NIH grant HL070241 to P.D.

All methods and procedures described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Tulane University School of Medicine.
1. Tools and instruments for AAB model creation
<ol>
	<li>Obtain disinfectants, such as 70% isopropyl alcohol and povidone-iodine.</li>
	<li>Obtain ketamine and xylazine for anesthesia and buprenorphine for analgesia.</li>
	<li>Obtain a heating pad and heavy absorbency disposable underpad with the dimensions of 18 inches x 30 inches.</li>
	<li>Obtain a 100% cotton twine roll, a tape and a hair clipper.</li>
	<li>Obtain a 20 cm x 25 cm plastic board, thickness range between 3-5 mm.</li>
	<li>Obtain a Z-LITE fiber optic illuminator.</li>
	<li>Obtain a mechanical ventilator for small animals (e.g., SAR-830/AP).</li>
	<li>Obtain 2-0 and 3-0 Vicryl taper sutures and nylon 3-0 monofilament suture, sterile gauze pads and sterile extra large cotton tips and sterile gloves.</li>
	<li>Obtain 16 G angiocath for intubation.</li>
	<li>Purchase the following surgical tools.
	<ol>
		<li>Obtain a Weck stainless steel Hemoclip ligation and stainless-steel ligating clips.</li>
		<li>Obtain hardened fine iris scissors.</li>
		<li>Obtain Adson forceps.</li>
		<li>Obtain two curved Graefe forceps.</li>
		<li>Obtain a Halsted-Mosquito Hemostats-straight forceps.</li>
		<li>Obtain a Mayo-Hegar needle holder.</li>
		<li>Obtain an Alm chest retractor with blunt teeth.</li>
	</ol>
	</li>
	<li>Utilize and obtain an autoclave and a bead sterilizer.</li>
</ol>
2. Ascending aortic banding surgical procedure
<ol>
	<li>Anesthetize the animal with an intraperitoneal injection of a mix of 75-100 mg/kg Ketamine and 10 mg/kg Xylazine. 
	NOTE: Allow a few minutes for the animal to be completely sedated and flaccid. If the anesthetic dose is not sufficient and the animal is still moving in the cage, re-inject the animal with the same anesthetic dose after allowing enough time, around 5-10 minutes between subsequent injections. Most animals require 1-2 injections to achieve deep sedation and anesthesia.</li>
	<li>Shave the hair on the surgical site located at the right lateral thoracic area under the right armpit.</li>
	<li>Stabilize the animal by gently taping all four limbs to the plastic board. Then perform endotracheal intubation with a 16 G angiocath. After the animal is successfully intubated, initiate mechanical ventilation with tidal volumes of 2 mL at 50 cycles/min and FiO2 of 21%. Look for the symmetrical rise in chest wall with each breath.</li>
	<li>Turn the animal slowly to lie on its left lateral side, and then bend the tail in a U-shape manner and stabilize it by gently taping it to the plastic board. Then go ahead and disinfect the shaved area with topical application of povidone-iodine.&#160;</li>
	<li>Infiltrate the skin at the incision site with 50/50 mix by volume of 1-2% Lidocaine/0.25-0.5 % Bupivacaine as preemptive analgesia before making the incision.</li>
	<li>Perform a right horizontal skin incision, 1-2 centimeters long, in the right axillary area 1 cm below the right armpit. Then, dissect the thoracic muscular layer until reaching the thoracic rib cage. Make a 1 cm thoracotomy between the 2nd and 3rd rib cage.
	<ol>
		<li>While dissecting the muscular layer of the chest, be careful and avoid injury of the right axillary artery, which runs underneath the right armpit. 
		NOTE: Thoracotomy performed between the 1st and the 2nd rib carries the risk of banding the right brachiocephalic artery instead of the ascending aorta. Thoracotomy between the 3rd and the fourth rib makes it hard to visualize and band the ascending aorta, as the operator will be looking at the right atrium. 
		NOTE: Avoid extending the thoracotomy too medially towards the sternum to avoid dissecting and injuring the right internal mammary artery.</li>
	</ol>
	</li>
	<li>Dissect the two lobes of the thymus gland gently and push them apart on the side. Then identify the ascending aorta and isolate it from the superior vena cava by blunt dissection via a curved Graefe forceps. 
	NOTE: Significant manipulation of the thymus gland will render it swollen and makes it hard to visualize the ascending aorta.
	<ol>
		<li>Dissect the superior vena cava from the aorta with extra caution to avoid injury or rupture of the superior vena cava, which is fatal. This may be the trickiest part of the procedure and is expected to happen from time to time even in most experienced hands, but often with beginners and learners.</li>
	</ol>
	</li>
	<li>Lift gently the ascending aorta with a curved Graefe forceps and place the vascular clip around the ascending aorta.
	<ol>
		<li>Adjust the vascular hemoclip ligation tool via a plastic pre-cut 7&#34; piece to obtain a vascular clip of the desired internal area of 1.5 mm2 or 2 mm2, depending on which HF model is desired.</li>
	</ol>
	</li>
	<li>Suture the thorax via a Vicryl 2-0 monofilament suture. Then suture the muscular layer of the chest via a 3-0 Vicryl taper suture. Then suture the skin incision via a Nylon 3-0 monofilament suture.</li>
	<li>Administer a combination of the following drugs after completion of the surgery for 48-72 hrs to serve as analgesia in the post-operative period: 1) Buprenorphine 0.01-0.05 mg/kg subcutaneously every 8-12h, 2) Meloxicam 2 mg/kg subcutaneously every 12h, and 3) Morphine 2.5 mg/kg subcutaneously every 2-4h as needed for severe pain. 
	NOTE: Leave the animal to recover on a heating pad under regular monitoring. Once the animal shows signs of recovery from anesthesia (able to breath spontaneously - without evidence of gasping or use of accessory muscles for more than two minutes - and has good reflexes, red and warm extremities), extubate the animal and return it to the cage.</li>
</ol>
3. Echocardiography
<ol>
	<li>Sedate the animal with intraperitoneal injection of 80-100 mg/kg ketamine. Ensure adequate sedation for proper acquisition of good quality echo images. 
	NOTE: The use of isoflurane as an anesthetic is discouraged for its cardiodepressor effect, especially in the setting of severe pressure overload and might give a false impression of LV dilatation and systolic dysfunction that resolves once animal is off anesthetic.
	<ol>
		<li>Be cautious and administer half or even one third of the dose of ketamine in animals that look dyspneic and tachypneic with suspicion that they have developed the HFrEF phenotype.</li>
	</ol>
	</li>
	<li>Shave the hair of the chest, anteriorly, in the completely sedated animal.</li>
	<li>Lay the animal on its back and stabilize it to the plastic board.</li>
	<li>Acquire 2D parasternal long axis and 2D parasternal short axis view clips at the level of the papillary muscle. Also, obtain M-mode images from the short parasternal axis view at the level of the papillary muscle to measure LV septal and posterior wall thickness in diastole as well as LV end-diastolic and end-systolic diameter.
	<ol>
		<li>Acquire images or clips at a heart rate of 370 - 420 beats per minute to ensure proper assessment of LV size and function. Acquisition of images at lower heart rates will lead to a false impression of depressed LV function and LV dilatation. 
		NOTE: Acquisition of foreshortened 2D long parasternal axis view images/clips lead to false measurements. For quality control purposes, make sure that the LV apex and the aorto-mitral angle are visualized within the same plane cut.</li>
		<li>Acquire 2D short parasternal axis view images/clips at the level of the mid papillary muscle. This will serve as a reference to obtain reliable serial and subsequent LV measurements while following the animals over time throughout the study period.</li>
	</ol>
	</li>
	<li>Obtain M-mode images in long parasternal axis view at the level of the aortic valve to assess the relative aortic to left atrium (LA) diameter at end systole. 
	NOTE: Animals with the MOD and HFrEF phenotypes should show evidence of LA dilatation with LA/Ao ratio being &#8805;1.25 and &#60;1.5 in MOD HF phenotype and &#8805;1.5 in the HFrEF phenotype10.</li>
</ol>

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A., Hill, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+load-induced+heart+disease.">Autophagy in load-induced heart disease.</a> Circulation Research. 103 (12), 1363-1369 (2008).</li><li>Barrick, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parent-of-origin+effects+on+cardiac+response+to+pressure+overload+in+mice.">Parent-of-origin effects on cardiac response to pressure overload in mice.</a> American Journal of Physiology-Heart and Circulatory Physiology. 297 (3), H1003-H1009 (2009).</li><li>Barrick, C. J., Rojas, M., Schoonhoven, R., Smyth, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+response+to+pressure+overload+in+129S1/SvImJ+and+C57BL/6J+mice:+temporal-+and+background-dependent+development+of+concentric+left+ventricular+hypertrophy.">Cardiac response to pressure overload in 129S1/SvImJ and C57BL/6J mice: temporal- and background-dependent development of concentric left ventricular hypertrophy.</a> American Journal of Physiology-Heart and Circulatory Physiology. 292 (5), H2119-H2130 (2007).</li><li>Lygate, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+high+resolution+3D-MRI+after+aortic+banding+in+mice:+band+internalization+is+a+source+of+variability+in+the+hypertrophic+response.">Serial high resolution 3D-MRI after aortic banding in mice: band internalization is a source of variability in the hypertrophic response.</a> Basic Research in Cardiology. 101 (1), 8-16 (2006).</li><li>Chaanine, A. H., Hajjar, 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=Characterization+of+the+Differential+Progression+of+Left+Ventricular+Remodeling+in+a+Rat+Model+of+Pressure+Overload+Induced+Heart+Failure.+Does+Clip+Size+Matter?.">Characterization of the Differential Progression of Left Ventricular Remodeling in a Rat Model of Pressure Overload Induced Heart Failure. Does Clip Size Matter?.</a> Methods in Molecular Biology (Clifton, N.J.). 1816, 195-206 (2018).</li><li>Chaanine, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Integrity+and+Function+in+the+Progression+of+Early+Pressure+Overload-Induced+Left+Ventricular+Remodeling.">Mitochondrial Integrity and Function in the Progression of Early Pressure Overload-Induced Left Ventricular Remodeling.</a> Journal of the American Heart Association. 6 (6), (2017).</li><li>Chaanine, A. 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All authors report no conflict of interest.

Following PO related to AAB in rat, the LV undergoes concentric remodeling by increasing LV wall thickness, known as concentric LVH, as a compensatory mechanism to counteract for the increase in LV wall stress. Increase in LV wall thickness becomes noticeable during the first week following AAB and reaches its maximum thickness at 2-3 weeks post-AAB. During this time period, activation of maladaptive signal transduction pathways lead to progressive enlargement of the LV with increases in LV volumes, a process known as ec...

Heart failure (HF) is a prevalent disease and is associated with high morbidity and mortality1. Rodent pressure-overload (PO) models of HF, produced by ascending or transverse aortic banding, are commonly used to explore molecular mechanisms leading to HF and to test potential novel therapeutic targets in HF. They also mimic changes seen in human HF secondary to prolonged systemic hypertension or severe aortic stenosis. Following PO, the left ventricular (LV) wall gradually increases in thickness, a process known as concentric LV hypertrophy (LVH), to compensate and adapt for the increase in LV wall stress. However, this is associated with the activation of a number of maladaptive signaling pathways, which lead to derangements in calcium cycling and homeostasis, metabolic and extracellular matrix remodeling and changes in gene expression as well as enhanced apoptosis and autophagy2,3,4,5,6. These molecular changes constitute the trigger for the initiation and propagation of myocardial remodeling and transition into a decompensated HF phenotype.
Despite the use of inbred rodent strains and standardization of clip size and surgical technique, there is tremendous phenotypic variability in LV chamber structure and function in aortic banding models7,8,9. The phenotypic variability encountered after PO in rat, Sprague-Dawley strain, is described elsewhere10,11. Of those, two HF phenotypes are encountered with evidence of myocardial remodeling and activation of signal transduction pathways leading to a state of heightened oxidative stress. This is associated with metabolic remodeling, altered gene expression and changes in posttranslational modification of proteins, altogether playing a role in the remodeling process10,12. The first is a phenotype of moderate remodeling and early systolic dysfunction (MOD) and the second is a phenotype of overt systolic HF (HFrEF).
The PO model of HF is advantageous over the myocardial infarction (MI) model of HF because the PO-induced circumferential and meridional wall stresses are homogeneously distributed across all segments of the myocardium. However, both models suffer from variability in the severity of PO10,11 and in infarct size13,14 along with intense inflammation and scarring at the infarct site15 as well as adhesion to the chest wall and surrounding tissues, which are observed in the MI model of HF. Moreover, the rat PO induced HF model is challenging to create as it is associated with high mortality and failure rates10, with only 20% of the operated rats developing the MOD HF phenotype10.
The MOD is an attractive HF phenotype and constitutes an evolution of the traditionally created HFrEF phenotype as it allows for early targeting of signal transduction pathways that play a role in myocardial remodeling, especially when it pertains to perturbations in mitochondrial dynamics and function, myocardial metabolism, calcium cycling and extracellular matrix remodeling. These pathophysiological processes are highly evident in the MOD HF phenotype11. In this manuscript, we describe how to create the MOD and HFrEF phenotypes and we address pitfalls while performing the ascending aortic banding (AAB) procedure. We also elaborate on how to best characterize by echocardiography the two HF phenotypes, MOD and HFrEF, and how to differentiate them from other phenotypes that fail to develop severe PO or that develop severe PO and concentric remodeling but without significant eccentric remodeling.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adson forceps</td>
			<td>F.S.T.</td>
			<td>11019-12</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Alm chest retractor with blunt teeth</td>
			<td>ROBOZ</td>
			<td>RS-6510</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Graefe forceps, curved</td>
			<td>F.S.T.</td>
			<td>11152-10</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito Hemostats, straight</td>
			<td>F.S.T.</td>
			<td>13010-12</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Hardened fine iris scissors, straight</td>
			<td>Fine Science Tools F.S.T.</td>
			<td>14090-11</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>hemoclip traditional-stainless steel ligating clips</td>
			<td>Weck</td>
			<td>523435</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Mayo-Hegar needle holder</td>
			<td>F.S.T.</td>
			<td>12004-18</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>mechanical ventilator</td>
			<td>CWE inc</td>
			<td>SAR-830/AP</td>
			<td>mechanical ventilator for small animals</td>
		</tr>
		<tr>
			<td>Weck stainless steel Hemoclip ligation</td>
			<td>Weck</td>
			<td>533140</td>
			<td>surgical tool</td>
		</tr>
	</tbody>
</table>

a rat model of pressure overload induced moderate remodeling and systolic dysfunction as opposed to overt systolic heart failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

Characterization of the HF phenotypes, that develop 8-12 weeks following AAB, could be easily performed via echocardiography. Representative M-mode images of Sham, Week 3 post-AAB, MOD and HFrEF phenotypes are presented in Figure 1A. Figure 1B and Figure 1C are showing the vascular clip size for the creation of the MOD HF phenotype and HFrEF phenotype, respective...

Watch this Scientific Journal Video about A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure at JoVE.com

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...

a-rat-model-pressure-overload-induced-moderate-remodeling-systolic

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6.4K Views.  Tulane University. We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

Video: A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to suboptimal clinical results. We set out to establish a chronic ovine model of FTR and right heart failure to investigate the mechanisms of FTR. Twenty adult male sheep (6-12 months old, 62 &#177; 7 kg) underwent a left thoracotomy and baseline echocardiography. A pulmonary artery band (PAB) was placed and cinched around the main pulmonary artery (PA) to at least double the systolic pulmonary artery pressure (SPAP), inducing right ventricular (RV) pressure overload and signs of RV dilatation. PAB acutely increased the SPAP from 21 &#177; 2 mmHg to 62 &#177; 2 mmHg. The animals were followed for 8 weeks, symptoms of heart failure were treated with diuretics, and surveillance echocardiography was used to assess for pleural and abdominal fluid collection. Three animals died during the follow-up period due to stroke, hemorrhage, and acute heart failure. After 2 months, a median sternotomy and epicardial echocardiography were performed. Of the surviving 17 animals, 3 developed mild tricuspid regurgitation, 3 developed moderate tricuspid regurgitation, and 11 developed severe tricuspid regurgitation. Eight weeks of pulmonary artery banding resulted in a stable chronic ovine model of right ventricular dysfunction and significant FTR. This large animal platform can be used to further investigate the structural and molecular basis of RV failure and functional tricuspid regurgitation.

Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments.

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...

Scopolamine-Induced Lacrimal Gland Dysfunction in Rats

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal tear production can lead to a disturbance in the ocular surface environment, including corneal damage and inflammation. In severe cases, ADDE can cause vision loss or even blindness. Currently, dry eye treatment is limited to eye drops or physical therapy, which can only alleviate eye discomfort symptoms and cannot fundamentally cure dry eye syndrome. To restore the function of the lacrimal gland in dry eye, we have created an animal model of lacrimal gland dysfunction in rats induced by scopolamine. Through the comprehensive evaluation of the lacrimal gland, corneas, conjunctivas, and other factors, we aim to provide a full understanding of the pathological changes of ADDE. Compared with the current dry eye mouse model, this ADDE animal model includes a functional evaluation of the lacrimal gland, providing a better platform for studying lacrimal gland dysfunction in ADDE.

Author Spotlight: Challenges in Developing Dry Eye Animal Models and Future Research Directions

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal ...

Investigating HFpEF Pathophysiology in a Hyperlipidemia&#45;Induced Mouse Model

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...