energy trauma includes injuries sustained during team sports, biking, and running. The primary outcome was the assessment of the degree of soft tissue involvement following closed fractures by comparing different techniques focusing on the ankle region and including ankle and pilon fractures. The variables of interest included the circumference of soft tissue swelling around the ankle, determined within a 5-mm range in the area of the medial and lateral malleolus and the bone-skin distance on a plain radiograph, determined by the largest distance from the malleolus to the border of the soft-tissue shadow. STS assessment included optical measures of local perfusion (O2C, Lea Inc. Germany) and tactile measures of mechanical characteristics (Myoton® tensiometer AS, Estonia). Measurements of Group Temp (temporary stabilization) and Group Def (definitive surgery) were taken on admission and prior to the treatment strategy decision. The contralateral non-injured ankle served as a control. The quality of assessment tools was quantified by calculating the smallest detectable change (SDC).ResultsIn total, 38 patients with a mean age of 40.4 (SD 17.8) years were included. The SDC was 3.2% (95%CI 2.5 to 3.8) for local blood flow and 1.1% (95%CI 0.4 to 1.7) for soft tissue stiffness. The circumference of the injured area at admission was significantly higher than that of the healthy site (28.2 [SD 3.4] cm versus 23.9 [SD 2.4] cm, p < 0.001). The local perfusion (blood flow 107.5 (SD 40.79 A.U. vs. 80.1 [SD 13.8] A.U., p = 0.009), and local dynamic stiffness of the skin (668.1 (SD 148.0) N/m vs 449.5 (SD 87.7) N/m, p < 0.001) were significantly higher at the injured site. In Group Temp, the local blood flow was significantly higher when compared with Group Def (109.6 [SD 39.8] vs. 94.5 [SD 13.0], p = 0.023). The dynamic stiffness of the soft tissue was significantly higher in Group Temp (679.4 N/m [SD 147.0] N/m vs. 573.0 N/m (SD 93.8) N/m, p < 0.001). The physical properties of STS were comparable among the fracture types. None of the included patients had local soft tissue complications.ConclusionClosed fractures of the ankle and the pilon are associated with an increase in local circulation and local soft tissue stiffness and tension. These changes of the STS following injury can be quantified in a standardized and reproducible manner.

Deep learning identification of stiffness markers in breast cancer

While essential to our understanding of solid tumor progression, the study of cell and tissue mechanics has yet to find traction in the clinic. Determining tissue stiffness, a mechanical property known to promote a malignant phenotype in vitro and in vivo, is not part of the standard algorithm for the diagnosis and treatment of breast cancer. Instead, clinicians routinely use mammograms to identify malignant lesions and radiographically dense breast tissue is associated with an increased risk of developing cancer. Whether breast density is related to tumor tissue stiffness, and what cellular and non-cellular components of the tumor contribute the most to its stiffness are not well understood. Through training of a deep learning network and mechanical measurements of fresh patient tissue, we create a bridge in understanding between clinical and mechanical markers. The automatic identification of cellular and extracellular features from hematoxylin and eosin (H&E)-stained slides reveals that global and local breast tissue stiffness best correlate with the percentage of straight collagen. Importantly, the percentage of dense breast tissue does not directly correlate with tissue stiffness or straight collagen content.

Region- and degeneration dependent stiffness distribution in intervertebral discs derived by shear wave elastography

Information on the local stiffness characteristics of the intervertebral disc (IVD) is crucial for the understanding of its structure-function properties in health and disease and may improve numerical modeling. Previous studies have attempted to map local tissue stiffness by sectioning the disc and performing mechanical testing on these discrete tissue units, which is technically challenging and may bias the results. Shear wave elastography (SWE) represents a nondestructive alternative that can provide spatially continuous elasticity estimates. We investigated the feasibility of SWE for human intervertebral disc elasticity mapping in a laboratory setting.To this end, global spinal segment mechanical behavior was determined in 6 loading directions and served as ground truth data for the validation of the approach. Subsequently, the cranial spinal vertebra was removed and shear wave elastographic scans of the IVD were acquired. SWE-measurements were reconstructed into three-dimensional elastographic maps, discretized into distinct IVD regions and correlated with global segment mechanical parameters. SWE-derived Young’s modulus estimates were compared among different regions and as a function of their state of degeneration.We found annulus shear wave speed to be moderately correlated with segment mechanical behavior irrespective of the loading direction whereas shear wave speed in the nucleus pulposus showed a very weak association (mean (SD) absolute Pearson correlation coefficients: 0.51 (0.14) and 0.17 (0.12), respectively). Young’s modulus mapping of the intervertebral disc revealed stiffness to be highest in the ventral annulus with a stiffness decrease both circumferentially towards the dorsal aspect as well as towards the center of the disc. SWE hence provides a valid alternative to disc sectioning and piecewise mechanical testing.

Palpation Sensitivity of an Embedded Nodule Using the Finite Element Method

Abstract A physician palpates a tissue to detect an embedded tumor nodule by sensing an increase in local tissue stiffness and nodule size. The Hertz contact model, however, is unable to predict the material or physical properties of a tumor nodule embedded in a healthy tissue of finite thickness. In this study, utilizing a hyperelastic material model, we propose a general methodology to analyze the extent to which the stiffness, size, and depth of a nodule embedded in a tissue affect its detectability. Using dimensional analysis, we generate simple power-law relations to predict physical and material properties of tumor nodules embedded in healthy tissue during indentation. Our results indicate that indenter radius and indentation depth are critical parameters in nodule detection and a thin indenter and large indentation depth increase detection sensitivity of an embedded tumor nodule. Our results also show that anisotropic material properties of either a tissue or an embedded nodule render the embedded tumor nodule undetectable using indentation. We define palpation sensitivity maps that can be used to predict material and physical properties of tumor nodules in healthy tissues. The analysis and results presented in this study might increase accuracy and precision in instrumented probe-based laparoscopic or robotic surgeries.

A Comparison of Clinical Spinal Mobility Measures to Experimentally Derived Lumbar Spine Passive Stiffness.

Spinal stiffness and mobility assessments vary between clinical and research settings, potentially hindering the understanding and treatment of low back pain. A total of 71 healthy participants were evaluated using 2 clinical assessments (posteroanterior spring and passive intervertebral motion) and 2 quantitative measures: lumped mechanical stiffness of the lumbar spine and local tissue stiffness (lumbar erector spinae and supraspinous ligament) measured via myotonometry. The authors hypothesized that clinical, mechanical, and local tissue measures would be correlated, that clinical tests would not alter mechanical stiffness, and that males would demonstrate greater lumbar stiffness than females. Clinical, lumped mechanical, and tissue stiffness were not correlated; however, gradings from the posteroanterior spring and passive intervertebral motion tests were positively correlated with each other. Clinical assessments had no effect on lumped mechanical stiffness. The males had greater lumped mechanical and lumbar erector spinae stiffness compared with the females. The lack of correlation between clinical, tissue, and lumped mechanical measures of spinal stiffness indicates that the use of the term "stiffness" by clinicians may require reevaluation; clinicians should be confident that they are not altering mechanical stiffness of the spine through segmental mobility assessments; and greater resting lumbar erector stiffness in males suggests that sex should be considered in the assessment and treatment of the low back.

How hydrogel inclusions modulate the local mechanical response in early and fully formed post-infarcted myocardium

Expansion of myocardium after myocardial infarction (MI) has long been identified as the primary mechanism that drives adverse left ventricular (LV) remodeling towards heart failure and death. Direct injection of hydrogels into the myocardium to mechanically constrain the infarct has demonstrated promise in limiting its remodeling and expansion. Despite early successes, there remain open questions in the determination of optimal hydrogel therapies, key application characteristics for which include injected polymer volume, stiffness, and spatial placement. Addressing these questions is complicated by the substantial variations in infarct type and extent, as well as limited understanding of the underlying mechanisms. Herein, we present an investigation on how hydrogel inclusions affect the effective tissue-level stiffness and strain fields in myocardium using full three-dimensional (3D) finite element simulations at early and late post-MI time points. We calibrated our simulations to triaxial mechanical and structural measurements of cuboidal LV myocardial specimens of post-infarcted myocardium, 0 and 4 weeks post-MI, injected with a dual-crosslinking hyaluronic acid-based hydrogel. Simulations included multiple deformation modes that spanned the anticipated physiological range in order to assess the effects of variations in inclusion size, location, and modulus on tissue-level myocardial mechanics. We observed significant local stiffening in the hydrogel-injected specimens that was highly dependent on the volume and mechanical properties of the injected hydrogel. Simulations revealed that the primary effect of the injections under physiological loading was a reduction in myocardial strain. This result suggests that hydrogel injections reduce infarct expansion by limiting the peak strains over the cardiac cycle. Overall, our study indicated that modulation of local effective tissue stiffness and corresponding strain reduction are governed by the volume and stiffness of the hydrogel, but relatively insensitive to its transmural placement. These findings provide important insights into mechanisms for ameliorating post-MI remodeling, as well as guidance for the future design of post-MI therapies.

Mechanical changes of peripheral nerve tissue microenvironment and their structural basis during development.

Peripheral nerves are constantly exposed to mechanical stresses associated with body growth and limb movements. Although some aspects of these nerves' biomechanical properties are known, the link between nerve biomechanics and tissue microstructures during development is poorly understood. Here, we used atomic force microscopy to comprehensively investigate the elastic modulus of living peripheral nerve tissue cross sections ex vivo at distinct stages of development and correlated these elastic moduli with various cellular and extracellular aspects of the underlying histological microstructure. We found that local nerve tissue stiffness is spatially heterogeneous and evolves biphasically during maturation. Furthermore, we found the intracellular microtubule network and the extracellular matrix collagens type I and type IV as major contributors to the nerves' biomechanical properties, but surprisingly not cellular density and myelin content as previously shown for the central nervous system. Overall, these findings characterize the mechanical microenvironment that surrounds Schwann cells and neurons and will further our understanding of their mechanosensing mechanisms during nerve development. These data also provide the design of artificial nerve scaffolds to promote biomedical nerve regeneration therapies by considering mechanical properties that better reflect the nerve microenvironment.

Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain.

Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that during development local tissue stiffness changes significantly within tens of minutes. Within this time frame, a stiffness gradient arose in the developing Xenopus brain, and retinal ganglion cell axons turned to follow this gradient. Changes in local tissue stiffness were largely governed by cell proliferation, as perturbation of mitosis diminished both the stiffness gradient and the caudal turn of axons found in control brains. Hence, we identified a close relationship between the dynamics of tissue mechanics and developmental processes, underpinning the importance of time-resolved stiffness measurements.

Fatigue loading of tendon results in collagen kinking and denaturation but does not change local tissue mechanics

Fatigue loading is a primary cause of tendon degeneration, which is characterized by the disruption of collagen fibers and the appearance of abnormal (e.g., cartilaginous, fatty, calcified) tissue deposits. The formation of such abnormal deposits, which further weakens the tissue, suggests that resident tendon cells acquire an aberrant phenotype in response to fatigue damage and the resulting altered mechanical microenvironment. While fatigue loading produces clear changes in collagen organization and molecular denaturation, no data exist regarding the effect of fatigue on the local tissue mechanical properties. Therefore, the objective of this study was to identify changes in the local tissue stiffness of tendons after fatigue loading. We hypothesized that fatigue damage would reduce local tissue stiffness, particularly in areas with significant structural damage (e.g., collagen denaturation). We tested this hypothesis by identifying regions of local fatigue damage (i.e., collagen fiber kinking and molecular denaturation) via histologic imaging and by measuring the local tissue modulus within these regions via atomic force microscopy (AFM). Counter to our initial hypothesis, we found no change in the local tissue modulus as a consequence of fatigue loading, despite widespread fiber kinking and collagen denaturation. These data suggest that immediate changes in topography and tissue structure – but not local tissue mechanics – initiate the early changes in tendon cell phenotype as a consequence of fatigue loading that ultimately culminate in tendon degeneration.

Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-β1 (TGF-β1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. We aimed to define an association between mtDNA and fibroblast responses in IPF. We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-β1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-β1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival. These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

The role of cell body density in ruminant retina mechanics assessed by atomic force and Brillouin microscopy

Cells in the central nervous system (CNS) respond to the stiffness of their environment. CNS tissue is mechanically highly heterogeneous, thus providing motile cells with region-specific mechanical signals. While CNS mechanics has been measured with a variety of techniques, reported values of tissue stiffness vary greatly, and the morphological structures underlying spatial changes in tissue stiffness remain poorly understood. We here exploited two complementary techniques, contact-based atomic force microscopy and contact-free Brillouin microscopy, to determine the mechanical properties of ruminant retinae, which are built up by different tissue layers. As in all vertebrate retinae, layers of high cell body densities (‘nuclear layers’) alternate with layers of low cell body densities (‘plexiform layers’). Different tissue layers varied significantly in their mechanical properties, with the photoreceptor layer being the stiffest region of the retina, and the inner plexiform layer belonging to the softest regions. As both techniques yielded similar results, our measurements allowed us to calibrate the Brillouin microscopy measurements and convert the Brillouin shift into a quantitative assessment of elastic tissue stiffness with optical resolution. Similar as in the mouse spinal cord and the developing Xenopus brain, we found a strong correlation between nuclear densities and tissue stiffness. Hence, the cellular composition of retinae appears to strongly contribute to local tissue stiffness, and Brillouin microscopy shows a great potential for the application in vivo to measure the mechanical properties of transparent tissues.

PP.25.43] CHANGES OF INTRINSIC STIFFNESS OF THE CAROTID ARTERIAL WALL DURING THE CARDIAC CYCLE MEASURED BY SHEAR WAVE ELASTOGRAPHY IN HYPERTENSIVES COMPARED TO NORMOTENSIVES

Objective: The question whether arterial stiffness in hypertension is increased solely because of increased arterial pressure is not solved. Because measurement of arterial stiffness is highly dependent on measurement of blood pressure (BP), the development of methods independent of BP is necessary for clarifying this question. Shear wave elastography (SWE, Supersonic Imagine, Aix-en-Provence, France) is an ultrasound technique which enables to quantitatively assess local tissue stiffness by remotely generating transient shear waves into the tissue (push mode) and tracking their propagation using a very fast sampling rate (up to 10 kHz). The elastic modulus of the material is directly related to the speed of the shear waves, with no need of BP. This method has never been tested against classical echotracking (Artlab, Esaote, Maastricht, NL) and carotid to femoral pulse wave velocity (cf-PWV, Sphygmocor, AtCor, Sydney, Australia). Design and method: We included 25 subjects, 14 normotensives (NT) and 11 essential hypertensives (HT), matched for age and sex. We compared SWE to echotracking and cf-PWV. We optimized SWE algorithms for carotid wall tracking and shear wave group velocity calculation for the anterior (a-SWV) and posterior wall (p-SWV). 8 ultrasonic pushes were triggered at intervals of 200 ms in order to study the variations of stiffness during the cardiac cycle. Results: p-SWV showed no association with carotid PWV, cf-PWV nor BP. Mean a-SWV over the cardiac cycle is strongly associated with carotid PWV measured by Echotracking (r = 0.56, p = 0.003) and cf-PWV (r = 0.66, p < 0.001). a-SWV strongly increases with BP level during the cardiac cycle (p < 10–6). Similar associations between a-SWV and BP were found in NT and HT although HT had higher values of a-SWV throughout all BP levels. However, when a common BP value (100 mmHg) was considered, no significant difference was found between NT and HT, therefore showing that the wall intrinsic material stiffness is not increased in HT. Conclusions: We have demonstrated with a method independent of BP that the increased arterial stiffness in HT is entirely due to the BP increase. SWE appears as a promising technique for assessing arterial stiffness.