This work was supported by a grant (N&#176; 32016) of the Swiss Cardiovascular Foundation to RT.

Male C57BL/6J mice (10 weeks, 25 - 30g, n=60) are used in this protocol. Animals receive humane care in compliance with the guidelines formulated by the French Ministry of Agriculture and of Higher Education and Research, and all procedures are performed in accordance with the European Community Council Directive of November 24, 1986 (86/609/EEC) and the French laws. The protocol was approved by the &#34;Regional Ethics Committee for Animal Experimentation CREMEAS&#34; (#2016092816207606).
1. Preparation for surgery
<ol>
	<li>Maintain the mice for one week after arrival in the animal facility on a 12h/12h light/dark cycle, in standard cages, with food (for details see the Table of Materials) and water available ad libitum.</li>
	<li>On the day of operation, place the mice in individual cages a few minutes prior to induction of anesthesia, in order to avoid any additional stress to the animal. Sterilize all surgical instruments the day before surgery.</li>
	<li>Inject intra-peritoneally a single dose of a mixture of ketamine (51.4 mg/kg) and xylazine (3.3 mg/kg) diluted in saline solution (0.9% NaCl).</li>
	<li>Make sure of the depth of the anesthesia by the absence of the toe-retreat reflex.</li>
	<li>Shave the neck and chest of the animal with a commercially available razor and disinfect the shaved area with 70% alcohol.</li>
	<li>Place the animal supine on a clean cork working pad and fix the paws with adhesive tape.</li>
</ol>
2. Surgery
<ol>
	<li>Sterile surgical technique is used throughout the procedures. In a spontaneously breathing animal, perform a longitudinal midline cervical incision over 10 mm with an 11-blade knife from supra-sternal notch to the mid-chest in order to expose the sternum (Figure 1).</li>
	<li>Retract the thyroid by passing a 4/0 monofilament polypropylene stay suture with a Crile-Wood needle holder and tape it to the working pad.</li>
	<li>Separate bluntly the pre-tracheal muscles with micro-surgical forceps to uncover the trachea.</li>
	<li>Slide gently the smooth-tipped curved micro-surgical forceps with the closed jaws over the trachea and behind the sternum.</li>
	<li>By carefully opening and closing the jaws of the smooth-tipped curved microsurgical forceps carry out a blunt dissection under the pre-tracheal muscles and behind the sternum to move the pleura away.</li>
	<li>Grasp the right supra-clavicular muscles with the smooth-tipped straight micro-surgical forceps and pull up gently the chest of the animal.</li>
	<li>Slide the inferior jaw of the bone nipper under the sternum and perform a 3-4 mm upper partial sternotomy (Figure 2). Direct the lower part of the mini-sternotomy slightly toward the left.</li>
	<li>Pass a 7/0 monofilament polypropylene stay suture from inside to outside through the second intercostal space on each side of the mini-sternotomy using a micro-surgical needle holder. Stay close to costo-sternal angle to avoid injury to intercostal and internal thoracic vessels or pleura.</li>
	<li>Spread the sternal edges using 7/0 monofilament polypropylene stay sutures on each side and fix them to the working pad with adhesive tape.</li>
	<li>Gently move aside the pre-tracheal muscles, mediastinal fat and thymus using smooth-tipped curved microsurgical forceps to visualize the aortic arch under low-power magnification (2 - 3X) (Figure 3). Take particular care not to touch or damage the parietal pleura to prevent pneumothorax development.</li>
	<li>Expose the soft tissue under the aortic arch by tying forceps (Figure 4A) and spread gently its jaws. Prepare a tunnel in the soft tissue under the aortic arch with second tying forceps by gently opening and closing the jaws in the soft tissue.</li>
	<li>Pass a segment of 6/0 silk ligature threaded through the eye of a ligation aid (Figure 4B) held in the left hand under the aortic arch and retrieve it by tying forceps held in the right hand between the origin of the right innominate and left common carotid arteries (Figure 5).</li>
	<li>Cut a 27-gauge needle to a length of 5 mm and blunt both ends by pressing them with a Crile needle holder. Place the blunted 27-gauge needle next to the aortic arch (Figure 6) with smooth-tipped straight micro-surgical forceps and tie the suture snugly around the needle and the aorta between the right innominate and left common carotid arteries using the two tying forceps (Figure 7). To tie snugly the suture, perform an initial double knot followed by four additional knots. Make sure that all knots are flat.</li>
	<li>After ligation, remove quickly but gently the needle to achieve a 0.4 mm diameter narrowing and a reproducible transverse 65-70% aortic constriction.</li>
	<li>Check for hemostasis of the soft tissue around the aortic arch, of the sternal edges and pre-tracheal muscles. Put resorbable hemostatic gauze wherever oozing of blood is observed.Remove the 7/0 monofilament polypropylene stay sutures used for spreading the sternal edges.</li>
	<li>Pass a simple 6/0 monofilament polypropylene suture with a micro-surgical needle holder from outside to inside of the left second intercostal space and then from inside to outside of the right second intercostal space. Stay close to costo-sternal angle to avoid injury to intercostal and internal thoracic vessels or pleura.</li>
	<li>Bring together the sternal edges by tying 6/0 monofilament polypropylene suture with a Crile-Wood needle holder.</li>
	<li>Close the skin with a 5/0 monofilament polypropylene running suture in one layer with a Crile-Wood needle holder.</li>
	<li>Perform the sham procedure identical to the constriction operation but without tying a suture around the aorta.</li>
</ol>
3. Post-operative recovery
<ol>
	<li>Monitor the animals very closely.&#160;Transfer the mouse to an individual cage and place it in a prone position.</li>
	<li>Allow the mouse to recover under a warming light until fully awake (less than 1 h after inducing anesthesia).</li>
	<li>For post-operative analgesia, inject 0.1 mg/kg of buprenorphine intraperitoneally.&#160;Repeat subcutaneous injections of 0.1 mg/kg of buprenorphine every 8 h for the first three days as indicated.</li>
	<li>Place the operated mice in standard cages (maximum 3 mice per cage and minimum 2 mice per cage).</li>
</ol>
4. Heart harvest
<ol>
	<li>On the day of analysis, euthanize the mouse with a solution of ketamine 300 mg/kg and xylazine 20 mg/kg in saline by intraperitoneal injection.</li>
	<li>First harvest the blood from the inferior vena cava and then through the same line inject 5 mL of solution of 2.6 mM EDTA in saline.</li>
	<li>Harvest the heart, remove the atria and weight the heart (left and right ventricles without atria).</li>
	<li>Separate the left from the right ventricle with the septum remaining to the left ventricle part. Weigh both tissue samples and freeze them in liquid nitrogen.</li>
</ol>

<ol>
	<li>Molinari, F., Malara, N., Mollace, V., Rosano, G., Ferraro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+cardiac+cachexia.">Animal models of cardiac cachexia.</a> Int. J. Cardiol. 219 (15), 105-110 (2016).</li><li>Tarnavski, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+surgical+models+in+cardiovascular+research.">Mouse surgical models in cardiovascular research.</a> Methods. Mol. Biol. 573, 115-137 (2009).</li><li>Verma, S. K., Krishnamurthy, P., Kishore, R., Ardehali, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+aortic+constriction:+a+model+to+study+heart+failure+in+small+animals.">Transverse aortic constriction: a model to study heart failure in small animals.</a> Manual of Research Techniques in Cardiovascular Medicine. , 164-169 (2014).</li><li>Patten, R. D., Hall-Porter, M. R. <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.+Development+of+novel+therapies,+past+and+present.">Small animal models of heart failure. Development of novel therapies, past and present.</a> Circ. Heart Fail. 2 (2), 138-144 (2009).</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> Proc. Natl. Acad. Sci. USA. 88 (18), 8277-8281 (1991).</li><li>Barrick, C. J., Rojas, M., Schoonhoven, R., Smyth, S. S., Threadgill, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+response+to+pressure+overload+in+129S1/SvImJ+and+C57BL/6J+mice:+temporal-+and+background-dependent+development+of+concentric+left+ventricular+hypertrophy.">Cardiac response to pressure overload in 129S1/SvImJ and C57BL/6J mice: temporal- and background-dependent development of concentric left ventricular hypertrophy.</a> Am. J. Physiol. Heart Circ. Physiol. 292 (5), 2119-2130 (2007).</li><li>Deschepper, C. F., Olson, J. L., Otis, M., Gallo-Payet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+blood+pressure+and+morphological+traits+in+cardiovascular-related+organs+in+13+different+inbred+mouse+strains.">Characterization of blood pressure and morphological traits in cardiovascular-related organs in 13 different inbred mouse strains.</a> J. Appl. Physiol. 97 (1), 369-376 (2004).</li><li>Almeida, A. C., van Oort, R. J., Wehrens, X. H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+aortic+constriction+in+mice.">Transverse aortic constriction in mice.</a> J. Vis. Exp. (38), (2010).</li><li>Hu, P., Zhang, D., Swenson, L., Chakrabarti, G., Abel, E. D., Litwin, S. E. <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> Am. J. Physiol. Heart Circ. Physiol. 285 (3), 1261-1269 (2003).</li><li>Faerber, G., 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=Induction+of+heart+failure+by+minimally+invasive+aortic+constriction+in+mice:+Reduced+peroxisome+proliferator-activated+receptor+&#978;+coactivator+levels+and+mitochondrial+dysfunction.">Induction of heart failure by minimally invasive aortic constriction in mice: Reduced peroxisome proliferator-activated receptor &#978; coactivator levels and mitochondrial dysfunction.</a> J. Thorac. Cardiovasc. Surg. 141 (2), 492-500 (2011).</li><li>Andersen, N. M., Tang, R., Li, L., Javan, H., Zhang, X. Q., Selzman, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IKK-&#946;+inhibition+prevents+adaptive+left+ventricular+hypertrophy.">IKK-&#946; inhibition prevents adaptive left ventricular hypertrophy.</a> J. Surg. Res. 178 (1), 105-109 (2012).</li><li>Nemska, S., Monassier, L., Gassmann, M., Frossard, N., Tavakoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+mRNA+profiling+in+a+rat+model+of+left+ventricular+hypertrophy+reveals+early+expression+of+chemokines+and+their+receptors.">Kinetic mRNA profiling in a rat model of left ventricular hypertrophy reveals early expression of chemokines and their receptors.</a> PLoS ONE. 11 (8), 0161273 (2016).</li><li>Liao, 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=Echocardiographic+assessment+of+LV+hypertrophy+and+function+in+aortic-banded+mice:+necropsy+validation.">Echocardiographic assessment of LV hypertrophy and function in aortic-banded mice: necropsy validation.</a> Am. J. Physiol. Heart Circ. Physiol. 282 (5), 1703-1708 (2002).</li><li>Stansfield, W. E., 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=Characterization+of+a+model+to+independently+study+regression+of+ventricular+hypertrophy.">Characterization of a model to independently study regression of ventricular hypertrophy.</a> J. Surg. Res. 142 (2), 387-393 (2007).</li><li>Beetz, N., 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=Ablation+of+biglycan+attenuates+cardiac+hypertrophy+and+fibrosis+after+left+ventricular+pressure+overload.">Ablation of biglycan attenuates cardiac hypertrophy and fibrosis after left ventricular pressure overload.</a> J. Mol. Cell. Cardiol. 101, 145-155 (2016).</li></ol>

The authors have no conflict of interest to disclose.

The aim of this protocol is to present a step-by-step illustration of the surgical technique for minimally invasive transverse aortic constriction in mice. Detailed technical description of transverse aortic constriction in mice has been reported by other authors2,8. However, these investigators perform surgery following intubation and ventilation of animals. The use of an additional step of intubation-ventilation increases the complexity and duration of the whole procedure and the global stress the animal is exposed to. For these reasons, the concept of minimally invasive transverse aortic constriction has received some attention. The minimally invasive transverse aortic constriction in mice is used to induce left ventricular hypertrophy and its progression to heart failure9,10,11. These studies focus on the pathways involved in the genesis of left ventricular hypertrophy and heart failure, but not on the description of the surgical technique9,10,11.
In this protocol, we report in details a simplified and reproducible technique of minimally invasive TAC in mice. A skilled surgeon can do the constriction operation in 20 minutes and the sham operation (without suture tying) in 15 minutes. During our initial technical proof, we found that the introduction of a key instrument, the ligation aid, allowed a very low operative mortality of 1.7%. This compares favorably to operative mortality of 4% reported by Rockman et. al.5, of 3.7% by Liao et al.13 and of 2.7% by Stansfield et al14. Further, the observation period up to 28 days, also shows a very low late post-operative mortality of 1.7%. Again, this compares well to the late mortality reported by Rockman et al (10%)5, Liao et al (19%)13 or Stansfield et al (2.6%)14.
The passage under the aortic arch is the most crucial step of the whole procedure. The reproducibility of this step was not described by Hu and co-workers who used a home-made wire with a snare at its end to pass under the aorta between the origin of the right innominate and left common carotid arteries9, nor by Tarnavski who positioned the curved forceps from the medial side under the ascending aorta to catch the 7/0 silk suture on the opposite side and move it underneath the aorta2. The ligation aid used in our technique allows a standardized and reproducible maneuver with low risk of aortic tear.
Another decisive step of the procedure is the tension applied to the tie over the 27-gauge needle to reduce efficiently and homogenously the lumen of the aortic arch. First, we use tying forceps, which help applying a uniform and reproducible tension on the suture around the aortic arch. The appropriate placement of the suture is verified during the harvest of the heart and aortic arch. Andersen and coworkers verified the appropriate placement of the band by evaluation of Doppler signals of the carotid arteries both before and after placement of the aortic band11. In their report, adequate banding was accepted when the Doppler velocity ratio doubled from right to left carotid arteries11. In our technique, we chose to measure the efficiency of TAC by the degree of induced left ventricular hypertrophy in banded compared to sham animals in order to validate the procedure, since it does not necessitate any increased duration of the procedure or supplementary anesthesia of the animals. In our technique, the degree of left ventricular hypertrophy and appropriate placement of banding are checked at the end of the experiment. The degree of left ventricular hypertrophy by our technique compares favorably with the results reported by other investigators at 3 weeks following TAC in mice15. In addition, the low variation of the ratio of heart to body weight observed in our banded animals attests the low fluctuation of the tension applied to the tie.
In conclusion, through avoidance of intubation-ventilation as presented in this protocol, our technique of minimally invasive TAC in mice provides a reliable and reproducible model. This model reduces the global strain put on the animals and is time- and cost-saving compared to TAC using intubation-ventilation of animals. The operative and late mortality rates of this procedure are very low and make this technique one of the methods of choice for induction of left ventricular hypertrophy in mice.

Over the past years, the study of heart failure has been conducted in viable animal models1. Compared to large animal models of heart failure, small animal models have numerous potential advantages. Beside lower costs of housing and maintenance, small animal models are accessible to more researchers due to the less complex facilities needed2.
Mouse heart failure models offer many of the same advantages as the rat models. In addition to reduced housing costs3, mouse models benefit from the availability of relevant transgenic and knockout (KO) strains. The possibility of cell type-specific, inducible KO or transgenic strategies make the mouse an invaluable tool to study the pathogenesis of heart failure and to try to identify novel therapeutic regimens3.
Among the mouse models of heart failure currently used4, transverse aortic constriction (TAC) which was first described by Rockman5 is the preferred model to generate pressure overload-induced left ventricular hypertrophy (LVH)1,3. The greatest advantage of this model is the ability to allow stratification of LVH2, although left ventricular remodeling in response to TAC is variable among different mouse strains. In particular, C57BL/6 mice develop rapid LV dilation after TAC that may not occur with other strains4,6,7.
The sudden onset of hypertension achieved with TAC causes an approximately 50% increase in LV mass within 2 weeks, allowing to rapidly examine the activity of pharmacological or molecular interventions aiming at modulating the development of LVH4. The acute induction of severe hypertension by TAC does not exactly reproduce the progressive left ventricular hypertrophy and remodeling observed in the clinical setting of aortic stenosis or arterial hypertension. Nevertheless, this model is used by many investigators to identify and modify novel therapeutic targets in heart failure4.
Performing TAC in mice requires greater surgical expertise than that required for other techniques used to induce LVH and subsequent heart failure2. Most authors perform this procedure by intubating and ventilating the animal2,8, which makes this procedure more demanding and time-consuming and adds to the surgical burden for the animal. Only few investigators have used minimally invasive TAC in their study with brief reference to the surgical procedure9,10,11.
The aim of this protocol is to describe step-by-step a simplified and user-friendly technique of minimally invasive transverse aortic constriction in mice, highlighting the critical stages of the procedure. By following these key steps, one can easily perform this technique.

<table><tbody><tr><td>Surgical microscope</td><td>Olympus</td><td>SZX2-TR30</td><td></td></tr><tr><td>Razor</td><td>Rowenta</td><td>Nomad TN3650FO</td><td></td></tr><tr><td>&lt;strong&gt;Sutures:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Polypropylene 7/0</td><td>Ethicon</td><td>BV-1X</td><td></td></tr><tr><td>Polypropylene 6/0</td><td>BBraun</td><td>C0862061</td><td></td></tr><tr><td>Silk 6/0 ligature&amp;nbsp;</td><td>FST</td><td>18020-60</td><td></td></tr><tr><td>Polypropylene 4/0</td><td>Ethicon</td><td>8683</td><td></td></tr><tr><td>Polypropylene 5/0</td><td>Ethicon</td><td>Z303</td><td></td></tr><tr><td>&lt;strong&gt;Drugs:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamin</td><td>Merial</td><td>Imalg&amp;egrave;ne 1000, LBM154AD</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>Rompun 2%, KP09PPC</td><td></td></tr><tr><td>Buprenorphine</td><td>Ceva</td><td>Vetergesic, 072013</td><td></td></tr><tr><td>&lt;strong&gt;Instruments:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bone nippers</td><td>Fine Surgical Tools</td><td>16101-10</td><td></td></tr><tr><td>Ligation aid</td><td>Fine Surgical Tools</td><td>18062-12</td><td></td></tr><tr><td>Tying forceps</td><td>Fine Surgical Tools</td><td>18026-10</td><td></td></tr><tr><td>Needle holder Crile-Wood</td><td>Fine Surgical Tools</td><td>12003-15</td><td></td></tr><tr><td>Microsurgery forceps&amp;nbsp;</td><td>Fine Surgical Tools</td><td>11003-12</td><td></td></tr><tr><td>Microsurgery forceps&amp;nbsp;</td><td>Fine Surgical Tools</td><td>11002-12</td><td></td></tr><tr><td>Tissue forceps</td><td>Fine Surgical Tools</td><td>11021-12</td><td></td></tr><tr><td>Microsurgery needle holder</td><td>Fine Surgical Tools</td><td>12076-12</td><td></td></tr><tr><td>Microsurgery scissors</td><td>Fine Surgical Tools</td><td>91501-09</td><td></td></tr><tr><td>Mayo scissors</td><td>Fine Surgical Tools</td><td>14511-15</td><td></td></tr><tr><td>11-blade knife</td><td>Fine Surgical Tools</td><td>10011-00</td><td></td></tr><tr><td>&lt;strong&gt;RNA extraction and qPCR:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TriReagent</td><td>Euromedex</td><td>TR-118-200</td><td></td></tr><tr><td>Rneasy Mini kit</td><td>Qiagen</td><td>74704</td><td></td></tr><tr><td>Qubit Fluorimetric RNA assay</td><td>Fisher Scientific</td><td>10034622</td><td></td></tr><tr><td>RNA 6000 Nano kit</td><td>Agilent</td><td>5067-1511</td><td></td></tr><tr><td>High Capacity cDNA kit</td><td>Fisher Scientific</td><td>10400745</td><td></td></tr><tr><td>Taqman Master Mix</td><td>Fisher Scientific</td><td>10157154</td><td></td></tr><tr><td>Taqman BNP primers</td><td>Fisher Scientific</td><td>Mm01255770_g1</td><td></td></tr><tr><td>Taqman ANP primers</td><td>Fisher Scientific</td><td>Mm01255747_g1&amp;nbsp;</td><td></td></tr><tr><td>Taqman ACE primers</td><td>Fisher Scientific</td><td>Mm00802048_m1</td><td></td></tr><tr><td>Taqman Col1a1 primers&amp;nbsp;</td><td>Fisher Scientific</td><td>Mm00801666_g1</td><td></td></tr><tr><td>Taqman TGFb primers</td><td>Fisher Scientific</td><td>Mm01178820_m1</td><td></td></tr><tr><td>Taqman Gapdh primers</td><td>Fisher Scientific</td><td>Mm99999915_g1</td><td></td></tr><tr><td>ABIPrism&amp;nbsp; Thermocycler</td><td>Applied Biosystems</td><td>7000</td><td></td></tr><tr><td>&lt;strong&gt;Software:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>Prism 7</td><td></td></tr><tr><td>&lt;strong&gt;Animal food&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Complete diet for adult rats/mice</td><td>Safe</td><td>UB220610R</td><td></td></tr></tbody></table>

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.

Operative and late survival 
The operative survival was very high, 98.3% (59 out of 60) for the entire series (TAC and sham-operated animals). The only operative death was due to a bleeding complication in a mouse planed for sham operation. Post-operative survival during the observation period of 28 days was also excellent, by 98.3% (58 out of 59). The only late post-operative death occurred in a TAC mouse on day (D) 16, possibly of cardiac origin.
Validation of the technique 
The presented technique is very reliable and reproducible. The correct placement of the suture between the right innominate and left common carotid arteries was confirmed during tissue harvest in all animals undergoing TAC.
The efficacy of the technique to induce left ventricular hypertrophy was validated by determination of heart weight/body weight ratios (HW/BW, mg/g) at 3, 7, 14 and 28 days post-surgery. The HW is the weight of the left and right ventricles without atria. The HW/BW ratio significantly increased in the banded compared to the sham groups from post-operative D7 (4.9&#177;0.2 versus 4.1&#177;0.05 mg/g, P&#60;0.01) on, and remained significantly higher up to D28 (5.8&#177;0.3 versus 4.1&#177;0.1 mg/g, P&#60;0.0001) post-surgery (Figure 8). The observed increase in HW/BW ratio was solely due to a rise in left ventricle/body weight ratio (Figure 9A) since the right ventricle/body weight ratio remained comparable between TAC and sham-operated animals during the whole observation period (Figure 9B).
Further, we measured in the left ventricle tissue the mRNA expression of the biomarkers of cardiac hypertrophy as previously described12. At D14, mRNA expression of brain natriuretic protein (BNP), atrial natriuretic protein (ANP), angiotensin converting enzyme (ACE), collagen 1a1 (Col1a1) and transforming growth factor &#223; (TGF&#223;) was significantly higher in aortic-banded compared to sham-operated animals (Figure 10). Hence, the observed left ventricular hypertrophy validates the efficiency of our TAC technique.
Mean and standard error of mean values were compared between TAC and sham groups using one-way ANOVA followed by Bonferroni&#39;s post-hoc test for comparison of paired data.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56231/56231fig1.jpg" /> 
Figure 1: Incision. 
The skin is incised over 10 mm from supra-sternal notch to mid-sternum and the thyroid is retracted with a stay suture. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56231/56231fig2.jpg" /> 
Figure 2: Bone nipper. 
This instrument allows a short and precise cut in the bone for a 3-4 mm upper partial superior mini-sternotomy. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56231/56231fig3.jpg" /> 
Figure 3: Exposure. 
Following retraction of the sternal edges with 7/0 stay sutures, the aortic arch, right innominate and left common carotid arteries together with the trachea are exposed. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56231/56231fig4.jpg" /> 
Figure 4: A. Tying forceps.&#160;These forceps are necessary to perform a gentle and blunt dissection behind the sternum and around the aortic arch. B. Ligation aid. This is the key instrument for realizing a delicate and atraumatic passage under the aortic arch both in TAC and sham-operated mice. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56231/56231fig5.jpg" /> 
Figure 5: Passage under the aortic arch. 
A segment of 6/0 silk ligature is passed under the aortic arch using the ligation aid and placed between the right innominate and left common carotid arteries. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56231/56231fig6.jpg" /> 
Figure 6: Preparation for ligation. 
A short segment 2-3 mm of a blunted 27-gauge needle is placed over the aortic arch. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56231/56231fig7.jpg" /> 
Figure 7: Transverse aortic constriction. 
The silk suture is tied over the needle and the aortic arch between the right innominate and left common carotid arteries using tying forceps. The silk instead of polypropylene suture is preferred for the aortic ligation because the knot will better hold. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56231/56231fig8.jpg" /> 
Figure 8: Validation of transverse aortic constriction. 
The induction of cardiac hypertrophy by our minimally invasive transverse aortic constriction is demonstrated by significant increase in heart weight/body weight ratio in banded (black bars) as compared to sham operated (white bars) mice. The cardiac hypertrophy is already present at D7 after surgery and increases progressively over time up to D28 (n=6-10 per group. **P&#60;0.01, ***P&#60;0.001, ****P&#60;0.0001). Data are presented as mean &#177; SEM (error bars). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56231/56231fig9v2.jpg" /> 
Figure 9: Left (A) and right (B) ventricle/ body weight ratio. 
During the observation period, the left ventricle/body weight ratio increases while the right ventricle/body weight ratio remains similar in TAC (black bars) compared to sham-operated (white bars) animals. This confirms left ventricular hypertrophy without modification in the right ventricle, and strengthens the validation of our technique (n=6-10 per group. **P&#60;0.01, ***P&#60;0.001, ****P&#60;0.0001).Data are presented as mean &#177; SEM (error bars). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig9v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56231/56231fig10.jpg" /> 
Figure 10: BNP-mRNA expression. 
mRNA expression of brain natriuretic protein (BNP), atrial natriuretic protein (ANP), angiotensin converting enzyme (ACE), collagen 1a1 (Col1a1) and transforming growth factor &#223; (TGF&#223;), positive controls for cardiac hypertrophy in aortic-banded (black bar) vs sham animals (white bar) (n=6 per group) at D14. Expression is calculated as 2(-&#916;Ct) where the calibrator is the mRNA level of the Gapdh reference gene. Data are presented as mean &#177; SEM (error bars). *P&#60;0.05, **P&#60;0.01, ***P&#60;0.001 compared to sham group (t-test). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56231/56231fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy at JoVE.com

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

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 ...

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24.2K Views.  University of Zurich. Het doel van dit protocol is te beschrijven stap voor stap de techniek van minimaal invasieve dwarse aorta vernauwing (TAC) in muizen. Door eliminatie van intubatie en ventilatie die verplicht is voor de gebruikte standaardprocedure zijn, minimaal invasieve TAC vereenvoudigt de operatieve procedure en vermindert de stam het dier op te zetten.

Video: Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular 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 ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Biology

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

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 ...

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 ...

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.

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, ...

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 Modified Technique for Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of pressure overload in mouse models. The main challenge of this procedure is to clearly visualize the transverse aortic arch and precisely band the target vessel. Classical approaches perform a partial thoracotomy to expose the transverse aortic arch. However, it is an open-chest model that causes a rather large surgical trauma and requires a ventilator during the surgery. To prevent unnecessary trauma and simplify the operating procedure, the aortic arch is approached via the proximal proportion of the sternum, reaching and binding the target vessel using a small self-made retractor that contains a snare. This procedure can be conducted without entering the pleura cavity and does not need a ventilator or microsurgical operation, which leaves the mice with physiological breathing patterns, simplifies the procedure, and significantly reduces operation time. Due to the less invasive approach and less operation time, mice can undergo fewer stress reactions and recover rapidly.

The present protocol describes a modified and simplified technique with a minimally invasive transverse aortic constriction (TAC) procedure using a self-made retractor. This procedure can be conducted without a ventilator or microscope and introduces pressure overload, eventually leading to cardiac hypertrophy or heart failure.

Transverse aortic constriction (TAC) is a frequently used surgery in research regarding heart failure and cardiac hypertrophy based on the formation of ...

Transverse Aortic Arch Banding in Mouse Model: A Minimally Invasive Technique in Mice for Induction of High Pressure in the Left Ventricle

Source: Zaw, A. M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55293" target="_blank">Minimally Invasive Transverse Aortic Constriction in Mice</a>. J. Vis. Exp. (2017)
In this video, we demonstrate an experimental protocol for minimally invasive transverse aortic constriction in mice to examine pressure overload-induced heart failure.

Source: Zaw, A. M. et al. Minimally Invasive Transverse Aortic Constriction in Mice. J. Vis. Exp. (2017)
In this video, we demonstrate an experimental ...

JoVE Encyclopedia of Experiments

Rodent Models

Adult

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://www-jove-com.remotexs.ntu.edu.sg/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.

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

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