Name: technique of minimally invasive transverse aortic constriction in mice for induction of left ventricular hypertrophy
Uploaded: 2017-09-25T12:30:00
Description: Watch this Scientific Journal Video about Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy at JoVE.com

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. Pr...

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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 whol...

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.

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	<tbody>
		<tr height="20">
			<td height="20" style="width:216px;height:20px;">Surgical microscope</td>
			<td style="width:108px;">Olympus</td>
			<td style="width:171px;">SZX2-TR30</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Razor</td>
			<td>Rowenta</td>
			<td>Nomad TN3650FO</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:495px;height:20px;">Sutures:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polypropylene 7/0</td>
			<td>Ethicon</td>
			<td>BV-1X</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polypropylene 6/0</td>
			<td>BBraun</td>
			<td>C0862061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silk 6/0 ligature&#160;</td>
			<td>FST</td>
			<td>18020-60</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polypropylene 4/0</td>
			<td>Ethicon</td>
			<td>8683</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polypropylene 5/0</td>
			<td>Ethicon</td>
			<td>Z303</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:495px;height:20px;">Drugs:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamin</td>
			<td>Merial</td>
			<td>Imalg&#232;ne 1000, LBM154AD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine</td>
			<td>Bayer</td>
			<td>Rompun 2%, KP09PPC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine</td>
			<td>Ceva</td>
			<td>Vetergesic, 072013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:495px;height:20px;">Instruments:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone nippers</td>
			<td>Fine Surgical Tools</td>
			<td>16101-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ligation aid</td>
			<td>Fine Surgical Tools</td>
			<td>18062-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tying forceps</td>
			<td>Fine Surgical Tools</td>
			<td>18026-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle holder Crile-Wood</td>
			<td>Fine Surgical Tools</td>
			<td>12003-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsurgery forceps&#160;</td>
			<td>Fine Surgical Tools</td>
			<td>11003-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsurgery forceps&#160;</td>
			<td>Fine Surgical Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue forceps</td>
			<td>Fine Surgical Tools</td>
			<td>11021-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsurgery needle holder</td>
			<td>Fine Surgical Tools</td>
			<td>12076-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsurgery scissors</td>
			<td>Fine Surgical Tools</td>
			<td>91501-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayo scissors</td>
			<td>Fine Surgical Tools</td>
			<td>14511-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">11-blade knife</td>
			<td>Fine Surgical Tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:495px;height:20px;">RNA extraction and qPCR:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TriReagent</td>
			<td>Euromedex</td>
			<td>TR-118-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rneasy Mini kit</td>
			<td>Qiagen</td>
			<td>74704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit Fluorimetric RNA assay</td>
			<td>Fisher Scientific</td>
			<td>10034622</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA 6000 Nano kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA kit</td>
			<td>Fisher Scientific</td>
			<td>10400745</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman Master Mix</td>
			<td>Fisher Scientific</td>
			<td>10157154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman BNP primers</td>
			<td>Fisher Scientific</td>
			<td>Mm01255770_g1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:216px;height:20px;">Taqman ANP primers</td>
			<td>Fisher Scientific</td>
			<td>Mm01255747_g1&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman ACE primers</td>
			<td>Fisher Scientific</td>
			<td>Mm00802048_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman Col1a1 primers&#160;</td>
			<td>Fisher Scientific</td>
			<td>Mm00801666_g1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman TGFb primers</td>
			<td>Fisher Scientific</td>
			<td>Mm01178820_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman Gapdh primers</td>
			<td>Fisher Scientific</td>
			<td>Mm99999915_g1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ABIPrism&#160; Thermocycler</td>
			<td>Applied Biosystems</td>
			<td>7000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:495px;height:20px;">Software:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GraphPad Prism</td>
			<td>GraphPad</td>
			<td>Prism 7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:495px;height:20px;">Animal food</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:216px;height:27px;">Complete diet for adult rats/mice</td>
			<td style="width:108px;">Safe</td>
			<td style="width:171px;">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.
<p class="jove...

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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