Name: studying left ventricular reverse remodeling by aortic debanding in rodents
Uploaded: 2021-07-14T13:00:00.000+00:00
Duration: 7 min 26 s
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.
1. Preparation of the surgical field
<ol>
	<li>Disinfect the operation site with 70% alcohol and place a disposable operating room table cover over the surgical area.</li>
	<li>Sterilize all the instruments before surgery. 
	NOTE: This procedure requires micro surgical scissors, 2 fine curved forceps, 3 fine straight forceps, a scalpel, small forceps, an angled dissector scissor, a needle holder, an ultrafine ligation aid, 2 hemostats and, lastly, a magnetic fixator retraction system is highly recommended (Figure 1A).</li>
	<li>Curve the tip of a 26 G blunted needle to 90&#176; for an easier approach to the aorta. A 26 G needle will create a 0.45 mm diameter aortic narrowing (Figure 1B).</li>
	<li>Adjust the heating pad temperature to 37 &#177; 0.1 &#176;C.</li>
</ol>
2. Mice preparation and intubation
<ol>
	<li>Anesthetize young C57B1/J6 mice (20-25 g) by inhalation of 8% sevoflurane with 0.5 - 1.0 L/min 100% O2 in a cone tube.</li>
	<li>Check the anesthesia depth using the toe pinch withdrawal reflex.</li>
	<li>Place the mouse at dorsal recumbency on an inclined plate and proceed to orotracheal intubation.</li>
	<li>Move the mouse to the heating pad and quickly connect the orotracheal tube to the ventilator to initiate the mechanical ventilation.</li>
	<li>Adjust the ventilator parameters to a frequency of 160 breaths/min and a tidal volume of 10 mL/kg.</li>
</ol>
3. Preparation for surgery (for both banding and debanding surgeries)
<ol>
	<li>Shave and apply the depilatory cream from the neckline to mid-chest level of the mice.</li>
	<li>Apply ophthalmic gel to the animals&#39;&#160;eyes to prevent drying out of the cornea.</li>
	<li>Place a rectal probe and the oximeter at the paw or tail for monitoring temperature and blood oxygenation, and heart rate, respectively. 
	NOTE: Anesthesia induces significant hypothermia, therefore, it is important to maintain normal body temperature during surgery to avoid a rapid decrease in heart rate.</li>
	<li>Maintain anesthesia with sevoflurane (2.0 - 3.0%). Check the correct level of anesthesia by the lack of the toe-pinch reflex.</li>
	<li>Place the mice in right-lateral decubitus on a heating pad and secure the limbs to the magnetic fixator retraction system with a tape to keep the animal in the correct position during the surgery (Figure 2, Figure 3A).</li>
	<li>Disinfect the mouse chest with 70% alcohol followed by providone-iodine solution.</li>
</ol>
4. Ascending aortic banding surgery
NOTE: For a detailed protocol description, consult 2,3,4,13. &#160;
<ol>
	<li>With a disposable blade, perform a small (~0.5 cm) skin incision on the left side immediately below the axilla level and dissect the skin.</li>
	<li>Gently dissect and separate the pectoralis muscle and other muscle layers until the ribs become visible. Use fine forceps and avoid cutting the muscle.</li>
	<li>Under a microscope, identify the intercostal spaces and open a small incision between the 2nd and 3rd intercostal space with fine forceps.</li>
	<li>Retract the ribs by placing the chest retractor (Figure 2A).</li>
	<li>Use small forceps to gently dissect and separate the thymic lobes until ascending aorta becomes visible. 
	NOTE: Cotton applicators should be handy in case of bleeding. Warm sterile saline should be given subcutaneously in case of significant bleeding (e.g., the mammary artery).</li>
	<li>Use small forceps to gently dissect the aorta. 
	NOTE: Aorta is considered to be dissected when there are no fat or other adhesions around it and it is possible to easily encircle the vessel with a small curve forceps.</li>
	<li>After aortic dissection, place a 7-0 polypropylene ligature around aorta by using ligation aid and curved forceps (Figure 2B).</li>
	<li>Position the blunted 26 G needle parallel to the aorta (tip pointed towards the mice head) (Figure 2B). For mice weighing 20-25 g, this needle induces a reproducible 65-70% aortic constriction.</li>
	<li>Make 2 loose knots around the aorta and the 26 G needle with the help of 2 forceps (Figure 2B).</li>
	<li>Tighten the 1st knot and, quickly after, the 2nd knot. Briefly confirm the right positioning of the constriction and quickly remove the needle to restore aortic blood flow. Finally, make a 3rd knot (BA group).</li>
	<li>Reposition the thymus and the muscles into their initial position.</li>
	<li>Perform the sham procedure identical to the constriction procedure but keeping the suture loose around the aorta (SHAM group).</li>
	<li>Cut the ends of the suture and remove the chest retractor. 
	NOTE: Short suture ends may increase the probability of knots loosening with aortic pressure, while long ends make the debanding procedure riskier since adhesions can occur between the suture and the left atrium.</li>
	<li>Close the chest wall using 6-0 polypropylene suture with a simple interrupted or continuous suture using the lowest number of stitches possible. Tighten the last chest knot with the lungs inflated at end inspiration by pinched off the outflow of the ventilator for 2s to re-inflate the lungs.</li>
	<li>Close the skin with a 6-0 silk/polypropylene suture in a continuous suture pattern.&#160; 
	NOTES: If a more recent ventilator is used, it is possible to programme it to pause in inspiration (Setup-Advanced-Pause-Inspiration)</li>
</ol>
5. Post-operative care
<ol>
	<li>Apply providone-iodine solution to the skin suture site.</li>
	<li>For proper analgesia, administer buprenorphine subcutaneously 0.1 mg/kg, twice daily, until the animal fully recovers (usually 2-3 days after surgery).</li>
	<li>Inject sterile saline intraperitoneally to prevent dehydration in case of significant bleeding during the surgery.</li>
	<li>Turn off the anesthesia (without deintubating the mouse) and wait until the animal recover the reflexes (whiskers movements are an awakening signal)&#160; and starts to breathe spontaneously.</li>
	<li>Remove the tracheal cannula.</li>
	<li>Let the animal recover in an incubator at 37 &#176;C.</li>
	<li>Return the animal to a 12 h light/dark cycle room after full recovery.</li>
</ol>
6. Aortic debanding surgery
<ol>
	<li>Seven weeks later, perform the debanding of the aorta in half of the BA&#160; animals and remove the loose suture from half of the SHAM mice, giving rise to 2 new groups --&#160;debanding (DEB) and debanding SHAMA (DESHAM), respectively. DESHAM&#160;represents the control for the DEB group (Figure 4).</li>
	<li>Repeat all the steps 2.1 to 3.6 mentioned above.</li>
	<li>Gently dissect the tissues, adhesions, and fibrosis around the aorta until its constriction becomes visible.</li>
	<li>Carefully dissect the aorta and separate the suture from the aorta. Cut the suture with angled one-probed spring scissors (Figure 3B).</li>
	<li>Close the chest wall using 6-0 polypropylene suture with a simple interrupted or continuous suture using the minimum number of stitches possible. 
	NOTE: Try to tighten the last chest suture when lungs are inflated to avoid pneumothorax.</li>
	<li>Close the skin with a 6-0 silk/polypropylene suture in a continuous suture pattern.&#160;</li>
	<li>Perform all post-operative care procedures as mentioned in 5.</li>
	<li>Sacrifice the animals 2 weeks later.</li>
</ol>
7. Echocardiography to assess cardiac function and left ventricular hypertrophy in vivo
<ol>
	<li>Perform the echocardiographic exam every 2-3 weeks to follow the progression of hypertrophy and cardiac function.</li>
	<li>Anesthetize the animals, as described, by inhalation of 5% sevoflurane with a nose cone. Adjust the anesthesia level by decreasing it to 2.5%.</li>
	<li>Shave and apply the depilatory cream from the neckline to mid-chest level.</li>
	<li>Place the animal on a heating pad and place the ECG electrodes. Assure a good ECG trace and maintain heart rate between 300 and 350 beats/min.</li>
	<li>Monitor the temperature (~37 &#176;C).</li>
	<li>Apply echo gel and position the animal at left lateral decubitus.</li>
	<li>Start the echocardiograph and adjust the settings.</li>
	<li>Position an ultrasound probe over the thorax.</li>
	<li>Assess the pressure gradient across the banding at 7 and 2 weeks after banding and debanding surgery, respectively. Position the probe at the LV long axis and place the beam over aorta. Press the button PW to activate pulsed wave Doppler echocardiography. After seven weeks of banding, aortic gradients will be &#62;25 mmHg in the banded animals.</li>
	<li>Record two-dimensional guided images of aorta showing the presence or absence of the ascending aorta constriction to anatomically visualize the efficacy of the banding and debanding. 
	NOTE: It is possible to visualize turbulent flow at the constriction level if the color mode is available.</li>
	<li>Assess hypertrophy by positioning the probe at an LV short axis, at papillary muscles level, and press M-mode tracing to visualize LV anterior wall (LVAW), LV diameter (LVD) and LV posterior wall (LVPW) in diastole (D) and systole (S) (Figure 5).</li>
	<li>Assess systolic function, calculate the ejection fraction and fractional shortening as previously described14,15.</li>
	<li>Assess diastolic function by 1) determining the peak of pulsed-wave Doppler of early and late mitral flow velocity (E and A waves, respectively) using an apical 4-chamber apical view just above the mitral leaflets; 2) recording lateral mitral annular myocardial early diastolic (E&#39;) and peak systolic (S&#39;) velocities using pulsed-TDI and apical 4-chamber apical view (Figure 5). &#160;</li>
	<li>Record at least three consecutive heartbeats to each parameter assessment. These values will be subsequently averaged.</li>
</ol>
8. Hemodynamic assessment
<ol>
	<li>At the end of the protocol (Figure 4), perform final echocardiography, as described in 7, before the terminal hemodynamic assessment.</li>
	<li>Repeat steps 2.1 to 3.6.</li>
	<li>Cannulate the right jugular vein and perfuse sterile saline at 64 mL/kg/h.</li>
	<li>Rotate slightly the animal to the left side and make a skin incision at the level of the xiphoid appendix.</li>
	<li>Separate the skin from the muscle with forceps or with a scissor.</li>
	<li>Make a lateral incision between the left ribs at the xiphoid appendix level.</li>
	<li>Perform a left lateral thoracotomy to expose the heart fully. 
	NOTE: To avoid bleeding and lung damage, insert a cotton swab into the thoracic cavity and push the lung gently while inserting two hemostats on the right and left side of the place to cut.</li>
	<li>Pre-heat the P-V loop catheters in a water-bath at 37 &#176;C.</li>
	<li>Calibrate the catheter (setup, channel setting, chose the correct channel for pressure and volume, units).</li>
	<li>Insert a catheter apically into the LV and assure the volume sensors are positioned between the aortic valve and the apex. Volumes can be assessed by echocardiography (Figure 5). Visualization of the pressure-volume loops helps to confirm the correct positioning of the catheter (Figure 6).</li>
	<li>Allow the animal to stabilize 20-30 min without significant changes in the shape of the pressure-volume loops.</li>
	<li>With ventilation suspended at end-expiration, acquire baseline recordings (Figure 6). Continuously acquire data at 1,000 Hz to be subsequently analyzed off-line by appropriate software.</li>
	<li>Compute parallel conductance after the hypertonic saline bolus (10%, 10 &#181;L).</li>
	<li>While anesthetized, sacrifice the animal by exsanguination, collect and centrifuge the blood.</li>
	<li>Lastly, excise and collect&#160;the heart. Weight the heart, the left, and the right ventricle separately and immediately store the samples in liquid nitrogen or formalin for subsequent molecular or histological studies, respectively.</li>
</ol>
9. Aortic banding/debanding procedure in rats
<ol>
	<li>Perform aortic banding in young Wistar (70-90 g) using a 22 G needle and 6-0 polypropylene ligature to constrict the aorta.</li>
	<li>Ensure a proper anesthetic and analgesic procedures with 3-4% of sevoflurane and 0.05&#8201;mg/kg of buprenorphine, respectively.</li>
	<li>During echocardiography, assure a heart rate always above 300 rate / min (ideally between 300 and 350).</li>
	<li>Before step 8.9, gently dissect the rat aorta, place a flow probe around it to measure cardiac output. The use of the aortic flow probe is the gold standard procedure for rats.</li>
	<li>For the hemodynamic evaluation, cannulate the jugular or femoral vein for fluid administration (32 mL/kg/h).</li>
	<li>Replace the pressure-volume catheter SPR-1035 by the SPR-847 or SPR-838, whose sizes better suit the rat ventricular dimensions.</li>
</ol>

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The authors have no conflict of interest.

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><tbody><tr><td>Absorption Spears</td><td>F.S.T</td><td>18105-03</td><td>To absorb fluids during the surgery</td></tr><tr><td>Blades</td><td>F.S.T</td><td>10011-00</td><td>To perform the skin incision</td></tr><tr><td>Buprenorphine</td><td>Buprelieve</td><td></td><td>Analgesia drug</td></tr><tr><td>Catutery</td><td>F.S.T</td><td>18010-00</td><td>To prevent exsanguination</td></tr><tr><td>Catutery tips</td><td>F.S.T</td><td>18010-01</td><td>To prevent exsanguination</td></tr><tr><td>cotton swab</td><td>Johnson&#039;s</td><td></td><td>To absorb fluids during the surgery</td></tr><tr><td>Depilatory cream</td><td>Veet</td><td></td><td>To delipate the animal</td></tr><tr><td>Disposable operating room table cover</td><td>MEDKINE</td><td>DYND4030SB</td><td>To cover the surgical area</td></tr><tr><td>Echo probe</td><td>Siemens</td><td>Sequoia 15L8W</td><td>Ultrasound signal aquisition</td></tr><tr><td>Echocardiograph</td><td>Siemens</td><td>Acuson Sequoia C512</td><td>Ultrasound signal aquisition</td></tr><tr><td>End-tidal CO&lt;sub&gt;2&lt;/sub&gt; monitor</td><td>Kent Scientific</td><td>CapnoStat</td><td>To control expiration gas saturation</td></tr><tr><td>Forcep/Tweezers</td><td>F.S.T</td><td>11255-20</td><td>To dissect the tissues and aorta</td></tr><tr><td>Forcep/Tweezers</td><td>F.S.T</td><td>11272-30</td><td>To dissect the tissues and aorta</td></tr><tr><td>Forcep/Tweezers</td><td>F.S.T</td><td>11151-10</td><td>To dissect the tissues and aorta</td></tr><tr><td>Forcep/Tweezers</td><td>F.S.T</td><td>11152-10</td><td>To dissect the tissues and aorta</td></tr><tr><td>Gas system</td><td>Penlon Sigma Delta</td><td></td><td>To anesthesia and mechanical ventilation</td></tr><tr><td>Hemostats</td><td>F.S.T</td><td>13010-12</td><td>To hold the suture before tight the aorta</td></tr><tr><td>Hemostats</td><td>F.S.T</td><td>13011-12</td><td>To hold the suture before tight the aorta</td></tr><tr><td>Ligation aids</td><td>F.S.T</td><td>18062-12</td><td>To place a suture around the aorta</td></tr><tr><td>Magnetic retractor</td><td>F.S.T</td><td>18200-20</td><td>To help keep the animal in a proper position</td></tr><tr><td>Needle holder</td><td>F.S.T</td><td>12503-15</td><td>To suture the animal</td></tr><tr><td>Needle 26G</td><td>B-BRAUN</td><td>4665457</td><td>To serve as a molde of aortic constriction diameter</td></tr><tr><td>Oxygen</td><td>Air Liquide</td><td></td><td>To anesthesia and mechanical ventilation</td></tr><tr><td>Polipropilene suture</td><td>Vycril</td><td>W8304/W8597</td><td>To suture the animal and to do the constriction</td></tr><tr><td>Povidone-iodine solution</td><td>Betadine&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td><td></td><td>Skin antiseptic</td></tr><tr><td>PowerLab</td><td>Millar instruments</td><td>ML880 PowerLab 16/30</td><td>PV loop Signal Aquisition</td></tr><tr><td>Pulse oximeter</td><td>Kent Scientific</td><td>MouseStat</td><td>To control heart rate and blood saturation</td></tr><tr><td>PVAN software</td><td>Millar Instruments</td><td></td><td>To analyse the haemodynamic data</td></tr><tr><td>PV loop cathether</td><td>Millar instruments</td><td>SPR-1035. 1.4 F</td><td>PV loop Signal Aquisition</td></tr><tr><td>Retractor</td><td>F.S.T</td><td>17000-01</td><td>To provide a better overview of the aorta</td></tr><tr><td>Scalpet handle</td><td>F.S.T</td><td>10003-12</td><td>To perform the skin incision</td></tr><tr><td>Scissors</td><td>F.S.T</td><td>15070-08</td><td>To cut the suture in debanding surgery</td></tr><tr><td>Scissors</td><td>F.S.T</td><td>14084-09</td><td>To cut other material during the surgery e.g. suture, papper</td></tr><tr><td>Sevoflurane</td><td>Baxter</td><td>533-CA2L9117</td><td></td></tr><tr><td>Temperature control module</td><td>Kent Scientific</td><td>RightTemp</td><td>To control animal corporal temperature</td></tr><tr><td>Ventilator</td><td>Kent Scientific</td><td>PhysioSuite</td><td>To ventilate the animal</td></tr><tr><td>Water-bath</td><td>Thermo Scientific&amp;trade;</td><td>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, ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

5.0K Views.  Universidade do Porto. 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.

Video: Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

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 ...

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.

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 Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary arterial hypertension (PAH). As a result, approved therapeutic interventions for the treatment or prevention of PH-LHD are missing. Medications used to treat PH in PAH patients are not recommended for treatment of PH-LHD, as reduced pulmonary vascular resistance (PVR) and increased pulmonary blood flow in the presence of increased left-sided filling pressures may cause left heart decompensation and pulmonary edema. New strategies need to be developed to reverse PH in LHD patients. In contrast to PAH, PH-LHD develops due to increased mechanical load caused by congestion of blood into the lung circulation during left heart failure. Clinically, mechanical unloading of the left ventricle (LV) by aortic valve replacement in aortic stenosis patients or by implantation of LV assist devices in end-stage heart failure patients normalizes not only pulmonary arterial and right ventricular (RV) pressures but also PVR, thus providing indirect evidence for reverse remodeling in the pulmonary vasculature. Using an established rat model of PH-LHD due to left heart failure triggered by pressure overload with subsequent development of PH, a model is developed to study the molecular and cellular mechanisms of this physiological reverse remodeling process. Specifically, an aortic debanding surgery was performed, which resulted in reverse remodeling of the LV myocardium and its unloading. In parallel, complete normalization of RV systolic pressure and significant but incomplete reversal of RV hypertrophy was detectable. This model may present a valuable tool to study the mechanisms of physiological reverse remodeling in the pulmonary circulation and the RV, aiming to develop therapeutic strategies for treating PH-LHD and other forms of PH.

The present protocol describes a surgical procedure to remove ascending-aortic banding in a rat model of pulmonary hypertension due to left heart disease. This technique studies endogenous mechanisms of reverse remodeling in the pulmonary circulation and the right heart, thus informing strategies to reverse pulmonary hypertension and/or right ventricular dysfunction.

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary ...

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor&#39;s cardiac function. This technique&#39;s key points are as follows: transecting the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart; perfusing the donor&#39;s coronary arteries with a cold cardioplegic solution; and topical cooling of the donor&#39;s heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate.
Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,&#160;which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor&#39;s heart is harvested, a stiff guidewire is inserted into the donor&#39;s brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient&#39;s circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Biology

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle pressure overload and progressive right heart failure (RHF). RHF can be identified by right-sided cardiac hypertrophy and dilation associated with abnormal myocardial function affecting the RV and the right atrium (RA). We recently demonstrated that severe RHF is accompanied by an increased risk of atrial inflammation, atrial fibrosis, and atrial fibrillation (AF), the most common type of cardiac arrhythmia (CA). Recent studies have shown that RV and RA inflammation plays an important role in the arrhythmogenesis of CA, including AF. However, the impact of inflammation in the development of CA and AF in RHF is poorly described.
Experimental models of RHF are required to better understand the association between right-sided myocardial inflammation and CA. The rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH) is well-established to provoke RHF. However, MCT triggers severe pneumo-toxicity and pulmonary inflammation. Hence, MCT-induced RHF does not help to distinguish whether the subsequent myocardial inflammation originates from the RHF per se or circulating inflammatory signals secreted by the injured lung.
In this article, a mechanical method involving pulmonary artery trunk banding (PAB) was used to provoke right-sided cardiac arrhythmogenesis. The PAB consists of performing a permanent suture of the pulmonary artery trunk for 3 weeks. Such an approach generates increased right-sided pressure overload. At D21 post-PAB, the suture results in hypertrophied, dilated, and inflamed RV and RA. The PAB-induced RHF is also accompanied by vulnerability to ventricular and atrial arrhythmias, including AF.

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle ...

Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. Although mice are widely applied to shed light on the mechanisms of cardiovascular disease, mouse models of AR, especially those induced by surgery, are still paucity. Here, a mouse model of AR was described in detail which is surgically induced by disruption of the aortic valves under high-resolution echocardiography. In accordance with regurgitated blood flow, AR mouse hearts present a distinctive and clinically relevant volume overload phenotype, which is characterized by eccentric hypertrophy and cardiac dysfunction, as evidenced by echocardiographic and invasive hemodynamic evaluation. Our proposal, in a reliable and reproducible manner, provides a practical guide on the establishment and assessment of a mouse model of AR for future studies on molecular mechanisms and therapeutic targets of volume overload cardiomyopathy.

This protocol presents a practical guide on the surgery for creation of aortic regurgitation (AR) in the mouse. Assessment of the AR mouse by echocardiography and invasive hemodynamic measurement recapitulates its clinically relevant characteristics of volume overload-induced eccentric hypertrophy, suggesting its promising application in the study of cardiac hypertrophy.

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. ...

Surgical Aortic Debanding: A Procedure to Study Left Ventricular Reverse Remodeling in Murine Model of Aortic Constriction

Source: Goncalves-Rodrigues, P. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/60036" target="_blank">Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents</a>. J. Vis. Exp. (2021)
This video demonstrates the technique of aortic debanding, which can lead to reverse remodeling to limit the progression towards heart failure in a murine model. Post debanding, a significant reduction of ventricular afterload triggers regression of ventricular hypertrophy and fibrosis, causing a recovery in cardiac function.