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 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.
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.
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
2. Surgery
3. Post-operative recovery
4. Heart harvest
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±0.2 versus 4.1±0.05 mg/g, P<0.01) on, and remained significantly higher up to D28 (5.8±0.3 versus 4.1±0.1 mg/g, P<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 ß (TGFß) 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's post-hoc test for comparison of paired data.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4: A. Tying forceps. 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 6: Preparation for ligation.
A short segment 2-3 mm of a blunted 27-gauge needle is placed over the aortic arch. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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<0.01, ***P<0.001, ****P<0.0001). Data are presented as mean ± SEM (error bars). Please click here to view a larger version of this figure.
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<0.01, ***P<0.001, ****P<0.0001).Data are presented as mean ± SEM (error bars). Please click here to view a larger version of this figure.
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 ß (TGFß), 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(-ΔCt) where the calibrator is the mRNA level of the Gapdh reference gene. Data are presented as mean ± SEM (error bars). *P<0.05, **P<0.01, ***P<0.001 compared to sham group (t-test). Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
This work was supported by a grant (N° 32016) of the Swiss Cardiovascular Foundation to RT.
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 |