Name: studying left ventricular reverse remodeling by aortic debanding in rodents
Uploaded: 2021-07-14T13:00:00
Description: Watch this Scientific Journal Video about Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents at JoVE.com

The authors thank Portuguese Foundation for Science and Technology (FCT), European Union, Quadro de Refer&#234;ncia Estrat&#233;gico Nacional (QREN), Fundo Europeu de Desenvolvimento Regional (FEDER) and Programa Operacional Factores de Competitividade (COMPETE) for funding UnIC (UID/IC/00051/2013) research unit. This project is supported by FEDER through COMPETE 2020 &#8211; Programa Operacional Competitividade E Internacionaliza&#231;&#227;o (POCI), the project DOCNET (NORTE-01-0145-FEDER-000003), supported by Norte Portugal regional operational programme (NORTE 2020), under the Portugal 2020 partnership agreement, through the European Regional Development Fund (ERDF), the project NETDIAMOND (POCI-01-0145-FEDER-016385), supported by European Structural And Investment Funds, Lisbon&#8217;s regional operational program 2020. Daniela Miranda-Silva and Patr&#237;cia Rodrigues are funded by Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (FCT) by fellowship grants (SFRH/BD/87556/2012 and SFRH/BD/96026/2013 respectively). &#160;

All animal experiments comply with the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85&#8211;23, revised 2011) and the Portuguese law on animal welfare (DL 129/92, DL 197/96; P 1131/97). The competent local authorities approved this experimental protocol (018833). Seven-week-old male C57B1/J6 mice were maintained in appropriate cages, with a regular 12/12 h light-dark cycle environment, a temperature of 22 &#176;C and 60% humidity with access to water and a standard diet ad libitum.
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The model proposed herein mimics the process of LV remodeling and RR after aortic banding and debanding, respectively. Therefore, it represents an excellent experimental model to advance our knowledge on the molecular mechanisms involved in the adverse LV remodeling and to test novel therapeutic strategies able to induce myocardial recovery of these patients. This protocol details steps on how to create a rodent animal model of aortic banding and debanding with a minimally invasive and highly conservative surgical techni...

The constriction of the transverse or ascending aorta in the mouse is a widely used experimental model for pressure overload-induced cardiac hypertrophy, diastolic and systolic dysfunction and heart failure1,2,3,4. Aortic-constriction initially leads to compensated left ventricle (LV) concentric hypertrophy to normalize wall stress1. However, under certain circumstances, such as prolonged cardiac overload, this hypertrophy is insufficient to decrease the wall stress, triggering diastolic and systolic dysfunction (pathological hypertrophy)5. In parallel, changes in extracellular matrix (ECM) lead to the collagen deposition and crosslinking in a process known as fibrosis, which can be subdivided into replacement fibrosis and reactive fibrosis. Fibrosis is, in most of the cases, irreversible and compromises myocardial recovery after overload relief6,7. Nevertheless, recent cardiac magnetic resonance imaging studies revealed that reactive fibrosis is able to regress in the long term8. Altogether, fibrosis, hypertrophy and cardiac dysfunction are part of a process known as myocardial remodeling that rapidly progresses towards heart failure (HF).
Understanding the features of myocardial remodeling has become a major objective for limiting or reversing its progression, the latter known as reverse remodeling (RR). The term RR includes any myocardial alteration chronically reversed by a given intervention, such pharmacological therapy (e.g., antihypertensive medication), valve surgery (e.g., aortic stenosis) or ventricular assist devices (e.g., chronic HF). However, RR is often incomplete due to the prevailing hypertrophy or systolic/diastolic dysfunction. Thus, the clarification of RR underlying mechanisms and novel therapeutic strategies are still missing, which is mostly due to the impossibility to access and study human myocardial tissue during RR in most of these patients.
To overcome this limitation, rodent models have played a significant role in advancing our understanding of the signaling pathways involved in HF progression. Specifically, aortic debanding of mice with an aortic constriction represents a useful model to study the molecular mechanisms underlying adverse LV remodeling9 and RR10,11 as it allows the collection of myocardial samples at different time points in these two phases. Moreover, it provides an excellent experimental setting to test potential novel targets that can promote/accelerate RR. For instance, in aortic stenosis context, this model might provide information about the molecular mechanisms involved in the vast diversity of myocardial response underlying the (in)completeness of the RR6,12, as well as, the optimal timing for valve replacement, which represents a major shortcoming of the current knowledge. Indeed, the optimal timing for this intervention is a subject of debate, mainly because it is defined based on the magnitude of aortic gradients. Several studies advocate that this time point might be too late for the myocardial recovery as fibrosis and diastolic dysfunction are often already present12.
To our knowledge, this is the only animal model that recapitulates the process of both myocardial remodeling and RR taking place in conditions such as aortic stenosis or hypertension before and after valve replacement or the onset of anti-hypertensive medication, respectively.
Seeking to address the challenges summarized above, we describe a surgical animal model that can be implemented both in mice and rats, addressing the differences between these two species. We describe the main steps and details involved when carrying out these surgeries. Finally, we report the most significant changes taking place in the LV immediately before and throughout RR.

<table border="0" cellpadding="0" cellspacing="0" style="width:965px;" width="964">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Absorption Spears</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">18105-03</td>
			<td style="width:393px;">To absorb fluids during the surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Blades</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">10011-00</td>
			<td>To perform the skin incision</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td style="width:145px;">Buprelieve</td>
			<td style="width:161px;"></td>
			<td>Analgesia drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Catutery</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">18010-00</td>
			<td style="width:393px;">To prevent exsanguination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Catutery tips</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">18010-01</td>
			<td style="width:393px;">To prevent exsanguination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">cotton swab</td>
			<td style="width:145px;">Johnson&#39;s</td>
			<td style="width:161px;"></td>
			<td style="width:393px;">To absorb fluids during the surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Depilatory cream</td>
			<td style="width:145px;">Veet</td>
			<td style="width:161px;"></td>
			<td>To delipate the animal</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:18px;">Disposable operating room table cover</td>
			<td style="width:145px;">MEDKINE</td>
			<td style="width:161px;">DYND4030SB</td>
			<td>To cover the surgical area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Echo probe</td>
			<td style="width:145px;">Siemens</td>
			<td>Sequoia 15L8W</td>
			<td>Ultrasound signal aquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Echocardiograph</td>
			<td style="width:145px;">Siemens</td>
			<td>Acuson Sequoia C512</td>
			<td>Ultrasound signal aquisition</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:265px;">End-tidal CO2 monitor</td>
			<td style="width:145px;">Kent Scientific</td>
			<td style="width:161px;">CapnoStat</td>
			<td>To control expiration gas saturation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Forcep/Tweezers</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">11255-20</td>
			<td>To dissect the tissues and aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Forcep/Tweezers</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">11272-30</td>
			<td>To dissect the tissues and aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Forcep/Tweezers</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">11151-10</td>
			<td>To dissect the tissues and aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Forcep/Tweezers</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">11152-10</td>
			<td>To dissect the tissues and aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Gas system</td>
			<td>Penlon Sigma Delta</td>
			<td style="width:161px;"></td>
			<td>To anesthesia and mechanical ventilation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Hemostats</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">13010-12</td>
			<td>To hold the suture before tight the aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Hemostats</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">13011-12</td>
			<td>To hold the suture before tight the aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Ligation aids</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">18062-12</td>
			<td>To place a suture around the aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Magnetic retractor</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">18200-20</td>
			<td>To help keep the animal in a proper position</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Needle holder</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">12503-15</td>
			<td>To suture the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Needle 26G</td>
			<td style="width:145px;">B-BRAUN</td>
			<td style="width:161px;">4665457</td>
			<td>To serve as a molde of aortic constriction diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Oxygen</td>
			<td style="width:145px;">Air Liquide</td>
			<td style="width:161px;"></td>
			<td>To anesthesia and mechanical ventilation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Polipropilene suture</td>
			<td style="width:145px;">Vycril</td>
			<td style="width:161px;">W8304/W8597</td>
			<td>To suture the animal and to do the constriction</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Povidone-iodine solution</td>
			<td style="width:145px;">Betadine&#174;</td>
			<td style="width:161px;"></td>
			<td>Skin antiseptic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">PowerLab</td>
			<td>Millar instruments</td>
			<td>ML880 PowerLab 16/30</td>
			<td>PV loop Signal Aquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Pulse oximeter</td>
			<td style="width:145px;">Kent Scientific</td>
			<td style="width:161px;">MouseStat</td>
			<td>To control heart rate and blood saturation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">PVAN software</td>
			<td style="width:145px;">Millar Instruments</td>
			<td style="width:161px;"></td>
			<td>To analyse the haemodynamic data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">PV loop cathether</td>
			<td>Millar instruments</td>
			<td>SPR-1035. 1.4 F</td>
			<td>PV loop Signal Aquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Retractor</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">17000-01</td>
			<td>To provide a better overview of the aorta</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Scalpet handle</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">10003-12</td>
			<td>To perform the skin incision</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Scissors</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">15070-08</td>
			<td>To cut the suture in debanding surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Scissors</td>
			<td style="width:145px;">F.S.T</td>
			<td style="width:161px;">14084-09</td>
			<td>To cut other material during the surgery e.g. suture, papper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Sevoflurane</td>
			<td style="width:145px;">Baxter</td>
			<td style="width:161px;">533-CA2L9117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Temperature control module</td>
			<td style="width:145px;">Kent Scientific</td>
			<td style="width:161px;">RightTemp</td>
			<td>To control animal corporal temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Ventilator</td>
			<td style="width:145px;">Kent Scientific</td>
			<td style="width:161px;">PhysioSuite</td>
			<td>To ventilate the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water-bath</td>
			<td style="width:145px;">Thermo Scientific&#8482;</td>
			<td style="width:161px;">TSGP02</td>
			<td>To maintain water temperature adequate to heat the P-V loop catethers</td>
		</tr>
	</tbody>
</table>

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.

Post-operative and late survival 
The perioperative survival of the banding procedure is 80% and the mortality during the first month is typically &#60;20%. As previously mentioned, the success of the debanding surgery is highly dependent on how invasive the previous surgery was. After a learning curve, the mortality rate during the debanding procedures is around 25%. For this mortality accounts mostly deaths during the surgery procedure, including aorta or left atrium rupture (in rats, the survival...

Watch this Scientific Journal Video about Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents at JoVE.com

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

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, ...

Aortic Debanding is a useful experimental model for studying myocardial reverse remodeling in rodents and for a reveling novel insights into the mechanisms of cardiomyocytes regression and iso lic functional recovery. These metal facilitates the evolution of critique reverse modeling in VIVO and the collection of biological samples for in-vitro and molecular studies at different time points during the disease progression. This technique mimics the progression of myocardial remodeling and reversely remodeling and allows exploring the mechanisms of incomplete myocardial recovery.This protocol provides insights into the mechanisms and the line complete and incomplete myocardial reverse remodeling in chronic pressure overload related pathologies such as eye pretension or aortic stenosis. Connect the orotracheal tube to the ventilator to initiate the mechanical ventilation and adjust the ventilator to a frequency of 160 breaths per minute and a tidal volume of 10 milliliters per kilogram. Before beginning the band procedure, confirm a lack of response to pain reflex in a six to eight week old C57 black six mouse, shave and applied depilatory cream from the neck line to the mid chest level of the mouse and apply ointment to the animal's eyes.Place a rectal probe and the oximeter for monitoring temperature blood oxygenation and heart rate. Place the mouse in the right lateral decubitus on a heating pad and tape the limbs to the magnetic fixator retraction system to maintain the animal in the correct position during the procedure. Then, disinfect the chest with consecutive 70%alcohol and povidone-iodine solution scrubs then and open the skin muscle and ribs in order to perform the band to the aorta.At the end of the procedure apply povidone-iodine solution to the skin suture site and administer analgesia twice daily for two to three days, Then inject sterile saline intraperitoneal Lea to prevent dehydration in cases of significant bleeding during the surgery and place the animal in an incubator with monitoring until full recovery. Seven weeks after the banding surgery, gently dissect to the tissues, adhesion and fibrosis around the aorta in half of the banded animals until the constriction becomes visible. Carefully dissect the aorta and separate the suture from the vessel, then use scissors to cut the suture.Note that's a prolonged occlusion of the ax indigo water during binding or demonic may lead to lung edema, an excessive activation of inflammatory pathways. Close the chest wall with a simple interrupted or continuous six old polypropylene sutures using the minimum number of stitches possible and close the skin with a six O silk polypropylene suture in a continuous suture pattern, then perform the postoperative care as demonstrated. Every two to three weeks after the surgery remove the fur from the neck line to the mid chest level as demonstrated and place the animal on a heating pad in the supine position Place ECG electrodes onto the exposed skin and confirm the presence of a good ECG trace and a heart rate between 300 and 350 beats per minute.Monitor the temperature and apply echo gel to the chest region. Place the mice in dorsal recumbency slightly turned to the right and start the echocardiograph using the appropriate settings. Next, position an ultrasound probe over the thorax and assess the pressure gradient across the aorta.Record two dimensional guided images of the aorta showing the presence or absence of the ascending aorta constriction to anatomically visualize the efficacy of the banding and debanding. To assess the hypertrophy, position the probe at a left ventricle short axis at the papillary muscle level and press the mode tracing button to visualize the left ventricle anterior wall, left ventricle diameter and left ventricle posterior wall in diacetyl and systole. Then assess the systolic function and calculate the ejection fraction and fractional shortening according to standard protocols.To assess the diastolic function, determine the peak of the post wave Doppler of the early and leaf mitral flow velocity using an atypical four chamber view just above the mitral leaflets and record the lateral mitral annular myocardial early diastolic and peak systolic velocities using post tissue doppler imaging and the apical four chamber view. The success of aortic constriction can be verified by an increased left ventricle and systolic pressure. And by Doppler aortic flow velocities greater than 2.5 meters per second.Compared to Sham operated animals, bending induced left ventricle hypertrophy is demonstrated by an increased in left ventricle mass and an impaired diastolic function as evidenced by higher filling pressures and left ventricular end diastolic pressure. Histologically, seven weeks of aortic banding induces significant cardiomyocytes hypertrophy and fibrosis. In my subjected to debanding, as successful removal of the aortic stenosis can be verified by echo Doppler velocities.Overall debanding promotes a significant decrease in the afterload and the left ventricle hypertrophy assessed by LV mass. Moreover, a normalization of the diastolic function is also observed. When you play it carefully the avoiding prolonged ethical collusion in order to guarantee that the blood oxygenation remains above 90%This minimum model allows us to study my cardiac remodeling and reverse remodeling at different time points of the disease progression and to even wake the most dysregulated pathways at each time point.This procedure allows to assess the therapeutic potential of promising drugs as well as to study the impact of several co-morbidities in myocardial reverse remolding and recovery.

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Medicine

Reverse Total Shoulder Arthroplasty

Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in patients who were not appropriate for tendon transfer reconstructions. Traditional surgical options for these patients were limited and functional results were sub-optimal and at times catastrophic. The use of reverse shoulder arthroplasty has been found to effectively restore these patients function and relieve symptoms associated with their disease. The procedure can be done through two approaches, the deltopectoral or the superolateral. Complication rates associated with the use of the prosthesis have ranged from 8-60% with more recent reports trending lower as experienced is gained. Salvage options for a failed reverse shoulder prosthesis are limited and often have significant associated disability. Indications for the use of this prosthesis continue to be evaluated including its use for revision arthroplasty, proximal humeral fracture and tumor. Careful patient selection is essential because of the significant risks associated with the procedure.

Reverse total shoulder arthroplasty is indicated for the treatment of conditions that cannot be treated with conventional arthroplasty or other procedures. These primarily include degenerative and incapacitating conditions with irreparable rotator cuff and loss of the normal biomechanical coupling of the shoulder.

Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in ...

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.

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 ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners&#39; expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions&#160;&#8212; radial, circumferential, longitudinal &#8212; of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane.
Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Speckle tracking echocardiography is an emerging diagnostic imaging technique for the quantitative assessment of global and regional myocardial performance. Standard view echocardiographic motion images are recorded and deformation parameters are subsequently measured by automated continuous frame-by-frame tracking and motion analysis of speckles within the B-mode images of the myocardium.

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Full-root Aortic Valve Replacement by Stentless Aortic Xenografts in Patients with Small Aortic Roots

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial valve might not meet the functional needs. The implantation of a too-small stented pericardial valve, leading to an effective orifice area indexed to a body surface area less than 0.85 cm2/m2, is regarded as prosthesis-patient mismatch (PPM). A PPM negatively affects the regression of left ventricular hypertrophy and thus the normalization of left ventricular function and the alleviation of symptoms. Persistent left ventricular hypertrophy is associated with an increased risk of arrhythmias and sudden cardiac death. In the case of predictable PPM, there are three options: 1) accept the PPM resulting from the implantation of a stented pericardial valve when comorbidities of the patient forbid the more technically demanding operative technique of implanting a larger prosthesis, 2) enlarge the aortic root to accommodate a larger stented valve substitute, or 3) implant a stentless biological valve or a homograft. Compared to classical aortic valve replacement with stented pericardial valves, the full-root implantation of stentless aortic xenografts offers the possibility of implanting a 3-4 mm larger valve in a given patient, thus allowing significant reduction in transvalvular gradients. However, a number of cardiac surgeons are reluctant to transform a classical aortic valve replacement with stented pericardial valves into the more technically challenging full-root implantation of stentless aortic xenografts. Given the potential hemodynamic advantages of stentless aortic xenografts, we have adopted full-root implantation to avoid PPM in patients with small aortic roots necessitating an aortic valve replacement. Here, we describe in detail a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses. Limitations of this technique and alternative options are discussed.

Full-root aortic valve replacement by stentless aortic xenograft is a viable option in patients with small aortic roots. We describe, a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses, and discuss its limitations and alternative options.

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial ...

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 ...

Utilizing Percutaneous Ventricular Assist Devices in Acute Myocardial Infarction Complicated by Cardiogenic Shock

Cardiogenic shock is defined as persistent hypotension, accompanied by evidence of end organ hypo-perfusion. Percutaneous ventricular assist devices (PVADs) are used for the treatment of cardiogenic shock in an effort to improve hemodynamics. Impella is currently the most common PVAD and actively pumps blood from the left ventricle into the aorta. PVADs unload the left ventricle, increase cardiac output and improve coronary perfusion. PVADs are typically placed in the cardiac catheterization laboratory under fluoroscopic guidance via the femoral artery when feasible. In cases of severe peripheral arterial disease, PVADs can be implanted through an alternative access. In this article, we summarize the mechanism of action of PVAD and the data supporting their use in the treatment of cardiogenic shock.

Percutaneous ventricular assist devices are increasingly being utilized in patients with acute myocardial infarction and cardiogenic shock. Herein, we discuss the mechanism of action and hemodynamic effects of such devices. We also review algorithms and best practices for the implantation, management and weaning of these complex devices.

Cardiogenic shock is defined as persistent hypotension, accompanied by evidence of end organ hypo-perfusion. Percutaneous ventricular assist devices ...

Use of a Percutaneous Ventricular Assist Device/Left Atrium to Femoral Artery Bypass System for Cardiogenic Shock

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the left ventricle by draining blood from the left atrium (LA) and returning it to the systemic arterial circulation via the femoral artery. It can provide flows ranging from 2.5-5 L/min depending on the size of the cannula. Here, we discuss the mechanism of action of LAFAB, available clinical data, indications for its use in cardiogenic shock, steps of implantation, post-procedural care, and complications associated with the use of this device and their management.
We also provide a brief video of the procedural component of device therapy, including the pre-placement preparation, percutaneous placement of the device via transseptal puncture under echocardiographic guidance and the post-operative management of device parameters.

The following article describes the stepwise procedure for placement of a device (e.g., Tandemheart) in cardiogenic shock (CS) that is a percutaneous left ventricular assist device (pLVAD) and a left atrial to femoral artery bypass (LAFAB) system that bypasses and supports the left ventricle (LV) in CS.

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the ...