Name: 3d whole-heart myocardial tissue analysis
Uploaded: 2017-04-12T13:00:00
Description: Watch this Scientific Journal Video about 3D Whole-heart Myocardial Tissue Analysis at JoVE.com

The authors would like to thank Marlijn Jansen, Joyce Visser, and Martijn van Nieuwburg for their assistance with the animal experiments. We greatly acknowledge Martijn Froeling and Anke Wassink for their assistance with the MRI imaging.

The in vivo experiment was conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Research. The experiment was approved by the local Animal Experiments Committee.
1. Preparation of Injectable and Embedding Solution
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	<li>Prepare the injectable gel.
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		<li>Prepare 1 mL of ureido-pyrimidinone (UPy) gel in accordance to previously-described protocols12,<sup class="xref"...

<ol>
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Whole-heart 3D myocardial tissue processing according to this protocol provides a structured method that enables the 3D analysis of the infarct, the IBZ, and the performed injections with respect to the cardiac anatomy. The filling volume of the heart depends on the desired analysis. In this study, to assess the injection accuracy, we aimed to fill the heart to resemble the end-diastolic geometry as closely as possible. To enforce this, the LV apex is fixed to the bottom of the container and the LV is filled with agar wh...

Ischemic heart disease has been the world&#39;s leading cause of death for the past decades1. Acute treatment after myocardial infarction aims to restore blood flow to the myocardium via percutaneous coronary intervention or coronary artery bypass grafting. In severe infarctions, a large area of the myocardium is scarred, and these cases often result in ischemic heart failure (HF)2. Current treatment options for HF focus on prevention and the preservation of cardiac function for the HF patients, but not on regeneration.
In the last decade, cardiac regenerative therapies have been investigated as a treatment option for HF3. This therapy aims to deliver biologicals, such as stem cells or growth factors, directly to the injured myocardium to induce revascularization, cardiomyocyte protection, differentiation, and growth4. For optimal therapeutic effect, it is hypothesized that the biological must be injected in the infarct border zone (IBZ) to facilitate good tissue perfusion for the survival of the biological and for optimal effect to the target zone5,6. Multiple techniques have been developed to perform identification and visualization of the IBZ to guide intramyocardial injections7,8,9,10,11. Besides identification and visualization of the IBZ, the delivery also relies on the biomaterials and injection catheters used. To validate the injection accuracy of the delivery techniques, an accurate and reproducible quantification method is required.
We have developed a protocol for whole-heart myocardial tissue processing that offers two-dimensional (2D) and three-dimensional (3D) imaging, which can be used for qualitative and quantitative study aims. The protocol covers the embedding process and the digital image analysis. In this paper, we demonstrate a protocol for the assessment of the targeting accuracy of intramyocardial injections in the IBZ in a large porcine model of chronic myocardial infarction.

<table border="0" cellpadding="0" cellspacing="0" width="610">
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">0.9% Saline</td>
			<td style="width:108px;">Braun</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Agarose</td>
			<td style="width:108px;">Roche Diagnostics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Scientific grade multipurpose agar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biomolecular fluorescence scanner Typhoon 9410&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Embedding container</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>Plastic, dimensions 17 x 14,5 x 14 cm</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">FluoSpheres Polystyrene Microspheres</td>
			<td style="width:108px;">Invitrogen</td>
			<td>F8834</td>
			<td>red, 10 &#181;m</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gadolinium</td>
			<td style="width:108px;">Gadovist</td>
			<td style="width:119px;"></td>
			<td>1,0 mmol/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">dS 32 channel head coil</td>
			<td style="width:108px;">Philips</td>
			<td style="width:119px;"></td>
			<td>Or similar</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Matlab</td>
			<td style="width:108px;">Mathworks</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To insure compatability 2015a or newer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Meat slicer</td>
			<td style="width:108px;">Berkel</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Myostar injection catheter</td>
			<td style="width:108px;">Biosense Webster</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Super paramagnetic iron oxide particles</td>
			<td style="width:108px;">Sinerem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Triphenyl-tetrazolium chloride</td>
			<td style="width:108px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UPy-PEG10k</td>
			<td style="width:108px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Vicryl 2-0</td>
			<td style="width:108px;">Ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

3d whole-heart myocardial tissue analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

Tissue Embedding
Through the embedding process, an end-diastolic-like geometry was established. The agar successfully adhered to the heart tissue, enabling the tissue to be sliced at the desired angulation with equal slice thicknesses (Figure 2A and 2C).
Scar- and Injection-site Assessment
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Watch this Scientific Journal Video about 3D Whole-heart Myocardial Tissue Analysis at JoVE.com

3D Whole-heart Myocardial Tissue Analysis

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Narrator}The overall goal of this protocol is to facilitate a structured and reproducible method for 3D whole-heart tissue processing that can be used to assess the targeting accuracy of intramyocardial injections into the infarct border zone. This method can help to answer key questions in the cardioregenerative medicine field, such as practice development and optimization of injection techniques. The main advantage of this technique is that it offers a standardized and reproducible method for 3D whole-heart tissue processing.The implications of this technique extend towards development of cardiac regenerative therapies and validating injection accuracy of normal delivery techniques. The aim of this cardiac stem cell therapy is to inject cells into the infarct border zone for stimulation of local vasculogenesis and cardiomyocytes protection. Capturing the heart in end diastolic phase requires practice and patience.A visual demonstration of these steps is critical. Begin by separating the pericardium from the microbead injected heart, keeping the atria and ventricles intact. When all of the tissue has been removed, use Klinkenberg scissors to dissect the ascending aorta about one centimeter above the aortic valve, and cut the inferior caval and pulmonary veins about one centimeter from the atrium.After fixing the apex of the heart to the bottom of the plastic embedding container to prevent flotation, place 2-0 sutures through the remaining part of the aorta to the rims of the container, and fixate the heart. Take care that the heart is centered and is not touching the walls of the container. When the organ has been secured in an end diastolic like geometry, use mosquito clamps to grip the inferior caval vein and use a 50 milliliter syringe to slowly inject freshly prepared 50 to 60 degree Celsius agar into the right atrium via the superior caval vein until both the right atrium and ventricle are completely filled.To obtain an embedded heart resembling the end diastolic geometry as closely as possible, fix the left ventricle apex to the bottom of the container and fill the right ventricle with agar while the pulmonary veins are clamped. Next, place a mosquito clamp on the pulmonary veins and gently pass a new agar filled 50 milliliter syringe in the retrograde direction through the aortic valves. Slowly inject the solution into the left ventricle until the ventricle and left atrium are completely full, clamping the aorta once the left ventricle has been filled.Now, pour the remaining agar into the container until the heart is fully covered and place two rigid plastic tubes within the embedding container to serve as reference structures during the image registration. Then place the heart at two to seven degrees Celsius for at least four hours to solidify the agar. After magnetic resonance imaging, turn the container upside down and allow air between the agar and the sides of the container to remove the solid agar solution and the heart from the container.Remove the plastic rods. Then use a meat slicer to section the agar block and the heart in five millimeter slices from the apex to the base of the heart, cutting parallel to the bottom of the agar block to keep the angulation of the slices the same as in the acquired magnetic resonance images. When all of the slices have been obtained, stain the tissue samples in TTC.After 15 minutes, photograph the slices on both sides at a perpendicular angle, then carefully rinse the sections in 0.9%saline. To image the samples by fluorescence microscopy, select fluorescence mode imaging on the variable mode scanner and set the pixel size to 100 by 100 microns. For the first filter block, select a band pass filter that overlaps with the emission wavelength of the bead fluorescence in channel one, then to band pass filter for the second filter block outside the emission wavelength in channel two.Next, select the appropriate filter block, set the photomultiplier tube to the appropriate voltage, and the excitation laser to the excitation wavelength closest to that of the injected microbeads. After all of the images have been obtained, in the appropriate post processing software, segment the myocardium and endo-and epicardial left ventricle borders in the magnetic resonance images followed by a similar segmentation of the myocardium and injection sites in the fluorescence images. Then linearly interpolate the image registered segments from both sides of the slices to reconstruct the original sample thickness and to create a 3D model of the data.With the heart secured in an end diastolic like geometry as just demonstrated, in this representative experiment, the agar successfully adhered to the heart tissue enabling the tissue to be sliced at the desired angulation with equal slice thicknesses. In both the 2D fluorescence and magnetic resonance images, the scar and injection sites are clearly distinct. TTC stained tissue and late gadolinium enhanced magnetic resonance images provide a control for scar assessment in fluorescence imaging.Post processing imaging enables reconstruction of the 3D geometry of the ex vivo heart based on the segmentations and fluorescence images, allowing assessment of the 3D injection accuracy. In this study, the injection depositions and the infarct border zone were protected onto the endocardial wall, and the distances between the projections on the endocardial surface were measured. Following this procedure, other methods like a surgical assessment, infarct size quantification, or validation of other three dimensional imaging modalities can be performed.We have used this technique to assess the accuracy of injection into the infarct border zone. This research was conducted to improve cardioregenerative therapy for heart failure. After watching this video, you should have a good understanding of how to prepare the heart, how to embed and process cardiac tissue, and how to perform fluorescence imaging of cardiac tissue samples.

3d-whole-heart-myocardial-tissue-analysis

Research

JoVE Journal

Medicine

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

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 ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Histological Quantification of Chronic Myocardial Infarct in Rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...