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 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.
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.
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 >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
2. Preoperative Doppler Measurement
3. Thoracotomy
4. Banding of the Transverse Aorta
5. Confirmation of Successful Ligation of the Transverse Aorta
6. Heart harvest
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 flow velocity in the left carotid artery.
The efficacy of TAC and its resulting hypertrophy was validated by calculation of heart weight/body weight ratios (HW/BW; mg/g) of C57BL/6J male mice at day 3, 6, and 21 days post-surgery. The HW/BW ratios significantly increased in TAC mice compared to non-banded mice 6 days after surgery (4.78 ± 0.18 vs7.66±1.43 mg/g, p <0.0001). This ratio was nearly constant after 21 days (4.8 ± 0.11 vs7.81 ± 0.65 mg/g, p <0.0001) (see Figure 3). The survival rate is mainly dependent on intra-operative bleeding: it can be reduced to under 5% through regular practice. The survival rate after 21 days depends mainly on the genotype. For mice not suffering from functional heart diseases the survival rate amounts to >85%. The survival rate in the presented C57BL/6J mice after 21 days amounted to 88%.
Systolic blood pressure and cardiac function was measured in intubation anesthesia and performed with a 1.4 French pressure conductance catheter8 as described by others.9 Heart rate (HR) has a significant effect on left ventricular (LV) contractility. There were no differences in the heart rates (HR) of aortic banded and non-aortic banded mice (p = 0.1456) after 21 days (see Figure 4A). A constant banding of the aorta (p =<0.0001) was proven by an increased systolic blood pressure measured after 21 days (see Figure 4B).
As has been discussed in the literature, C57BL/6J mice are commonly known to develop eccentric hypertrophy with systolic dysfunction10 after TAC. An increase of left ventricular diameter was found, which also appears significant in pressure volume measurements. End-systolic volume increased from 16.25 µL (± 1.935 µL) to 23.31 µL (± 1.617µL). This change was significant (p = 0.0131) (see Figure 4C). End-diastolic volume increased from 25.81 µl (± 1.852 µL) to 31.24 µl ± (1.093 µL). This change was significant (p = 0.0268) (see Figure 4D).
One-way ANOVA followed by Bonferroni's posthoc testing was performed to compare TAC and sham groups. In case of pressure volume measurements, groups were compared using an unpaired t-test with Welch's correction. All data has been presented as mean ± SEM (error bars).
Figure 1: The surgical approach via the 2nd intercostal space at 200% magnification. This picture was taken with the surgical microscope and displays the aortic arch with a thread between the brachiocephalic artery and left common carotid artery. Please click here to view a larger version of this figure.
Figure 2: Representative pulsed-wave Doppler imaging from both carotid arteries (sham vs. TAC mice). A) Pulsed-wave Doppler imaging of the left carotid artery before TAC. B) Pulsed-wave Doppler imaging of the right carotid artery before TAC. C) Image ofPulsed-wave Doppler of the left carotid artery after TAC.The blood flow velocity is reduced compared to figure 2A. D) Pulsed-wave Doppler of the right carotid artery after TAC. The blood flow velocity is increased in comparison to figure 2B. Please click here to view a larger version of this figure.
Figure 3: Heart weight / body weight ratio. Cardiac hypertrophy is induced due to TAC. This is demonstrated by a significant increase in heart weight/body weight ratio. Mice without aortic banding (i.e. sham mice; white bars) were compared to TAC operated mice (black bars) after 3, 6, and 21 days. 6 days after TAC the heart weight /body weight ratio increased significantly in TAC mice. This effect is only slightly pronounced after 21 days. Significance was set to p =<0.05. ns = not significant; ****p <0.0001. Data are presented as mean ± SEM (error bars). n = 6 – 9 per group. Please click here to view a larger version of this figure.
Figure 4: Hemodynamic parameters measured via pressure–volume catheter in mice (C57BL/6J) with and without TAC 21 days after surgery: A) Heart rate (HR) in beats per minute (bpm). There was no difference in HR in both groups indicating a comparable narcosis during the invasive measurements. B) Systolic blood pressure in the right common carotid artery (sBP). The significant increase of sBP after 21 days indicates a constant constriction of the aortic arch. C) End-systolic volumes (ESV) are significantly increased (p = 0.0131) after 21 days and show an increased afterload due to the TAC induced dilatation of the ventricle. D) End-diastolic volume (ESV) is increased (p =0 .0268). Significance was set to p =<0.05. ns = not significant; *p <0.05; ****p <0.0001. Data are presented as mean ± SEM (error bars); n = 8 – 13 per group. Please click here to view a larger version of this figure.
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 under the aortic arch. A too tight suture around the aorta may cause a fatal reduction of blood flow to important organs such as the kidneys. According to the law of Hagen-Poiseuille, flow is mainly dependent on the radius. Therefore, some weight-adapted spacers were used in our protocol. This procedure makes this model more universally applicable, particularly in regard to very young or old mice, depending on the individual experimental setup.
Surgical trauma itself induces an immune response and should be reduced to an absolute minimum to prevent distorting effects. Fast recovery and high survival rates are mandatory, especially in complex animal models. Historically, unlike thoracotomy in human patients, the rib cage in mice is not restored after TAC surgery. Therefore, restitution to physiological breathing movements is limited due to the free floating ribs, which are not reconnected to the sternum.
Minimally invasive techniques for TAC are also used by others12,13. In both models, the aortic arch is reached through a midline incision and an upper partial sternotomy. Although both models are less invasive than open chest models, surgeons have to remove ribs or parts of the sternum to reach the aorta. We believe that maintaining the physiology of the whole rib cage aids faster recovery. Therefore, this protocol improves already existing protocols and helps minimize the surgical trauma itself.
Due to the more apical surgical access, a post-surgical hyperinflation of the lungs for prevention of atelectasis or pneumothoraces, as has been sometimes described4,14, is not required. This access prevents a barotrauma of the lungs, which can be induced by clamping the expiratory tube to open up atelectasis in existing models. This protocol also includes an individualized physiological ventilation strategy. It is tempting to speculate that an individually adapted ventilation aids in reducing ventilator-associated complications such as barotrauma. A weight adapted ventilation strategy was used to avoid effects on the systemic cytokine production by the ventilation itself15.
In conclusion, these techniques represent an alternative and improved model for inducing cardiac hypertrophy in mice.
Although trauma is minimized by avoiding thoracotomy, the superior effect regarding the reduction of inflammation is not shown in this publication. Unfortunately, limitations set by animal protection laws did not allow us to perform open chest TAC in parallel with minimal invasive TAC for comparison because this minimally invasive model has been established for years already. Therefore, these statements are based on the previous experiences of our group.
The authors have nothing to disclose.
We thank Stilla Frede and Susanne Schulz for their technical assistance. This study received no funding.
Pressure-volume catheter | Millar Instruments, USA | SPR-839 | |
Mouse ventilator | Harvard Apparatus GmbH, Germany | Minivent – TYPE 845 | |
Mouse ventilator | Harvard Apparatus GmbH, Germany | Y-connection with intubation cannula OD 1.2mm 73-2844 | |
Vaporizer | Dräger Medical AG&CO.KG, Germany | 19.3 Isofluran-Vaporizer (a newer version is available under catalog number D-877-2010) | |
Microscope | Leica Microsystems, Germany | MZ 7.5 | |
Light source | Schott AG, Germany | KL 1500 LCD | |
6-0 Prolene | Ethicon, USA | Polypropylene suture BV-1 9.3 mm 3/8c | suture for surgery |
Seraflex | Serag Wiessner, Germany | USP 5/0 schwarz; IC108000 | suture for constriction |
Homoeothermic Controlled Operating Tables | Harvard Apparatus GmbH, Germany | Typ 872/3 HT with tripod stand and homoeothermic controller Type 874; 73-4233 | |
Flexible Rectal Probe | Harvard Apparatus GmbH, Germany | 1.6 mm OD; 55-7021 | |
Doppler Signal Visualisation Instrument | Indus Instruments, USA | Doppler Signal Processing Workstation (DSWP) with 20MHz Pulsed Doppler Module | |
Doppler Probe | Indus Instruments, USA | 20MHz Tubing-mounted Probe |