This study was supported by National Heart, Lung, and Blood Institute Grants R01-HL-77921 and -86400 (to J. M. Guccione).

1. Algisyl-LVR Implant Procedure (Refer to video)
<ol>
	<li>Algisyl-LVR (LoneStar Heart, Inc. Laguna Hills, CA) is a Calcium-Alginate hydrogel consisting of two components. The Na+-Alginate component is a sterile aqueous solution with 4.6% mannitol and the Ca2+-Alginate component consists of water insoluble particles suspended in a sterile 4.6% mannitol solution (00:27).</li>
	<li>The procedure for implant placement can be performed using either a standard sternotomy or a small limited anterior thoracotomy on the beating heart. Cardiopulmonary bypass is not needed for the procedure.</li>
	<li>Right before use, mix the Na+-Alginate component and the Ca+-Alginate component by combining these two components into one syringe for intramyocardial injections (00:33).</li>
	<li>After 2 minutes, the two different alginate types will cross-link and form a gel that is ready for implant and placement in the myocardium via an injection.</li>
	<li>Identify the free wall of the LV at the mid-ventricular level halfway between the LV apex and base (00:44)</li>
	<li>Starting from the antero-septal groove at the mid-ventricular level, insert the needle at an approximately 45-degree angle and inject 0.3cc of Algisyl-LVR slowly (0.1 ml per second) in one uninterrupted motion (01:15).</li>
	<li>Repeat the injection (step 6) at 10 to 19 sites in a single line (circumferentially) along the mid-ventricular level, starting from the antero-septal groove and ending at the postero-septal groove. The number of implants is dictated by the size of the ventricle, spacing the implants approximately 1 cm apart.</li>
</ol>
2. Quantification of Left Ventricular Stress Using Mathematical Modeling
<ol>
	<li>Before using mathematical modeling to quantify LV wall stress, one must have already obtained the short axis and long axis view of the magnetic resonance images (MRI) containing the LV of the patient. Acquisition of these images can be performed using standard MRI protocol (e.g. Zhang et al.20).</li>
	<li>Digitize the endocardial surface and epicardial surface of the LV from the MR images containing the short-axis (SA) view of the LV. This can be done using the Contour Segmentation Object (CSO) library found in the freely-available software MevisLab.
	<ol class="lalpha">
		<li>In our laboratory, we have created a program based on the modules found in the CSO library of MevisLab so that one simply "contours" the endocardial and epicardial boundary found in the SA view of the MR images containing the LV. Points of the epicardium and the endocardium in real three-dimensional (3D) space are then automatically generated from these contours.</li>
	</ol>
	</li>
	<li>Import the 3D points from step 2 into a commercial software, Rapidform (INUS Technology, Inc, Sunnyvale, CA), to create surfaces of the LV epicardium and the endocardium in the Initial Graphics Exchange Specification (IGES) format. The steps used in creating these IGES surfaces in Rapidform are:
	<ol class="lalpha">
		<li>Insert/Import.</li>
		<li>Create polygon mesh.</li>
		<li>Output IGES surfaces.</li>
	</ol>
	</li>
	<li>Import the IGES surfaces into the commercial software TrueGrid to create an FE mesh of the LV.
	<ol class="lalpha">
		<li>Fill the space between the endocardial and epicardial surface with the eight-node trilinear brick element. In general, a mesh containing about 3,000 elements with 3 elements through the wall thickness is sufficient to model the LV21.</li>
		<li>Once this is completed, export the mesh as an input deck for the FE solver LS-DYNA (LSTC, Livermore, CA). More details of this process can be found in Guccione et al.23.</li>
	</ol>
	</li>
	<li>Assign the myofiber directions using our in-house software "Closer" that modifies the input deck exported from TrueGrid. Closer assigns the myofiber direction in each element as a vector that is parallel to the local epicardial tangent plane. This vector is oriented at an angle measured with respect to the local circumferential direction. In human LV, this angle is set to vary linearly across the wall thickness from -60 &deg; at the epicardium to 60 &deg; at the endocardium23.</li>
	<li>Write the boundary conditions and assign the myocardial material model to the elements in the input deck from step 5.
	<ol class="lalpha">
		<li>Impose nodal displacements at the LV base with the keyword "SPC" in LS-DYNA. The nodes in the epicardial-basal ring are fixed and the rest of the nodes at the LV base are constrained to move only on the basal plane.</li>
		<li>Assign a constitutive law or stress-strain relationship described earlier (see "Introduction") to all the elements using the keyword "MAT" with material identity 128 in LS-DYNA.</li>
		<li>Define the elemental surfaces making up the endocardium and impose pressure boundary conditions with the keyword "LOAD_SEGMENTS".</li>
		<li>Define a pressure-time load curve using the keyword "DEFINE_CURVE".
		<ol class="lroman">
			<li>To simulate end of diastole, prescribe a pressure that increases rapidly with time to a prescribed end-diastolic pressure (EDP) of 20mmHg. The pressure is kept constant at EDP and sufficient time is then allowed for the LV to reach steady-state.</li>
			<li>To simulate end of systole, prescribe a pressure that increases rapidly with time from the end-diastolic state until a prescribed end-systolic pressure (ESP) of 125mmHg is achieved. The pressure is kept constant at ESP and sufficient time is then allowed for the LV to reach steady-state.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Import the completed input deck into the commercial FE solver LS-DYNA to compute the ventricular wall stresses and the LV cavity volume at end of diastole and at end of systole.</li>
	<li>Adjust the material parameters reflecting the passive stiffness and the contractility of the myocardium until the computed LV cavity volume matches the MRI-measured volume at end of diastole and end of systole.</li>
</ol>

<ol>
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Rev. 66 (3), 710-771 (1986).</li><li>Komuro, I., Kurabayashi, M., Takaku, F., Yazaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+ofcellular+oncogenes+in+the+myocardium+during+the+developmental+stage+and+pressure-overloaded+hypertrophy+of+the+rat+heart.">Expression ofcellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat heart.</a> Circ. Res. 62 (6), 1075-1079 (1988).</li><li>Schunkert, H., Dzau, V. J., Tang, S. S., Hirsch, A. T., Apstein, C. S., Lorell, B. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STICH+Hypothesis+2+Investigators.+Coronary+bypass+surgery+with+or+without+surgical+ventricular+reconstruction.">STICH Hypothesis 2 Investigators. Coronary bypass surgery with or without surgical ventricular reconstruction.</a> N. Engl. J. Med. 360 (17), 1705-1717 (2009).</li><li>Buckberg, G. D., Athanasuleas, C. L., Wechsler, A. S., Beyersdorf, F., Conte, J. V., Strobeck, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STICH+trial+unravelled.">The STICH trial unravelled.</a> Eur. J. Heart Fail. 12 (10), 1024-1027 (2010).</li><li>Sabbah, H. N., Wang, M., Jiang, A., Ilsar, I., Sabbah, M. S., Helgerson, S., Peterson, R., Tarazona, N., Lee, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumferential+mid-ventricular+intramyocardial+injections+of+alginate+hydrogel+improve+left+ventricular+function+and+prevent+progressive+remodeling+in+dogs+with+chronic+heart+failure.">Circumferential mid-ventricular intramyocardial injections of alginate hydrogel improve left ventricular function and prevent progressive remodeling in dogs with chronic heart failure.</a> Circulation. 120, S912 .</li><li>Yu, J., Christman, K. L., Chin, E., Sievers, R. E., Saeed, M., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+left+ventricular+geometry+and+improvement+of+left+ventricular+function+in+a+rodent+model+of+chronic+ischemic+cardiomyopathy.">Restoration of left ventricular geometry and improvement of left ventricular function in a rodent model of chronic ischemic cardiomyopathy.</a> J. Thorac. Cardiovasc. Surg. 137 (1), 180-187 (2009).</li><li>Lee, R. J., Hinson, A., Helgerson, S., Bauerschmitt, R., Sabbah, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-based+restoration+of+left+ventricular+mechanics.">Polymer-based restoration of left ventricular mechanics.</a> Cell Transplant. 22 (3), 529-533 (2013).</li><li>Huisman, R. M., Elzinga, G., Westerhof, N., Sipkema, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+left+ventricular+wall+stress.">Measurement of left ventricular wall stress.</a> Cardiovasc. Res. 14 (3), 142-153 (1980).</li><li>Zhang, Z., Tendulkar, A., Sun, K., Saloner, D. A., Wallace, A. W., Ge, L., Guccione, J. M., Ratcliffe, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+Young-Laplace+law+and+finite+element+based+calculation+of+ventricular+wall+stress:+implications+for+post+infarct+and+surgical+ventricular+remodeling.">Comparison of the Young-Laplace law and finite element based calculation of ventricular wall stress: implications for post infarct and surgical ventricular remodeling.</a> Ann. Thorac. Surg. 91 (1), 150-156 (2011).</li><li>Guccione, J. M., McCulloch, A. D., Waldman, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+material+properties+of+intact+ventricular+myocardium+determined+from+a+cylindrical+model.">Passive material properties of intact ventricular myocardium determined from a cylindrical model.</a> J. Biomech. Eng. 113 (1), 42-55 (1991).</li><li>Guccione, J. M., Waldman, L. K., McCulloch, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+active+contraction+in+cardiac+muscle:+Part+II--Cylindrical+models+of+the+systolic+left+ventricle.">Mechanics of active contraction in cardiac muscle: Part II--Cylindrical models of the systolic left ventricle.</a> J. Biomech. Eng. 115 (1), 82-90 (1993).</li><li>Zhang, P., Guccione, J. M., Nicholas, S. I., Walker, J. C., Crawford, P. C., Shamal, A., Acevedo-Bolton, G., Guttman, M. A., Ozturk, C., McVeigh, E. R., Saloner, D. A., Wallace, A. W., Ratcliffe, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoventricular+patch+plasty+for+dyskinetic+anteroapical+left+ventricular+aneurysm+increases+systolic+circumferential+shortening+in+sheep.">Endoventricular patch plasty for dyskinetic anteroapical left ventricular aneurysm increases systolic circumferential shortening in sheep.</a> J. Thorac. Cardiovasc. Surg. 134 (4), 1017-1024 (2007).</li><li>Wenk, J. F., Klepach, D., Lee, L. C., Zhang, Z., Ge, L., Tseng, E. E., Martin, A., Kozerke, S., Gorman, J. H. 3. r. d., Gorman, R. C., Guccione, J. 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Mr. Hinson is an employee of LoneStar Heart, Inc.

Algisyl-LVR injection therapy
Injection of material into the LV free wall to reduce ventricular wall stress is a novel treatment designed for patients with dilated cardiomyopathy. This treatment has shown great promise in both preclinical and clinical studies 15. A randomized, controlled study to evaluate this treatment as a method of LV augmentation for patients with severe heart failure (AUGMENT-HF) has been underway since February 2012.
Multiple iterations of the product are being developed that target different physician segments and clinical needs. In the surgical product version for cardiothoracic surgeons, the alginate is delivered in a standard syringe and custom needle system designed for surgeons to perform the injections through a small surgical incision in the chest (minimal thoracotomy). The implant procedure is performed on a beating heart. The physical properties of the alginate hydrogel upon injection into the myocardium are similar to that of the diastolic myocardium, and become a permanent implant. The duration of the total operative procedure is expected to be less than 60 minutes in most cases, limiting exposure of the patient to a minimal anesthesia time. A second product version may offer patients with heart failure a procedure that can be performed by interventional cardiologists and, in some isolated cases, other specialists in a noninvasive or hybrid cardiology laboratory. It also would allow acute effects to be studied.
Quantification of left ventricular stress using mathematical modeling
The method of using mathematical modeling with the FE method is currently the only way to precisely quantify in-vivo regional wall stress in the ventricles. Combining mathematical modeling with medical imaging such as MRI enables one to compute in-vivo regional wall stress in patient-specific ventricles so as to help understand the functional state of these ventricles and quantify the mechanical effects of the injection treatment in patients.
Though we have treated the LV as a homogeneous material here, this method can be (and has been) extended to quantify in-vivo ventricular wall stress in non-homogeneous ventricles, particularly, when myocardial infarction is present. In such cases, the boundaries of the infarct and its adjacent borderzone have to be identified from MRI using gadolinium as a contrast agent. These boundaries are imported into TrueGrid to create elements that purely reside within each distinct region, namely, the infarct, the borderzone and the remote region. Material parameters reflecting pathological changes in each region can be assigned through the respective elements in LS-DYNA. These parameters have been found in a patient with a myocardial infarction using in-vivo myocardial strain measured from tagged MRI 21. Patients who require surgical revascularization will often experience atrial fibrillation during the post-operative period, which is associated with very poor quality tagged MRI data. Such patients also require a few days to recuperate from surgery. Thus, 3D echocardiography and speckle-tracking may be a more appropriate imaging modality and myocardial strain measurement technique than tagged MRI to study acute effects of surgical procedures.
Lastly, we used the commercial software Rapidform, Truegrid and LS-DYNA in the process of generating patient-specific mathematical models of the ventricles because we have found them to be generally efficient in accomplishing their respective tasks. However, other software is available, such as Cubit (for generating FE mesh) and Abaqus (a FE solver) that may also be suitable for creating mathematical models of the ventricles.

Reduction of ventricular wall stress is considered a cornerstone in the treatment of heart failure1. In its simplest form given by Laplace's law, ventricular wall stress is directly proportional to the diameter of the ventricle and the ventricular pressure, and is inversely proportional to the wall thickness of the ventricle. It is widely believed that increased ventricular wall stress is responsible for the adverse remodeling process in which the ventricles progressively become dilated, eventually leading to heart failure2. Clinical and animal studies have shown that increased wall stress induces changes in proteins, contractile element synthesis and gene expression that support the remodeling process3,4,5. Increased wall stress has also been shown to be an independent predictor of subsequent LV remodeling6,7.
Many new surgical treatments and devices have been developed with a central aim of reducing ventricular wall stress in a bid to prevent and reverse the progression of heart failure in patients8,9,10. Though these treatments share the same aim, they achieve it differently. For example, the surgical ventricular reduction procedure10 seeks to reduce ventricular wall stress by surgically reducing the size of a dilated left ventricle, but its result is a subject of controversy11,12.
Recently, the injection of a biocompatible material, Algisyl-LVR, into the left ventricle as a treatment for dilated cardiomyopathy has garnered considerable attention in the medical community. This treatment has been shown to be effective in preventing or even reversing the progression of heart failure in animal studies13,14 and, most recently, in a human clinical trial15. Contrary to other devices, this treatment seeks to reduce the ventricular wall stress by injecting material into the left ventricular wall to thicken it.
Detailed knowledge of ventricular wall stress, particularly in humans, however, remains elusive. This lack of knowledge is primarily because forces or stresses cannot be measured directly in the intact ventricles16. Though closed-form analytical equations such as Laplace's law can estimate left ventricular wall stress, they were developed based on restrictive assumptions which include axis-symmetry of the LV, material isotropy and homogeneity within the LV. Due to these factors, the prediction of ventricular wall stress in actual LV using Laplace's law is inaccurate17. To remove these restrictions and to obtain more accurate prediction of the ventricular wall stress, mathematical modeling using the finite element (FE) method with patient-specific ventricular geometry should be used in place of the simplified Laplace's law17.
The FE method is a numerical technique that is frequently used to solve a set of partial differential equations (PDE) describing a boundary value problem. This method is particularly useful when a closed form solution is difficult or cannot be obtained analytically. In the context of a mathematical LV model used to quantify ventricular wall stress, the set of PDEs are the governing equations of mechanical equilibrium (balance of linear momentum) that describe the LV motion when pressure or load is applied at the LV's endocardial surface. When the FE method is used, the LV wall is split into inter-connected sub-domains or elements (usually hexahedron with 8 corner nodes) that deform according to a prescribed stress-strain relationship of the myocardium.
Stress-strain relationships describing large deformation of the LV during passive filling in diastole and during active contraction in systole have been previously validated in large animal studies. The LV is modeled to be approximately three times stiffer in the myofiber direction than in directions perpendicular to the myofiber direction during diastole18. Active contraction during systole is modeled by increasing the stiffness of the LV along the myofiber direction. This increase in stiffness is a function of time and depends on experimentally determined variables such as the intracellular calcium concentration and the sarcomere length19.
Using this prescribed stress-strain relationship of the myocardium, the FE method calculates the new nodal positions based on load(s) applied to the LV. Once the new nodal positions are calculated, the resultant strain (a measure of deformation) and stress can be determined in each element to produce the strain and stress distribution within the LV.
Here, we outline the steps required to implant Algisyl-LVR in patients and to create the corresponding patient-specific LV mathematical models before and after treatment to quantify the LV wall stress.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REGENTS</td>
 </tr>
 <tr>
 <td>Na+-Alginate </td>
 <td>LoneStar Heart, Inc</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ca2+-Alginate </td>
 <td>LoneStar Heart, Inc</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>MevisLab</td>
 <td>Mevis Medical Solution</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>TrueGrid</td>
 <td>XYZ Scientific Application, Inc</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Rapidform</td>
 <td>Inus Technology, Inc</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>LS-Dyna</td>
 <td>Livermore Software Technology Corporation</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

reduction in left ventricular wall stress and improvement in function in failing hearts using algisyl-lvr

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

Injection of Algisyl-LVR into the LV free wall thickens it and continuously reduces the size of the LV over time. The thickening of the LV wall and the reduction in size of the LV is evident in the MRI of the LV in a patient at end of systole, before and 6 months after receiving Algisyl-LVR (Figure 1).
Figure 2 shows the outcome of each step involved in the quantification of the left ventricular wall stress. In Figure 2a, the epicardial and the endocardial edges were identified from a short-axis view of the LV in the MRI and contoured using MevisLab. The resultant IGES endocardial surface (blue) and the epicardial surface (red) created from Rapidform using the contoured points are shown in Figure 2b. Following this, the space between the endocardial and epicardial surfaces was filled with 8-noded trilinear brick element using TrueGrid (Figure 2c). Vectors defining the myofiber direction in each element were then computed with Closer. These vectors are shown as black arrows in Figure 2d and a slice of the LV wall (blue) shows the variation of myofiber direction across the LV wall as described earlier. In Figure 2e, the boundary conditions, namely, the pressure and the nodal displacement imposed on the LV are shown. The applied pressure is shown as arrows pointing towards the endocardial wall. Nodes at the epicardial-basal ring (shown as spheres) were constrained from moving in all directions whereas the rest of the basal nodes (shown as cubes) were constrained to move only in the basal plane (Figure 2e). Lastly, Figure 2f shows the computed ventricular wall stress in the myofiber direction at end of diastole from a patient before treatment. It is obvious from the figure that elevated stresses are found at the endocardium and at regions where the LV wall is thin.
<img src="/files/ftp_upload/50096/50096fig1.jpg" alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50096/50096fig1highres.jpg"/> 
Figure 1. Effects of Algisyl on a patient's LV (indicated by arrow) after 6 months, as seen on magnetic resonance images. The images show that the LV has shrunk and the wall thickness has increased after 6 months.
<img src="/files/ftp_upload/50096/50096fig2.jpg" alt="Figure 2" fo:content-width="4.71in" fo:src="/files/ftp_upload/50096/50096fig2highres.jpg" /> 
Figure 2. Steps involved in the quantification of left ventricular wall stress. (a) Digitizing the MR images. (b) Creating IGES surfaces. (c) Creating the FE mesh. (d) Assigning the myofiber orientation. (e) Imposing the boundary conditions. (f) Calculating the ventricular wall stress (shown here at end of diastole). Refer to text for explanation.

Watch this Scientific Journal Video about Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR at JoVE.com

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

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24.2K Views.  UCSF/VA Medical Center. This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Video: Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, mice undergo RR upon removal of the aortic constriction. In this paper, we describe a step-by-step procedure to perform a minimally invasive surgical aortic debanding in mice. Echocardiography was subsequently used to assess the degree of cardiac hypertrophy and dysfunction during LV remodeling and RR and to determine the best timing for aortic debanding. At the end of the protocol, terminal hemodynamic evaluation of the cardiac function was conducted, and samples were collected for histological studies. We showed that debanding is associated with surgical survival rates of 70-80%. Moreover, two weeks after debanding, the significant reduction of ventricular afterload triggers the regression of ventricular hypertrophy (~20%) and fibrosis (~26%), recovery of diastolic dysfunction as assessed by the normalization of left ventricular filling and end-diastolic pressures (E/e&#39;&#160;and LVEDP). Aortic debanding is a useful experimental model to study LV RR in rodents. The extent of myocardial recovery is variable between subjects, therefore, mimicking the diversity of RR that occurs in the clinical context, such as aortic valve replacement. We conclude that the aortic banding/debanding model represents a valuable tool to unravel novel insights into the mechanisms of RR, namely the regression of cardiac hypertrophy and the recovery of diastolic dysfunction.

Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery.

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...