This work was supported by the Bundesministerium f&#252;r Bildung und Forschung (BMBF) to L.L., N.F., and O.J.M. (IVOLADMT-HF; FKZ 01KC2006A).

The animal experiments were carried out under the principles of the regional committee (Ministerium f&#252;r Energiewende, Landwirtschaft, Umwelt, Natur und Digitalisierung des Landes Schleswig-Holstein, permission number: V242-21249/2020 [38-4/20]). The mice used for the present study were obtained from a commercial source (see Table of Materials). The animals were kept under standard conditions with a 12 h light, 12 h night cycle; water and food were offered ad libitum.
1. Animal care
<ol>
	<li>House the mice in specialized cages with bedding, nesting material, a hiding place, and proper access to drinking water and food.</li>
	<li>Keep the animals under continuous specialized veterinary control and treatment. 
	NOTE: For mice ordered by external suppliers, please assure 7 days of acclimation before starting the procedure.</li>
</ol>
2. Preparation of the o-ring
NOTE: An o-ring with a fixed diameter of 0.4 mm is recommended to induce cardiac hypertrophy after 2 weeks. The extent and severity of the cardiac phenotype induced depend on the size of the o-ring diameter.
<ol>
	<li>First, perform one cut of the o-ring (see Table of Materials) under the microscope using scissors or a scalpel to enable the placement around the aorta (Figure 1A, B).</li>
	<li>Pierce each ring side close to the cut with a needle connected with an 8-0 non-absorbable suture and pull the thread. Cut and leave 2-3 cm on one side and 2 cm on the other side to fasten the o-ring around the aorta in the final step (Figure 1C, D).</li>
	<li>Before surgery, take the ligation aid (species instrument, see Table of Materials) and pull the end of the thread (which is kept longer) of one ring side through the hole of the constriction (Figure 1E, F). Put the ligation aid with the attached o-ring aside for placement in the following step (step 6).</li>
	<li>For the disinfection&#160;of the ring with the threads, place the ring in an alcohol solution for half an hour. Afterward, lay it on cellulose to dry. Keep the dried ring in a closed tub or case until use. During the surgery, after pulling the thread through the ligation aid, place the ring on a clean surface until use.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64455/64455fig01.jpg" /> 
Figure 1: Performing the o-ring preparation for ligation. (A) An o-ring with a fixed diameter is cut with scissors or a scalpel on one side. (B)&#160;Image of an o-ring. (C)&#160;Each o-ring side is pierced with an 8-0 prolene thread. (D) O-ring pierced with two threads. (E) The threads of one ring side of the o-ring are pulled through the hole of the ligation aid. (F)&#160;Final position before placement: the threads of one side are placed through the hole of the ligation aid, while the threads of the other side are kept loose. <a href="https://www.jove.com/files/ftp_upload/64455/64455fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Premedication of the mice and preparation of the operating field
<ol>
	<li>To receive sufficient analgesia during the surgery, inject the analgesic buprenorphine (0.1 mg/kg, see Table of Materials) intraperitoneally 20 min before proceeding to the surgery. 
	NOTE: For the present study, the pain medication was used following the recommendations of the Gesellschaft f&#252;r Versuchstierkunde/Society of Laboratory Animal Science (GV-SOLAS).</li>
	<li>After premedication, anesthetize the mouse in an induction chamber with 2%-4% isoflurane mixed with 0.5-1.0 L/min of 100% O2.</li>
	<li>Shave the fur on the left thorax side of the sedated mouse. After shaving, put the mouse back into the isoflurane-filled chamber and wait for sufficient sedation before intubating the animal. 
	NOTE: The right time point of the sedation shows slow breathing but avoids snap breathing. Depending on the isoflurane gas setting, it takes 2-3 min to achieve the right level of sedation.</li>
	<li>Turn the heating pad on before the surgery to maintain the animal&#39;s body temperature (37 &#176;C). Connect the heating pad to a rectal probe (see Table of Materials) to maintain the mouse&#39;s body temperature automatically.</li>
</ol>
4. Intubation of the mice
<ol>
	<li>Prepare the required instruments (Figure 2A). Disinfect&#160;the laryngoscope before the surgery by placing it in alcohol for about 1-3 minutes and then keeping it for drying overnight.</li>
	<li>Stretch a rubber band around the heating pad to fix the mouse with the front teeth on the plate. Place the sedated mouse on the heating pad in a supine position.</li>
	<li>Place the rubber band over the animal&#39;s front teeth to extend the neck on the plate.</li>
	<li>Focus a light source on the throat for good visibility of the opening of the trachea for the endotracheal intubation (Figure 2B).</li>
	<li>Open the mouth gently with one hand positioning an intubation aid (handmade laryngoscope, see Table of Materials) (Figure 2A[3]).</li>
	<li>With the other hand and small forceps, gently move the tongue to clear the opening of the trachea.</li>
	<li>Use this hand also to introduce the endotracheal tube into the trachea. On the other hand, still hold the intubation aid. For intubation, use a 22 G cannula (see Table of Materials) (Figure 2A[1]).</li>
	<li>Connect the endotracheal tube position via a three-way stopcock to a ventilator (see Table of Materials) for mice to confirm the correct endotracheal tube position (endotracheal).</li>
	<li>Monitor the right ventilation according to the manufacturer&#39;s instructions (tidal volume of 200 &#181;L and respiration rate between 100-150 breaths/min) (Figure 2C).</li>
	<li>Confirm sufficient anesthetic depth by a toe pinch reflex check (no reflex response).</li>
	<li>Turn the anesthesia setting to 2% isoflurane mixed with 0.5-1.0 L/min 100% O2.</li>
	<li>Apply ophthalmic&#160;ointment on the eyes to avoid dryness during the surgery.</li>
	<li>Using a&#160;cotton swab,&#160;disinfect the surgery area 3 times with&#160;a commercially available disinfectant solution (see Table of Materials).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64455/64455fig02.jpg" /> 
Figure 2: Intubation of the mouse. (A) Intubation instruments: (1) A 22 G i.v. cannula is used as an endotracheal tube (without mandarin); (2) Forceps; (3) Handmade laryngoscope (deformed/flattened cannula glued with wooden sticks and tape). (B) Performing intubation on the positioned heating pad. (C) Intubated mouse connected to a ventilator. <a href="https://www.jove.com/files/ftp_upload/64455/64455fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Surgery and preparation for ring positioning
NOTE: Use sterile materials and instruments to avoid infections.
<ol>
	<li>Use scissors to make a 0.5-1 cm long skin incision in the middle of a line between the xyphoid process and left axilla. Use forceps to separate the muscle layer from the underlying ribs and place two retractors (5 mm length, see Table of Materials) into the incision to expose the rib cage.</li>
	<li>To start the left thoracotomy, perform a small incision (~1-2 mm) in the intercostal muscles between the second and third rib using micro spring scissors. Open the thoracic cavity and spread the incision with 45&#176; angled forceps.</li>
	<li>Place three chest retractors (1.0-2.5 mm length) into the incision for opening the thoracic cavity to improve visualization.</li>
	<li>To expose the aortic arch, try to lift&#160;up and gently separate the thymus and fat tissue from the arch with fine tip 45&#176; angled forceps.</li>
</ol>
6. Ligation of the transverse aorta with the o-ring
<ol>
	<li>Expose the aortic arch with 45&#176; angled forceps in one hand. Position, with the other hand, the o-ring connected with the ligation aid via the threads of one side (step 2).</li>
	<li>Pass the threads using the ligation aid under the aortic arch from the caudal side to the cranial side of the transverse aorta between the brachiocephalic and left common carotid arteries (Figure 3A).</li>
	<li>Take both threads between the ligation aid and the aortic arch with the forceps carefully. Retract and remove the ligation aid and gently position the o-ring around the arch by pulling the threads on each side (Figure 3B).</li>
	<li>After successful positioning, fix the o-ring with the threads and a surgical knot. Make an additional one to avoid opening the knot on each side (Figure 3C).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64455/64455fig03.jpg" /> 
Figure 3: Performing the o-ring implantation. (A) The aortic arch is exposed by three retractors of 1.0-2.5 mm. Both long threads of one ring side are passed under the aorta. (B) The o-ring will be placed by gently pushing the ring and pulling the threads. (C)&#160;The is o-ring is in the right position, and one cranial thread is knotted with caudal thread on each side. <a href="https://www.jove.com/files/ftp_upload/64455/64455fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Suture and post-operative recovery
<ol>
	<li>Remove the three chest retractors (2.5 mm length) from the incision.</li>
	<li>If needed, eliminate the residual air from the thorax by filling it with a warm 0.9% isotonic salt solution.</li>
	<li>To expose the thorax incision for suture, take two retractors (5 mm length) again to hold the skin on the side.</li>
	<li>Close the thorax with two or three 6-0 non-absorbable sutures (see Table of Materials) and pinch off the outflow of the ventilator for 2 s to reinflate the lungs.</li>
	<li>Remove the two retractors and close the skin with three to five 4-0- absorbable sutures.</li>
	<li>Turn off the isoflurane and monitor. When the animal starts self-breathing, the whiskers are moving, and the toe pinch reflexes can be triggered, extubate the mouse. Lay the mouse on the left side under the heat lamp in its care unit and observe it until it is completely awake.</li>
	<li>Do not leave an animal unattended until it has regained sufficient consciousness. 
	NOTE: An animal that has undergone surgery should get its own care unit (cage) for better recovery.</li>
	<li>Perform pain management with tramadol (1 mg/mL) in drinking water for 7 days and buprenorphine (0.1 mg/kg, 3x daily) by intraperitoneal injection for 3 days after surgery if needed. 
	NOTE: Follow local animal ethics committee recommendations for postoperative analgesia.</li>
	<li>Check medication by weighing the water bottles daily and watch the animal&#39;s behavior.</li>
</ol>
8. Confirmation of successful constriction and right position of the ring
<ol>
	<li>One day after surgery, verify the stenosis using ultrasound by measuring the maximal flow velocity over the stenosis.</li>
	<li>For measurements, use echocardiography with an ultrasound system and a transducer probe with a frequency of 30 MHz (see Table of Materials).</li>
	<li>As described above, maintain anesthesia using a mask at 1.5%-2% isoflurane with 0.5-1.0 L/min of 100% O2.</li>
	<li>Place the anesthetized animal on the heating pad in a supine position. Connect the heating pad with a rectal probe to maintain the body temperature at 37 &#176;C &#177; 1 &#176;C, and monitor the heart rate with an ECG using four mouse paw sensors (see Table of Materials).</li>
	<li>For better visualization, use depilation creme.</li>
	<li>A successfully performed ORAB results in an increased flow velocity over the stenosis as measured by ultrasound (~2,400 mm/s) (Figure 4C). For this measurement, position the transducer head parasternally on the right thorax side to localize the aortic arch by two-dimensional (2D) imaging (&#34;B-mode&#34;).
	<ol>
		<li>Use the color Doppler to visualize the blood flow in the aorta and measure with the pulsed wave Doppler blood flow velocity over the stenosis. 
		NOTE: Sham-operated mice (control surgery without a constriction) show a blood flow velocity of ~600-900 mm/s. In addition, a successful ORAB also results in an increased velocity flow ratio between the right carotid (~150 mm/s) (RC, Figure 4A) and the left carotid (~300 mm/s) LC,&#160;Figure 4B) in the mouse.</li>
	</ol>
	</li>
	<li>Visualize the right and left arteria carotis interna by two-dimensional (2D) imaging (B-mode). Position the transducer head horizontal on the left and right sides of the neck at a 45&#176; angle and use the pulsed wave Doppler to determine the blood flow velocity. 
	NOTE: In sham-operated mice, the velocity flow in both arteries is similar.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64455/64455fig04.jpg" /> 
Figure 4: Confirmation of transverse aorta ligation using pulse wave Doppler velocity measurement in the carotid arteries. (A) Representative pulsed wave Doppler velocity signals of the right carotid artery. (B)&#160;The stenosis results in a higher flow velocity in the right carotid artery than in the left. (C) The stenosis induced by constriction results in a flow velocity in the descending aorta of more than 2,400 mm/s. Sham mice show a flow velocity of 600-900 mm/s. <a href="https://www.jove.com/files/ftp_upload/64455/64455fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+risk+profile+of+heart+failure.">Epidemiology and risk profile of heart failure.</a> Nature Reviews Cardiology. 8 (1), 30-41 (2011).</li><li>Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+initiatives+to+improve+the+rigor+and+reproducibility+of+articles+published+in+Circulation+Research.">New initiatives to improve the rigor and reproducibility of articles published in Circulation Research.</a> Circulation Research. 121 (5), 472-479 (2017).</li><li>Dunlay, S. M., Weston, S. A., Jacobsen, S. J., Roger, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+heart+failure:+A+population-based+case-control+study.">Risk factors for heart failure: A population-based case-control study.</a> The American Journal of Medicine. 122 (11), 1023-1028 (2009).</li><li>Riehle, C., Bauersachs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+of+heart+failure.">Small animal models of heart failure.</a> Circulation Research. 115 (13), 1838-1849 (2019).</li><li>Rockman, H. 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=Segregation+of+atrial-specific+and+inducible+expression+of+an+atrial+natriuretic+factor+transgene+in+an+in+vivo+murine+model+of+cardiac+hypertrophy.">Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.</a> Proceedings of the National Academy of Sciences of the United States of America. 88 (18), 8277-8281 (1991).</li><li>Hu, P., 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=Minimally+invasive+aortic+banding+in+mice:+effects+of+altered+cardiomyocyte+insulin+signaling+during+pressure+overload.">Minimally invasive aortic banding in mice: effects of altered cardiomyocyte insulin signaling during pressure overload.</a> American Journal of Physiology-Heart and Circulatory Physiology. 285 (3), 1261-1269 (2003).</li><li>Mohammed, S. F., 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=Variable+phenotype+in+murine+transverse+aortic+constriction.">Variable phenotype in murine transverse aortic constriction.</a> Cardiovascular Pathology. 21 (3), 188-198 (2012).</li><li>Nakao, Y., 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=O-ring-induced+transverse+aortic+constriction+(OTAC)+is+a+new+simple+method+to+develop+cardiac+hypertrophy+and+heart+failure+in+mice.">O-ring-induced transverse aortic constriction (OTAC) is a new simple method to develop cardiac hypertrophy and heart failure in mice.</a> Scientific Reports. 12, 85 (2022).</li><li>Melleby, A. O., 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=A+novel+method+for+high+precision+aortic+constriction+that+allows+for+generation+of+specific+cardiac+phenotypes+in+mice.">A novel method for high precision aortic constriction that allows for generation of specific cardiac phenotypes in mice.</a> Cardiovascular Research. 114 (12), 1680-1690 (2018).</li><li>Lindsey, M. L., Kassiri, Z., Virag, J. A. I., de Castro Bras, L. E., Scherrer-Crosbie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+measuring+cardiac+physiology+in+mice.">Guidelines for measuring cardiac physiology in mice.</a> American Journal of Physiology-Heart and Circulatory Physiology. 314 (4), 733-752 (2018).</li><li>Garcia-Menendez, L., Karamanlidis, G., Kolwicz, S., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrain+specific+response+to+cardiac+pressure+overload+in+C57BL/6+mice.">Substrain specific response to cardiac pressure overload in C57BL/6 mice.</a> American Journal of Physiology-Heart and Circulatory Physiology. 305 (3), 397-402 (2013).</li><li>deAlmeida, A. C., van Oort, R. J., Wehrens, X. 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The authors have nothing to disclose.

Thread/suture-based aortic banding has been used for many years to induce pressure-overload cardiac hypertrophy in mice. It is an established method to investigate the pathomechanisms of cardiac remodeling and disease progression in vivo. The limitations are the relatively high variance in the degree of stenosis and, consequently, the remodeling. The recently introduced ORAB technique first described by Melleby et al.10 optimizes the conventional method by using a rubber o-ring.
<p cl...

While physiological cardiac hypertrophy can be observed during development, exercise, and pregnancy, pathological cardiac hypertrophy responds to hemodynamic stress conditions like arterial hypertension, valvular heart disease, or gene mutations. Initially, the heart undergoes a remodeling characterized by increased cardiomyocyte size and thickening of the ventricular walls to maintain cardiac function1,2. On the other hand, pathological cardiac remodeling is associated with an increased risk for arrhythmia, sudden death, and high mortality. Finally, with time, it results in ventricular dilation, a strong decrease in contractile function, and eventual progression to heart failure (HF), which is associated with high morbidity, mortality, and societal costs3. Therefore, there is an urgent need to understand the molecular background in order to develop new therapeutic strategies4.
Aortic banding is a model that mimics pressure overload-induced left ventricular (LV) hypertrophy and heart failure in mice5. With this method, it is possible to examine the pathomechanisms of pressure overload-induced cardiac remodeling in vivo. The first aortic banding procedure in mice was reported by Rockman et al.6. Pressure overload is induced by a thread suture-based ligation around the aorta (between the brachiocephalic and left common carotid artery). To create a 0.4 mm diameter stenosis, a suture is placed around a 27 G needle and the aorta. After ligation, the needle is removed6,7.
Even though the needle diameter is fixed, the tightness of the thread is highly dependent on the surgeon and, therefore, affects the induced phenotype of cardiac hypertrophy. In addition, in the thread/suture-based method, there is a variable degree of stenosis diameter after surgery, associated with a high variance in mortality8,9. Moreover, training this method is challenging, especially regarding finding the right level and consistency in tightening the thread. Finally, at the beginning of training, high intra- and post-operative mortality due to disruption of the aorta or other tissue injury occurs, as well as high variation in the extent of stenosis in the surviving animals.
Recently, an optimized procedure of aortic banding was described by Melleby et al.10. They presented the ORAB (o-ring aortic banding) method with less variance in stenosis and highly reproducible levels of pressure overload by using a non-slip rubber o-ring with a fixed inner diameter of 0.71 mm, 0.66 mm, and 0.61 mm. In short, the o-ring is cut open, placed around the ascending arch, and closed again by threads. Other scientists using these o-rings reported less variability in the induced cardiac hypertrophy9. They also observed intra- and post-operative mortality, as well as better reproducibility and less variance in the induced hypertrophic phenotype9,11. The present article describes the procedure of this unique strategy in a step-by-step protocol. The expertise shared in this report will help other scientists to improve their techniques in this area.
To induce cardiac hypertrophy resulting in heart failure after 6 weeks, 12-week-old C57BL/6N male mice are recommended for surgery. A comparison 2 weeks after aortic banding between the mouse substrains C57BL/6N and C57BL/6J showed severe cardiac dysfunction and associated increased mortality in C57BL/6N mice. Therefore, these are better suited for models of heart failure12. Twelve-week-old male and female mice have an optimal size for exposure of the aorta and placement of the o-ring with special instruments.

<table><tbody><tr><td>1 x long Fixators, 6 cm</td><td></td><td>&amp;nbsp;18200-01</td><td></td></tr><tr><td>2 x Blunt Retractors, 5 mm wide&amp;nbsp;</td><td></td><td>18200-11</td><td></td></tr><tr><td>2 x Short Fixators, 3 cm</td><td></td><td>&amp;nbsp;18200-02</td><td></td></tr><tr><td>2 x fine tip 45&amp;deg; angled forceps&amp;nbsp;</td><td>FST (fine sience tools)</td><td>11160-10</td><td></td></tr><tr><td>3 x Blunt Retractors, 2.5 mm wide&amp;nbsp;</td><td></td><td>18200-10</td><td></td></tr><tr><td>3 x Retractor Wires&amp;nbsp;</td><td></td><td>18200-05</td><td></td></tr><tr><td>4-0 absorbable suture (Vicryl)</td><td>ETHICON</td><td>SABBKLT0</td><td>Used to suture skin</td></tr><tr><td>6-0 suture (Prolene), needle size:13 mm</td><td>ETHICON</td><td>JDP879</td><td>Used to suture chest muscle</td></tr><tr><td>8-0 suture (Prolene), needle size: 6.5 mm</td><td>ETHICON</td><td>RHBECH</td><td>Used to fast the Ring&amp;nbsp;</td></tr><tr><td>Anesthetizing Box, Small</td><td>Havard apparatus&amp;nbsp;</td><td>50-0108</td><td></td></tr><tr><td>C57BL/6N mice</td><td>Charles River</td><td></td><td></td></tr><tr><td>Fluosorber Activated Charcoal Filter Canister</td><td>Havard apparatus&amp;nbsp;</td><td>34-0415</td><td>Used to induce and maintain anesthesia</td></tr><tr><td>Handmade laryngsopcope&amp;nbsp;</td><td></td><td></td><td>Intubationshelp</td></tr><tr><td>Harvard Apparatus Anesthetic Vaporizer</td><td>Havard apparatus&amp;nbsp;</td><td></td><td>Used to induce and maintain anesthesia</td></tr><tr><td>Heating pad + rectal probe (LSI Letica Scientific Instruments:Temperature control unit HB 101/2 )</td><td>Panlab/ Havard apparatus&amp;nbsp;</td><td></td><td>Used to control and maintain body temperature&amp;nbsp;</td></tr><tr><td>i.v. cannula blue 22-gauge (Vasofix Braun&amp;uuml;le 0.9 x 25 mm)</td><td>B/Braunsharing Expertise&amp;nbsp;</td><td>4268091B</td><td>intubation&amp;nbsp;</td></tr><tr><td>isofluran</td><td>Baxter</td><td></td><td>Anesthesia</td></tr><tr><td>Kodan (betadine solution)</td><td>Sch&amp;uuml;lke</td><td>20003960-A</td><td>Desinfection&amp;nbsp;</td></tr><tr><td>ligation aid</td><td>FST (fine sience tools)</td><td>18062-12</td><td>Used to perform liagtion with O-ring&amp;nbsp;</td></tr><tr><td>Microscope Lighting: Schott VisiLED Set MC1500/S80-55 (+ controller)</td><td>SCHOTT</td><td></td><td>Ligth</td></tr><tr><td>Microscope&amp;nbsp;camera&amp;nbsp;(Leica IC80 HD)</td><td>Leica</td><td></td><td>Used for visualiation operating field&amp;nbsp;</td></tr><tr><td>MiniVac Complete Anesthesia Systems&amp;nbsp;for small rodents</td><td>Havard apparatus&amp;nbsp;</td><td>75-0233</td><td>Used to induce and maintain anesthesia</td></tr><tr><td>Mouse Ventilator MiniVent Type 845</td><td>Havard Apparatus&amp;nbsp;</td><td>73-0044</td><td>Used to ventilation during surgery&amp;nbsp;</td></tr><tr><td>Needle holder&amp;nbsp;</td><td>FST (fine sience tools)</td><td>&amp;nbsp;TE-10804</td><td></td></tr><tr><td>O-ring, non-slip rubber (0.0018 mm x 0.020 mm)</td><td>Apple Rubber Products&amp;nbsp;</td><td></td><td>Liagtion of the aortic arch</td></tr><tr><td>Scissors</td><td>FST (fine sience tools)</td><td>14040-09</td><td>Used to cut the skin and threads&amp;nbsp;</td></tr><tr><td>Small Animal Retraction System (Kit for Animals up to 200 g)</td><td>FST (fine sience tools)</td><td>18200-20</td><td></td></tr><tr><td>Small Base Plate, 20 x 30 cm&amp;nbsp;</td><td></td><td>&amp;nbsp;18200-03</td><td></td></tr><tr><td>Table intgerated with heating pad + rectal probe + ECG and&amp;nbsp; transducer tripod</td><td>FujiFilm Visual Sonics Imaging System</td><td></td><td>Echocardiography&amp;nbsp;</td></tr><tr><td>Temgesic (Buprenorphin)</td><td>Indivior UK Limited</td><td>997.00.00</td><td>Pain pre-medication&amp;nbsp;</td></tr><tr><td>three-way stop cock (blue)</td><td></td><td></td><td></td></tr><tr><td>Tramal (Tamadol)</td><td>Gr&amp;uuml;nental</td><td></td><td>Pain post-medication&amp;nbsp;</td></tr><tr><td>transducer probe MS400 (Visual Sonics)&amp;nbsp;</td><td>FujiFilm Visual Sonics Imaging System</td><td></td><td>Echocardiography&amp;nbsp;</td></tr><tr><td>Ultrasound system with cardioligy package&amp;nbsp;</td><td>FujiFilm Visual Sonics Imaging System</td><td></td><td>Echocardiography&amp;nbsp;</td></tr><tr><td>Vannas Spring Scissors - 2.5 mm Cutting Edge</td><td>FST (fine sience tools)</td><td>15000-08</td><td>Used to cut intercostal chest muscle&amp;nbsp;</td></tr><tr><td>vet ointment&amp;nbsp;</td><td>Bepanten</td><td></td><td>Used to prevent eyes from drying out</td></tr></tbody></table>

o-ring aortic banding versus traditional transverse aortic constriction for modeling pressure overload-induced cardiac hypertrophy

Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of heart failure. The previously used technique is based on a threaded suture around the aortic arch tied over a blunted 27 G needle to create stenosis. This method depends on the surgeon manually tightening the thread and, thus, leads to high variance in the diameter size. A newly refined method described by Melleby et al. promises less variance and more reproducibility after surgery. The new technique, o-ring- aortic banding (ORAB), uses a non-slip rubber ring instead of a suture with a thread, resulting in reduced variation in pressure overload and reproducible phenotypes of cardiac hypertrophy. During surgery, the o-ring is placed between the brachiocephalic and left carotid arteries. Successful constriction is confirmed by echocardiography. After 1 day, correct placement of the ring results in an increased flow velocity in the transverse aorta over the o-ring-induced stenosis. After 2 weeks, impaired cardiac function is proven by decreased ejection fraction and increased wall thickness. Importantly, besides less variance in the diameter size, ORAB is associated with lower intra- and post-operative mortality rates compared with transverse aortic constriction (TAC). Thus, ORAB represents a superior method to the commonly used TAC surgery, resulting in more reproducible results and a possible reduction in the number of animals needed.

Generally, aortic banding mimics human aortic stenosis and induces cardiac hypertrophy in mice. A successful procedure is characterized by heart tissue remodeling reflected by cardiac hypertrophy and reduced heart function5,6.
Directly 1 day after the operation, the effect of o-ring constriction of the transverse aorta can be determined in vivo by echocardiography13. Successful induction of aortic steno...

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O-Ring Aortic Banding Versus Traditional Transverse Aortic Constriction for Modeling Pressure Overload-Induced Cardiac Hypertrophy

The present protocol describes a new technique of aortic banding in mice to induce pressure overload cardiac hypertrophy. For banding, a rubber ring with a fixed inner diameter is used. This new technique promises less variance and more reproducible data for future experiments.

Aortic banding in mice is one of the most commonly used experimental models for cardiac pressure overload-induced cardiac hypertrophy and the induction of ...

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Research

JoVE Journal

Medicine

3.4K Views.  University Hospital Kiel, Partner Site Hamburg/Kiel/Lübeck. Deze nieuwe techniek van aortabanding maakt inductie van drukoverbelasting cardiale hypertrofie mogelijk bij muizen met minder variantie en hogere reproduceerbaarheid. Het plaatsen van een rubberen ring met een vaste diameter rond de aorta induceert een consistente verhoogde stroomsnelheid in de transversale aorta, waardoor reproduceerbare cardiale hypertrofie wordt gegenereerd. Deze procedure moet worden uitgevoerd door iemand die al ervaring heeft met anesthesie- en microchirurgietechnieken...

Video: O-Ring Aortic Banding Versus Traditional Transverse Aortic Constriction for Modeling Pressure Overload-Induced Cardiac Hypertrophy

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 ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

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 Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

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 ...

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 ...

Operating Transverse Aortic Constriction with Absorbable Suture to Obtain Transient Myocardial Hypertrophy

Based on twice transverse aortic constrictions (TACs) in mice, it is proved that myocardial hypertrophic preconditioning (MHP) could attenuate cardiomyocyte hypertrophy and slow down progression to heart failure. For novices, however, the MHP model is usually quite difficult to establish because of the technical obstacles in ventilator operation, opening the chest repeatedly, and bleeding caused by debanding. To facilitate this model, to increase the surgical success rate and to reduce the incidence of bleeding, we switched to absorbable sutures for the first TAC combing with a ventilator-free technique. Using a 2-week absorbable suture, we demonstrated that this procedure could cause significant myocardial hypertrophy in 2 weeks; and 4 weeks after surgery, myocardial hypertrophy was almost completely regressed to the baseline. Using this protocol, the operators could master the MHP model easily with a lower operation mortality.

This protocol presents an improved method to obtain transient myocardial hypertrophy with absorbable suture, simulating left ventricular hypertrophy decrease after removing pressure overload. It could be valuable for the studies on myocardial hypertrophic preconditioning.

Based on twice transverse aortic constrictions (TACs) in mice, it is proved that myocardial hypertrophic preconditioning (MHP) could attenuate ...

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 ...

Ascending Aortic Banding for Studying Pressure-Overload Induced Heart Failure

A Minimally Invasive Model of Aortic Stenosis in Swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.

Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological ...

O-Ring Aortic Banding Versus Traditional Transverse Aortic Constriction for Modeling Pressure Overload-Induced Cardiac Hypertrophy

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