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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 "Regional Ethics Committee for Animal Experimentation CREMEAS" (#2016092816207606).

1. Preparation for surgery

1. Maintain the mice for one week after arrival in the animal facility on a 12h/12h light/dark cycle, in standard cag....

Results

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.

Discussion

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 authors^2,8. 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.......

Materials

List of materials used in this article

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