This work was supported by the American Heart Association Scientist Development Grant (16SDG27760143) and the Presbyterian Health Foundation. KMC was supported in part by the University of Oklahoma (OU) Undergraduate Research Opportunity Program and Honors Research Apprenticeship Program. DWL was supported in part by the National Science Foundation Graduate Research Fellowship (GRF 2019254233) and the American Heart Association/Children&#39;s Heart Foundation Predoctoral Fellowship (Award #821298). All of this support is gratefully acknowledged.

<ol><li>Vesely, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+elastin+in+aortic+valve+mechanics%5BTitle%5D)+AND+%22Journal+of+Biomechanics%22%5BJournal%5D)">The role of elastin in aortic valve mechanics.</a> Journal of Biomechanics. 31 (2), 115-123 (1998).</li>
<li>Zhang, W., Ayoub, S., Liao, J., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+meso-scale+layer-specific+structural+constitutive+model+of+the+mitral+heart+valve+leaflets%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">A meso-scale layer-specific structural constitutive model of the mitral heart valve leaflets.</a> Acta Biomaterialia. 32, 238-255 (2016).</li>
<li>Stella, J. A., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+biaxial+mechanical+properties+of+the+layers+of+the+aortic+valve+leaflet%5BTitle%5D)+AND+%22Journal+of+Biomechanical+Engineering%22%5BJournal%5D)">On the biaxial mechanical properties of the layers of the aortic valve leaflet.</a> Journal of Biomechanical Engineering. 129 (5), 757-766 (2007).</li>
<li>Khoiy, K. A., Amini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+biaxial+mechanical+response+of+porcine+tricuspid+valve+leaflets%5BTitle%5D)+AND+%22Journal+of+Biomechanical+Engineering%22%5BJournal%5D)">On the biaxial mechanical response of porcine tricuspid valve leaflets.</a> Journal of Biomechanical Engineering. 138 (10), 104504(2016).</li>
<li>Jett, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+investigation+of+the+anisotropic+mechanical+properties+and+anatomical+structure+of+porcine+atrioventricular+heart+valves%5BTitle%5D)+AND+%22Journal+of+the+Mechanical+Behavior+of+Biomedical+Materials%22%5BJournal%5D)">An investigation of the anisotropic mechanical properties and anatomical structure of porcine atrioventricular heart valves.</a> Journal of the Mechanical Behavior of Biomedical Materials. 87, 155-171 (2018).</li>
<li>Meador, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+detailed+mechanical+and+microstructural+analysis+of+ovine+tricuspid+valve+leaflets%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">A detailed mechanical and microstructural analysis of ovine tricuspid valve leaflets.</a> Acta Biomaterialia. 102, 100-113 (2020).</li>
<li>Hudson, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pilot+study+on+linking+tissue+mechanics+with+load-dependent+collagen+microstructures+in+porcine+tricuspid+valve+leaflets%5BTitle%5D)+AND+%22Bioengineering%22%5BJournal%5D)">A pilot study on linking tissue mechanics with load-dependent collagen microstructures in porcine tricuspid valve leaflets.</a> Bioengineering. 7 (2), 60(2020).</li>
<li>Pant, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pressure-induced+microstructural+changes+in+porcine+tricuspid+valve+leaflets%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Pressure-induced microstructural changes in porcine tricuspid valve leaflets.</a> Acta Biomaterialia. 67, 248-258 (2018).</li>
<li>Kramer, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+investigation+of+layer-specific+tissue+biomechanics+of+porcine+atrioventricular+heart+valve+leaflets%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">An investigation of layer-specific tissue biomechanics of porcine atrioventricular heart valve leaflets.</a> Acta Biomaterialia. 96, 368-384 (2019).</li>
<li>Ross, C. J., Laurence, D. W., Wu, Y., Lee, C. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biaxial+mechanical+characterizations+of+atrioventricular+heart+valves%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Biaxial mechanical characterizations of atrioventricular heart valves.</a> Journal of Visualized Experiments: JoVE. (146), e59170(2019).</li>
<li>Goth, W., Lesicko, J., Sacks, M. S., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical-based+analysis+of+soft+tissue+structures%5BTitle%5D)+AND+%22Annual+Review+of+Biomedical+Engineering%22%5BJournal%5D)">Optical-based analysis of soft tissue structures.</a> Annual Review of Biomedical Engineering. 18, 357-385 (2016).</li>
<li>Jett, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+polarized+spatial+frequency+domain+imaging+%28pSFDI%29+with+a+biaxial+mechanical+testing+system+for+quantification+of+load-dependent+collagen+architecture+in+soft+collagenous+tissues%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Integration of polarized spatial frequency domain imaging (pSFDI) with a biaxial mechanical testing system for quantification of load-dependent collagen architecture in soft collagenous tissues.</a> Acta Biomaterialia. 102, 149-168 (2020).</li>
<li>Reddy, J. N. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aReddy%2C+JN+%22An+Introduction+to+Continuum+Mechanics%22">An Introduction to Continuum Mechanics</a>. , Cambridge University Press. (2013).</li>
<li>Duginski, G. A., Ross, C. J., Laurence, D. W., Johns, C. H., Lee, C. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+investigation+of+the+effect+of+freezing+storage+on+the+biaxial+mechanical+properties+of+excised+porcine+tricuspid+valve+anterior+leaflets%5BTitle%5D)+AND+%22Journal+of+the+Mechanical+Behavior+of+Biomedical+Materials%22%5BJournal%5D)">An investigation of the effect of freezing storage on the biaxial mechanical properties of excised porcine tricuspid valve anterior leaflets.</a> Journal of the Mechanical Behavior of Biomedical Materials. 101, 103438(2020).</li>
<li>Salinas, S. D., Clark, M. M., Amini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+response+changes+in+porcine+tricuspid+valve+anterior+leaflet+under+osmotic-induced+swelling%5BTitle%5D)+AND+%22Bioengineering%22%5BJournal%5D)">Mechanical response changes in porcine tricuspid valve anterior leaflet under osmotic-induced swelling.</a> Bioengineering. 6 (3), 70(2019).</li>
<li>Pokutta-Paskaleva, A., Sulejmani, F., DelRocini, M., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+mechanical%2C+morphological%2C+and+microstructural+characterization+of+porcine+mitral+and+tricuspid+leaflets+and+chordae+tendineae%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Comparative mechanical, morphological, and microstructural characterization of porcine mitral and tricuspid leaflets and chordae tendineae.</a> Acta Biomaterialia. 85, 241-252 (2019).</li>
<li>Ross, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+investigation+of+the+glycosaminoglycan+contribution+to+biaxial+mechanical+behaviors+of+porcine+atrioventricular+heart+valve+leaflets%5BTitle%5D)+AND+%22Journal+of+the+Royal+Society+Interface%22%5BJournal%5D)">An investigation of the glycosaminoglycan contribution to biaxial mechanical behaviors of porcine atrioventricular heart valve leaflets.</a> Journal of the Royal Society Interface. 16 (156), 0069(2019).</li>
<li>Sommer, G., Regitnig, P., Költringer, L., Holzapfel, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biaxial+mechanical+properties+of+intact+and+layer-dissected+human+carotid+arteries+at+physiological+and+supraphysiological+loadings%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Biaxial mechanical properties of intact and layer-dissected human carotid arteries at physiological and supraphysiological loadings.</a> American Journal of Physiology-Heart and Circulatory Physiology. 298 (3), 898-912 (2009).</li>
<li>Holzapfel, G. A., Sommer, G., Gasser, C., Regitnig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+the+layer-specific+mechanical+properties+ofhuman+coronary+arteries+with+intimal+thickening%2C+and+related+constitutive+modelling%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Determination of the layer-specific mechanical properties ofhuman coronary arteries with intimal thickening, and related constitutive modelling.</a> American Journal of Physiology-Heart and Circulatory Physiology. 289 (5), 2048-2058 (2005).</li>
<li>Sommer, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiaxial+mechanical+response+and+constitutive+modeling+of+esophageal+tissues%3A+Impact+on+esophageal+tissue+engineering%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">Multiaxial mechanical response and constitutive modeling of esophageal tissues: Impact on esophageal tissue engineering.</a> Acta Biomaterialia. 9 (12), 9379-9391 (2013).</li>
</ol>

The authors have no conflicts of interest to disclose.

Critical steps for the protocol include: (i) the layer microdissection, (ii) the tissue mounting, (iii) the fiducial marker placement, and (iv) the pSFDI setup. Appropriate layer microdissection is the most important and difficult aspect of the method described herein. Prior to launching an investigation utilizing this technique, the dissector(s) should have long-term practice with the microdissection technique and all three TV leaflets. The dissector should ensure the composite layer specimens are sufficiently large (&#...

The TV is located between the right atrium and right ventricle of the heart. Throughout the cardiac cycle, the TV regulates the unidirectional blood flow via cyclic opening and closing of the TV anterior leaflet (TVAL), the TV posterior leaflet (TVPL), and the TV septal leaflet (TVSL). These leaflets are complex and have four distinct anatomical layers&#8212;the atrialis (A), the spongiosa (S), the fibrosa (F), and the ventricularis (V)&#8212;with unique microstructural constituents. The elastin fibers in the atrialis and ventricularis help restore the tissue to its undeformed geometry after mechanical loading1. In contrast, the fibrosa contains a dense network of undulated collagen fibers that contribute to the load-bearing capacity of the leaflets2. Mainly consisting of glycosaminoglycans, the spongiosa has been hypothesized to enable shearing between leaflet layers during heart valve function3. While all three leaflet types have the same anatomical layers, there are variations in the thicknesses of the layers and constituent ratios that have implications for leaflet-specific mechanical behaviors.
Researchers have explored the properties of the TV leaflets using planar mechanical characterizations, histomorphological assessments, and optical characterizations of the collagen fiber architecture. For example, planar biaxial mechanical characterizations seek to emulate physiological loading by applying perpendicular displacements to the tissue and recording the associated forces. The resulting force-displacement (or stress-stretch) observations have revealed that all three TV leaflets exhibit nonlinear, direction-specific mechanical behaviors with more apparent leaflet-specific responses in the radial tissue direction4,5,6. These leaflet-specific behaviors are believed to stem from differences in the microstructural properties observed using standard histological techniques6,7. Further, second harmonic generation imaging6, small-angle light scattering8, and polarized spatial frequency domain imaging7&#160;(pSFDI) aim to understand these microstructural properties and have shown leaflet-specific differences in the collagen fiber orientation and fiber crimp that have implications for the observed tissue-level mechanical behaviors. These studies have significantly advanced our understanding of the tissue microstructure and its role in tissue-level behaviors. However, much remains to be addressed in experimentally connecting the tissue mechanics and the underlying microstructure.
Recently, this laboratory performed mechanical characterizations of the TV leaflet layers separated into two composite layers (A/S and F/V) using a microdissection technique9. That earlier work highlighted differences in the mechanical properties of the layers and helped provide insight into how the layered microstructure contributes to the tissue mechanical behaviors. Although this investigation improved our understanding of the TV leaflet microstructure, the technique had several limitations. First, the properties of the composite layers were not directly compared to the intact tissue, leading to a lack of complete understanding of the mechanics-microstructure relationship. Second, the collagen fiber architecture of the composite layers was not examined. Third, only the layers of the TVAL were investigated due to difficulties with collecting the composite layers from the other two TV leaflets. The method described herein provides a holistic characterization framework that overcomes these limitations and provides complete characterizations of the TV leaflets and their composite layers.
This paper describes the microdissection technique that separates the three TV leaflets into their composite layers (A/S and F/V) for biaxial mechanical and microstructural characterizations10,11,12. This iterative protocol includes (i) biaxial mechanical testing and pSFDI characterization of the intact leaflet, (ii) a novel and reproducible microdissection technique to reliably obtain the composite TV layers, and (iii) biaxial mechanical testing and pSFDI characterization of the composite TV layers. The tissue was exposed to biaxial tensile loading with various force ratios for mechanical testing. Then, pSFDI was used to determine the collagen fiber orientation and alignment at various loaded configurations. pSFDI preserves the native collagen fiber architecture, allows load-dependent analysis, and circumvents the typical need to fix or clear tissue for collagen fiber architecture analysis, such as in second harmonic generation imaging or small-angle light scattering. Finally, the tissues were prepared using standard histology techniques to visualize the tissue microstructure. This iterative and holistic framework allows for the direct comparison of the mechanical and microstructural properties of the TV leaflet to its composite layers.

<table><tbody><tr><td>10% Formalin Solution, Neutral Buffered</td><td>Sigma-Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Alconox Detergent</td><td>Alconox</td><td></td><td>cleaning compound</td></tr><tr><td>BioTester - Biaxial Tester</td><td>CellScale Biomaterials Testing</td><td></td><td>1.5 N Load Cell Capacity</td></tr><tr><td>Cutting Mat</td><td>Dahle</td><td>B0027RS8DU</td><td></td></tr><tr><td>Deionized Water</td><td>N/A</td><td></td><td></td></tr><tr><td>Fine-Tipped Tool</td><td>HTI INSTRUMENTS</td><td>NSPLS-12</td><td></td></tr><tr><td>Forceps - Curved</td><td>Scientific Labwares</td><td>16122</td><td></td></tr><tr><td>Forceps - Thick</td><td>Scientific Labwares</td><td>161001078</td><td></td></tr><tr><td>Forceps - Thin</td><td>Scientific Labwares</td><td>16127</td><td></td></tr><tr><td>LabJoy</td><td>CellScale Biomaterials Testing</td><td>Version 10.66</td><td></td></tr><tr><td>Laser Displacement Sensor</td><td>Keyence</td><td>IL-030</td><td></td></tr><tr><td>Liquid Cyanoacrylate Glue</td><td>Loctite</td><td>2436365</td><td></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>Version 2020a</td><td></td></tr><tr><td>Micro Scissors</td><td>HTI Instruments</td><td>CAS55C</td><td></td></tr><tr><td>Pipette</td><td>Belmaks</td><td>360758081051Y4</td><td></td></tr><tr><td>Polarized Spatial Frequency Domain Imaging Device</td><td>N/A</td><td></td><td>Made in-house using a digital light projector, linear polarizer, rotating polarizer mount, and charge-coupled device camera.&lt;br/&gt; See doi.org/10.1016/j.actbio.2019.11.028 (PMCID: PMC8101699) for more details.</td></tr><tr><td>Scalpel</td><td>THINKPRICE</td><td>TP-SCALPEL-3010</td><td></td></tr><tr><td>Single Edge Industrial Razor Blades (Surgical Carbon Steel)</td><td>VWR International</td><td>H3515541105024</td><td></td></tr><tr><td>Surgical Pen</td><td>LabAider</td><td>LAB-Skin-6</td><td></td></tr><tr><td>T-Pins</td><td>Business Source</td><td>BSN32351</td><td></td></tr><tr><td>Wax Board</td><td>N/A</td><td></td><td>Made in-house using modeling wax and baking tray</td></tr><tr><td>Weigh Boat</td><td>Pure Ponta</td><td>mdo-azoc-1030</td><td></td></tr></tbody></table>

layer microdissection of tricuspid valve leaflets for biaxial mechanical characterization and microstructural quantification

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three leaflets, each with unique mechanical behaviors. These variations among the three TV leaflets can be further understood by examining their four anatomical layers, which are the atrialis (A), spongiosa (S), fibrosa (F), and ventricularis (V). While these layers are present in all three TV leaflets, there are differences in their thicknesses and microstructural constituents that further influence their respective mechanical behaviors. 
This protocol includes four steps to elucidate the layer-specific differences: (i) characterize the mechanical and collagen fiber architectural behaviors of the intact TV leaflet, (ii) separate the composite layers (A/S and F/V) of the TV leaflet, (iii) carry out the same characterizations for the composite layers, and (iv) perform post-hoc histology assessment. This experimental framework uniquely allows the direct comparison of the intact TV tissue to each of its composite layers. As a result, detailed information regarding the microstructure and biomechanical function of the TV leaflets can be collected with this protocol. Such information can potentially be used to develop TV computational models that seek to provide guidance for the clinical treatment of TV disease.

The microdissection will yield A/S and F/V specimens with relatively uniform thicknesses that can be mounted to a (commercial) biaxial testing device. Histology analysis of the intact leaflet and the two dissected layers will verify if the tissue was correctly separated along the border between the spongiosa and fibrosa (Figure 7). Additionally, the histology micrographs can be used to determine the tissue layer thicknesses and constituent mass fractions using ImageJ software. A failed disse...

Watch this Scientific Journal Video about Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification at JoVE.com

Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

This protocol describes the biaxial mechanical characterization, polarized spatial frequency domain imaging-based collagen quantification, and microdissection of tricuspid valve leaflets. The provided method elucidates how the leaflet layers contribute to the holistic leaflet behaviors.

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three ...

layer-microdissection-tricuspid-valve-leaflets-for-biaxial-mechanical

Research

JoVE Journal

Bioengineering

1.9K Views.  The University of Oklahoma. Deze methode kan de bijdragen van weefsellagen aan het algehele weefselgedrag isoleren, waardoor nieuw inzicht ontstaat in de weefselmicrostructuur. Ons microdissectie- en testraamwerk stelt ons in staat om de mechanische en microstructurele eigenschappen van hartklepfolders direct te vergelijken met hun eigen composietlagen. De microdissectie kan moeilijk onder de knie te krijgen zijn, daarom is het ten zeerste aan te raden om zoveel mogelijk te oefenen voordat je aan je studie begint.

Video: Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

Medicine

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (&#181;CT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. &#181;CT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...

Engineering

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Biology

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. Mechanistic studies to improve our&#160;understanding of the underlying pathophysiology responsible for the development of premature TV failure are lacking. Due to the inability to conduct these studies in humans, an animal model is required. In this manuscript, we describe the protocols for a novel chronic recovery infant piglet heart model for the study of changes in the TV when placed under combined volume and pressure stress. In this model, volume loading of the right ventricle&#160;and the TV is achieved through the disruption of the pulmonary valve. Then pressure loading is accomplished through the placement of a pulmonary artery band. The success of this model is assessed at four weeks post intervention surgery through echocardiography, intracardiac pressure measurement, and pathologic examination of the heart specimens.

A novel recovery piglet heart model with combined pressure and volume overload on the right ventricle is described for the study of tricuspid valve function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Mechanical Control of Relaxation Using Intact Cardiac Trabeculae

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.

Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation.

Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

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