We thank Stilla Frede and Susanne Schulz for their technical assistance. This study received no funding.

This protocol was approved by the Ethics Committee for Animal Experimentation LANUV Recklinghausen (#84-02.04.2016.A374). Generally, this procedure is performed on adult mice &#62;10 weeks of age. However, it is possible to perform this surgery on younger animals as well. Surgical tools must be sterilized before use and all steps are to be performed under aseptic conditions.
1. Induction of Anesthesia and Intubation
<ol>
	<li>Inject buprenorphine 0.1 &#181;g/g body weight intraperitoneally f...

<ol>
	<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 Clifton NJ. 573, 115-137 (2009).</li><li>Tarnavski, O., McMullen, J. R., Schinke, M., Nie, Q., Kong, S., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cardiac+surgery:+comprehensive+techniques+for+the+generation+of+mouse+models+of+human+diseases+and+their+application+for+genomic+studies.">Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies.</a> Physiol Genomics. 16 (3), 349-360 (2004).</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 U S A. 88 (18), 8277-8281 (1991).</li><li>deAlmeida, 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 JoVE. (38), (2010).</li><li>Grossman, W., Jones, D., McLaurin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wall+stress+and+patterns+of+hypertrophy+in+the+human+left+ventricle.">Wall stress and patterns of hypertrophy in the human left ventricle.</a> J Clin Invest. 56 (1), 56-64 (1975).</li><li>Hartley, C. J., Reddy, A. K., Madala, S., Michael, L. H., Entman, M. L., Taffet, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doppler+estimation+of+reduced+coronary+flow+reserve+in+mice+with+pressure+overload+cardiac+hypertrophy.">Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy.</a> Ultrasound Med Biol. 34 (6), 892-901 (2008).</li><li>Reddy, A. K., 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=Pulsed+Doppler+signal+processing+for+use+in+mice:+applications.">Pulsed Doppler signal processing for use in mice: applications.</a> IEEE Trans Biomed Eng. 52 (10), 1771-1783 (2005).</li><li>Shioura, K. M., Geenen, D. L., Goldspink, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cardiac+function+with+the+pressure-volume+conductance+system+following+myocardial+infarction+in+mice.">Assessment of cardiac function with the pressure-volume conductance system following myocardial infarction in mice.</a> Am J Physiol - Heart Circ Physiol. 293 (5), H2870-H2877 (2007).</li><li>Zhang, B., Davis, J. P., Ziolo, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Catheterization+in+Mice+to+Measure+the+Pressure+Volume+Relationship:+Investigating+the+Bowditch+Effect.">Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect.</a> JoVE J Vis Exp. (100), e52618 (2015).</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), H2119-H2130 (2007).</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>Zaw, A. M., Williams, C. M., Law, H. K. W., Chow, B. K. C. <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+Transverse+Aortic+Constriction+in+Mice.">Minimally Invasive Transverse Aortic Constriction in Mice.</a> J Vis Exp JoVE. (121), (2017).</li><li>Tavakoli, R., Nemska, S., Jamshidi, P., Gassmann, M., Frossard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+of+Minimally+Invasive+Transverse+Aortic+Constriction+in+Mice+for+Induction+of+Left+Ventricular+Hypertrophy.">Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy.</a> J Vis Exp. (127), (2017).</li><li>Kim, S. -. C., Boehm, O., Meyer, R., Hoeft, A., Kn&#252;fermann, P., Baumgarten, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Murine+Closed-chest+Model+of+Myocardial+Ischemia+and+Reperfusion.">A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion.</a> J Vis Exp JoVE. (65), (2012).</li><li>Veldhuizen, R. A., Slutsky, A. S., Joseph, M., McCaig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+ventilation+of+isolated+mouse+lungs+on+surfactant+and+inflammatory+cytokines.">Effects of mechanical ventilation of isolated mouse lungs on surfactant and inflammatory cytokines.</a> Eur Respir J. 17 (3), 488-494 (2001).</li></ol>

The rapid onset of hypertension due to TAC differs from clinically relevant hypertrophy caused by aortic stenosis or hypertension. Nevertheless, the use of small animal models to induce heart failure has many advantages and is, therefore, chosen by many investigators11. This closed chest-model improves the already existing models of the surgical technique to induce transverse aortic constriction in mice4.
The most critical step is the passage und...

Mouse models are often used to mimic human diseases1. Transverse aortic constriction (TAC) is used to induce pressure overload and left ventricular hypertrophy2. The open-chest TAC model in mice was validated by Rockman et al.3 and the surgical procedure is described in detail by DeAlmeida et al.4. Banding of the transverse aorta is more favorable in comparison to abdominal aortic constriction because a larger portion of the circulation can compensate negative effects of this latter procedure2.
The banding of the transverse aorta leads to an increased arterial pressure in the ascending aorta and brachiocephalic artery but leaves sufficient perfusion of the organs via the distal vessels (i.e. the left common carotid artery, the left subclavian artery, and descending aorta). This leads to an increased cardiac afterload and an elevated cardiac wall stress. The wall stress subsequently decreases due to fiber thickening5. The chronic change in cardiac hemodynamics results in maladaptation and dilatation of the left ventricle. This way the TAC creates a reproducible model of cardiac hypertrophy eventually leading to heart failure.
The standard procedure for TAC as described by DeAlmeide et al.4 approaches the aortic arch via a partial upper thoracotomy via dissection of the ribs or the sternum and entering the mediastinum as well as the pleural cavity. This allows for a good view of the aortic arch and its side branches. Unfortunately, the dissected ribs cannot be reattached, which leaves them floating freely and thereby altering the breathing dynamics.
We, therefore, established a minimally invasive closed-chest approach to the aortic arch using a lateral surgical approach via the 2nd intercostal space. The greatest advantage of this model is the ability to perform TAC without even cutting through the ribs. The surgical trauma is limited to the incision of the skin and the dissection of the intercostal muscles. This procedure minimizes the trauma itself and helps to maintain adequate chest stability.
Here we describe a detailed step-by-step procedure to perform TAC surgery in mice without performing the total or the upper thoracotomy. High frequency Doppler was used to ensure the success of TAC as previously described 6,7.

<table border="0" cellpadding="0" cellspacing="0" width="1303">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:312px;">Pressure-volume catheter</td>
			<td style="width:243px;">Millar Instruments, USA</td>
			<td style="width:591px;">SPR-839</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse ventilator</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>Minivent - TYPE 845</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse ventilator</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>Y-connection with intubation cannula OD 1.2mm 73-2844</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vaporizer</td>
			<td>Dr&#228;ger Medical AG&#38;CO.KG, Germany</td>
			<td>19.3 Isofluran-Vaporizer (a newer version is available under catalog number&#160; D-877-2010)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope&#160;</td>
			<td>Leica Microsystems, Germany</td>
			<td>MZ 7.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light source&#160;</td>
			<td>Schott AG, Germany</td>
			<td>KL 1500 LCD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-0 Prolene</td>
			<td>Ethicon, USA</td>
			<td>Polypropylene suture BV-1 9.3 mm 3/8c</td>
			<td>suture for surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Seraflex</td>
			<td>Serag Wiessner, Germany</td>
			<td>USP 5/0 schwarz;&#160; IC108000&#160;</td>
			<td>suture for constriction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Homoeothermic Controlled Operating Tables</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>Typ 872/3 HT with tripod stand and homoeothermic controller Type 874; 73-4233</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flexible Rectal Probe</td>
			<td>Harvard Apparatus GmbH, Germany</td>
			<td>1.6 mm OD; 55-7021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Doppler Signal Visualisation Instrument</td>
			<td>Indus Instruments, USA</td>
			<td>Doppler Signal Processing Workstation (DSWP) with 20MHz Pulsed Doppler Module</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doppler Probe</td>
			<td>Indus Instruments, USA</td>
			<td>20MHz Tubing-mounted Probe</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A successful TAC guarantees the induction of pressure overload and left ventricular hypertrophy. An ad hoc validation of pressure overload can be achieved using Doppler flow velocity measurement as shown in Figure 2. While preoperative blood flow velocity is equal in both carotid arteries, TAC causes an augmented blood velocity in the right carotid artery due to elevated pressure in the left ventricle and aorta while causing post-stenotic attenuated blood flo...

Watch this Scientific Journal Video about A Closed-chest Model to Induce Transverse Aortic Constriction in Mice at JoVE.com

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

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 ...

The overall goal of this procedure, is to perform transverse aortic constriction, in a murine closed chest model, inducing left ventricular myocardial hypertrophy. This minimal invasive technique, helps to reduce respiratory problems, and minimize the inflammatory response. This method can help us answer key questions, in the field of immunology, for example, the response of cardiac macrophagia in the development of left ventricular hypertrophy.The main advantage of this technique is, that the rib cage remains intact, and therefore the trauma is minimized. Since spaces and tidal volumes are adapted to body weight, this protocol can be used in different sized mice. It also reduces surgical trauma, and decreases deviation in measurement values.After anasthetizing a mouse, according to the text protocol, move the mouse to a temperature controlled operating table, and detach the nosecone that is connected to the anesthesia induction box to maintain narcosis. With a nylon suture, fix the upper incisors of the mouse. Pinch the tail to insure the absence of a reflex.Use adhesive tape to fixate the extremities. After inserting a rectal probe, apply depilatory cream to the throat and upper chest, according to the manufacturer's instructions, and allow it to sit for one minute. Then, wipe off the cream and repeat if necessary.With the ventilator settings adjusted to physiological parameters, and the animal under a surgical microscope, incise the skin at the mid line about three millimeters under the mandibular down to the second rib. Identify the midline and connective tissues of the submandibular gland. Then, use two angled forceps to gently and bluntly divide the gland at the midline, and explore the tracheal muscle.Gently prepare the trachea, by bluntly pulling the paratracheal muscles apart. Use forceps to pull on the tongue to straighten the throat for easier intubation, and gently insert an intubation canula inside the trachea. Confirm the intubation by direct visualization of the tube inside the trachea, and by checking for proper chest movement.Prepare both carotid arteries, which lay adjunct to the trachea, by using forceps to gently pull the connective tissue apart. Apply ultrasound gel to the tip of the 20 hertz doppler probe, and place the tip of the probe on the right and the left carotid artery, at an angle of less that 45 degrees. Slowly rotate the probe to move it lateral and medial to find a doppler signal, and then tilt the probe to optimize the signal.Using a fresh set of sterile gloves and scissors, expand the skin incision down to the second intercostal space. Then, with the tip of the forceps, open the second intercostal space, and insert the retractors. At 200%magnification, identify midline and connective tissues.Then, use angled forceps to gently divide the thymus. Remove the fatty tissue until the aortic arch can be clearly seen. With angled thumb forceps, prepare a tunnel under the transverse aorta, between the brachiocephalic artery, and left common carotid artery.Then, using fine forceps, hold a 6-0 suture, and pass the thread under the aortic arch. With the second forceps, take the thread from the other side of the arch. Carefully place the spacer parallel to the transverse aorta.Prepare a loose double knot on the spacer and insure optimal placement of the spacer in parallel to the aorta. Then, tie the first knot, and quickly make a second contrary knot, before promptly removing the spacer. The most critical step of the whole procedure is to tie both knots quickly and tightly, and remove the spacer immediately.Close the second intercostal space, with a 6-0 polypropylene suture, and pay special attention to the subclavian vessels when ligating. Then, using a 6-0 polypropylene suture, stitch the skin in a continuous suture pattern. Confirm ligation, and carry out post operative care, according to the text protocol.These doppler flow velocity measurements validate a successful TAC, which causes an augmented blood velocity in the right carotid artery, due to elevated pressure in the left ventricle and aorta, while causing post stenotic attenuated blood flow velocity in the left carotid artery. As shown in this graph, heart weight and body weight ratios significantly increased in TAC mice, compared to non-banded mice, six days after surgery. This ratio was nearly constant after 21 days.Heart rate has a significant effect on left ventricular contractility and cardiac output. This graph reveals that there were no differences in the heart rates of aortic banded and non aortic banded mice, after 21 days. As demonstrated here, a constant banding of the aorta, was proven by an increased systolic blood pressure, measured via a left ventricular catheter after 21 days.C57 black six mice, are commonly known to develop eccentric hypertrophy with systolic dysfunction after TAC. An increase of left ventricular diameter was found, which also appears significant in pressure volume measurements. As a result, end systolic and diastolic volumes increased significantly.Once mastered, this technique can be performed in under 10 minutes, if it's done properly. Finally, this procedure provides an optimized analysis of the immune response in left ventricular hypertrophy, because the minimal surgical trauma restricts the unspecific induction of the immune system. After watching this video, you should have a good understanding of how to perform standardized, minimal invasive transverse aortic constriction.

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18.5K 조회수.  University Hospital Bonn. The overall goal of this procedure, is to perform transverse aortic constriction, in a murine closed chest model, inducing left ventricular myocardial hypertrophy. This minimal invasive technique, helps to reduce respiratory problems, and minimize the inflammatory response. This method can help us answer key questions, in the field of immunology, for example, the response of cardiac macrophagia in the development of left ventricular hypertrophy.The main advantage of this technique is, ...